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Posts Tagged ‘Warburg and Pateur Effect’


Natural Drug Target Discovery and Translational Medicine in Human Microbiome

Author and Curator: Demet Sag, PhD

 

Remember Ecology 101, simple description of ecosystem includes both living, biotic, and non-living, abiotic, that response to differentiation based on external and internal factors.  Hence, biodiversity changes since living systems are open systems and always try to reach stability. Both soil and human body are rich in microbial life against ever changing conditions. Previously, discovery of marine microorganisms for treatment of complex diseases especially cancer and drug discovery for pharmaceutical applications was discussed. (https://pharmaceuticalintelligence.com/2014/03/20/without-the-past-no-future-but-learn-and-move-genomics-of-microorganisms-to-translational-medicine/)

Here, the focus will be given to clinical drug discovery based on how lactose intolerance and human microbiome related to treat cancer patients or other diseases. In sum, creating clinical relevance with human microbiome require knowledge of both of the worlds to make best of it to solve complex diseases naturally.

The huge undertake as a roadmap to biomedical research originated by NIH under The Human Microbiome Project (HMP) (http://nihroadmap.nih.gov) with 250 healthy individuals as a starting point.  Recent developments opened the doors to pursue us to understand how human microbiome reflects on metabolism, drug interactions and numerous diseases.  Finally, association between clinical states and microbiome are improving with advanced algorithms, bioinformatics and genomics. In classical reading tests questions finding the simile between two groups of words can well relate how microbiome- human and soil-earth relates.  Both are rich in microbial life with quite changing characters to survive through commensal living.

Thus, it is also good to talk about how we can synthesize existing info on interactions between soil microorganisms and decomposers for human diseases and human microbiome. Epidemiology of living organisms is diverse but they all share common interest. In soil, for example, radioactively contaminated soil can’t support plant growth well so Nitrosomonas may support to bring the life to soil through supplying nitrogen. And others can be added to bring a favorable enriched soil.

In human microbiome nutrition-diseases interacts in such a harmony with genetic make up (the information received at time of birth germline- or acquired later in life due to mutations by various reasons). For example, the simplest example is lactose intolerance and the other is development of diabetes.  Generally, it is described as If person is missing a gene to metabolize lactose (sugar) this person become Lactose intolerant yet this can be gained before birth or after. The fix is easy since avoiding certain food groups i.e. milk products.

Yet, this is not that simple!

In human microbiome, the rich gastrointestinal (GI) tract contains many organisms and one of the most important ones is Enterococci that are often simply described as lactic-acid–producing bacteria—by under- appreciation of their power of microbial physiology and outcomes as well as their ubiquitous nature of enterococci.  Schleifer & Kilpper-Bälz, 1984 also reported that the Group D streptococci, such as Streptococcus faecalis and Streptococcus faecium, were included in the new genus called Enterococcus.

The importance of this genius, consists of 37 species, coming from their spectrum of  habitats that include the gastrointestinal microbiota of nearly every animal phylum and flexibility with ability to widely colonize, intrinsic resistance to many inhabitable conditions even though they don’t have spores but they can survive against desiccation and can persist for months on dried surfaces.  Furthermore, they can tolerate extreme conditions such as pH changes, ionizing radiation, osmotic and oxidative stresses, high heavy metal concentrations, and antibiotics.

There is a double sword application as these organisms used as probiotics to improve immune system of the host.  If it is human to prevent contaminated food related diseases or in animals prevent transmitting them to the consumers. Thus, E. faecium and E. faecalis strains are used as probiotics and are ingested in high numbers, generally in the form of pharmaceutical preparations to treat diarrhea, antibiotic-associated diarrhea or irritable bowel syndrome, to lower cholesterol levels or to improve host immunity.

When it comes to human body within each system specific organs may create distinct values.  For example the pH values of GI tract vary and during diseases since pH levels are not at at correct levels.  As a result, due to mal-absorption of nutrients and elements such as food, vitamins and minerals body can’t heal itself. This changing microbial genomics on the surface of GI reflects on general health.  Entrococcus family among the other GI’s natural flora has the microbial physiology adopt these various pH conditions well. 

