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Archive for the ‘Discovery process’ Category

Outline of Medical Discoveries between 1880 and 1980

Curator: Larry H Bernstein, MD, FCAP

This is the first of a two part series tracing the developments in medical diagnosis and treatment, and herein, tracing the scientific events of the 19th century that accelerated and created the emergent events that brought together physics, organic and physical chemistry, electronics, computational biology.

Part I. Anatomy and Physiology

The first Nobel Prize in Physiology was awarded to Ivan Pavlov for work on digestion in 1904.  The presentation speech refers to the groundbreaking work of Vesalius and Harvey in his presentation address, citing their passionate pursuit of knowledge.  He credits the work of a young American physician, William Beaumont, who served as the only doctor on Michigan’s Mackinac Island in the French and Indian war in 1822, and who observed the gastric secretion from the gastric fistula of a wounded soldier. (see John Karlawish, Open Wound, University of Michigan Press, 2011). This was the basis for the work by Pavlov on dogs that extends our understanding of the telationship of the central nervous system to the digestive processes.

The Nobel Prize in Physiology or Medicine 1906 was awarded jointly to Camillo Golgi and Santiago Ramón y Cajal “in recognition of their work on the structure of the nervous system”. Golgi first opened the field of neuroanatomy with the silver staining method, and Cajal contributed equally to establishing the foundation for this research of great complexity.

The Nobel Prize in Physiology or Medicine 1909 was awarded to Emil Theodor Kocher for his work on the physiology, pathology, and surgery  of the thyroid gland. It had already been established that the enlargement of the thyroid compresses the trachea, and that complete removal has morbid effects. It was expressed by Kocher in 1883 that removal of the thyroid as a consequence of surgery must leave behind a functioning portion of the gland.

This was later followed by the establishment of a great medical institution Dr. William Worrall Mayo, a frontier doctor, and his two sons, Dr. William J. Mayo and Dr. Charles H. Mayo, Mayo Clinic.

The elder Dr. Mayo emigrated from his native England to the United States in 1846. He became a doctor in 1850. In 1863 he was appointed a surgeon for the enrollment board in southern Minnesota, to examine recruits for the Union Army, and settled in Rochester, Minn. His dedication to medicine became a family tradition when his sons, Drs. William James Mayo and Charles Horace Mayo, joined his practice in 1883 and 1888, respectively.

In 1883, a tornado swept through Rochester leaving in its wake many deaths and injuries. Temporary hospital quarters were set up in offices and hotels. Nuns from the Sisters of St. Francis, a teaching order, were recruited as nurses. The experience inspired Mother Alfred Moes to request that the Drs. Mayo join with the Sisters to build the first general hospital in southeastern Minnesota. The 27-bed Saint Mary’s Hospital opened in 1889 as a result of this partnership.

mayo-brothers

mayo-brothers

As the demand for their services increased, they asked other doctors and basic science researchers to join them in the world’s first private integrated group practice. In 1919, the Mayo brothers dissolved their partnership and turned the clinic’s name and assets, including the bulk of their life savings, to a private, not-for-profit, charitable organization now known as Mayo Foundation. It is worth noting that the Mayo Clinic became a favored place to have thyroid surgery, as its location is in the “goiter belt”.

Patients discovered the advantages to a “pooled resource” of knowledge and skills among doctors. In fact, the group practice concept that the Mayo family originated has influenced the structure and function of medical practice throughout the world.

The Nobel Prize in Physiology or Medicine 1912 was awarded to Alexis Carrel “in recognition of his work on vascular suture and the transplantation of blood vessels and organs”. He demonstrated the technique used to suture together open vessels, and even to transplant whole organs from one animal to another with excellent results.

The Nobel Prize in Physiology or Medicine 1920 was awarded to August Krogh “for his discovery of the capillary motor regulating mechanism”.  Harvey had shown in 1628 that the blood traverses the circulation returning to the heart in one minute. Malpighi showed that blood passes from the artery to the vein by capillaries  in 1661.  Krogh demonstrated by very elegant experiments that the quantity of gas that diffuses across the pulmonary alveoli is the same amount of gas that is released to the alveolar space. The importance of this is that the investigations having the aim to determine the process by which the oxygen requirement of the tissues is satisfied.

The Nobel Prize in Physiology or Medicine 1922 was divided equally between Archibald Vivian Hill “for his discovery relating to the production of heat in the muscle” and Otto Fritz Meyerhof “for his discovery of the fixed relationship between the consumption of oxygen and the metabolism of lactic acid in the muscle”. One need not be a physiologist to recognize that muscular activity is essentially bound up with the development of heat, or even with combustion. AV Hill determined the time relationships of heat production in muscle contraction measured galvanometrically, and Otto Meyerhof determined the oxygen consumption in the production of lactic acid. The muscle is regarded as a machine that converts chemical energy to mechanical energy (tension) with the production of heat. The development of heat entirely fails to appear if the supply of oxygen to the muscle is cut off, while the development of heat during the actual twitch, is independent of the presence of oxygen (consistent with Meyerhof’s glycolysis). The relaxation phase is consistent with oxygen uptake during recovery.

Fletcher and Hopkins had shown earlier that muscle not only forms, but also uses lactic acid in the presence of oxygen. Meyerhof determined by parallel determination of the lactic acid metabolism and the oxygen consumption during the recovery of the muscle, which yielded the result that the oxygen consumption does not account for more than1/3 – 1/4 of the lactic acid formed. When lactic
acid is formed an equivalent amount of glycogen in muscle disappears, and when lactic acid disappears, the quantity of
carbohydrate increases by the difference between lactic acid and quantity used in oxygen consumption.

The Nobel Prize in Physiology or Medicine 1923 was awarded jointly to Frederick Grant Banting and John James Rickard
Macleod “for the discovery of insulin”.  In 1857, Claude Bernard discovered that the liver contains glycogen, which converted to glucose, enters the blood stream (and thereby, the urine). Glycosuria became a starting point for the study of diabetes. It is of interest that he could not produce glycosuria by ligation of the pancreatic duct. But in 1889 Mering and Minkowsky did an operation on dogs that removed the pancreas, resulting in glycosuria, and creating a disease comparable to diabetes in humans. If part of the pancreas was left behind, it failed to produce diabetes. Brown-Sequard had called attention to ductless organs in the 1880s that are glands. These were
endocrine glands secreting hormones. Langerhans had shown in 1869 that the pancreas has glands that have no secretion into the pancreatic ducts, and in the beginning of the 1890s Languese surmised that these glands were involved in diabetes mellitus. Schulze and Ssobolev had shown that ligation of the duct resulted in atrophy of the pancreas sparing the islets. Frederick Banting at this time postulated that trypsin degraded the hormone, and with Best and Collip, under MacLeod’s guidance, Banting pursued his idea, and the effective extract was obtained in 1921, and demonstrated in 1922.

Arch Anat Histol Embryol. 1993-1994;75:151-82.

[History of histology in Strasbourg].

Le Minor JM.

Since the cellular theory was formulated in 1839, the University of Strasbourg has held a pioneer place in histology. This new morphological science has had, since its origin, close relations with physiology, and from 1846 to 1871, an original histophysiological school was organized in Strasbourg. The microscope and the study of tissues were considered as a fundamental approach for the progress of biological and medical knowledge. After the German annexation of Alsace, the scientists from this school participated in the renewal of histology in Nancy, Montpellier, and Paris. In 1872, when the new German university was created, an anatomical institute regrouped all aspects of normal morphology: anatomy, histology, and embryology. This was the case until 1918. In 1919, when the Faculty of Medicine was reorganized after Alsace was restored to France, a specific chair and institute of histology were created. This was the beginning of a school of histophysiology which was internationally renowned in the rise of experimental endocrinology. Great discoveries followed one after another: folliculin in 1924 and demonstration of the duality of ovarian hormones, the prominent place of the anterior part of the hypophysis and the demonstration of prolactin in 1928, thyreostimulin in 1929, then study of the other stimulins. In 1946 a chair and institute of medical biology were created. In 1948, a service of electron microscopy was opened.
P. Bouin (1870-1962), M. Aron (1892-1974), J. Benoit (1896-1982), R. Courrier (1895-1986) et M. Klein (1905-1975), were among the famous scientists who worked in histology in Strasbourg in the
period after the French restoration.
The Nobel Prize in Physiology or Medicine 1947

Bernardo Alberto Houssay

“for his discovery of the part played by the hormone of the anterior pituitary lobe in the metabolism of sugar”

He had already begun studying medicine and, in 1907, before completing his studies, he took up a post in the Department of Physiology. He began here his research on the hypophysis which resulted in his M.D.-thesis (1911), a thesis which earned him a University prize.

In 1919 he became Professor of Physiology in the Medical School at Buenos Aires University. He also organized the Institute of Physiology at the Medical School, making it a center with an international reputation. He remained Professor and Director of the Institute until 1943.  He made a lifelong study of the hypophysis and his most important discovery concerns the role of the anterior lobe of the hypophysis in carbohydrate metabolism and the onset of diabetes.

The Nobel Prize in Physiology or Medicine 1950

Edward Calvin Kendall, Tadeus Reichstein and Philip Showalter Hench

“for their discoveries relating to the hormones of the adrenal cortex, their structure and biological effects”

As late as in 1854 the German anatomist, Kölliker, was able to claim in a review of the subject that although the function of the adrenals was still unknown, yet in certain respects great advances had been made. Two quite different parts were now distinguished, an outer part, a fairly firm cortex, and an inner, softer medulla. Kölliker classified the adrenal cortices as ductless glands, which we now call the endocrine organs.

Thomas Addison, the English doctor, observed a rare disease with a fatal course, which was characterized chiefly by anemia, general weakness and fatigue, disturbances in the digestive apparatus, enfeebled heart activity and a peculiar dark pigmentation of the skin. He published a paper 1n 1855, suggesting that this morbid picture made its appearance in persons the greater part of whose adrenals was destroyed. Subsequent experiments in animals showed that removal of the adrenals led to speedy death, the symptoms recalling those known from Addison’s disease.

In 1894 Oliver and Schäfer proved that the injection of a watery extract from the adrenals had extremely pronounced effects. Within a few years adrenaline had been produced from the extract, its composition had been ascertained, and its artificial production accomplished. The more detailed analysis showed effects of the same kind as those resulting on increased activity of the so-called sympathetic nervous system, which innervates internal organs such as the heart and vessels, the intestinal canal, etc.  Attempts to prevent by means of adrenaline the deficiency symptoms following on the removal of the adrenals failed completely. The explanation of this was given when Biedl and others showed that it is the cortex which is of vital importance, not the medulla.

The isolation of the cortin proved to be a difficult task, calling for the combined efforts of a number of research workers. Particularly important contributions were made in this field by Wintersteiner and Pfiffner, and also by Edward Kendall at the Mayo Clinic in Rochester, and Tadeus Reichstein in Basel, and their co-workers. As early as in 1934, Kendall and his group succeeded in preparing from cortex extract what was at first assumed to be pure cortin in crystalline form. They found that it contained carbon, hydrogen, and oxygen, and indicated its empirical formula. But that was only a beginning. There was no reason to suspect that the cortin was not homogeneous; as further experiments proved. In reality Kendall and his co-workers had produced a mixture of different substances closely related to one another, and their work represents the early steps in the crystallization of a whole series of cortin substances. There is at least one active cortical substance – the best known of them all, first named Compound E and now called cortisone or cortone – which was isolated at four different laboratories, among them Kendall’s and Reichstein’s.

As all the cortin substances are closely related to one another, Reichstein’s finding implies that, like the sex hormones, they belong to the large and important group of steroids. The D vitamins and the bile acids, like our most important heart remedies, the active substances in Digitalis leaves and Strophanthus seeds, are also intimately associated with the steroids

The six definitely active cortical hormones are characterized, inter alia, by a double bond in the steroid skeleton; if this double bond disappears, inactive substances are obtained. They differ very inconsiderably from each other chemically. They are built up of 21 carbon atoms, but the number of oxygen atoms in the molecule is three, four, or five. The position of the additional oxygen atoms in the molecule was first established by Reichstein and Kendall, and thus a way was opened for semisynthetic production e.g. from the more easily obtainable bile acids or material from a certain species of Strophanthus. This is of particular importance, since the yield from the adrenals is very poor, at most about 1:1,000,000.

Thanks to the work of Kendall and his school, it has emerged that the comparatively inconsiderable dissimilarities in the matter of the structure of the cortical hormones are accompanied by material differences in respect of the effect. Thus some act especially strongly on the metabolism of sugar, others on the salt and fluid balances, and there are also several other differences. This was illustrated when Compound E was first tested. Pfiffner and Wintersteiner, like the Reichstein group, found that the substance had no, or extremely inconsiderable, life-prolonging effects on animals deprived of the adrenals. On the other hand, Ingle, Kendall’s coworker, observed that it stimulated the muscular work of such animals very strongly.

In the April of 1949, Hench, Kendall, Slocumb and Polley published their experiences in respect of the dramatic effects of cortisone in cases of chronic rheumatoid arthritis. A rapid improvement set in, pains and tenderness in the joints abated or disappeared, mobility increased, so that patients who had previously been complete invalids could walk about freely, and their general condition was also favourably affected. Similar results were obtained with a preparation from the anterior lobe of the pituitary, the so-called ACTH (Adreno-Cortico-Tropic Hormone), which, as the name indicates, stimulates the adrenal cortex to increased activity.

The value of a discovery lies not only in the immediate practical results, but equally much in the fact that it points out new lines of research. This is strikingly illustrated by the research during the last few decades into the cortical hormones, which has already led to unexpected and important new results within widely different spheres.

Nobel Prize in Physiology or Medicine 1966

Charles Huggins

Endocrine-Induced Regression of Cancers

The net increment of mass of a cancer is a function of the interaction of the tumor and its soil. Self-control of cancers results from a highly advantageous competition of host with his tumor. There are multiple factors which restrain cancer – enzymatic, nutritional, immunologic, the genotype and others.Prominent among them is the endocrine status, both of tumor and host – the subjects of this discourse.

The second quarter of our century found the biological sciences much pre-occupied with two noble topics :

  • chemistry and physiology of steroids and
  • biochemistry of organo-phosphorus compounds.

The key to the puzzle of the steroid hormones in cancer was the isolation of crystalline estrone by Doisy et al.2 from extracts of urine of pregnant women. In the phosphorus field there were magnificent findings of hexose phosphates, nucleotides, coenzymes and high-energy phosphate intermediates. These wonderful discoveries provided the Zeitgeist for our work.

Through the portal of phosphorus metabolism we entered on a series of interconnected observations in steroid endocrinology. A program was not prepared in advance for this basic physiologic study. The work was fascinating and informative so that it provided its own momentum and served as an end in itself.

The prostatic cell does not die in the absence of testosterone, it merely shrivels. But the hormone-dependent cancer cell is entirely different. It grows in the presence of supporting hormones but it dies in their absence and for this reason it cannot participate in growth cycles.

A remarkable effect of testosterone is the promotion of growth of its target cells during complete deprival of food. Androstane derivatives conferred on the prostate of puppies a selective nutritional advantage during starvation of 3 weeks whereby abundant growth of this gland-occurred while there was serious cell breakdown in most of the tissues of the body.

At first it was vexatious to encounter a dog with a prostatic tumor during a metabolic study but before long such dogs were sought. It was soon observed that orchiectomy or the administration of restricted amounts of phenolic estrogens caused a rapid shrinkage of canine prostatic tumors.

The experiments on canine neoplasia proved relevant to human prostate cancer; there had been no earlier reports indicating any relationship of hormones to this malignant growth.

Kutscher and Wolbergs9 discovered that acid phosphatase is rich in concentration in the prostate of adult human males. Gutman and Gutman10 found that many patients with metastatic prostate cancer have significant increases of acid phosphatase in their blood serum. Cancer of the prostate frequently metastasizes to bone.

Human prostate cancer which had metastasized to bone was studied at first. The activities of acid and alkaline phosphatases in the blood were measured concurrently at frequent intervals. The methods are reproducible and not costly in time or materials; both enzymes were measured in duplicate in a small quantity (0.5 ml) of serum. The level of acid phosphatase indicated activity of the disseminated cancer cells in all metastatic loci. The titer of alkaline phosphatase revealed the function of the osteoblasts as influenced by the presence of the prostatic cancer cells that were their near neighbors. By periodic measurement of the two enzymes one obtains a view of overall activity of the cancer and the reaction of non-malignant cells of the host to the presence of that cancer. Thereby the great but opposing influences of, respectively, the administration or deprival of androgenic hormones upon prostate cancer cells were revealed with precision and simplicity. Orchiectomy or the administration of phenolic estrogens resulted in regression of cancer of the human prostate whereas, in untreated cases, testosterone enhanced the rate of growth of the neoplasm.

The first indication that advanced cancer can be induced to regress was the beneficial effect of oöphorectomy on cancer of the breast of two women. This empirical observation17 of Beatson in 1896 was remarkable since it was made before the concept of hormones had been developed. The beneficial action of removal of ovaries was not understood until steroid hormones had been isolated 4 decades later.

But why does breast cancer thrive in folks who do not possess ovarian function – in men, old women, and females who have had oöphorectomy?

Farrow and Adair observed that benefits of great magnitude frequently follow orchiectomy in mammary cancer in the human male. Thereby, they established that testis function can sustain mammary cancer.

A half century after the classic invention of Beatson it was found out that adrenal function can maintain and promote growth of human mammary cancer. The adrenal factor supporting growth of cancer was identified when it was shown that bilateral adrenalectomy (with glucocorticoids as substitution therapy) can result in profound and prolonged regression of mammary carcinoma in men and women who do not possess gonadal function. In developing the idea of adrenalectomy for treatment of advanced cancer in man we were considerably influenced by the discovery of Woolley et al. that adrenals can evoke cancer of the breast in the mouse.

Mammary cancers induced in the male rat by aromatics were not influenced by orchiectomy and hypophysectomy; by definition, these neoplasms are hormone-independent. In contrast to male rat, most mammary cancers of men wither impressively after deprival of supporting hormones.

The hormone-responsiveness of established mammary cancers induced in female rat by aromatics or ionizing radiation is identical; it was a newly recognized property of experimental breast cancers. Prior to this finding, clinical study of patients with mammary cancer was the only material available for investigation of hormonal-restraint of neoplasms of the breast.

In female rat, many but far from all of the induced mammary cancers vanished after removal of ovaries or the pituitary. In our experiments hypophysectomy was the most efficient of all methods to cure rat’s mammary cancer.

Malignant cells which succumb to hormone-deprival, by definition, are hormone-dependent. The quality of hormone-dependence resides in the tumor cells whereas their growth is determined by the host’s endocrine status.

Both man and the animals can have some of their cancer cells which are hormone-dependent while other neoplastic cells in the same organism are not endocrine-responsive.

The cure of a cancer after hormone-deprival results from death of the cancer cells whereas their normal analogues in the same animal shrivel but survive. It is a basic proposition in endocrine-restraint of malignant disease that cancer cells can differ in a crucial way from ancestral normal cells in response to modification of the hormonal milieu intérieur of the body.

Cancer is not necessarily autonomous and intrinsically self-perpetuating. Its growth can be sustained and propagated by hormonal function in the host which is not unusual in kind or exaggerated in rate but which is operating at normal or even subnormal levels.

The control of cancer by endocrine methods can be described in three propositions:

  • Some types of cancer cells differ in a cardinal way from the cells from which they arose in their response to change in their hormonal environment.
  • Certain cancers are hormone-dependent and these cells die when supporting hormones are eliminated.
  • Certain cancers succumb when large amounts of hormones are administered.

The Nobel Prize in Physiology or Medicine 1971

Earl W. Sutherland, Jr.

“for his discoveries concerning the mechanisms of the action of hormones”

Part II. Vitamins

The Nobel Prize in Physiology or Medicine 1929

Christiaan Eijkman “for his discovery of the antineuritic vitamin”

Sir Frederick Gowland Hopkins “for his discovery of the growth-stimulating vitamins”

When the 20th century began, the prevailing thought about nutrition rested on the importance of energy requirements, as elucidated by  Rubner, Benedict and others, in the United States, that entails the quantitative measurement of the food value of carbohydrates, fats, and proteins. But there was a misconception of the process in its detail. The quantitative studies of the energetics and of respiratory exchange were not sufficient to explain problems that arise as a result of deficiencies of micronutrients in food intake.  The complexity of these nutritional needs as we now view them is indeed astonishing.

There is a need for indispensable organic substances specific in nature and function of which the quantitative supply is so small as to contribute little or nothing to the energy factor in nutrition. These substances, following the suggestion of Casimir Funk, we have agreed to call vitamins.

In 1881, Lunin, and associate of Bungel noted that a diet of milk was not sufficient to sustain the life of mice, even if the caloric nutrients were adequate. The main lesson taken from the findings was concerned with inorganic nutrients had not been determined that would answer the question. A decade later, Socin, in Bunge’s group, concluded that the deficiency was in the quality of protein.  In an important paper by Professor Pekelharing in 1905 published an astonishing paper following on the work in Bungel’s lab. He noted that there is a substance in milk in small quantities that he was unable to identify that is essential for life.  It is noteworthy that Pekelharing records prolonged endeavours towards the isolation of a vitamin.

Eikman’s work came in the 1880s. He did not at first visualize beriberi clearly as a deficiency disease. The view that the cortical substance in rice supplied a need rather than neutralized a poison was soon after put forward by Grijns and ultimately accepted by Professor Eijkman himself.  The prevailing thinking about nutritional requirements was preoccupied by the methods of calorimetry at the turn of the century.  The idea of “deficiency diseases” was obscured as a result. There was no concept of an indispensable portion of the food supply other than calories, proteins and minerals until 1911-1912.  Hopkins was convinced that the science of nutrition had to come to terms with an explanation for scurvy and rickets, and he needed to use the new science of biochemistry, which was ongoing at Cambridge.

In 1906-1907, he carried out studies of feeding rats casein, along the lines of Bungel.s experiments, and he found variability in the results with different casein preparations.  He next washed the casein so that any soluble substance was extracted and the rats died, but if he added the extract they grew.  He also used butter, with results more favorable than casein, and lard, with unfavorable results.  At the same time he was studying polyneuritis in birds, which took up much time.  He know that he had to extract the substance, but was unaware of the fat solubility in 1910. He published his work in 1912. Soon after the publication of his work, and duting WWI, much research was done in US, by Osborn and Mendel at Harvard, and by McCollum at Johns Hopkins, and the vitamins were separated into “water soluble” and “fat soluble”.

The Nobel Prize in Physiology or Medicine 1937

Albert von Szent-Györgyi Nagyrápolt

“for his discoveries in connection with the biological combustion processes, with special reference to vitamin C and the catalysis of fumaric acid”

http://pharmaceuticalintelligence.com/2014/08/18/studies-of-respiration-lead-to-acetyl-coa/

Szent Gyorgyi was a biochemist who worked with Otto Warburg and others, and had a special interest in muscle metabolism. He delineated a portion of the Krebs cycle (Krebs was also associated with Warburg), that which involves the conversion of fumaric acid to succinate.  He also purified vitamin C (ascorbic acid) from paprika in his native region of Hungary. He later turned his interest to cancer research, for which he was honored by the MD Anderson Cancer Center.

The Nobel Prize in Physiology or Medicine 1934

George Hoyt Whipple, George Richards Minot and William Parry Murphy

“for their discoveries concerning liver therapy in cases of anaemia”

The Nobel Prize in Physiology or Medicine 1943

Henrik Carl Peter Dam “for his discovery of vitamin K”

Edward Adelbert Doisy “for his discovery of the chemical nature of vitamin K”

To further his studies of the metabolism of sterols, Dam obtained a Rockefeller Fellowship and worked in Rudolph Schoenheimer’s Laboratory in Freiburg, Germany, during 1932-1933, and later worked with P. Karrer, of Zurich, in 1935. He discovered vitamin K while studying the sterol metabolism of chicks in Copenhagen. When he returned to Denmark after WWII in 1946, Dam’s main research subjects were vitamin K, vitamin E, fats, cholesterol.

Part III.  Microbiology and Plague

The Nobel Prize in Physiology or Medicine 1901

Emil Adolf von Behring

“for his work on serum therapy, especially its application against diphtheria, by which he has opened a new road in the domain of medical science and thereby placed in the hands of the physician a victorious weapon against illness and deaths”

The Nobel Prize in Physiology or Medicine 1902

Ronald Ross

“for his work on malaria, by which he has shown how it enters the organism and thereby has laid the foundation for successful research on this disease and methods of combating it”

The Nobel Prize in Physiology or Medicine 1905

Robert Koch

“for his investigations and discoveries in relation to tuberculosis”

The Nobel Prize in Physiology or Medicine 1908

The Nobel Prize in Physiology or Medicine 1928

Charles Jules Henri Nicolle

“for his work on typhus”

The Nobel Prize in Physiology or Medicine 1939

Gerhard Domagk

“for the discovery of the antibacterial effects of prontosil”

The Nobel Prize in Physiology or Medicine 1945

Sir Alexander Fleming, Ernst Boris Chain and Sir Howard Walter Florey

“for the discovery of penicillin and its curative effect in various infectious diseases”

The Nobel Prize in Physiology or Medicine 1951

Max Theiler

“for his discoveries concerning yellow fever and how to combat it”

The Nobel Prize in Physiology or Medicine 1952

Selman Abraham Waksman

“for his discovery of streptomycin, the first antibiotic effective against tuberculosis”

The Nobel Prize in Physiology or Medicine 1954

John Franklin Enders, Thomas Huckle Weller and Frederick Chapman Robbins

“for their discovery of the ability of poliomyelitis viruses to grow in cultures of various types of tissue”

The Nobel Prize in Physiology or Medicine 1976

Baruch S. Blumberg and D. Carleton Gajdusek

“for their discoveries concerning new mechanisms for the origin and dissemination of infectious diseases”

Part IV.

Ilya Ilyich Mechnikov and Paul Ehrlich

“in recognition of their work on immunity”

The Nobel Prize in Physiology or Medicine 1919

Jules Bordet

“for his discoveries relating to immunity”

The Nobel Prize in Physiology or Medicine 1930 was awarded to Karl Landsteiner “for his discovery of human blood groups”.

In 1901, in the course of his serological studies Landsteiner observed that when, under normal physiological conditions, blood serum of a human was added to normal blood of another human the red corpuscles in some cases coalesced into larger or smaller clusters. This observation of Landsteiner was the starting-point of his discovery of the human blood groups. In the following year, i.e. 1901, Landsteiner published his discovery that in man, blood types could be classified into three groups according to their different agglutinating properties. These agglutinating properties were identified more closely by two specific blood-cell structures, which can occur either singly or simultaneously in the same individual.

Landsteiner’s discovery of the blood groups was immediately confirmed but it was a long time before anyone began to realize the great importance of the discovery. The first incentive to pay greater attention to this discovery was provided by von Dungern and Hirszfeld when in 1910 they published their investigations into the hereditary transmission of blood groups. Thereafter the blood groups became the subject of exhaustive studies, on a scale increasing year by year, in more or less all civilized countries. In order to avoid, in the publication of research on this subject, detailed descriptions which would otherwise be necessary – of the four blood groups and their appropriate cell structures, certain short designations for the blood groups and corresponding specific cell structures have been introduced. Thus, one of the two specific cell structures, characterizing the agglutinating properties of human blood is designated by the letter A and another by B, and accordingly we speak of «blood group A» and «blood group B». These two cell structures can also occur simultaneously in the same individual, and this structure as well as the corresponding blood group is described as AB.

The fourth blood-cell structure and the corresponding blood group is known as O, which is intended to indicate that people belonging to this group lack the specific blood characteristics typical of each of the other blood groups. Landsteiner had shown that under normal physiological conditions the blood serum will not agglutinate the erythrocytes of the same individual or those of other individuals with the same structure. Thus, the blood serum of people whose erythrocytes have group structure A will not agglutinate erythrocytes of this structure but it will agglutinate those of group structure B, and where the erythrocytes have group structure B the corresponding serum does not agglutinate these erythrocytes but it does agglutinate those with group structure A. Blood serum of persons whose erythrocytes have structures A as well as B, i.e. who have structure AB, does not agglutinate erythrocytes having structures A, B, or AB. Blood serum of persons belonging to blood group O agglutinates erythrocytes of persons belonging to any of the group.

