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Mitochondria are present in almost all human cells, and vary in number from a few tens to many thousands. They generate the majority of a cell’s energy supply which powers every part of our body. Mitochondria have their own separate DNA, which carries just a few genes. All of these genes are involved in energy production but determine no other characteristics. And so, any faults in these genes lead only to problems in energy production. Around 1 in 6500 children is thought to be born with a serious mitochondrial disorder due to faults in mitochondrial DNA.
Unlike nuclear genes, mitochondrial DNA is inherited only from our mothers. Mothers can carry abnormal mitochondria and be at risk of passing on serious disease to their children, even if they themselves show only mild or no symptoms. It is for such women who by chance have a high proportion of faulty mitochondrial DNA in their eggs for which the methods of mitochondrial replacement or “donation” have been developed. This technique is also referred as the three parent technique and it involves a couple and a donor.
Mitochondrial Donation
The most developed techniques, maternal spindle transfer (MST) and pro-nuclear transfer (PNT), are based on an IVF cycle but have additional steps. Other techniques are being developed.
In both MST and PNT, nuclear DNA is moved from a patient’s egg or embryo containing unhealthy mitochondria to a donor’s egg or embryo containing healthy mitochondria, from which the donor’s nuclear DNA has been removed.
Maternal spindle transfer Bredenoord, A and P. Braude (2010) “Ethics of mitochondrial gene replacement: from bench to bedside” BMJ 341.
Pronuclear transfer Bredenoord, A and P. Braude (2010) “Ethics of mitochondrial gene replacement: from bench to bedside” BMJ 341.
Research Carried Out and Safety Issues
There have been many experiments conducted using MST and PNT in animals. PNT has been carried out since the mid-1980s in mice. MST has been carried out in a wide range of animals. More recently mice, monkeys and human embryos have been created with the specific aim of developing MST and PNT for avoiding mitochondrial disease.
There is no evidence to show that mitochondrial donation is unsafe
Research is progressing well and the recommended further experiments are expected to confirm this view.
The main area of research needed is to observe cells derived from embryos created by MST and PNT, to see how mitochondria behave.
Concerns about Mitochondrial Donation
The scientific evidence raises some potential concerns about mitochondrial donation. Just as we all have different blood groups, we also have different types of mitochondria, called haplotypes. Some scientists have suggested that if the patient and the mitochondria donor have different mitochondrial haplotypes, there is a theoretical risk that the donor’s mitochondria won’t be able to ‘talk’ properly to the patient’s nuclear DNA, which could cause problems in the embryo and resulting child. So, mitochondria haplotype matching in the process of selecting donors may be done to avoid problems.
Another potential concern is that a small amount of unhealthy mitochondrial DNA may be transferred into the donor’s egg along with the mother’s nuclear DNA. Studies carried out on MST and PNT show that some so-called mitochondrial ‘carry-over’ occurs. However, the carry-over is lower than 2% of the mitochondria in the resulting embryo, an amount which is very unlikely to be problematic for the children born.
Somatic, germ-cell, and whole sequence DNA in cell lineage and disease profiling
Curator: Larry H Bernstein, MD, FCAP
In humans, mitochondrial DNA spans about 16,500 DNA building blocks (base pairs), representing a small fraction of the total DNA in cells. Mitochondrial DNA contains 37 genes, essential for normal mitochondrial function and thirteen of them provide instructions for making enzymes involved in inner membrane function. The remaining 24 genes are transcribed into transfer RNA (tRNA) and ribosomal RNA (rRNA), which are needed to transfer amino acids into proteins.
Somatic mutations occur in the DNA of certain cells during a person’s lifetime and typically are not passed to future generations. They differ from germ-line mutations that have a lineal descent from the maternal parent, and they occur later in life. Mutations in the sperm DNA are not carried on to future generations, as the sperm mitochondria are destroyed after the egg is fertilized.
There is limited evidence linking somatic mutations in mitochondrial DNA with certain cancers, including breast, colon, stomach, liver, and kidney tumors. These mutations might also be associated with cancer of blood-forming tissue (leukemia) and cancer of immune system cells (lymphoma). There are many heritable diseases that are related to germ-line mutations, and germ-line mutations have a role in many common diseases. Mitochondrial DNA is particularly vulnerable to the effects of reactive oxygen species (ROS), and with a limited ability of the mitochondrion to repair itself, ROS easily damage mitochondrial DNA. The repair mechanism is tied to ubiquitinylation system. A list of disorders associated with mitochondrial genes is provided from Wikipedia.
Inherited changes in mitochondrial DNA may be associated with pathologies in growth and development, and multiorgan system disorders, as mutations disrupt the mitochondria’s ability to generate the cell’s energy. The effects of these conditions are most pronounced in organs and tissues with high energy requirements (such as the heart, brain, and muscles). Although the health consequences of inherited mitochondrial DNA mutations vary widely, some frequently observed features include muscle weakness and wasting, problems with movement, diabetes, kidney failure, heart disease, loss of intellectual functions (dementia), hearing loss, and abnormalities involving the eyes and vision.
A buildup of somatic mutations in mitochondrial DNA has been considered to have a role in or associated with increased risk of certain age-related disorders such as heart disease, Alzheimer disease, and Parkinson disease, and the severity of many mitochondrial disorders is thought to be associated with the percentage of mitochondria affected by a particular genetic change. Consequently, the progressive accumulation of these mutations over a person’s lifetime may play a role in aging.
Mitochondrial DNA is typically diagrammed as a circular structure with genes and regulatory regions labeled.
Mutations are a cumulative dossier of our own maternal prehistory. The main task of DNA is to copy itself to each new generation. We can use these mutations to reconstruct a genetic tree of mtDNA, because each new mtDNA mutation in a prospective mother’s ovum will be transferred in perpetuity to all her descendants down the female line. Each new female line is thus defined by the old mutations as well as the new ones.
By looking at the DNA code in a sample of people alive today, and piecing together the changes in the code that have arisen down the generations, biologists can trace the line of descent back in time to a distant shared ancestor. Because we inherit mtDNA only from our mother, this line of descent is a picture of the female genealogy of the human species.
formation of gene trees
The diagram above shows the drawing of gene trees using single mutations
Not only can we retrace the tree, but by taking into account here the sampled people came from, we can see where certain mutations occurred – for example, whether in Europe, or Asia, or Africa. What’s more, because the changes happen at a statistically consistent (though random) rate, we can approximate the time when they happened. This has made it possible, during the late 1990s and in the new century, for us to do something that anthropologists of the past could only have dreamt of: we can now trace the migrations of modern humans around our planet.
It turns out that the oldest changes in our mtDNA took place in Africa 150,000 – 190,000 years ago. Then new mutations start to appear in Asia, about 60,000 – 80,000 years ago. This tells us that modern humans evolved in Africa, and that some of us migrated out of Africa into Asia after 80,000 years ago. A method established in 1996, which dates each branch of the gene tree by averaging the number of new mutations in daughter types of that branch, has stood the test of time.
A final point on the methods of genetic tracking of migrations: it is important to distinguish this new approach to tracing the history of molecules on a DNA tree, known as phylogeography (literally ‘tree-geography’), from the mathematical study of the history of whole human populations, which has been used for decades and is known as classical population genetics.
The two disciplines are based on the same Mendelian biological principles, but have quite different aims and assumptions, and the difference is the source of much misunderstanding and controversy. The simplest way of explaining it is that phylogeography studies the prehistory of individual DNA molecules, while population genetics studies the prehistory of populations. Put another way, each human population contains multiple versions of any particular DNA molecule, each with its own history and different origin.
David Moskowitz, MD, PhD
Founder and President, GenoMed
Germline genes make the best drug targets
They operate earliest in the disease pathway
Unlike tissue-expressed genes, which operate years after the disease began
But which everybody else is using as drug targets
Variation in germline DNA is where all disease starts
Cancer patients overexpress oncogenes and underexpress tumor suppressors
beginning in their germline DNA
Mutations in tumor DNA are “private”
Each tumor is a “snowflake”
Tumor-expressed genes can be compensatory, not causative
“Passengers, not drivers”
We have the drivers
Tumorigenesis SNPs
Using a SNPnet™ covering only 1/3 of the genome, we found about
2,500 genes associated with each of 6 different cancers in whites
Nobody else has found any yet
This will change in 2-3 years
We estimate 10,000 genes per cancer
What cellular program takes up 1/3-1/2 of the genome?
What program takes up >1/3 of the genome?
Differentiation…
Does sporadic cancer arise when a tissue stem cell fails to differentiate?
In the embryo, the surrounding tissue expresses “fields”
Lent C. Johnson published a “field” based hypothesis of bone tumors that coincides with differentiation at the
METAPHYSIS
HYPOPHYSIS
and the type CELL – chondroblast, osteoblast, giant cell (osteoclast), fibroblast
Orthopedic surgeons use magnetic fields for healing
of powerful transcription factors.
Not so in adult life: a proliferating tissue stem cell is literally on its own.
Germlines hold the key to effective “differentiation therapy”
Ideal for patients with stage 3-4 cancer
Examples of differentiation therapy:
1,25-vitamin D and
retinoic acid
Non-toxic but more effective treatment for late stage disease,
GenoMed’s 2,500 cancer-causing genes:
½ are oncogenes,
½ are tumor suppressors
Design inhibitors to oncogenes
Screen 1st for toxicity;
genomic epidemiology guarantees clinical efficacy
Jewish Heritage Written in DNA
By Kate Yandell | Sept 9, 2014
Fully sequenced genomes of more than 100 Ashkenazi people clarify the group’s history and provide a reference for researchers and physicians trying to pinpoint disease-associated genes.
A whole-genome sequence study from 128 healthy Jewish people is aimed at identifying disease-associated variants in the jewish population of Ashkenazi ancestry, according to a study published Sept 9 in Nature Communications. The library of sequences confirms earlier conclusions about Ashkenazi history hinted at by more limited DNA sequencing studies. The sequences point to an approximate 350-person bottleneck in the Ashkenazi population as recently as 700 years ago (1400 A.D.), and suggest that the population has a mixture of European and Middle Eastern ancestry.
The study “provides a very nice reference panel for the very unique population of Ashkenazi Jews,” said Alon Keinan, who studies human population genomics at Cornell University in New York. Keinan
is acknowledged in the study but was not involved in the research.
“One might have thought that, after many years of genetic studies relating to Ashkenazi Jews . . . there would be little room for additional insights,” Karl Skorecki of the Rambam Healthcare Campus
in Israel who also was not involved in the study wrote in an e-mail to The Scientist. The study, he added, provides “a powerful further validation and further resolution of the demographic history of
the Ashkenazi Jews in relation to non-Jewish Europeans that is reassuringly consistent with inferences drawn from two decades of studies using uniparental regions . . . and from array-based data.”
Itsik Pe’er, coauthor of the new study and an associate professor of computer science at Columbia University in New York City, recalled that several years ago, he and his colleagues kept running into the same problem as they tried to understand the genetics of disease in Ashkenazi populations. They were comparing their Ashkenazi samples to the only control genomes that were available, which were of largely non-Jewish European origin. The Ashkenazi genomes had variation that was absent in these general European genomes, making it hard to distinguish rare variants in Ashkenazi people.
“Technology is there to tell us everything in that [Ashkenazi] patient’s genome, but the genome was not there to distinguish the variants that are there and to tell us whether they are normal or whether we should get worried,” said Pe’er. Pe’er’s group teamed up with researchers from additional universities and hospitals in the U.S., Belgium, and Israel to sequence a collection of healthy Ashkenazi people’s genomes. The panel of reference sequences performs better than a group of European genomes at filtering out harmless variants from Ashkenazi Jewish genomes, thereby making it easier to identify potentially harmful ones. According to Pe’er, researchers will also be able to use the panel to infer
more complete sequences from partially sequenced genomes by looking for familiar sequences from the reference genomes.
The team also used its data to better understand the history of the Ashkenazi Jewish people through analyzing both level of similarity within Ashkenazi genomes and between Ashkenazi and non-Jewish
European genomes. By analyzing the length of identical DNA sequences that Ashkenazi individuals share, the researchers were able to estimate that 25 to 32 generations ago, the Ashkenazi Jewish population shrunk to just several hundred people, before expanding rapidly to eventually include the millions of Ashkenazi Jews alive today. Further, the researchers concluded that modern Ashkenazi Jews likely have an approximately even mixture of European and Middle Eastern ancestry. This suggests that after the Jewish people migrated from the Middle East to Europe, they recruited people from local European populations.
These results are compatible with those of prior work on mitochondrial DNA (mtDNA), which is passed on maternally. This prior work suggested that Ashkenazi men from the Middle East intermarried with local European women. The Ashkenazi population “hasn’t been likely as isolated as at least some researchers considered,” said Keinan.
Finally, the newly sequenced genomes shed light on the deeper history of Europe, showing that the European and Middle Eastern portions of Ashkenazi ancestry diverged just around 20,000 years ago.
“This is, I think, the first evidence from whole human genomes that the most important wave of settlement from the Near East was most likely shortly after the Last Glacial Maximum . . . and, notably, before the Neolithic transition—which is what researchers working on mitochondrial DNA have been arguing for some years,” Martin Richards, an archeogeneticist at the University of Huddersfield in the U.K., told The Scientist in an e-mail.
Skorecki noted that the new study “demonstrates the utility of sequencing whole genomes in a diverse population… with sufficient numbers of samples, parent population information, and
computational analytic power, we can expect important and surprising utilities for personal genomic and insights in terms of human demographic history from whole genomes.”
Carmi et al., “Sequencing an Ashkenazi reference panel supports population-targeted personal genomics and illuminates Jewish and European origins,” Nature
Communications, http://dx.doi.org:/10.1038/ncomms5835, 2014.
Added Layers of Proteome Complexity
By Anna Azvolinsky | July 17, 2014
Scientists discover a broad spectrum of alternatively spliced human protein variants within a well-studied family of genes.
There may be more to the human proteome than previously thought. Some genes are known to have several different alternatively spliced protein variants, but the Scripps Research Institute’s Paul Schimmel and his colleagues have now uncovered almost 250 protein splice variants of an essential, evolutionarily conserved family of human genes. The results were published today (July 17) in Science.
Focusing on the 20-gene family of aminoacyl tRNA synthetases (AARSs), the team captured AARS transcripts from human tissues—some fetal, some adult—and showed that many of these messenger RNAs (mRNAs) were translated into proteins. Previous studies have identified
several splice variants of these enzymes that have novel functions, but uncovering so many more variants was unexpected, Schimmel said. Most of these new protein products lack the catalytic domain but retain other AARS non-catalytic functional domains. “The main point is that a vast new area of biology, previously missed, has been uncovered,”
said Schimmel.
“This is an incredible study that fundamentally changes how we look at the protein-synthesis machinery,” Michael Ibba, a protein translation researcher at Ohio State University who was not involved in the work, told The Scientist in an e-mail. “The unexpected and potentially vast
expanded functional networks that emerge from this study have the potential to influence virtually any aspect of cell growth.”
The team—including researchers at the Hong Kong University of Science and Technology, Stanford University, and aTyr Pharma, a San Diego-based biotech company that Schimmel co-founded—comprehensively captured and sequenced the AARS mRNAs from six human tissue types using high-throughput deep sequencing. While many of the transcripts were expressed in each of the tissues, there was also some tissue specificity.
Next, the team showed that a proportion of these transcripts, including those missing the catalytic domain, indeed resulted in stable protein products: 48 of these splice variants associated with polysomes. In vitro translation assays and the expression of more than 100 of these variants in cells confirmed that many of these variants could be made into
stable protein products.
The AARS enzymes—of which there’s one for each of the 20 amino acids—bring together an amino acid with its appropriate transfer RNA (tRNA) molecule. This reaction allows a ribosome to add the amino acid to a growing peptide chain during protein translation. AARS
enzymes can be found in all living organisms and are thought to be among the first proteins to have originated on Earth.
To understand whether these non-catalytic proteins had unique biological activities, the researchers expressed and purified recombinant AARS fragments, testing them in cell-based assays for proliferation, cell differentiation, and transcriptional regulation, among other
phenotypes. “We screened through dozens of biological assays and found that these variants operate in many signaling pathways,” said Schimmel.
“This is an interesting finding and fits into the existing paradigm that, in many cases, a single gene is processed in various ways [in the cell] to have alternative functions,” said Steven Brenner, a computational genomics researcher at the University of California, Berkeley.
The team is now investigating the potentially unique roles of these protein splice variants in greater detail—in both human tissue as well as in model organisms. For example, it is not yet clear whether any of these variants directly bind tRNAs.
“I do think [these proteins] will play some biological roles,” said Tao Pan, who studies the functional roles of tRNAs at the University of Chicago. “I am very optimistic that interesting biological functions will come out of future studies on these variants.”
Brenner agreed. “There could be very different biological roles [for some of these proteins]. Biology is very creative that way, [it’s] able to generate highly diverse new functions using combinations of existing protein domains.” However, the low abundance of these variants
is likely to constrain their potential cellular functions, he noted.
Because AARSs are among the oldest proteins, these ancient enzymes were likely subject to plenty of change over time, said Karin Musier-Forsyth, who studies protein translational
at the Ohio State University. According to Musier-Forsyth, synthetases are already known to have non-translational functions and differential localizations. “Like the addition of post-translational modifications, splicing variation has evolved as another way to repurpose protein function,” she said.
One of the protein variants was able to stimulate skeletal muscle fiber formation ex vivo and upregulate genes involved in muscle cell differentiation and metabolism in primary human skeletal myoblasts. “This was really striking,” said Musier-Forsyth. “This suggests
that, perhaps, peptides derived from these splice variants could be used as protein-based therapeutics for a variety of diseases.”