 

Our body has its own standards to function, such as  pH, temperature, oxygen etc these are basics so that enzymatic reactions may happen to metabolize,synthesizing (making) or catalyzing (breaking) what we eat.  The pH is the measure of hydrogen-ion concentration  in solution.  For example, human blood has a narrow pH (7.35 – 7.45 ) and below or above this range means symptoms and disease yet if blood pH moves to much below 6.8 or above 7.8, cells stop functioning and the patient dies since the ideal pH for blood is 7.4.  This value is unified.  On the other hand, the pH in the human digestive tract or GI changes tremendously to adopt and carry on its function, the pH of saliva (6.5 – 7.5), upper portion of the stomach (4.0 – 6.5) where “predigestion” occurs, the lower portion of the stomach is secreting hydrochloric acid (HCI) and pepsin until it reaches a pH between 1.5 – 4.0; duodenum, small intestine, (7.0 – 8.5) where 90% of the absorption of nutrients is taken in by the body while the waste products are passed out through the colon (pH 4.0 – 7.0).

 

Why is pH important and how related to anything?

Development and presence of cancer always require an acid pH and lack of oxygen.  Thus, prevention of these two factors may be the key for treatment of cancer as it progress the acidity increases such that the level raises even up to 1000 more than normal levels.

Mainly, due to Warburg effect body opt to get its energy from fermentation of glucose and produce lactic acid that decreases the body pH from 7.3 down to 7 then to 6.5 in advanced stages of cancer.  Furthermore, during metastases this level even reaches to 6.0 and even 5.7 where body can’t fight back with the disease. (Warburg effect is well explained previously by Dr. Larry Berstein (www.linkedin.com/pub/larry-bernstein/38/94b/3aa).

How to bypass the lack of oxygen naturally?

One of the many solution can be a natural solution. The nature made the hemoglobin carrying bacteria, Vitreoscilla hemoglobin (VHb), which is first described by Dale Webster in 1966. The gram negative and obligate aerobic bacterium, Vitreoscilla synthesizes elevated quantities of a homodimeric hemoglobin (VHb) under hypoxic growth conditions.   The main role is likely the binding of oxygen at low concentrations and its direct delivery to the terminal respiratory oxidase(s) such as cytochrome o.  Then, after 1986 with detailed description of the molecule other hemoglobins and flavohemoglobins were identified in a variety of microbes, indicating the widespread occurrence of Hb-like proteins.   Currently, it is the most studied bacterial hemoglobin with application potentials in biotechnology.

It is a plausible solution to integrate Vitroscilla and Enterobacter powers for cancer detection and treatment naturally with body’s own microbiome.

However, there are many microbial organisms and differ person to person based on gender, age, background, genetic make-up, food intake, habits, location etc.  The huge undertake as a roadmap to biomedical research originated by NIH under The Human Microbiome Project (HMP) (http://nihroadmap.nih.gov) with 250 healthy individuals as a starting point.

There were three goals in the agenda of The Human Microbiome Project (HMP) simply:

 1. Utilize advanced high throughput technology,

2. Identify any association between microbiome and disease/health stages,

3. Initiate scientific studies to collect more data.

In sum, creating clinical relevance with human microbiome require knowledge of both of the worlds to make best of it to solve complex diseases naturally.

Previously  Discussed:

AMPK Is a Negative Regulator of the Warburg Effect and Suppresses Tumor Growth In Vivo
Reporter-Curator: Stephen J. Williams, Ph.D.
https://pharmaceuticalintelligence.com/2013/03/12/ampk-is-a-negative-regulator-of-the-warburg-effect-and-suppresses-tumor-growth-in-vivo/

Is the Warburg Effect the Cause or the Effect of Cancer: A 21st Century View?
Author: Larry H. Bernstein, MD, FCAP
https://pharmaceuticalintelligence.com/2012/10/17/is-the-warburg-effect-the-cause-or-the-effect-of-cancer-a-21st-century-view/

Otto Warburg, A Giant of Modern Cellular Biology
Reporter: Larry H Bernstein, MD, FCAP
https://pharmaceuticalintelligence.com/2012/11/02/otto-warburg-a-giant-of-modern-cellular-biology/

Targeting Mitochondrial-bound Hexokinase for Cancer Therapy
Author: Ziv Raviv, PhD
https://pharmaceuticalintelligence.com/2013/04/06/targeting-mito…cancer-therapy

Nitric Oxide has a ubiquitous role in the regulation of glycolysis -with a concomitant influence on mitochondrial function
Curator, Larry H. Bernstein, MD, FCAP
https://pharmaceuticalintelligence.com/2012/09/16/nitric-oxide-has-a-ubiquitous-role-in-the-regulation-of-glycolysis-with-a-concomitant-influence-on-mitochondrial-function/

Potential Drug Target: Glucolysis Regulation – Oxidative stress-responsive microRNA-320
Reporter: Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2012/07/25/potential-drug-target-glucolysis-regulation-oxidative-stress-responsive-microrna-320/

