Archive for the ‘Cell Biology, Signaling & Cell Circuits’ Category

Genomic Diagnostics: Three Techniques to Perform Single Cell Gene Expression and Genome Sequencing Single Molecule DNA Sequencing

Curator: Aviva Lev-Ari, PhD, RN


This article presents Three Techniques to Perform Single Cell Gene Expression and Genome Sequencing Single molecule DNA sequencing


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Ido Sagi – PhD Student @HUJI, 2017 Kaye Innovation Award winner for leading research that yielded the first successful isolation and maintenance of haploid embryonic stem cells in humans.

Reporter: Aviva Lev-Ari, PhD, RN


Ido Sagi – PhD Student, Silberman Institute of Life Sciences, HUJI, Israel

  • Ido Sagi’s research focuses on studying genetic and epigenetic phenomena in human pluripotent stem cells, and his work has been published in leading scientific journals, including NatureNature Genetics and Cell Stem Cell.
  • Ido Sagi received BSc summa cum laude in Life Sciences from the Hebrew University, and currently pursues a PhD at the laboratory of Prof. Nissim Benvenisty at the university’s Department of Genetics in the Alexander Silberman Institute of Life Sciences.

The Kaye Innovation Awards at the Hebrew University of Jerusalem have been awarded annually since 1994. Isaac Kaye of England, a prominent industrialist in the pharmaceutical industry, established the awards to encourage faculty, staff and students of the Hebrew University to develop innovative methods and inventions with good commercial potential, which will benefit the university and society.

Publications – Ido Sagi

Comparable frequencies of coding mutations and loss of imprinting in human pluripotent cells derived by nuclear transfer and defined factors.
Cell Stem Cell 2014 Nov 6;15(5):634-42. Epub 2014 Nov 6.
The New York Stem Cell Foundation Research Institute, New York, NY 10032, USA; Naomi Berrie Diabetes Center & Department of Pediatrics, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA. Electronic address:

November 2014


Stem cells: Aspiring to naivety.
Nature 2016 12 30;540(7632):211-212. Epub 2016 Nov 30.
The Azrieli Center for Stem Cells and Genetic Research, Department of Genetics, Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel.
November 2016

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Other related articles on Genetic and Epigenetic phenomena in human pluripotent stem cells published by LPBI Group can be found in the following e-Books on

e-Books in Medicine

9 results for Kindle Store : “Aviva Lev-Ari”

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    Etiologies of Cardiovascular Diseases: Epigenetics, Genetics and Genomics

    Nov 28, 2015 | Kindle eBook

    by Justin D. Pearlman MD ME PhD MA FACC and Stephen J. Williams PhD
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    Cancer Therapies: Metabolic, Genomics, Interventional, Immunotherapy and Nanotechnology in Therapy Delivery (Series C Book 2)

    May 13, 2017 | Kindle eBook

    by Larry H. Bernstein and Demet Sag
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    Perspectives on Nitric Oxide in Disease Mechanisms (Biomed e-Books Book 1)

    Jun 20, 2013 | Kindle eBook

    by Margaret Baker PhD and Aviva Lev-Ari PhD RN
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    Cancer Biology and Genomics for Disease Diagnosis (Series C: e-Books on Cancer & Oncology Book 1)

    Aug 10, 2015 | Kindle eBook

    by Larry H Bernstein MD FCAP and Prabodh Kumar Kandala PhD
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    Genomics Orientations for Personalized Medicine (Frontiers in Genomics Research Book 1)

    Nov 22, 2015 | Kindle eBook

    by Sudipta Saha PhD and Ritu Saxena PhD
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    Metabolic Genomics & Pharmaceutics (BioMedicine – Metabolomics, Immunology, Infectious Diseases Book 1)

    Jul 21, 2015 | Kindle eBook

    by Larry H. Bernstein MD FCAP and Prabodah Kandala PhD
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    Milestones in Physiology: Discoveries in Medicine, Genomics and Therapeutics (Series E: Patient-Centered Medicine Book 3)

    Dec 26, 2015 | Kindle eBook

    by Larry H. Bernstein MD FACP and Aviva Lev-Ari PhD RN
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    Regenerative and Translational Medicine: The Therapeutic Promise for Cardiovascular Diseases

    Dec 26, 2015 | Kindle eBook

    by Justin D. Pearlman MD ME PhD MA FACC and Ritu Saxena PhD
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    Cardiovascular Original Research: Cases in Methodology Design for Content Co-Curation: The Art of Scientific & Medical Curation

    Nov 29, 2015 | Kindle eBook

    by Larry H. Bernstein MD FCAP and Aviva Lev-Ari PhD RN
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Reporter and Curator: Dr. Sudipta Saha, Ph.D.


Scientists think excessive population growth is a cause of scarcity and environmental degradation. A male pill could reduce the number of unintended pregnancies, which accounts for 40 percent of all pregnancies worldwide.


But, big drug companies long ago dropped out of the search for a male contraceptive pill which is able to chemically intercept millions of sperm before they reach a woman’s egg. Right now the chemical burden for contraception relies solely on the female. There’s not much activity in the male contraception field because an effective solution is available on the female side.


Presently, male contraception means a condom or a vasectomy. But researchers from Center for Drug Discovery at Baylor College of Medicine, USA are renewing the search for a better option—an easy-to-take pill that’s safe, fast-acting, and reversible.


The scientists began with lists of genes active in the testes for sperm production and motility and then created knockout mice that lack those genes. Using the gene-editing technology called CRISPR, in collaboration with Japanese scientists, they have so far made more than 75 of these “knockout” mice.


