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Posts Tagged ‘Nobel Prize’


How to win the Nobel prize

Larry H Bernstein, MD, FCAP, Curator

LPBI

Behind closed doors: How to win the Nobel prize

5 October 2015 Chemistry World

http://www.rsc.org/chemistryworld/2015/09/how-win-nobel-prize-chemistry

The Nobel prize, now in its 120th year, remains the most sought-after of scientific prizes, to many the pinnacle of scientific achievement. But few know the process by which the winner or winners are chosen.Chemistry World goes behind closed doors to find out how the Nobel committee make their selection.

Our guide on this voyage of discovery is Bengt Norden, chair professor of physical chemistry of Chalmers University of Technology. Norden was a member of the Nobel committee for chemistry – the body responsible for recommending potential laureates – from 1995 to 2004, and counts a great number of Nobel laureates amongst his personal friends. To celebrate his 70th birthday, he held the ‘Amazing week’ at Chalmers University, inviting 40 leading scientists – including twelve Nobel laureates – to speak about their research.

In this series of videos, Norden walks us through the selection criteria, the nomination process and the investigations they undertake (keep an eye out for Swedes hiding in the bushes outside your lab). He also addresses some of the myths and rumours surrounding the prize and takes us through his favourite examples, as well as some who sadly missed their opportunity.

Criteria

‘The achievement should somehow open a door, or open our eyes. We will see things in a different way’

Is the Nobel prize awarded for a lifetime of scientific achievement? Norden discusses the critera against which research is judged

https://youtu.be/dqllfpmhKf4

Nominations

‘It never, ever happens that one who gets nominated for the first time gets the prize.’

Who nominates people for the Nobel prize? In the second in our series of videos, Bengt explains the nomination process.

https://youtu.be/dSMKkg7ewvU

Investigations

‘Then we dig until we are satisfied’

In this video, Bengt reveals how the committee investigates nominees to make sure the prize goes to the right person.

https://youtu.be/VXKCKGcU6wg

Chemistry on rotation?

‘Our artificial division into the five parts of chemistry, for instance, is irrelevant’

Should the chemistry prize only be given to chemists? And should we rotate between different chemistry disciplines? This time, Bengt tackles the perception that different fields ‘take it in turns’.

https://youtu.be/PuXoHR8G2jA

Awards to the many

‘One person is ideal … you make somebody an icon’

According to the rules, the Nobel prize should only be awarded to a maximum of three people. The peace prize has twisted this rule. Will we soon see the other prizes follow suit?

https://youtu.be/n6rvM9MsQfE

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DNA Repair Pioneers Win Nobel

Larry H. Bernstein, MD, FCAP, Curator

LPBI

Tomas Lindahl, Paul Modrich, and Aziz Sancar have won this year’s Nobel Prize in Chemistry for their work elucidating mechanisms of DNA repair.

By Tracy Vence | October 7, 2015

http://www.the-scientist.com//?articles.view/articleNo/44182/title/DNA-Repair-Pioneers-Win-Nobel/

Tomas Lindahl, Paul Modrich, and Aziz Sancar today (October 7) took the 2015 Nobel Prize in Chemistry for their seminal research on DNA repair. The three scientists share equally in the prize “for having mapped, at a molecular level, how cells repair damaged DNA and safeguard the genetic information,” according to a Nobel Foundation statement.

“All living cells have repair mechanisms . . . to counter DNA damage,” Lindahl said during a Nobel Foundation press conference following the prize announcement. “[DNA damage] can result in a number of diseases, including cancer.” Asked about sharing in a Nobel Prize, he told reporters: “I feel very lucky and proud to be selected.”

Lindahl is a member of the Nobel Prize in Chemistry’s selection committee but did not participate in this year’s prize selection. He is a professor emeritus at the U.K.’s Francis Crick Institute. In the early 1970s, he was among the first to describe base excision repair, a process that works to patch decaying DNA throughout the cell cycle.

“Tomas was a kind of giant in the field and he has made really, really profound contributions to all aspects of DNA repair and DNA decay,” said Lindahl’s colleague Peter Karran of the Francis Crick Institute.

Later, Modrich, a professor of biochemistry and genomics at the Duke University Medical Center in Durham, North Carolina, described mismatch repair, which reduces the frequency of DNA replication-related errors. Sancar, a professor of biochemistry and biophysics at the University of North Carolina School of Medicine in Chapel Hill, described nucleotide excision repair, which cells use to counteract the effects of mutagens.

This year’s prize “is a very timely recognition of the field,” Karran said. “The field has made tremendous contributions to understanding how cancer develops, for example, and these people have made enormous contributions to understanding cancer, as well as to the basic science of DNA.”

Update (7:06 a.m.): Sancar “spent his entire career working in DNA repair,” Christopher Selby,  a research assistant professor at the University of North Carolina who has worked in Sancar’s lab since 1987, told The Scientist. “It’s exciting and interesting to see what it takes to get an award like that. . . . I’ve been fairly close with him throughout his career and I [have now] seen firsthand what goes into generating a body of work that goes into that sort of thing.”

Update (7:26 a.m.): “These guys have [made] tremendous, longstanding contributions to DNA repair, which is what keeps us all alive,” David Lilley, a professor of molecular biology at the University of Dundee, U.K., told The Scientist. “DNA is your library in the cell—you’ve gotta repair it, and it’s under massive onslaught. These [DNA repair] mechanisms are tremendously important.”

Update (7:34 a.m.): “I am absolutely thrilled. Paul was really there at the ground level. He really discovered these proteins in the DNA mismatch repair pathway,” said Lorena Beese, a professor of biochemistry at Duke University who has collaborated with Modrich since the mid-1990s. DNA repair “is such an essential area [of research],” Beese told The Scientist. “Mismatch repair, in particular—there are so many questions left on how this essential process works.”

GEN News Highlights

Oct 7, 2015

Nobel Prize in Chemistry Awarded to DNA Repair Researchers

Thomas Lindahl, Ph.D., Paul Modrich, Ph.D., and Aziz Sancar, M.D., Ph.D., have made fundamental contributions to the study of how cells repair DNA and maintain genomic integrity. [N. Elmehed. © Nobel Media 2015]

http://www.genengnews.com/media/images/GENHighlight/Oct7_2015_NElmehedNobelMedia2015_ChemistryNobelPrize3312717921.jpg

Recipients of the Nobel Prize in Chemistry were announced today acknowledging three scientists that  made fundamental contributions to the study of how cells repair DNA and maintain genomic integrity.

Each day our DNA is damaged by UV radiation, free radicals, and other carcinogenic substances. However, even without such external attacks, a DNA molecule within the cell is inherently unstable. Thousands of spontaneous changes to a cell’s genome occur on a daily basis and defects can arise when DNA is copied during cell division, a process that occurs several million times every day in the human body.

The reason our genetic material does not disintegrate into complete chemical chaos is that a host of molecular systems continuously monitor and repair DNA.Most cells use three main pathways to repair damage incurred to genetic material.

Thomas Lindahl, Ph.D., emeritus scientist at the Francis Crick Institute in London, was recognized for his discoveries in base excision repair—the pathway that constitutes the bulk of DNA restoration during the cell cycle from alkylation, methylation, and oxidative stress. In the early 1970s, many scientists believed that DNA was an extremely stable molecule, but Dr. Lindahl demonstrated that DNA decays at a rate that ought to have made the development of life on Earth impossible. This insight led him to discover the base excision repair mechanisms.