Blood stem cells have the potential to turn into any type of blood cell, whether it is the oxygen-carrying red blood cells or the immune system’s many types of white blood cells that help fight infection. How exactly is the fate of these stem cells regulated? Preliminary findings from research conducted by scientists from the Weizmann Institute of Science and the Hebrew University are starting to reshape the conventional understanding of the way blood stem cell fate decisions are controlled, thanks to a new technique for epigenetic analysis developed at these institutions. Understanding epigenetic mechanisms (environmental influences other than genetics) of cell fate could lead to the deciphering of the molecular mechanisms of many diseases,
including immunological disorders, anemia, leukemia, and many more. The study of epigenetics also lends strong support to findings that environmental factors and lifestyle play a more prominent
role in shaping our destiny than previously realized.
The process of differentiation – in which a stem cell becomes a specialized mature cell – is controlled by a cascade of events in which specific genes are turned “on” and “off” in a highly regulated and accurate order. The instructions for this process are contained within the DNA itself in short regulatory sequences.
These regulatory regions are normally in a “closed” state, masked by special proteins called histones to ensure against unwarranted activation. Therefore, to access and “activate”
the instructions,
this DNA mask needs to be “opened” by epigenetic modifications of the histones so it can be read by the necessary machinery.
In a paper published in Science, Dr. Ido Amit and David Lara-Astiaso of the Weizmann Institute’s Department of Immunology, along with Prof. Nir Friedman and Assaf Weiner of the Hebrew University of Jerusalem, charted – for the first time – histone dynamics during blood development. Thanks to the new technique for epigenetic profiling they developed, in which just a handful of cells – as few as 500 – can be sampled and analyzed accurately, they have identified the exact
DNA sequences, as well as the various regulatory proteins, that are involved in regulating the process of blood stem cell fate.
This research has also yielded unexpected results: As many as
50% of these regulatory sequences are established and opened during intermediate stages of cell development.
The meaning of the research is that epigenetics can be active at stages in which it had been thought that cell destiny was already set. “This changes our whole understanding of the process of blood stem cell fate decisions,” says Lara-Astiaso, “suggesting that the process is more
dynamic and flexible than previously thought.”
Although this research was conducted on mouse blood stem cells, the scientists believe that the mechanism may hold true for other types of cells. “This research creates a lot of excitement in the field, as it sets the groundwork to study these regulatory elements in humans,” says Weiner.
Largest Cancer Genetic Analysis Reveals New Way of Classifying Cancer
Researchers with The Cancer Genome Atlas (TCGA) Research Network have completed the largest, most diverse tumor genetic analysis ever conducted, revealing a new approach to classifying cancers. The work, led by researchers at the UNC Lineberger Comprehensive
Cancer Center at the University of North Carolina at Chapel Hill and other TCGA sites, not only
revamps traditional ideas of how cancers are diagnosed and treated, but could also have
a profound impact on the future landscape of drug development.
“We found that one in 10 cancers analyzed in this study would be classified differently using this new approach,” said Chuck Perou, PhD, professor of genetics and pathology, UNC Lineberger member and senior author of the paper, which appears online Aug. 7 in Cell.
“That means that
10 percent of the patients might be better off getting a different therapy—that’s huge.”
Since 2006, much of the research has identified cancer as not a single disease, but many types and subtypes and has defined these disease types based on the tissue—breast, lung, colon, etc.—in which it originated. In this scenario, treatments were tailored to which
tissue was affected, but questions have always existed because some treatments work, and fail for others, even when a single tissue type is tested.
In their work, TCGA researchers analyzed more than 3,500 tumors across 12 different tissue types to see how they compared to one another — the largest data set of tumor genomics ever assembled, explained Katherine Hoadley, PhD, research assistant professor
in genetics and lead author. They found that
cancers are more likely to be genetically similar based on the type of cell in which the cancer originated, compared to the type of tissue in which it originated.
This is fundamental premise of pathology! (Larry Bernstein) It goes back to Rudolph Virchow.
“In some cases, the cells in the tissue from which the tumor originates are the same,” said Hoadley. “But in other cases, the tissue in which the cancer originates is made up of multiple types of cells that can each give rise to tumors. Understanding the cell in which the cancer originates appears to be very important in determining the subtype of a tumor
and, in turn, how that tumor behaves and how it should be treated.”
Perou and Hoadley explain that the new approach may also shift how cancer drugs are developed, focusing more on the development of drugs targeting larger groups of cancers with genomic similarities, as opposed to a single tumor type as they are currently developed.
One striking example of the genetic differences within a single tissue type is breast cancer.
The breast, a highly complex organ with multiple types of cells, gives rise to multiple types of breast cancer; luminal A, luminal B, HER2-enriched and basal-like, which was previously known. In this analysis, the basal-like breast cancers looked more like ovarian cancer
and cancers of a squamous-cell type origin, a type of cell that composes the lower-layer of a tissue, rather than other cancers that arise in the breast.
“This latest research further solidifies that basal-like breast cancer is an entirely unique disease and is completely distinct from other types of breast cancer,” said Perou. In addition, bladder cancers were also quite diverse and might represent at least three different disease types that also showed differences in patient survival.
As part of the Alliance for Clinical Trials in Oncology, a national network of researchers conducting clinical trials, UNC researchers are already testing the effectiveness of carboplatin—a common treatment for ovarian cancer—on top of standard of care chemotherapy for triple-negative breast cancer (TNBC) patients, of which 80 percent are the basal-like subtype. The results of this study (called CALGB40603)
were just published on Aug. 6 in the Journal of Clinical Oncology and showed a benefit of carboplatin in TNBC patients. This new clinical trial result suggests that there may be great value in comparing clinical results across tumor types for which this study highlights as having common genomic similarities.
As participants in TCGA, UNC Lineberger scientists have been involved in multiple individual tissue type studies including most recently an analysis of a comprehensive genomic profile of lung adenocarcinoma. Perou’s seminal work in 2000 led to the first discovery of breast
cancer as not one, but in fact, four distinct subtypes of disease. These most recent findings should continue to lay the groundwork for what could be the next generation of cancer diagnostics.
Source: University of North Carolina at Chapel Hill School of Medicine
New Gene Tied to Breast Cancer Risk
Wed, 08/06/2014
Marilynn Marchione – AP Chief Medical Writer – Associated Press
It’s long been known that faulty BRCA genes greatly raise the risk for breast cancer. Now, scientists say a more recently identified, less common gene can do the same.
Mutations in the gene can make breast cancer up to nine times more likely to develop, an international team of researchers reports in this week’s New England Journal of Medicine.
About 5 to 10 percent of breast cancers are thought to be due to bad BRCA1 or BRCA2 genes. Beyond those, many other genes are thought to play a role but how much each one raises risk has not been known, said Dr. Jeffrey Weitzel, a genetics expert at City of Hope Cancer Center
in Duarte, Calif.
The new study on the gene- called PALB2 – shows “this one is serious,” and probably is the most dangerous in terms of breast cancer after the BRCA genes, said Weitzel, one of leaders of the study.
It involved 362 members of 154 families with PALB2 mutations – the largest study of its kind. The faulty gene seems to give a woman a 14 percent chance of breast cancer by age 50 and 35 percent by age 70 and an even greater risk if she has two or more close relatives with the disease.
That’s nearly as high as the risk from a faulty BRCA2 gene, Dr. Michele Evans of the National Institute on Aging and Dr. Dan Longo of the medical journal staff write in a commentary in the journal.
The PALB2 gene works with BRCA2 as a tumor suppressor, so when it is mutated, cancer can flourish.
How common the mutations are isn’t well known, but it’s “probably more than we thought because people just weren’t testing for it,” Weitzel said. He found three cases among his own breast cancer
patients in the last month alone.
Among breast cancer patients, BRCA mutations are carried by 5 percent of whites and 12 percent of Eastern European (Ashkenazi) Jews. PALB2 mutations have been seen in up to 4 percent of families with a history of breast cancer.
Men with a faulty PALB2 gene also have a risk for breast cancer that is eight times greater than men in the general population.
Testing for PALB2 often is included in more comprehensive genetic testing, and the new study should give people with the mutation better information on their risk, Weitzel said. Doctors say that people with faulty cancer genes should be offered genetic counseling and may want to consider more frequent screening and prevention options, which can range from hormone-blocking pills to breast removal.
The actress Angelina Jolie had her healthy breasts removed last year after learning she had a defective BRCA1 gene.
The study was funded by many government and cancer groups around the world and was led by Dr. Marc Tischkowitz of the University of Cambridge in England. The authors include Mary-Clare King, the University of Washington scientist who discovered the first breast
cancer predisposition gene, BRCA1.
In the 1950s, the drug thalidomide, administered as a sedative to pregnant women, led to the birth of thousands of children with multiple defects. Despite the teratogenicity of thalidomide and its derivatives lenalidomide and pomalidomide,
these immunomodulatory drugs (IMiDs) recently emerged as effective treatments for
multiple myeloma and 5q-deletion-associated dysplasia.
IMiDs target the E3 ubiquitin ligase CUL4–RBX1–DDB1–CRBN (known as CRL4CRBN) and
promote the ubiquitination of the IKAROS family transcription factors IKZF1 and IKZF3 by CRL4CRBN.
Here we present crystal structures of the DDB1–CRBN complex bound to thalidomide,
lenalidomide and pomalidomide. The structure establishes that
CRBN is a substrate receptor within CRL4CRBN and enantioselectively binds IMiDs.
Using an unbiased screen, we identified the
homeobox transcription factor MEIS2 as an endogenous substrate of CRL4CRBN.
Our studies suggest that IMiDs block endogenous substrates (MEIS2) from binding to CRL4CRBN while the ligase complex is recruiting IKZF1 or IKZF3 for degradation.
This dual activity implies that
small molecules can modulate an E3 ubiquitin ligase and thereby upregulate or downregulate the ubiquitination of proteins.
Curator’s Viewpoint:
The short pieces may not appear to be so closely connected, except for the last subject on the pharmaceutical targeting of an E3 ubiquitin ligase ubiquitination of proteins, but even in that case, we have to keep in mind that protein formation by amino acid transcription, remodeling, and recapture of amino acids are in equilibrium through ubiquitylation. So I put it there. The DNA in populations ties some mutations to disease that is tied specifically to populations, not only the sephardic population, but in Asia as well.
The next article for consideration is methodological considerations. The BRCA2 in the sephardic population is one of a number of mutations we can identify, extending to Tay Sachs disease, for instance. How this might have occurred in the history of the jewish people is not so obvious, except perhaps in the segregation of the jewish population for centuries. The mutation would be confined within the population with limited marriage outside of the jewish community. It has been known for some time that there is a Cohen gene that traces back to the priests (Kohanim) of the Holy Temple, the descendents of Aaron (Aharon), the brother of Moses. The priests would stand at the Ark and bless the congregation in the most holy convocation of Yom Kippur, according to tradition. Marriages were arranged between the bride and the groom. Of course, arranged marriages were also the case in other ethnic communities, and between the privileged.
That was dramatically the case during the reign of Queen Victoria of England, with Royal arrangements across Europe.
That would be a factor in the transmission of hemophilia, and in mental disorders in the Royal families. Haemophilia figured prominently in the history of European royalty in the 19th and 20th centuries. Britain’s Queen Victoria, through two of her five daughters (Princess Alice and Princess Beatrice), passed the mutation to various royal houses across the continent, including the royal families of Spain, Germany and Russia. Victoria’s son Prince Leopold, Duke of Albany suffered from the disease. The Prince Leopold, Duke of AlbanyKGKTGCSIGCMGGCStJ (Leopold George Duncan Albert; 7 April 1853 – 28 March 1884) was the eighth child and fourth son of Queen Victoria and Prince Albert of Saxe-Coburg and Gotha. Leopold was later created Duke of Albany, Earl of Clarence, and Baron Arklow. He had haemophilia, which led to his death at the age of 30. The sex-linkedX chromosome disorder manifests almost entirely in males, although the gene for the disorder is located on the X chromosome and may be inherited from either mother or father. Expression of the disorder is much more common in males than in females. This is because, although the trait is recessive, males only inherit one X chromosome, from their mothers. Of course, this is classical Mendelian genetics. Victoria appears to have been a spontaneous or de novomutation and is usually considered the source of the disease in modern cases of haemophilia among royalty. The mutation would probably be assumed today to have occurred at the conception of Princess Alice, as she was the only known carrier among Victoria and Albert’s first seven children. Leopold was a sufferer of haemophilia and her daughters Alice and Beatrice were confirmed carriers of the gene.
Cousin marriage is marriage between people with a common grandparent or other more distant ancestor. In various cultures and legal jurisdictions, Marriages between first and second cousins account for over 10% of marriages worldwide, and they are common in the Middle East, where in some nations they account for over half of all marriages. Proportions of first-cousin marriage in the United States, Europe and other Western countries like Brazil have declined since the 19th century, though even during that period they were not more than 3.63 percent of all unions in Europe. Cousin marriage is allowed throughout the Middle East for all recorded history, and is used mostly in Syria. It has often been chosen to keep cultural values intact through many generations and preserve familial wealth. In Iraq the right of the cousin has also traditionally been followed and a girl breaking the rule without the consent of the ibn ‘amm could have ended up murdered by him. The Syrian city of Aleppo during the 19th century featured a rate of cousin marriage among the elite of 24% according to one estimate, a figure that masked widespread variation: some leading families had none or only one cousin marriage, while others had rates approaching 70%. Cousin marriage rates were highest among women, merchant families, and older well-established families. The percentage of Iranian cousin marriages increased from 34 to 44% between the 1940s and 1970s. Cousin marriage among native Middle Eastern Jews is generally far higher than among the European Ashkenazim, who assimilated European marital practices after the diaspora.
The essential elements of the marriage contract were now an offer by the man, an acceptance by the woman, and the performance of such conditions as the payment of dowry. According to anthropologist Ladislav Holý, cousin marriage is not an independent phenomenon but rather one expression of a wider Middle Eastern preference for agnatic solidarity, or solidarity with one’s father’s lineage.
A 2009 study found that many Arab countries display some of the highest rates of consanguineous marriages in the world, and that first cousin marriages which may reach 25-30% of all marriages. Research among Arabs and worldwide has indicated that consanguinity could have an effect on some reproductive health parameters such as postnatal mortality and rates of congenital malformations.
In the terraced streets of Bradford, Yorkshire, a child’s death is anything but rare. At the boy’s inquest, coroner Mark Hinchliffe said Hamza Rehman had died because his Pakistan-born parents (shopkeeper Abdul and housewife Rozina) are first cousins. Muslims have practiced marriages between first cousins in non-prohibited countries since the time of the Quran.
Four years before, Hamza’s older sister, three-month-old Khadeja, had died of the same brain disorder which causes fits, sickness and chest infections. The couple had another baby born with equally devastating neurological problems.
A heartbroken Mr Rehman told the inquest that he and his wife were unsure whether to have any more children. The coroner expressed deep sympathy before saying that Hamza’s death should serve as a warning to others.
I have diverged somewhat onto the genetic risks of consanguinous marriages, which George Darwin, son of Charles Darwin, argues were had a small effect in then English society. But most importantly, we see the larger factor here of social and familial inheritance, and also the concept of cultural identity.
Insofar as the somatic and mitochondrial mutations are concerned, I call attention to the finding in the GWAS study above discussed that the results were supportive of the conclusions from mtDNA. This gives some reason to consider whether sufficient information is obtained from the mtDNA, without the more robust GWAS. One cannot fully consider this without some knowledge of the methodology of specimen preparation.
It is not difficult to prepare mitochondria from cells and obtain a very good preparation before further analysis, whether of the membrane structures, the enzymatic activity, or of the DNA and RNA polynucleotides. The separation is easily achieved with differential centrifugation. On the other hand, the finding of the basal layer of epithelium having a different signature than the superficial layer, established by the genomic studies, but known histologically for non-neoplastic tissue, is a matter for cell separation methods that are not easy. It is from the lower layer of cells that we derive carcinoma in-situ. These cells were identified in breast, are expected to be found in uterus, and were like the cells in ovarian-cancer, which suggested the use of a common treatment regimen as adjunct in triple negative breast cancer and ovarian cancer. The importance of a suuficiently prepared cellular specimen as opposed to tissue specimen can’t be taken for granted.
This article is the THIRD in a four-article Series covering the topic of the Roles of the Mitochondria in Cardiovascular Diseases. They include the following;
Mitochondria and Cardiovascular Disease: A Tribute to Richard Bing, Larry H Bernstein, MD, FACP
Mitochondrial Metabolism in Impaired Cardiac Function
Mitochondrial Dysfunction and the Heart
Chronically elevated plasma free fatty acid levels in heart failure are associated with
decreased metabolic efficiency and cellular insulin resistance.
The mitochondrial theory of aging (MTA) and the free-radical theory of aging (FRTA) are closely related.
They were in fact proposed by the same researcher about 20 years apart. MTA adds
the mitochondria and its production of free radicals
into the concept that free-radicals damage DNA over time.
Tissue hypoxia, resulting from low cardiac output with or independent of endothelial impairment,
increases oxidative stress and leads to apoptosis and mitochondrial DNA damage.
This dysfunctional state causes loss of mitochondrial mass. Therapies aimed at protecting mitochondrial function
have shown promise in patients and animal models with heart failure that will be the subject of Chapter III.
Myocardial function in hypertension
Genetic variation in vitamin D-dependent signaling
is associated with congestive heart failure in human subjects with hypertension.
Functional polymorphisms were selected from five candidate genes:
CYP27B1,
CYP24A1,
VDR,
REN and
ACE.
Using the Marshfield Clinic Personalized Medicine Research Project,
205 subjects with hypertension and congestive heart failure,
206 subjects with hypertension alone and
206 controls (frequency matched by age and gender) were genotyped.