They allowed these mice to mate with normal (wild type) female mice, and if their female partners don’t get pregnant after three to six months, it means the gene might be a target for a contraceptive. Out of 2300 genes that are particularly active in the testes of mice, the researchers have identified 30 genes whose deletion makes the male infertile. Next the scientists are planning a novel screening approach to test whether any of about two billion chemicals can disable these genes in a test tube. Promising chemicals could then be fed to male mice to see if they cause infertility.


Female birth control pills use hormones to inhibit a woman’s ovaries from releasing eggs. But hormones have side effects like weight gain, mood changes, and headaches. A trial of one male contraceptive hormone was stopped early in 2011 after one participant committed suicide and others reported depression. Moreover, some drug candidates have made animals permanently sterile which is not the goal of the research. The challenge is to prevent sperm being made without permanently sterilizing the individual.


As a better way to test drugs, Scientists at University of Georgia, USA are investigating yet another high-tech approach. They are turning human skin cells into stem cells that look and act like the spermatogonial cells in the testes. Testing drugs on such cells might provide more accurate leads than tests on mice.


The male pill would also have to start working quickly, a lot sooner than the female pill, which takes about a week to function. Scientists from University of Dundee, U.K. admitted that there are lots of challenges. Because, a women’s ovary usually release one mature egg each month, while a man makes millions of sperm every day. So, the male pill has to be made 100 percent effective and act instantaneously.



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Regulatory MicroRNAs in Aberrant Cholesterol Transport and Metabolism

Curator: Marzan Khan, B.Sc

Aberrant levels of lipids and cholesterol accumulation in the body lead to cardiometabolic disorders such as atherosclerosis, one of the leading causes of death in the Western World(1). The physical manifestation of this condition is the build-up of plaque along the arterial endothelium causing the arteries to constrict and resist a smooth blood flow(2). This obstructive deposition of plaque is merely the initiation of atherosclerosis and is enriched in LDL cholesterol (LDL-C) as well foam cells which are macrophages carrying an overload of toxic, oxidized LDL(2). As the condition progresses, the plaque further obstructs blood flow and creates blood clots, ultimately leading to myocardial infarction, stroke and other cardiovascular diseases(2). Therefore, LDL is referred to as “the bad cholesterol”(2).

Until now, statins are most widely prescribed as lipid-lowering drugs that inhibit the enzyme 3-hydroxy-3methylgutaryl-CoA reductase (HMGCR), the rate-limiting step in de-novo cholesterol biogenesis (1). But some people cannot continue with the medication due to it’s harmful side-effects(1). With the need to develop newer therapeutics to combat cardiovascular diseases, Harvard University researchers at Massachusetts General Hospital discovered 4 microRNAs that control cholesterol, triglyceride, and glucose homeostasis(3)

MicroRNAs are non-coding, regulatory elements approximately 22 nucleotides long, with the ability to control post-transcriptional expression of genes(3). The liver is the center for carbohydrate and lipid metabolism. Stringent regulation of endogenous LDL-receptor (LDL-R) pathway in the liver is crucial to maintain a minimal concentration of LDL particles in blood(3). A mechanism whereby peripheral tissues and macrophages can get rid of their excess LDL is mediated by ATP-binding cassette, subfamily A, member 1 (ABCA1)(3). ABCA1 consumes nascent HDL particles- dubbed as the “good cholesterol” which travel back to the liver for its contents of triglycerides and cholesterol to be excreted(3).

Genome-wide association studies (GWASs) meta-analysis carried out by the researchers disclosed 4 microRNAs –(miR-128-1, miR-148a, miR-130b, and miR-301b) to lie close to single-nucleotide polymorphisms (SNPs) associated with abnormal metabolism and transport of lipids and cholesterol(3) Experimental analyses carried out on relevant cell types such as the liver and macrophages have proven that these microRNAs bind to the 3’ UTRs of both LDL-R and ABCA1 transporters, and silence their activity. Overexpression of miR-128-1 and miR148a in mice models caused circulating HDL-C to drop. Corroborating the theory under investigation further, their inhibition led to an increased clearance of LDL from the blood and a greater accumulation in the liver(3).

That the antisense inhibition of miRNA-128-1 increased insulin signaling in mice, propels us to hypothesize that abnormal expression of miR-128-1 might cause insulin resistance in metabolic syndrome, and defective insulin signaling in hepatic steatosis and dyslipidemia(3)

Further examination of miR-148 established that Liver-X-Receptor (LXR) activation of the Sterol regulatory element-binding protein 1c (SREBP1c), the transcription factor responsible for controlling  fatty acid production and glucose metabolism, also mediates the expression of miR-148a(4,5) That the promoter region of miR-148 contained binding sites for SREBP1c was shown by chromatin immunoprecipitation combined with massively parallel sequencing (ChIP-seq)(4). More specifically, SREBP1c attaches to the E-box2, E-box3 and E-box4 elements on miR-148-1a promoter sites to control its expression(4).

Earlier, the same researchers- Andres Naars and his team had found another microRNA called miR-33 to block HDL generation, and this blockage to reverse upon antisense targeting of miR-33(6).

These experimental data substantiate the theory of miRNAs being important regulators of lipoprotein receptors and transporter proteins as well as underscore the importance of employing antisense technologies to reverse their gene-silencing effects on LDL-R and ABCA1(4). Such a therapeutic approach, that will consequently lower LDL-C and promote HDL-C seems to be a promising strategy to treat atherosclerosis and other cardiovascular diseases(4).