Paul Modrich, Ph.D., investigator at the Howard Hughes Medical Institute and professor of biochemistry Duke University School of Medicine, was honored for uncovering how cells resolve errors that occur during DNA replication. This so called mismatch repair pathway rectifies base-pairing errors within DNA and defects within this molecular machinery has been shown to increase genomic mutations up to 1,000-fold. Moreover, mismatch repair errors are the cause of the most common form of hereditary colon cancer (HNPCC) and are believed to contribute to the development of a subset of sporadic tumors that occur in a variety of tissues.

Aziz Sancar, M.D., Ph.D., professor of biochemistry and biophysics at the University of North Carolina School of Medicine, was acknowledged for his seminal work on the nucleotide excision repair pathway. Cells use this pathway to repair UV damage to DNA. Individuals born with defects in this repair system will develop skin cancer when exposed to sunlight. Additionally, the cell also utilizes nucleotide excision repair to correct defects caused by mutagenic substances and DNA lesions that create aberrant bulky regions within the helical strand.

These scientists have provided the essential insights into how cells function and maintain their genomic stability—knowledge that integral for the development of new cancer treatments.

sjwilliamspa

Dr. Lindahl was one of my first influencers during research into glycosylases and DNA repair mechanisms. I still remember the academic debates occurring in hallways concerning the existence of the FAPY glycosylase. Very good to see Dr. Lindahl, Dr. Sancar (who contributed greatly) and Dr. Modrich being recognized for their lifelong works. I would wonder why Erol Friedberg was not mentioned though for his contributions to complement factors. However nice to see DNA repair recognized as a pivotal research area.

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Heroes in Medical Research: The Postdoctoral Fellow

Writer: Stephen J. Williams, Ph.D

Thank your Postdoc

The National Postdoctoral Association (NPA) had its Fifth Annual Celebration Of National Postdoc Appreciation Week (NPAW) in September and I wanted to focus a posting on curating stories from postdoctoral fellows as well as private investigators (PIs) and mentors on the impacts that postdoctoral fellows had in research and to recognize the critical and tremendous contributions which postdocs make to science.

During our postdoctoral years, we develop deep friendships which last a lifetime, a close bonding to our kindred scientists different in nature than our bonding with our mentors.  Nothing can replace a great mentor but our fellow postdocs make a huge difference in our complete scientific training.

                                   It’s always the little things that stand out in our fondest memories

Unfortunately I have a plethora of fond, little memories; too many for this posting but just want to ad in a few things:

  • Thank you!    –  To all those postdocs who worked tirelessly to make a memorable PostDoc Day!
  • Thank you!  –  To all my postdoc colleagues who stayed late n the lab with me giving each other moral and scientific support
  • Thank you!  – All my postdoc friends who would give up their time to show me how to make and use a text box correctly in Word
  • Thank you!  =-  for your friendship and understanding in those rough times we had experienced

To enliven the discussion, I ask that postdocs, past, present, and future, as well as PI’s and postdoc mentors comment on their postdoc experience. I also would like PI’s to share the stories how their postdocs made an impact to their labs.

A few interesting links and articles from the web on the importance and struggles of postdocs are included below:

Keith Micoli, from New York University Langone Medical Center states in an Elsevier article on The Academic Executive Brief

Consequently, it’s very difficult to come up with accurate numbers. Current estimates on number of postdocs come between 40,000 and 90,000 — a range that is unacceptable. A solid bet is that there are 60,000 postdocs and that more than half, if not two thirds or higher, are international.

– from US research enterprise powered by international postdocs by Keith Micoli at NYU

Survey Methodology

Since Science started conducting annual surveys seven years ago, alternating between polling postdocs and postdoc advisors, the attributes that survey respondents select as being most important to a successful postdoc have not varied much.This year’s survey was launched on March 15, 2011, with e-mail invitations sent out to about 40,000 current and former postdoc advisors worldwide. Of the 798 completed surveys that were collected, 71 percent came from Europe (39 percent) and North America (32 percent). The remaining respondents were located in Asia/Australia/Pacific Rim (20 percent) or other areas of the world (9 percent). Most were males (72 percent) 40 years of age and older (76 percent) and worked in academic institutions (70 percent) and government organizations (13 percent). The primary area represented was the life sciences (57 percent).

However only a handful of institutions were featured.

An open letter to AAAS journal “Science”: Postdocs need to address the “The Future of Research”

https://thewinnower.com/papers/an-open-letter-to-aaas-journal-science-postdocs-need-to-address-the-the-future-of-research?jm.npa=

This letter, posted on the Winnower.com, was a response to Callier’s article “Ailing academia needs culture change”1 and discussed how postdoctoral fellows have to lead in effecting change if the US research enterprise is to flourish in the future. In addition, the authors have been organizing Boston area postdoctoral associations and are sponsoring a symposium to be held at Boston University October 2-3 2014, focusing on the challenges facing graduate students and postdoctoral fellows: the “Future of Research” symposium (futureofresearch.org, @FORsymp).

  1. V. Callier, N. L. Vanderford. “Ailing academia needs culture change.” Science, 2014: 345; 6199: 885. DOI: 10.1126/science.345.6199.885-b

On the surface, many acknowledge the importance of postdoctoral fellows to the US research effort,

HOWEVER, the QUESTION remains DO POSTDOCS FEEL APPRECIATED FOR THEIR EFFORTS?

Please read Jacquelyn Gil, Ph.D.’s GREAT blog post

Have you hugged your postdoc today?

in The Contemplative Mammoth about her surviving postdoctoral life.

For some postdoc humor go to

http://phdcomics.com/comics.php where Jorge Cham, Ph.D. has been satiring the Ph.D. life since he was a graduate student in the late 90’s.

and see if you could be a star in their movie about Ph.D.’s: The PhD Movie and the sequel.

Don’t Underestimate Your Postdoc

Dr. Thomas C. Sudhof, MD is an example of a postdoctoral fellow making great contributions to a lab. A summary of his work is seen below and obtained from the site thebestschools.org on the “50 Most Influential Scientists”.

http://www.thebestschools.org/features/50-influential-scientists-world-today/#S%C3%BCdhof

Thomas C. Südhof

Thomas C. Südhof is a biochemist and professor in the School of Medicine in the Department of Molecular and Cellular Physiology at Stanford University. He is best known for his work in the area of synaptic transmission, which is the process by which signaling chemicals known as neurotransmitters are released by one neuron and bind to and activate the receptors of another neuron.

Südhof won the 1985 Nobel Prize in Physiology or Medicine, along with Randy Schekman and James Rothman.

Südhof, a native of Germany, obtained his MD from the University of Göttingen and conducted his postdoctoral training in the department of molecular genetics at the University of Texas’s Health Science Center. During his postdoctoral training, he worked on describing the role of the LDL receptor in cholesterol metabolism, for which Michael S. Brown and Joseph L. Goldstein were awarded the Nobel Prize in Physiology or Medicine in 1985.

 

Another example from the site includes Dr. Craig Mello (Craig C. Mello’s Home Page.) who, along with Dr., Andrew Fire discovered RNAi when both at Carnegie Institute. Both received a Nobel for their work.

So again would love to hear and curate personal stories highlighting how postdocs make a great contribution to US science.