In the context of hypertension, a SNP in CYP27B1 was associated with congestive heart failure
(odds ratio: 2.14 for subjects homozygous for the C allele; 95% CI: 1.05–4.39).
Genetic variation in vitamin D biosynthesis is associated with increased risk of heart failure.
While initially unregulated, a gradual decline was observed over time (from day 45 to 90), in
hypoxic-inducible factor 1 and
stromal-derived factor 1 mRNA expression .
On serial assessment, endothelial progenitor cell migration was progressively impaired in response to chemo-attractant gradients of:
vascular endothelial growth factor (10-200 ng/mL)
and stromal cell-derived factor-1 (10-100 ng/mL) .
Decreased circulating levels and migratory dysfunction of bone marrow derived endothelial progenitor cells
were documented in a reproducible clinically relevant model of myocardial ischemia.
Nitric Oxide (NO) in Myocardial Ischemia and Infarct
Nitric oxide (NO) is a free radical with an unpaired electron; it is an important physiologic messenger,
produced by nitric oxide synthases, which catalyze the reaction l-arginine to citrulline and NO.
The constitutive isoforms exists in neuronal and endothelial cells and is calcium dependent. Calcium binds to calmodulin and
the calcium calmodulin complex activates the constitutive NO synthase that releases NO,
relaxing smooth muscle cells through activation of guanylate cyclase and the production cGMP.
Therefore, the NO produced has a negative inotropic effect on the heart and is instrumental in the autoregulation of the coronary circulation.
The inducible form of NO synthase (iNOS), mostly produced in macrophages, is activated by cytokines and endotoxin. It eliminates intracellular pathogens,
damaging cells by inhibiting
ATP production
oxidative phosphorylation
DNA synthesis.
In infection, lipopolysaccharide released from bacterial walls, stimulates production of iNOS. The large amount of NO produced
causes extensive vasodilation and hypotension.
We sought to assess whether oxidation products of
nitric oxide (NO), nitrite (NO2−) and nitrate (NO3−), referred to as NOx,
are released by the heart of patients after acute myocardial infarction (AMI) and
whether NOx can be determined in peripheral blood of these patients.
Previously we reported that in experimental myocardial infarction (rabbits) NOx is released mainly by inflammatory cells
(macrophages) in the myocardium 3 days after onset of ischemia.
NOx is formed in heart muscle from NO; It originates through the activity of the inducible form of nitric oxide synthase (iNOS).
Eight patients with acute anterior MI and an equal number of controls were studied. Coronary venous blood was obtained by
coronary sinus catheterization; NOx concentrations in coronary sinus, in arterial and peripheral venous plasma were measured.
Left ventricular end-diastolic pressure was determined. Measurements were carried out 24, 48 and 72 h after onset of symptoms.
The type and location of coronary arterial lesions were determined by coronary angiography. Plasma NO3− was reduced to NO2−
by nitrate reductase before determination of NO2− concentration by chemiluminescence.
The results provided evidence that in patients with acute anterior MI, the myocardial production of nitrite and nitrate (NOx) was increased,
as well as the coronary arterial–venous difference.
Increased NOx production by the infarcted heart accounted for the increase of NOx concentration in arterial and the peripheral venous plasma.
The peak elevation of NOx occurred on days 2 and 3 after onset of the symptoms, suggesting that NOx production was at least in part the result of
production of NO by inflammatory cells (macrophages) in the heart.
The appearance of oxidative products of NO (NO2− and NO3−) in peripheral blood of patients with acute MI is
the result of their increased release from infarcted heart during the inflammatory phase of myocardial ischemia.
Further studies are needed to define the clinical value of these observations.
EUK-8, a superoxide dismutase and catalase mimetic improved survival and contractile parameters in a mutant mouse model
of pressure overload-induced oxidative stress and heart failure and in wild-type mice subjected to pressure overload.
In addition, mitochondria show
functional impairment and
morphological disorganization
in the left ventricle of Hypertrophic Cardiomyopathy (HCM) patients without baseline systolic dysfunction.
These mitochondrial changes were associated with impaired myocardial contractile and relaxation reserves.
A strategy to protect the heart against oxidative stress could lie with
the modulation of mitochondrial electron transport itself.
Mild mitochondrial uncoupling may offer a potential cardioprotective effect by decreasing ROS production
preventing electron accumulation at complex III and
the Fe–S centres of complex I, and may therefore
mtDNA, Autophagy, and Heart Failure
Mitochondria are evolutionary endosymbionts derived from bacteria and contain DNA similar to bacterial DNA.
Mitochondria damaged by external haemodynamic stress are degraded by the autophagy/lysosome system in cardiomyocytes.
Mitochondrial DNA (mtDNA) that escapes from autophagy cell-autonomously leads to Toll-like receptor (TLR) 9-mediated
inflammatory responses in cardiomyocytes and
is capable of inducing myocarditis and dilated cardiomyopathy.
Cardiac-specific deletion of lysosomal deoxyribonuclease (DNase) II showed no cardiac phenotypes under baseline conditions,
but increased mortality and caused severe myocarditis and dilated cardiomyopathy 10 days after treatment with pressure overload.
Early in the pathogenesis, DNase II-deficient hearts showed
infiltration of inflammatory cells
increased messenger RNA expression of inflammatory cytokines
accumulation of mitochondrial DNA deposits in autolysosomes in the myocardium.
Administration of inhibitory oligodeoxynucleotides against TLR9, which is known to be activated by bacterial DNA6, or ablation of Tlr9
attenuated the development of cardiomyopathy in DNase II-deficient mice.
Furthermore, Tlr9 ablation
improved pressure overload-induced cardiac dysfunction and
inflammation even in mice with wild-type Dnase2a alleles.
These data provide new perspectives on the mechanism of genesis of chronic inflammation in failing hearts.
T Oka, S Hikoso, O Yamaguchi, M Taneike, T Takeda, T Tamai, et al. Mitochondrial DNA that escapes from autophagy causes inflammation and heart failure.
Clinical Trials Results for Endothelin System: Pathophysiological role in Chronic Heart Failure, Acute Coronary Syndromes and MI – Marker of Disease Severity or Genetic Determination? Aviva Lev-Ari, PhD, RN 10/19/2012
Inhibition of ET-1, ETA and ETA-ETB, Induction of NO production, stimulation of eNOS and Treatment Regime with PPAR-gamma agonists (TZD): cEPCs Endogenous Augmentation for Cardiovascular Risk Reduction – A Bibliography, Aviva Lev-Ari, PhD, RN 10/4/2012
Genomics & Genetics of Cardiovascular Disease Diagnoses: A Literature Survey of AHA’s Circulation Cardiovascular Genetics, 3/2010 – 3/2013, L H Bernstein, MD, FACP and Aviva Lev-Ari,PhD, RN 3/7/2013
Cardiovascular Disease (CVD) and the Role of agent alternatives in endothelial Nitric Oxide Synthase (eNOS) Activation and Nitric Oxide Production, Aviva Lev-Ari, PhD, RN 7/19/2012
Hypertriglyceridemia concurrent Hyperlipidemia: Vertical Density Gradient Ultracentrifugation a Better Test to Prevent Undertreatment of High-Risk Cardiac Patients, Aviva Lev-Ari, PhD, RN 4/4/2013
Fight against Atherosclerotic Cardiovascular Disease: A Biologics not a Small Molecule – Recombinant Human lecithin-cholesterol acyltransferase (rhLCAT) attracted AstraZeneca to acquire AlphaCore, Aviva Lev-Ari, PhD, RN 4/3/2013
High-Density Lipoprotein (HDL): An Independent Predictor of Endothelial Function & Atherosclerosis, A Modulator, An Agonist, A Biomarker for Cardiovascular Risk, Aviva Lev-Ari, PhD, RN 3/31/2013
Peroxisome proliferator-activated receptor (PPAR-gamma) Receptors Activation: PPARγ transrepression for Angiogenesis in Cardiovascular Disease and PPARγ transactivation for Treatment of Diabetes, Aviva Lev-Ari, PhD, RN 11/13/2012
This article is the FOURTH in a four-article Series covering the topic of the Roles of the Mitochondria in Cardiovascular Diseases. They include the following;
Mitochondria and Cardiovascular Disease: A Tribute to Richard Bing, Larry H Bernstein, MD, FACP
Alpha lipoic acid is known to be a mitochondrial antioxidant that preserves or improves mitochondrial function.
lipoic acid can prevent arterial calcification, and
arterial calcification may be related to mitochondrial dysfunction
methods are under study to increase lipoic acid synthase production, the enzyme responsible for making lipoic acid in the body.
Co-Enzyme Q10
It is well known that statin drugs taken for high cholesterol severely reduce CoQ10 levels, and causes other negative cardiovascular side effects.
A study on CAD patients has shown that over 8 weeks of supplementing with 300mg of CoQ10 reversed
mitochondrial dysfunction (as measured by a reduced lactate:pyruvate ratio) and
improved endothelial function (as measured by increased flow-mediated dilation)
Other Mitochondrial Antioxidants
Other natural compounds that have been shown to have antioxidant effects in the mitochondria include
resveratrol, found in wine and grapes,
curcumin from turmeric and
EGCG, found abundantly in green tea extract.
But no studies have been conducted for these compounds in CVD.
Metabolic syndrome and serum carotenoids: findings of a cross-sectional study
in Queensland, Australia
Metabolic syndrome and serum carotenoids.
T Coyne, TI Ibiebele, PD Baade, CS McClintock and JE Shaw.
Viertel Center for Research in Cancer Control, Cancer Council Queensland, and School of Public Health,
Queensland University of Technology and University of Queensland, Brisbane, Australia
Several components of the metabolic syndrome are known to be oxidative stress-related conditions
diabetes and
cardiovascular disease,
Carotenoids are compounds derived primarily from plants and several have been shown to be potent antioxidant nutrients.
Both diabetes and cardiovascular disease are known to be oxidative stress-related conditions such that
antioxidant nutrients may play a protective role in these conditions.
Several cross–sectional surveys have found lower levels of serum carotenoids among those with impaired glucose metabolism or type 2 diabetes.
Carotenoids are compounds derived primarily from plants, several of which are known to be potent antioxidants.
Epidemiological evidence indicates that some serum carotenoids may play a protective role against the development of chronic diseases such as
atherosclerosis,
stroke,
hypertension,
certain cancers,
inflammatory diseases and
diabetic retinopathy.
The primary carotenoids found in human serum are
α-carotene
β-carotene
β-cryptoxanthin
lutein/zeaxanthin
lycopene.
The aim of this study was to examine the associations between metabolic syndrome status and major serum carotenoids in adult Australians.
Data on the presence of the metabolic syndrome, based on International Diabetes Federation 2005 criteria, were collected from 1523 adults
aged 25 years and over in six randomly selected urban centers in Queensland, Australia, using a cross sectional study design.
The following were determined:
Weight
height
BMI
waist circumference
blood pressure
fasting and 2-34 hour blood glucose
lipids
five serum carotenoids.
Criteria used to assess the number of metabolic syndrome components present in a 171 participant using the
2005 International Diabetes Federation definition are as follows:
Components = 0 -none of the metabolic syndrome components (i.e. abdominal obesity, raised triglyceride,
reduced HDL-cholesterol, raised blood pressure, and impaired fasting plasma glucose) are present;
Components = any 1 one of the five metabolic syndrome components is present ;
Components = 2 – any two of the five components are present;
Components = 3 any three of the components are present;
Components = 4 – any four of the components are present;
Components = 5 = all five metabolic syndrome components are present.
This study investigated the relationships between these five primary serum carotenoids and the metabolic syndrome
in a cross-sectional population-based study in Queensland, Australia. Distributions of serum carotenoids were skewed
and therefore natural logarithmically transformed to better approximate the normal distribution for regression analyses.
Association between log transformed serum carotenoids as dependent variables and metabolic syndrome status were
assessed using multiple linear regression analysis. Results are reported as back transformed geometric means.
Analysis was performed for each serum carotenoid separately, and the sum of the five carotenoids,
adjusting for the following potential confounders:
age
sex
education
BMI
smoking
alcohol intake
physical activity
vitamin use.
Mean serum alpha-carotene, beta-carotene and the sum of the five carotenoid concentrations were significantly lower (p<0.05)
in persons with the metabolic syndrome (after adjusting for age,sex, education, BMI status, alcohol intake, smoking, physical activity
status and vitamin/mineral use) than persons without the syndrome. Alpha, beta and total carotenoids also decreased significantly
(p<0.05) with increased number of components of the metabolic syndrome, after adjusting for these confounders. These differences
were significant among former smokers and non-smokers, but not in current smokers. Low concentrations of serum
alpha-carotene,
beta carotene and
the sum of five carotenoids
appear to be associated with metabolic syndrome status.
The overall prevalence of the syndrome was 24% and was significantly higher among males than females. As would be expected, significant
differences in prevalence of the syndrome were seen with
body mass index
waist circumference
systolic and diastolic blood pressure
blood lipids.
Significant differences were also evident by
age group, smoking status, educational status and income.
Income was marginally inversely associated. The prevalence increased with age, and was lower in those with post graduate education.
No significant differences were seen by alcohol intake, physical activity levels, vitamin usage, or fruit intake. There was actually an
inverse relationship between vegetable intake (not fruit) and serum carotenoids.
Those who consumed 4 serves or more of vegetable were less likely to have the metabolic syndrome
compared to those who consumed 1 serve or less of vegetables.
The mean concentrations of serum alpha-carotene, beta-carotene and the sum of the five carotenoids were significantly lower for participants
with the metabolic syndrome present compared with those without the syndrome, after adjusting for potential confounding variables.
Concentrations of alpha-carotene, beta-carotene and the sum of the five carotenoids decreased significantlyas
the number of components of the metabolic syndrome increased after adjusting for potential confounding variables.
Similarly there was an inverse association between quartiles of
individual and total serum carotenoids and metabolic syndrome status and each of its components.
This study was designed to investigate the association between several serum carotenoids and the metabolic syndrome.
The data from the present population study suggest that several serum carotenoids are inversely related to the metabolic syndrome.
The study showed significantly lower concentrations present among those with the metabolic syndrome of
α-carotene,
β-carotene and
the sum of the five carotenoids
compared to those without.We also found decreasing concentrations of all the carotenoids tested as
the number of the metabolic syndrome components increased.
This was significant for
α-carotene,
β-carotene,
β-cryptoxanthin
total carotenoids.
(not lycopenes)
These findings are consistent with data reported from the third National Health and Nutrition Examination Survey (NHANES III).
In the NHANES III study, significantly lower concentrations of all the carotenoids, except lycopene, were found among persons
with the metabolic syndrome compared with those without, after adjusting for confounding factors similar to those in our study.
Carnitine: A novel health factor-An overview.
CD Dayanand, N Krishnamurthy, S Ashakiran, KN Shashidhar
Int J Pharm Biomed Res 2011; 2(2): 79-89. ISSN No: 0976-0350
Carnitine comprises L-carnitine, acetyl –L-carnitine and Propionyl –L-carnitine. Carnitine is
obtained in greater amount from animal dietary sources than from plant sources.
The endogenous synthesis of carnitine takes place in animal tissues like
liver
kidney
brain
using precursor amino acids lysine and methionine by a pathway
dependent on iron, vitamin C, niacin, pyridoxine .
This is the basis of vegans generally depending on carnitine in larger proportion
through in vivo synthesis than omnivorous subjects.
The concentration of tri-methyl lysine residues and the tissue specificity of butyro-betaine dehydrogenase
plays a significant role in regulating the carnitine biosynthesis.
Carnitine transport from the site of synthesis to target tissue occurs via blood.
The measurement of plasma carnitine concentration represents –
the balance between the rate of synthesis and rate of excretion
through specific transporter proteins.
The cellular functional role of carnitine depends on the uptake into cells through
carnitine transport proteins and
transport into mitochondrial matrix.
The function of carnitine is to traverse Long-chain Fatty Acids across inner mitochondrial membrane
for β-oxidation, thereby, generating ATP.
Carnitine deficiency results in muscle disorders. The deficiency states are primary and secondar.
The primary is of systemic or myopathic, characterized by a defect of high affinity organic cation transporter protein (CTP)
present on the plasma membrane of liver and kidney and
also due to dysfunction of carnitine reabsorbtion through
similar transport proteins in renal tubules.
Secondary carnitine deficiency is associated with
mitochondrial disorders and also
defective β-oxidation such as CPT-II and acyl CoA dehydrogenase.
In recent times, carnitine has been extensively studied in various research activities to explore the therapeutic benefit.
Thus, carnitine justifies as a novel health factor.
PLC improved the insulin-resistant state and reversed the increased total cholesterol
but not the increase in free fatty acid, triglyceride and HDL/LDL ratio induced by high-fat diet.
Vehicle-HF exhibited a reduced
cardiac output/body weight ratio,
endothelial dysfunction and
tissue decrease of NO production,
all of them being improved by PLC treatment.
The decrease of hepatic mitochondrial activity by high-fat diet was reversed by PLC.
Oral administration of PLC improves the insulin-resistant state developed by obese animals and
decreases the cardiovascular risk associated with the metabolically impaired mitochondrial function.
Omega-3 Fatty Acid and cardioprotection
The Benefits of Flaxseed
By Elaine Magee, MPH, RD WebMD Expert Column
Some call it one of the most powerful plant foods on the planet. There’s some evidence it may help reduce your risk of
heart disease, cancer, stroke, and diabetes.
That’s quite a tall order for a tiny seed that’s been around for centuries.
Flaxseed was cultivated in Babylon as early as 3000 BC. In the 8th century, King Charlemagne believed so strongly in the
health benefits of flaxseed that he passed laws requiring his subjects to consume it. Now, thirteen centuries later, some
experts say we have preliminary research to back up what Charlemagne suspected.