1.Goedeke L1,Wagschal A2,Fernández-Hernando C3, Näär AM4. miRNA regulation of LDL-cholesterol metabolism. Biochim Biophys Acta. 2016 Dec;1861(12 Pt B):. Biochim Biophys Acta. 2016 Dec;1861(12 Pt B):2047-2052

2.MedicalNewsToday. Joseph Nordgvist. Atherosclerosis:Causes, Symptoms and Treatments. 13.08.2015

3.Wagschal A1,2, Najafi-Shoushtari SH1,2, Wang L1,2, Goedeke L3, Sinha S4, deLemos AS5, Black JC1,6, Ramírez CM3, Li Y7, Tewhey R8,9, Hatoum I10, Shah N11, Lu Y11, Kristo F1, Psychogios N4, Vrbanac V12, Lu YC13, Hla T13, de Cabo R14, Tsang JS11, Schadt E15, Sabeti PC8,9, Kathiresan S4,6,8,16, Cohen DE7, Whetstine J1,6, Chung RT5,6, Fernández-Hernando C3, Kaplan LM6,10, Bernards A1,6,16, Gerszten RE4,6, Näär AM1,2. Genome-wide identification of microRNAs regulating cholesterol and triglyceride homeostasis. . Nat Med.2015 Nov;21(11):1290

4.Goedeke L1,2,3,4, Rotllan N1,2, Canfrán-Duque A1,2, Aranda JF1,2,3, Ramírez CM1,2, Araldi E1,2,3,4, Lin CS3,4, Anderson NN5,6, Wagschal A7,8, de Cabo R9, Horton JD5,6, Lasunción MA10,11, Näär AM7,8, Suárez Y1,2,3,4, Fernández-Hernando C1,2,3,4. MicroRNA-148a regulates LDL receptor and ABCA1 expression to control circulating lipoprotein levels. Nat Med. 2015 Nov;21(11):1280-9.

5.Eberlé D1, Hegarty B, Bossard P, Ferré P, Foufelle F. SREBP transcription factors: master regulators of lipid homeostasis. Biochimie. 2004 Nov;86(11):839-48.

6.Harvard Medical School. News. MicoRNAs and Metabolism.

7. MGH – Four microRNAs identified as playing key roles in cholesterol, lipid metabolism


Other related articles published in this Open Access Online Scientific Journal include the following:


  • Cardiovascular Diseases, Volume Three: Etiologies of Cardiovascular Diseases: Epigenetics, Genetics and Genomics,

on Amazon since 11/29/2015


HDL oxidation in type 2 diabetic patients

Larry H. Bernstein, MD, FCAP, Curator


HDL-C: Target of Therapy – Steven E. Nissen, MD, MACC, Cleveland Clinic vs Peter Libby, MD, BWH

Reporter: Aviva Lev-Ari, PhD, RN


High-Density Lipoprotein (HDL): An Independent Predictor of Endothelial Function & Atherosclerosis, A Modulator, An Agonist, A Biomarker for Cardiovascular Risk

Curator: Aviva Lev-Ari, PhD, RN


Risk of Major Cardiovascular Events by LDL-Cholesterol Level (mg/dL): Among those treated with high-dose statin therapy, more than 40% of patients failed to achieve an LDL-cholesterol target of less than 70 mg/dL.

Reporter: Aviva Lev-Ari, PhD., RN


LDL, HDL, TG, ApoA1 and ApoB: Genetic Loci Associated With Plasma Concentration of these Biomarkers – A Genome-Wide Analysis With Replication

Reporter: Aviva Lev-Ari, PhD, RN


Two Mutations, in the PCSK9 Gene: Eliminates a Protein involved in Controlling LDL Cholesterol

Reporter: Aviva Lev-Ari, PhD, RN

Artherogenesis: Predictor of CVD – the Smaller and Denser LDL Particles

Reporter: Aviva Lev-Ari, PhD, RN


A Concise Review of Cardiovascular Biomarkers of Hypertension

Curator: Larry H. Bernstein, MD, FCAP


Triglycerides: Is it a Risk Factor or a Risk Marker for Atherosclerosis and Cardiovascular Disease ? The Impact of Genetic Mutations on (ANGPTL4) Gene, encoder of (angiopoietin-like 4) Protein, inhibitor of Lipoprotein Lipase

Reporters, Curators and Authors: Aviva Lev-Ari, PhD, RN and Larry H. Bernstein, MD, FCAP


Excess Eating, Overweight, and Diabetic

Larry H Bernstein, MD, FCAP, Curator


Obesity Issues

Larry H. Bernstein, MD, FCAP, Curator


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Researchers determine how part of the endoplasmic reticulum gets its TUBULAR shape

Reporter: Aviva Lev-Ari, PhD, RN


It’s Tubular

Researchers determine how part of the endoplasmic reticulum gets its shape

From the double membrane enclosing the cell nucleus to the deep infolds of the mitochondria, each organelle in our cells has a distinctive silhouette that makes it ideally suited to do its job. How these shapes arise, however, is largely a mystery.

Harvard Medical School cell biologists have now cracked the code for part of the endoplasmic reticulum (ER), a protein- and fat-making organelle that consists of stacked sheets in some parts and a complex network of tubules in others.

Producing the ER’s tubular network is “surprisingly simple,” requiring just three ingredients, principal investigator Tom Rapoport, professor of cell biology at HMS, and colleagues report Feb. 22 in Nature.