More articles in this “Heroes in Medical Research” series and posts on Scientific Careers from this site include:

Heroes in Medical Research: Green Fluorescent Protein and the Rough Road in Science

Heroes in Medical Research: Developing Models for Cancer Research

Heroes in Medical Research: Dr. Carmine Paul Bianchi Pharmacologist, Leader, and Mentor

Heroes in Medical Research: Dr. Robert Ting, Ph.D. and Retrovirus in AIDS and Cancer

Heroes in Medical Research: Barnett Rosenberg and the Discovery of Cisplatin

Science Budget FY’14: Stakeholders’ Reactions on Selective Budget Drops and Priorities Shift

Careers for Researchers Beyond Academia

BEYOND THE “MALE MODEL”: AN ALTERNATIVE FEMALE MODEL OF SCIENCE, TECHNOLOGY AND INNOVATION

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


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

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

Pentose Shunt, Electron Transfer, Galactose, and other Lipids in brief

This is a continuation of the series of articles that spans the horizon of the genetic
code and the progression in complexity from genomics to proteomics, which must
be completed before proceeding to metabolomics and multi-omics.  At this point
we have covered genomics, transcriptomics, signaling, and carbohydrate metabolism
with considerable detail.In carbohydrates. There are two topics that need some attention –
(1) pentose phosphate shunt;
(2) H+ transfer
(3) galactose.
(4) more lipids
Then we are to move on to proteins and proteomics.

Summary of this series:

The outline of what I am presenting in series is as follows:

  1. Signaling and Signaling Pathways
    https://pharmaceuticalintelligence.com/2014/08/12/signaling-and-signaling-pathways/
  2. Signaling transduction tutorial.
    https://pharmaceuticalintelligence.com/2014/08/12/signaling-transduction-tutorial/
  3. Carbohydrate metabolism
    https://pharmaceuticalintelligence.com/2014/08/13/carbohydrate-metabolism/

Selected References to Signaling and Metabolic Pathways published in this Open Access Online Scientific Journal, include the following: 

https://pharmaceuticalintelligence.com/2014/08/14/selected-references-to-signaling-
and-metabolic-pathways-in-leaders-in-pharmaceutical-intelligence/

  1. Lipid metabolism

4.1  Studies of respiration lead to Acetyl CoA
https://pharmaceuticalintelligence.com/2014/08/18/studies-of-respiration-lead-to-acetyl-coa/

4.2 The multi-step transfer of phosphate bond and hydrogen exchange energy
https://pharmaceuticalintelligence.com/2014/08/19/the-multi-step-transfer-of-phosphate-
bond-and-hydrogen-exchange-energy/

5.Pentose shunt, electron transfers, galactose, and other lipids in brief

6. Protein synthesis and degradation

7.  Subcellular structure

8. Impairments in pathological states: endocrine disorders; stress
hypermetabolism; cancer.

Section I. Pentose Shunt

Bernard L. Horecker’s Contributions to Elucidating the Pentose Phosphate Pathway

Nicole Kresge,     Robert D. Simoni and     Robert L. Hill

The Enzymatic Conversion of 6-Phosphogluconate to Ribulose-5-Phosphate
and Ribose-5-Phosphate (Horecker, B. L., Smyrniotis, P. Z., and Seegmiller,
J. E.      J. Biol. Chem. 1951; 193: 383–396

Bernard Horecker

Bernard Leonard Horecker (1914) began his training in enzymology in 1936 as a
graduate student at the University of Chicago in the laboratory of T. R. Hogness.
His initial project involved studying succinic dehydrogenase from beef heart using
the Warburg manometric apparatus. However, when Erwin Hass arrived from Otto
Warburg’s laboratory he asked Horecker to join him in the search for an enzyme
that would catalyze the reduction of cytochrome c by reduced NADP. This marked
the beginning of Horecker’s lifelong involvement with the pentose phosphate pathway.

During World War II, Horecker left Chicago and got a job at the National Institutes of
Health (NIH) in Frederick S. Brackett’s laboratory in the Division of Industrial Hygiene.
As part of the wartime effort, Horecker was assigned the task of developing a method
to determine the carbon monoxide hemoglobin content of the blood of Navy pilots
returning from combat missions. When the war ended, Horecker returned to research
in enzymology and began studying the reduction of cytochrome c by the succinic
dehydrogenase system.

Shortly after he began these investigation changes, Horecker was approached by
future Nobel laureate Arthur Kornberg, who was convinced that enzymes were the
key to understanding intracellular biochemical processes
. Kornberg suggested
they collaborate, and the two began to study the effect of cyanide on the succinic
dehydrogenase system. Cyanide had previously been found to inhibit enzymes
containing a heme group, with the exception of cytochrome c. However, Horecker
and Kornberg found that

  • cyanide did in fact react with cytochrome c and concluded that
  • previous groups had failed to perceive this interaction because
    • the shift in the absorption maximum was too small to be detected by
      visual examination.

Two years later, Kornberg invited Horecker and Leon Heppel to join him in setting up
a new Section on Enzymes in the Laboratory of Physiology at the NIH. Their Section on Enzymes eventually became part of the new Experimental Biology and Medicine
Institute and was later renamed the National Institute of Arthritis and Metabolic
Diseases.

Horecker and Kornberg continued to collaborate, this time on

  • the isolation of DPN and TPN.

By 1948 they had amassed a huge supply of the coenzymes and were able to
present Otto Warburg, the discoverer of TPN, with a gift of 25 mg of the enzyme
when he came to visit. Horecker also collaborated with Heppel on 

  • the isolation of cytochrome c reductase from yeast and 
  • eventually accomplished the first isolation of the flavoprotein from
    mammalian liver.

Along with his lab technician Pauline Smyrniotis, Horecker began to study

  • the enzymes involved in the oxidation of 6-phosphogluconate and the
    metabolic intermediates formed in the pentose phosphate pathway.

Joined by Horecker’s first postdoctoral student, J. E. Seegmiller, they worked
out a new method for the preparation of glucose 6-phosphate and 6-phosphogluconate, 
both of which were not yet commercially available.
As reported in the Journal of Biological Chemistry (JBC) Classic reprinted here, they

  • purified 6-phosphogluconate dehydrogenase from brewer’s yeast (1), and 
  • by coupling the reduction of TPN to its reoxidation by pyruvate in
    the presence of lactic dehydrogenase
    ,
  • they were able to show that the first product of 6-phosphogluconate oxidation,
  • in addition to carbon dioxide, was ribulose 5-phosphte.
  • This pentose ester was then converted to ribose 5-phosphate by a
    pentose-phosphate isomerase.

They were able to separate ribulose 5-phosphate from ribose 5- phosphate and demonstrate their interconversion using a recently developed nucleotide separation
technique called ion-exchange chromatography. Horecker and Seegmiller later
showed that 6-phosphogluconate metabolism by enzymes from mammalian
tissues also produced the same products
.8

Bernard Horecker

Bernard Horecker

http://www.jbc.org/content/280/29/e26/F1.small.gif

Over the next several years, Horecker played a key role in elucidating the

  • remaining steps of the pentose phosphate pathway.