The group characteristics are handed down in accordance with Mendel’s laws. The characteristics of blood groups A, B, and AB are dominant, and opposing these dominant characteristics are the recessive ones which characterize blood group O. An individual cannot belong to blood group A, B, or AB, unless the specific characteristics of these groups are present in the parents, whereas the recessive characteristics of blood group O can occur if the parents belong to any one of the four groups. If both parents belong to group O, then the children never have the characteristics of A, B, or AB. The children must then likewise belong to blood group O. If one of the parents belongs to group A and the other to group B, then the child may belong to group A or B or it may possess both characteristics and therefore belong to group AB. If one of the parents belongs to group AB and the other to group O, then in accordance with Mendel’s law of segregation the AB characteristic can be segregated and the components can occur as separate characteristics in the children.

Even while he was a student he had begun to do biochemical research and in 1891 he published a paper on the influence of diet on the composition of blood ash. To gain further knowledge of chemistry he spent the next five years in the laboratories of Hantzsch at Zurich, Emil Fischer at Wurzburg, and E. Bamberger at Munich.

In 1896 he became an assistant under Max von Gruber in the Hygiene Institute at Vienna. Even at this time he was interested in the mechanisms of immunity and in the nature of antibodies. From 1898 till 1908 he held the post of assistant in the University Department of Pathological Anatomy in Vienna, the Head of which was Professor A. Weichselbaum, who had discovered the bacterial cause of meningitis, and with Fraenckel had discovered the pneumococcus. Here Landsteiner worked on morbid physiology rather than on morbid anatomy. In this he was encouraged by Weichselbaum, in spite of the criticism of others in this Institute.

Up to the year 1919, after twenty years of work on pathological anatomy, Landsteiner with a number of collaborators had published many papers on his findings in morbid anatomy and on immunology. He discovered new facts about the immunology of syphilis, added to the knowledge of the Wassermann reaction, and discovered the immunological factors which he named haptens (it then became clear that the active substances in the extracts of normal organs used in this reaction were, in fact, haptens). He made fundamental contributions to our knowledge of paroxysmal haemoglobinuria.

He also showed that the cause of poliomyelitis could be transmitted to monkeys by injecting into them material prepared by grinding up the spinal cords of children who had died from this disease, and, lacking in Vienna monkeys for further experiments, he went to the Pasteur Institute in Paris, where monkeys were available. His work there, together with that independently done by Flexner and Lewis, laid the foundations of our knowledge of the cause and immunology of poliomyelitis.

http://www.nobelprize.org/nobel_prizes/medicine/laureates/1930/landsteiner-bio.html

His discovery of the differences and identification of the groups that were alike made it possible for blood transfusions to become a routine procedure.  This paved the way for many other medical procedures that we don’t even think twice about today, such as surgery, blood banks, and transplants.

While in medical school, Landsteiner began experimental work in chemistry, as he was greatly inspired by Ernst Ludwig, one of his professors. After receiving his medical degree, Landsteiner spent the next five years doing advanced research in organic chemistry for Emil Fischer, although medicine remained his chief interest. During 1886-1897, he combined these interests at the Institute of Hygiene at the University of Vienna where he researched immunology and serology. These fields were developing rapidly in the late 1800s as scientists explored numerous physiological changes associated with bacterial infection. Immunology and serology then became Landsteiner’s lifelong focus. Landsteiner was primarily interested in the lack of safety and effectiveness of blood transfusions.

Landsteiner is known as the “melancholy genius” because he was so sad and intense, yet he was so systematic, thorough, and dedicated. He wrote 346 papers during his long career contributing to many areas of scientific knowledge. He is considered the father of Hematology (the study of blood), Immunology (the study of the immune system), Polio research, and Allergy research.

The fundamental contribution of Robert A. Good to the discovery of the crucial role of thymus in mammalian immunity

Domenico Ribatti

Immunology. Nov 2006; 119(3): 291–295.

http://dx.doi.org:/10.1111/j.1365-2567.2006.02484.x

Robert Alan Good was a pioneer in the field of immunodeficiency diseases. He and his colleagues defined the cellular basis and functional consequences of many of the inherited immunodeficiency diseases. His was one of the groups that discovered the pivotal role of the thymus in the immune system development and defined the separate development of the thymus-dependent and bursa-dependent lymphoid cell lineages and their responsibilities in cell-mediated and humoral immunity.

Keywords: bursa of Fabricius, history of medicine, immunology, thymus

Robert A. Good (Fig. 1) began his intellectual and experimental queries related to the thymus in 1952 at the University of Minnesota, initially with paediatric patients. However, his interest in the plasma cell, antibodies and the immune response began in 1944, while still in Medical School at the University of Minnesota in Minneapolis, with his first publication appearing in 1945.

Robert Good

Robert Good

Figure 1

Robert A. Good with two young patients. Source: http://www.robertagoodarchives.com.

Good described a new syndrome that would carry his name: ‘Good syndrome: thymoma with immunodeficiency’.7 The clinical characteristics of Good syndrome are increased susceptibility to bacterial infections by encapsulated organisms and opportunistic viral and fungal infections. Subsequently, Good saw several patients with thymic tumours, which regularly presented with immunodeficiencies, leukopenia, lymphopenia and eosinophylopenia. Plasma cells, however, were not completely absent: the patient was severely hypogammaglobulinaemic rather than agammaglobulinaemic.

The association of thymoma with profound and broadly based immunodeficiency provoked Good’s group to ask what role the thymus plays in immunity.

Good and others found that the patients lacked all of the subsequently described immunoglobulins. These patients were found not to have plasma cells or germinal centres in their haematopoietic and lymphoid tissues. They possessed circulating lymphocytes in normal numbers.

In the mouse and other rodents, immunological depression is profound after thymectomy in neonatal animals, resulting in considerable depression of antibody production, plus deficient transplantation immunity and delayed-type hypersensitivity. Speculation on the reason for immunological failure following neonatal thymectomy has centred on the thymus as a source of cells or humoral factors essential to normal lymphoid development and immunological maturation.

Three independent groups of experiments showed that neonatal thymectomy has a significant effect on immunological reactivity: (i) the studies of Fichtelius et al. in young guinea-pigs showed that the depression of antibody response is slight, but significant; (ii) the experiments of Archer, Good and co-workers in rabbits and mice; and (iii) the studies by Miller at the Chester Beatty Research Institute in London.

Stutman, in Good’s laboratory, demonstrated that non-lymphoid thymomas induced the restoration of immunological functions in neonatally thymectomized mice and that when thymomas were grafted into allogenic hosts, immunological restoration was mediated by lymphoid cells of host type. Comparable results were obtained with free thymus grafts.

Cooper et al. postulated that a lymphoid stem cell population exists that is induced to differentiate along two distinct and separate cell lines related to two central lymphoid organs. In birds this developmental influence is exercised by the thymus and the bursa of Fabricius. Removal of one or both in the early post-hatching period has strikingly different influences on immunological function in the maturing animals. The thymus in the chicken functions exactly as does the thymus of the mouse. It represents the site of differentiation of a population of lymphocytes that subserve largely the functions of cell-mediated immunity.

The athymic children described by Di George, who lacked lymphoid cells in the deep cortical areas of the nodes but not at the peripheral areas, seemed the equivalent of the neonatally thymectomized mice and chickens. These patients had severe deficiencies of small T lymhocytes and profound deficiencies of all cell-mediated immunities, including delayed allergies, deficient allograft immunities and deficiencies in resistance to viruses, fungi and opportunistic infections.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1819567/

The Nobel Prize in Physiology or Medicine 1960

Sir Frank Macfarlane Burnet and Peter Brian Medawar

“for discovery of acquired immunological tolerance”

The Nobel Prize in Physiology or Medicine 1980

Baruj Benacerraf, Jean Dausset and George D. Snell

“for their discoveries concerning genetically determined structures on the cell surface that regulate immunological reactions”

Part V.

Biochemistry and Molecular Biology

The Nobel Prize in Physiology or Medicine 1922

Archibald Vivian Hill

“for his discovery relating to the production of heat in the muscle”

Otto Fritz Meyerhof

“for his discovery of the fixed relationship between the consumption of oxygen and the metabolism of lactic acid in the muscle”

The Nobel Prize in Physiology or Medicine 1931

Otto Heinrich Warburg

“for his discovery of the nature and mode of action of the respiratory enzyme”

http://pharmaceuticalintelligence.com/2012/11/02/otto-warburg-a-giant-of-modern-cellular-biology/

http://pharmaceuticalintelligence.com/2013/11/28/warburg-effect-revisited/

http://pharmaceuticalintelligence.com/2013/03/12/ampk-is-a-negative-regulator-of-the-warburg-effect-and-suppresses-tumor-growth-in-vivo/

http://pharmaceuticalintelligence.com/2012/10/17/is-the-warburg-effect-the-cause-or-the-effect-of-cancer-a-21st-century-view/

The Nobel Prize in Physiology or Medicine 1933

Thomas Hunt Morgan

“for his discoveries concerning the role played by the chromosome in heredity”

The Nobel Prize in Physiology or Medicine 1947

Carl Ferdinand Cori and Gerty Theresa Cori, née Radnitz

“for their discovery of the course of the catalytic conversion of glycogen”

The Nobel Prize in Physiology or Medicine 1953

Hans Adolf Krebs

“for his discovery of the citric acid cycle”

http://pharmaceuticalintelligence.com/2014/10/22/introduction-to-metabolic-pathways/

Fritz Albert Lipmann

“for his discovery of co-enzyme A and its importance for intermediary metabolism”

http://pharmaceuticalintelligence.com/2014/10/22/introduction-to-metabolic-pathways/

http://pharmaceuticalintelligence.com/2014/11/07/summary-of-cell-structure-anatomic-correlates-of-metabolic-function-2/

http://pharmaceuticalintelligence.com/2014/08/18/studies-of-respiration-lead-to-acetyl-coa/

http://pharmaceuticalintelligence.com/2013/01/26/portrait-of-a-great-scientist-and-mentor-nathan-oram-kaplan/

The Nobel Prize in Physiology or Medicine 1955

Axel Hugo Theodor Theorell

“for his discoveries concerning the nature and mode of action of oxidation enzymes”

http://pharmaceuticalintelligence.com/2014/08/18/studies-of-respiration-lead-to-acetyl-coa/

The Nobel Prize in Physiology or Medicine 1958

George Wells Beadle and Edward Lawrie Tatum

“for their discovery that genes act by regulating definite chemical events”

The Nobel Prize in Physiology or Medicine 1959

Severo Ochoa and Arthur Kornberg

“for their discovery of the mechanisms in the biological synthesis of ribonucleic acid and deoxyribonucleic acid”

Joshua Lederberg

“for his discoveries concerning genetic recombination and the organization of the genetic material of bacteria”

The Nobel Prize in Physiology or Medicine 1962

Francis Harry Compton Crick, James Dewey Watson and Maurice Hugh Frederick Wilkins

“for their discoveries concerning the molecular structure of nucleic acids and its significance for information transfer in living material”

The Nobel Prize in Physiology or Medicine 1963

Sir John Carew Eccles, Alan Lloyd Hodgkin and Andrew Fielding Huxley

“for their discoveries concerning the ionic mechanisms involved in excitation and inhibition in the peripheral and central portions of the nerve cell membrane”

The Nobel Prize in Physiology or Medicine 1964

Konrad Bloch and Feodor Lynen

“for their discoveries concerning the mechanism and regulation of the cholesterol and fatty acid metabolism”
http://pharmaceuticalintelligence.com/2014/10/25/oxidation-and-synthesis-of-fatty-acids/

The Nobel Prize in Physiology or Medicine 1965

François Jacob, André Lwoff and Jacques Monod

“for their discoveries concerning genetic control of enzyme and virus synthesis”

http://pharmaceuticalintelligence.com/2014/10/06/isoenzymes-in-cell-metabolic-pathways/

The Nobel Prize in Physiology or Medicine 1967

Ragnar Granit, Haldan Keffer Hartline and George Wald

“for their discoveries concerning the primary physiological and chemical visual processes in the eye”

The Nobel Prize in Physiology or Medicine 1968

Robert W. Holley, Har Gobind Khorana and Marshall W. Nirenberg

“for their interpretation of the genetic code and its function in protein synthesis”

The Nobel Prize in Physiology or Medicine 1969

Max Delbrück, Alfred D. Hershey and Salvador E. Luria

“for their discoveries concerning the replication mechanism and the genetic structure of viruses”

The Nobel Prize in Physiology or Medicine 1970

Sir Bernard Katz, Ulf von Euler and Julius Axelrod

“for their discoveries concerning the humoral transmittors in the nerve terminals and the mechanism for their storage, release and inactivation”

The Nobel Prize in Physiology or Medicine 1972

Gerald M. Edelman and Rodney R. Porter

“for their discoveries concerning the chemical structure of antibodies”

The Nobel Prize in Physiology or Medicine 1974

Albert Claude, Christian de Duve and George E. Palade

“for their discoveries concerning the structural and functional organization of the cell”

The Nobel Prize in Physiology or Medicine 1975

David Baltimore, Renato Dulbecco and Howard Martin Temin

“for their discoveries concerning the interaction between tumour viruses and the genetic material of the cell”
The Nobel Prize in Physiology or Medicine 1977

Rosalyn Yalow

“for the development of radioimmunoassays of peptide hormones”

The Nobel Prize in Physiology or Medicine 1978

Werner Arber, Daniel Nathans and Hamilton O. Smith

“for the discovery of restriction enzymes and their application to problems of molecular genetics”

Read Full Post »

Summary and Perspectives: Impairments in Pathological States: Endocrine Disorders, Stress Hypermetabolism and Cancer

Summary and Perspectives: Impairments in Pathological States: Endocrine Disorders, Stress Hypermetabolism and Cancer

Author and Curator: Larry H. Bernstein, MD, FCAP

Article ID #160: Summary and Perspectives: Impairments in Pathological States: Endocrine Disorders, Stress Hypermetabolism and Cancer. Published on 11/9/2014

WordCloud Image Produced by Adam Tubman

This summary is the last of a series on the impact of transcriptomics, proteomics, and metabolomics on disease investigation, and the sorting and integration of genomic signatures and metabolic signatures to explain phenotypic relationships in variability and individuality of response to disease expression and how this leads to  pharmaceutical discovery and personalized medicine.  We have unquestionably better tools at our disposal than has ever existed in the history of mankind, and an enormous knowledge-base that has to be accessed.  I shall conclude here these discussions with the powerful contribution to and current knowledge pertaining to biochemistry, metabolism, protein-interactions, signaling, and the application of the -OMICS to diseases and drug discovery at this time.

The Ever-Transcendent Cell

Deriving physiologic first principles By John S. Torday | The Scientist Nov 1, 2014
http://www.the-scientist.com/?articles.view/articleNo/41282/title/The-Ever-Transcendent-Cell/

Both the developmental and phylogenetic histories of an organism describe the evolution of physiology—the complex of metabolic pathways that govern the function of an organism as a whole. The necessity of establishing and maintaining homeostatic mechanisms began at the cellular level, with the very first cells, and homeostasis provides the underlying selection pressure fueling evolution.

While the events leading to the formation of the first functioning cell are debatable, a critical one was certainly the formation of simple lipid-enclosed vesicles, which provided a protected space for the evolution of metabolic pathways. Protocells evolved from a common ancestor that experienced environmental stresses early in the history of cellular development, such as acidic ocean conditions and low atmospheric oxygen levels, which shaped the evolution of metabolism.

The reduction of evolution to cell biology may answer the perennially unresolved question of why organisms return to their unicellular origins during the life cycle.

As primitive protocells evolved to form prokaryotes and, much later, eukaryotes, changes to the cell membrane occurred that were critical to the maintenance of chemiosmosis, the generation of bioenergy through the partitioning of ions. The incorporation of cholesterol into the plasma membrane surrounding primitive eukaryotic cells marked the beginning of their differentiation from prokaryotes. Cholesterol imparted more fluidity to eukaryotic cell membranes, enhancing functionality by increasing motility and endocytosis. Membrane deformability also allowed for increased gas exchange.

Acidification of the oceans by atmospheric carbon dioxide generated high intracellular calcium ion concentrations in primitive aquatic eukaryotes, which had to be lowered to prevent toxic effects, namely the aggregation of nucleotides, proteins, and lipids. The early cells achieved this by the evolution of calcium channels composed of cholesterol embedded within the cell’s plasma membrane, and of internal membranes, such as that of the endoplasmic reticulum, peroxisomes, and other cytoplasmic organelles, which hosted intracellular chemiosmosis and helped regulate calcium.

As eukaryotes thrived, they experienced increasingly competitive pressure for metabolic efficiency. Engulfed bacteria, assimilated as mitochondria, provided more bioenergy. As the evolution of eukaryotic organisms progressed, metabolic cooperation evolved, perhaps to enable competition with biofilm-forming, quorum-sensing prokaryotes. The subsequent appearance of multicellular eukaryotes expressing cellular growth factors and their respective receptors facilitated cell-cell signaling, forming the basis for an explosion of multicellular eukaryote evolution, culminating in the metazoans.

Casting a cellular perspective on evolution highlights the integration of genotype and phenotype. Starting from the protocell membrane, the functional homolog for all complex metazoan organs, it offers a way of experimentally determining the role of genes that fostered evolution based on the ontogeny and phylogeny of cellular processes that can be traced back, in some cases, to our last universal common ancestor.  ….

As eukaryotes thrived, they experienced increasingly competitive pressure for metabolic efficiency. Engulfed bacteria, assimilated as mitochondria, provided more bioenergy. As the evolution of eukaryotic organisms progressed, metabolic cooperation evolved, perhaps to enable competition with biofilm-forming, quorum-sensing prokaryotes. The subsequent appearance of multicellular eukaryotes expressing cellular growth factors and their respective receptors facilitated cell-cell signaling, forming the basis for an explosion of multicellular eukaryote evolution, culminating in the metazoans.

Casting a cellular perspective on evolution highlights the integration of genotype and phenotype. Starting from the protocell membrane, the functional homolog for all complex metazoan organs, it offers a way of experimentally determining the role of genes that fostered evolution based on the ontogeny and phylogeny of cellular processes that can be traced back, in some cases, to our last universal common ancestor.

Given that the unicellular toolkit is complete with all the traits necessary for forming multicellular organisms (Science, 301:361-63, 2003), it is distinctly possible that metazoans are merely permutations of the unicellular body plan. That scenario would clarify a lot of puzzling biology: molecular commonalities between the skin, lung, gut, and brain that affect physiology and pathophysiology exist because the cell membranes of unicellular organisms perform the equivalents of these tissue functions, and the existence of pleiotropy—one gene affecting many phenotypes—may be a consequence of the common unicellular source for all complex biologic traits.  …

The cell-molecular homeostatic model for evolution and stability addresses how the external environment generates homeostasis developmentally at the cellular level. It also determines homeostatic set points in adaptation to the environment through specific effectors, such as growth factors and their receptors, second messengers, inflammatory mediators, crossover mutations, and gene duplications. This is a highly mechanistic, heritable, plastic process that lends itself to understanding evolution at the cellular, tissue, organ, system, and population levels, mediated by physiologically linked mechanisms throughout, without having to invoke random, chance mechanisms to bridge different scales of evolutionary change. In other words, it is an integrated mechanism that can often be traced all the way back to its unicellular origins.

The switch from swim bladder to lung as vertebrates moved from water to land is proof of principle that stress-induced evolution in metazoans can be understood from changes at the cellular level.

http://www.the-scientist.com/Nov2014/TE_21.jpg

A MECHANISTIC BASIS FOR LUNG DEVELOPMENT: Stress from periodic atmospheric hypoxia (1) during vertebrate adaptation to land enhances positive selection of the stretch-regulated parathyroid hormone-related protein (PTHrP) in the pituitary and adrenal glands. In the pituitary (2), PTHrP signaling upregulates the release of adrenocorticotropic hormone (ACTH) (3), which stimulates the release of glucocorticoids (GC) by the adrenal gland (4). In the adrenal gland, PTHrP signaling also stimulates glucocorticoid production of adrenaline (5), which in turn affects the secretion of lung surfactant, the distension of alveoli, and the perfusion of alveolar capillaries (6). PTHrP signaling integrates the inflation and deflation of the alveoli with surfactant production and capillary perfusion.  THE SCIENTIST STAFF

From a cell-cell signaling perspective, two critical duplications in genes coding for cell-surface receptors occurred during this period of water-to-land transition—in the stretch-regulated parathyroid hormone-related protein (PTHrP) receptor gene and the β adrenergic (βA) receptor gene. These gene duplications can be disassembled by following their effects on vertebrate physiology backwards over phylogeny. PTHrP signaling is necessary for traits specifically relevant to land adaptation: calcification of bone, skin barrier formation, and the inflation and distention of lung alveoli. Microvascular shear stress in PTHrP-expressing organs such as bone, skin, kidney, and lung would have favored duplication of the PTHrP receptor, since sheer stress generates radical oxygen species (ROS) known to have this effect and PTHrP is a potent vasodilator, acting as an epistatic balancing selection for this constraint.

Positive selection for PTHrP signaling also evolved in the pituitary and adrenal cortex (see figure on this page), stimulating the secretion of ACTH and corticoids, respectively, in response to the stress of land adaptation. This cascade amplified adrenaline production by the adrenal medulla, since corticoids passing through it enzymatically stimulate adrenaline synthesis. Positive selection for this functional trait may have resulted from hypoxic stress that arose during global episodes of atmospheric hypoxia over geologic time. Since hypoxia is the most potent physiologic stressor, such transient oxygen deficiencies would have been acutely alleviated by increasing adrenaline levels, which would have stimulated alveolar surfactant production, increasing gas exchange by facilitating the distension of the alveoli. Over time, increased alveolar distension would have generated more alveoli by stimulating PTHrP secretion, impelling evolution of the alveolar bed of the lung.

This scenario similarly explains βA receptor gene duplication, since increased density of the βA receptor within the alveolar walls was necessary for relieving another constraint during the evolution of the lung in adaptation to land: the bottleneck created by the existence of a common mechanism for blood pressure control in both the lung alveoli and the systemic blood pressure. The pulmonary vasculature was constrained by its ability to withstand the swings in pressure caused by the systemic perfusion necessary to sustain all the other vital organs. PTHrP is a potent vasodilator, subserving the blood pressure constraint, but eventually the βA receptors evolved to coordinate blood pressure in both the lung and the periphery.

Gut Microbiome Heritability

Analyzing data from a large twin study, researchers have homed in on how host genetics can shape the gut microbiome.
By Tracy Vence | The Scientist Nov 6, 2014

Previous research suggested host genetic variation can influence microbial phenotype, but an analysis of data from a large twin study published in Cell today (November 6) solidifies the connection between human genotype and the composition of the gut microbiome. Studying more than 1,000 fecal samples from 416 monozygotic and dizygotic twin pairs, Cornell University’s Ruth Ley and her colleagues have homed in on one bacterial taxon, the family Christensenellaceae, as the most highly heritable group of microbes in the human gut. The researchers also found that Christensenellaceae—which was first described just two years ago—is central to a network of co-occurring heritable microbes that is associated with lean body mass index (BMI).  …

Of particular interest was the family Christensenellaceae, which was the most heritable taxon among those identified in the team’s analysis of fecal samples obtained from the TwinsUK study population.

While microbiologists had previously detected 16S rRNA sequences belonging to Christensenellaceae in the human microbiome, the family wasn’t named until 2012. “People hadn’t looked into it, partly because it didn’t have a name . . . it sort of flew under the radar,” said Ley.

Ley and her colleagues discovered that Christensenellaceae appears to be the hub in a network of co-occurring heritable taxa, which—among TwinsUK participants—was associated with low BMI. The researchers also found that Christensenellaceae had been found at greater abundance in low-BMI twins in older studies.

To interrogate the effects of Christensenellaceae on host metabolic phenotype, the Ley’s team introduced lean and obese human fecal samples into germ-free mice. They found animals that received lean fecal samples containing more Christensenellaceae showed reduced weight gain compared with their counterparts. And treatment of mice that had obesity-associated microbiomes with one member of the Christensenellaceae family, Christensenella minuta, led to reduced weight gain.   …

Ley and her colleagues are now focusing on the host alleles underlying the heritability of the gut microbiome. “We’re running a genome-wide association analysis to try to find genes—particular variants of genes—that might associate with higher levels of these highly heritable microbiota.  . . . Hopefully that will point us to possible reasons they’re heritable,” she said. “The genes will guide us toward understanding how these relationships are maintained between host genotype and microbiome composition.”

J.K. Goodrich et al., “Human genetics shape the gut microbiome,” Cell,  http://dx.doi.org:/10.1016/j.cell.2014.09.053, 2014.

Light-Operated Drugs

Scientists create a photosensitive pharmaceutical to target a glutamate receptor.
By Ruth Williams | The Scentist Nov 1, 2014
http://www.the-scientist.com/?articles.view/articleNo/41279/title/Light-Operated-Drugs/

light operated drugs MO1

light operated drugs MO1

http://www.the-scientist.com/Nov2014/MO1.jpg

The desire for temporal and spatial control of medications to minimize side effects and maximize benefits has inspired the development of light-controllable drugs, or optopharmacology. Early versions of such drugs have manipulated ion channels or protein-protein interactions, “but never, to my knowledge, G protein–coupled receptors [GPCRs], which are one of the most important pharmacological targets,” says Pau Gorostiza of the Institute for Bioengineering of Catalonia, in Barcelona.

Gorostiza has taken the first step toward filling that gap, creating a photosensitive inhibitor of the metabotropic glutamate 5 (mGlu5) receptor—a GPCR expressed in neurons and implicated in a number of neurological and psychiatric disorders. The new mGlu5 inhibitor—called alloswitch-1—is based on a known mGlu receptor inhibitor, but the simple addition of a light-responsive appendage, as had been done for other photosensitive drugs, wasn’t an option. The binding site on mGlu5 is “extremely tight,” explains Gorostiza, and would not accommodate a differently shaped molecule. Instead, alloswitch-1 has an intrinsic light-responsive element.

In a human cell line, the drug was active under dim light conditions, switched off by exposure to violet light, and switched back on by green light. When Gorostiza’s team administered alloswitch-1 to tadpoles, switching between violet and green light made the animals stop and start swimming, respectively.

The fact that alloswitch-1 is constitutively active and switched off by light is not ideal, says Gorostiza. “If you are thinking of therapy, then in principle you would prefer the opposite,” an “on” switch. Indeed, tweaks are required before alloswitch-1 could be a useful drug or research tool, says Stefan Herlitze, who studies ion channels at Ruhr-Universität Bochum in Germany. But, he adds, “as a proof of principle it is great.” (Nat Chem Biol, http://dx.doi.org:/10.1038/nchembio.1612, 2014)

Enhanced Enhancers

The recent discovery of super-enhancers may offer new drug targets for a range of diseases.
By Eric Olson | The Scientist Nov 1, 2014
http://www.the-scientist.com/?articles.view/articleNo/41281/title/Enhanced-Enhancers/

To understand disease processes, scientists often focus on unraveling how gene expression in disease-associated cells is altered. Increases or decreases in transcription—as dictated by a regulatory stretch of DNA called an enhancer, which serves as a binding site for transcription factors and associated proteins—can produce an aberrant composition of proteins, metabolites, and signaling molecules that drives pathologic states. Identifying the root causes of these changes may lead to new therapeutic approaches for many different diseases.

Although few therapies for human diseases aim to alter gene expression, the outstanding examples—including antiestrogens for hormone-positive breast cancer, antiandrogens for prostate cancer, and PPAR-γ agonists for type 2 diabetes—demonstrate the benefits that can be achieved through targeting gene-control mechanisms.  Now, thanks to recent papers from laboratories at MIT, Harvard, and the National Institutes of Health, researchers have a new, much bigger transcriptional target: large DNA regions known as super-enhancers or stretch-enhancers. Already, work on super-enhancers is providing insights into how gene-expression programs are established and maintained, and how they may go awry in disease.  Such research promises to open new avenues for discovering medicines for diseases where novel approaches are sorely needed.