Fibroblast growth factor 21 (FGF21) is a distinctive member of the FGF family with potent beneficial effects on
lipid
body weight
glucose metabolism
A monoclonal antibody, mimAb1, binds to βKlotho with high affinity and specifically
activates signaling from the βKlotho/FGFR1c (FGF receptor 1c) receptor complex.
Injection of mimAb1 into obese cynomolgus monkeys led to FGF21-like metabolic effects:
decreases in body weight,
plasma insulin,
triglycerides, and
glucose during tolerance testing.
Mice with adipose-selective FGFR1 knockout were refractory to FGF21-induced improvements
in glucose metabolism and body weight.
mimAb1 depends on βKlotho to activate FGFR1c, but
it is not expected to induce side effects caused by activating FGFR1c alone.
The results in obese monkeys (with mimAb1) and in FGFR1 knockout mice (with FGF21) demonstrated
the essential role of FGFR1c in FGF21 function and
suggest fat as a critical target tissue for the cytokine and antibody.
This antibody activates FGF21-like signaling through cell surface receptors, and provided
preclinical validation for an innovative therapeutic approach to diabetes and obesity.
Influencing Factors on Cardiac Structure and Function Beyond Glycemic Control
in Patients With Type 2 Diabetes Mellitus (T2DM)
R Ichikawa, M Daimon, T Miyazaki, T Kawata, et al. Cardiovasc Diabetol. 2013;12(38)
We studied 148 asymptomatic patients with T2DM without overt heart disease.
Early (E) and late (A) diastolic mitral flow velocity and early diastolic mitral annular velocity (e’)
were measured for assessing left ventricular (LV) diastolic function.
In addition
insulin resistance,
non-esterified fatty acid,
high-sensitive CRP,
estimated glomerular filtration rate,
waist/hip ratio,
abdominal visceral adipose tissue (VAT),
subcutaneous adipose tissue (SAT)
In T2DM (compared to controls),
E/A and e’ were significantly lower, and
E/e’, left atrial volume and LV mass were significantly greater
VAT and age were independent determinants of
left atrial volume (β =0.203, p=0.011),
E/A (β =−0.208, p=0.002), e’ (β =−0.354, p<0.001) and
E/e’ (β=0.220, p=0.003).
Independent determinants of LV mass were
systolic blood pressure,
waist-hip ratio (β=0.173, p=0.024)
VAT/SAT ratio (β=0.162, p=0.049)
Excessive visceral fat accompanied by adipocyte dysfunction may play a greater role than
glycemic control in the development of diastolic dysfunction and LV hypertrophy in T2DM
Proposed mechanisms through which overexpression of the
mitochondrial transcription factor A (TFAM) gene prevents
mitochondrial DNA (mtDNA) damage,
oxidative stress, and
myocardial remodelling and failure.
In wild-type mice, mitochondrial transcription factor A
directly interacts with mitochondrial DNA to form nucleoids.
Stress such as ischaemia causes mitochondrial DNA damage, which
increases the production of reactive oxygen species (ROS)
leading to a catastrophic cycle of mitochondrial electron transport impairment,
further reactive oxygen species generation, and mitochondrial dysfunction.
TFAM overexpression may protect mitochondrial DNA from damage by
directly binding and stabilizing mitochondrial DNA and
increasing the steady-state levels of mitochondrial DNA
ameliorating mitochondrial dysfunction and thus the development and progression of heart failure.
Conclusion
Heart failure is a multifactorial syndrome that is characterized by
abnormal energetics and substrate metabolism in heart and skeletal muscle.
Although existing therapies have been beneficial, there is a clear need for new approaches to treatment.
Pharmacological targeting of the cellular stresses underlying mitochondrial dysfunction, such as
elevated fatty acid levels,
tissue hypoxia and oxidative stress and
metabolic modulation of heart and skeletal muscle mitochondria,
appears to offer a promising therapeutic strategy for tackling heart failure.
Murray AJ, Anderson RE, Watson GC, et al. Uncoupling proteins in human heart. Lancet 2004; 364:1786.
Delarue J, Magnan C. Free fatty acids and insulin resistance. Curr Opin ClinNutr Metab Care 2007; 10:142
Lee L, Campbell R, Scheuermann-Freestone M, et al. Metabolic modulation with perhexiline in chronic heart failure: a randomized, controlled trialof short-term use of a novel treatment. Circulation 2005; 112:3280
Tsutsui H, Kinugawa S, Matsushima S. Mitochondrial oxidative stress and dysfunction in myocardial remodelling. Cardiovasc Res. 2009;81(3):449-56. http://dxdoi.org/10.1093/cvr/cvn280.
Writer, Author, ResponderClinical Pathologist, Biochemist, and Transfusion Physician _____________________________________________________________________________________________________________________________________________
Heterogeneity The heterogeneity is a problem that will take at least another decade to unravel because of the number of signaling pathways and the crosstalk that is specifically at issue. I must refer back to the work of Frank Dixon, Herschel Sidransky, and others, who did much to develop a concept of neoplasia occurring in several stages – minimal deviation and fast growing. These have differences in growth rates, anaplasia, and biochemical. This resembles the multiple “hit” theory that is described in “systemic inflammatory” disease leading to a final stage, as in sepsis and septic shock.
Tumor heterogeneity is problematic because of differences among the metabolic variety among types of gastrointestinal (GI) cancers, confounding treatment response and prognosis. A group of investigators from Sunnybrook Health Sciences Centre, University of Toronto, Ontario, Canada who evaluated the feasibility and safety of magnetic resonance (MR) imaging–controlled transurethral ultrasound therapy for prostate cancer in humans. Their study’s objective was to prove that using real-time MRI guidance of HIFU treatment is possible and it guarantees that the location of ablated tissue indeed corresponds to the locations planned for treatment. The real-time MRI guidance is an improvement in imaging technology.
The ability to allow resection with removal of the tumor, and adjacent tissue at risk is unproved, and is related to the length of remission.
See comment written for :
Knowing the tumor’s size and location, could we target treatment to THE ROI by applying…..
The Response vs. Recurrence Free Interval Conundrum
There is a difference between expected response to esophageal or gastric neoplasms both biologically and in expected response, even given variability within a class. The expected time to recurrence is usually longer in the latter case, but the confounders are –
There is a long latent period in abdominal cancers before discovery, unless a lesion is found incidentally in surgery for another reason. The undeniable reality is that it is not difficult to identify the main lesion, but it is difficult to identify adjacent epithelium that is at risk (transitional or pretransitional). Pathologists have a very good idea about precancerous cervical neoplasia.
The heterogeneity rests within each tumor and between the primary and metastatic sites, which is expected to be improved by targeted therapy directed by tumor-specific testing. Despite rapid advances in our understanding of targeted therapy for GI cancers, the impact on cancer survival has been marginal. Brücher BLDM, Bilchik A, Nissan A, Avital I & Stojadinovic A. Can tumor response to therapy be predicted, thereby improving the selection of patients for cancer treatment? Future Oncology 2012; 8(8): 903-906 , DOI 10.2217/fon.12.78 (doi:10.2217/fon.12.78) The heterogeneity is a problem that will take at least another decade to unravel because of the number of signaling pathways and the crosstalk that is specifically at issue.
Anaerobic Glycolysis and Respiratory Impairment In 1920, Otto Warburg received the Nobel Prize for his work on respiration. He postulated that cancer cells become anaerobic compared with their normal counterpart that uses aerobic respiration to meet most energy needs. He attributed this to “mitochondrial dysfunction. In fact, we now think that in response to oxidative stress, the mitochondrion relies on the Lynen Cycle to make more cells and the major source of energy becomes glycolytic, which is at the expense of the lean body mass (muscle), which produces gluconeogenic precursors from muscle proteolysis (cancer cachexia).
There is a loss of about 26 ATP ~Ps in the transition. The mitochondrial gene expression system includes the mitochondrial genome, mitochondrial ribosomes, and the transcription and translation machinery needed to regulate and conduct gene expression as well as mtDNA replication and repair. Machinery involved in energetics includes the enzymes of the Kreb’s citric acid or TCA (tricarboxylic acid) cycle, some of the enzymes involved in fatty acid catabolism (β-oxidation), and the proteins needed to help regulate these systems. The inner membrane is central to mitochondrial physiology and, as such, contains multiple protein systems of interest. These include the protein complexes involved in the electron transport component of oxidative phosphorylation and proteins involved in substrate and ion transport. ________________________________________________________________________________________________________________________________________________________________________________ Mitochondrial Roles in Cellular Homeostasis Mitochondrial roles in, and effects on, cellular homeostasis extend far beyond the production of ATP, but the transformation of energy is central to most mitochondrial functions. Reducing equivalents are also used for anabolic reactions. The energy produced by mitochondria is most commonly thought of to come from the pyruvate that results from glycolysis, but it is important to keep in mind that the chemical energy contained in both fats and amino acids can also be converted into NADH and FADH2 through mitochondrial pathways.
The major mechanism for harvesting energy from fats is β-oxidation; the major mechanism for harvesting energy from amino acids and pyruvate is the TCA cycle. Once the chemical energy has been transformed into NADH and FADH2 (also discovered by Warburg and the basis for a second Nobel nomination in 1934), these compounds are fed into the mitochondrial respiratory chain. The hydroxyl free radical is extremely reactive. It will react with most, if not all, compounds found in the living cell (including DNA, proteins, lipids and a host of small molecules).
The hydroxyl free radical is so aggressive that it will react within 5 (or so) molecular diameters from its site of production. The damage caused by it, therefore, is very site specific. The reactions of the hydroxyl free radical can be classified as hydrogen abstraction, electron transfer, and addition. The formation of the hydroxyl free radical can be disastrous for living organisms. Unlike superoxide and hydrogen peroxide, which are mainly controlled enzymatically, the hydroxyl free radical is far too reactive to be restricted in such a way – it will even attack antioxidant enzymes. Instead, biological defenses have evolved that reduce the chance that the hydroxyl free radical will be produced and, as nothing is perfect, to repair damage. ________________________________________________________________________________________________________________________________________________________________________________ Oxidative Stress and Mitochondrial Impairment Currently, some endogenous markers are being proposed as useful measures of total “oxidative stress” e.g., 8-hydroxy-2’deoxyguanosine in urine. The ideal scavenger must be non-toxic, have limited or no biological activity, readily reach the site of hydroxyl free radical production (i.e., pass through barriers such as the blood-brain barrier), react rapidly with the free radical, be specific for this radical, and neither the scavenger nor its product(s) should undergo further metabolism. Nitric oxide has a single unpaired electron in its π*2p antibonding orbital and is therefore paramagnetic. This unpaired electron also weakens the overall bonding seen in diatomic nitrogen molecules so that the nitrogen and oxygen atoms are joined by only 2.5 bonds. The structure of nitric oxide is a resonance hybrid of two forms. In living organisms nitric oxide is produced enzymatically. Microbes can generate nitric oxide by the reduction of nitrite or oxidation of ammonia.
In mammals nitric oxide is produced by stepwise oxidation of L-arginine catalyzed by nitric oxide synthase (NOS). Nitric oxide is formed from the guanidino nitrogen of the L-arginine in a reaction that consumes five electrons and requires flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN) tetrahydrobiopterin (BH4), and iron protoporphyrin IX as cofactors. The primary product of NOS activity may be the nitroxyl anion that is then converted to nitric oxide by electron acceptors. The thiol-disulfide redox couple is very important to oxidative metabolism. GSH is a reducing cofactor for glutathione peroxidase, an antioxidant enzyme responsible for the destruction of hydrogen peroxide.
Thiols and disulfides can readily undergo exchange reactions, forming mixed disulfides. Thiol-disulfide exchange is biologically very important. For example, GSH can react with protein cystine groups and influence the correct folding of proteins, and it GSH may play a direct role in cellular signaling through thiol-disulfide exchange reactions with membrane bound receptor proteins (e.g., the insulin receptor complex), transcription factors (e.g., nuclear factor κB), and regulatory proteins in cells. Conditions that alter the redox status of the cell can have important consequences on cellular function. So the complexity of life is not yet unravelled.
Cells seem to be well-adjusted to glycolysis. While Otto Warburg first proposed that cancer cells show increased levels of glucose consumption and lactate fermentation even in the presence of ample oxygen (known as “Warburg Effect”), which requires oxidative phosphorylation to switch to glycolysis promoting the proliferation of cancer cells., many studies have demonstrated glycolysis as the main metabolic pathway in cancer cells. It is now accepted that glycolysis provides cancer cells with the most abundant extracellular nutrient, glucose, to make ample ATP metabolic intermediates, such as ribose sugars, glycerol and citrate, nonessential amino acids, and the oxidative pentose phosphate pathway, which serve as building blocks for cancer cells.
Dampened Mitochondrial Respiration
Since, cancer cells have increased rates of aerobic glycolysis, investigators argue over the function of mitochondria in cancer cells. Mitochondrion, a one of the smaller organelles, produces most of the energy in the form of ATP to supply the body. In Warburg’s theory, the function of cellular mitochondrial respiration is dampened and mitochondria are not fully functional. There are many studies backing this theory. A recent review on hypoxia nicely summarizes some current studies and speculates that the “Warburg Effect” provides a benefit to the tumor not by increasing glycolysis but by decreasing mitochondrial activity.
Glycolysis
Glycolysis is enhanced and beneficial to cancer cells. The mammalian target of rapamycin (mTOR) has been well discussed in its role to promote glycolysis; recent literature has revealed some new mechanisms of how glycolysis is promoted during skin cancer development.
On the other hand, Akt is not only involved in the regulation of mitochondrial metabolism in skin cancer but also of glycolysis. Activation of Akt has been found to phosphorylate FoxO3a, a downstream transcription factor of Akt, which promotes glycolysis by inhibiting apoptosis in melanoma. In addition, activated Akt is also associated with stabilized c-Myc and activation of mTOR, which both increase glycolysis for cancer cells.
Nevertheless, ras mutational activation prevails in skin cancer. Oncogenic ras induces glycolysis. In human squamous cell carcinoma, the c-Jun NH(2)-terminal Kinase (JNK) is activated as a mediator of ras signaling, and is essential for ras-induced glycolysis, since pharmacological inhibitors if JNK suppress glycolysis. CD147/basigin, a member of the immunoglobulin superfamily, is high expressed in melanoma and other cancers.
Glyoxalase I (GLO1) is a ubiquitous cellular defense enzyme involved in the detoxification of methylglyoxal, a cytotoxic byproduct of glycolysis. In human melanoma tissue, GLO1 is upregulated at both the mRNA and protein levels.
Knockdown of GLO1 sensitizes A375 and G361 human metastatic melanoma cells to apoptosis.
The transcription factor HIF-1 upregulates a number of genes in low oxygen conditions including glycolytic enzymes, which promotes ATP synthesis in an oxygen independent manner. Studies have demonstrated that hypoxia induces HIF-1 overexpression and its transcriptional activity increases in parallel with the progression of many tumor types. A recent study demonstrated that in malignant melanoma cells, HIF-1 is upregulated, leading to elevated expression of Pyruvate Dehydrogenase Kinase 1 (PDK1), and downregulated mitochondrial oxygen consumption.
The M2 isoform of Pyruvate Kinase (PKM2), which is required for catalyzing the final step of aerobic glycolysis, is highly expressed in cancer cells; whereas the M1 isoform (PKM1) is expressed in normal cells. Studies using the skin cell promotion model (JB6 cells) demonstrated that PKM2 is activated whereas PKM1 is inactivated upon tumor promoter treatment. Acute increases in ROS inhibited PKM2 through oxidation of Cys358 in human lung cancer cells. The levels of ROS and stage of tumor development may be pivotal for the role of PKM2.
Warburg effect is both, a cause and effect of cancer…Review article mentioned in link below explains how different factors can contribute to metabolic reprogramming and Warburg effect….The Supply-based model and Traditional model clearly explains how the cancer cells will progress during different availability of growth factors and nutrients…And recent studies including my project (under process of getting published) will also suggest that growth factors can drive cancer cells to undergo Warburg effect regardless of the presence of oxygen…
Otto Warburg proposed that “EVEN IN THE PRESENCE OF OXYGEN, cancer cells can reprogram their glucose metabolism, and thus their energy production, by limiting their energy metabolism largely to glycolysis” . http://www.ncbi.nlm.nih.gov/pubmed
The autophagic tumor stroma model of cancer metabolism.
Cancer cells induce oxidative stress in adjacent cancer-associated fibroblasts (CAFs). This activates reactive oxygen species (ROS) production and autophagy. ROS production in CAFs, via the bystander eff ect, serves to induce random mutagenesis in epithelial cancer cells, leading to double-strand DNA breaks and aneuploidy. Cancer cells mount an anti-oxidant defense and upregulate molecules that protect them against ROS and autophagy, preventing them from undergoing apoptosis. So, stromal fibroblasts conveniently feed and mutagenize cancer cells, while protecting them against death. See the text for more details. A+, autophagy positive; A-, autophagy negative; AR, autophagy resistant.
1. Recycled Nutrients
2. Random Mutagenesis
3. Protection Against Apoptosis
The reverse Warburg effect.
Via oxidative stress, cancer cells activate two major transcription factors in adjacent stromal fibroblasts (hypoxia-inducible factor (HIF)1α and NFκB).
This leads to the onset of both autophagy and mitophagy, as well as aerobic glycolysis, which then produces recycled nutrients (such as lactate, ketones, and glutamine).
These high-energy chemical building blocks can then be transferred and used as fuel in the tricarboxylic acid cycle (TCA) in adjacent cancer cells.
The outcome is high ATP production in cancer cells, and protection against cell death. ROS, reactive oxygen species.