His total contributions included the discovery of three new sugar phosphate esters,
ribulose 5-phosphate, sedoheptulose 7-phosphate, and erythrose 4-phosphate, and
three new enzymes, transketolase, transaldolase, and pentose-phosphate 3-epimerase.
The outline of the complete pentose phosphate cycle was published in 1955
(2). Horecker’s personal account of his work on the pentose phosphate pathway can
be found in his JBC Reflection (3).1

Horecker’s contributions to science were recognized with many awards and honors
including the Washington Academy of Sciences Award for Scientific Achievement in
Biological Sciences (1954) and his election to the National Academy of Sciences in
1961. Horecker also served as president of the American Society of Biological
Chemists (now the American Society for Biochemistry and Molecular Biology) in 1968.

Footnotes

  • 1 All biographical information on Bernard L. Horecker was taken from Ref. 3.
  • The American Society for Biochemistry and Molecular Biology, Inc.

References

  1. ↵Horecker, B. L., and Smyrniotis, P. Z. (1951) Phosphogluconic acid dehydrogenase
    from yeast. J. Biol. Chem. 193, 371–381FREE Full Text
  2. Gunsalus, I. C., Horecker, B. L., and Wood, W. A. (1955) Pathways of carbohydrate
    metabolism in microorganisms. Bacteriol. Rev. 19, 79–128  FREE Full Text
  3. Horecker, B. L. (2002) The pentose phosphate pathway. J. Biol. Chem. 277, 47965–
    47971 FREE Full Text

The Pentose Phosphate Pathway (also called Phosphogluconate Pathway, or Hexose
Monophosphate Shunt) is depicted with structures of intermediates in Fig. 23-25
p. 863 of Biochemistry, by Voet & Voet, 3rd Edition. The linear portion of the pathway
carries out oxidation and decarboxylation of glucose-6-phosphate, producing the
5-C sugar ribulose-5-phosphate.

Glucose-6-phosphate Dehydrogenase catalyzes oxidation of the aldehyde
(hemiacetal), at C1 of glucose-6-phosphate, to a carboxylic acid in ester linkage
(lactone). NADPserves as electron acceptor.

6-Phosphogluconolactonase catalyzes hydrolysis of the ester linkage (lactone)
resulting in ring opening. The product is 6-phosphogluconate. Although ring opening
occurs in the absence of a catalyst, 6-Phosphogluconolactonase speeds up the
reaction, decreasing the lifetime of the highly reactive, and thus potentially
toxic, 6-phosphogluconolactone.

Phosphogluconate Dehydrogenase catalyzes oxidative decarboxylation of
6-phosphogluconate, to yield the 5-C ketose ribulose-5-phosphate. The
hydroxyl at C(C2 of the product) is oxidized to a ketone. This promotes loss
of the carboxyl at C1 as CO2.  NADP+ again serves as oxidant (electron acceptor).

pglucose hd

pglucose hd

https://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb2/part1/images/pglucd.gif

Reduction of NADP+ (as with NAD+) involves transfer of 2e- plus 1H+ to the
nicotinamide moiety.

nadp

NADPH, a product of the Pentose Phosphate Pathway, functions as a reductant in
various synthetic (anabolic) pathways, including fatty acid synthesis.

NAD+ serves as electron acceptor in catabolic pathways in which metabolites are
oxidized. The resultant NADH is reoxidized by the respiratory chain, producing ATP.

nadnadp

https://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb2/part1/images/nadnadp.gif

Regulation: 
Glucose-6-phosphate Dehydrogenase is the committed step of the Pentose
Phosphate Pathway. This enzyme is regulated by availability of the substrate NADP+.
As NADPH is utilized in reductive synthetic pathways, the increasing concentration of
NADP+ stimulates the Pentose Phosphate Pathway, to replenish NADPH.

The remainder of the Pentose Phosphate Pathway accomplishes conversion of the
5-C ribulose-5-phosphate to the 5-C product ribose-5-phosphate, or to the 3-C
glyceraldehyde -3-phosphate and the 6-C fructose-6-phosphate (reactions 4 to 8
p. 863).

Transketolase utilizes as prosthetic group thiamine pyrophosphate (TPP), a
derivative of vitamin B1.

tpp

tpp

https://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb2/part1/images/tpp.gif

Thiamine pyrophosphate binds at the active sites of enzymes in a “V” conformation.The amino group of the aminopyrimidine moiety is close to the dissociable proton,
and serves as the proton acceptor. This proton transfer is promoted by a glutamate
residue adjacent to the pyrimidine ring.

The positively charged N in the thiazole ring acts as an electron sink, promoting
C-C bond cleavage. The 3-C aldose glyceraldehyde-3-phosphate is released.
2-C fragment remains on TPP.

FASEB J. 1996 Mar;10(4):461-70.   http://www.ncbi.nlm.nih.gov/pubmed/8647345

Reviewer

The importance of this pathway can easily be underestimated.  The main source for
energy in respiration was considered to be tied to the

  • high energy phosphate bond in phosphorylation and utilizes NADPH, converting it to NADP+.

glycolysis n skeletal muscle in short term, dependent on muscle glycogen conversion
to glucose, and there is a buildup of lactic acid – used as fuel by the heart.  This
pathway accounts for roughly 5% of metabolic needs, varying between tissues,
depending on there priority for synthetic functions, such as endocrine or nucleic
acid production.

The mature erythrocyte and the ocular lens both are enucleate.  85% of their
metabolic energy needs are by anaerobic glycolysis.  Consider the erythrocyte
somewhat different than the lens because it has iron-based hemoglobin, which
exchanges O2 and CO2 in the pulmonary alveoli, and in that role, is a rapid
regulator of H+ and pH in the circulation (carbonic anhydrase reaction), and also to
a lesser extent in the kidney cortex, where H+ is removed  from the circulation to
the urine, making the blood less acidic, except when there is a reciprocal loss of K+.
This is how we need a nomogram to determine respiratory vs renal acidosis or
alkalosis.  In the case of chronic renal disease, there is substantial loss of
functioning nephrons, loss of countercurrent multiplier, and a reduced capacity to
remove H+.  So there is both a metabolic acidosis and a hyperkalemia, with increased
serum creatinine, but the creatinine is only from muscle mass – not accurately
reflecting total body mass, which includes visceral organs.  The only accurate
measure of lean body mass would be in the linear relationship between circulating
hepatic produced transthyretin (TTR).

The pentose phosphate shunt is essential for

  • the generation of nucleic acids, in regeneration of red cells and lens – requiring NADPH.

Insofar as the red blood cell is engaged in O2 exchange, the lactic dehydrogenase
isoenzyme composition is the same as the heart. What about the lens of and cornea the eye, and platelets?  The explanation does appear to be more complex than
has been proposed and is not discussed here.

Section II. Mitochondrial NADH – NADP+ Transhydrogenase Reaction

There is also another consideration for the balance of di- and tri- phospopyridine
nucleotides in their oxidized and reduced forms.  I have brought this into the
discussion because of the centrality of hydride tranfer to mitochondrial oxidative
phosphorylation and the energetics – for catabolism and synthesis.

The role of transhydrogenase in the energy-linked reduction of TPN 

Fritz HommesRonald W. Estabrook∗∗

The Wenner-Gren Institute, University of Stockholm
Stockholm, Sweden
Biochemical and Biophysical Research Communications 11, (1), 2 Apr 1963, Pp 1–6
http://dx.doi.org:/10.1016/0006-291X(63)90017-2

In 1959, Klingenberg and Slenczka (1) made the important observation that incubation of isolated

  • liver mitochondria with DPN-specific substrates or succinate in the absence of phosphate
    acceptor resulted in a rapid and almost complete reduction of the intramitochondrial TPN.