Super-enhancers cover stretches of DNA that are 10- to 100-fold longer and about 10-fold less abundant in the genome than typical enhancer regions (Cell, 153:307-19, 2013). They also appear to bind a large percentage of the transcriptional machinery compared to typical enhancers, allowing them to better establish and enforce cell-type specific transcriptional programs (Cell, 153:320-34, 2013).

Super-enhancers are closely associated with genes that dictate cell identity, including those for cell-type–specific master regulatory transcription factors. This observation led to the intriguing hypothesis that cells with a pathologic identity, such as cancer cells, have an altered gene expression program driven by the loss, gain, or altered function of super-enhancers.

Sure enough, by mapping the genome-wide location of super-enhancers in several cancer cell lines and from patients’ tumor cells, we and others have demonstrated that genes located near super-enhancers are involved in processes that underlie tumorigenesis, such as cell proliferation, signaling, and apoptosis.

Super-enhancers cover stretches of DNA that are 10- to 100-fold longer and about 10-fold less abundant in the genome than typical enhancer regions.

Genome-wide association studies (GWAS) have found that disease- and trait-associated genetic variants often occur in greater numbers in super-enhancers (compared to typical enhancers) in cell types involved in the disease or trait of interest (Cell, 155:934-47, 2013). For example, an enrichment of fasting glucose–associated single nucleotide polymorphisms (SNPs) was found in the stretch-enhancers of pancreatic islet cells (PNAS, 110:17921-26, 2013). Given that some 90 percent of reported disease-associated SNPs are located in noncoding regions, super-enhancer maps may be extremely valuable in assigning functional significance to GWAS variants and identifying target pathways.

Because only 1 to 2 percent of active genes are physically linked to a super-enhancer, mapping the locations of super-enhancers can be used to pinpoint the small number of genes that may drive the biology of that cell. Differential super-enhancer maps that compare normal cells to diseased cells can be used to unravel the gene-control circuitry and identify new molecular targets, in much the same way that somatic mutations in tumor cells can point to oncogenic drivers in cancer. This approach is especially attractive in diseases for which an incomplete understanding of the pathogenic mechanisms has been a barrier to discovering effective new therapies.

Another therapeutic approach could be to disrupt the formation or function of super-enhancers by interfering with their associated protein components. This strategy could make it possible to downregulate multiple disease-associated genes through a single molecular intervention. A group of Boston-area researchers recently published support for this concept when they described inhibited expression of cancer-specific genes, leading to a decrease in cancer cell growth, by using a small molecule inhibitor to knock down a super-enhancer component called BRD4 (Cancer Cell, 24:777-90, 2013).  More recently, another group showed that expression of the RUNX1 transcription factor, involved in a form of T-cell leukemia, can be diminished by treating cells with an inhibitor of a transcriptional kinase that is present at the RUNX1 super-enhancer (Nature, 511:616-20, 2014).

Fungal effector Ecp6 outcompetes host immune receptor for chitin binding through intrachain LysM dimerization 
Andrea Sánchez-Vallet, et al.   eLife 2013;2:e00790 http://elifesciences.org/content/2/e00790#sthash.LnqVMJ9p.dpuf

LysM effector

LysM effector

http://img.scoop.it/ZniCRKQSvJOG18fHbb4p0Tl72eJkfbmt4t8yenImKBVvK0kTmF0xjctABnaLJIm9

While host immune receptors

  • detect pathogen-associated molecular patterns to activate immunity,
  • pathogens attempt to deregulate host immunity through secreted effectors.

Fungi employ LysM effectors to prevent

  • recognition of cell wall-derived chitin by host immune receptors

Structural analysis of the LysM effector Ecp6 of

  • the fungal tomato pathogen Cladosporium fulvum reveals
  • a novel mechanism for chitin binding,
  • mediated by intrachain LysM dimerization,

leading to a chitin-binding groove that is deeply buried in the effector protein.

This composite binding site involves

  • two of the three LysMs of Ecp6 and
  • mediates chitin binding with ultra-high (pM) affinity.

The remaining singular LysM domain of Ecp6 binds chitin with

  • low micromolar affinity but can nevertheless still perturb chitin-triggered immunity.

Conceivably, the perturbation by this LysM domain is not established through chitin sequestration but possibly through interference with the host immune receptor complex.

Mutated Genes in Schizophrenia Map to Brain Networks
From www.nih.gov –  Sep 3, 2013

Previous studies have shown that many people with schizophrenia have de novo, or new, genetic mutations. These misspellings in a gene’s DNA sequence

  • occur spontaneously and so aren’t shared by their close relatives.

Dr. Mary-Claire King of the University of Washington in Seattle and colleagues set out to

  • identify spontaneous genetic mutations in people with schizophrenia and
  • to assess where and when in the brain these misspelled genes are turned on, or expressed.

The study was funded in part by NIH’s National Institute of Mental Health (NIMH). The results were published in the August 1, 2013, issue of Cell.

The researchers sequenced the exomes (protein-coding DNA regions) of 399 people—105 with schizophrenia plus their unaffected parents and siblings. Gene variations
that were found in a person with schizophrenia but not in either parent were considered spontaneous.

The likelihood of having a spontaneous mutation was associated with

  • the age of the father in both affected and unaffected siblings.

Significantly more mutations were found in people

  • whose fathers were 33-45 years at the time of conception compared to 19-28 years.

Among people with schizophrenia, the scientists identified

  • 54 genes with spontaneous mutations
  • predicted to cause damage to the function of the protein they encode.

The researchers used newly available database resources that show

  • where in the brain and when during development genes are expressed.

The genes form an interconnected expression network with many more connections than

  • that of the genes with spontaneous damaging mutations in unaffected siblings.

The spontaneously mutated genes in people with schizophrenia

  • were expressed in the prefrontal cortex, a region in the front of the brain.

The genes are known to be involved in important pathways in brain development. Fifty of these genes were active

  • mainly during the period of fetal development.

“Processes critical for the brain’s development can be revealed by the mutations that disrupt them,” King says. “Mutations can lead to loss of integrity of a whole pathway,
not just of a single gene.”

These findings support the concept that schizophrenia may result, in part, from

  • disruptions in development in the prefrontal cortex during fetal development.

James E. Darnell’s “Reflections”

A brief history of the discovery of RNA and its role in transcription — peppered with career advice
By Joseph P. Tiano

James Darnell begins his Journal of Biological Chemistry “Reflections” article by saying, “graduate students these days

  • have to swim in a sea virtually turgid with the daily avalanche of new information and
  • may be momentarily too overwhelmed to listen to the aging.

I firmly believe how we learned what we know can provide useful guidance for how and what a newcomer will learn.” Considering his remarkable discoveries in

  • RNA processing and eukaryotic transcriptional regulation

spanning 60 years of research, Darnell’s advice should be cherished. In his second year at medical school at Washington University School of Medicine in St. Louis, while
studying streptococcal disease in Robert J. Glaser’s laboratory, Darnell realized he “loved doing the experiments” and had his first “career advancement event.”
He and technician Barbara Pesch discovered that in vivo penicillin treatment killed streptococci only in the exponential growth phase and not in the stationary phase. These
results were published in the Journal of Clinical Investigation and earned Darnell an interview with Harry Eagle at the National Institutes of Health.

Darnell arrived at the NIH in 1956, shortly after Eagle  shifted his research interest to developing his minimal essential cell culture medium, still used. Eagle, then studying cell metabolism, suggested that Darnell take up a side project on poliovirus replication in mammalian cells in collaboration with Robert I. DeMars. DeMars’ Ph.D.
adviser was also James  Watson’s mentor, so Darnell met Watson, who invited him to give a talk at Harvard University, which led to an assistant professor position
at the MIT under Salvador Luria. A take-home message is to embrace side projects, because you never know where they may lead: this project helped to shape
his career.

Darnell arrived in Boston in 1961. Following the discovery of DNA’s structure in 1953, the world of molecular biology was turning to RNA in an effort to understand how
proteins are made. Darnell’s background in virology (it was discovered in 1960 that viruses used RNA to replicate) was ideal for the aim of his first independent lab:
exploring mRNA in animal cells grown in culture. While at MIT, he developed a new technique for purifying RNA along with making other observations

  • suggesting that nonribosomal cytoplasmic RNA may be involved in protein synthesis.

When Darnell moved to Albert Einstein College of Medicine for full professorship in 1964,  it was hypothesized that heterogenous nuclear RNA was a precursor to mRNA.
At Einstein, Darnell discovered RNA processing of pre-tRNAs and demonstrated for the first time

  • that a specific nuclear RNA could represent a possible specific mRNA precursor.

In 1967 Darnell took a position at Columbia University, and it was there that he discovered (simultaneously with two other labs) that

  • mRNA contained a polyadenosine tail.

The three groups all published their results together in the Proceedings of the National Academy of Sciences in 1971. Shortly afterward, Darnell made his final career move
four short miles down the street to Rockefeller University in 1974.

Over the next 35-plus years at Rockefeller, Darnell never strayed from his original research question: How do mammalian cells make and control the making of different
mRNAs? His work was instrumental in the collaborative discovery of

  • splicing in the late 1970s and
  • in identifying and cloning many transcriptional activators.

Perhaps his greatest contribution during this time, with the help of Ernest Knight, was

  • the discovery and cloning of the signal transducers and activators of transcription (STAT) proteins.

And with George Stark, Andy Wilks and John Krowlewski, he described

  • cytokine signaling via the JAK-STAT pathway.

Darnell closes his “Reflections” with perhaps his best advice: Do not get too wrapped up in your own work, because “we are all needed and we are all in this together.”

Darnell Reflections - James_Darnell

Darnell Reflections – James_Darnell

http://www.asbmb.org/assets/0/366/418/428/85528/85529/85530/8758cb87-84ff-42d6-8aea-96fda4031a1b.jpg

Recent findings on presenilins and signal peptide peptidase

By Dinu-Valantin Bălănescu

γ-secretase and SPP

γ-secretase and SPP

Fig. 1 from the minireview shows a schematic depiction of γ-secretase and SPP

http://www.asbmb.org/assets/0/366/418/428/85528/85529/85530/c2de032a-daad-41e5-ba19-87a17bd26362.png

GxGD proteases are a family of intramembranous enzymes capable of hydrolyzing

  • the transmembrane domain of some integral membrane proteins.

The GxGD family is one of the three families of

  • intramembrane-cleaving proteases discovered so far (along with the rhomboid and site-2 protease) and
  • includes the γ-secretase and the signal peptide peptidase.

Although only recently discovered, a number of functions in human pathology and in numerous other biological processes

  • have been attributed to γ-secretase and SPP.

Taisuke Tomita and Takeshi Iwatsubo of the University of Tokyo highlighted the latest findings on the structure and function of γ-secretase and SPP
in a recent minireview in The Journal of Biological Chemistry.

  • γ-secretase is involved in cleaving the amyloid-β precursor protein, thus producing amyloid-β peptide,

the main component of senile plaques in Alzheimer’s disease patients’ brains. The complete structure of mammalian γ-secretase is not yet known; however,
Tomita and Iwatsubo note that biochemical analyses have revealed it to be a multisubunit protein complex.

  • Its catalytic subunit is presenilin, an aspartyl protease.

In vitro and in vivo functional and chemical biology analyses have revealed that

  • presenilin is a modulator and mandatory component of the γ-secretase–mediated cleavage of APP.

Genetic studies have identified three other components required for γ-secretase activity:

  1. nicastrin,
  2. anterior pharynx defective 1 and
  3. presenilin enhancer 2.

By coexpression of presenilin with the other three components, the authors managed to

  • reconstitute γ-secretase activity.

Tomita and Iwatsubo determined using the substituted cysteine accessibility method and by topological analyses, that

  • the catalytic aspartates are located at the center of the nine transmembrane domains of presenilin,
  • by revealing the exact location of the enzyme’s catalytic site.

The minireview also describes in detail the formerly enigmatic mechanism of γ-secretase mediated cleavage.

SPP, an enzyme that cleaves remnant signal peptides in the membrane

  • during the biogenesis of membrane proteins and
  • signal peptides from major histocompatibility complex type I,
  • also is involved in the maturation of proteins of the hepatitis C virus and GB virus B.

Bioinformatics methods have revealed in fruit flies and mammals four SPP-like proteins,

  • two of which are involved in immunological processes.

By using γ-secretase inhibitors and modulators, it has been confirmed

  • that SPP shares a similar GxGD active site and proteolytic activity with γ-secretase.

Upon purification of the human SPP protein with the baculovirus/Sf9 cell system,

  • single-particle analysis revealed further structural and functional details.

HLA targeting efficiency correlates with human T-cell response magnitude and with mortality from influenza A infection

From www.pnas.org –  Sep 3, 2013 4:24 PM

Experimental and computational evidence suggests that

  • HLAs preferentially bind conserved regions of viral proteins, a concept we term “targeting efficiency,” and that
  • this preference may provide improved clearance of infection in several viral systems.

To test this hypothesis, T-cell responses to A/H1N1 (2009) were measured from peripheral blood mononuclear cells obtained from a household cohort study
performed during the 2009–2010 influenza season. We found that HLA targeting efficiency scores significantly correlated with

  • IFN-γ enzyme-linked immunosorbent spot responses (P = 0.042, multiple regression).

A further population-based analysis found that the carriage frequencies of the alleles with the lowest targeting efficiencies, A*24,

  • were associated with pH1N1 mortality (r = 0.37, P = 0.031) and
  • are common in certain indigenous populations in which increased pH1N1 morbidity has been reported.

HLA efficiency scores and HLA use are associated with CD8 T-cell magnitude in humans after influenza infection.
The computational tools used in this study may be useful predictors of potential morbidity and

  • identify immunologic differences of new variant influenza strains
  • more accurately than evolutionary sequence comparisons.

Population-based studies of the relative frequency of these alleles in severe vs. mild influenza cases

  • might advance clinical practices for severe H1N1 infections among genetically susceptible populations.

Metabolomics in drug target discovery

J D Rabinowitz et al.

Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ.
Cold Spring Harbor Symposia on Quantitative Biology 11/2011; 76:235-46.
http://dx.doi.org:/10.1101/sqb.2011.76.010694 

Most diseases result in metabolic changes. In many cases, these changes play a causative role in disease progression. By identifying pathological metabolic changes,

  • metabolomics can point to potential new sites for therapeutic intervention.

Particularly promising enzymatic targets are those that

  • carry increased flux in the disease state.

Definitive assessment of flux requires the use of isotope tracers. Here we present techniques for

  • finding new drug targets using metabolomics and isotope tracers.

The utility of these methods is exemplified in the study of three different viral pathogens. For influenza A and herpes simplex virus,

  • metabolomic analysis of infected versus mock-infected cells revealed
  • dramatic concentration changes around the current antiviral target enzymes.

Similar analysis of human-cytomegalovirus-infected cells, however, found the greatest changes

  • in a region of metabolism unrelated to the current antiviral target.

Instead, it pointed to the tricarboxylic acid (TCA) cycle and

  • its efflux to feed fatty acid biosynthesis as a potential preferred target.

Isotope tracer studies revealed that cytomegalovirus greatly increases flux through

  • the key fatty acid metabolic enzyme acetyl-coenzyme A carboxylase.
  • Inhibition of this enzyme blocks human cytomegalovirus replication.

Examples where metabolomics has contributed to identification of anticancer drug targets are also discussed. Eventual proof of the value of

  • metabolomics as a drug target discovery strategy will be
  • successful clinical development of therapeutics hitting these new targets.

 Related References

Use of metabolic pathway flux information in targeted cancer drug design. Drug Discovery Today: Therapeutic Strategies 1:435-443, 2004.

Detection of resistance to imatinib by metabolic profiling: clinical and drug development implications. Am J Pharmacogenomics. 2005;5(5):293-302. Review. PMID: 16196499

Medicinal chemistry, metabolic profiling and drug target discovery: a role for metabolic profiling in reverse pharmacology and chemical genetics.
Mini Rev Med Chem.  2005 Jan;5(1):13-20. Review. PMID: 15638788 [PubMed – indexed for MEDLINE] Related citations

Development of Tracer-Based Metabolomics and its Implications for the Pharmaceutical Industry. Int J Pharm Med 2007; 21 (3): 217-224.

Use of metabolic pathway flux information in anticancer drug design. Ernst Schering Found Symp Proc. 2007;(4):189-203. Review. PMID: 18811058

Pharmacological targeting of glucagon and glucagon-like peptide 1 receptors has different effects on energy state and glucose homeostasis in diet-induced obese mice. J Pharmacol Exp Ther. 2011 Jul;338(1):70-81. http://dx.doi.org:/10.1124/jpet.111.179986. PMID: 21471191

Single valproic acid treatment inhibits glycogen and RNA ribose turnover while disrupting glucose-derived cholesterol synthesis in liver as revealed by the
[U-C(6)]-d-glucose tracer in mice. Metabolomics. 2009 Sep;5(3):336-345. PMID: 19718458

Metabolic Pathways as Targets for Drug Screening, Metabolomics, Dr Ute Roessner (Ed.), ISBN: 978-953-51-0046-1, InTech, Available from: http://www.intechopen.com/books/metabolomics/metabolic-pathways-as-targets-for-drug-screening

Iron regulates glucose homeostasis in liver and muscle via AMP-activated protein kinase in mice. FASEB J. 2013 Jul;27(7):2845-54.
http://dx.doi.org:/10.1096/fj.12-216929. PMID: 23515442

Metabolomics and systems pharmacology: why and how to model the human metabolic network for drug discovery

Drug Discov. Today 19 (2014), 171–182     http://dx.doi.org:/10.1016/j.drudis.2013.07.014

Highlights

  • We now have metabolic network models; the metabolome is represented by their nodes.
  • Metabolite levels are sensitive to changes in enzyme activities.
  • Drugs hitchhike on metabolite transporters to get into and out of cells.
  • The consensus network Recon2 represents the present state of the art, and has predictive power.
  • Constraint-based modelling relates network structure to metabolic fluxes.

Metabolism represents the ‘sharp end’ of systems biology, because changes in metabolite concentrations are

  • necessarily amplified relative to changes in the transcriptome, proteome and enzyme activities, which can be modulated by drugs.

To understand such behaviour, we therefore need (and increasingly have) reliable consensus (community) models of

  • the human metabolic network that include the important transporters.

Small molecule ‘drug’ transporters are in fact metabolite transporters, because

  • drugs bear structural similarities to metabolites known from the network reconstructions and
  • from measurements of the metabolome.

Recon2 represents the present state-of-the-art human metabolic network reconstruction; it can predict inter alia:

(i) the effects of inborn errors of metabolism;

(ii) which metabolites are exometabolites, and

(iii) how metabolism varies between tissues and cellular compartments.

However, even these qualitative network models are not yet complete. As our understanding improves

  • so do we recognise more clearly the need for a systems (poly)pharmacology.

Introduction – a systems biology approach to drug discovery

It is clearly not news that the productivity of the pharmaceutical industry has declined significantly during recent years

  • following an ‘inverse Moore’s Law’, Eroom’s Law, or
  • that many commentators, consider that the main cause of this is
  • because of an excessive focus on individual molecular target discovery rather than a more sensible strategy
  • based on a systems-level approach (Fig. 1).
drug discovery science

drug discovery science

Figure 1.

The change in drug discovery strategy from ‘classical’ function-first approaches (in which the assay of drug function was at the tissue or organism level),
with mechanistic studies potentially coming later, to more-recent target-based approaches where initial assays usually involve assessing the interactions
of drugs with specified (and often cloned, recombinant) proteins in vitro. In the latter cases, effects in vivo are assessed later, with concomitantly high levels of attrition.

Arguably the two chief hallmarks of the systems biology approach are:

(i) that we seek to make mathematical models of our systems iteratively or in parallel with well-designed ‘wet’ experiments, and
(ii) that we do not necessarily start with a hypothesis but measure as many things as possible (the ’omes) and

  • let the data tell us the hypothesis that best fits and describes them.

Although metabolism was once seen as something of a Cinderella subject,

  • there are fundamental reasons to do with the organisation of biochemical networks as
  • to why the metabol(om)ic level – now in fact seen as the ‘apogee’ of the ’omics trilogy –
  •  is indeed likely to be far more discriminating than are
  • changes in the transcriptome or proteome.

The next two subsections deal with these points and Fig. 2 summarises the paper in the form of a Mind Map.

metabolomics and systems pharmacology

metabolomics and systems pharmacology

http://ars.els-cdn.com/content/image/1-s2.0-S1359644613002481-gr2.jpg

Metabolic Disease Drug Discovery— “Hitting the Target” Is Easier Said Than Done

David E. Moller, et al.   http://dx.doi.org:/10.1016/j.cmet.2011.10.012

Despite the advent of new drug classes, the global epidemic of cardiometabolic disease has not abated. Continuing

  • unmet medical needs remain a major driver for new research.

Drug discovery approaches in this field have mirrored industry trends, leading to a recent

  • increase in the number of molecules entering development.

However, worrisome trends and newer hurdles are also apparent. The history of two newer drug classes—

  1. glucagon-like peptide-1 receptor agonists and
  2. dipeptidyl peptidase-4 inhibitors—

illustrates both progress and challenges. Future success requires that researchers learn from these experiences and

  • continue to explore and apply new technology platforms and research paradigms.

The global epidemic of obesity and diabetes continues to progress relentlessly. The International Diabetes Federation predicts an even greater diabetes burden (>430 million people afflicted) by 2030, which will disproportionately affect developing nations (International Diabetes Federation, 2011). Yet

  • existing drug classes for diabetes, obesity, and comorbid cardiovascular (CV) conditions have substantial limitations.

Currently available prescription drugs for treatment of hyperglycemia in patients with type 2 diabetes (Table 1) have notable shortcomings. In general,

Therefore, clinicians must often use combination therapy, adding additional agents over time. Ultimately many patients will need to use insulin—a therapeutic class first introduced in 1922. Most existing agents also have

  • issues around safety and tolerability as well as dosing convenience (which can impact patient compliance).

Pharmacometabolomics, also known as pharmacometabonomics, is a field which stems from metabolomics,

  • the quantification and analysis of metabolites produced by the body.

It refers to the direct measurement of metabolites in an individual’s bodily fluids, in order to

  • predict or evaluate the metabolism of pharmaceutical compounds, and
  • to better understand the pharmacokinetic profile of a drug.

Alternatively, pharmacometabolomics can be applied to measure metabolite levels

  • following the administration of a pharmaceutical compound, in order to
  • monitor the effects of the compound on certain metabolic pathways(pharmacodynamics).

This provides detailed mapping of drug effects on metabolism and

  • the pathways that are implicated in mechanism of variation of response to treatment.

In addition, the metabolic profile of an individual at baseline (metabotype) provides information about

  • how individuals respond to treatment and highlights heterogeneity within a disease state.

All three approaches require the quantification of metabolites found

relationship between -OMICS

relationship between -OMICS

http://upload.wikimedia.org/wikipedia/commons/thumb/e/eb/OMICS.png/350px-OMICS.png

Pharmacometabolomics is thought to provide information that

Looking at the characteristics of an individual down through these different levels of detail, there is an

  • increasingly more accurate prediction of a person’s ability to respond to a pharmaceutical compound.
  1. the genome, made up of 25 000 genes, can indicate possible errors in drug metabolism;
  2. the transcriptome, made up of 85,000 transcripts, can provide information about which genes important in metabolism are being actively transcribed;
  3. and the proteome, >10,000,000 members, depicts which proteins are active in the body to carry out these functions.

Pharmacometabolomics complements the omics with

  • direct measurement of the products of all of these reactions, but with perhaps a relatively
  • smaller number of members: that was initially projected to be approximately 2200 metabolites,

but could be a larger number when gut derived metabolites and xenobiotics are added to the list. Overall, the goal of pharmacometabolomics is

  • to more closely predict or assess the response of an individual to a pharmaceutical compound,
  • permitting continued treatment with the right drug or dosage
  • depending on the variations in their metabolism and ability to respond to treatment.

Pharmacometabolomic analyses, through the use of a metabolomics approach,

  • can provide a comprehensive and detailed metabolic profile or “metabolic fingerprint” for an individual patient.

Such metabolic profiles can provide a complete overview of individual metabolite or pathway alterations,

This approach can then be applied to the prediction of response to a pharmaceutical compound

  • by patients with a particular metabolic profile.

Pharmacometabolomic analyses of drug response are

Pharmacogenetics focuses on the identification of genetic variations (e.g. single-nucleotide polymorphisms)

  • within patients that may contribute to altered drug responses and overall outcome of a certain treatment.

The results of pharmacometabolomics analyses can act to “inform” or “direct”

  • pharmacogenetic analyses by correlating aberrant metabolite concentrations or metabolic pathways to potential alterations at the genetic level.

This concept has been established with two seminal publications from studies of antidepressants serotonin reuptake inhibitors

  • where metabolic signatures were able to define a pathway implicated in response to the antidepressant and
  • that lead to identification of genetic variants within a key gene
  • within the highlighted pathway as being implicated in variation in response.

These genetic variants were not identified through genetic analysis alone and hence

  • illustrated how metabolomics can guide and inform genetic data.

en.wikipedia.org/wiki/Pharmacometabolomics

Benznidazole Biotransformation and Multiple Targets in Trypanosoma cruzi Revealed by Metabolomics

Andrea Trochine, Darren J. Creek, Paula Faral-Tello, Michael P. Barrett, Carlos Robello
Published: May 22, 2014   http://dx.doi.org:/10.1371/journal.pntd.0002844

The first line treatment for Chagas disease, a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi,

  • involves administration of benznidazole (Bzn).

Bzn is a 2-nitroimidazole pro-drug which requires nitroreduction to become active. We used a

  • non-targeted MS-based metabolomics approach to study the metabolic response of T. cruzi to Bzn.

Parasites treated with Bzn were minimally altered compared to untreated trypanosomes, although the redox active thiols

  1. trypanothione,
  2. homotrypanothione and
  3. cysteine

were significantly diminished in abundance post-treatment. In addition, multiple Bzn-derived metabolites were detected after treatment.

These metabolites included reduction products, fragments and covalent adducts of reduced Bzn

  • linked to each of the major low molecular weight thiols:
  1. trypanothione,
  2. glutathione,
  3. g-glutamylcysteine,
  4. glutathionylspermidine,
  5. cysteine and
  6. ovothiol A.

Bzn products known to be generated in vitro by the unusual trypanosomal nitroreductase, TcNTRI,

  • were found within the parasites,
  • but low molecular weight adducts of glyoxal, a proposed toxic end-product of NTRI Bzn metabolism, were not detected.

Our data is indicative of a major role of the

  • thiol binding capacity of Bzn reduction products
  • in the mechanism of Bzn toxicity against T. cruzi.

 

 

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Summary to Metabolomics

Summary to Metabolomics

Author and Curator: Larry H. Bernstein, MD, FCAP 

This concludes a long step-by-step journey into rediscovering biological processes from the genome as a framework to the remodeled and reconstituted cell through a number of posttranscription and posttranslation processes that modify the proteome and determine the metabolome.  The remodeling process continues over a lifetime. The process requires a balance between nutrient intake, energy utilization for work in the lean body mass, energy reserves, endocrine, paracrine and autocrine mechanisms, and autophagy.  It is true when we look at this in its full scope – What a creature is man?

http://masspec.scripps.edu/metabo_science/recommended_readings.php
 Recommended Readings and Historical Perspectives

Metabolomics is the scientific study of chemical processes involving metabolites. Specifically, metabolomics is the “systematic study of the unique chemical fingerprints that specific cellular processes leave behind”, the study of their small-molecule metabolite profiles.[1] The metabolome represents the collection of all metabolites in a biological cell, tissue, organ or organism, which are the end products of cellular processes.[2] mRNA gene expression data and proteomic analyses reveal the set of gene products being produced in the cell, data that represents one aspect of cellular function. Conversely, metabolic profiling can give an instantaneous snapshot of the physiology of that cell. One of the challenges of systems biology and functional genomics is to integrate proteomic, transcriptomic, and metabolomic information to provide a better understanding of cellular biology.

The term “metabolic profile” was introduced by Horning, et al. in 1971 after they demonstrated that gas chromatography-mass spectrometry (GC-MS) could be used to measure compounds present in human urine and tissue extracts. The Horning group, along with that of Linus Pauling and Arthur B. Robinson led the development of GC-MS methods to monitor the metabolites present in urine through the 1970s.