Differentiation Therapy – Epigenetics Tackles Solid Tumors
Author-Writer: Stephen J. Williams, Ph.D.
https://pharmaceuticalintelligence.com/2013/01/03/differentiation-therapy-epigenetics-tackles-solid-tumors/

Prostate Cancer Cells: Histone Deacetylase Inhibitors Induce Epithelial-to-Mesenchymal Transition
Reporter-Curator: Stephen J. Williams, Ph.D.
https://pharmaceuticalintelligence.com/2012/11/30/histone-deacetylase-inhibitors-induce-epithelial-to-mesenchymal-transition-in-prostate-cancer-cells/

Mitochondrial Damage and Repair under Oxidative Stress
Curator: Larry H Bernstein, MD, FCAP
https://pharmaceuticalintelligence.com/2012/10/28/mitochondrial-damage-and-repair-under-oxidative-stress/

Mitochondria: Origin from oxygen free environment, role in aerobic glycolysis, metabolic adaptation
Curator: Larry H Bernsatein, MD, FCAP
https://pharmaceuticalintelligence.com/2012/09/26/mitochondria-origin-from-oxygen-free-environment-role-in-aerobic-glycolysis-metabolic-adaptation/

Expanding the Genetic Alphabet and Linking the Genome to the Metabolome
Reporter& Curator: Larry Bernstein, MD, FCAP
https://pharmaceuticalintelligence.com/2012/09/24/expanding-the-genetic-alphabet-and-linking-the-genome-to-the-metabolome/

What can we expect of tumor therapeutic response?
Author: Larry H. Bernstein, MD, FCAP
https://pharmaceuticalintelligence.com/2012/12/05/what-can-we-expect-of-tumor-therapeutic-response/

A Second Look at the Transthyretin Nutrition Inflammatory Conundrum
Larry H. Bernstein, MD, FACP
https://pharmaceuticalintelligence.com/2012/12/03/a-second-look-at-the-transthyretin-nutrition-inflammatory-conundrum/

 

Further  Readings and References:

Palmer KL, van Schaik W, Willems RJL, Gilmore MS. “Enterococcal Genomics Enterococci: From Commensals to Leading Causes of Drug Resistant Infection.” 2014-.2014 Feb 8

Franz CM, Holzapfel WH, Stiles ME. Enterococci at the crossroads of food safety?

Int J Food Microbiol.” 1999 Mar 1; 47(1-2):1-24.

Franz CM, Huch M, Abriouel H, Holzapfel W, Gálvez A.Int J Food Microbiol. “Enterococci as probiotics and their implications in food safety.” 2011 Dec 2; 151(2):125-40. Epub 2011 Sep 8.

Kayser FH.”Safety aspects of enterococci from the medical point of view.” Int J Food Microbiol. 2003 Dec 1; 88(2-3):255-62.

Webster DA, Hackett DP (1966). “The purification and properties of cytochrome o fromVitreoscilla“. J Biol Chem 241 (14): 3308–3315

Stark BC, Dikshit KL, Pagilla KR (2011). “Recent advances in understanding the structure, function, and biotechnological usefulness of the hemoglobin from the bacterium Vitreoscilla“. Biotechnol Lett 33 (9): 1705–1714

Stark BC, Dikshit KL, Pagilla KR (2012). “The Biochemistry  of Vitreoscillahemoglobin“. Computational and Structural Biotechnology Journal 3 (4): e201210002.

Brenner K, You L, Arnold F. (2008). “Engineering microbial consortia: A new frontier in synthetic biology.” Trends in Biotechnology 26: 483489.

Dunbar J, White S, Forney L. (1997). “Genetic diversity through the looking glass: Effect of enrichment bias.Applied and Environmental Microbiology 63: 13261331.

Foster J. (2001). “Evolutionary computation Nature Reviews Genetics 2: 428436.

Dinsdale EA, et al. 2008. “Functional metagenomic profiling of nine biomes.” Nature452: 629632.

Gudelj I, Beardmore RE, Arkin SS, MacLean RC. (2007). “Constraints on microbial metabolism drive evolutionary diversification in homogeneous environments.” Journal of Evolutionary Biology 20: 1882–1889.

Haack SK, Garchow H, Klug MJ, Forney L. (1995). “Analysis of factors affecting the accuracy, reproducibility, and interpretation of microbial community carbon source utilization patterns.” Applied and Environmental Microbiology 61: 14581468.

Lozupone C, Knight R. (2007). “Global patterns in bacterial diversity.” Proceedings of the National Academy of Sciences 104: 1143611440.

Thurnheer T, Gmr R, Guggenheim B,  (2004). “Multiplex FISH analysis of a six-species bacterial biofilm. “Journal of Microbiological Methods 56: 3747.