These and related findings led Klingenberg and co-workers (1-3) to postulate

  • the occurrence of an ATP-controlled transhydrogenase reaction catalyzing the reduction of
    mitochondrial TPN by DPNH. A similar conclusion was reached by Estabrook and Nissley (4).

The present paper describes the demonstration and some properties of an

  • energy-dependent reduction of TPN by DPNH, catalyzed by submitochondrial particles.

Preliminary reports of some of these results have already appeared (5, 6 ) , and a
complete account is being published elsewhere (7).We have studied the energy- dependent reduction of TPN by PNH with submitochondrial particles from both
rat liver and beef heart. Rat liver particles were prepared essentially according to
the method of Kielley and Bronk (8), and beef heart particles by the method of
Low and Vallin (9).

PYRIDINE NUCLEOTIDE TRANSHYDROGENASE  II. DIRECT EVIDENCE FOR
AND MECHANISM OF THE
 TRANSHYDROGENASE REACTION*

BY  NATHAN 0. KAPLAN, SIDNEY P. COLOWICK, AND ELIZABETH F. NEUFELD
(From the McCollum-Pratt Institute, The Johns Hopkins University, Baltimore,
Maryland)  J. Biol. Chem. 1952, 195:107-119.
http://www.jbc.org/content/195/1/107.citation

NO Kaplan

NO Kaplan

Sidney Colowick

Sidney Colowick

Elizabeth Neufeld

Elizabeth Neufeld

Kaplan studied carbohydrate metabolism in the liver under David M. Greenberg at the
University of California, Berkeley medical school. He earned his Ph.D. in 1943. From
1942 to 1944, Kaplan participated in the Manhattan Project. From 1945 to 1949,
Kaplan worked with Fritz Lipmann at Massachusetts General Hospital to study
coenzyme A. He worked at the McCollum-Pratt Institute of Johns Hopkins University
from 1950 to 957. In 1957, he was recruited to head a new graduate program in
biochemistry at Brandeis University. In 1968, Kaplan moved to the University of
California, San Diego
, where he studied the role of lactate dehydrogenase in cancer. He also founded a colony of nude mice, a strain of laboratory mice useful in the study
of cancer and other diseases. [1] He was a member of the National Academy of
Sciences.One of Kaplan’s students at the University of California was genomic
researcher Craig Venter.[2]3]  He was, with Sidney Colowick, a founding editor of the scientific book series Methods
in Enzymology
.[1]

http://books.nap.edu/books/0309049768/xhtml/images/img00009.jpg

Colowick became Carl Cori’s first graduate student and earned his Ph.D. at
Washington University St. Louis in 1942, continuing to work with the Coris (Nobel
Prize jointly) for 10 years. At the age of 21, he published his first paper on the
classical studies of glucose 1-phosphate (2), and a year later he was the sole author on a paper on the synthesis of mannose 1-phosphate and galactose 1-phosphate (3). Both papers were published in the JBC. During his time in the Cori lab,

Colowick was involved in many projects. Along with Herman Kalckar he discovered
myokinase (distinguished from adenylate kinase from liver), which is now known as
adenyl kinase. This discovery proved to be important in understanding transphos-phorylation reactions in yeast and animal cells. Colowick’s interest then turned to
the conversion of glucose to polysaccharides, and he and Earl Sutherland (who
will be featured in an upcoming JBC Classic) published an important paper on the
formation of glycogen from glucose using purified enzymes (4). In 1951, Colowick
and Nathan Kaplan were approached by Kurt Jacoby of Academic Press to do a
series comparable to Methodem der Ferment Forschung. Colowick and Kaplan
planned and edited the first 6 volumes of Methods in Enzymology, launching in 1955
what became a series of well known and useful handbooks. He continued as
Editor of the series until his death in 1985.

The Structure of NADH: the Work of Sidney P. Colowick

Nicole KresgeRobert D. Simoni and Robert L. Hill

On the Structure of Reduced Diphosphopyridine Nucleotide

(Pullman, M. E., San Pietro, A., and Colowick, S. P. (1954)

J. Biol. Chem. 206, 129–141)

Elizabeth Neufeld
·  Born: September 27, 1928 (age 85), Paris, France
·  EducationQueens College, City University of New YorkUniversity of California,
Berkeley

http://fdb5.ctrl.ucla.edu/biological-chemistry/institution/photo?personnel%5fid=45290&max_width=155&max_height=225

In Paper I (l), indirect evidence was presented for the following transhydrogenase
reaction, catalyzed by an enzyme present in extracts of Pseudomonas
fluorescens:

TPNHz + DPN -+ TPN + DPNHz

The evidence was obtained by coupling TPN-specific dehydrogenases with the
transhydrogenase and observing the reduction of large amounts of diphosphopyridine nucleotide (DPN) in the presence of catalytic amounts of triphosphopyridine
nucleotide (TPN).

In this paper, data will be reported showing the direct

  • interaction between TPNHz and DPN, in thepresence of transhydrogenase alone,
  • to yield products having the propertiesof TPN and DPNHZ.

Information will be given indicating that the reaction involves

  • a transfer of electrons (or hydrogen) rather than a phosphate 

Experiments dealing with the kinetics and reversibility of the reaction, and with the
nature of the products, suggest that the reaction is a complex one, not fully described
by the above formulation.

Materials and Methods [edited]

The TPN and DPN used in these studies were preparations of approximately 75
percent purity and were prepared from sheep liver by the chromatographic procedure
of Kornberg and Horecker (unpublished). Reduced DPN was prepared enzymatically with alcohol dehydrogenase as described elsewhere (2). Reduced TPN was prepared by treating TPN with hydrosulfite. This treated mixture contained 2 pM of TPNHz per ml.
The preparations of desamino DPN and reduced desamino DPN have been
described previously (2, 3). Phosphogluconate was a barium salt which was kindly
supplied by Dr. B. F. Horecker. Cytochrome c was obtained from the Sigma Chemical Company.

Transhydrogenase preparations with an activity of 250 to 7000 units per mg. were
used in these studies. The DPNase was a purified enzyme, which was obtained
from zinc-deficient Neurospora and had an activity of 5500 units per mg. (4). The
alcohol dehydrogenase was a crystalline preparation isolated from yeast according to the procedure of Racker (5).

Phosphogluconate dehydrogenase from yeast and a 10 per cent pure preparation of the TPN-specific cytochrome c reductase from liver (6) were gifts of Dr. B. F.
Horecker.

DPN was assayed with alcohol and crystalline yeast alcohol dehydrogenase. TPN was determined By the specific phosphogluconic acid dehydrogenase from yeast and also by the specific isocitric dehydrogenase from pig heart. Reduced DPN was
determined by the use of acetaldehyde and the yeast alcohol dehydrogenase.
All of the above assays were based on the measurement of optical density changes
at 340 rnp. TPNHz was determined with the TPN-specific cytochrome c reductase system. The assay of the reaction followed increase in optical density at 550 rnp  as a measure of the reduction of the cytochrome c after cytochrome c
reductase was added to initiate the reaction. The changes at 550 rnp are plotted for different concentrations of TPNHz in Fig. 3, a. The method is an extremely sensitive and accurate assay for reduced TPN.