The choline dependent methylation of PP2A is the brake, the “antidote”, which limits “the poison” resulting from an excess of insulin signaling. Moreover, it seems that choline deficiency is involved in the L to M2 transition of PK isoenzymes. The negative regulation of Ras/MAP kinase signals mediated by PP2A phosphatase seems to be complex.
The serine-threonine phosphatase does more than simply counteracting kinases; it binds to the intermediate Shc protein on the signaling cascade, which is inhibited. The targeting of PP2A towards proteins of the signaling pathway depends of the assembly of the different holoenzymes.
The relative decrease of methylated PP2A in the cytosol, not only cancels the brake over the signaling kinases, but also favors the inactivation of PK and PDH, which remain phosphorylated, contributing to the metabolic anomaly of tumor cells. In order to prevent tumors, one should then favor the methylation route rather than the phosphorylation route for choline metabolism.
the produced peroxide via free radicals over activate the cyclooxigenase and consequently the PI3K pathway, thereby activating the most important protein-kinase. This brakes the Warburg effect, and stops the PI3K activation.
Then all the cancer protein related with the generation of tumor (pAKT,pP70S6K, Cyclin D1, HIF1, VEGF, EGFrc, GSK, Myc, etc, etc, etc) get down regulated. That is what happens when one knocks down the new protein-kinase in pancreatic cancer cell lines. These pancreatic cancer cell lines divide very-very-very slowly.
I now transition from what is understood about the metabolic signatures of cancer that tend to behave more alike than the cell of origin, but not initially. This is perhaps a key to therapeutics. >>>
Can tumor response to therapy be predicted, thereby improving the selection of patients for cancer treatment? The goal is not just complete response. Histopathological response seems to be related post-treatment histopathological assessment but it is not free from the challenge of accurately determining treatment response, as this method cannot delineate whether or not there are residual cancer cells. Functional imaging to assess metabolic response by 18-fluorodeoxyglucose PET also has its limits, as the results are impacted significantly by several variables:
tumor type
sizing
doubling time
anaplasia?
extent of tumor necrosis
presence of tumor at the margin of biopsy
lymph node and/or distant metastasis
vascular involvement
type of antitumor therapy and the time when response was determined.
The new modality should be based on individualized histopathology as well as tumor molecular, genetic and functional characteristics, and individual patients’ characteristics, a greater challenge in an era of ‘minimally invasive treatment’. This has been pointed out by Brücher et al. if the International Consortium on Cancer with respect to the shortcoming of MIS as follows: Minimally Invasive Surgery (MIS) vs. conventional surgery dissection applied to cancer tissue with the known pathophysiology of recurrence and remission cycles has its short term advantages.
in many cases MIS is not the right surgical decision
predicting the uncertain future behavior of the tumor with respect to its response to therapeutics bears uncertain outcomes.
An increase in the desirable outcomes of MIS as a modality of treatment, will be assisted in the future, when anticipated progress is made in the field of
Cancer Research,
Translational Medicine and
Personalized Medicine,
when each of the cancer types, above, will already have a Genetic Marker allowing the Clinical Team to use the marker(s) for:
prediction of Patient’s reaction to Drug induction
design of Clinical Trials to validate drug efficacy on small subset of patients predicted to react favorable to drug regimen, increasing validity and reliability
Genetical identification of patients at no need to have a drug administered if non sensitivity to the drug has been predicted by the genetic marker.
See listing of cancers provided by Dr. Aviva Lev-Ari.
That is an optimistic order to effectively carry out in the face of the statistical/mathematical challenge imposed for any real success.
Brücher BLDM, Bilchik A, Nissan A, Avital I & Stojadinovic A. Can tumor response to therapy be predicted, thereby improving the selection of patients for cancer treatment? Future Oncology 2012; 8(8): 903-906 , DOI 10.2217/fon.12.78 (doi:10.2217/fon.12.78) _________________________________________________________________________________________________________________________________________________________________________________ A Model Based on Kullback Entropy and Identifying and Classifying Anomalies This listing suggests that for every cancer the following data has to be collected (except doubling time). If there are 8 variables, the classification based on these alone would calculate to be very sizable based on Eugene Rypka’s feature extraction and classification. But looking forward,
The critical question encountered by the pathologist is that key histological stains have been used for some time, such as Her2, and a number of others to establish tumor cell type, and differences with cell types. The number will grow as the genomic identifiers are explored and put to use. It doesn’t appear that the pathologist will be displaced any time soon. This is separate from older observations of nuclear polymorphism, anaplastic changes related to cell adhesion, etc. These do not displace the information gained from staging criteria. Clearly, there is much information that is used for individual decisions about therapeutic approach, which will undergo further refinement even before the end of this decade.
_________________________________________________________________________________________________________________________________________________________________________________ Melanoma Example A marker for increased glycolysis in melanoma is the elevated levels of Lactate Dehydrogenase (LDH) in the blood of patients with melanoma, which has proven to be an accurate predictor of prognosis and response to treatments. LDH converts pyruvate, the final product of glycolysis, to lactate when oxygen is absent. High concentrations of lactate, in turn, negatively regulate LDH. Therefore, targeting acid excretion may provide a feasible and effective therapeutic approach for melanoma. For instance, JugloSne, a main active component in walnut, has been used in traditional medicines.
Studies have shown that Juglone causes cell membrane damage and increased LDH levels in a concentration-dependent manner in cultured melanoma cells. As one of the rate-limiting enzyme of glycolysis, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isozyme 3 (PFKFB3) is activated in neoplastic cells. Studies have confirmed that an inhibitor of PFKFB3, 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), suppresses glycolysis in neoplastic cells. In melanoma cell lines, the concentrations of Fru-2, 6-BP, lactate, ATP, NAD+, and NADH are diminished by 3PO. Therefore, targeting PFKFB3 using 3PO and other PFKFB3 specific inhibitors could be effective in melanoma chemotherapy.
This is only one example of the encouraging results from targeted therapy. An unexplored idea was provided to me that is interesting and be highly conditional, by loading with high concentrations of ketones to offset the glycolytic pathway redirected bypass of mitochondrial pathways. There is an inherent problem with muscle proteolysis raising the glucose level from gluconeogenesis. The effect is uncertain with respect to TCA cycle intermediates. It seems plausible that cure is not necessarily attainable due to inability to identify portions of proximate local tumor, modification and drug resistance. The reliable extension of disease free survival and maintaining a patient acceptable quality of life is improvable. __________________________________________________________________________________________________________________________________________________________________________________
Quiescent versus Proliferating Cells: Both Use Mitochondria, but to Different Ends
Altered Metabolism Is a Direct Response to Growth-Factor Signaling
PI3K/Akt/mTORC1 Activation: Driving Anabolic Metabolism and Tumorigenesis by Reprogramming Mitochondria
Bhowmick NA. Metastatic Ability: Adapting to a Tissue Site Unseen. Cancer Cell 2012; 22(5): 563-564. _____________________________________________________________________________________________________________________________________________________________________________ Therapeutic strategies that target glycolysis and biosynthetic pathways in cancer cells are currently the main focus of research in the field of cancer metabolism. In this issue of Cancer Cell, Hitosugi and colleagues show that targeting PGAM1 could be a way of “killing two birds with one stone”. Chaneton B, Gottlieb E. PGAMgnam Style: A Glycolytic Switch Controls Biosynthesis. Cancer Cell 2012; 22(5): 565-566. ______________________________________________________________________________________________________________________________________________________________________________ The Polycomb epigenetic silencing protein EZH2 is affected by gain-of-function somatic mutations in B cell lymphomas. Two recent reports describe the development of highly selective EZH2 inhibitors and reveal mutant EZH2 as playing an essential role in maintaining lymphoma proliferation. EZH2 inhibitors are thus a promising new targeted therapy for lymphoma. Melnick A. Epigenetic Therapy Leaps Ahead with Specific Targeting of EZH2. Cancer Cell 22(5): 569-570. _______________________________________________________________________________________________________________________________________________________________________________ The microenvironment of the primary as well as the metastatic tumor sites can determine the ability for a disseminated tumor to progress. In this issue of Cancer Cell, Calon and colleagues find that systemic TGF-β can facilitate colon cancer metastatic engraftment and expansion. Calon A, Espinet E, Palomo-Ponce S, Tauriello DVF, et al. Dependency of Colorectal Cancer on a TGF-β-Driven Program in Stromal Cells for Metastasis Initiation. Cancer Cell 2012;22(5): 571-584.
_______________________________________________________________________________________________________________________________________________________________________________ An analysis of what is possible, but who knows how far into the accelerating future? Tumor response criteria: are they appropriate? The International Consortium is centered at the Billroth Institute, in Munich. Interesting it is that Billroth was the father of abdominal surgery and performed the first esophagectomy and the firat gastrectomy. He also pioneered in keeping a record of treatments and outcomes in the 19th century, which Halsted studied. I need not repeat what has been stated in the post. The pathologist’s role is still important, as the editorial in Future Oncology gets at. This also requires necessary and sufficient features to extract differentiating classifiers. I don’t think we shall see pathologists the likes of many who were masters until the 1990′s. The surgical pathologist today cannot have complete command of the large knowledge base, but the tumor registry and the cancer committee has evolved to a better stage than in the 1960′s. Surgical grand rounds have been used for teaching and evaluating the practice since at least the 1960′s. What is asked is that we go beyond that.
See comment written for:
Knowing the tumor’s size and location, could we target treatment to THE ROI by applying…..
________________________________________________________________________________________________________________________________________________________________________________ Evidence-based medicine Evidence-based medicine is substantially flawed because of reliance on meta-analysis to arrive at conclusions from underpowered and inconsistent studies, discarding more than half of the studies examined that don’t meet the inclusion criteria.
– There can be no movement forward without the systematic collection of data into a functionally well designed repository.
– The current construct of the EMR probably has to be “remodeled” if not “remade”.
– The studies will have to use real data, not aggregates of studies with “missing information”.
– Bioinformatics is an emerging field that is only supported in the top two tiers of academic medical centers, which would include the well known cancer centers in Boston, Houston, and New York.
I don’t place much hope in “Watson” coming to the rescue, because you have to collect both a lot of information and “sufficient” information.
-”Sufficient” information has been precluded by years of cost-elimination without paying attention to the real impact of “technologies” on costs, and an inherent competition between labor and “capital” investment.
– Despite the progress in genomics, the heterogeneity of these solid tumors is a natural adaptation that occurs in carcinogenesis.
The heterogeneity traced over a time-span should have information about stage in carcinogenesis.
The pathologist can see and interpret histologic grades in the evolution that may have a better relationship to the evolutionary studies of genomics and signaling pathways than to stage of disease, but by combining the best available evidence, you move to a better classification. Without good classification, I don’t see how you can arrive at “science based” personalized medicine.
–there is still a Rubicon to cross in going from genomics to translational medicine, which extends to diet and lifestyle.
Mitochondria, double membranous and semi-autonomous organelles, are known to convert energy into forms that are usable to the cell. Apart from being sites of cellular respiration, multiple roles of mitochondria have been emphasized in processes such as cell division, growth and cell death. Mitochondria are semi-autonomous in that they are only partially dependent on the cell to replicate and grow. They have their own DNA, ribosomes, and can make their own proteins. Mitochondria have been discussed in several posts published in the Pharmaceutical Intelligence blog.
Mitochondria do not exist as lone organelles, but are part of a dynamic network that continuously undergoes fusion and fission in response to various metabolic and environmental stimuli. Nucleoids, the assemblies of mitochondrial DNA (mtDNA) with its associated proteins, are distributed during fission in such a way that each mitochondrion contains at least one nucleoid. Mitochondrial fusion and fission within a cell is speculated to be involved in several functions including mtDNA DNA protection, alteration of cellular energetics, and regulation of cell division.
Proteins involved in mitochondrial fission & fusion
Multiple mitochondrial membrane GTPases that regulate mitochondrial networking have recently been identified. They are classified as fission and fusion proteins:
Fusion proteins: Members of dynamin family of protein, mitofusin 1 (Mfn-1) and mitofusin 2 (Mfn-2), are involved in fusion between mitochondria by tethering adjacent mitochondria. These proteins have two transmembrane segments that anchor them in the mitochondrial outer membrane. Mutations in Mitofusin proteins gives rise to fragmented mitochondria, but this can be reversed by mutations in mammalian Drp1. Mitochondrial inner membranes are fused by dynamin family members called Opa1.
Fission proteins: Another member of the dynamin family of proteins, dynamin-related protein 1 (Drp-1) mediates fission of mitochondria. Drp-1 is activated by phosphorylation. Drp-1 proteins are largely cytosolic, but cycle on and off of mitochondria as needed for fission. Fission is a complex process and involves a series of well-defined stages and proteins. Cytosolic Drp-1 is activated by calcineurin or other cytosolic signaling proteins after which it translocates to the mitochondrial tubules where it assembles into foci through its interaction with another protein, hFis1. Once Drp-1 rings assemble on the constricted spots, outer membrane of mitochondria undergoes fission through GTP hydrolysis. Drp-1 is now left bound to one of the newly formed mitochondrial ends after which it slowly disassembles before returning to the cytoplasm.
Control of mitochondrial fission & fusion
Mitochondrial fission and fusion are controlled by several regulatory mechanisms. Few of which are mentioned as follows:
Drp-1 activation by Cdk1/Cyclin B mediated phosphorylation during mitosis – triggers fission
Drp-1 inactivation by cAMP-dependent protein kinase (PKA) in quiescent cells- prevents fission
Drp-1 activation after reversal of PKA phosphorylation by Calcineurin- triggers fission
Ubiquination of fission and fusion proteins by E3 ubiquitin ligase- alters fission
Sumoylation of fission proteins – regulates fission
Imparied mitochondrial fission leads to loss of mtDNA
Mitochondrial fission plays an important role in mitochondrial and cellular homeostasis. It was reported by Parone et al (2008) that preventing mitochondrial fission by down-regulating expression of Drp-1 lead to loss of mtDNA and mitochondrial dysfunction. An increase in cellular reactive oxygen species (ROS) was observed. Other cellular implications included depletion of cellular ATP, inhibition of cell proliferation and autophagy. The observations were made in HeLa cells.
MicroRNA regulation of mitochondrial fission
Although several factors have been attributed to the regulation of mitochondrial fission, the mechanism still remains poorly understood. Recently, regulation of mitochondrial fission via miRNAs has become a topic of interest. Following miRNAs have been found to be involved in mitochondrial fission:
miR-484: Wang et al (2012) demonstrated that miR-484 was able to regulate mitochondrial fission by suppressing the translation of a fission protein Fis1, leading to inhibition of Fis1-mediated fission and apoptosis in cardiomyocytes and in the adrenocortical cancer cells. The authors showed that Fis1 is necessary for mitochondrial fission and apoptosis, and is upregulated during anoxia, whereas miR-484 is downregulated. Underlying mechanism involved transactivation of miR-484 by a transcription factor, Foxo3a and miR-484 is able to attenuate Fis1 upregulation and mitochondrial fission, by binding to the amino acid coding sequence of Fis1 and inhibiting its translation.
miR-499: miR-499 was reported by Wang et al (2011) to be able to directly target both the α- and β-isoforms of the calcineurin catalytic subunit. Suppression of calcineurin-mediated dephosphorylation of Drp-1 lead to inhibition of the fission machinery ultimately resulting in the inhibition of cardiomyocyte apoptosis. miR-499 levels, by altering mitochondrial fusion were able affect the severity of myocardial infarction and cardiac dysfunction induced by ischemia-reperfusion. Modulation of miR-499 expression could provide a therapeutic approach for myocardial infarction treatment.
miR-30: It was reported by Li et al (2010) that miR-30 family members were able to inhibit mitochondrial fission and also the resulting apoptosis. While exploring the underlying molecular mechanism, the authors identified that miR-30 family members can suppress p53 expression. When cell received apoptotic stimulation, p53 was found to transcriptionally activate the fission protein, Drp-1. Drp-1 was able to induce mitochondrial fission. miR-30 family members were observed to inhibit mitochondrial fission through attenuation of p53 expression and its downstream target Drp-1.
Mitochondrial fission & fusion as a therapeutic target
Since alteration of mitochondrial fission and fusion have been reported to affect various cellular processes including apoptosis, proliferation, ATP consumption, the proteins involved in the process of fission and fusion might be harnessed as therapeutic target.
Mentioned below is a description of research where dynamics of the mitochondrial organelle has been utilized as a therapeutic target:
Inhibition of mitochondrial fission prevents cell cycle progression in lung cancer
A recent article published by Rehman et al (2012) in the FASEB journal drew much attention after interesting observations were made in the mitochondria of lung adenocarcinoma cells. The mitochondrial network of these cells exhibited both impaired fusion and enhanced fission. It was also found that the fragmented phenotype in multiple lung adenocarcinoma cell lines was associated with both a down-regulation of the fusion protein, Mfn-2 and an upregulation of expression of fission protein, Drp-1. The imbalance of Drp-1/Mfn-2 expression in human lung cancer cell lines was reported to promote a state of mitochondrial fission. Similar increase in Drp-1 and decrease in Mfn-2 was observed in the tissue samples from patients compared to adjacent healthy lung. Authors used complementary approaches of Mfn-2 overexpression, Drp-1 inhibition, or Drp-1 knockdown and were able to observe reduction of cancer cell proliferation and an increase spontaneous apoptosis. Thus, the study identified mitochondrial fission and Drp-1 activation as a novel therapeutic target in lung cancer.