VijayKumar M, Aitken JD, Carvalho FA, Cullender TC, Mwangi S, Srinivasan S,Sitaraman S, Knight R, Ley RE, Gewirtz AT. (2010). “Metabolic syndrome and altered gut microbiota in mice lacking Toll-like receptor 5.” Science 328: 228231

Williams HTP, Lenton TM. (2007). “Artificial selection of simulated microbial ecosystems.” Proceedings of the National Academy of Sciences 104: 89188923.

 

 

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Larry H. Bernstein, MD, FCAP, Reviewer and Curator

http://pharmaceuticalinnovation.com/2012-12-09/larryhbern/Silencing Cancers with Synthetic siRNAs

The challenge of cancer drug development has been marker by less than a century of development of major insights into the know of biochemical pathways and the changes in those pathways in a dramatic shift in enrgy utilization and organ development, and the changes in those pathways with the development of malignant neoplasia.  The first notable change is the Warburg Effect (attributed to the 1860 obsevation by Pasteur that yeast cells use glycolysis under anaerobic conditions).  Warburg also referred to earlier work by Meyerhoff, in a ratio of CO2 release to O2 consumption, a Meyerhoff ratio.  Much more was elucidated after the discovery of the pyridine nucleotides, which gave understanding of glycolysis and lactate production with a key two enzyme separation at the forward LDH reaction and the back reentry to the TCA cycle.  But the TCA cycle could be used for oxidative energy utilization in the mitochondria by oxidative phosphorylation elucidated by Peter Mitchell, or it can alternatively be used for syntheses, like proteins and lipid membrane structures.

A brilliant student in Leloir’s laboratory in Brazil undertook a study of isoenzyme structure in 1971, at a time that I was working under Nathan O. Kaplan on the mechanism of inhibition of mitochondrial malate dehydrogenase. In his descripton, taking into account the effect of substrates upon protein stability (FEBS) could be, in a prebiotic system, the form required in order to select protein and RNA in parallel or in tandem in a way that generates the genetic code (3 bases for one amino acid). Later, other proteins like reverse transcriptase, could transcribe it into the more stable DNA. Leloir had just finished ( a few years before 1971 but, not published by these days yet) a somehow similar reasoning about metabolic regions rich in A or in C or .. G or T.  He later spent time in London to study the early events in the transition of growing cells linked to ion fluxes, which he was attracted to by the idea that life is so strongly associated with the K (potassium) and Na (sodium) asymmetry.   Moreover, he notes that while DNA is the same no matter the cell is dead or alive,  and therefore,  it is a huge mistake to call DNA the molecule of life. In all life forms, you will find K reach inside and Na rich outside its membrane. On his return to Brazil, he accepted a request to collaborate with the Surgery department in energetic metabolism of tissues submitted to ischemia and reperfusion. This led me back to Pasteur and Warburg effects and like in Leloir´s time, he worked with a dimorphic yeast/mold that was considered a morphogenetic presentation of the Pasteur Effect.  His findings were as follows. In absence of glucose, a condition that prevents the yeast like cell morphology, which led to the study of an enzyme “half reaction”. The reaction that on the half, “seen in our experimental conditions did not followed classical thermodynamics” (According to Collowick & Kaplan (of your personal knowledge) vol. I See Utter and Kurahashi in it). This somehow contributed to a way of seeing biochemistry with modesty. The second and more strongly related to the Pasteur Effect was the use an entirely designed and produced in our Medical School Coulometer spirometer that measures oxygen consumption in a condition of constant oxygen supply. At variance with Warburg apparatus and Clark´s electrode, this oxymeters uses decrease in partial oxygen pressure and decrease electrical signal of oxygen polarography to measure it (Leite, J.V.P. Research in Physiol. Kao, Koissumi, Vassali eds Aulo Gaggi Bologna, 673-80-1971). “With this, I was able to measure the same mycelium in low and high “cell density” inside the same culture media. The result shows, high density one stops mitochondrial function while low density continues to consume oxygen (the internal increase or decrease in glycogen levels shows which one does or does not do it). Translation for today: The same genome in the same chemical environment behave differently mostly likely by its interaction differences. This previous experience fits well with what  I have to read by that time of my work with surgeons.  Submitted to total ischemia tissues mitochondrial function is stopped when they already have enough oxyhemoglobin (1) Epstein, Balaban and Ross Am J Physiol.243, F356-63 (1982) 2) Bashford , C. L, Biological membranes a practical approach Oxford Was. P 219-239 (1987).”