Results
[No Figures or Table shown]

Formation of DPNHz from TPNHz and DPN-Fig. 1, a illustrates the direct reaction between TPNHz and DPN to form DPNHZ. The reaction was carried out by incubating TPNHz with DPN in the presence of the
transhydrogenase, yeast alcohol dehydrogenase, and acetaldehyde. Since the yeast dehydrogenase is specific for DPN,

  • a decrease in absorption at340 rnp can only be due to the formation of reduced DPN. It can
    be seen from the curves in Fig. 1, a that a decrease in optical density occurs only in the
    presence of the complete system.

The Pseudomonas enzyme is essential for the formation of DPNH2. It is noteworthy
that, under the conditions of reaction in Fig. 1, a,

  • approximately 40 per cent of theTPNH, reacted with the DPN.

Fig. 1, a also indicates that magnesium is not required for transhydrogenase activity.  The reaction between TPNHz and DPN takes place in the absence of alcohol
dehydrogenase and acetaldehyde
. This can be demonstrated by incubating the
two pyridine nucleotides with the transhydrogenase for 4 8 12 16 20 24 28 32 36
minutes

FIG. 1. Evidence for enzymatic reaction of TPNHt with DPN.

  • Rate offormation of DPNH2.

(b) DPN disappearance and TPN formation.

(c) Identification of desamino DPNHz as product of reaction of TPNHz with desamino DPN.  (assaying for reduced DPN by the yeast alcohol dehydrogenase technique.

Table I (Experiment 1) summarizes the results of such experiments in which TPNHz was added with varying amounts of DPN.

  • In the absence of DPN, no DPNHz was formed. This eliminates the possibility that TPNH 2 is
    converted to DPNHz
  • by removal ofthe monoester phosphate grouping.

The data also show that the extent of the reaction is

  • dependent on the concentration of DPN.

Even with a large excess of DPN, only approximately 40 per cent of the TPNHzreacts to form reduced DPN. It is of importance to emphasize that in the above
experiments, which were carried out in phosphate buffer, the extent of  the reaction

  • is the same in the presence or absence of acetaldehyde andalcohol dehydrogenase.

With an excess of DPN and different  levels of TPNHZ,

  • the amount of reduced DPN which is formed is
  • dependent on the concentration of TPNHz(Table I, Experiment 2).
  • In all cases, the amount of DPNHz formed is approximately
    40 per cent of the added reduced TPN.

Formation of TPN-The reaction between TPNHz and DPN should yield TPN as well as DPNHz.
The formation of TPN is demonstrated in Table 1. in Fig. 1, b. In this experiment,
TPNHz was allowed to react with DPN in the presence of the transhydrogenase
(PS.), and then alcohol and alcohol dehydrogenase were added . This
would result in reduction of the residual DPN, and the sample incubated with the
transhydrogenase contained less DPN. After the completion of the alcohol
dehydrogenase reaction, phosphogluconate and phosphogluconic dehydrogenase (PGAD) were added to reduce the TPN. The addition of this TPN-specific
dehydrogenase results in an

  • increase inoptical density in the enzymatically treated sample.
  • This change represents the amount of TPN formed.

It is of interest to point out that, after addition of both dehydrogenases,

  • the total optical density change is the same in both

Therefore it is evident that

  • for every mole of DPN disappearing  a mole of TPN appears.

Balance of All Components of Reaction

Table II (Experiment 1) shows that,

  • if measurements for all components of the reaction are made, one can demonstrate
    that there is
  • a mole for mole disappearance of TPNH, and DPN, and
  • a stoichiometric appearance of TPN and DPNH2.
  1. The oxidized forms of the nucleotides were assayed as described
  2. the reduced form of TPN was determined by the TPNHz-specific cytochrome c reductase,
  3. the DPNHz by means of yeast alcohol dehydrogenase plus

This stoichiometric balance is true, however,

  • only when the analyses for the oxidized forms are determined directly on the reaction

When analyses are made after acidification of the incubated reaction mixture,

  • the values found forDPN and TPN are much lower than those obtained by direct analysis.

This discrepancy in the balance when analyses for the oxidized nucleotides are
carried out in acid is indicated in Table II (Experiment 2). The results, when
compared with the findings in Experiment 1, are quite striking.

Reaction of TPNHz with Desamino DPN

Desamino DPN

  • reacts with the transhydrogenase system at the same rate as does DPN (2).

This was of value in establishing the fact that

  • the transhydrogenase catalyzesa transfer of hydrogen rather than a phosphate transfer reaction.

The reaction between desamino DPN and TPNHz can be written in two ways.

TPN f desamino DPNHz

TPNH, + desamino DPN

DPNH2 + desamino TPN

If the reaction involved an electron transfer,

  • desamino DPNHz would be
  • Phosphate transfer would result in the production of reduced

Desamino DPNHz can be distinguished from DPNHz by its

  • slowerrate of reaction with yeast alcohol dehydrogenase (2, 3).

Fig. 1, c illustrates that, when desamino DPN reacts with TPNH2, 

  • the product of the reaction is desamino DPNHZ.

This is indicated by the slow rate of oxidation of the product by yeast alcohol
dehydrogenase and acetaldehyde.

From the above evidence phosphate transfer 

  • has been ruled out as a possible mechanism for the transhydrogenase reaction.

Inhibition by TPN

As mentioned in Paper I and as will be discussed later in this paper,

  • the transhydrogenase reaction does not appear to be readily reversible.

This is surprising, particularly since only approximately 

  • 40 per cent of the TPNHz undergoes reaction with DPN
    under the conditions described above. It was therefore thought that
  • the TPN formed might inhibit further transfer of electrons from TPNH2.

Table III summarizes data showing the

  • strong inhibitory effect of TPN on thereaction between TPNHz and DPN.

It is evident from the data that

  • TPN concentration is a factor in determining the extent of the reaction.

Effect of Removal of TPN on Extent of Reaction

A purified DPNase from Neurospora has been found

  • to cleave the nicotinamide riboside linkagesof the oxidized forms of both TPN and DPN
  • without acting on thereduced forms of both nucleotides (4).

It has been found, however, that

  • the DPNase hydrolyzes desamino DPN at a very slow rate (3).

In the reaction between TPNHz and desamino DPN, TPN and desamino DPNH:,

  • TPNis the only component of this reaction attacked by the Neurospora enzyme
    at an appreciable rate

It was  thought that addition of the DPNase to the TPNHZ-desamino DPN trans-
hydrogenase reaction mixture

  • would split the TPN formed andpermit the reaction to go to completion.

This, indeed, proved to be the case, as indicated in Table IV, where addition of
the DPNase with desamino DPN results in almost

  • a stoichiometric formation of desamino DPNHz
  • and a complete disappearance of TPNH2.

Extent of Reaction in Buffers Other Than Phosphate

All the reactions described above were carried out in phosphate buffer of pH 7.5.
If the transhydrogenase reaction between TPNHz and DPN is run at the same pH
in tris(hydroxymethyl)aminomethane buffer (TRIS buffer)

  • with acetaldehydeand alcohol dehydrogenase present,
  • the reaction proceeds muchfurther toward completion 
  • than is the case under the same conditions ina phosphate medium (Fig. 2, a).

The importance of phosphate concentration in governing the extent of the reaction
is illustrated in Fig. 2, b.

In the presence of TRIS the transfer reaction

  • seems to go further toward completion in the presence of acetaldehyde
    and 
    alcohol dehydrogenase
  • than when these two components are absent.

This is not true of the reaction in phosphate,

  • in which the extent is independent of the alcoholdehydrogenase system.