Concurrently, NMR spectroscopy, which was discovered in the 1940s, was also undergoing rapid advances. In 1974, Seeley et al. demonstrated the utility of using NMR to detect metabolites in unmodified biological samples.This first study on muscle highlighted the value of NMR in that it was determined that 90% of cellular ATP is complexed with magnesium. As sensitivity has improved with the evolution of higher magnetic field strengths and magic angle spinning, NMR continues to be a leading analytical tool to investigate metabolism. Efforts to utilize NMR for metabolomics have been influenced by the laboratory of Dr. Jeremy Nicholson at Birkbeck College, University of London and later at Imperial College London. In 1984, Nicholson showed 1H NMR spectroscopy could potentially be used to diagnose diabetes mellitus, and later pioneered the application of pattern recognition methods to NMR spectroscopic data.

In 2005, the first metabolomics web database, METLIN, for characterizing human metabolites was developed in the Siuzdak laboratory at The Scripps Research Institute and contained over 10,000 metabolites and tandem mass spectral data. As of September 2012, METLIN contains over 60,000 metabolites as well as the largest repository of tandem mass spectrometry data in metabolomics.

On 23 January 2007, the Human Metabolome Project, led by Dr. David Wishart of the University of Alberta, Canada, completed the first draft of the human metabolome, consisting of a database of approximately 2500 metabolites, 1200 drugs and 3500 food components. Similar projects have been underway in several plant species, most notably Medicago truncatula and Arabidopsis thaliana for several years.

As late as mid-2010, metabolomics was still considered an “emerging field”. Further, it was noted that further progress in the field depended in large part, through addressing otherwise “irresolvable technical challenges”, by technical evolution of mass spectrometry instrumentation.

Metabolome refers to the complete set of small-molecule metabolites (such as metabolic intermediates, hormones and other signaling molecules, and secondary metabolites) to be found within a biological sample, such as a single organism. The word was coined in analogy with transcriptomics and proteomics; like the transcriptome and the proteome, the metabolome is dynamic, changing from second to second. Although the metabolome can be defined readily enough, it is not currently possible to analyse the entire range of metabolites by a single analytical method. The first metabolite database(called METLIN) for searching m/z values from mass spectrometry data was developed by scientists at The Scripps Research Institute in 2005. In January 2007, scientists at the University of Alberta and the University of Calgary completed the first draft of the human metabolome. They catalogued approximately 2500 metabolites, 1200 drugs and 3500 food components that can be found in the human body, as reported in the literature. This information, available at the Human Metabolome Database (www.hmdb.ca) and based on analysis of information available in the current scientific literature, is far from complete.

Each type of cell and tissue has a unique metabolic ‘fingerprint’ that can elucidate organ or tissue-specific information, while the study of biofluids can give more generalized though less specialized information. Commonly used biofluids are urine and plasma, as they can be obtained non-invasively or relatively non-invasively, respectively. The ease of collection facilitates high temporal resolution, and because they are always at dynamic equilibrium with the body, they can describe the host as a whole.

Metabolites are the intermediates and products of metabolism. Within the context of metabolomics, a metabolite is usually defined as any molecule less than 1 kDa in size.
A primary metabolite is directly involved in the normal growth, development, and reproduction. A secondary metabolite is not directly involved in those processes.  By contrast, in human-based metabolomics, it is more common to describe metabolites as being either endogenous (produced by the host organism) or exogenous. Metabolites of foreign substances such as drugs are termed xenometabolites. The metabolome forms a large network of metabolic reactions, where outputs from one enzymatic chemical reaction are inputs to other chemical reactions.

Metabonomics is defined as “the quantitative measurement of the dynamic multiparametric metabolic response of living systems to pathophysiological stimuli or genetic modification”. The word origin is from the Greek μεταβολή meaning change and nomos meaning a rule set or set of laws. This approach was pioneered by Jeremy Nicholson at Imperial College London and has been used in toxicology, disease diagnosis and a number of other fields. Historically, the metabonomics approach was one of the first methods to apply the scope of systems biology to studies of metabolism.

There is a growing consensus that ‘metabolomics’ places a greater emphasis on metabolic profiling at a cellular or organ level and is primarily concerned with normal endogenous metabolism. ‘Metabonomics’ extends metabolic profiling to include information about perturbations of metabolism caused by environmental factors (including diet and toxins), disease processes, and the involvement of extragenomic influences, such as gut microflora. This is not a trivial difference; metabolomic studies should, by definition, exclude metabolic contributions from extragenomic sources, because these are external to the system being studied.

Toxicity assessment/toxicology. Metabolic profiling (especially of urine or blood plasma samples) detects the physiological changes caused by toxic insult of a chemical (or mixture of chemicals).

Functional genomics. Metabolomics can be an excellent tool for determining the phenotype caused by a genetic manipulation, such as gene deletion or insertion. Sometimes this can be a sufficient goal in itself—for instance, to detect any phenotypic changes in a genetically-modified plant intended for human or animal consumption. More exciting is the prospect of predicting the function of unknown genes by comparison with the metabolic perturbations caused by deletion/insertion of known genes.

Nutrigenomics is a generalised term which links genomics, transcriptomics, proteomics and metabolomics to human nutrition. In general a metabolome in a given body fluid is influenced by endogenous factors such as age, sex, body composition and genetics as well as underlying pathologies. The large bowel microflora are also a very significant potential confounder of metabolic profiles and could be classified as either an endogenous or exogenous factor. The main exogenous factors are diet and drugs. Diet can then be broken down to nutrients and non- nutrients.

http://en.wikipedia.org/wiki/Metabolomics

Jose Eduardo des Salles Roselino

The problem with genomics was it was set as explanation for everything. In fact, when something is genetic in nature the genomic reasoning works fine. However, this means whenever an inborn error is found and only in this case the genomic knowledge afterwards may indicate what is wrong and not the completely way to put biology upside down by reading everything in the DNA genetic as well as non-genetic problems.

Coordination of the transcriptome and metabolome by the circadian clock PNAS 2012

Coordination of the transcriptome and metabolome by the circadian clock PNAS 2012

analysis of metabolomic data and differential metabolic regulation for fetal lungs, and maternal blood plasma

conformational changes leading to substrate efflux.img

conformational changes leading to substrate efflux.img

The cellular response is defined by a network of chemogenomic response signatures.

The cellular response is defined by a network of chemogenomic response signatures.

Dynamic Construct of the –Omics

Dynamic Construct of the –Omics

 genome cartoon

genome cartoon

central dogma phenotype

central dogma phenotype

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Introduction to Protein Synthesis and Degradation

Curator: Larry H. Bernstein, MD, FCAP

Updated 8/31/2019

 

Introduction to Protein Synthesis and Degradation

This chapter I made to follow signaling, rather than to precede it. I had already written much of the content before reorganizing the contents. The previous chapters on carbohydrate and on lipid metabolism have already provided much material on proteins and protein function, which was persuasive of the need to introduce signaling, which entails a substantial introduction to conformational changes in proteins that direct the trafficking of metabolic pathways, but more subtly uncovers an important role for microRNAs, not divorced from transcription, but involved in a non-transcriptional role.  This is where the classic model of molecular biology lacked any integration with emerging metabolic concepts concerning regulation. Consequently, the science was bereft of understanding the ties between the multiple convergence of transcripts, the selective inhibition of transcriptions, and the relative balance of aerobic and anaerobic metabolism, the weight of the pentose phosphate shunt, and the utilization of available energy source for synthetic and catabolic adaptive responses.

The first subchapter serves to introduce the importance of transcription in translational science.  The several subtitles that follow are intended to lay out the scope of the transcriptional activity, and also to direct attention toward the huge role of proteomics in the cell construct.  As we have already seen, proteins engage with carbohydrates and with lipids in important structural and signaling processes.  They are integrasl to the composition of the cytoskeleton, and also to the extracellular matrix.  Many proteins are actually enzymes, carrying out the transformation of some substrate, a derivative of the food we ingest.  They have a catalytic site, and they function with a cofactor – either a multivalent metal or a nucleotide.

The amino acids that go into protein synthesis include “indispensable” nutrients that are not made for use, but must be derived from animal protein, although the need is partially satisfied by plant sources. The essential amino acids are classified into well established groups. There are 20 amino acids commonly found in proteins.  They are classified into the following groups based on the chemical and/or structural properties of their side chains :

  1. Aliphatic Amino Acids
  2. Cyclic Amino Acid
  3. AAs with Hydroxyl or Sulfur-containing side chains
  4. Aromatic Amino Acids
  5. Basic Amino Acids
  6. Acidic Amino Acids and their Amides

Examples include:

Alanine                  aliphatic hydrophobic neutral
Arginine                 polar hydrophilic charged (+)
Cysteine                polar hydrophobic neutral
Glutamine             polar hydrophilic neutral
Histidine                aromatic polar hydrophilic charged (+)
Lysine                   polar hydrophilic charged (+)
Methionine            hydrophobic neutral
Serine                   polar hydrophilic neutral
Tyrosine                aromatic polar hydrophobic

Transcribe and Translate a Gene

  1. For each RNA base there is a corresponding DNA base
  2. Cells use the two-step process of transcription and translation to read each gene and produce the string of amino acids that makes up a protein.
  3. mRNA is produced in the nucleus, and is transferred to the ribosome
  4. mRNA uses uracil instead of thymine
  5. the ribosome reads the RNA sequence and makes protein
  6. There is a sequence combination to fit each amino acid to a three letter RNA code
  7. The ribosome starts at AUG (start), and it reads each codon three letters at a time
  8. Stop codons are UAA, UAG and UGA

 

protein synthesis

protein synthesis

http://learn.genetics.utah.edu/content/molecules/transcribe/images/TandT.png

mcell-transcription-translation

mcell-transcription-translation

http://www.vcbio.science.ru.nl/images/cellcycle/mcell-transcription-translation_eng_zoom.gif

transcription_translation

transcription_translation

 

http://www.biologycorner.com/resources/transcription_translation.JPG

 

What about the purine inosine?

Inosine triphosphate pyrophosphatase – Pyrophosphatase that hydrolyzes the non-canonical purine nucleotides inosine triphosphate (ITP), deoxyinosine triphosphate (dITP) as well as 2′-deoxy-N-6-hydroxylaminopurine triposphate (dHAPTP) and xanthosine 5′-triphosphate (XTP) to their respective monophosphate derivatives. The enzyme does not distinguish between the deoxy- and ribose forms. Probably excludes non-canonical purines from RNA and DNA precursor pools, thus preventing their incorporation into RNA and DNA and avoiding chromosomal lesions.

Gastroenterology. 2011 Apr;140(4):1314-21.  http://dx.doi.org:/10.1053/j.gastro.2010.12.038. Epub 2011 Jan 1.

Inosine triphosphate protects against ribavirin-induced adenosine triphosphate loss by adenylosuccinate synthase function.

Hitomi Y1, Cirulli ET, Fellay J, McHutchison JG, Thompson AJ, Gumbs CE, Shianna KV, Urban TJ, Goldstein DB.

Genetic variation of inosine triphosphatase (ITPA) causing an accumulation of inosine triphosphate (ITP) has been shown to protect patients against ribavirin (RBV)-induced anemia during treatment for chronic hepatitis C infection by genome-wide association study (GWAS). However, the biologic mechanism by which this occurs is unknown.

Although ITP is not used directly by human erythrocyte ATPase, it can be used for ATP biosynthesis via ADSS in place of guanosine triphosphate (GTP). With RBV challenge, erythrocyte ATP reduction was more severe in the wild-type ITPA genotype than in the hemolysis protective ITPA genotype. This difference also remains after inhibiting adenosine uptake using nitrobenzylmercaptopurine riboside (NBMPR).

ITP confers protection against RBV-induced ATP reduction by substituting for erythrocyte GTP, which is depleted by RBV, in the biosynthesis of ATP. Because patients with excess ITP appear largely protected against anemia, these results confirm that RBV-induced anemia is due primarily to the effect of the drug on GTP and consequently ATP levels in erythrocytes.

Ther Drug Monit. 2012 Aug;34(4):477-80.  http://dx.doi.org:/10.1097/FTD.0b013e31825c2703.

Determination of inosine triphosphate pyrophosphatase phenotype in human red blood cells using HPLC.

Citterio-Quentin A1, Salvi JP, Boulieu R.

Thiopurine drugs, widely used in cancer chemotherapy, inflammatory bowel disease, and autoimmune hepatitis, are responsible for common adverse events. Only some of these may be explained by genetic polymorphism of thiopurine S-methyltransferase. Recent articles have reported that inosine triphosphate pyrophosphatase (ITPase) deficiency was associated with adverse drug reactions toward thiopurine drug therapy. Here, we report a weak anion exchange high-performance liquid chromatography method to determine ITPase activity in red blood cells and to investigate the relationship with the occurrence of adverse events during azathioprine therapy.

The chromatographic method reported allows the analysis of IMP, inosine diphosphate, and ITP in a single run in <12.5 minutes. The method was linear in the range 5-1500 μmole/L of IMP. Intraassay and interassay precisions were <5% for red blood cell lysates supplemented with 50, 500, and 1000 μmole/L IMP. Km and Vmax evaluated by Lineweaver-Burk plot were 677.4 μmole/L and 19.6 μmole·L·min, respectively. The frequency distribution of ITPase from 73 patients was investigated.

The method described is useful to determine the ITPase phenotype from patients on thiopurine therapy and to investigate the potential relation between ITPase deficiency and the occurrence of adverse events.

 

System wide analyses have underestimated protein abundances and the importance of transcription in mammals

Jingyi Jessica Li1, 2, Peter J Bickel1 and Mark D Biggin3

PeerJ 2:e270; http://dx.doi.org:/10.7717/peerj.270

Using individual measurements for 61 housekeeping proteins to rescale whole proteome data from Schwanhausser et al. (2011), we find that the median protein detected is expressed at 170,000 molecules per cell and that our corrected protein abundance estimates show a higher correlation with mRNA abundances than do the uncorrected protein data. In addition, we estimated the impact of further errors in mRNA and protein abundances using direct experimental measurements of these errors. The resulting analysis suggests that mRNA levels explain at least 56% of the differences in protein abundance for the 4,212 genes detected by Schwanhausser et al. (2011), though because one major source of error could not be estimated the true percent contribution should be higher.We also employed a second, independent strategy to determine the contribution of mRNA levels to protein expression.We show that the variance in translation rates directly measured by ribosome profiling is only 12% of that inferred by Schwanhausser et al. (2011), and that the measured and inferred translation rates correlate poorly (R2 D 0.13). Based on this, our second strategy suggests that mRNA levels explain 81% of the variance in protein levels. We also determined the percent contributions of transcription, RNA degradation, translation and protein degradation to the variance in protein abundances using both of our strategies. While the magnitudes of the two estimates vary, they both suggest that transcription plays a more important role than the earlier studies implied and translation a much smaller role. Finally, the above estimates only apply to those genes whose mRNA and protein expression was detected. Based on a detailed analysis by Hebenstreit et al. (2012), we estimat that approximately 40% of genes in a given cell within a population express no mRNA. Since there can be no translation in the ab-sence of mRNA, we argue that differences in translation rates can play no role in determining the expression levels for the 40% of genes that are non-expressed.

 

Related studies that reveal issues that are not part of this chapter:

  1. Ubiquitylation in relationship to tissue remodeling
  2. Post-translational modification of proteins
    1. Glycosylation
    2. Phosphorylation
    3. Methylation
    4. Nitrosylation
    5. Sulfation – sulfotransferases
      cell-matrix communication
    6. Acetylation and histone deacetylation (HDAC)
      Connecting Protein Phosphatase to 1α (PP1α)
      Acetylation complexes (such as CBP/p300 and PCAF)
      Sirtuins
      Rel/NF-kB Signal Transduction
      Homologous Recombination Pathway of Double-Strand DNA Repair
    7. Glycination
    8. cyclin dependent kinases (CDKs)
    9. lyase
    10. transferase

 

This year, the Lasker award for basic medical research went to Kazutoshi Mori (Kyoto University) and Peter Walter (University of California, San Francisco) for their “discoveries concerning the unfolded protein response (UPR) — an intracellular quality control system that

detects harmful misfolded proteins in the endoplasmic reticulum and signals the nucleus to carry out corrective measures.”

About UPR: Approximately a third of cellular proteins pass through the Endoplasmic Reticulum (ER) which performs stringent quality control of these proteins. All proteins need to assume the proper 3-dimensional shape in order to function properly in the harsh cellular environment. Related to this is the fact that cells are under constant stress and have to make rapid, real time decisions about survival or death.

A major indicator of stress is the accumulation of unfolded proteins within the Endoplasmic Reticulum (ER), which triggers a transcriptional cascade in order to increase the folding capacity of the ER. If the metabolic burden is too great and homeostasis cannot be achieved, the response shifts from

damage control to the induction of pro-apoptotic pathways that would ultimately cause cell death.

This response to unfolded proteins or the UPR is conserved among all eukaryotes, and dysfunction in this pathway underlies many human diseases, including Alzheimer’s, Parkinson’s, Diabetes and Cancer.

 

The discovery of a new class of human proteins with previously unidentified activities

In a landmark study conducted by scientists at the Scripps Research Institute, The Hong Kong University of Science and Technology, aTyr Pharma and their collaborators, a new class of human proteins has been discovered. These proteins [nearly 250], called Physiocrines belong to the aminoacyl tRNA synthetase gene family and carry out novel, diverse and distinct biological functions.

The aminoacyl tRNA synthetase gene family codes for a group of 20 ubiquitous enzymes almost all of which are part of the protein synthesis machinery. Using recombinant protein purification, deep sequencing technique, mass spectroscopy and cell based assays, the team made this discovery. The finding is significant, also because it highlights the alternate use of a gene family whose protein product normally performs catalytic activities for non-catalytic regulation of basic and complex physiological processes spanning metabolism, vascularization, stem cell biology and immunology

 

Muscle maintenance and regeneration – key player identified

Muscle tissue suffers from atrophy with age and its regenerative capacity also declines over time. Most molecules discovered thus far to boost tissue regeneration are also implicated in cancers.  During a quest to find safer alternatives that can regenerate tissue, scientists reported that the hormone Oxytocin is required for proper muscle tissue regeneration and homeostasis and that its levels decline with age.

Oxytocin could be an alternative to hormone replacement therapy as a way to combat aging and other organ related degeneration.

Oxytocin is an age-specific circulating hormone that is necessary for muscle maintenance and regeneration (June 2014)

 

Proc Natl Acad Sci U S A. 2014 Sep 30;111(39):14289-94.   http://dx.doi.org:/10.1073/pnas.1407640111. Epub 2014 Sep 15.

Role of forkhead box protein A3 in age-associated metabolic decline.

Ma X1, Xu L1, Gavrilova O2, Mueller E3.

Aging is associated with increased adiposity and diminished thermogenesis, but the critical transcription factors influencing these metabolic changes late in life are poorly understood. We recently demonstrated that the winged helix factor forkhead box protein A3 (Foxa3) regulates the expansion of visceral adipose tissue in high-fat diet regimens; however, whether Foxa3 also contributes to the increase in adiposity and the decrease in brown fat activity observed during the normal aging process is currently unknown. Here we report that during aging, levels of Foxa3 are significantly and selectively up-regulated in brown and inguinal white fat depots, and that midage Foxa3-null mice have increased white fat browning and thermogenic capacity, decreased adipose tissue expansion, improved insulin sensitivity, and increased longevity. Foxa3 gain-of-function and loss-of-function studies in inguinal adipose depots demonstrated a cell-autonomous function for Foxa3 in white fat tissue browning. Furthermore, our analysis revealed that the mechanisms of Foxa3 modulation of brown fat gene programs involve the suppression of peroxisome proliferator activated receptor γ coactivtor 1 α (PGC1α) levels through interference with cAMP responsive element binding protein 1-mediated transcriptional regulation of the PGC1α promoter.

 

Asymmetric mRNA localization contributes to fidelity and sensitivity of spatially localized systems

RJ Weatheritt, TJ Gibson & MM Babu
Nature Structural & Molecular Biology 24 Aug, 2014; 21: 833–839 http://dx.do.orgi:/10.1038/nsmb.2876

Although many proteins are localized after translation, asymmetric protein distribution is also achieved by translation after mRNA localization. Why are certain mRNA transported to a distal location and translated on-site? Here we undertake a systematic, genome-scale study of asymmetrically distributed protein and mRNA in mammalian cells. Our findings suggest that asymmetric protein distribution by mRNA localization enhances interaction fidelity and signaling sensitivity. Proteins synthesized at distal locations frequently contain intrinsically disordered segments. These regions are generally rich in assembly-promoting modules and are often regulated by post-translational modifications. Such proteins are tightly regulated but display distinct temporal dynamics upon stimulation with growth factors. Thus, proteins synthesized on-site may rapidly alter proteome composition and act as dynamically regulated scaffolds to promote the formation of reversible cellular assemblies. Our observations are consistent across multiple mammalian species, cell types and developmental stages, suggesting that localized translation is a recurring feature of cell signaling and regulation.

 

An overview of the potential advantages conferred by distal-site protein synthesis, inferred from our analysis.

 

An overview of the potential advantages conferred by distal-site protein synthesis

An overview of the potential advantages conferred by distal-site protein synthesis

 

Turquoise and red filled circle represents off-target and correct interaction partners, respectively. Wavy lines represent a disordered region within a distal site synthesis protein. Grey and red line in graphs represents profiles of t…

http://www.nature.com/nsmb/journal/v21/n9/carousel/nsmb.2876-F5.jpg

 

Tweaking transcriptional programming for high quality recombinant protein production

Since overexpression of recombinant proteins in E. coli often leads to the formation of inclusion bodies, producing properly folded, soluble proteins is undoubtedly the most important end goal in a protein expression campaign. Various approaches have been devised to bypass the insolubility issues during E. coli expression and in a recent report a group of researchers discuss reprogramming the E. coli proteostasis [protein homeostasis] network to achieve high yields of soluble, functional protein. The premise of their studies is that the basal E. coli proteostasis network is insufficient, and often unable, to fold overexpressed proteins, thus clogging the folding machinery.

By overexpressing a mutant, negative-feedback deficient heat shock transcription factor [σ32 I54N] before and during overexpression of the protein of interest, reprogramming can be achieved, resulting in high yields of soluble and functional recombinant target protein. The authors explain that this method is better than simply co-expressing/over-expressing chaperones, co-chaperones, foldases or other components of the proteostasis network because reprogramming readies the folding machinery and up regulates the essential folding components beforehand thus  maintaining system capability of the folding machinery.

The Heat-Shock Response Transcriptional Program Enables High-Yield and High-Quality Recombinant Protein Production in Escherichia coli (July 2014)

 

 Unfolded proteins collapse when exposed to heat and crowded environments

Proteins are important molecules in our body and they fulfil a broad range of functions. For instance as enzymes they help to release energy from food and as muscle proteins they assist with motion. As antibodies they are involved in immune defence and as hormone receptors in signal transduction in cells. Until only recently it was assumed that all proteins take on a clearly defined three-dimensional structure – i.e. they fold in order to be able to assume these functions. Surprisingly, it has been shown that many important proteins occur as unfolded coils. Researchers seek to establish how these disordered proteins are capable at all of assuming highly complex functions.

Ben Schuler’s research group from the Institute of Biochemistry of the University of Zurich has now established that an increase in temperature leads to folded proteins collapsing and becoming smaller. Other environmental factors can trigger the same effect.

Measurements using the “molecular ruler”

“The fact that unfolded proteins shrink at higher temperatures is an indication that cell water does indeed play an important role as to the spatial organisation eventually adopted by the molecules”, comments Schuler with regard to the impact of temperature on protein structure. For their studies the biophysicists use what is known as single-molecule spectroscopy. Small colour probes in the protein enable the observation of changes with an accuracy of more than one millionth of a millimetre. With this “molecular yardstick” it is possible to measure how molecular forces impact protein structure.

With computer simulations the researchers have mimicked the behaviour of disordered proteins.
(Courtesy of Jose EDS Roselino, PhD.

 

MLKL compromises plasma membrane integrity

Necroptosis is implicated in many diseases and understanding this process is essential in the search for new therapies. While mixed lineage kinase domain-like (MLKL) protein has been known to be a critical component of necroptosis induction, how MLKL transduces the death signal was not clear. In a recent finding, scientists demonstrated that the full four-helical bundle domain (4HBD) in the N-terminal region of MLKL is required and sufficient to induce its oligomerization and trigger cell death.

They also found a patch of positively charged amino acids on the surface of the 4HBD that bound to phosphatidylinositol phosphates (PIPs) and allowed the recruitment of MLKL to the plasma membrane that resulted in the formation of pores consisting of MLKL proteins, due to which cells absorbed excess water causing them to explode. Detailed knowledge about how MLKL proteins create pores offers possibilities for the development of new therapeutic interventions for tolerating or preventing cell death.

MLKL compromises plasma membrane integrity by binding to phosphatidylinositol phosphates (May 2014)

 

Mitochondrial and ER proteins implicated in dementia

Mitochondria and the endoplasmic reticulum (ER) form tight structural associations that facilitate a number of cellular functions. However, the molecular mechanisms of these interactions aren’t properly understood.

A group of researchers showed that the ER protein VAPB interacted with mitochondrial protein PTPIP51 to regulate ER-mitochondria associations and that TDP-43, a protein implicated in dementia, disturbs this interaction to regulate cellular Ca2+ homeostasis. These studies point to a new pathogenic mechanism for TDP-43 and may also provide a potential new target for the development of new treatments for devastating neurological conditions like dementia.

ER-mitochondria associations are regulated by the VAPB-PTPIP51 interaction and are disrupted by ALS/FTD-associated TDP-43. Nature (June 2014)

 

A novel strategy to improve membrane protein expression in Yeast

Membrane proteins play indispensable roles in the physiology of an organism. However, recombinant production of membrane proteins is one of the biggest hurdles facing protein biochemists today. A group of scientists in Belgium showed that,

by increasing the intracellular membrane production by interfering with a key enzymatic step of lipid synthesis,

enhanced expression of recombinant membrane proteins in yeast is achieved.

Specifically, they engineered the oleotrophic yeast, Yarrowia lipolytica, by

deleting the phosphatidic acid phosphatase, PAH1 gene,

which led to massive proliferation of endoplasmic reticulum (ER) membranes.

For all 8 tested representatives of different integral membrane protein families, they obtained enhanced protein accumulation.

 

An unconventional method to boost recombinant protein levels

MazF is an mRNA interferase enzyme in E.coli that functions as and degrades cellular mRNA in a targeted fashion, at the “ACA” sequence. This degradation of cellular mRNA causes a precipitous drop in cellular protein synthesis. A group of scientists at the Robert Wood Johnson Medical School in New Jersey, exploited the degeneracy of the genetic code to modify all “ACA” triplets within their gene of interest in a way that the corresponding amino acid (Threonine) remained unchanged. Consequently, induction of MazF toxin caused degradation of E.coli cellular mRNA but the recombinant gene transcription and protein synthesis continued, causing significant accumulation of high quality target protein. This expression system enables unparalleled signal to noise ratios that could dramatically simplify structural and functional studies of difficult-to-purify, biologically important proteins.

 

Tandem fusions and bacterial strain evolution for enhanced functional membrane protein production

Membrane protein production remains a significant challenge in its characterization and structure determination. Despite the fact that there are a variety of host cell types, E.coli remains the popular choice for producing recombinant membrane proteins. A group of scientists in Netherlands devised a robust strategy to increase the probability of functional membrane protein overexpression in E.coli.

By fusing Green Fluorescent Protein (GFP) and the Erythromycin Resistance protein (ErmC) to the C-terminus of a target membrane protein they wer e able to track the folding state of their target protein while using Erythromycin to select for increased expression. By increasing erythromycin concentration in the growth media and testing different membrane targets, they were able to identify four evolved E.coli strains, all of which carried a mutation in the hns gene, whose product is implicated in genome organization and transcriptional silencing. Through their experiments the group showed that partial removal of the transcriptional silencing mechanism was related to production of proteins that were essential for functional overexpression of membrane proteins.

 

The role of an anti-apoptotic factor in recombinant protein production

In a recent study, scientists at the Johns Hopkins University and Frederick National Laboratory for Cancer Research examined an alternative method of utilizing the benefits of anti-apoptotic gene expression to enhance the transient expression of biotherapeutics, specifically, through the co-transfection of Bcl-xL along with the product-coding target gene.