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3D Liver Model in a Droplet

Curator: Marzan Khan, BSc

Recently, a Harvard University Professor of Physics and Applied Physics, David Weitz and his team of researchers have successfully generated 3D models of liver tissue composed of two different kinds of liver cells, precisely compartmentalized in a core-shell droplet, using the microfluidics approach(1). Compared to alternative in-vitro methods, this approach comes with more advantages – it is cost-effective, can be quickly assembled and produces millions of organ droplets in a second(1). It is the first “organ in a droplet” technology that enables two disparate liver cells to physically co-exist and exchange biochemical information, thus making it a good mimic of the organ in vivo(1).

Liver tissue models are used by researchers to investigate the effect of drugs and other chemical compounds, either alone or in combination on liver toxicity(2). The liver is the primary center of drug metabolism, detoxification and removal and all of these processes need to be carried out systematically in order to maintain a homeostatic environment within the body(2) Any deviation from the steady state will shift the dynamic equilibrium of metabolism, leading to production of reactive oxygen species (ROS)(2). These are harmful because they will exert oxidative stress on the liver, and ultimately cause the organ to malfunction. Drug-induced liver toxicity is a critical problem – 10% of all cases of acute hepatitis, 5% of all hospital admissions, and 50% of all acute liver failures are caused by it(2).

Before any novel drug is launched into the market, it is tested in-vitro, in animal models, and then progresses onto human clinical trials(1). Weitz’s system can produce up to one-thousand organ droplets per second, each of which can be used in an experiment to test for drug toxicity(1). Clarifying further, he asserts that “Each droplet is like a mini experiment. Normally, if we are running experiments, say in test tubes, we need a milliliter of fluid per test tube. If we were to do a million experiments, we would need a thousand liters of fluid. That’s the equivalent of a thousand milk jugs! Here, each droplet is only a nanoliter, so we can do the whole experiment with one milliliter of fluid, meaning we can do a million more experiments with the same amount of fluid.”

Testing hepatocytes alone on a petri dish is a poor indicator of liver-specific functions because the liver is made up of multiple cells systematically arranged on an extracellular matrix and functionally interdependent(3). The primary hepatocytes, hepatic stellate cells, Kupffer cells, endothelial cells and fibroblasts form the basic components of a functioning liver(3). Weitz’s upgraded system contains hepatocytes (that make up the majority of liver cells and carry out most of the important functions) supported by a network of fibroblasts(3). His microfluidic chip is comprised of a network of constricted, circular channels spanning the micrometer range, the inner phase of which contains hepatocytes mixed in a cell culture solution(3). The surrounding middle phase accommodates fibroblasts in an alginate solution and the two liquids remain separated due to differences in their chemical properties as well as the physics of fluids travelling in narrow channels. Addition of a fluorinated carbon oil interferes with the two aqueous layers, forcing them to become individual monodisperse droplets(3). The hydrogel shell is completed when a 0.15% solution of acetic acid facilitates the cross-linking of alginate to form a gelatinous shell, locking the fibroblasts in place(3). Thus, the aqueous core of hepatocytes are encapsulated by fibroblasts confined to a strong hydrogel network, creating a core-shell hydrogel scaffold of 3D liver micro-tissue in a droplet(3). Using empirical analysis, scientists have shown that albumin secretion and urea synthesis (two important markers of liver function) were significantly higher in a co-culture of hepatocytes and fibroblasts 3D core-shell spheroids compared to a monotypic cell-culture of hepatocyte-only spheroids(3). These results validate the theory that homotypic as well as heterotypic communication between cells are important to achieve optimal organ function in vitro(3).

This system of creating micro-tissues in a droplet with enhanced properties is a step-forward in biomedical science(3). It can be used in experiments to test for a myriad of drugs, chemicals and cosmetics on different human tissue samples, as well as to understand the biological connectivity of contrasting cells(3).


Image source: DOI: 10.1039/c6lc00231

A simple demonstration of the microfluidic chip that combines different solutions to create a core-shell droplet consisting of two different kinds of liver cells.


  1. National Institute of Biomedical Imaging and Bioengineering. (2016, December 13). New device creates 3D livers in a droplet.ScienceDaily. Retrieved February 9, 2017 from
  2. Singh, D., Cho, W. C., & Upadhyay, G. (2015). Drug-Induced Liver Toxicity and Prevention by Herbal Antioxidants: An Overview.Frontiers in Physiology,6, 363.
  3. Qiushui Chen, Stefanie Utech, Dong Chen, Radivoje Prodanovic, Jin-Ming Lin and David A. Weitz; Controlled assembly of heterotypic cells in a core– shell scaffold: organ in a droplet; Lab Chip, 2016, 16, 1346; DOI: 10.1039/c6lc00231

Other related articles on 3D on a Chip published in this Open Access Online Scientific Journal include the following:


What could replace animal testing – ‘Human-on-a-chip’ from Lawrence Livermore National Laboratory

Reporter: Aviva Lev-Ari, PhD, RN

AGENDA for Second Annual Organ-on-a-Chip World Congress & 3D-Culture Conference, July 7-8, 2016, Wyndham Boston Beacon Hill by SELECTBIO US

Reporter: Aviva Lev-Ari, PhD, RN

Medical MEMS, BioMEMS and Sensor Applications

Curator and Reporter: Aviva Lev-Ari, PhD, RN

Contribution to Inflammatory Bowel Disease (IBD) of bacterial overgrowth in gut on a chip