Removal of one of the products of the reaction (DPNHp) in TRIS thus

  • appears to permit the reaction to approach completion,whereas
  • in phosphate this removal is without effect on the finalcourse of the reaction.

The extent of the reaction in TRIS in the absence of alcohol dehydrogenase
and acetaldehyde
 is

  • somewhat greater than when the reaction is run in phosphate.

TPN also inhibits the reaction of TPNHz with DPN in TRIS medium, but the inhibition

  • is not as marked as when the reaction is carried out in phosphate buffer.

Reversibility of Transhydrogenase Reaction;

Reaction between DPNHz and TPN

In Paper I, it was mentioned that no reversal of the reaction could be achieved in a system containing alcohol, alcohol dehydrogenase, TPN, and catalytic amounts of
DPN.

When DPNH, and TPN are incubated with the purified transhydrogenase, there is
also

  • no evidence for reversibility.

This is indicated in Table V which shows that

  • there is no disappearance of DPNHz in such a system.

It was thought that removal of the TPNHz, which might be formed in the reaction,
could promote the reversal of the reaction. Hence,

  • by using the TPNHe-specific cytochrome c reductase, one could
  1. not only accomplishthe removal of any reduced TPN,
  2. but also follow the course of the reaction.

A system containing DPNH2, TPN, the transhydrogenase, the cytochrome c
reductase, and cytochrome c, however, gives

  • no reduction of the cytochrome

This is true for either TRIS or phosphate buffers.2

Some positive evidence for the reversibility has been obtained by using a system
containing

  • DPNH2, TPNH2, cytochrome c, and the cytochrome creductase in TRIS buffer.

In this case, there is, of course, reduction of cytochrome c by TPNHZ, but,

  • when the transhydrogenase is present.,there is
  • additional reduction over and above that due to the added TPNH2.

This additional reduction suggests that some reversibility of the reaction occurred
under these conditions. Fig. 3, b shows

  • the necessity of DPNHzfor this additional reduction.

Interaction of DPNHz with Desamino DPN-

If desamino DPN and DPNHz are incubated with the purified Pseudomonas enzyme,
there appears

  • to be a transfer of electrons to form desamino DPNHz.

This is illustrated in Fig. 4, a, which shows the

  • decreased rate of oxidation by thealcohol dehydrogenase system
  • after incubation with the transhydrogenase.
  • Incubation of desamino DPNHz with DPN results in the formation of DPNH2,
  • which is detected by the faster rate of oxidation by the alcohol dehydrogenase system
  • after reaction of the pyridine nucleotides with thetranshydrogenase (Fig. 4, b).

It is evident from the above experiments that

the transhydrogenase catalyzes an exchange of hydrogens between

  • the adenylic and inosinic pyridine nucleotides.

However, it is difficult to obtain any quantitative information on the rate or extent of
the reaction by the method used, because

  • desamino DPNHz also reacts with the alcohol dehydrogenase system,
  • although at a much slower rate than does DPNH2.

DISCUSSION

The results of the balance experiments seem to offer convincing evidence that
the transhydrogenase catalyzes the following reaction.

TPNHz + DPN -+ DPNHz + TPN

Since desamino DPNHz is formed from TPNHz and desamino DPN,

  • thereaction appears to involve an electron (or hydrogen) transfer
  • rather thana transfer of the monoester phosphate grouping of TPN.

A number of the findings reported in this paper are not readily understandable in
terms of the above simple formulation of the reaction. It is difficult to understand
the greater extent of the reaction in TRIS than in phosphate when acetaldehyde
and alcohol dehydrogenase are present.

One possibility is that an intermediate may be involved which is more easily converted
to reduced DPN in the TRIS medium. The existence of such an intermediate is also
suggested by the discrepancies noted in balance experiments, in which

  • analyses of the oxidized nucleotides after acidification showed
  • much lower values than those found by direct analysis.

These findings suggest that the reaction may involve

  • a 1 electron ratherthan a 2 electron transfer with
  • the formation of acid-labile free radicals as intermediates.

The transfer of hydrogens from DPNHz to desamino DPN

  • to yield desamino DPNHz and DPN and the reversal of this transfer
  • indicate the unique role of the transhydrogenase
  • in promoting electron exchange between the pyridine nucleotides.

In this connection, it is of interest that alcohol dehydrogenase and lactic
dehydrogenase cannot duplicate this exchange  between the DPN and
the desamino systems.3  If one assumes that desamino DPN behaves
like DPN,

  • one might predict that the transhydrogenase would catalyze an
    exchange of electrons (or hydrogen) 3.

Since alcohol dehydrogenase alone

  • does not catalyze an exchange of electrons between the adenylic
    and inosinic pyridine nucleotides, this rules out the possibility
  • that the dehydrogenase is converted to a reduced intermediate
  • during electron between DPNHz and added DPN.

It is hoped to investigate this possibility with isotopically labeled DPN.
Experiments to test the interaction between TPN and desamino TPN are
also now in progress.

It seems likely that the transhydrogenase will prove capable of

  • catalyzingan exchange between TPN and TPNH2, as well as between DPN and

The observed inhibition by TPN of the reaction between TPNHz and DPN may
therefore

  • be due to a competition between DPN and TPNfor the TPNH2.

SUMMARY

  1. Direct evidence for the following transhydrogenase reaction. catalyzedby an
    enzyme from Pseudomonas fluorescens, is presented.

TPNHz + DPN -+ TPN + DPNHz

Balance experiments have shown that for every mole of TPNHz disappearing
1 mole of TPN appears and that for each mole of DPNHz generated 1 mole of
DPN disappears. The oxidized nucleotides found at the end of the reaction,
however, show anomalous lability toward acid.

  1. The transhydrogenase also promotes the following reaction.

TPNHz + desamino DPN -+ TPN + desamino DPNH,

This rules out the possibility that the transhydrogenase reaction involves a
phosphate transfer and indicates that the

  • enzyme catalyzes a shift of electrons (or hydrogen atoms).

The reaction of TPNHz with DPN in 0.1 M phosphate buffer is strongly
inhibited by TPN; thus

  • it proceeds only to the extent of about40 per cent or less, even
  • when DPNHz is removed continuously by meansof acetaldehyde
    and alcohol dehydrogenase.
  • In other buffers, in whichTPN is less inhibitory, the reaction proceeds
    much further toward completion under these conditions.
  • The reaction in phosphate buffer proceedsto completion when TPN
    is removed as it is formed.
  1. DPNHz does not react with TPN to form TPNHz and DPN in the presence
    of transhydrogenase. Some evidence, however, has been obtained for
    the reversibility by using the following system:
  • DPNHZ, TPNHZ, cytochromec, the TPNHz-specific cytochrome c reductase,
    and the transhydrogenase.
  1. Evidence is cited for the following reversible reaction, which is catalyzed
    by the transhydrogenase.

DPNHz + desamino DPN fi DPN + desamino DPNHz

  1. The results are discussed with respect to the possibility that the
    transhydrogenase reaction may
  • involve a 1 electron transfer with theformation of free radicals as intermediates.