Chinese Hamster Ovary(CHO) cells were co-transfected with the product-coding gene and a vector containing Bcl-xL, using Polyethylenimine (PEI) reagent. They found that the cells co-transfected with Bcl-xL demonstrated reduced apoptosis, increased specific productivity, and an overall increase in product yield.

B-cell lymphoma-extra-large (Bcl-xL) is a mitochondrial transmembrane protein and a member of the Bcl-2 family of proteins which are known to act as either pro- or anti-apoptotic proteins. Bcl-xL itself acts as an anti-apoptotic molecule by preventing the release of mitochondrial contents such as cytochrome c, which would lead to caspase activation. Higher levels of Bcl-xL push a cell toward survival mode by making the membranes pores less permeable and leaky.

Introduction to Protein Synthesis and Degradation Updated 8/31/2019

N-Terminal Degradation of Proteins: The N-End Rule and N-degrons

In both prokaryotes and eukaryotes mitochondria and chloroplasts, the ribosomal synthesis of proteins is initiated with the addition of the N-formyl methionine residue.  However in eukaryotic cytosolic ribosomes, the N terminal was assumed to be devoid of the N-formyl group.  The unformylated N-terminal methionine residues of eukaryotes is then  often N-acetylated (Ac) and creates specific degradation signals, the Ac N-end rule.  These N-end rule pathways are proteolytic systems which recognize these N-degrons resulting in proteosomal degradation or autophagy.  In prokaryotes this system is stimulated by certain amino acid deficiencies and in eukaryotes is dependent on the Psh1 E3 ligase.

Two papers in the journal Science describe this N-degron in more detail.

Structured Abstract
INTRODUCTION

In both bacteria and eukaryotic mitochondria and chloroplasts, the ribosomal synthesis of proteins is initiated with the N-terminal (Nt) formyl-methionine (fMet) residue. Nt-fMet is produced pretranslationally by formyltransferases, which use 10-formyltetrahydrofolate as a cosubstrate. By contrast, proteins synthesized by cytosolic ribosomes of eukaryotes were always presumed to bear unformylated N-terminal Met (Nt-Met). The unformylated Nt-Met residue of eukaryotic proteins is often cotranslationally Nt-acetylated, a modification that creates specific degradation signals, Ac/N-degrons, which are targeted by the Ac/N-end rule pathway. The N-end rule pathways are a set of proteolytic systems whose unifying feature is their ability to recognize proteins containing N-degrons, thereby causing the degradation of these proteins by the proteasome or autophagy in eukaryotes and by the proteasome-like ClpAP protease in bacteria. The main determinant of an N‑degron is a destabilizing Nt-residue of a protein. Studies over the past three decades have shown that all 20 amino acids of the genetic code can act, in cognate sequence contexts, as destabilizing Nt‑residues. The previously known eukaryotic N-end rule pathways are the Arg/N-end rule pathway, the Ac/N-end rule pathway, and the Pro/N-end rule pathway. Regulated degradation of proteins and their natural fragments by the N-end rule pathways has been shown to mediate a broad range of biological processes.

RATIONALE

The chemical similarity of the formyl and acetyl groups and their identical locations in, respectively, Nt‑formylated and Nt-acetylated proteins led us to suggest, and later to show, that the Nt-fMet residues of nascent bacterial proteins can act as bacterial N-degrons, termed fMet/N-degrons. Here we wished to determine whether Nt-formylated proteins might also form in the cytosol of a eukaryote such as the yeast Saccharomyces cerevisiae and to determine the metabolic fates of Nt-formylated proteins if they could be produced outside mitochondria. Our approaches included molecular genetic techniques, mass spectrometric analyses of proteins’ N termini, and affinity-purified antibodies that selectively recognized Nt-formylated reporter proteins.

RESULTS

We discovered that the yeast formyltransferase Fmt1, which is imported from the cytosol into the mitochondria inner matrix, can generate Nt-formylated proteins in the cytosol, because the translocation of Fmt1 into mitochondria is not as efficacious, even under unstressful conditions, as had previously been assumed. We also found that Nt‑formylated proteins are greatly up-regulated in stationary phase or upon starvation for specific amino acids. The massive increase of Nt-formylated proteins strictly requires the Gcn2 kinase, which phosphorylates Fmt1 and mediates its retention in the cytosol. Notably, the ability of Gcn2 to retain a large fraction of Fmt1 in the cytosol of nutritionally stressed cells is confined to Fmt1, inasmuch as the Gcn2 kinase does not have such an effect, under the same conditions, on other examined nuclear DNA–encoded mitochondrial matrix proteins. The Gcn2-Fmt1 protein localization circuit is a previously unknown signal transduction pathway. A down-regulation of cytosolic Nt‑formylation was found to increase the sensitivity of cells to undernutrition stresses, to a prolonged cold stress, and to a toxic compound. We also discovered that the Nt-fMet residues of Nt‑formylated cytosolic proteins act as eukaryotic fMet/N-degrons and identified the Psh1 E3 ubiquitin ligase as the recognition component (fMet/N-recognin) of the previously unknown eukaryotic fMet/N-end rule pathway, which destroys Nt‑formylated proteins.

CONCLUSION

The Nt-formylation of proteins, a long-known pretranslational protein modification, is mediated by formyltransferases. Nt-formylation was thought to be confined to bacteria and bacteria-descended eukaryotic organelles but was found here to also occur at the start of translation by the cytosolic ribosomes of a eukaryote. The levels of Nt‑formylated eukaryotic proteins are greatly increased upon specific stresses, including undernutrition, and appear to be important for adaptation to these stresses. We also discovered that Nt-formylated cytosolic proteins are selectively destroyed by the eukaryotic fMet/N-end rule pathway, mediated by the Psh1 E3 ubiquitin ligase. This previously unknown proteolytic system is likely to be universal among eukaryotes, given strongly conserved mechanisms that mediate Nt‑formylation and degron recognition.

The eukaryotic fMet/N-end rule pathway.

(Top) Under undernutrition conditions, the Gcn2 kinase augments the cytosolic localization of the Fmt1 formyltransferase, and possibly also its enzymatic activity. Consequently, Fmt1 up-regulates the cytosolic fMet–tRNAi (initiator transfer RNA), and thereby increases the levels of cytosolic Nt-formylated proteins, which are required for the adaptation of cells to specific stressors. (Bottom) The Psh1 E3 ubiquitin ligase targets the N-terminal fMet-residues of eukaryotic cytosolic proteins, such as Cse4, Pgd1, and Rps22a, for the polyubiquitylation-mediated, proteasome-dependent degradation.

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The eukaryotic fMet/N-end rule pathway.

(Top) Under undernutrition conditions, the Gcn2 kinase augments the cytosolic localization of the Fmt1 formyltransferase, and possibly also its enzymatic activity. Consequently, Fmt1 up-regulates the cytosolic fMet–tRNAi (initiator transfer RNA), and thereby increases the levels of cytosolic Nt-formylated proteins, which are required for the adaptation of cells to specific stressors. (Bottom) The Psh1 E3 ubiquitin ligase targets the N-terminal fMet-residues of eukaryotic cytosolic proteins, such as Cse4, Pgd1, and Rps22a, for the polyubiquitylation-mediated, proteasome-dependent degradation.

 

A glycine-specific N-degron pathway mediates the quality control of protein N-myristoylation. Richard T. Timms1,2, Zhiqian Zhang1,2, David Y. Rhee3, J. Wade Harper3, Itay Koren1,2,*,†, Stephen J. Elledge1,2

Science  05 Jul 2019: Vol. 365, Issue 6448

The second paper describes a glycine specific N-degron pathway in humans.  Specifically the authors set up a screen to identify specific N-terminal degron motifs in the human.  Findings included an expanded repertoire for the UBR E3 ligases to include substrates with arginine and lysine following an intact initiator methionine and a glycine at the extreme N-terminus, which is a potent degron.

Glycine N-degron regulation revealed

For more than 30 years, N-terminal sequences have been known to influence protein stability, but additional features of these N-end rule, or N-degron, pathways continue to be uncovered. Timms et al. used a global protein stability (GPS) technology to take a broader look at these pathways in human cells. Unexpectedly, glycine exposed at the N terminus could act as a potent degron; proteins bearing N-terminal glycine were targeted for proteasomal degradation by two Cullin-RING E3 ubiquitin ligases through the substrate adaptors ZYG11B and ZER1. This pathway may be important, for example, to degrade proteins that fail to localize properly to cellular membranes and to destroy protein fragments generated during cell death.

Science, this issue p. eaaw4912

Structured Abstract

INTRODUCTION

The ubiquitin-proteasome system is the major route through which the cell achieves selective protein degradation. The E3 ubiquitin ligases are the major determinants of specificity in this system, which is thought to be achieved through their selective recognition of specific degron motifs in substrate proteins. However, our ability to identify these degrons and match them to their cognate E3 ligase remains a major challenge.

RATIONALE

It has long been known that the stability of proteins is influenced by their N-terminal residue, and a large body of work over the past three decades has characterized a collection of N-end rule pathways that target proteins for degradation through N-terminal degron motifs. Recently, we developed Global Protein Stability (GPS)–peptidome technology and used it to delineate a suite of degrons that lie at the extreme C terminus of proteins. We adapted this approach to examine the stability of the human N terminome, allowing us to reevaluate our understanding of N-degron pathways in an unbiased manner.

RESULTS

Stability profiling of the human N terminome identified two major findings: an expanded repertoire for UBR family E3 ligases to include substrates that begin with arginine and lysine following an intact initiator methionine and, more notably, that glycine positioned at the extreme N terminus can act as a potent degron. We established human embryonic kidney 293T reporter cell lines in which unstable peptides that bear N-terminal glycine degrons were fused to green fluorescent protein, and we performed CRISPR screens to identify the degradative machinery involved. These screens identified two Cul2 Cullin-RING E3 ligase complexes, defined by the related substrate adaptors ZYG11B and ZER1, that act redundantly to target substrates bearing N-terminal glycine degrons for proteasomal degradation. Moreover, through the saturation mutagenesis of example substrates, we defined the composition of preferred N-terminal glycine degrons specifically recognized by ZYG11B and ZER1.

We found that preferred glycine degrons are depleted from the native N termini of metazoan proteomes, suggesting that proteins have evolved to avoid degradation through this pathway, but are strongly enriched at annotated caspase cleavage sites. Stability profiling of N-terminal peptides lying downstream of all known caspase cleavages sites confirmed that Cul2ZYG11Band Cul2ZER1 could make a substantial contribution to the removal of proteolytic cleavage products during apoptosis. Last, we identified a role for ZYG11B and ZER1 in the quality control of N-myristoylated proteins. N-myristoylation is an important posttranslational modification that occurs exclusively on N-terminal glycine. By profiling the stability of the human N-terminome in the absence of the N-myristoyltransferases NMT1 and NMT2, we found that a failure to undergo N-myristoylation exposes N-terminal glycine degrons that are otherwise obscured. Thus, conditional exposure of glycine degrons to ZYG11B and ZER1 permits the selective proteasomal degradation of aberrant proteins that have escaped N-terminal myristoylation.

CONCLUSION

These data demonstrate that an additional N-degron pathway centered on N-terminal glycine regulates the stability of metazoan proteomes. Cul2ZYG11B– and Cul2ZER1-mediated protein degradation through N-terminal glycine degrons may be particularly important in the clearance of proteolytic fragments generated by caspase cleavage during apoptosis and in the quality control of protein N-myristoylation.

The glycine N-degron pathway.

Stability profiling of the human N-terminome revealed that N-terminal glycine acts as a potent degron. CRISPR screening revealed two Cul2 complexes, defined by the related substrate adaptors ZYG11B and ZER1, that recognize N-terminal glycine degrons. This pathway may be particularly important for the degradation of caspase cleavage products during apoptosis and the removal of proteins that fail to undergo N-myristoylation.

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The glycine N-degron pathway.

Stability profiling of the human N-terminome revealed that N-terminal glycine acts as a potent degron. CRISPR screening revealed two Cul2 complexes, defined by the related substrate adaptors ZYG11B and ZER1, that recognize N-terminal glycine degrons. This pathway may be particularly important for the degradation of caspase cleavage products during apoptosis and the removal of proteins that fail to undergo N-myristoylation.

 

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Summary of Signaling and Signaling Pathways

Summary of Signaling and Signaling Pathways

Author and Curator: Larry H Bernstein, MD, FCAP

In the imtroduction to this series of discussions I pointed out JEDS Rosalino’s observation about the construction of a complex molecule of acetyl coenzyme A, and the amount of genetic coding that had to go into it.  Furthermore, he observes –  Millions of years later, or as soon as, the information of interaction leading to activity and regulation could be found in RNA, proteins like reverse transcriptase move this information to a more stable form (DNA). In this way it is easier to understand the use of CoA to make two carbon molecules more reactive.

acetylCoA

acetylCoA

In the tutorial that follows we find support for the view that mechanisms and examples from the current literature, which give insight into the developments in cell metabolism, are achieving a separation from inconsistent views introduced by the classical model of molecular biology and genomics, toward a more functional cellular dynamics that is not dependent on the classic view.  The classical view fits a rigid framework that is to genomics and metabolomics as Mendelian genetics if to multidimentional, multifactorial genetics.  The inherent difficulty lies in two places:

  1. Interactions between differently weighted determinants
  2. A large part of the genome is concerned with regulatory function, not expression of the code

The goal of the tutorial was to achieve an understanding of how cell signaling occurs in a cell.  Completion of the tutorial would provide

  1. a basic understanding signal transduction and
  2. the role of phosphorylation in signal transduction.
Regulation of the integrity of endothelial cell–cell contacts by phosphorylation of VE-cadherin

Regulation of the integrity of endothelial cell–cell contacts by phosphorylation of VE-cadherin

In addition – detailed knowledge of –

  1. the role of Tyrosine kinases and
  2. G protein-coupled receptors in cell signaling.
serine

serine

threonine

threonine

protein kinase

protein kinase

We are constantly receiving and interpreting signals from our environment, which can come

  • in the form of light, heat, odors, touch or sound.

The cells of our bodies are also

  • constantly receiving signals from other cells.

These signals are important to

  • keep cells alive and functioning as well as
  • to stimulate important events such as
  • cell division and differentiation.

Signals are most often chemicals that can be found

  • in the extracellular fluid around cells.

These chemicals can come

  • from distant locations in the body (endocrine signaling by hormones), from
  • nearby cells (paracrine signaling) or can even
  • be secreted by the same cell (autocrine signaling).

Notch-mediated juxtacrine signal between adjacent cells. 220px-Notchccr

Signaling molecules may trigger any number of cellular responses, including

  • changing the metabolism of the cell receiving the signal or
  • result in a change in gene expression (transcription) within the nucleus of the cell or both.
controlling the output of ribosomes.

controlling the output of ribosomes.

To which I would now add..

  • result in either an inhibitory or a stimulatory effect

The three stages of cell signaling are:

Cell signaling can be divided into 3 stages:

Reception: A cell detects a signaling molecule from the outside of the cell.

Transduction: When the signaling molecule binds the receptor it changes the receptor protein in some way. This change initiates the process of transduction. Signal transduction is usually a pathway of several steps. Each relay molecule in the signal transduction pathway changes the next molecule in the pathway.

Response: Finally, the signal triggers a specific cellular response.

signal transduction

signal transduction

http://www.hartnell.edu/tutorials/biology/images/signaltransduction_simple.jpg

The initiation is depicted as follows:

Signal Transduction – ligand binds to surface receptor

Membrane receptors function by binding the signal molecule (ligand) and causing the production of a second signal (also known as a second messenger) that then causes a cellular response. These types of receptors transmit information from the extracellular environment to the inside of the cell.

  • by changing shape or
  • by joining with another protein
  • once a specific ligand binds to it.

Examples of membrane receptors include

  • G Protein-Coupled Receptors and
Understanding these receptors and identifying their ligands and the resulting signal transduction pathways represent a major conceptual advance.

Understanding these receptors and identifying their ligands and the resulting signal transduction pathways represent a major conceptual advance.

  • Receptor Tyrosine Kinases.
intracellular signaling

intracellular signaling

http://www.hartnell.edu/tutorials/biology/images/membrane_receptor_tk.jpg

Intracellular receptors are found inside the cell, either in the cytopolasm or in the nucleus of the target cell (the cell receiving the signal).

Note that though change in gene expression is stated, the change in gene expression does not here imply a change in the genetic information – such as – mutation.  That does not have to be the case in the normal homeostatic case.

This point is the differentiating case between what JEDS Roselino has referred as

  1. a fast, adaptive reaction, that is the feature of protein molecules, and distinguishes this interaction from
  2. a one-to-one transcription of the genetic code.

The rate of transcription can be controlled, or it can be blocked.  This is in large part in response to the metabolites in the immediate interstitium.

This might only be

  • a change in the rate of a transcription or a suppression of expression through RNA.
  • Or through a conformational change in an enzyme
 Swinging domains in HECT E3 enzymes

Swinging domains in HECT E3 enzymes

Since signaling systems need to be

  • responsive to small concentrations of chemical signals and act quickly,
  • cells often use a multi-step pathway that transmits the signal quickly,
  • while amplifying the signal to numerous molecules at each step.

Signal transduction pathways are shown (simplified):

Signal Transduction

Signal Transduction

Signal transduction occurs when an

  1. extracellular signaling molecule activates a specific receptor located on the cell surface or inside the cell.
  2. In turn, this receptor triggers a biochemical chain of events inside the cell, creating a response.
  3. Depending on the cell, the response alters the cell’s metabolism, shape, gene expression, or ability to divide.
  4. The signal can be amplified at any step. Thus, one signaling molecule can cause many responses.

In 1970, Martin Rodbell examined the effects of glucagon on a rat’s liver cell membrane receptor. He noted that guanosine triphosphate disassociated glucagon from this receptor and stimulated the G-protein, which strongly influenced the cell’s metabolism. Thus, he deduced that the G-protein is a transducer that accepts glucagon molecules and affects the cell. For this, he shared the 1994 Nobel Prize in Physiology or Medicine with Alfred G. Gilman.

Guanosine monophosphate structure

Guanosine monophosphate structure

In 2007, a total of 48,377 scientific papers—including 11,211 e-review papers—were published on the subject. The term first appeared in a paper’s title in 1979. Widespread use of the term has been traced to a 1980 review article by Rodbell: Research papers focusing on signal transduction first appeared in large numbers in the late 1980s and early 1990s.

Signal transduction involves the binding of extracellular signaling molecules and ligands to cell-surface receptors that trigger events inside the cell. The combination of messenger with receptor causes a change in the conformation of the receptor, known as receptor activation.

This activation is always the initial step (the cause) leading to the cell’s ultimate responses (effect) to the messenger. Despite the myriad of these ultimate responses, they are all directly due to changes in particular cell proteins. Intracellular signaling cascades can be started through cell-substratum interactions; examples are the integrin that binds ligands in the extracellular matrix and steroids.

Integrin

Integrin

Most steroid hormones have receptors within the cytoplasm and act by stimulating the binding of their receptors to the promoter region of steroid-responsive genes.

steroid hormone receptor

steroid hormone receptor

Various environmental stimuli exist that initiate signal transmission processes in multicellular organisms; examples include photons hitting cells in the retina of the eye, and odorants binding to odorant receptors in the nasal epithelium. Certain microbial molecules, such as viral nucleotides and protein antigens, can elicit an immune system response against invading pathogens mediated by signal transduction processes. This may occur independent of signal transduction stimulation by other molecules, as is the case for the toll-like receptor. It may occur with help from stimulatory molecules located at the cell surface of other cells, as with T-cell receptor signaling. Receptors can be roughly divided into two major classes: intracellular receptors and extracellular receptors.

Signal transduction cascades amplify the signal output

Signal transduction cascades amplify the signal output

Signal transduction cascades amplify the signal output

G protein-coupled receptors (GPCRs) are a family of integral transmembrane proteins that possess seven transmembrane domains and are linked to a heterotrimeric G protein. Many receptors are in this family, including adrenergic receptors and chemokine receptors.

Arrestin binding to active GPCR kinase (GRK)-phosphorylated GPCRs blocks G protein coupling

signal transduction pathways

signal transduction pathways

Arrestin binding to active GPCR kinase (GRK)-phosphorylated GPCRs blocks G protein coupling

Arrestin binding to active GPCR kinase (GRK)-phosphorylated GPCRs blocks G protein coupling

Signal transduction by a GPCR begins with an inactive G protein coupled to the receptor; it exists as a heterotrimer consisting of Gα, Gβ, and Gγ. Once the GPCR recognizes a ligand, the conformation of the receptor changes to activate the G protein, causing Gα to bind a molecule of GTP and dissociate from the other two G-protein subunits.

The dissociation exposes sites on the subunits that can interact with other molecules. The activated G protein subunits detach from the receptor and initiate signaling from many downstream effector proteins such as phospholipases and ion channels, the latter permitting the release of second messenger molecules.

Receptor tyrosine kinases (RTKs) are transmembrane proteins with an intracellular kinase domain and an extracellular domain that binds ligands; examples include growth factor receptors such as the insulin receptor.

 insulin receptor and and insulin receptor signaling pathway (IRS)

insulin receptor and and insulin receptor signaling pathway (IRS)

To perform signal transduction, RTKs need to form dimers in the plasma membrane; the dimer is stabilized by ligands binding to the receptor.

RTKs

RTKs

The interaction between the cytoplasmic domains stimulates the autophosphorylation of tyrosines within the domains of the RTKs, causing conformational changes.

Allosteric_Regulation.svg

Subsequent to this, the receptors’ kinase domains are activated, initiating phosphorylation signaling cascades of downstream cytoplasmic molecules that facilitate various cellular processes such as cell differentiation and metabolism.

Signal-Transduction-Pathway

Signal-Transduction-Pathway

As is the case with GPCRs, proteins that bind GTP play a major role in signal transduction from the activated RTK into the cell. In this case, the G proteins are

  • members of the Ras, Rho, and Raf families, referred to collectively as small G proteins.

They act as molecular switches usually

  • tethered to membranes by isoprenyl groups linked to their carboxyl ends.

Upon activation, they assign proteins to specific membrane subdomains where they participate in signaling. Activated RTKs in turn activate

  • small G proteins that activate guanine nucleotide exchange factors such as SOS1.

Once activated, these exchange factors can activate more small G proteins, thus

  • amplifying the receptor’s initial signal.

The mutation of certain RTK genes, as with that of GPCRs, can result in the expression of receptors that exist in a constitutively activate state; such mutated genes may act as oncogenes.

Integrin

 

Integrin

Integrin

Integrin-mediated signal transduction

An overview of integrin-mediated signal transduction, adapted from Hehlgens et al. (2007).

Integrins are produced by a wide variety of cells; they play a role in

  • cell attachment to other cells and the extracellular matrix and
  • in the transduction of signals from extracellular matrix components such as fibronectin and collagen.

Ligand binding to the extracellular domain of integrins

  • changes the protein’s conformation,
  • clustering it at the cell membrane to
  • initiate signal transduction.

Integrins lack kinase activity; hence, integrin-mediated signal transduction is achieved through a variety of intracellular protein kinases and adaptor molecules, the main coordinator being integrin-linked kinase.

As shown in the picture, cooperative integrin-RTK signaling determines the

  1. timing of cellular survival,
  2. apoptosis,
  3. proliferation, and
  4. differentiation.
integrin-mediated signal transduction

integrin-mediated signal transduction

Integrin signaling

Integrin signaling

ion channel

A ligand-gated ion channel, upon binding with a ligand, changes conformation

  • to open a channel in the cell membrane
  • through which ions relaying signals can pass.

An example of this mechanism is found in the receiving cell of a neural synapse. The influx of ions that occurs in response to the opening of these channels

  1. induces action potentials, such as those that travel along nerves,
  2. by depolarizing the membrane of post-synaptic cells,
  3. resulting in the opening of voltage-gated ion channels.
RyR and Ca+ release from SR

RyR and Ca+ release from SR

An example of an ion allowed into the cell during a ligand-gated ion channel opening is Ca2+;

  • it acts as a second messenger
  • initiating signal transduction cascades and
  • altering the physiology of the responding cell.

This results in amplification of the synapse response between synaptic cells

  • by remodelling the dendritic spines involved in the synapse.

In eukaryotic cells, most intracellular proteins activated by a ligand/receptor interaction possess an enzymatic activity; examples include tyrosine kinase and phosphatases. Some of them create second messengers such as cyclic AMP and IP3,

cAMP

cAMP

Inositol_1,4,5-trisphosphate.svg

Inositol_1,4,5-trisphosphate.svg

  • the latter controlling the release of intracellular calcium stores into the cytoplasm.

Many adaptor proteins and enzymes activated as part of signal transduction possess specialized protein domains that bind to specific secondary messenger molecules. For example,

  • calcium ions bind to the EF hand domains of calmodulin,
  • allowing it to bind and activate calmodulin-dependent kinase.
calcium movement and RyR2 receptor

calcium movement and RyR2 receptor

PIP3 and other phosphoinositides do the same thing to the Pleckstrin homology domains of proteins such as the kinase protein AKT.

Signals can be generated within organelles, such as chloroplasts and mitochondria, modulating the nuclear
gene expression in a process called retrograde signaling.

Recently, integrative genomics approaches, in which correlation analysis has been applied on transcript and metabolite profiling data of Arabidopsis thaliana, revealed the identification of metabolites which are putatively acting as mediators of nuclear gene expression.

http://fpls.com/unraveling_retrograde_signaling_pathways:_finding_candidate_signaling_molecules_via_metabolomics_and_systems_biology_driven_approaches

Related articles

  1. Systems Biology Approach Reveals Genome to Phenome Correlation in Type 2 Diabetes (plosone.org)
  2. Gene Expression and Thiopurine Metabolite Profiling in Inflammatory Bowel Disease – Novel Clues to Drug Targets and Disease Mechanisms? (plosone.org)
  3. Activation of the Jasmonic Acid Plant Defence Pathway Alters the Composition of Rhizosphere

Nutrients 2014, 6, 3245-3258; http://dx.doi.org:/10.3390/nu6083245

Omega-3 (ω-3) fatty acids are one of the two main families of long chain polyunsaturated fatty acids (PUFA). The main omega-3 fatty acids in the mammalian body are

  • α-linolenic acid (ALA), docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA).

Central nervous tissues of vertebrates are characterized by a high concentration of omega-3 fatty acids. Moreover, in the human brain,

  • DHA is considered as the main structural omega-3 fatty acid, which comprises about 40% of the PUFAs in total.

DHA deficiency may be the cause of many disorders such as depression, inability to concentrate, excessive mood swings, anxiety, cardiovascular disease, type 2 diabetes, dry skin and so on.

On the other hand,

  • zinc is the most abundant trace metal in the human brain.

There are many scientific studies linking zinc, especially

  • excess amounts of free zinc, to cellular death.

Neurodegenerative diseases, such as Alzheimer’s disease, are characterized by altered zinc metabolism. Both animal model studies and human cell culture studies have shown a possible link between

  • omega-3 fatty acids, zinc transporter levels and
  • free zinc availability at cellular levels.

Many other studies have also suggested a possible

  • omega-3 and zinc effect on neurodegeneration and cellular death.

Therefore, in this review, we will examine

  • the effect of omega-3 fatty acids on zinc transporters and
  • the importance of free zinc for human neuronal cells.

Moreover, we will evaluate the collective understanding of

  • mechanism(s) for the interaction of these elements in neuronal research and their
  • significance for the diagnosis and treatment of neurodegeneration.

Epidemiological studies have linked high intake of fish and shellfish as part of the daily diet to

  • reduction of the incidence and/or severity of Alzheimer’s disease (AD) and senile mental decline in

Omega-3 fatty acids are one of the two main families of a broader group of fatty acids referred to as polyunsaturated fatty acids (PUFAs). The other main family of PUFAs encompasses the omega-6 fatty acids. In general, PUFAs are essential in many biochemical events, especially in early post-natal development processes such as

  • cellular differentiation,
  • photoreceptor membrane biogenesis and
  • active synaptogenesis.