Mitochondrial Damage and Repair under Oxidative Stress
Curator: Larry H Bernstein, MD, FCAP
Keywords: Mitochondria, mitochondrial dysfunction, electron transport chain, mtDNA, oxidative stress, oxidation-reduction, NO, DNA repair, lipid peroxidation, thiols, ROS, RNS, sulfur,base excision repair, ferredoxin. Summary: The mitochondrion is the energy source for aerobic activity of the cell, but it also has regulatory functions that will be discussed. The mitochondrion has been discussed in other posts at this site. It has origins from organisms that emerged from an anaerobic environment, such as the bogs and marshes, and may be related to the chloroplast. The aerobic cell was an advance in evolutionary development, but despite the energetic advantage of using oxygen, the associated toxicity of oxygen abundance required adaptive changes. Most bacteria that reduce nitrate (producing nitrite, nitrous oxide or nitrogen) are called facultative anaerobes use electron acceptors such as ferric ions, sulfate or carbon dioxide which become reduced to ferrous ions, hydrogen sulfide and methane, respectively, during the oxidation of NADH (reduced nicotinamide adenine dinucleotide is a major electron carrier in the oxidation of fuel molecules).
The underlying problem we are left with is oxidation-reduction reactions that are necessary for catabolic and synthetic reactions, and that cumulatively damage the organism associated with cancer, cardiovascular disease, neurodegerative disease, and inflammatory overload. Aerobic organisms tolerate have evolved mechanisms to repair or remove damaged molecules or to prevent or deactivate the formationof toxic species that lead to oxidative stress and disease. However, the normal balance between production of pro-oxidant species and destruction by the antioxidant defenses is upset in favor of overproduction of the toxic species, which leads to oxidative stress and disease. How this all comes together is the topic of choice.
The transformation of energy is central to mitochondrial function. The system of energetics includes:
the enzymes of the Kreb’s citric acid or TCA cycle,
some of the enzymes involved in fatty acid catabolism (β-oxidation), and
the proteins needed to help regulate these systems,
central to mitochondrial physiology through the production of reducing equivalents. Reducing equivalents are also used for anabolic reactions. Electron Transport Chain
It also houses the protein complexes involved in the electron transport component of oxidative phosphorylation and proteins involved in substrate and ion transport. The chemical energy contained in both fats and amino acids can also be converted into NADH and FADH2 through mitochondrial pathways. The major mechanism for harvesting energy from fats is β-oxidation; the major mechanism for harvesting energy from amino acids and pyruvate is the TCA cycle. Once the chemical energy has been transformed into NADH and FADH2, these compounds are fed into the mitochondrial respiratory chain.
Under physiological conditions, electrons generally enter either through complex I (NADH-mediated, examined in vitro using substrates such as glutamate/malate) or complex II (FADH2-mediated, examined in vitro using succinate).
Electrons are then sequentially passed through a series of electron carriers.
The progressive transfer of electrons (and resultant proton pumping) converts the chemical energy stored in carbohydrates, lipids, and amino acids into potential energy in the form of the proton gradient. The potential energy stored in this gradient is used to phosphorylate ADP forming ATP. Redox-Cycling
In redox cycling the reductant is continuously regenerated, thereby providing substrate for the “auto-oxidation” reaction.
When partially oxidized compounds are enzymatically reduced, the auto-oxidative generation of superoxide and other ROS to start again. Several enzymes
NADPH-cytochrome P450 reductase,
NADPH-cytochrome b5 reductase [EC 1.6.2.2]
NADPH-ubiquinone oxidoreductase [EC 1.6.5.3], and
xanthine oxidase [EC 1.2.3.2]),
can reduce quinones into semiquinones in a single electron process.
The semiquinone can then reduce dioxygen to superoxide during its oxidation to a quinone.
Redox cycling is thought to play a role in carcinogenesis. The naturally occurring estrogen metabolites (the catecholestrogens) have been implicated in hormone-induced cancer, possibly as a result of their redox cycling and production of ROS. It is thought that diethylstilbestrol causes the production of the mutagenic lesion 8-hydroxy-2’deoxyguanosine. It can also cause DNA strand breakage.
Another oxidative reaction that is associated with H2O2 is a significant problem for living organisms as a consequence of the reaction between hydrogen peroxide and oxidizable metals, the Fenton reaction [originally described in the oxidation of an α-hydroxy acid to an α-keto acid in the presence of hydrogen peroxide (or hypochlorite) and low levels of iron salts (Fenton (1876, 1894)). Chemical Reactions and Biological Significance
The hydroxyl free radical is so aggressive that it will react within 5 (or so) molecular diameters from its site of production. The damage caused by it, therefore, is very site specific. Biological defenses have evolved that reduce the chance that the hydroxyl free radical will be produced to repair damage. An antioxidant would have to occur at the site of hydroxyl free radical production and be at sufficient concentration to be effective.
Some endogenous markers have been proposed as a useful measures of total “oxidative stress” e.g., 8-hydroxy-2’deoxyguanosine in urine. The ideal scavenger
must be non-toxic,
have limited or no biological activity,
readily reach the site of hydroxyl free radical production,
react rapidly with the free radical, be specific for this radical, and
neither the scavenger nor its product(s) should undergo further metabolism.
Unlike oxygen, nitrogen does not possess unpaired electrons and is therefore considered diamagnetic. Nitrogen does not possess available d orbitals so it is limited to a valency of 3. In the presence of oxygen, nitrogen can produce Nitric oxide which occurs physiologically with the immune system which, when activated, can produce large quantities of nitric oxide.
Nitric oxide is produced by stepwise oxidation of L-arginine catalyzed by nitric oxide synthase (NOS). Nitric oxide is formed from the guanidino nitrogen of the L-arginine in a reaction that
consumes five electrons and
requires flavin adenine dinucleotide (FAD),
flavin mononucleotide (FMN) tetrahydrobiopterin (BH4), and
iron protoporphyrin IX as cofactors.
The primary product of NOS activity may be the nitroxyl anion that is then converted to nitric oxide by electron acceptors.
NOS cDNAs show homology with the cytochrome P450 reductase family. Based on molecular genetics there appears to be at least three distinct forms of NOS:
A Ca2+/calmodulin-requiring constitutive enzyme (c-NOS; ncNOS or type I)
A calcium-independent inducible enzyme (i-NOS; type II), which is primarily involved in the mediation of the cellular immune response; and
A second Ca2+/calmodulin-requiring constitutive enzyme found in aortic and umbilical endothelia (ec-NOS or type III)
This has been discussed extensively in this series of posts. Recently, a mitochondrial form of the enzyme, which appears to be similar to the endothelial form, has been found in brain and liver tissue. Although the exact role of nitric oxide in the mitochondrion remains elusive, it may play a role in the regulation of cytochrome oxidase. Nitric Oxide
Nitric oxide appears to regulate its own production through a negative feedback loop. The binding of nitric oxide to the heme prosthetic group of NOS inhibits this enzyme, and c-NOS and ec-NOS are much more sensitive to this regulation than i-NOS. It appears that in the brain, NO can regulate its own synthesis and therefore the neurotransmission process.
On the one hand, inhibition of ec-NOS will prevent the cytotoxicity associated with excessive nitric oxide production.
On the other, the insensitivity of i-NOS to nitric oxide will enable high levels of nitric oxide to be produced for cytotoxic effects.
Endogenous inhibitors of NOS (guanidino-substituted derivatives of arginine) occur in vivo as a result of post-translational modification of protein contained arginine residues by S-adenosylmethionine. The dimethylarginines (NG,NG-dimethyl-L-arginine and NG,N’G-dimethyl-L-arginine) occurs in tissue proteins, plasma, and urine of humans and they are thought to act as both regulators of NOS activity and reservoirs of arginine for the synthesis of nitric oxide.
It has been calculated that even though membrane makes up about 3% of the total tissue volume, 90% of the reaction of nitric oxide with oxygen occurs within this compartment. Thus the membrane is an important site for nitric oxide chemistry.
There are two major aspects to nitric oxide chemistry.
It can undergo single electron oxidation and reduction reactions producing nitrosonium and nitroxyl
Having a single unpaired electron in its π*2p molecular orbital it will react readily with other molecules that also have unpaired electrons, such as free radicals and transition metals.
Examples of the reaction of nitric oxide with radical species include:
Nitric oxide will react with oxygen to form the peroxynitrite (nitrosyldioxyl) radical (ONO2)
and with superoxide to form the powerful oxidizing and nitrating agent, peroxynitrite anion (ONO2-). Peroxynitrite causes damage to many important biomolecules
Importance:
nitrosothiols that are important in the regulation of blood pressure terminates lipid peroxidation
3-nitrosotyrosine and/or 4-O-nitrosotyrosine can affect the activity of enzymes that utilize tyrosyl radicals
rapidly reacts with oxyhemoglobin, the primary route of its destruction in vivo
the reaction between nitric oxide and transition metal complexes
During the last reaction a “ligand” bond is formed (the unpaired electron of nitric oxide is partially transferred to the metal cation),
resulting in a nitrosated (nitrosylated) complex.
For example, such complexes can be formed with free iron ions,
iron bound to heme or iron located in iron-sulfur clusters.
Ligand formation allows nitric oxide to act as a signal, activating some enzymes while inhibiting others. Thus, the binding of nitric oxide to the Fe (II)-heme of guanylate (guanalyl) cyclase [GTP-pyrophosphate lyase: cyclizing] is the signal transduction mechanism. Guanylate cyclase exists as cytosolic and membrane-bound isozymes. Thiol-Didulfide Redox Couple
The thiol-disulfide redox couple is very important to oxidative metabolism. For example, GSH is a reducing cofactor for glutathione peroxidase, an antioxidant enzyme responsible for the destruction of hydrogen peroxide.
The importance of the antioxidant role of the thiol-disulfide redox couple:
Thiols and disulfides can readily undergo exchange reactions, forming mixed disulfides. Thiol-disulfide exchange is biologically very important. For example,
GSH can react with protein cystine groups and influence the correct folding of proteins.
GSH may also play a direct role in cellular signaling through thiol-disulfide exchange reactions with membrane bound receptor proteins
the insulin receptor complex)
transcription factors (e.g., nuclear factor κB)
and regulatory proteins in cells
Conditions that alter the redox status of the cell can have important consequences on cellular function.
The generation of ROS by redox cycling is only one possible explanation for the action of many drugs. Rifamycin not only owes its activity to ROS generation but also to its ability to block bacterial RNA synthesis as well. Quinones (and/or semiquinones) can also form adducts with nucleophiles, especially thiols. These adducts may act as toxins directly or indirectly through the inhibition of key enzymes (e.g., by reacting with essential cysteinyl residues) or the depletion of GSH. DNA Adduct Formation
By far the most intense research in this field has been directed towards the chemistry and biology of DNA adduct formation. Attack of the free bases and nucleosides by pro-oxidants can yield a wide variety of adducts and DNA-protein cross-links. Such attack usually occurs
at the C-4 and C-8 position of purines and
C-5 and C-6 of pyrimidines.
Hydroxyl free radical-induced damage to purine bases and nucleosides can proceed through a C-8-hydroxy N-7 radical intermediate, and then either undergo oxidation with the production of an 8-hydroxy purine, or reduction, probably by cellular thiols, followed by ring opening and the formation of FAPy (formamido-pyrimidine) metabolites (hydroxyl free radical-induced damage to guanosine). Although most research has focused on 8-hydroxy-purine adducts a growing number of publications are attempting to measure the FAPy derivative.
Nitrosation of the Amines of the Nucleic Acid Bases.
Primary aromatic amines produce deaminated products, while secondary amines form N-nitroso compounds. Formation of Peroxynitrite from Nitric Oxide.
Peroxynitrite shows complex reactivity
with DNA initiating DNA strand breakage, oxidation (e.g., formation of 8-hydroxyguanine, 8-OH2’dG, (5-hydroxymethyl)-uracil, and FAPyGua),
nitration (e.g., 8-nitroguanine), and
deamination of bases.
Peroxynitrite can also promote the production of lipid peroxidation related active carbonyls and cause the activation of NAD+ ADP-ribosyltransferase.
Modification of Guanine
Although all DNA bases can be oxidatively damaged, it is the modification of guanine that is the most frequent. 8OH2’dG is the most abundant DNA adduct. This can affect its hydrogen bonding between base-pairs. These base-pair substitutions are usually found clustered into areas called “hot spots”. Guanine normally binds to cytosine.
8OH2’dG, however, can form hydrogen bonds with adenine. The formation of 8OH2’dG in DNA can therefore result in a G→T transversion.
8-Hydroxyguanine was also shown to induce codon 12 activation of c-Ha-ras and K-ras in mammalian systems. G→T transversions are also the most frequent hot spot mutations found in the p53 supressor gene which is associated with human tumors.
Other mechanisms by which ROS/RNS can lead to mutations have been
proposed. Direct mechanisms include:
conformational changes in the DNA template that reduces the accuracy of replication by DNA polymerases
altered methylation of cytosine that affects gene control
Indirect mechanisms include:
Oxidative damage to proteins, including DNA polymerases and repair enzymes.
Damage to lipids causes the production of mutagenic carbonyl compounds
Misalignment mutagenesis (“slippery DNA”)
DNA Mismatch Repair 5 (Photo credit: Allen Gathman)
Repair of ROS/RNS-induced DNA Damage
The repair of damaged DNA is an ongoing and continuous process involving a
number of repair enzymes. Damaged DNA appears to be mended by two major mechanisms:
base excision repair (BER) and
nucleotide excision repair (NER)
Isolated DNA is found to contain low levels of damaged bases, so it appears that these repair processes are not completely effective. Base Excision Repair
BER is first started by DNA glycosylases which recognize specific base
modifications (e.g., 8OH2’dG). For example,
Formamido-pyrimidine-DNA glycosylase (Fpg protein) recognizes damaged purines such as 8-oxoguanine and FAPyGua.
Damaged pyrimidines are recognized by endonuclease III, which acts as both a glycosylase and AP endonuclease.
Glycosylases cleave the N-glycosylic bond between the damaged base and the sugar
Following the glycosylase step, AP endonucleases then remove the 3′-deoxyribose moiety by cleavage of the phosphodiester bonds thereby generating a 3’-hydroxyl group that can then be extended by DNA polymerase.
The final step in mending damaged DNA is the rejoining of the free ends of DNA by a DNA ligase. It also appears that the presence of 8-oxoguanine modified bases in DNA is not only a result of ROS attack on this macromolecule. Oxidized nucleosides and nucleotides from free cellular pools can also be incorporated into DNA by polymerases and cause AT to CG base substitution mutations.
Mitochondrial DNA Repair
The mitochondrion genome encodes the various complexes of the electron transport chain, but contains no genetic information for DNA repair enzymes. These enzymes must be obtained from the nucleus. As mitochondria are continuously producing DNA damaging pro-oxidant species, effective DNA repair mechanisms must exist within the mitochondrial matrix in order for these organelles to function. Mitochondria have a short existence, and excessively damaged mitochondria will be quickly removed. Mitochondria contain many BER enzymes and are proficient at repair, but they do not appear to repair damaged DNA by NER mechanisms.
Single Strand DNA Damage and PARP Activation
Single strand DNA breakage activates NAD+ ADP-ribosyltransferase (PARP). PARP is a protein-modifying, nucleotide-polymerizing enzyme and is found at high levels in the nucleus. Activated PARP
cleaves NAD+ into ADP-ribose and nicotinamide
then attaches the ADP-ribose units to a variety of nuclear proteins (including histones and its own automodification domain).
then polymerizes the initial ADP-ribose modification with other ADP-ribose units to form the nucleic acid-like polymer, poly (ADP) ribose.
PARP only appears to be involved with BER and not NER. In BER PARP does not appear to play a direct role but rather it probably helps by keeping the chromatin in a conformation that enables other repair enzymes to be effective. It may also provide temporary protection to DNA molecules while it is being repaired. Conflicting evidence suggests that PARP may not be an important DNA repair enzyme as cells from a PARP knockout mouse model have normal repair characteristics.
Activation of PARP can be dangerous to the cell. For each mole of ADP-ribose transferred, one mole of NAD+ is consumed, and through the regeneration of NAD+ four ATP molecules are wasted. Thus the activation of PARP can rapidly deplete a cell’s energy store and even lead to cell death. Some researchers suggest that this may be one mechanism whereby cells with excessive DNA damage are effectively removed. However, a variety of diseases may involve PARP overactivation including
circulatory shock,
CNS injury,
diabetes,
drug-induced cytotoxicity, and
inflammation.
The Indirect Pathway.
This (mutation) pathway does not involve oxidative damage to the protein per se. This process involves oxidative damage to the DNA molecule encoding the protein. Thus pro-oxidants can cause changes in the base sequence of the DNA molecule. If such base modification is in a coding region of DNA (exon) and not corrected, the DNA molecule may be transcribed incorrectly. Translation of the mutant mRNA can result in a mutant protein containing a wrong amino acid in its primary sequence. If this modified amino acid occurs in an essential part of the protein (e.g., the active site of an enzyme or a portion that alters folding), the function of that protein may be impaired. Fortunately, unlike modified DNA
that can pass from cell to cell during mitosis thereby continuing the production of mutant protein, damage to a protein is non-replicating and stops with its destruction.
The Direct Pathway
This (post-translational) pathway involves the action of a pro-oxidant on a protein resulting in
modification of amino acid residues,
the formation of carbonyl adducts,
cross-linking and
polypeptide chain fragmentation.
Such changes often result in altered protein conformation and/or activity. Proteins will produce a variety of carbonyl products when exposed to metal-based systems (metal/ascorbate and metal/hydrogen peroxide) in vitro. For example, histidine yields aspartate, asparagine and 2-oxoimidazoline, while proline produces glutamate, pyroglutamate, 4-hydroxyproline isomers, 2-pyrrolidone and γ-aminobutyric acid. Metal-based systems and other pro-oxidant conditions can oxidize methionine to its sulfoxide.