Larry H. Bernstein, MD, FCAP, Curator

Current Advances in Medical Technology

Larry H. Bernstein, MD, FCAP, Curator


Other related articles on Liver published in this Open Access Online Scientific Journal include the following:


Alnylam down as it halts development for RNAi liver disease candidate

by Stacy Lawrence

LIVE 9/21 8AM to 2:40PM Targeting Cardio-Metabolic Diseases: A focus on Liver Fibrosis and NASH Targets at CHI’s 14th Discovery On Target, 9/19 – 9/22/2016, Westin Boston Waterfront, Boston

Reporter: Aviva Lev-Ari, PhD, RN

2016 Nobel in Economics for Work on The Theory of Contracts to winners: Oliver Hart and Bengt Holmstrom

Reporter: Aviva Lev-Ari, PhD, RN

LIVE 9/20 2PM to 5:30PM New Viruses for Therapeutic Gene Delivery at CHI’s 14th Discovery On Target, 9/19 – 9/22/2016, Westin Boston Waterfront, Boston

Reporter: Aviva Lev-Ari, PhD, RN

Seven Cancers: oropharynx, larynx, oesophagus, liver, colon, rectum and breast are caused by Alcohol Consumption

Reporter: Aviva Lev-Ari, PhD, RN


Other related articles on 3D on a Chip published in this Open Access Online Scientific Journal include the following:


Liquid Biopsy Chip detects an array of metastatic cancer cell markers in blood – R&D @Worcester Polytechnic Institute,  Micro and Nanotechnology Lab

Reporters: Tilda Barliya, PhD and Aviva Lev-Ari, PhD, RN

Trovagene’s ctDNA Liquid Biopsy urine and blood tests to be used in Monitoring and Early Detection of Pancreatic Cancer

Reporters: David Orchard-Webb, PhD and Aviva Lev-Ari, PhD, RN

Liquid Biopsy Assay May Predict Drug Resistance

Curator: Larry H. Bernstein, MD, FCAP

One blood sample can be tested for a comprehensive array of cancer cell biomarkers: R&D at WPI

Curator: Marzan Khan, B.Sc

Real Time Coverage of the AGENDA for Powering Precision Health (PPH) with Science, 9/26/2016, Cambridge Marriott Hotel, Cambridge, MA

Reporter: Aviva Lev-Ari, PhD, RN



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ATP – the universal energy carrier in the living cell: Reflections on the discoveries and applications in Medicine

Curators: Larry H. Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN


This article has two parts:

Part One: Reflections on the discoveries and applications in Medicine

Part Two: ATP – the universal energy carrier in the living cell: Reflections on the discoveries


Part One:

Reflections on the discoveries and applications in Medicine


From: “Dr. Larry Bernstein” <>

Reply-To: “Dr. Larry Bernstein” <>

Date: Friday, December 23, 2016 at 1:15 PM

To: Aviva Lev-Ari <>

Cc: Harry Maisel <>, Jose E S Roselino <>

Thank you Larry,

This is a very interesting topic addressed by the chemistry Nobel laureates in their lecture. It is rather important to stress the numbers presented in this lecture. ATP use and production in 24 hours in a 70 Kg bw human may vary from 35 Kg  to 1 ton in two extreme conditions (Basal metabolism or very heavy work). This very large range calls for the very fast regulatory mechanism that I always call attention upon, or better saying those regulatory mechanisms that must occur without any change in gene expression.

Furthermore, the effect of Oligomicin shows a clear mechanism of induced fit ince it binds to FO and affects F1.

It is interesting that there was more than a decade of debate about high energy phosphate bond and the role of ATP, which Boyer tried to moderate.  Britten Chance proposed a complicated mechanism, but even though he was not awarded a Nobel Prize, few biochemists made so many large contributions to our understanding of respiration.  My medical school biochemistry exam required a response to whether Chance deserved a Nobel Prize.  My mentor who identified that the liver adenylate kinase was different than muscle AK (myokinase) surmised that Chance’s work was phenomenally on instrumentation.

The electron transport chain was proposed by Peter Mitchell, who did work in a home laboratory.

On Wed, Dec 21, 2016 at 1:44 PM, Aviva Lev-Ari <> wrote:

From: “Dr. Larry Bernstein” <>

Reply-To: “Dr. Larry Bernstein” <>

Date: Thursday, December 22, 2016 at 7:32 PM

To: Aviva Lev-Ari <>

Cc: Harry Maisel <>, Jose E S Roselino <>

An important role was that played by the 1953 Nobel laureate in Medicine Fritz Lipmann when he during the years 1939-41 showed that ATP is the universal carrier of chemical energy in the cell and coined the expression “energy-rich phosphate bonds”.

Kaplan had a significant role in the discovery. He was subsequently recognized for his contribution, not in the Nobel Prize.  In 1970, he rivaled Art Karmen at Stanford.  His Harvard lecture on the transhydrogenases in 1971 was hugely important.

Part Two:

ATP – the universal energy carrier in the living cell: Reflections on the discoveries


1997 Nobel Prize in Chemistry – for their elucidation of the enzymatic mechanism underlying the synthesis of adenosine triphosphate (ATP)

Press Release

15 October 1997

The Royal Swedish Academy of Sciences has decided to award the 1997 Nobel Prize in Chemistry with one half to

Professor Paul D. Boyer, University of California, Los Angeles, USA, and

Dr. John E. Walker, Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom

for their elucidation of the enzymatic mechanism underlying the synthesis of adenosine triphosphate (ATP)

and with one half to

Professor Jens C. Skou, Aarhus University, Denmark

for the first discovery of an ion-transporting enzyme, Na + , K + -ATPase.