Of course, the world of medical and pharmaceutical engagement with this problem, though changed in focus, has benefitted hugely from “The Human Genome Project”, and the events since the millenium, because of technology advances in instrumental analysis, and in bioinformatics and computational biology.  This has lead to recent advances in regenerative biology with stem cell “models”, to advances in resorbable matrices, and so on.  We proceed to an interesting work that applies synthetic work with nucleic acid signaling to pharmacotherapy of cancer.

Synthetic RNAs Designed to Fight Cancer

Fri, 12/06/2013 Biosci Technology
Xiaowei Wang and his colleagues have designed synthetic molecules that combine the advantages of two experimental RNA therapies against cancer. (Source: WUSTL/Robert J. Boston)In search of better cancer treatments, researchers at Washington University School of Medicine in St. Louis have designed synthetic molecules that combine the advantages of two experimental RNA therapies.  The study appears in the December issue of the journal RNA.
 RNAs play an important role in how genes are turned on and off in the body. Both siRNAs and microRNAs are snippets of RNA known to modulate a gene’s signal or shut it down entirely. Separately, siRNA and microRNA treatment strategies are in early clinical trials against cancer, but few groups have attempted to marry the two.   “These are preliminary findings, but we have shown that the concept is worth pursuing,” said Xiaowei Wang, assistant professor of radiation oncology at the School of Medicine and a member of the Siteman Cancer Center. “We are trying to merge two largely separate fields of RNA research and harness the advantages of both.”
 “We designed an artificial RNA that is a combination of siRNA and microRNA, The showed that the artificial RNA combines the functions of the two separate molecules, simultaneously inhibiting both cell migration and proliferation. They designed and assembled small interfering” RNAs, or siRNAs,  made to shut down– or interfere with– a single specific gene that drives cancer.  The siRNA molecules work extremely well at silencing a gene target because the siRNA sequence is made to perfectly complement the target sequence, thereby
  • silencing a gene’s expression.
Though siRNAs are great at turning off the gene target, they also have potentially dangerous side effects:
  • siRNAs inadvertently can shut down other genes that need to be expressed to carry out tasks that keep the body healthy.
 According to Wang and his colleagues, siRNAs interfere with off-target genes that closely complement their “seed region,” a short but important
  • section of the siRNA sequence that governs binding to a gene target.
 “We can never predict all of the toxic side effects that we might see with a particular siRNA,” said Wang. “In the past, we tried to block the seed region in an attempt to reduce the side effects. Until now,
  • we never tried to replace the seed region completely.”
 Wang and his colleagues asked whether
  • they could replace the siRNA’s seed region with the seed region from microRNA.
Unlike siRNA, microRNA is a natural part of the body’s gene expression. And it can also shut down genes. As such, the microRNA seed region (with its natural targets) might reduce
  • the toxic side effects caused by the artificial siRNA seed region. Plus,
  • the microRNA seed region would add a new tool to shut down other genes that also may be driving cancer.
 Wang’s group started with a bioinformatics approach, using a computer algorithm to design
  • siRNA sequences against a common driver of cancer,
  • a gene called AKT1 that encourages uncontrolled cell division.
They used the program to select siRNAs against AKT1 that also had a seed region highly similar to the seed region of a microRNA known to inhibit a cell’s ability to move, thus
  • potentially reducing the cancer’s ability to spread.
In theory, replacing the siRNA seed region with the microRNA seed region also would combine their functions
  • reducing cell division and
  • movement with a single RNA molecule.
 Of more than 1,000 siRNAs that can target AKT1,
  • they found only three that each had a seed region remarkably similar to the seed region of the microRNA that reduces cell movement.
 They then took the microRNA seed region and
  • used it to replace the seed region in the three siRNAs that target AKT1.
The close similarity between the two seed regions is required because
  • changing the original siRNA sequence too much would make it less effective at shutting down AKT1.
 They dubbed the resulting combination RNA molecule “artificial interfering” RNA, or aiRNA. Once they arrived at these three sequences using computer models,
  1. they assembled the aiRNAs and
  2. tested them in cancer cells.
 One of the three artificial RNAs that they built in the lab
  • combined the advantages of the original siRNA and the microRNA seed region that was transplanted into it.
This aiRNA greatly reduced both
  1. cell division (like the siRNA) and
  2. movement (like the microRNA).
And to further show proof-of-concept, they also did the reverse, designing an aiRNA that
  1. both resists chemotherapy and
  2. promotes movement of the cancer cells.
 “Obviously, we would not increase cell survival and movement for cancer therapy, but we wanted to show how flexible this technology can be, potentially expanding it to treat diseases other than cancer,” Wang said.
Source: WUSTL

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