This portion of the presentation is endebted to THE HANDBOOK OF REDOX
BIOCHEMISTRY, Ian N. Acworth, August 2003, esa. (inacworth@esainc.com).
We shall now identify more recent work related to this presentation.
Oxygen and Oxidative Stress
The reduction of oxygen to water proceeds via one electron at a time. In the mitochondrial respiratory chain, Complex IV (cytochrome oxidase) retains all partially reduced intermediates until full reduction is achieved. Other redox centres in the electron transport chain, however, may leak electrons to oxygen, partially reducing this molecule to superoxide anion (O2_•). Even though O2_• is not a strong oxidant, it is a precursor of most other reactive oxygen species, and it also becomes involved in the propagation of oxidative chain reactions. Despite the presence of various antioxidant defences, the mitochondrion appears to be the main intracellular source of these oxidants. This review describes the main mitochondrial sources of reactive species and the antioxidant defences that evolved to prevent oxidative damage in all the mitochondrial compartments.
Reactive oxygen species (ROS) is a phrase used to describe a variety of molecules and free radicals (chemical species with one unpaired electron) derived from molecular oxygen. Molecular oxygen in the ground state is a bi-radical, containing two unpaired electrons in the outer shell (also known as a triplet state).
Since the two single electrons have the same spin, oxygen can only react with one electron at a time and therefore it is not very reactive with the two electrons in a chemical bond.
On the other hand, if one of the two unpaired electrons is excited and changes its spin, the resulting species (known as singlet oxygen) becomes a powerful oxidant as the two electrons with opposing spins can quickly react with other pairs of electrons, especially double bonds.
The formation of OH• is catalysed by reduced transition metals, which in turn may be re-reduced by O2 -•, propagating this process. In addition, O2-• may react with other radicals including nitric oxide (NO•) in a reaction controlled by the rate of diffusion of both radicals. The product, peroxynitrite, is also a very powerful oxidant. The oxidants derived from NO• have been recently called reactive nitrogen species (RNS).
‘Oxidative stress’ is an expression used to describe various deleterious processes resulting from an imbalance between the excessive formation of ROS and/or RNS and limited antioxidant defences.
Whilst small fluctuations in the steady-state concentration of these oxidants may actually play a role in intracellular signalling,
uncontrolled increases in the steady-state concentrations of these oxidants lead to free radical mediated chain reactions
which indiscriminately target
proteins,
lipids,
polysaccharides.
In vivo, O2-• is produced both enzymatically and nonenzymatically.
Enzymatic sources include
NADPH oxidases located on the cell membrane of
polymorphonuclear cells,
macrophages and
endothelial cells and
cytochrome P450-dependent oxygenases.
The proteolytic conversion of xanthine dehydrogenase to xanthine oxidase provides another enzymatic source of both O2 -• and H2O2 (and therefore constitutes a source of OH•) and has been proposed to mediate deleterious processes in vivo.
Given the highly reducing intramitochondrial environment, various respiratory components, including flavoproteins, iron–sulfur clusters and ubisemiquinone, are thermodynamically capable of transferring one electron to oxygen. Moreover, most steps in the respiratory chain involve single-electron reactions, further favouring the monovalent reduction of oxygen. On the other hand, the mitochondrion possesses various antioxidant defences designed to eliminate both O2- • and H2O2.
The rate of O2 -• formation by the respiratory chain is controlled primarily by mass action, increasing both when electron flow slows down (increasing the concentration of electron donors, R•) and when the concentration of oxygen increases (eqn (1); Turrens et al. 1982).
d[O2]/dt = k [O2] [R•].
The energy released as electrons flow through the respiratory chain is converted into a H+ gradient through the inner mitochondrial membrane (Mitchell, 1977). This gradient, in turn, dissipates through the ATP synthase complex (Complex V) and is responsible for the turning of a rotor-like protein complex required for ATP synthesis. In the absence of ADP,
the movement of H+ through ATP synthase ceases and
the H+ gradient builds up
causing electron flow to slow down and
the respiratory chain to become more reduced (State IV respiration).
Mitochondrial Antioxidant Defences
The deleterious effects resulting from the formation of ROS in the mitochondrion are, to a large extent, prevented by various antioxidant systems. Superoxide is enzymatically converted to H2O2 by a family of metalloenzymes called superoxide dismutases (SOD). Since O2-• may either reduce transition metals, which in turn can react with H2O2 producing OH• or spontaneously react with NO• to produce peroxynitrite, it is important to maintain the steady-state concentration of O2-• at the lowest possible level. Thus, although the dismutation of O2-• to H2O2 and O2 can also occur spontaneously, the role of SODs is to increase the rate of the reaction to that of a diffusion-controlled process.
The mitochondrial matrix contains a specific form of SOD, with manganese in the active site, which eliminates the O2 -• formed in the matrix or on the inner side of the inner membrane. The expression of MnSOD is further induced by agents that cause oxidative stress, including radiation and hyperoxia, in a process mediated by the oxidative activation of the nuclear transcription factor NFkB .
Reactive oxygen species (ROS) are products of normal metabolism and xenobiotic exposure, and depending on their concentration, ROS can be beneficial or harmful to cells and tissues.
At physiological low levels, ROS function as “redox messengers” in intracellular signaling and regulation, whereas
excess ROS induce oxidative modification of cellular macromolecules, inhibit protein function, and promote cell death.
Additionally, various redox systems, such as
the glutathione,
thioredoxin, and
pyridine nucleotide redox couples,
NADPH and antioxidant defense
NAD+ and the function of sirtuin proteins
participate in cell signaling and modulation of cell function, including apoptotic cell death. Cell apoptosis is initiated by extracellular and intracellular signals via two main pathways,
the death receptor and
the mitochondria-mediated pathways.
ROS and JNK-mediated apoptotic signaling
GSH redox status and apoptotic signaling
Various pathologies can result from oxidative stress-induced apoptotic signaling that is consequent to
ROS increases and/or antioxidant decreases,
disruption of intracellular redox homeostasis, and
irreversible oxidative modifications of lipid, protein, or DNA.
We focus on several key aspects of ROS and redox mechanisms in apoptotic signaling and highlight the gaps in knowledge and potential avenues for further investigation. A full understanding of the redox control of apoptotic initiation and execution could underpin the development of therapeutic interventions targeted at oxidative stress-associated disorders.
Iron-sulfur (FeyS) cluster-containing proteins catalyze a number of electron transfer and metabolic reactions. The components and molecular mechanisms involved in the assembly of the FeyS clusters have been identified only partially. In eukaryotes, mitochondria have been proposed to execute a crucial task in the generation of intramitochondrial and extramitochondrial FeyS proteins. Herein, we identify the essential ferredoxin Yah1p of Saccharomyces cerevisiae mitochondria as a central component of the FeyS protein biosynthesis machinery. Depletion of Yah1p by regulated gene expression resulted in a
30-fold accumulation of iron within mitochondria,
similar to what has been reported for other components involved in FeyS protein biogenesis. Yah1p was shown to be required for the assembly of FeyS proteins both inside mitochondria and in the cytosol. Apparently, at least one of the steps of FeyS cluster biogenesis within mitochondria requires reduction by ferredoxin. Our findings lend support to the idea of a primary function of mitochondria in the biosynthesis of FeyS proteins outside the organelle. To our knowledge, Yah1p is the first member of the ferredoxin family for which a function in FeyS cluster formation has been established. A similar role may be predicted for the bacterial homologs that are encoded within iron-sulfur cluster assembly (isc) operons of prokaryotes.
H Lange, A Kaut, G Kispal, and R Lill. A mitochondrial ferredoxin is essential for biogenesis of cellular iron-sulfur proteins. PNAS 2000; 97(3): 1050–1055.
DNA Charge Transport
Damaged bases in DNA are known to lead to errors in replication and transcription, compromising the integrity of the genome. The authors proposed a model where repair proteins containing redoxactive [4Fe-4S] clusters utilize DNA charge transport (CT) as a first step in finding lesions. In this model, the population of sites to search is reduced by a localization of protein in the vicinity of lesions. Here, we examine this model using single-molecule atomic force microscopy (AFM). XPD, a 5′-3′ helicase involved in nucleotide
excision repair, contains a [4Fe-4S] cluster and exhibits a DNA bound redox potential that is physiologically relevant.
In AFM studies, they observe the redistribution of XPD onto kilobase DNA strands containing a single base mismatch, which is not a specific substrate for XPD but, like a lesion, inhibits CT. They also provide evidence for DNA-mediated signaling between XPD and Endonuclease III (EndoIII), a base excision repair glycosylase that also contains a [4Fe-4S] cluster.
When XPD and EndoIII are mixed together, they coordinate in relocalizing onto the mismatched strand.
However, when a CT-deficient mutant of either repair protein is combined with the CT-proficient repair partner, no relocalization occurs.
The data presented here indicate that XPD, an archaeal protein from the NER pathway, may cooperate with other proteins that are proficient at DNA CT to localize in the vicinity of damage. XPD, a superfamily 2 DNA helicase with 5′-3′ polarity, is a component of TFIIH that is essential for repair of bulky lesions generated by exogenous sources such as UV light and chemical carcinogens. XPD contains a conserved [4Fe-4S] cluster suggested to be conformationally controlled by ATP binding and hydrolysis.
Mutations in the iron-sulfur domain of XPD can lead to diseases including TTD and XP, yet the function of the [4Fe-4S] cluster appears to be unknown.
Electrochemical studies have shown that when BER proteins MutY and EndoIII bind to DNA, their [4Fe-4S] clusters are activated toward one electron oxidation. XPD exhibits a DNA-bound midpoint potential similar to that of EndoIII and MutY when bound to DNA (approximately 80 mV vs. NHE), indicative of a possible role for the [4Fe-4S] cluster in DNA-mediated CT.
For EndoIII we have also already determined a direct correlation between the ability of proteins to redistribute in the vicinity of mismatches as measured by AFM, and the CT proficiency of the proteins measured electrochemically. Thus, we may utilize single-molecule AFM as a tool to probe the redistribution of proteins in the vicinity of base lesions and in so doing, the proficiency of the protein to carry out DNA CT.
Here we show that, like the BER protein EndoIII, XPD, involved both in transcription and NER, redistributes in the vicinity of a lesion. Importantly, this ability to relocalize is associated with the ability of XPD to carry out DNA CT. The mutant L325V is defective in its ability to carry out DNA CTand this XPD mutant also does not redistribute effectively onto the mismatched strand.
These data not only indicate a general link between the ability of a repair protein to carry out DNA CT and its ability to redistribute onto DNA strands near lesions but also provide evidence for coordinated DNA CT between different repair proteins in their search for damage in the genome. These data also provide evidence that two different repair proteins, each containing a [4Fe-4S] cluster at similar DNA bound potential, can communicate with one another through DNA-mediated CT.
The signaling function of mitochondria is considered with a special emphasis on their role in the regulation of redox status of the cell, possibly determining a number of pathologies including cancer and aging. The review summarizes the transport role of mitochondria in energy supply to all cellular compartments (mitochondria as an electric cable in the cell), the role of mitochondria in plastic metabolism of the cell including synthesis of
heme,
steroids,
iron-sulfur clusters, and
reactive oxygen and nitrogen species.
Mitochondria also play an important role in the Ca2+-signaling and the regulation of apoptotic cell death. Knowledge of mechanisms responsible for apoptotic cell death is important for the strategy for prevention of unwanted degradation of postmitotic cells such as cardiomyocytes and neurons.
In accordance with P. Mitchell’s chemiosmotic concept, vectorial transmembrane transfer of electrons and protons is accompanied by generation of electrochemical difference of proton electrochemical potential on the inner mitochondrial membrane; its utilization by ATP synthase induces conformational rearrangements resulting in ATP synthesis from ADP and inorganic phosphate. Details of the mechanism responsible for ATP synthesis are given elsewhere.
Membrane potential (DY) generated across the inner mitochondrial membrane is the component of the transmembrane electrochemical potential of H+ ions (DμH+), which provides ATP synthesis together with the concentration component (DpH). Maintenance of constant membrane potential is a vitally important precondition for functioning of mitochondria and the cell. Under conditions of limited supply of the cell with oxygen (hypoxia) and inability to carry out aerobic ATP synthesis, mitochondria become ATP consumers (rather than generators) and ATP is hydrolyzed by mitochondrial ATPase, and this is accompanied by generation of membrane potential.
Redox homeostasis, i.e. the sum of redox components (including proteins, low molecular weight redox components such as NAD/NADH, flavins, coenzymes Q, oxidized and reduced substrates, etc.) is one of important preconditions for normal cell functioning.
Single-strand and double-strand DNA damage (Photo credit: Wikipedia)
Mitochondria generate such potent regulators of redox potential as
superoxide anion,
hydrogen peroxide,
nitric oxide,
peroxynitrite, etc.
They are actively involved in regulation of cell redox potential and consequently
control proteolysis,
activation of transcription,
changes in mitochondrial DNA (mDNA),
cell metabolism, and
cell differentiation.
Zorov DB, Isaev NK, Plotnikov EY, Zorova LD, et al. The Mitochondrion as Janus Bifrons. Biochemistry (Moscow) 2007; 72(10): 1115-1126. ISSN 0006-2979.
DOI: 10.1134/S0006297907100094
Structure of the human mitochondrial genome. (Photo credit: Wikipedia)
Gene Expression Associated with Oxidoreduction and Mitochondria
The naked mole-rat (Heterocephalus glaber) is a long-lived, cancer resistant rodent and there is a great interest in identifying the adaptations responsible for these and other of its unique traits. We employed RNA sequencing to compare liver gene expression profiles between naked mole-rats and wild-derived mice. Our results indicate that genes associated with oxidoreduction and mitochondria were expressed at higher relative levels in naked mole-rats. The largest effect is nearly
300-fold higher expression of epithelial cell adhesion molecule (Epcam), a tumour-associated protein.
Also of interest are the
protease inhibitor, alpha2-macroglobulin (A2m), and the
mitochondrial complex II subunit Sdhc,
both ageing-related genes found strongly over-expressed in the naked mole-rat.
These results hint at possible candidates for specifying species differences in ageing and cancer, and in particular suggest complex alterations in mitochondrial and oxidation reduction pathways in the naked mole-rat. Our differential gene expression analysis obviated the need for a reference naked mole-rat genome by employing a combination of Illumina/Solexa and 454 platforms for transcriptome sequencing and assembling transcriptome contigs of the non-sequenced species. Overall, our work provides new research foci and methods for studying the naked mole-rat’s fascinating characteristics.
The complete set of viable deletion strains in Saccharomyces cerevisiae was screened for sensitivity of mutants to five oxidants to identify cell functions involved in resistance to oxidative stress. This screen identified a unique set of mainly constitutive functions providing the first line of defense against a particular oxidant; these functions are very dependent on the nature of the oxidant. Most of these functions are distinct from those involved in repair and recovery from damage, which are generally induced in response to stress, because there was little correlation between mutant sensitivity and the reported transcriptional response to oxidants of the relevant gene. The screen identified 456 mutants sensitive to at least one of five different types of oxidant, and these were ranked in order of sensitivity. Many genes identified were not previously known to have a role in resistance to reactive oxygen species. These encode functions including
protein sorting,
ergosterol metabolism,
autophagy, and
vacuolar acidification.
two mutants were sensitive to all oxidants examined, 12 were sensitive to at least four,
Different oxidants had very different spectra of deletants that were sensitive. These findings highlight the specificity of cellular responses to different oxidants:
No single oxidant is representative of general oxidative stress.
Mitochondrial respiratory functions were overrepresented in mutants sensitive to H2O2, and
vacuolar protein-sorting mutants were enriched in mutants sensitive to diamide.
Core functions required for a broad range of oxidative-stress resistance include
Isolated complex I deficiency is the most common enzymatic defect of the oxidative phosphorylation (OXPHOS) system, causing a wide range of clinical phenotypes. Th authers reported before that the rates at which reactive oxygen species (ROS)-sensitive dyes are converted into their fluorescent oxidation products are markedly increased in cultured skin fibroblasts of patients with nuclear-inherited isolated complex I deficiency.
Using videoimaging microscopy we show here that these cells also display a marked increase in NAD(P)H autofluorescence. Linear regression analysis revealed a negative correlation with the residual complex I activity and a positive correlation with the oxidation rates of the ROS sensitive dyes (5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein and hydroethidine for a large cohort of 10 patient cell lines.
On the other hand, video-imaging microscopy of cells selectively expressing reduction-oxidation sensitive GFP1 in either the mitochondrial matrix or cytosol showed the absence of any detectable change in thiol redox state. In agreement with this result, neither the glutathione nor the glutathione disulfide content differed significantly between patient and healthy fibroblasts.
Finally, video-rate confocal microscopy of cells loaded with C11-BODIPY581/591 demonstrated that the extent of lipid peroxidation, which is regarded as a measure of oxidative damage, was not altered in patient fibroblasts. Our results indicate that fibroblasts of patients with isolated complex I deficiency maintain their thiol redox state despite marked increases in ROS production.
Mitochondrial research has recently been driven by the
identification of mitochondria-associated diseases and the role of mitochondria in apoptosis.
Moreover, mitochondria have been implicated in the process of carcinogenesis because of their vital role in
energy production,
nuclear-cytoplasmic signal integration and
control of metabolic pathways.
At some point during neoplastic transformation, there is an increase in reactive oxygen species (ROS), which damage the mitochondrial genome. This accelerates the somatic mutation rate of mitochondrial DNA.
Mitochondrial characteristics
There are several biological characteristics which cast mitochondria and, in particular, the mitochondrial genome, as a biological tool for early detection and monitoring of neoplasia and its potential progression. These vital characteristics are important in cancer research, as not all neoplasias become malignant. Mitochondria are archived in the cytoplasm of the ovum and as such do not recombine.