The three laureates have performed pioneering work on enzymes that participate in the conversion of the “high-energy” compound adenosine triphosphate (ATP).

Paul D. Boyer and John E. Walker receive half the prize for their work on how the enzyme ATP synthase catalyses the formation of ATP. Boyer and his co-workers have proposed, on the basis of biochemical data, a mechanism for how ATP is formed from adenosine diphosphate (ADP) and inorganic phosphate. Walker and his co-workers have established the structure of the enzyme and verified the mechanism proposed by Boyer.

Jens C. Skou receives his half of the prize for the discovery of the enzyme sodium, potassium-stimulated adenosine triphosphatase (Na + , K + -ATPase). This enzyme maintains the balance of sodium and potassium ions in the living cell.

Both enzymes are bound to membranes in the cell and linked with the transport of ions through these – but for different reasons.

ATP – the universal energy carrier in the living cell

The German chemist Karl Lohmann discovered ATP in 1929. Its structure was clarified some years later and in 1948 the Scottish Nobel laureate of 1957 Alexander Todd synthesised ATP chemically. An important role was that played by the 1953 Nobel laureate in Medicine Fritz Lipmann when he during the years 1939-41 showed that ATP is the universal carrier of chemical energy in the cell and coined the expression “energy-rich phosphate bonds”.

ATP functions as a carrier of energy in all living organisms from bacteria and fungi to plants and animals including humans. ATP captures the chemical energy released by the combustion of nutrients and transfers it to reactions that require energy, e.g. the building up of cell components, muscle contraction, transmission of nerve messages and many other functions. ATP has been termed the cell’s energy currency.

Adenosine triphosphate (ATP) consists of the nucleoside adenosine linked to three phosphate groups. On removal of the outermost phosphate group, adenosine diphosphate (ADP) is formed while at the same time the energy released can be employed for other reactions. Conversely, with the help of energy, an inorganic phosphate group can be bound to ADP and form ATP. Considerable quantities of ATP are formed and consumed. At rest, an adult converts daily a quantity of ATP corresponding to about one half body-weight, and during hard work the quantity can rise to almost a ton. Most of the ATP synthesis is carried out by the enzyme ATP synthase. At rest Na + , K + -ATPase uses up a third of all ATP formed.

ATP synthase – an exceptional molecular machine

During the 1940s and 1950s it was clarified that the bulk of ATP is formed in cell respiration in the mitochondria and photosynthesis in the chloroplasts of plants. In 1960 the American scientist Efraim Racker and co-workers isolated, from mitochondria, the enzyme “F o F 1 ATPase” which we now call ATP synthase. The enzyme can be divided into an F 1 part containing the catalytic center and the F o part coupling the F 1 part to the membrane. The same enzyme exists in chloroplasts and bacteria. In 1961 Peter Mitchell presented what is termed the chemiosmotic hypothesis for which he received the Nobel Prize in 1978. He showed that cell respiration leads to a difference in hydrogen ion concentration (pH) inside and outside the mitochondrial membrane, and that a stream of hydrogen ions drives the formation of ATP. The same applies to the chloroplast membrane. The coupling of ATP synthase to hydrogen ion transport takes place via the F opart.

Paul D. Boyer began his studies of ATP formation in the early 1950s and is still highly active as a scientist. His chief interest has been to find out by isotope techniques how ATP synthase functions and particularly how it uses energy to create new ATP. His work has been crowned with unusual success in the past few years. ATP synthase has a mode of function that is unusual for enzymes, and this required much time and extensive studies to establish. John E. Walker made his first studies of ATP synthase at the beginning of the 1980s. His starting point was that a detailed chemical and structural knowledge of an enzyme is required to understand in detail how it functions. He therefore determined the amino acid sequences in the constituent protein units. During the 1990s he has collaborated with crystallographers to clarify the three-dimensional structure of ATP synthase. So far, the structure of the enzyme’s F 1 part has been established. Walker’s work complements Boyer’s in a remarkable manner and further studies based on this structure demonstrate the correctness of the mechanism proposed by Boyer.

Figure 1.

Simplified picture of ATP syntase

The Fo part through which hydrogen ions (H+ ) stream is located in the membrane. The F1part which synthesises ATP is outside the membrane. When the hydrogen ions flow through the membrane via the disc of c subunits in the Fo part, the disc is forced to twist around. The gamma subunit in the F1 part is attached to the disc and therefore rotates with it. The three alpha and three beta subunits in the F1 part cannot rotate, however. They are locked in a fixed position by the b subunit. This in turn is anchored in the membrane. Thus the gamma subunit rotates inside the cylinder formed by the six alpha and beta subunits. Since the gamma subunit is asymmetrical it compels the beta subunits to undergo structural changes. This leads to the beta subunits binding ATP and ADP with differing strengths (see Figure 2).

As mentioned above, ATP synthase (Fig. 1) consists of a membrane-bound part, F o , which transports hydrogen ions, and a protruding part (F 1 ) which can be released from the membrane. (The terms are historical, and F 1stands for factor 1 and F o for oligomycin-sensitive factor). Each F o part consists of three types of subunits in differing numbers, the proteins a (1), b (2) and c (9-12). The F 1 part consists of five subunits, alpha, beta, gamma, delta and epsilon. While there are three each of alpha and beta, there is only one unit of each of the others. It has been shown that it is on the beta units where the synthesis of ATP occurs. The analysis of the amino acid sequences that Walker and co-workers did at the beginning of the 1980s showed that subunits gamma, delta and epsilon are not symmetrical, a feature of importance for our understanding of how ATP synthase functions.