Despite the significance of these

two families, mammals cannot synthesize PUFA de novo, so they must be ingested from dietary sources. Though belonging to the same family, both

  • omega-3 and omega-6 fatty acids are metabolically and functionally distinct and have
  • opposing physiological effects. In the human body,
  • high concentrations of omega-6 fatty acids are known to increase the formation of prostaglandins and
  • thereby increase inflammatory processes [10].

the reverse process can be seen with increased omega-3 fatty acids in the body.

Many other factors, such as

  1. thromboxane A2 (TXA2),
  2. leukotriene
  3. B4 (LTB4),
  4. IL-1,
  5. IL-6,
  6. tumor necrosis factor (TNF) and
  7. C-reactive protein,

which are implicated in various health conditions, have been shown to be increased with high omega-6 fatty acids but decreased with omega-3 fatty acids in the human body.

Dietary fatty acids have been identified as protective factors in coronary heart disease, and PUFA levels are known to play a critical role in

  • immune responses,
  • gene expression and
  • intercellular communications.

omega-3 fatty acids are known to be vital in

  • the prevention of fatal ventricular arrhythmias, and
  • are also known to reduce thrombus formation propensity by decreasing platelet aggregation, blood viscosity and fibrinogen levels

.Since omega-3 fatty acids are prevalent in the nervous system, it seems logical that a deficiency may result in neuronal problems, and this is indeed what has been identified and reported.

The main

In another study conducted with individuals of 65 years of age or older (n = 6158), it was found that

  • only high fish consumption, but
  • not dietary omega-3 acid intake,
  • had a protective effect on cognitive decline

In 2005, based on a meta-analysis of the available epidemiology and preclinical studies, clinical trials were conducted to assess the effects of omega-3 fatty acids on cognitive protection. Four of the trials completed have shown

a protective effect of omega-3 fatty acids only among those with mild cognitive impairment conditions.

A  trial of subjects with mild memory complaints demonstrated

  • an improvement with 900 mg of DHA.

We review key findings on

  • the effect of the omega-3 fatty acid DHA on zinc transporters and the
  • importance of free zinc to human neuronal cells.

DHA is the most abundant fatty acid in neural membranes, imparting appropriate

  • fluidity and other properties,

and is thus considered as the most important fatty acid in neuronal studies. DHA is well conserved throughout the mammalian species despite their dietary differences. It is mainly concentrated

  • in membrane phospholipids at synapses and
  • in retinal photoreceptors and
  • also in the testis and sperm.

In adult rats’ brain, DHA comprises approximately

  • 17% of the total fatty acid weight, and
  • in the retina it is as high as 33%.

DHA is believed to have played a major role in the evolution of the modern human –

  • in particular the well-developed brain.

Premature babies fed on DHA-rich formula show improvements in vocabulary and motor performance.

Analysis of human cadaver brains have shown that

  • people with AD have less DHA in their frontal lobe
  • and hippocampus compared with unaffected individuals

Furthermore, studies in mice have increased support for the

  • protective role of omega-3 fatty acids.

Mice administrated with a dietary intake of DHA showed

  • an increase in DHA levels in the hippocampus.

Errors in memory were decreased in these mice and they demonstrated

  • reduced peroxide and free radical levels,
  • suggesting a role in antioxidant defense.

Another study conducted with a Tg2576 mouse model of AD demonstrated that dietary

  • DHA supplementation had a protective effect against reduction in
  • drebrin (actin associated protein), elevated oxidation, and to some extent, apoptosis via
  • decreased caspase activity.

 

Zinc

Zinc is a trace element, which is indispensable for life, and it is the second most abundant trace element in the body. It is known to be related to

  • growth,
  • development,
  • differentiation,
  • immune response,
  • receptor activity,
  • DNA synthesis,
  • gene expression,
  • neuro-transmission,
  • enzymatic catalysis,
  • hormonal storage and release,
  • tissue repair,
  • memory,
  • the visual process

and many other cellular functions. Moreover, the indispensability of zinc to the body can be discussed in many other aspects,  as

  • a component of over 300 different enzymes
  • an integral component of a metallothioneins
  • a gene regulatory protein.

Approximately 3% of all proteins contain

  • zinc binding motifs .

The broad biological functionality of zinc is thought to be due to its stable chemical and physical properties. Zinc is considered to have three different functions in enzymes;

  1. catalytic,
  2. coactive and

Indeed, it is the only metal found in all six different subclasses

of enzymes. The essential nature of zinc to the human body can be clearly displayed by studying the wide range of pathological effects of zinc deficiency. Anorexia, embryonic and post-natal growth retardation, alopecia, skin lesions, difficulties in wound healing, increased hemorrhage tendency and severe reproductive abnormalities, emotional instability, irritability and depression are just some of the detrimental effects of zinc deficiency.

Proper development and function of the central nervous system (CNS) is highly dependent on zinc levels. In the mammalian organs, zinc is mainly concentrated in the brain at around 150 μm. However, free zinc in the mammalian brain is calculated to be around 10 to 20 nm and the rest exists in either protein-, enzyme- or nucleotide bound form. The brain and zinc relationship is thought to be mediated

  • through glutamate receptors, and
  • it inhibits excitatory and inhibitory receptors.

Vesicular localization of zinc in pre-synaptic terminals is a characteristic feature of brain-localized zinc, and

  • its release is dependent on neural activity.

Retardation of the growth and development of CNS tissues have been linked to low zinc levels. Peripheral neuropathy, spina bifida, hydrocephalus, anencephalus, epilepsy and Pick’s disease have been linked to zinc deficiency. However, the body cannot tolerate excessive amounts of zinc.

The relationship between zinc and neurodegeneration, specifically AD, has been interpreted in several ways. One study has proposed that β-amyloid has a greater propensity to

  • form insoluble amyloid in the presence of
  • high physiological levels of zinc.

Insoluble amyloid is thought to

  • aggregate to form plaques,

which is a main pathological feature of AD. Further studies have shown that

  • chelation of zinc ions can deform and disaggregate plaques.

In AD, the most prominent injuries are found in

  • hippocampal pyramidal neurons, acetylcholine-containing neurons in the basal forebrain, and in
  • somatostatin-containing neurons in the forebrain.

All of these neurons are known to favor

  • rapid and direct entry of zinc in high concentration
  • leaving neurons frequently exposed to high dosages of zinc.

This is thought to promote neuronal cell damage through oxidative stress and mitochondrial dysfunction. Excessive levels of zinc are also capable of

  • inhibiting Ca2+ and Na+ voltage gated channels
  • and up-regulating the cellular levels of reactive oxygen species (ROS).

High levels of zinc are found in Alzheimer’s brains indicating a possible zinc related neurodegeneration. A study conducted with mouse neuronal cells has shown that even a 24-h exposure to high levels of zinc (40 μm) is sufficient to degenerate cells.

If the human diet is deficient in zinc, the body

  • efficiently conserves zinc at the tissue level by compensating other cellular mechanisms

to delay the dietary deficiency effects of zinc. These include reduction of cellular growth rate and zinc excretion levels, and

  • redistribution of available zinc to more zinc dependent cells or organs.

A novel method of measuring metallothionein (MT) levels was introduced as a biomarker for the

  • assessment of the zinc status of individuals and populations.

In humans, erythrocyte metallothionein (E-MT) levels may be considered as an indicator of zinc depletion and repletion, as E-MT levels are sensitive to dietary zinc intake. It should be noted here that MT plays an important role in zinc homeostasis by acting

  • as a target for zinc ion binding and thus
  • assisting in the trafficking of zinc ions through the cell,
  • which may be similar to that of zinc transporters

Zinc Transporters

Deficient or excess amounts of zinc in the body can be catastrophic to the integrity of cellular biochemical and biological systems. The gastrointestinal system controls the absorption, excretion and the distribution of zinc, although the hydrophilic and high-charge molecular characteristics of zinc are not favorable for passive diffusion across the cell membranes. Zinc movement is known to occur

  • via intermembrane proteins and zinc transporter (ZnT) proteins

These transporters are mainly categorized under two metal transporter families; Zip (ZRT, IRT like proteins) and CDF/ZnT (Cation Diffusion Facilitator), also known as SLC (Solute Linked Carrier) gene families: Zip (SLC-39) and ZnT (SLC-30). More than 20 zinc transporters have been identified and characterized over the last two decades (14 Zips and 8 ZnTs).

Members of the SLC39 family have been identified as the putative facilitators of zinc influx into the cytosol, either from the extracellular environment or from intracellular compartments (Figure 1).

The identification of this transporter family was a result of gene sequencing of known Zip1 protein transporters in plants, yeast and human cells. In contrast to the SLC39 family, the SLC30 family facilitates the opposite process, namely zinc efflux from the cytosol to the extracellular environment or into luminal compartments such as secretory granules, endosomes and synaptic vesicles; thus decreasing intracellular zinc availability (Figure 1). ZnT3 is the most important in the brain where

  • it is responsible for the transport of zinc into the synaptic vesicles of
  • glutamatergic neurons in the hippocampus and neocortex,

Figure 1: Subcellular localization and direction of transport of the zinc transporter families, ZnT and ZIP. Arrows show the direction of zinc mobilization for the ZnT (green) and ZIP (red) proteins. A net gain in cytosolic zinc is achieved by the transportation of zinc from the extracellular region and organelles such as the endoplasmic reticulum (ER) and Golgi apparatus by the ZIP transporters. Cytosolic zinc is mobilized into early secretory compartments such as the ER and Golgi apparatus by the ZnT transporters. Figures were produced using Servier Medical Art, http://www.servier.com/.   http://www.hindawi.com/journals/jnme/2012/173712.fig.001.jpg

Figure 2: Early zinc signaling (EZS) and late zinc signaling (LZS). EZS involves transcription-independent mechanisms where an extracellular stimulus directly induces an increase in zinc levels within several minutes by releasing zinc from intracellular stores (e.g., endoplasmic reticulum). LSZ is induced several hours after an external stimulus and is dependent on transcriptional changes in zinc transporter expression. Components of this figure were produced using Servier Medical Art, http://www.servier.com/ and adapted from Fukada et al. [30].

omega-3 fatty acids in the mammalian body are

  1. α-linolenic acid (ALA),
  2. docosahexenoic acid (DHA) and
  3. eicosapentaenoic acid (EPA).

In general, seafood is rich in omega-3 fatty acids, more specifically DHA and EPA (Table 1). Thus far, there are nine separate epidemiological studies that suggest a possible link between

  • increased fish consumption and reduced risk of AD
  • and eight out of ten studies have reported a link between higher blood omega-3 levels

DHA and Zinc Homeostasis

Many studies have identified possible associations between DHA levels, zinc homeostasis, neuroprotection and neurodegeneration. Dietary DHA deficiency resulted in

  • increased zinc levels in the hippocampus and
  • elevated expression of the putative zinc transporter, ZnT3, in the rat brain.

Altered zinc metabolism in neuronal cells has been linked to neurodegenerative conditions such as AD. A study conducted with transgenic mice has shown a significant link between ZnT3 transporter levels and cerebral amyloid plaque pathology. When the ZnT3 transporter was silenced in transgenic mice expressing cerebral amyloid plaque pathology,

  • a significant reduction in plaque load
  • and the presence of insoluble amyloid were observed.

In addition to the decrease in plaque load, ZnT3 silenced mice also exhibited a significant

  • reduction in free zinc availability in the hippocampus
  • and cerebral cortex.

Collectively, the findings from this study are very interesting and indicate a clear connection between

  • zinc availability and amyloid plaque formation,

thus indicating a possible link to AD.

DHA supplementation has also been reported to limit the following:

  1. amyloid presence,
  2. synaptic marker loss,
  3. hyper-phosphorylation of Tau,
  4. oxidative damage and
  5. cognitive deficits in transgenic mouse model of AD.

In addition, studies by Stoltenberg, Flinn and colleagues report on the modulation of zinc and the effect in transgenic mouse models of AD. Given that all of these are classic pathological features of AD, and considering the limiting nature of DHA in these processes, it can be argued that DHA is a key candidate in preventing or even curing this debilitating disease.

In order to better understand the possible links and pathways of zinc and DHA with neurodegeneration, we designed a study that incorporates all three of these aspects, to study their effects at the cellular level. In this study, we were able to demonstrate a possible link between omega-3 fatty acid (DHA) concentration, zinc availability and zinc transporter expression levels in cultured human neuronal cells.

When treated with DHA over 48 h, ZnT3 levels were markedly reduced in the human neuroblastoma M17 cell line. Moreover, in the same study, we were able to propose a possible

  • neuroprotective mechanism of DHA,

which we believe is exerted through

  • a reduction in cellular zinc levels (through altering zinc transporter expression levels)
  • that in turn inhibits apoptosis.

DHA supplemented M17 cells also showed a marked depletion of zinc uptake (up to 30%), and

  • free zinc levels in the cytosol were significantly low compared to the control

This reduction in free zinc availability was specific to DHA; cells treated with EPA had no significant change in free zinc levels (unpublished data). Moreover, DHA-repleted cells had

  • low levels of active caspase-3 and
  • high Bcl-2 levels compared to the control treatment.

These findings are consistent with previous published data and further strengthen the possible

  • correlation between zinc, DHA and neurodegeneration.

On the other hand, recent studies using ZnT3 knockout (ZnT3KO) mice have shown the importance of

  • ZnT3 in memory and AD pathology.

For example, Sindreu and colleagues have used ZnT3KO mice to establish the important role of

  • ZnT3 in zinc homeostasis that modulates presynaptic MAPK signaling
  • required for hippocampus-dependent memory

Results from these studies indicate a possible zinc-transporter-expression-level-dependent mechanism for DHA neuroprotection.

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Introduction to Signaling

Curator: Larry H. Bernstein, MD, FCAP

 

We have laid down a basic structure and foundation for the remaining presentations.  It was essential to begin with the genome, which changed the course of teaching of biology and medicine in the 20th century, and introduced a central dogma of translation by transcription.  Nevertheless, there were significant inconsistencies and unanswered questions entering the twenty first century, accompanied by vast improvements in technical advances to clarify these issues. We have covered carbohydrate, protein, and lipid metabolism, which function in concert with the development of cellular structure, organ system development, and physiology.  To be sure, the progress in the study of the microscopic and particulate can’t be divorced from the observation of the whole.  We were left in the not so distant past with the impression of the Sufi story of the elephant and the three blind men, who one at a time held the tail, the trunk, and the ear, each proclaiming that it was the elephant.

I introduce here a story from the Brazilian biochemist, Jose

Eduardo des Salles Rosalino, on a formativr experience he had with the Nobelist, Luis Leloir.

Just at the beginning, when phosphorylation of proteins is presented, I assume you must mention that some proteins are activated by phosphorylation. This is fundamental in order to present self –organization reflex upon fast regulatory mechanisms. Even from an historical point of view. The first observation arrived from a sample due to be studied on the following day of glycogen synthetase. It was unintended left overnight out of the refrigerator. The result was it has changed from active form of the previous day to a non-active form. The story could have being finished here, if the researcher did not decide to spent this day increasing substrate levels (it could be a simple case of denaturation of proteins that changes its conformation despite the same order of amino acids). He kept on trying and found restoration of maximal activity. This assay was repeated with glycogen phosphorylase and the result was the opposite – it increases its activity. This led to the discovery

  • of cAMP activated protein kinase and
  • the assembly of a very complex system in the glycogen granule
  • that is not a simple carbohydrate polymer.

Instead, it has several proteins assembled and

  • preserves the capacity to receive from a single event (rise in cAMP)
  • two opposing signals with maximal efficiency,
  • stops glycogen synthesis,
  • as long as levels of glucose 6 phosphate are low
  • and increases glycogen phosphorylation as long as AMP levels are high).

I did everything I was able to do by the end of 1970 in order to repeat the assays with PK I, PKII and PKIII of M. Rouxii and using the Sutherland route to cAMP failed in this case. I then asked Leloir to suggest to my chief (SP) the idea of AA, AB, BB subunits as was observed in lactic dehydrogenase (tetramer) indicating this as his idea. The reason was my “chief”(SP) more than once, had said to me: “Leave these great ideas for the Houssay, Leloir etc…We must do our career with small things.” However, as she also had a faulty ability for recollection she also used to arrive some time later, with the very same idea but in that case, as her idea.
Leloir, said to me: I will not offer your interpretation to her as mine. I think it is not phosphorylation, however I think it is glycosylation that explains the changes in the isoenzymes with the same molecular weight preserved. This dialogue explains why during the reading and discussing “What is life” with him he asked me if as a biochemist in exile, talking to another biochemist, I expressed myself fully. I had considered that Schrödinger would not have confronted Darlington & Haldane because he was in U.K. in exile. This might explain why Leloir could have answered a bad telephone call from P. Boyer, Editor of The Enzymes, in a way that suggested that the pattern could be of covalent changes over a protein. Our FEBS and Eur J. Biochemistry papers on pyruvate kinase of M. Rouxii is wrongly quoted in this way on his review about pyruvate kinase of that year (1971).

 

Another aspect I think you must call attention to the following. Show in detail with different colors what carbons belongs to CoA, a huge molecule in comparison with the single two carbons of acetate that will produce the enormous jump in energy yield

  • in comparison with anaerobic glycolysis.

The idea is

  • how much must have been spent in DNA sequences to build that molecule in order to use only two atoms of carbon.

Very limited aspects of biology could be explained in this way. In case we follow an alternative way of thinking, it becomes clearer that proteins were made more stable by interaction with other molecules (great and small). Afterwards, it’s rather easy to understand how the stability of protein-RNA complexes where transmitted to RNA (vibrational +solvational reactivity stability pair of conformational energy).

Millions of years later, or as soon as, the information of interaction leading to activity and regulation could be found in RNA, proteins like reverse transcriptase move this information to a more stable form (DNA). In this way it is easier to understand the use of CoA to make two carbon molecules more reactive.

The discussions that follow are concerned with protein interactions and signaling.

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Introduction to Metabolic Pathways

Author: Larry H. Bernstein, MD, FCAP

 

Humans, mammals, plants and animals, and eukaryotes and prokaryotes all share a common denominator in their manner of existence.  It makes no difference whether they inhabit the land, or the sea, or another living host. They exist by virtue of their metabolic adaptation by way of taking in nutrients as fuel, and converting the nutrients to waste in the expenditure of carrying out the functions of motility, breakdown and utilization of fuel, and replication of their functional mass.

There are essentially two major sources of fuel, mainly, carbohydrate and fat.  A third source, amino acids which requires protein breakdown, is utilized to a limited extent as needed from conversion of gluconeogenic amino acids for entry into the carbohydrate pathway. Amino acids follow specific metabolic pathways related to protein synthesis and cell renewal tied to genomic expression.

Carbohydrates are a major fuel utilized by way of either of two pathways.  They are a source of readily available fuel that is accessible either from breakdown of disaccharides or from hepatic glycogenolysis by way of the Cori cycle.  Fat derived energy is a high energy source that is metabolized by one carbon transfers using the oxidation of fatty acids in mitochondria. In the case of fats, the advantage of high energy is conferred by chain length.

Carbohydrate metabolism has either of two routes of utilization.  This introduces an innovation by way of the mitochondrion or its equivalent, for the process of respiration, or aerobic metabolism through the tricarboxylic acid, or Krebs cycle.  In the presence of low oxygen supply, carbohydrate is metabolized anaerobically, the six carbon glucose being split into two three carbon intermediates, which are finally converted from pyruvate to lactate.  In the presence of oxygen, the lactate is channeled back into respiration, or mitochondrial oxidation, referred to as oxidative phosphorylation. The actual mechanism of this process was of considerable debate for some years until it was resolved that the mechanism involve hydrogen transfers along the “electron transport chain” on the inner membrane of the mitochondrion, and it was tied to the formation of ATP from ADP linked to the so called “active acetate” in Acetyl-Coenzyme A, discovered by Fritz Lipmann (and Nathan O. Kaplan) at Massachusetts General Hospital.  Kaplan then joined with Sidney Colowick at the McCollum Pratt Institute at Johns Hopkins, where they shared tn the seminal discovery of the “pyridine nucleotide transhydrogenases” with Elizabeth Neufeld,  who later established her reputation in the mucopolysaccharidoses (MPS) with L-iduronidase and lysosomal storage disease.

This chapter covers primarily the metabolic pathways for glucose, anaerobic and by mitochondrial oxidation, the electron transport chain, fatty acid oxidation, galactose assimilation, and the hexose monophosphate shunt, essential for the generation of NADPH. The is to be more elaboration on lipids and coverage of transcription, involving amino acids and RNA in other chapters.

The subchapters are as follows:

1.1      Carbohydrate Metabolism

1.2      Studies of Respiration Lead to Acetyl CoA

1.3      Pentose Shunt, Electron Transfer, Galactose, more Lipids in brief

1.4      The Multi-step Transfer of Phosphate Bond and Hydrogen Exchange Energy

Complex I or NADH-Q oxidoreductase

Complex I or NADH-Q oxidoreductase

Fatty acid oxidation and ETC

Fatty acid oxidation and ETC

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Metformin, Thyroid-Pituitary Axis, Diabetes Mellitus, and Metabolism

Metformin, Thyroid-Pituitary Axis, Diabetes Mellitus, and Metabolism

Larry H, Bernstein, MD, FCAP, Author and Curator
and Aviva Lev-Ari, PhD, RN

http://pharmaceuticalintelligence.com/9/27/2014/Metformin,_thyroid-pituitary_ axis,_diabetes_mellitus,_and_metabolism

The following article is a review of the central relationship between the action of
metformin as a diabetic medication and its relationship to AMPK, the important and
essential regulator of glucose and lipid metabolism under normal activity, stress, with
its effects on skeletal muscle, the liver, the action of T3 and more.

We start with a case study and a publication in the J Can Med Assoc.  Then we shall look
into key literature on these metabolic relationships.

Part I.  Metformin , Diabetes Mellitus, and Thyroid Function

Hypothyroidism, Insulin resistance and Metformin
May 30, 2012   By Janie Bowthorpe
The following was written by a UK hypothyroid patient’s mother –
Sarah Wilson.

My daughter’s epilepsy is triggered by unstable blood sugars. And since taking
Metformin to control her blood sugar, she has significantly reduced the number of
seizures. I have been doing research and read numerous academic medical journals,
which got me thinking about natural thyroid hormone and Hypothyroidism. My hunch
was that when patients develop hypothyroid symptoms, they are actually becoming
insulin resistant (IR). There are many symptoms in common between women with
polycystic ovaries and hypothyroidism–the hair loss, the weight gain, etc.
(http://insulinhub.hubpages.com/hub/PCOS-and-Hypothyroidism).

A hypothyroid person’s body behaves as if it’s going into starvation mode and so, to
preserve resources and prolong life, the metabolism changes. If hypothyroid is prolonged
or pronounced, then perhaps, chemical preservation mode becomes permanent even
with the reintroduction of thyroid hormones. To get back to normal, they need
a “jump-start” reinitiate a higher rate of metabolism. The kick start is initiated through
AMPK, which is known as the “master metabolic regulating enzyme.”
(http://en.wikipedia.org/wiki/AMP-activated protein kinase).

Guess what? This is exactly what happens to Diabetes patients when Metformin is
introduced. http://en.wikipedia.org/wiki/Metformin
Suggested articles: http://www.springerlink.com/content/r81606gl3r603167/  and
http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2265.2011.04029.x/pdf

Note the following comments/partial statements:
“Hypothyroidism is characterized by decreased insulin responsiveness”;
“the pivotal regulatory role of T3 in major metabolic pathways”.

The community knows that T3/NTH (natural thyroid hormone [Armour]) makes
hypothyroid patients feel better – but the medical establishment is averse to T3/NTH
(treating subclinical hypoT (T3/T4 euthyroid) with natural dessicated thyroid (NDT).
The medical establishment might find an alternative view about impaired metabolism
more if shown real proof that the old NDT **was/is** having the right result –i.e., the
T3 is jump-starting the metabolism by re-activating
 AMPK.

If NDT also can be used for hypothyroidism without the surmised “dangers” of NTH,
then they should consider it. [The reality in the choice is actually recombinant TH
(Synthroid)]. Metformin is cheap, stable and has very few serious side effects. I use the
car engine metaphor, and refer to glucose as our petrol, AMPK as the spark plug and
both T3 and Metformin as the ignition switches. Sometimes if you have flat batteries in
the car, it doesn’t matter how much you turn the ignition switch or pump the petrol
pedal, all it does is flatten the battery and flood the engine.

Dr. Skinner in the UK has been treating “pre-hypothyroidism” the way that some
doctors treat “pre-diabetes”. Those hypothyroid patients who get treated early
might not have had their AMPK pathways altered and the T4-T3 conversion still works.
There seems to be no reason why thyroid hormone replacement therapy shouldn’t
logically be given to ward off a greater problem down the line.

It’s my belief that there is clear and abundant academic evidence that the AMPK/
Metformin research should branch out to also look at thyroid disease.

Point – direct T3 is kicking the closed -down metabolic process back into life,
just like Metformin does for insulin resistance.
http://www.hotthyroidology.com/editorial_79.html
There is serotonin resistance! http://www.ncbi.nlm.nih.gov/pubmed/17250776

Metformin Linked to Risk of Low Levels of Thyroid Hormone

CMAJ (Canadian Medical Association Journal) 09/22/2014

Metformin, the drug commonly for treating type 2 diabetes,

  • is linked to an increased risk of low thyroid-stimulating hormone
    (TSH) levels
  • in patients with underactive thyroids (hypothyroidism),

according to a study in CMAJ (Canadian Medical Association Journal).

Metformin is used to lower blood glucose levels

  • by reducing glucose production in the liver.

previous studies have raised concerns that

  • metformin may lower thyroid-stimulating hormone levels.

Study characteristics:

  1. Retrospective  long-term
  2. 74 300 patient who received metformin and sulfonylurea
  3. 25-year study period.
  4. 5689 had treated hypothyroidism
  5. 59 937 had normal thyroid function.

Metformin and low levels of thyroid-stimulating hormone in
patients with type 2 diabetes mellitus

Jean-Pascal Fournier,  Hui Yin, Oriana Hoi Yun Yu, Laurent Azoulay  +
Centre for Clinical Epidemiology (Fournier, Yin, Yu, Azoulay), Lady Davis Institute,
Jewish General Hospital; Department of Epidemiology, Biostatistics and Occupational
Health (Fournier), McGill University; Division of Endocrinology (Yu), Jewish General
Hospital; Department of Oncology (Azoulay), McGill University, Montréal, Que., Cananda

CMAJ Sep 22, 2014,   http://dx.doi.org:/10.1503/cmaj.140688

Background:

  • metformin may lower thyroid-stimulating hormone (TSH) levels.

Objective:

  • determine whether the use of metformin monotherapy, when compared with
    sulfonylurea monotherapy,
  • is associated with an increased risk of low TSH levels(< 0.4 mIU/L)
  • in patients with type 2 diabetes mellitus.

Methods:

  • Used the Clinical Practice Research Datalink,
  • identified patients who began receiving metformin or sulfonylurea monotherapy
    between Jan. 1, 1988, and Dec. 31, 2012.
  • 2 subcohorts of patients with treated hypothyroidism or euthyroidism,

followed them until Mar. 31, 2013.

  • Used Cox proportional hazards models to evaluate the association of low TSH
    levels with metformin monotherapy, compared with sulfonylurea monotherapy,
    in each subcohort.

Results:

  • 5689 patients with treated hypothyroidism and 59 937 euthyroid patients were
    included in the subcohorts.

For patients with treated hypothyroidism:

  1. 495 events of low TSH levels were observed (incidence rate 0.1197/person-years).
  2. 322 events of low TSH levels were observed (incidence rate 0.0045/person-years)
    in the euthyroid group.
  • metformin monotherapy was associated with a 55% increased risk of low TSH
    levels 
    in patients with treated hypothyroidism (incidence rate 0.0795/person-years
    vs.0.1252/ person-years, adjusted hazard ratio [HR] 1.55, 95% confidence
    interval [CI] 1.09– 1.20), compared with sulfonylurea monotherapy,
  • the highest risk in the 90–180 days after initiation (adjusted HR 2.30, 95% CI
    1.00–5.29).
  • No association was observed in euthyroid patients (adjusted HR 0.97, 95% CI 0.69–1.36).

Interpretation: The clinical consequences of this needs further investigation.