This genome has an accelerated mutation rate, by comparison with the nucleus, and accrues somatic mutations in tumour tissue. Moreover, mitochondrial DNA (mtDNA) has a high copy number in comparison with the nuclear archive of DNA. There are potentially thousands of mitochondrial genomes per cell, which enables detection of important biomarkers, even at low levels. In addition, mtDNA can be heteroplasmic, which means that disease-associated mutations occur in a subset of the genomes.
The presence of heteroplasmy is an indication of disease and is found in many human tumours. Identification of low levels of heteroplasmy may allow unprecedented early identification and monitoring of neoplastic progression to malignancy.
Coding for just 13 enzyme complex subunits, 22 transfer RNAs and two ribosomal RNAs, the mitochondrial genome is packaged in a compact 16,569 base pair (bp) circular molecule. These products participate in the critical electron transport process of ATP production. Collectively, mitochondria generate 80 per cent of the chemical fuel which fires cellular metabolism.
As a result, nuclear investment in the mitochondria is high — that is, several thousand nuclear genes control this organelle in order to accomplish the complex interactions required to maintain a network of pathways, which coordinate energy demand and supply.
It has been proposed that these mutations may serve as an early indication of potential cancer development and may represent a means for tracking tumour progression.
Does this provide a potential utility in that these mutations may be used for the identification and monitoring of neoplasia and malignant transformation where appropriate body fluids or non-invasive tissue access is available for mtDNA recovery? Specifically discussed are:
prostate,
breast,
colorectal,
skin and
lung cancers
There are many important questions yet to be addressed: such as
the relationship between mtDNA and the actual disease;
are mutations causative or merely a reflection of nuclear instability?
And, are these processes independent events?
Alterations in the non-coding D-loop suggest genome instability;
however, as studies focus more on the coding regions of the
mitochondrial genome,
Particularly in the case of nonsynonymous mutations in the genes
contributing products to the electron transport process, metabolic
implications are evident. Moreover, mutations in mitochondrial transfer RNAs indicate the possibility of a global mitochondrial translational shut down.
RL Parr, GD Dakubo, RE Thayer, K McKenney, MA Birch-Machin. Mitochondrial DNA as a potential tool for early cancer detection. HUMAN GENOMICS 2006; 2(4). 252–257.
Mitochondrial DNA (mtDNA) is particularly prone to oxidation due to the lack of histones and a deficient mismatch repair system. This explains an increased mutation rate of mtDNA that results in heteroplasmy, e.g., the coexistence of the mutant and wild-type mtDNA molecules within the same mitochondrion. Hyperglycemia is a key risk factor not only for diabetes-related disease, but also for cardiovascular and all-cause mortality. One can assume an increase in the risk of cardiovascular disease by 18% for each unit (%) glycated hemoglobin HbA1c. In the Glucose Tolerance in Acute Myocardial Infarction study of patients with acute coronary syndrome, abnormal glucose tolerance was the strongest independent predictor of subsequent cardiovascular complications and death. In the Asian Pacific Study, fasting plasma glucose was shown to be an independent predictor of cardiovascular events up to a level of 5.2 mmol/L.
Glucose level fluctuations and hyperglycemia are triggers for inflammatory responses via increased mitochondrial superoxide production and endoplasmic reticulum stress. Inflammation leads to insulin resistance and β-cell dysfunction, which further aggravates hyperglycemia. The molecular pathways that integrate hyperglycemia, oxidative stress, and diabetic vascular complications have been most clearly described in the pathogenesis of endothelial dysfunction, which is considered as the first step in atherogenesis according to the response to injury hypothesis.
In diabetes mellitus,
glycotoxicity,
advanced oxidative stress,
collagen cross-linking, and
accumulation of lipid peroxides
in foam macrophage cells and arterial wall cells may significantly
decrease the mutation threshold,
endothelial dysfunction,
promoting atherosclerosis.
Alterations in mitochondrial DNA (mtDNA), known as homoplasmic and heteroplasmic mutations, may influence mitochondrial OXPHOS capacity, and in turn contribute to the magnitude of oxidative stress in micro- and macrovascular networks in diabetic patients.
The authors critically consider the impact of mtDNA mutations on the pathogenesis of cardiovascular diabetic complications.
Mutation Threshhold
Although cells may harbor mutant mtDNA, the expression of disease is dependent on the percent of alleles bearing mutations. Modeling confirms that an upper threshold level might exist for mutations beyond which the mitochondrial population collapses, with a subsequent decrease in ATP. This decrease in ATP results in the phenotypic expression of disease. It is estimated that in many patients with clinical manifestations of mitochondrial disorders, the proportion of mutant DNA exceeds 50%.
For the MELAS (mitochondrial encephalopathy, lactic acidosis and stroke-like syndrome)-causing mutation m.3243 A>G in the mitochondrial gene encoding tRNALeu, which is also associated with diabetes plus deafness, a strong correlation between the level of mutational heteroplasmy and documented disease has been found. Increased percentages of mutant mtDNA in muscle cells (up to 71%) can lead to mitochondrial myopathy. Levels of heteroplasmy of over 80% may lead to recurrent stroke and mutation levels of 95% have been associated with MELAS.
Regardless of the type of mutation or the level of heteroplasmy in affected mitochondria, unrepaired damage leads to a decrease in ATP, which in turn causes the phenotypic manifestation of disease. The manifestation of disease not only depends on the ATP level but also on the tissue affected. Various tissues have differing levels of demand on OXPHOS capacity. To evaluate a tissue threshold, Leber’s hereditary optic neuropathy can be used as a model for mitochondrial neurodegenerative disease. For neural and skeletal muscle tissues, the tissue threshold should be as high as or higher than 90% of
damaged (mutated) mtDNA. To induce mitochondrial malfunctions, the tissue threshold of the cardiac muscle is estimated to be significantly lower (approximately 64%-67%). In chronic vascular disease such as atherosclerosis, a mutation threshold in the affected vessel wall (e.g., in the postmortem aortic atherosclerotic plaques) was observed to be significantly lower. For example, for mutations m.3256 C>T, m.12315 G>A, m.15059 G>A, and m.15315 G>A, the heteroplasmy range of 18%-66% in the atherosclerotic lesions was 2-3.5-fold that in normal vascular tissue.
Mitochondrial stress and insulin resistance
Mitochondrial damage precedes the development of atherosclerosis and tracks the extent of the lesion in apoE-null mice, and
mitochondrial dysfunction caused by heterozygous deficiency of a superoxide dismutase increases atherosclerosis and vascular mitochondrial damage in the same model.
Blood vessels destined to develop atherosclerosis may be characterized by inefficient ATP production due to the uncoupling of respiration and OXPHOS. Blood vessels have regions of hypoxia, which lower the ratio of state 3 (phosphorylating) to state 4 (nonphosphorylating) respiration. Human atherosclerotic lesions have been known for decades to be deficient in essential fatty acids, a condition that causes respiratory uncoupling and atherosclerosis.
The finding by Kokaze et al. helps to explain, at least in part, the anti-atherogenic effect of the allele m. 5178A due to its relation with the favorable lipid profile. The nucleotide change causes leucine-to-methionine substitution at codon 237 (Leu-237Met) of the NADH dehydrogenase subunit 2 located in the loop between 7th and 8th transmembrane domains of the mitochondrial protein. Given that this methionine residue is exposed at the surface of respiratory Complex I, this residue may be available as an efficient oxidant scavenger. Complex I
accepts electrons from NADH,
transfers them to ubiquinone, and
uses the energy released to pump protons across the mitochondrial inner membrane.
Thus, the Leu237Met replacement in the ND2 subunit might have a protective effect against oxidative damage to mitochondria.
Most fatty acid oxidation, which is promoted by peroxisome proliferator-activated receptor α (PPARα) activation, occurs in the mitochondria. Mitochondrial effects could explain why PPARα- deficient mice are protected from diet-induced insulin resistance and atherosclerosis as well as glucocorticoid induced insulin resistance and hypertension. Caloric restriction,
Researchers from Germany, Denmark, and the US sequenced a hyper-variable portion of the koala’s mitochondrial genome sequence using DNA from more than a dozen museum samples. The samples, obtained from museums in Australia and beyond, represented koalas that had been collected in different parts of Australia from the late 1800s to the 1980s.
The team found surprisingly similar mitochondrial profiles in the historical koala samples and samples from modern day koalas. And all four of the mitochondrial haplotypes identified in the older museum samples persist in modern koala populations, the researchers said. That hints that relatively low genetic diversity has been present in koalas for at least 120 years — prior to dramatic population declines at the end of the 19th century, which have been attributed to factors such as hunting, habitat loss, and disease.
“The event which reduced the genetic diversity of koalas must have happened a long time ago, perhaps during the late Pleistocene when the larger species of koala, P. stirtoni, became extinct,” Leibniz-Institute for Zoo and Wildlife Research’s Alex Greenwood, the study’s corresponding author, said in a statement.
The koala (Phascolarctos cinereus) is an arboreal marsupial that was historically widespread across eastern Australia until the end of the 19th century when it suffered a steep population decline. Hunting for the fur trade, habitat conversion, and disease contributed to a precipitous reduction in koala population size during the late 1800s and early 1900s. To examine the effects of these reductions in population size on koala genetic diversity, we sequenced part of the hypervariable region of mitochondrial DNA (mtDNA) in koala museum specimens collected in the 19th and 20th centuries, hypothesizing that the historical samples would exhibit greater genetic diversity.
Results
The mtDNA haplotypes present in historical museum samples were identical to haplotypes found in modern koala populations, and no novel haplotypes were detected. Rarefaction analyses suggested that the mtDNA genetic diversity present in the museum samples was similar to that of modern koalas.
Conclusions
Low mtDNA diversity may have been present in koala populations prior to recent population declines. When considering management strategies, low genetic diversity of the mtDNA hypervariable region may not indicate recent inbreeding or founder events but may reflect an older historical pattern for koalas.
Learn about Koalas:
The Koala (Phascolarctos cinereus) is a thickset arboreal marsupial herbivore native to Australia, and the only extant representative of the family Phascolarctidae.
The Koala is found in coastal regions of eastern and southern Australia, from near Adelaide to the southern part of Cape York Peninsula. Populations also extend for considerable distances inland in regions with enough moisture to support suitable woodlands. The Koalas of South Australia were largely exterminated during the early part of the 20th century, but the state has since been repopulated with Victorian stock. The Koala is not found in Tasmania or Western Australia.
Contrary to (un)popular belief: A koala is NOT a bear!
The US Government have declared the koala a threatened species, however the Australian Government has not. A review of the species national conservation status concluded that the koala are not threatened at a national scale, with a population that numbers in the hundreds of thousands
As with most native Australian animals, the Koala cannot legally be kept as a pet in Australia without a permit.
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About this photo
This was my first photo in Flickr Explore! Check this photo’s Explore history.
Highest recorded Explore: 16 on Saturday, March 29, 2008!
It is currently the number one hit if you search for “koala” on Flickr, and the number one google-hit for “cutest koala”. I get a LOT of views for this one, so thanks goes out to each and ever one of you for having a look at it!
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Mitochondria are responsible for more than 90% of a cell’s energy production via ATP (adenosine triphosphate) generation, in addition to playing a significant role in respiration and many signaling events within most eukaryotic cells. These intracellular powerhouses range in size and quantity within each cell depending on the organism and overall cell function.
Mitochondria consist of a semi-permeable outer membrane, a thin inter-membrane space where oxidative phosphorylation occurs, an impermeable inner membrane that is intricately folded to create layered compartments—or christae—and the matrix that contains ATP-producing enzymes and the organelle’s own independent genome. Each section has a highly specialized function, and any impairment within the organelle can lead to disease or disorders within the overall organism.
Mitochondrial dysfunction may be due to:
1. Hereditary:
Inherited mitochondrial disorders can play a role in prevalent diseases such as cardiac disease and diabetes, and can also result in rare diseases such as Pearson syndrome or Leigh’s disease.
2. Drug Toxicity:
Mitochondrial toxicity as a result of pharmaceutical use may damage key organs, such as the liver and heart. For example:
nefazodone—a depression treatment—was withdrawn from the U.S. market after it was shown to significantly inhibit mitochondrial respiration in liver cells, leading to liver failure.
Troglitazone, an anti-diabetic and anti-inflammatory, was withdrawn from all markets after research concluded that it caused acute mitochondrial membrane depolarization, also leading to liver failure.
Drug recalls are costly to a manufacturer’s bottom line and reputation, and more importantly, can be harmful or even fatal to users. As drug discovery continues to evolve, much lead compound research now includes careful review of its interaction and potential toxicity with mitochondria.
Cell-based mitochondrial assays in microplate format may include mitochondrial membrane potential, total energy metabolism, oxygen consumption, and metabolic activity; and offer a truer environment for mitochondrial function in the presence of drug compounds compared to isolated mitochondria-based tests. Combining more than one assay in a multiplex format increases the amount of data per well while decreasing data variability arising from running the assays separately. The aggregated data also provides a more encompassing analysis of the drug’s effect on mitochondria than a single test.
One example, when testing compound effects on mitochondria, would be to measure cell membrane integrity as a function of cytotoxicity and mitochondrial function via ATP production concurrently, thus distinguishing between compounds that exhibit mitochondrial toxicity versus overt cytotoxicity.
General cytotoxicity is characterized by a decrease in ATP production and a loss of membrane integrity whereas mitochondrial toxicity results in decreased ATP production with little to no change in membrane integrity.
The assay’s efficiency is further enhanced via automation.
Robotic instrumentation ensures repeatable operation within the microplate wells when performing tasks such as cell dispensing, serial titration and transfer of compounds, and reagent dispensing. Additionally, by automating tasks within the assay process, researchers are free to attend to other tasks, reducing overall active time spent on the assay. Multi-mode microplate readers are compact instruments that can detect both fluorescent and luminescent signals. In addition, an automated process—including liquid handling and detection—can increase throughput capacity compared to manual methods.
Multiplexed cell-based mitochondrial assays increase sample throughput and decrease variability, costs, and overall time for project completion. Automating the process with robotic instrumentation allows for rapid compound profiling, repeatability, further throughput increase, and decreased per-assay and overall project time.
Mitochondria are found in every cell in the human body.1 Known as the “power plant of the cell,” mitochondria are central to the conversion of fatty acids and glucose to usable energy in the form of ATP (adenosine triphosphate).1, 2 A growing body of evidence now demonstrates a link between various disturbances in mitochondrial functioning and type 2 diabetes.1
In patients with type 2 diabetes, the size, number, and efficiency of mitochondria are reduced.3 This can have pathogenic effects in the tissues central to glucose metabolism — the pancreas, liver, and skeletal muscle.
In pancreatic beta cells, mitochondria are central to insulin secretion. As the amount of glucose in the circulation increases, so does the mitochondrial production of ATP inside the cell. When this occurs, ATP-sensitive channels open, leading to membrane depolarization and the secretion of insulin.1
Much data support the concept that mitochondrial function is required for appropriate glucose-induced insulin secretion.4 Studies in beta cell lines have shown that when mitochondrial function is experimentally decreased, insulin secretion shows a similar reduction.4 Supporting studies in humans have shown that individuals with disabling mutations in mitochondrial DNA (i.e., the A32433G mutation) demonstrate impaired pancreatic insulin secretion in response to glucose challenge.
Mitochondrial dysfunction in skeletal muscle and the liver might also contribute to the development of diabetes. As part of its cellular respiratory function, mitochondria utilize (and break down) fatty acids. When mitochondrial function is reduced, intracellular fats may accumulate.2
One hypothesis is that excessive accumulation of intracellular fat may have a central role in insulin resistance. This hypothesis is supported by the observation that excessive lipids lead to reductions in numbers and function of insulin receptors.2
The link between obesity, inactivity, and type 2 diabetes is well established — and weight loss remains a cornerstone of diabetes management.3 The role of mitochondria as cellular “power plant” makes a compelling case for a causative relationship between mitochondrial dysfunction and clinical disease.3
Reduced mitochondrial capacity has been demonstrated in patients with type 2 diabetes.3 In one study, patients who lost weight demonstrated an increase in mitochondrial density and insulin sensitivity. Patients achieved an average weight loss of 7.1% and experienced a decrease in mean HbA1c from 7.9 to 6.5, as well as significant improvements in both fasting and postprandial blood glucose.3
Strategies that focus on increasing mitochondrial function could represent important new approaches in the treatment of diabetes.
One agent under investigation is coenzyme Q10 (CoQ10). In animal studies, CoQ10 significantly reduced fasting and 2-hour postprandial glucose levels. In humans, early, uncontrolled studies of diabetic patients receiving CoQ10 have demonstrated improvements in blood glucose and insulin synthesis and secretion. Furthermore, the clinical benefit of CoQ10 has been evident in a number of therapeutic trials in patients with maternally inherited mitochondrial defects like MELAS (Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like episodes).1 The therapeutic advantage of supplementary CoQ10 may be especially helpful in patients taking statins, as these patients have been shown to have decreased production of endogenous CoQ10.5
Impaired mitochondrial function in tissues central to glucose metabolism (pancreas, muscle, liver) may be partly responsible for diabetes pathogenesis.2 The failure to appropriately manage cellular energy needs may result in impaired insulin secretion and/or insulin resistance.2 Targeting mitochondrial dysfunction may represent a promising path forward in the development of novel treatments for diabetes.
REFERENCES
Lamson DW, et al. Mitochondrial Factors in the Pathogenesis of Diabetes: A Hypothesis for Treatment. Altern Med Rev. 2002;7:94-111.
Toledo FG, et al. Effects of Physical Activity and Weight Loss on Skeletal Muscle Mitochondria and Relationship With Glucose Control in Type 2 Diabetes. Diabetes. 2007;56:2142-2147.
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