The most detailed studies of ATP synthase concern the F 1 part and how it functions. Boyer and co-workers clarified that the enzyme functions in a very particular way. They found that, as opposed to the view generally held, the step requiring energy was not the synthesis of ATP from ADP and inorganic phosphate, but that energy was required to bind ADP and the phosphate to the enzyme and to release ATP. Nevertheless an energy surplus was stored in the ATP. In this respect ATP synthase differs from the majority of all enzymes, which bind and release substrates and products spontaneously, but for which the overall catalytic reaction requires energy. A further observation was that despite the asymmetrical character of F 1 , there is only one way for the enzyme to react. But how then can the three beta subunits function in the same way if they have different couplings to subunits gamma, delta and epsilon? Boyer found the answer to this question by clarifying that gamma, delta and epsilon rotate in a cylinder formed of alternating alpha and beta subunits. This rotation induces structural changes in beta which lead to differences in bonding ability during a cyclical course (see Figure 2). This mechanism is called Boyer’s “Binding Change Mechanism”. Boyer also proposed that this rotation is driven by the above-mentioned hydrogen ion flow through the membrane.

Figure 2.

Boyer’s “Binding Change Mechanism”

The picture shows the cylinder with alternating alpha and beta subunits at four different stages of ATP synthesis. The asymmetrical gamma subunit that causes changes in the structure of the beta subunits can be seen in the centre. The structures are termed open betaO (light grey sector), loose betaL (grey sector) and tight betaT (black sector). At stage A we see an already-fully-formed ATP molecule bound to betaT. In the step to stage B betaLbinds ADP and inorganic phosphate (Pi ). At the next stage, C, we see how the gamma subunit has twisted due to the flow of hydrogen ions (see Figure 1). This brings about changes in the structure of the three beta subunits. The tight beta subunit now becomes open and the bound ATP molecule is released. The loose beta subunit becomes tight and the open becomes loose. In the last stage the chemical reaction takes place in which phosphate ions react with the ADP molecule to form a new ATP molecule. We are back at the first stage.

Boyer has called ATP synthase a molecular machine. It may be compared to a water-driven hammer minting coins. The F o part is the wheel, the flow of protons is the waterfall and the structural changes in F 1 lead to three coins in the ATP currency being minted for each turn of the wheel.

Walker clarified the structural conditions of the enzyme’s molecular machinery and thereby verified Boyer’s mechanism. The crystallographic structure of the F 1 part of ATP synthase from cows, determined chiefly in collaboration with the Dutchman J.P. Abrahams and the Englishman A. Leslie, shows partly that the alpha and beta subunits are related in terms of structure and evolution and partly that they have clearly differing structures and therefore differing abilities to bind ADP and ATP. The gamma subunit is placed as an asymmetrical axle in the cylinder formed by the three alpha and the three beta subunits and has unique contacts with the beta units and forces their active surfaces to assume different three-dimensional structures. These results can be interpreted according to Boyer’s mechanism to mean that the enzyme functions through rotation of the gamma subunits. It has been difficult to demonstrate this rotation experimentally but several groups have now succeeded. Wolfgang Junge in Germany used spectroscopic techniques and the American scientist Richard Cross chemical cross-bonding. Recently a Japanese group under Masasuke Yoshida succeeded in visualising the rotation in the F 1 part of ATP synthase. They attached a fibre of the muscle protein actin to the gamma subunit, and the beta units were attached to the substratum. Depending on the ATP concentration in the surrounding liquid it was possible to show under a microscope how the actin fibre rotated at increasing speed with increasing ATP concentration.

Na+, K+-ATPase, the first molecular pump to be discovered
It was known as early as the 1920s that the ion composition within living cells is different from that in the surroundings. Within the cells the sodium concentration is lower and the potassium concentration higher than in the liquid outside. Through the work of the Englishmen Richard Keynes and Alan Hodgkin at the beginning of the 1950s (Hodgkin received the Nobel Prize in 1963) it was known that when a nerve is stimulated sodium ions pour into the nerve cell. The difference in concentration is restored by sodium being transported out once again. That this transport required ATP was probable since the transport could be inhibited in the living cell by inhibiting the formation of ATP.

With this as the starting point Jens C. Skou searched for an ATP-degrading enzyme in the nerve membrane that could be associated with ion transport. In 1957 he published the first article on an ATPase, which was activated by sodium and potassium ions (Na + , K + -ATPase). He was the first to describe an enzyme that can promote directed (vectored) transport of substances through a cell membrane, a fundamental property of all living cells. Numerous enzymes have since been demonstrated to have essentially similar functions.

Skou used as experimental material finely ground crab nerve membranes. The ATP-degrading enzyme found in the preparation required the presence of magnesium ions and was stimulated with increasing quantities of sodium ions up to a certain limit. Above this Skou was able to obtain further stimulation if he added small quantities of potassium ions. An indication that the enzyme was coupled to the ion pump was that maximal stimulation was obtained at the concentrations of sodium and potassium that normally occur in the nerve. In his further studies of the enzyme mechanism Skou showed that sodium ions and potassium ions bind with high affinity to different places in the enzyme. In addition he showed that the phosphate group separated from ATP also binds to ATPase. This is described as a phosphorylation of the enzyme. The enzyme is dependent on sodium ions when it is phosphorylated and on potassium ions when it is dephosphorylated. Substances known to inhibit sodium/potassium transport are certain digitalis alkaloids, e.g. oubain, and Skou showed that oubain interferes in the enzyme’s activation by sodium.