 

Crude and adjusted hazard ratios for suppressed thyroid-stimulating hormone
levels (< 0.1 mIU/L) associated with the use metformin monotherapy, compared
with sulfonylurea monotherapy, in patients with treated hypothyroidism or
euthyroidism and type 2 diabetes
Variable No. events
suppressed
TSH levels
Person-years of
exposure
Incidence rate,
per 1000 person-years (95% CI)
Crude
HR
Adjusted HR*(95% CI)
Patients with treated hypothyroidism, n = 5689
Sulfonylure,
n = 762
18 503 35.8
(21.2–56.6)
1.00 1.00
(reference)
Metformin,
n = 4927
130 3 633 35.8
(29.9–42.5)
1.05 0.99
(0.57–1.72)
Euthyroid patients, n = 59 937
Sulfonylurea,
n = 7980
12 8 576 1.4
(0.7–2.4)
1.00 1.00
(reference)
Metformin,
n = 51 957
75 63 047 1.2
(0.9–1.5)
0.85 1.03
(0.52–2.03)

 

Part II. Metabolic Underpinning 
(Source: Wikipedia, AMPK and thyroid)

5′ AMP-activated protein kinase or AMPK or 5′ adenosine monophosphate-activated protein kinase
is an enzyme that plays a role in cellular energy homeostasis.
It consists of three proteins (subunits) that

  1. together make a functional enzyme, conserved from yeast to humans.
  2. It is expressed in a number of tissues, including the liver, brain, and skeletal
    muscle.
  3. The net effect of AMPK activation is stimulation of
    1. hepatic fatty acid oxidation and ketogenesis,
    2. inhibition of cholesterol synthesis,
    3. lipogenesis, and triglyceride synthesis,
    4. inhibition of adipocyte lipolysis and lipogenesis,
    5. stimulation of skeletal muscle fatty acid oxidation and muscle
      glucose uptake, and
    6. modulation of insulin secretion by pancreatic beta-cells.

The heterotrimeric protein AMPK is formed by α, β, and γ subunits. Each of these three
subunits takes on a specific role in both the stability and activity of AMPK.

  • the γ subunit includes four particular Cystathionine beta synthase (CBS) domains
    giving AMPK its ability to sensitively detect shifts in the AMP:ATP ratio.
  • The four CBS domains create two binding sites for AMP commonly referred to as
    Bateman domains. Binding of one AMP to a Bateman domain cooperatively
    increases the binding affinity of the second AMP to the other Bateman domain.
  • As AMP binds both Bateman domains the γ subunit undergoes a conformational
    change which exposes the catalytic domain found on the α subunit.
  • It is in this catalytic domain where AMPK becomes activated when
    phosphorylation takes place at threonine-172by an upstream AMPK kinase
    (AMPKK). The α, β, and γ subunits can also be found in different isoforms.

AMPK acts as a metabolic master switch regulating several intracellular systems

  1. the cellular uptake of glucose,
  2. the β-oxidation of fatty acids and
  3. the biogenesis of glucose transporter 4 (GLUT4) and
  4. mitochondria

The energy-sensing capability of AMPK can be attributed to

  • its ability to detect and react to fluctuations in the AMP:ATP ratio that take
    place during rest and exercise (muscle stimulation).

During muscle stimulation,

  • AMP increases while ATP decreases, which changes AMPK into a good substrate
    for activation.
  • AMPK activity increases while the muscle cell experiences metabolic stress
    brought about by an extreme cellular demand for ATP.
  • Upon activation, AMPK increases cellular energy levels by
    • inhibiting anabolic energy consuming pathways (fatty acid synthesis,
      protein synthesis, etc.) and
    • stimulating energy producing, catabolic pathways (fatty acid oxidation,
      glucose transport, etc.).

A recent JBC paper on mice at Johns Hopkins has shown that when the activity of brain
AMPK was pharmacologically inhibited,

  • the mice ate less and lost weight.

When AMPK activity was pharmacologically raised (AICAR see below)

  • the mice ate more and gained weight.

Research in Britain has shown that the appetite-stimulating hormone ghrelin also
affects AMPK levels.

The antidiabetic drug metformin (Glucophage) acts by stimulating AMPK, leading to

  1. reduced glucose production in the liver and
  2. reduced insulin resistance in the muscle.

(Metformin usually causes weight loss and reduced appetite, not weight gain and
increased appetite, ..opposite of expected from the Johns Hopkins mouse study results.)

Triggering the activation of AMPK can be carried out provided two conditions are met.

First, the γ subunit of AMPK

  • must undergo a conformational change so as to
  • expose the active site(Thr-172) on the α subunit.

The conformational change of the γ subunit of AMPK can be accomplished

  • under increased concentrations of AMP.

Increased concentrations of AMP will

  • give rise to the conformational change on the γ subunit of AMPK
  • as two AMP bind the two Bateman domains located on that subunit.
  • It is this conformational change brought about by increased concentrations
    of  AMP that exposes the active site (Thr-172) on the α subunit.

This critical role of AMP is further substantiated in experiments that demonstrate

  • AMPK activation via an AMP analogue 5-amino-4-imidazolecarboxamide
    ribotide (ZMP) which is derived fromthe familiar
  • 5-amino-4-imidazolecarboxamide riboside (AICAR)

AMPK is a good substrate for activation via an upstream kinase complex, AMPKK
AMPKK is a complex of three proteins,

  1. STE-related adaptor (STRAD),
  2. mouse protein 25 (MO25), and
  3. LKB1 (a serine/threonine kinase).

The second condition that must be met is

  • the phosphorylation/activation of AMPK on its activating loop at
    Thr-172of the α subunit
  • brought about by an upstream kinase (AMPKK).

The complex formed between LKB1 (STK 11), mouse protein 25 (MO25), and the
pseudokinase STE-related adaptor protein (STRAD) has been identified as

  • the major upstream kinase responsible for phosphorylation of AMPK
    on its activating loop at Thr-172

Although AMPK must be phosphorylated by the LKB1/MO25/STRAD complex,

  • it can also be regulated by allosteric modulators which
  • directly increase general AMPK activity and
  • modify AMPK to make it a better substrate for AMPKK
  • and a worse substrate for phosphatases.

It has recently been found that 3-phosphoglycerate (glycolysis intermediate)

  • acts to further pronounce AMPK activation via AMPKK

Muscle contraction is the main method carried out by the body that can provide
the conditions mentioned above needed for AMPK activation

  • As muscles contract, ATP is hydrolyzed, forming ADP.
  • ADP then helps to replenish cellular ATP by donating a phosphate group to
    another ADP,

    • forming an ATP and an AMP.
  • As more AMP is produced during muscle contraction,
    • the AMP:ATP ratio dramatically increases,
  • leading to the allosteric activation of AMPK

For over a decade it has been known that calmodulin-dependent protein kinase
kinase-beta (CaMKKbeta) can phosphorylate and thereby activate AMPK,

  • but it was not the main AMPKK in liver.

CaMKK inhibitors had no effect on 5-aminoimidazole-4-carboxamide-1-beta-4-
ribofuranoside (AICAR) phosphorylation and activation of AMPK.

  • AICAR is taken into the celland converted to ZMP,
  • an AMP analogthat has been shown to activate AMPK.

Recent LKB1 knockout studies have shown that without LKB1,

  • electrical and AICAR stimulation of muscleresults in very little
    phosphorylation of AMPK and of ACC, providing evidence that
  • LKB1-STRAD-MO25 is the major AMPKK in muscle.

Two particular adipokines, adiponectin and leptin, have even been demonstrated
to regulate AMPK. A main functions of leptin in skeletal muscle is

  • the upregulation of fatty acid oxidation.

Leptin works by way of the AMPK signaling pathway, and adiponectin also
stimulates the oxidation of fatty acids via the AMPK pathway, and

  • Adiponectin also stimulates the uptake of glucose in skeletal muscle.

An increase in enzymes which specialize in glucose uptake in cells such as GLUT4
and hexokinase II are thought to be mediated in part by AMPK when it is activated.
Increases in AMPK activity are brought about by increases in the AMP:ATP ratio
during single bouts of exercise and long-term training.

One of the key pathways in AMPK’s regulation of fatty acid oxidation is the

  • phosphorylation and inactivation of acetyl-CoA carboxylase.
  1. Acetyl-CoA carboxylase (ACC) converts acetyl-CoA (ACA) to malonyl-CoA
    (MCA), an inhibitor of carnitine palmitoyltransferase 1 (CPT-1).
  2. CPT-1 transports fatty acids into the mitochondria for oxidation.
  3. Inactivation of ACC results in increased fatty acid transport and oxidation.
  4. the AMPK induced ACC inactivation  and reduced conversion to MCA
    may occur as a result of malonyl-CoA decarboxylase (MCD)
  5. MCD as an antagonist to ACC, decarboxylatesmalonyl-CoA to acetyl-CoA
    (reversal of ACC conversion of ACA to MCA)
  6. This resultsin decreased malonyl-CoA and increased CPT-1 and fatty acid oxidation.

AMPK also plays an important role in lipid metabolism in the liver. It has long been
known that hepatic ACC has been regulated in the liver.

  1. It phosphorylates and inactivates 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR)
  2. acetyl-CoA(ACA) is converted to mevalonic acid (MVA) by ACC
    with inhibition of CPT-1
  3. HMGR converts 3-hydroxy-3-methylglutaryl-CoA, which is made from MVA
  4. which then travels down several more metabolic steps to become cholesterol.

Insulin facilitates the uptake of glucose into cells via increased expression and
translocation of glucose transporter GLUT-4. In addition, glucose is phosphorylated
by hexokinase wheni iot enters the cell. The phosphorylated form keeps glucose from
leaving the cell,

  • The decreasedthe concentration of glucose molecules creates a gradient for more
    glucose to be transported into the cell.
AMPK and thyroid hormone regulate some similar processes. Knowing these similarities,
Winder and Hardie et al. designed an experiment to see if AMPK was influenced by thyroid
hormone. They found that all of the subunits of AMPK were increased in skeletal muscle,
especially in the soleus and red quadriceps, with thyroid hormone treatment. There was
also an increase in phospho-ACC, a marker of AMPK activity.
  •  Winder WW, Hardie DG (July 1999). “AMP-activated protein kinase,
    a metabolic master switch: possible roles in type 2 diabetes”. J. Physiol. 277
    (1 Pt 1): E1–10. PMID 10409121.
  • Winder WW, Hardie DG (February 1996). “Inactivation of acetyl-CoA
    carboxylase and activation of AMP-activated protein kinase in muscle
    during exercise”. J. Physiol. 270 (2 Pt 1): E299–304. PMID 8779952.
  • Hutber CA, Hardie DG, Winder WW (February 1997). “Electrical stimulation
    inactivates muscle acetyl-CoA carboxylase and increases AMP-activated
    protein kinase”. Am. J. Physiol. 272 (2 Pt 1): E262–6. PMID 9124333
  • Durante PE, Mustard KJ, Park SH, Winder WW, Hardie DG (July 2002).
    “Effects of endurance training on activity and expression of AMP-activated
    protein kinase isoforms in rat muscles”. Am. J. Physiol. Endocrinol.
    Metab. 283 (1): E178–86. doi:10.1152/ajpendo.00404.2001. PMID 12067859
  • Corton JM, Gillespie JG, Hardie DG (April 1994). “Role of the AMP-activated
    protein kinase in the cellular stress response”. Curr. Biol. 4 (4):
    315–24. doi:10.1016/S0960-9822(00)00070-1. PMID 7922340
  • Winder WW (September 2001). “Energy-sensing and signaling by
    AMP-activated protein kinase in skeletal muscle”. J. Appl. Physiol. 91 (3):
    1017–28. PMID 11509493
  • Suter M, Riek U, Tuerk R, Schlattner U, Wallimann T, Neumann D (October
    2006). “Dissecting the role of 5′-AMP for allosteric stimulation, activation,
    and deactivation of AMP-activated protein kinase”.  J. Biol. Chem.
    281 (43): 32207–6. doi:10.1074/jbc.M606357200. PMID 16943194

 

Part III. Pituitary-thyroid axis and diabetes mellitus
The Interface Between Thyroid and Diabetes Mellitus

Leonidas H. Duntas, Jacques Orgiazzi, Georg Brabant   Clin Endocrinol. 2011;75(1):1-9.
Interaction of Metformin and Thyroid Function

Metformin acts primarily by

  • suppressing hepatic gluconeogenesis via activation of AMPK
  • It has the opposite effects on hypothalamic AMPK,
    • inhibiting activity of the enzyme.
  • the metformin effects on hypothalamic AMPK activity will
    • counteractT3 effects at the hypothalamic level.
  • AMPK therefore represents a direct target for dual regulation
    • in the hypothalamic partitioning of energy homeostasis.
  • metformin crossesthe blood–brain barrier and
    • levels in the pituitary gland are substantially increased.
  • It convincinglysuppresses TSH

A recent study recruiting 66 patients with benign thyroid nodules furthermore
demonstrated that metformin significantly decreases nodule size in patients with
insulin resistance.[76] The effect of metformin, which was produced over a
6-month treatment period, parallelled a fall in TSH concentrations and achieved a
shrinkage amounting to 30% of the initial nodule size when metformin was
administered alone and up to 55% when it was added to ongoing LT4 treatment.

These studies reveal a

  • suppressive effect of metformin on TSH secretion patterns in
    hypothyroid patients, an effect that is apparently
  • independent of T4 treatment and does not alter the TH profile.
  • A rebound of TSH secretion occurs at about 3 months following metformin
    withdrawal.

It appears that recommendations for more frequent testing, on an annual to
biannual basis, seems justified in higher risk groups like patients over 50 or 55,
particularly with suggestive symptoms, raised antibody titres or dylipidaemia.
We thus would support the suggestion of an initial TSH and TPO antibody testing
which, as discussed, will help to predict the development of hypothyroidism in
patients with diabetes.

Hypothalamic AMPK and fatty acid metabolism mediate thyroid
regulation of energy 
balance
M López,  L Varela,  MJ Vázquez,  S Rodríguez-Cuenca, CR González, …, & Vidal-Puig
Nature Medicine  29 Aug 2010; 16: 1001–1008 http://dx.doi.org:/10.1038/nm.2207

Thyroid hormones have widespread cellular effects; however it is unclear whether
their effects on the central nervous system (CNS) contribute to global energy balance.
Here we demonstrate that either

  • whole-body hyperthyroidism or central administration of triiodothyronine
    (T3) decreases

    • the activity of hypothalamic AMP-activated protein kinase (AMPK),
    • increases sympathetic nervous system (SNS) activity and
    • upregulates thermogenic markers in brown adipose tissue (BAT).

Inhibition of the lipogenic pathway in the ventromedial nucleus of the hypothalamus
(VMH) prevents CNS-mediated activation of BAT by thyroid hormone and reverses
the weight loss associated with hyperthyroidism. Similarly, inhibition of thyroid
hormone receptors in the VMH reverses the weight loss associated with hyperthyroidism.

This regulatory mechanism depends on AMPK inactivation, as genetic inhibition of this
enzyme in the VMH of euthyroid rats induces feeding-independent weight loss and
increases expression of thermogenic markers in BAT. These effects are reversed by
pharmacological blockade of the SNS. Thus, thyroid hormone–induced modulation
of AMPK activity and lipid metabolism in the hypothalamus is a major regulator of
whole-body energy homeostasis.

Metabolic Basis for Thyroid Hormone Liver Preconditioning:
Upregulation of AMP-Activated Protein Kinase Signaling
  
LA Videla,1 V Fernández, P Cornejo, and R Vargas
1Molecular and Clinical Pharmacology Program, Institute of Biomedical Sciences,
Faculty of Medicine, University of Chile, 2Faculty of Medicine, Diego Portales University,
Santiago, Chile
Academic Editors: H. M. Abu-Soud and D. Benke
The Scientific World Journal 2012; 2012, ID 475675, 10 pp
http://dx.doi.org/10.1100/2012/475675

The liver is a major organ responsible for most functions of cellular metabolism and

  • a mediator between dietary and endogenous sources of energy for extrahepatic tissues.
  • In this context, adenosine-monophosphate- (AMP-) activated protein kinase (AMPK)
    constitutes an intrahepatic energy sensor
  • regulating physiological energy dynamics by limiting anabolism and stimulating
    catabolism, thus increasing ATP availability.
  • This is achieved by mechanisms involving direct allosteric activation and
    reversible phosphorylation of AMPK, in response to signals such as

    • energy status,
    • serum insulin/glucagon ratio,
    • nutritional stresses,
    • pharmacological and natural compounds, and
    • oxidative stress status.

Reactive oxygen species (ROS) lead to cellular AMPK activation and

  • downstream signaling under several experimental conditions.

Thyroid hormone (L-3,3′,5-triiodothyronine, T3) administration, a condition
that enhances liver ROS generation,

  • triggers the redox upregulation of cytoprotective proteins
    • affording preconditioning against ischemia-reperfusion (IR) liver injury.

Data discussed in this work suggest that T3-induced liver activation of AMPK

  • may be of importance in the promotion of metabolic processes
  • favouring energy supply for the induction and operation of preconditioning
    mechanisms.

These include

  1. antioxidant,
  2. antiapoptotic, and
  3. anti-inflammatory mechanisms,
  4. repair or resynthesis of altered biomolecules,
  5. induction of the homeostatic acute-phase response, and
  6. stimulation of liver cell proliferation,

which are required to cope with the damaging processes set in by IR.

The liver functions as a mediator between dietary and endogenous sources
of energy and extrahepatic organs that continuously require energy, mainly
the brain and erythrocytes, under cycling conditions between fed and fasted states.

In the fed state, where insulin action predominates, digestion-derived glucose is
converted to pyruvate via glycolysis, which is oxidized to produce energy, whereas
fatty acid oxidation is suppressed. Excess glucose can be either stored as hepatic
glycogen or channelled into de novo lipogenesis.

In the fasted state, considerable liver fuel metabolism changes occur due to decreased
serum insulin/glucagon ratio, with higher glucose production as a consequence of
stimulated glycogenolysis and gluconeogenesis (from alanine, lactate, and glycerol).

Major enhancement in fatty acid oxidation also occurs to provide energy for liver
processes and ketogenesis to supply metabolic fuels for extrahepatic tissues. For these
reasons, the liver is considered as the metabolic processing organ of the body, and
alterations in liver functioning affect whole-body metabolism and energy homeostasis.

In this context, adenosine-monophosphate- (AMP-) activated protein kinase (AMPK)
is the downstream component of a protein kinase cascade acting as an

  • intracellular energy sensor regulating physiological energy dynamics by
  • limiting anabolic pathways, to prevent excessive adenosine triphosphate (ATP)
    utilization, and
  • by stimulating catabolic processes, to increase ATP production.

Thus, the understanding of the mechanisms by which liver AMPK coordinates hepatic
energy metabolism represents a crucial point of convergence of regulatory signals
monitoring systemic and cellular energy status

Liver AMPK: Structure and Regulation

AMPK, a serine/threonine kinase, is a heterotrimeric complex comprising

  1. a catalytic subunit α and
  2. two regulatory subunits β and γ .

The α subunit has a threonine residue (Thr172) within the activation loop of the kinase
domain, with the C-terminal region being required for association with β and γ subunits.
The β subunit associates with α and γ by means of its C-terminal region , whereas

  • the γ subunit has four cystathionine β-synthase (CBS) motifs, which
  • bind AMP or ATP in a competitive manner.

75675.fig.001 (not shown)

Figure 1: Regulation of AMP-activated protein kinase (AMPK) by
(A) direct allosteric activation and
(B) reversible phosphorylation and downstream responses maintaining
intracellular energy balance.

Regulation of liver AMPK activity involves both direct allosteric activation and
reversible phosphorylation. AMPK is allosterically activated by AMP through

  • binding to the regulatory subunit-γ, which induces a conformational change in
    the kinase domain of subunit α that protects AMPK from dephosphorylation
    of Thr172, probably by protein phosphatase-2C.

Activation of AMPK requires phosphorylation of Thr172 in its α subunit, which can be
attained by either

(i) tumor suppressor LKB1 kinase following enhancement in the AMP/ATP ratio, a
kinase that plays a crucial role in AMPK-dependent control of liver glucose and
lipid metabolism;

(ii) Ca2+-calmodulin-dependent protein kinase kinase-β (CaMKKβ) that
phosphorylates AMPK in an AMP-independent, Ca2+-dependent manner;

(iii) transforming growth-factor-β-activated kinase-1 (TAK1), an important
kinase in hepatic Toll-like receptor 4 signaling in response to lipopolysaccharide.

Among these kinases, the relevance of CaMKKβ and TAK1 in liver AMPK activation
remains to be established in metabolic stress conditions. Both allosteric and
phosphorylation mechanisms are able to elicit

  • over 1000-fold increase in AMPK activity, thus allowing
  • the liver to respond to small changes in energy status in a highly sensitive fashion.

In addition to rapid AMPK regulation through allosterism and reversible phosphorylation

  • long-term effects of AMPK activation induce changes in hepatic gene expression.

This was demonstrated for

(i) the transcription factor carbohydrate-response element-binding protein (ChREBP),

  • whose Ser568 phosphorylation by activated AMPK
  • blocks its DNA binding capacity and glucose-induced gene transcription
  • under hyperlipidemic conditions;(ii) liver sterol regulatory element-binding
    protein-1c (SREBP-1c), whose mRNA and protein expression and those of
    its target gene for fatty acid synthase (FAS)
  • are reduced by metformin-induced AMPK activation,
  • decreasing lipogenesis and increasing fatty acid oxidation due to
    malonyl-CoA depletion;

(iii) transcriptional coactivator transducer of regulated CREB activity-2 (TORC2),
a crucial component of the hepatic gluconeogenic program, was reported
to be phosphorylated by activated AMPK.

This modification leads to subsequent cytoplasmatic sequestration of TORC2 and
inhibition of gluconeogenic gene expression, a mechanism underlying

  • the plasma glucose-lowering effects of adiponectin and metformin
  • through AMPK activation by upstream LKB1.

Activation of AMPK in the liver is a key regulatory mechanism controlling glucose
and lipid metabolism,

  1. inhibiting anabolic processes, and
  2. enhancing catabolic pathways in response to different signals, including
    1. energy status,
    2. serum insulin/glucagon ratio,
    3. nutritional stresses,
    4. pharmacological and natural compounds, and
    5. oxidative stress status

Reactive Oxygen Species (ROS) and AMPK Activation

The high energy demands required to cope with all the metabolic functions
of the liver are met by

  • fatty acid oxidation under conditions of both normal blood glucose levels and
    hypoglycemia, whereas
  • glucose oxidation is favoured in hyperglycemic states, with consequent
    generation of ROS.

Under normal conditions, ROS occur at relatively low levels due to their fast processing
by antioxidant mechanisms, whereas at acute or prolonged high ROS levels, severe
oxidation of biomolecules and dysregulation of signal transduction and gene expression
is achieved, with consequent cell death through necrotic and/or apoptotic-signaling
pathways.

Thyroid Hormone (L-3,3′,5-Triiodothyronine, T3), Metabolic Regulation,
and ROS Production

T3 is important for the normal function of most mammalian tissues, with major actions
on O2 consumption and metabolic rate, thus

  • determining enhancement in fuel consumption for oxidation processes
  • and ATP repletion.

T3 acts predominantly through nuclear receptors (TR) α and β, forming

  • functional complexes with retinoic X receptor that
  • bind to thyroid hormone response elements (TRE) to activate gene expression.

T3 calorigenesis is primarily due to the

  • induction of enzymes related to mitochondrial electron transport and ATP
    synthesis, catabolism, and
  • some anabolic processes via upregulation of genomic mechanisms.

The net result of T3 action is the enhancement in the rate of O2 consumption of target
tissues such as liver, which may be effected by secondary processes induced by T3

(i) energy expenditure due to higher active cation transport,

(ii) energy loss due to futile cycles coupled to increase in catabolic and anabolic pathways, and

(iii) O2 equivalents used in hepatic ROS generation both in hepatocytes and Kupffer cells

In addition, T3-induced higher rates of mitochondrial oxidative phosphorylation are
likely to induce higher levels of ATP, which are partially balanced by intrinsic uncoupling
afforded by induction of uncoupling proteins by T3. In agreement with this view, the
cytosolic ATP/ADP ratio is decreased in hyperthyroid tissues, due to simultaneous
stimulation of ATP synthesis and consumption.

Regulation of fatty acid oxidation is mainly attained by carnitine palmitoyltransferase Iα (CPT-Iα),

  • catalyzing the transport of fatty acids from cytosol to mitochondria for β-oxidation,
    and acyl-CoA oxidase (ACO),
  • catalyzing the first rate-limiting reaction of peroxisomal β-oxidation, enzymes that are
    induced by both T3 and peroxisome proliferator-activated receptor α (PPAR-α).

Furthermore, PPAR-α-mediated upregulation of CPT-Iα mRNA is enhanced by PPAR-γ
coactivator 1α (PGC-1α), which in turn

  • augments T3 induction of CPT-Iα expression.

Interestingly, PGC-1α is induced by

  1. T3,
  2. AMPK activation, and
  3. ROS,

thus establishing potential links between

  • T3 action, ROS generation, and AMPK activation

with the onset of mitochondrial biogenesis and fatty acid β-oxidation.

Liver ROS generation leads to activation of the transcription factors

  1. nuclear factor-κB (NF-κB),
  2. activating protein 1 (AP-1), and
  3. signal transducer and activator of transcription 3 (STAT3)

at the Kupffer cell level, with upregulation of cytokine expression (TNF-α, IL-1, IL-6),
which upon interaction with specific receptors in hepatocytes trigger the expression of

  1. cytoprotective proteins (Figure 3(A)).

These responses and the promotion of hepatocyte and Kupffer-cell proliferation
represent hormetic effects reestablishing

  1. redox homeostasis,
  2. promoting cell survival, and
  3. protecting the liver against ischemia-reperfusion injury.

T3 liver preconditioning also involves the activation of the

  1. Nrf2-Keap1 defense pathway
  • upregulating antioxidant proteins,
  • phase-2 detoxifying enzymes, and
  • multidrug resistance proteins, members of the ATP binding cassette (ABC)
    superfamily of transporters (Figure 3(B))

In agreement with T3-induced liver preconditioning, T3 or L-thyroxin afford
preconditioning against IR injury in the heart, in association with

  • activation of protein kinase C and
  • attenuation of p38 and
  • c-Jun-N-terminal kinase activation ,

and in the kidney, in association with

  • heme oxygenase-1 upregulation.

475675.fig.002

http://www.hindawi.com/journals/tswj/2012/floats/475675/thumbnails/475675.fig.002_th.jpg

Figure 2: Calorigenic response of thyroid hormone (T3) and its relationship with O2
consumption, reactive oxygen species (ROS) generation, and antioxidant depletion in the liver.
Abbreviations: CYP2E1, cytochrome P450 isoform 2E1; GSH, reduced glutathione; QO2, rate
of O2 consumption; SOD, superoxide dismutase.

475675.fig.003

genomic signaling in T3 calorigenesis and ROS production 475675.fig.003

genomic signaling in T3 calorigenesis and ROS production 475675.fig.003

http://www.hindawi.com/journals/tswj/2012/floats/475675/thumbnails/475675.fig.003_th.jpg

Figure 3: Genomic signaling mechanisms in T3 calorigenesis and liver reactive oxygen
species (ROS) production leading to
(A) upregulation of cytokine expression in Kupffer cells and hepatocyte activation of genes
conferring cytoprotection,
(B) Nrf2 activation controling expression of antioxidant and detoxication proteins, and
(C) activation of the AMPK cascade regulating metabolic functions.Abbreviations: AP-1, activating protein 1; ARE, antioxidant responsive element; CaMKKβ,
Ca2+-calmodulin-dependent kinase kinase-β; CBP, CREB binding protein; CRC, chromatin
remodelling complex; EH, epoxide hydrolase; HO-1, hemoxygenase-1; GC-Ligase,
glutamate cysteine ligase; GPx, glutathione peroxidase; G-S-T, glutathione-S-transferase;
HAT, histone acetyltransferase; HMT, histone arginine methyltransferase; IL1,
interleukin 1; iNOS, inducible nitric oxide synthase; LKB1, tumor suppressor LKB1 kinase;
MnSOD, manganese superoxide dismutase; MRPs, multidrug resistance proteins; NF-κB,
nuclear factor-κB; NQO1, NADPH-quinone oxidoreductase-1; NRF-1, nuclear respiratory
factor-1; Nrf2, nuclear receptor-E2-related factor 2; PCAF, p300/CBP-associated
factor; RXR, retinoic acid receptor; PGC-1, peroxisome proliferator-activated receptor-γ
coactivator-1; QO2, rate of O2 consumption; STAT3, signal transducer and activator
of transcription 3; TAK1, transforming-growth-factor-β-activated kinase-1; TNF-α, tumor
necrosis factor-α; TR, T 3 receptor; TRAP, T3-receptor-associated protein; TRE,  T3 responsive element; UCP, uncoupling proteins; (—), reported mechanisms;
(- - - -), proposed mechanisms.