The picture that slowly emerged from Skou’s and others’ work is that the enzyme consists of two subunits, alpha and beta. The first carries the enzyme’s activity and the other presumably stabilises the structure. The enzyme molecules are located in the cell membrane, often in twos, and expose surfaces to the outside as well as the inside. Three sodium ions and ATP bind to the interior surface. A phosphate is then transferred from ATP to an amino acid in the enzyme, aspartic acid, whereupon the ADP is liberated and the enzyme changes form so that the sodium ions are transported to the outside. Here they are released and two potassium ions attach instead. When the phosphorus that is bound to the enzyme is removed the potassium ions are transported into the cell and when new ATP binds to the enzyme they are rejected.

As a result of the action of the Na + , K + -ATPase, the cell keeps a high concentration of potassium in its inside. As the cell membrane is rather permeable for potassium ions, a few of these potassium ions leak out, leaving unpermeable, negative charges on the inside of the cell. Therefore, the inside of the cell membrane becomes electrically negatively charged, as compared to the outside.

This difference in potential across the membrane is necessary for a nerve stimulation to propagate along a nerve fibre or a muscle cell. This is why a shortage of nourishment or oxygen in the brain rapidly leads to unconsciousness since the ATP formation ceases and the ion pump stops. The pump is also important for maintaining cell volume. If the pump stops, the cell swells. The difference in sodium concentration between the interior and the exterior is the driving force in the uptake of important nutrients necessary to the cell, e.g. glucose and amino acids. It can also be used for transport of other ions through the cell membrane. Thus sodium ions that enter can be exchanged for calcium ions that exit.

Following the discovery of Na + , K + -ATPase other ion pumps have been discovered with similar structures and functions. Examples are Ca 2+ >-ATPase in skeletal muscle, which participates in the control of muscle contraction and H + , K + -ATPase which produces hydrochloric acid in the stomach. It is the latter enzyme that is specifically inhibited in modern treatment of stomach ulcers. Corresponding enzymes are also found in lower organisms, for example in yeast where an H + -ATPase secretes hydrogen ions formed during fermentation. As a common name these enzymes are nowadays termed P-type ATPases since they are phosphorylated during the course of the reaction.


Further reading


Paul D. Boyer and John E. Walker

Boyer, P.D., The binding change mechanism for ATP synthase – Some probabilities and possibilities, Biochimica et Biophysica Acta (1993) 1140, 215-250.

Abrahams, J.P., Leslie, A.G., Lutter, R., and Walker J.E., Structure at 2.8 Å resolution of F 1 -ATPase from bovine heart mitochondria, Nature (1994) 370, 621-628.

Boyer, P.D., The ATP synthase – a splendid molecular machine, Annual Review in Biochemistry (1997) 66, 717-749.


Jens C. Skou

Skou, J.C., The influence of some cations on an adenosine triphosphatase from peripheral nerves, Biochimica et Biophysica Acta (1957) 23, 394-401.

Skou, J.C., and Esmann, M., The Na, K-ATPase, Journal of Bioenergetics and Biomembranes (1992) 24, 249-261.

Lingrel, J.B., Na-K-ATPase: Isoform Structure, Function, and Expression, Journal of Bioenergetics and Biomembranes (1992) 24, 263-270.

Möller, J.V., Juul, B., and le Maire, M., Structural organization, ion transport, and energy transduction of P-type ATPases, Biochimica et Biophysica Acta (1996) 1286, 1-51.

Lutsenko, S. and Kaplan, J.H., Organization of P-type ATPases: Significance of structural diversity, Biochemistry (1996) 34, 15607-15613.


Professor Paul D. Boyer was born 1918 in Provo, Utah, USA. Ph.D. in Biochemistry 1943, University of Wisconsin, Madison, USA. From 1963 to 1989 he was Professor of Chemistry at Department of Chemistry and Biochemistry, University of California at Los Angeles (UCLA), and from 1965 to 1983 Director of the Molecular Biology Institute, UCLA. Since 1990 he has been Professor Emeritus at Department of Chemistry and Biochemistry, UCLA. Boyer has been a member of the National Academy of Sciences since 1970. He received an honorary doctorate from Stockholm University in 1974 and in 1989 he was awarded the Rose Award of the American Society of Biochemistry and Molecular Biology.

Professor Paul D. Boyer

Department of Chemistry and Biochemistry

University of California

Los Angeles, CA 90024, USA

Dr. John E. Walker was born 1941 in Halifax, Great Britain. He received M.A. and Dr.Phil. at Oxford University, Great Britain. Since 1982 Walker has been Senior Scientist at the Medical Research Council Laboratory of Molecular Biology, Cambridge, Great Britain. He was elected to the Royal Society, London, in 1995.

Dr. John E. Walker

Medical Research Council Laboratory of Molecular Biology

Hills Road

Cambridge, CB2 2QH


Professor Jens C. Skou was born 1918 in Denmark. He received his medical training at Copenhagen University. In 1954 Skou received his doctoral degree at Aarhus University, where he became Professor of Physiology in 1963. He was appointed Professor of Biophysics in 1977 at the same university. Skou is a member of the Danish Academy of Sciences.

Professor Jens C. Skou

Aarhus University

Nordre Ringgade

DK-8000 Aarhus



“The Nobel Prize in Chemistry 1997”. Nobel Media AB 2014. Web. 27 Dec 2016. <>

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