 

T3 is a key metabolic regulator coordinating short-term and long-term energy needs,
with major actions on liver metabolism. These include promotion of

(i) gluconeogenesis and hepatic glucose production, and

(ii) fatty acid oxidation coupled to enhanced adipose tissue lipolysis, with

  • higher fatty acid flux to the liver and
  • consequent ROS production (Figure 2) and
  • redox upregulation of cytoprotective proteins

affording liver preconditioning (Figure 3).

Thyroid Hormone and AMPK Activation: Skeletal Muscle and Heart

In skeletal muscle, T3 increases the levels of numerous proteins involved in

  1. glucose uptake (GLUT4),
  2. glycolysis (enolase, pyruvate kinase, triose phosphate isomerase),
  3. fatty acid oxidation (carnitine palmitoyl transferase-1, mitochondrial thioesterase I),
    and uncoupling protein-3,

effects that are achieved through enhanced transcription of TRE-containing genes

Skeletal muscle AMPK activation is characterized by

(i) being a rapid and transient response,

(ii) upstream activation by Ca2+-induced mobilization and CaMKKβ activation,

(iii) upstream upregulation of LKB1 expression, which requires association with STRAD
and MO25 for optimal phosphorylation/activation of AMPK, and

(iv) stimulation of mitochondrial fatty acid β-oxidation.

T3-induced muscle AMPK activation was found to trigger two major downstream

signaling pathways, namely,

(i) peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) mRNA
expression and phosphorylation, a transcriptional regulator for genes related to

  • mitochondrial biogenesis,
  • fatty acid oxidation, and
  • gluconeogenesis and

(ii) cyclic AMP response element binding protein (CREB) phosphorylation, which

  • in turn induces PGC-1α expression in liver tissue, thus
  • reinforcing mechanism (i).

These data indicate that AMPK phosphorylation of PGC-1α initiates many of the
important gene regulatory functions of AMPK in skeletal muscle.

In heart, hyperthyroidism increased glycolysis and sarcolemmal GLUT4 levels by the
combined effects of AMPK activation and insulin stimulation, with concomitant increase
in fatty acid oxidation proportional to enhanced cardiac mass and contractile function.

Thyroid Hormone, AMPK Activation, and Liver Preconditioning

Recent studies by our group revealed that administration of a single dose of 0.1 mg T3/kg
to rats activates liver AMPK (Figure 4; unpublished work).

  1. enhancement in phosphorylated AMPK/nonphosphorylated AMPK ratios in T3-
    treated rats over control values thatis significant in the time period of 1 to 48
    hours after hormone treatment
  2. Administration of a substantially higher dose (0.4 mg T3/kg) resulted in
    decreased liver AMPK activation at 4 h to return to control values at 6 h
    after treatment

Activation of liver AMPK by T3 may be of relevance in terms of

  • promotion of fatty acid oxidation for ATP supply,
  • supporting hepatoprotection against IR injury (Figure 3(C)).

This proposal is based on the high energy demands underlying effective liver
preconditioning for full operation of hepatic

  • antioxidant, antiapoptotic, and anti-inflammatory mechanisms,
  • oxidized biomolecules repair or resynthesis,
  • induction of the homeostatic acute-phase response, and
  • promotion of hepatocyte and Kupffer cell proliferation,

mechanisms that are needed to cope with the damaging processes set in by IR.
T3 liver preconditioning , in addition to that afforded by

  • n-3 long-chain polyunsaturated fatty acids given alone or
  • combined with T3 at lower dosages, or
  • by iron supplementation,

constitutes protective strategies against hepatic IR injury.

Studies on the molecular mechanisms underlying T3-induced liver AMPK
activation (Figure 4) are currently under assessment in our laboratory.

References

Fernández and L. A. Videla, “Kupffer cell-dependent signaling in thyroid hormone
calorigenesis: possible applications for liver preconditioning,” Current Signal
Transduction Therapy 2009; 4(2): 144–151.

Viollet, B. Guigas, J. Leclerc et al., “AMP-activated protein kinase in the regulation
of  hepatic energy metabolism: from physiology to therapeutic perspectives,” Acta
Physiologica 2009; 196(1): 81–98.

Carling, “The AMP-activated protein kinase cascade – A unifying system
for energy control,” Trends in Biochemical Sciences, 2004;. 29(1): 18–24.

E. Kemp, D. Stapleton, D. J. Campbell et al., “AMP-activated protein kinase,
super 
metabolic regulator,” Biochemical Society Transactions 2003; 31(1):
162–168
.

G. Hardie, “AMP-activated protein kinase-an energy sensor that
regulates all ;aspects of cell function,” Genes and Development,
2011; 25(18): 1895–1908.

Woods, P. C. F. Cheung, F. C. Smith et al., “Characterization of AMP-activated
protein kinase βandγ subunits Assembly of the heterotrimeric complex in vitro,”
Journal of Biological Chemistry 1996;271(17): 10282–10290.

Xiao, R. Heath, P. Saiu et al., “Structural basis for AMP binding to mammalian AMP-
activated protein kinase,” Nature 2007; 449(7161): 496–500.

more…

Impact of Metformin and compound C on NIS expression and iodine uptake in vitro and in vivo: a role for CRE in AMPK modulation of thyroid function.
Abdulrahman RM1, Boon MR, Sips HC, Guigas B, Rensen PC, Smit JW, Hovens GC.
Author information 
Thyroid. 2014 Jan;24(1):78-87.  Epub 2013 Sep 25.  PMID: 23819433
http://dx.doi.org:/10.1089/thy.2013.0041.

Although adenosine monophosphate activated protein kinase (AMPK) plays a crucial role
in energy metabolism, a direct effect of AMPK modulation on thyroid function has only
recently been reported, and much of its function in the thyroid is currently unknown.

The aim of this study was

  1. to investigate the mechanism of AMPK modulation in iodide uptake.
  2. to investigate the potential of the AMPK inhibitor compound C as an enhancer of
    iodide uptake by thyrocytes.

Metformin reduced NIS promoter activity (0.6-fold of control), whereas compound C
stimulated its activity (3.4-fold) after 4 days. This largely coincides with

  • CRE activation (0.6- and 3.0-fold).

These experiments show that AMPK exerts its effects on iodide uptake, at least partly,
through the CRE element in the NIS promoter. Furthermore, we have used AMPK-alpha1
knockout mice to determine the long-term effects of AMPK inhibition without chemical compounds.
These mice have a less active thyroid, as shown by reduced colloid volume and reduced
responsiveness to thyrotropin.

NIS expression and iodine uptake in thyrocytes

  • can be modulated by metformin and compound C.

These compounds exert their effect by

  • modulation of AMPK, which, in turn, regulates
  • the activation of the CRE element in the NIS promoter.

Overall, this suggests that AMPK modulating compounds may be useful for the
enhancement of iodide uptake by thyrocytes, which could be useful for the
treatment of thyroid cancer patients with radioactive iodine.

AMPK: Master Metabolic Regulator

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@ themedicalbiochemistrypage.org

AMPK-activating drugs metformin or phenformin might provide protection against cancer 1741-7007-11-36-5

AMPK-activating drugs metformin or phenformin might provide protection against cancer 1741-7007-11-36-5

 

AMPK and AMPK-related kinase (ARK) family 1741-7007-11-36-4

AMPK and AMPK-related kinase (ARK) family 1741-7007-11-36-4

 

central role of AMPK in the regulation of metabolism

 

 

AMP-activated protein kinase (AMPK) was first discovered as an activity that

AMPK induces a cascade of events within cells in response to the ever changing energy
charge of the cell. The role of AMPK in regulating cellular energy charge places this
enzyme at a central control point in maintaining energy homeostasis.

More recent evidence has shown that AMPK activity can also be regulated by physiological stimuli, independent of the energy charge of the cell, including hormones and nutrients.

 

Once activated, AMPK-mediated phosphorylation events

These events are rapidly initiated and are referred to as

  • short-term regulatory processes.

The activation of AMPK also exerts

  • long-term effects at the level of both gene expression and protein synthesis.

Other important activities attributable to AMPK are

  1. regulation of insulin synthesis and
  2. secretion in pancreatic islet β-cells and
  3. modulation of hypothalamic functions involved in the regulation of satiety.

How these latter two functions impact obesity and diabetes will be discussed below.

Regulation of AMPK

In the presence of AMP the activity of AMPK is increased approximately 5-fold.
However, more importantly is the role of AMP in regulating the level of phosphorylation
of AMPK. An increased AMP to ATP ratio leads to a conformational change in the γ-subunit
leading to increased phosphorylation and decreased dephosphorylation of AMPK.

The phosphorylation of AMPK results in activation by at least 100-fold. AMPK is
phosphorylated by at least three different upstream AMPK kinases (AMPKKs).
Phosphorylation of AMPK occurs in the α subunit at threonine 172 (T172) which

  • lies in the activation loop.

One kinase activator of AMPK is

  • Ca2+-calmodulin-dependent kinase kinase β (CaMKKβ)
  • which phosphorylates and activates AMPK in response to increased calcium.

The distribution of CaMKKβ expression is primarily in the brain with detectable levels
also found in the testes, thymus, and T cells. As described for the Ca2+-mediated
regulation of glycogen metabolism,

  • increased release of intracellular stores of Ca2+ create a subsequent demand for
    ATP.

Activation of AMPK in response to Ca fluxes

  • provides a mechanism for cells to anticipate the increased demand for ATP.

Evidence has also demonstrated that the serine-threonine kinase, LKB1 (also called
serine-threonine kinase 11, STK11) which is encoded by the Peutz-Jeghers syndrome
tumor suppressor gene, is required for activation of AMPK in response to stress.

The active LKB1 kinase is actually a complex of three proteins:

  1. LKB1,
  2. Ste20-related adaptor (STRAD) and
  3. mouse protein 25 (MO25).

Thus, the enzyme complex is often referred to as LKB1-STRAD-MO25. Phosphorylation
of AMPK by LKB1 also occurs on T172. Unlike the limited distribution of CaMKKβ,

  • LKB1 is widely expressed, thus making it the primary AMPK-regulating kinase.

Loss of LKB1 activity in adult mouse liver leads to

  • near complete loss of AMPK activity and
  • is associated with hyperglycemia.

The hyperglycemia is, in part, due to an increase in the transcription of gluconeogenic
genes. Of particular significance is the increased expression of

  • the peroxisome proliferator-activated receptor-γ (PPAR-γ) coactivator 1α
    (PGC-1α), which drives gluconeogenesis.
  • Reduction in PGC-1α activity results in normalized blood glucose levels in
    LKB1-deficient mice.

The third AMPK phosphorylating kinase is transforming growth factor-β-activated
kinase 1 (TAK1). However, the normal physiological conditions under which TAK1
phosphorylates AMPK are currently unclear.

The effects of AMP are two-fold:

  1. a direct allosteric activation and making AMPK a poorer substrate for
    dephosphorylation.

Because AMP affects both
the rate of AMPK phoshorylation in the positive direction and
dephosphorylation in the negative direction,

the cascade is ultrasensitive. This means that

  1. a very small rise in AMP levels can induce a dramatic increase in the activity of
    AMPK.

The activity of adenylate kinase, catalyzing the reaction shown below, ensures that

  • AMPK is highly sensitive to small changes in the intracellular [ATP]/[ADP] ratio.

2 ADP ——> ATP + AMP

Negative allosteric regulation of AMPK also occurs and this effect is exerted by
phosphocreatine. As indicated above, the β subunits of AMPK have a glycogen-binding domain, GBD. In muscle, a high glycogen content

  • represses AMPK activity and
  • this is likely the result of interaction between the GBD and glycogen,
  • the GBD of AMPK allows association of the enzyme with the regulation of glycogen metabolism
  • by placing AMPK in close proximity to one of its substrates glycogen synthase.

AMPK has also been shown to be activated by receptors that are coupled to

  • phospholipase C-β (PLC-β) and by
  • hormones secreted by adipose tissue (termed adipokines) such as leptinand adiponectin (discussed below).

Targets of AMPK

The signaling cascades initiated by the activation of AMPK exert effects on

  • glucose and lipid metabolism,
  • gene expression and
  • protein synthesis.

These effects are most important for regulating metabolic events in the liver, skeletal
muscle, heart, adipose tissue, and pancreas.

Demonstration of the central role of AMPK in the regulation of metabolism in response
to events such as nutrient- or exercise-induced stress. Several of the known physiologic
targets for AMPK are included as well as several pathways whose flux is affected by
AMPK activation. Arrows indicate positive effects of AMPK, whereas, T-lines indicate
the resultant inhibitory effects of AMPK action.

The uptake, by skeletal muscle, accounts for >70% of the glucose removal from the
serum in humans. Therefore, it should be obvious that this event is extremely important
for overall glucose homeostasis, keeping in mind, of course, that glucose uptake by
cardiac muscle and adipocytes cannot be excluded from consideration. An important fact
related to skeletal muscle glucose uptake is that this process is markedly impaired in
individuals with type 2 diabetes.

The uptake of glucose increases dramatically in response to stress (such as ischemia) and
exercise and is stimulated by insulin-induced recruitment of glucose transporters
to the plasma membrane, primarily GLUT4. Insulin-independent recruitment of glucose
transporters also occurs in skeletal muscle in response to contraction (exercise).

The activation of AMPK plays an important, albeit not an exclusive, role in the induction of
GLUT4 recruitment to the plasma membrane. The ability of AMPK to stimulate
GLUT4 translocation to the plasma membrane in skeletal muscle is by a different mechanism
than that stimulated by insulin and insulin and AMPK effects are additive.

Under ischemic/hypoxic conditions in the heart the activation of AMPK leads to the
phosphorylation and activation of the kinase activity of phosphofructokinase-2, PFK-2
(6-phosphofructo-2-kinase). The product of the action of PFK-2 (fructose-2,6-bisphosphate,
F2,6BP) is one of the most potent regulators of the rate of flux through
glycolysis and gluconeogenesis.

In liver the PKA-mediated phosphorylation of PFK-2 results in conversion of the
enzyme from a kinase that generates F2,6BP to a phosphatase that removes the
2-phosphate thus reducing the levels of the potent allosteric activator of the glycolytic
enzyme 6-phosphfructo-1-kinase, PFK-1 and the potent allosteric inhibitor
of the gluconeogenic enzyme fructose-1,6-bisphosphatase (F1,-6BPase).

It is important to note that like many enzymes, there are multiple isoforms of PFK-2
(at least 4) and neither the liver or the skeletal muscle isoforms contain the AMPK
phosphorylation sites found in the cardiac and inducible (iPFK2) isoforms of PFK-2.

Inducible PFK-2 is expressed in the monocyte/macrophage lineage in response to pro-
inflammatory stimuli. The ability to activate the kinase activity by phosphorylation of
PFK-2 in cardiac tissue and macrophages in response to ischemic conditions allows these
cells to continue to have a source of ATP via anaerobic glycolysis. This phenomenon is
recognized as the Pasteur effect: an increased rate of glycolysis in response to hypoxia.

Of pathological significance is the fact that the inducible form of PFK-2 is commonly
expressed in many tumor cells and this may allow AMPK to play an important role in
protecting tumor cells from hypoxic stress. Indeed, techniques for depleting AMPK in
tumor cells have shown that these cells become sensitized to nutritional stress upon loss
of AMPK activity.

Whereas, stress and exercise are powerful inducers of AMPK activity in skeletal muscle,
additional regulators of its activity have been identified.

Insulin-sensitizing drugs of the thiazolidinedione family (activators of PPAR-γ, see
below) as well as the hypoglycemia drug metformin exert a portion of their effects
through regulation of the activity of AMPK.

As indicated above, the activity of the AMPK activating kinase, LKB1, is critical for
regulating gluconeogenic flux and consequent glucose homeostasis. The action of
metformin in reducing blood glucose levels

  • requires the activity of LKB1 in the liver for this function.

Also, several adipokines (hormones secreted by adipocytes) either stimulate or inhibit
AMPK activation:

  1. leptin and adiponectin have been shown to stimulate AMPK activation, whereas,
  2. resistininhibits AMPK activation.

Cardiac effects exerted by activation of AMPK also include

AMPK-mediated phosphorylation of eNOS leads to increased activity and consequent
NO production and provides a link between metabolic stresses and cardiac function.

In platelets, insulin action leads to an increase in eNOS activity that is

  • due to its phosphorylation by AMPK.

Activation of NO production in platelets leads to

  • a decrease in thrombin-induced aggregation, thereby,
  • limiting the pro-coagulant effects of platelet activation.

The response of platelets to insulin function clearly indicates why disruption in insulin
action is a major contributing factor in the development of the metabolic syndrome

Activation of AMPK leads to a reduction in the level of SREBP

  • a transcription factor &regulator of the expression of numerous
    lipogenic enzymes

Another transcription factor reduced in response to AMPK activation is

  • hepatocyte nuclear factor 4α, HNF4α
    • a member of the steroid/thyroid hormone superfamily.
    • HNF4α is known to regulate the expression of several liver and
      pancreatic β-cell genes such as GLUT2, L-PK and preproinsulin.
  • Of clinical significance is that mutations in HNF4α are responsible for
    • maturity-onset diabetes of the young, MODY-1.

Recent evidence indicates that the gene for the carbohydrate-response-element-
binding protein (ChREBP) is a target for AMPK-mediated transcriptional regulation
in the liver. ChREBP is rapidly being recognized as a master regulator of lipid
metabolism in liver, in particular in response to glucose uptake.

The target of the thiazolidinedione (TZD) class of drugs used to treat type 2 diabetes is
the peroxisome proliferator-activated receptor γ, PPARγ which

  • itself may be a target for the action of AMPK.

The transcription co-activator, p300, is phosphorylated by AMPK

  • which inhibits interaction of p300 with not only PPARγ but also
  • the retinoic acid receptor, retinoid X receptor, and
  • thyroid hormone receptor.

PPARγ is primarily expressed in adipose tissue and thus it was difficult to reconcile how
a drug that was apparently acting only in adipose tissue could lead to improved insulin
sensitivity of other tissues. The answer to this question came when it was discovered that the TZDs stimulated the expression and release of the adipocyte hormone (adipokine),
adiponectin. Adiponectin stimulates glucose uptake and fatty acid oxidation in skeletal
muscle. In addition, adiponectin stimulates fatty acid oxidation in liver while inhibiting
expression of gluconeogenic enzymes in this tissue.

These responses to adiponectin are exerted via activation of AMPK. Another
transcription factor target of AMPK is the forkhead protein, FKHR (now referred to as
FoxO1). FoxO1 is involved in the activation of glucose-6-phosphatase expression and,
therefore, loss of FoxO1 activity in response to AMPK activation will lead to reduced
hepatic output of glucose.

This concludes a very complicated perspective that ties together the thyroid hormone
activity, the hypophysis, diabetes mellitus, and AMPK tegulation of metabolism in the
liver, skeletal muscle, adipose tissue, and heart.  I also note at this time that there
nongenetic points to be made here:

  1. The tissue specificity of isoenzymes
  2. The modulatory role of AMP:ATP ratio in phosphorylation/dephosphorylation
    effects on metabolism tied to AMPK
  3. The tie in of stress or ROS with fast reactions to protect harm to tissues
  4. The relationship of cytokine activation and release to the above metabolic events
  5. The relationship of effective and commonly used diabetes medications to AMPK
    mediated processes
  6. The preceding presentation is notable for the importance of proteomic and
    metabolomic invetigations in elucidation common chronic and nongenetic diseases

 

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Imaging-Biomarkers; from discovery to validation

Author: Dror Nir, PhD.

Preface

Recent technology advances such as miniaturization and improvement in electronic-processing components is driving increased introduction of innovative medical-imaging devices into critical nodes of major-diseases’ management pathways. Similarly, medical imaging bears outstanding potential to improve the process of drugs development and regulation (e.g. companion diagnostics and imaging surrogate markers. In; The Role of Medical Imaging in Personalized Medicine I discussed in length the role medical imaging assumes in drugs development.  Integrating imaging into drug development processes, specifically at the early stages of drug discovery, as well as for monitoring drug delivery and the response of targeted processes to the therapy is a growing trend. A nice (and short) review highlighting the processes, opportunities, and challenges of medical imaging in new drug development is: Medical imaging in new drug clinical development. An important aspect of drug development that is largely discussed is facilitating testing of the new drug through clinical studies. A major hurdle in development of many anti-cancer drugs is the long time that is required to determine the efficacy of the new drug through measurement of clinically meaningful endpoints; e.g. overall survival. Imaging is offering the opportunity to determine surrogate markers of clinical outcome (as a substitute for a clinically meaningful endpoints). The need for surrogate outcome markers is especially great with newer agents that may act by tumour stabilization as opposed to shrinkage.

To comply with current trends; e.g. personalized medicine and evidence-based medicine, medical imaging must support quantification of meaningful pathological phenomena; e.g. morphological deformations, enhanced/reduced chemical reactions, presence/absence of biological substances etc….

 

Two examples: 

Molecular imaging (e.g. PET, MRS) allows the visual representation, characterization, and quantification of biological processes at the cellular and subcellular levels within intact living organisms. In oncology, it can be used to depict the abnormal molecules as well as the aberrant interactions of altered molecules on which cancers depend. An established biological process is neoplastic angiogenesis is associated with a number of detectable changes at molecular and microcirculatory levels. In Positron emission tomographic imaging of angiogenesis and vascular function the authors are offering that direct study of angiogenic molecular biology and tumour circulation before during and after treatment may offer useful surrogate markers for vascular-targeted therapies. The paper reviews two main areas: (a) the methodology behind PET imaging of tumour blood supply with 15O-oxygen labelled compounds; and (b) newer tracers in development as markers of angiogenetic biology.

A largely sought-for application for medical imaging is Monitoring quality of surgery: Cancer patients could benefit from a surgical procedure that helps the surgeon to determine adequate tumor resection margins. Variety of applications and work-flows; e.g. Systemic injection of tumor-specific fluorescence agents with subsequent intraoperative optical imaging to guide the surgeon in the process are offered. Recently, in order to overcome the problem of tumor heterogeneity it was proposed to shift the focus of tumor targeting towards the follicle-stimulating hormone receptor (FSHR).

Imaging bio-markers

Being able to discover and clinically validate fundamental finger-prints of cancer which can be detected and quantified through medical-imaging modalities is key to transforming the potential presented by medical imaging into clinical reality. Such specific finger-prints/characteristics are usually referred to as imaging bio-markers.

A critical step in the discovery and validation of imaging bio-markers is the matching of tissue location as depicted by imaging-products (most commonly images) to their histology, as underlined by a pathologist under the microscope.

Since histology requires extraction of organ tissue and some processing, it is impossible to achieve such matching in real time. Therefore, different techniques were developed to support the retrospective matching between histology and imaging. The most prevalent one rely on image registration: i.e. the products of medical imaging are registered to images of pathology slides. The main limitation of such methods has to do with:

  1. The fact that the two images poses largely different image resolution.
  2. The form-factor (shape and dimensions) of Histological tissue-slides are distorted in comparison to their in-vivo state.
  3. Histology-reading is subjective; i.e. the concordance between readings of different pathologist is far from being satisfactory. It gets worse when it comes to staging of the cancer.
  4. There is large variation in the quality of medical imaging products.

A Workflow to Improve the Alignment of Prostate Imaging with Whole-mount Histopathology presents a robust methodology validating imaging biomarkers in the case of prostate cancer. In this paper we describe a workflow for three-dimensional alignment of prostate imaging data against whole-mount prostatectomy reference specimens and assess its performance against a standard workflow. We hypothesized that integration of image registration principles into the histological workflow for radical prostatectomy specimens would increase the alignment accuracy. In this post I will include only few excerpts from this paper which I strongly recommend to read in full.

Materials and Methods

Ethical approval was granted. Patients underwent motorized transrectal ultrasound (Prostate Histoscanning) to generate a three-dimensional image of the prostate before radical prostatectomy. The test workflow incorporated steps for axial alignment between imaging and histology, size adjustments following formalin fixation, and use of custom-made parallel cutters and digital caliper instruments. The control workflow comprised freehand cutting and assumed homogeneous block thicknesses at the same relative angles between pathology and imaging sections. The basic requirements of image registration were incorporated within the pathological protocol.

We demonstrate that the use of a simple, custom-made tissue-planer to slice the formalin-fixed prostate results in more uniform and parallel tissue blocks than conventional freehand techniques, and increases the accuracy of image alignment.  We also show that accounting for dimensional change due to formalin fixation is essential during image alignment.

Figure 1: Suggested workflow for registration of scanned histopathological data with radiological imaging

 fig1

 Figure 3

A sketch of the tissue cutting device is shown (A).  The formalin-fixed prostate was placed on the space marked “X” on the device with its flat posterior surface facing down.  With the probe in the urethra to align the AP axis with the device, the base of the gland was gently pressed onto “Y”.  The probe was then removed, and a mounted microtome blade was lowered along the 4mm raised edge of the device from top to bottom to cut away the block (B).  The sliced block was put aside with its apical face facing down, and the process was repeated by gently pressing the cut surface flush against the device before each cut (C).  The thickness of each block was measured in 5 locations marked (D).

fig3

Results

Thirty radical prostatectomy specimens were histologically and radiologically processed, either by an alignment-optimized workflow (n = 20) or a control workflow (n = 10). The optimized workflow generated tissue blocks of heterogeneous thicknesses but with no significant drifting in the cutting plane. The control workflow resulted in significantly nonparallel blocks, accurately matching only one out of four histology blocks to their respective imaging data. The image-to-histology alignment accuracy was 20% greater in the optimized workflow (P < .0001), with higher sensitivity (85% vs. 69%) and specificity (94% vs. 73%) for margin prediction in a 5 × 5-mm grid analysis.

Figure 5. Assessment of alignment accuracy between radiological images and pathological sections

The method of assessing alignment accuracy between radiological images and pathological slides is shown using an example.  Each square within the grids overlaid onto histology and radiological images were scored either as a “1”, indicating the presence of a histological or radiological margin, respectively, or “0”.  Scored pathology grids were used as the reference, and scored radiology grids were used as the index.  Hence, we determined true positives i.e. grid points score “1” in both histology and radiology (yellow squares, n=25), false positives i.e. grid points on the radiology scores “1” but not on histology (green squares, n=4), false negatives i.e. grid points on the histology scores “0” but not on radiology (red squares, n=3), and true negatives (grey squares, n=38).

 fig5

Conclusions

A significantly better alignment was observed in the optimized workflow. Evaluation of prostate imaging biomarkers using whole-mount histology references should include a test-to-reference spatial alignment workflow.

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Leaders in Pharmaceutical Business Intelligence Announced New Cardiovascular Series of e-Books at SACHS Associates 14th Annual Biotech In Europe Forum

Reporter: Aviva Lev-Ari, PhD, RN

 

 

Please see Further Titles at

http://pharmaceuticalintelligence.com/biomed-e-books/

Please see Further Information on the Sachs Associates 14th Annual Biotech in Europe Forum for Global Investing & Partnering at:

http://pharmaceuticalintelligence.com/2014/03/25/14th-annual-biotech-in-europe-forum-for-global-partnering-investment-930-1012014-%E2%80%A2-congress-center-basel-sachs-associates-london/

AND

http://www.sachsforum.com/basel14/index.html

why-is-twitter-s-logo-named-after-larry-bird--b8d70319daON TWITTER Follow at

@SachsAssociates

#Sachs14thBEF

@pharma_BI

@AVIVA1950 

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