 

BIBLIOGRAPHY

  1. Coiowick, S. P., Kaplan, N. O., Neufeld, E. F., and Ciotti, M. M., J. Biol. Chem.,196, 95 (1952).
  2. Pullman, 111. E., Colowick, S. P., and Kaplan, N. O., J. Biol. Chem., 194, 593(1952).
  3. Kaplan, N. O., Colowick, S. P., and Ciotti, M. M., J. Biol. Chem., 194, 579 (1952).
  4. Kaplan, N. O., Colowick, S. P., and Nason, A., J. Biol. Chem., 191, 473 (1951).
  5. Racker, E., J. Biol. Chem., 184, 313 (1950).
  6. Horecker, B. F., J. Biol. Chem., 183, 593 (1950).

Section !II. 

Luis_Federico_Leloir_-_young

The Leloir pathway: a mechanistic imperative for three enzymes to change
the stereochemical configuration of a single carbon in galactose.

Frey PA.
FASEB J. 1996 Mar;10(4):461-70.    http://www.fasebj.org/content/10/4/461.full.pdf
PMID:8647345

The biological interconversion of galactose and glucose takes place only by way of
the Leloir pathway and requires the three enzymes galactokinase, galactose-1-P
uridylyltransferase, and UDP-galactose 4-epimerase.
The only biological importance of these enzymes appears to be to

  • provide for the interconversion of galactosyl and glucosyl groups.

Galactose mutarotase also participates by producing the galactokinase substrate
alpha-D-galactose from its beta-anomer. The galacto/gluco configurational change takes place at the level of the nucleotide sugar by an oxidation/reduction
mechanism in the active site of the epimerase NAD+ complex. The nucleotide portion
of UDP-galactose and UDP-glucose participates in the epimerization process in two ways:

1) by serving as a binding anchor that allows epimerization to take place at glycosyl-C-4 through weak binding of the sugar, and

2) by inducing a conformational change in the epimerase that destabilizes NAD+ and
increases its reactivity toward substrates.

Reversible hydride transfer is thereby facilitated between NAD+ and carbon-4
of the weakly bound sugars.

The structure of the enzyme reveals many details of the binding of NAD+ and
inhibitors at the active site
.

The essential roles of the kinase and transferase are to attach the UDP group
to galactose, allowing for its participation in catalysis by the epimerase. The
transferase is a Zn/Fe metalloprotein
, in which the metal ions stabilize the
structure rather than participating in catalysis. The structure is interesting
in that

  • it consists of single beta-sheet with 13 antiparallel strands and 1 parallel strand
    connected by 6 helices.

The mechanism of UMP attachment at the active site of the transferase is a double
displacement
, with the participation of a covalent UMP-His 166-enzyme intermediate
in the Escherichia coli enzyme. The evolution of this mechanism appears to have
been guided by the principle of economy in the evolution of binding sites.

PMID: 8647345 Free full text

Section IV.

More on Lipids – Role of lipids – classification

  • Energy
  • Energy Storage
  • Hormones
  • Vitamins
  • Digestion
  • Insulation
  • Membrane structure: Hydrophobic properties

Lipid types

lipid types

lipid types

nat occuring FAs in mammals

nat occuring FAs in mammals

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Heroes in Medical Research: Green Fluorescent Protein and the Rough Road in Science

Curator: Stephen J. Williams, Ph.D.

In this series, “Heroes in Medical Research”, I like to discuss the people who made some important contributions to science and medicine which underlie the great transformative changes but don’t usually get the notoriety given to Nobel Laureates or who seem to fly under the radar of popular news. Their work may be the development of research tools which allowed a great discovery leading to a line of transformative research, a moment of serendipity leading to discovery of a cure, or just contributions to the development of a new field or the mentoring of a new generation of scientists and clinicians. One such discovery, which has probably been pivotal in many of our research, is the discovery of the green fluorescent protein (GFP), commonly used as an invaluable tool to monitor protein for cellular expression and localization studies. Although the development of research tools, whether imaging tools, vectors, animal models, cell lines, and such are not heralded, they always assist in the pivotal discoveries of our time. The following is a heartwarming story by Discover Magazine’s Yudhijit Bhattacharjee behind Dr. Douglas Prasher’s discovery of the green fluorescent protein, his successful efforts to sequence the gene and subsequent struggles in science and finally scientific recognition for his work. In addition the story describes Dr. Prather’s perseverance, a trait necessary for every scientist.

http://discovermagazine.com/2011/apr/30-how-bad-luck-networking-cost-prasher-nobel

 

The following is a wonderful entry into Wikipedia about Dr. Prasher at:

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

including a listing of his publications including the seminal Science and PNAS publications1,2.

 

prasher

 

 

(Photo: Dr. Prasher in the lab at UCSD. Photo credit UCSD and John Galstaldo)

 

 

 

In summary, Dr. Prather had been working at Wood’s Hole in Massachusetts trying to discover, isolate, then clone the protein which allowed a species of jellyfish living in the cold waters of the North Pacific, Aequorea victoria, to emit a green glow. Eventually he cloned the GFP gene, but gave up on work to express the gene in mammalian cells. Before leaving Wood’s Hole he gave the gene to Dr. Roger Tsien, who with Dr. Martin Chalfie and Osamu Shimomura showed the utility of GFP as an intracellular tracer to visualize, in real time, the expression and localization of GFP-tagged proteins (all three shared the 2008 Nobel Prize for this work). Dr. Tsien however realized the importance of Douglas’s cloning work as pivotal for their research, contacted Douglas (who now due to the bad economy was working at a Toyota dealership in Alabama) and invited him to the Nobel Prize Award Ceremony in Sweden as his guest. Although Dr. Prasher had “left academic science” he never really stopped his quest for a scientific career, using his spare time to review manuscripts.

Other researchers have invited their colleagues who made important contributions to the ultimate Nobel work. One such guest was one of my colleagues Dr. Leonard Cohen, who worked with Dr. Irwin Rose and Avram Hershko at the Institute for Cancer Research in Philadelphia a cell-free system from clams to discover the mechanism how cyclin B is degraded during the exit from the cell cycle (from A. Hershko’s Nobel speech). Dr. Hershko had acknowledged a slew of colleagues and highlighted their contributions to the ultimate work. It shows how even small discoveries can contribute to the sphere of scientific knowledge and breakthrough.

Luckily, in the end, perseverance has paid off as Dr. Prasher is now using his talents in Roger Tsien‘s group at the University of California in San Diego.

References:

1. Chalfie, M., Tu, Y., Euskirchen, G., Ward, W.W., Prasher, D.C., Green fluorescent protein as a marker for gene expression. Science, 263(5148), 802-805 (1994).

 

2. Heim, R., Prasher, D.C., Tsien, R.Y., Wavelength mutations and posttranslational autoxidation of green fluorescent protein. Proc. Natl. Acad. Sci. USA, 91(26), 12501-12504 (1994).

More posts on this site on Heroes in Medical Research series include:

Heroes in Medical Research: Developing Models for Cancer Research

Heroes in Medical Research: Dr. Carmine Paul Bianchi Pharmacologist, Leader, and Mentor

Heroes in Medical Research: Dr. Robert Ting, Ph.D. and Retrovirus in AIDS and Cancer

Heroes in Medical Research: Barnett Rosenberg and the Discovery of Cisplatin

 

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Image

Curation is Uniquely Distinguished by the Historical Exploratory Ties that Bind

Author and Curator: Larry H Bernstein, MD, FCAP

The description and definition of curation has been introduced in a Forward to Series A: e-Books on Cardiovascular DiseasesVolume Two, by Dr. Aviva Lev-Ari, PhD, RN, the Founder of Leaders in Pharmaceutical Business Intelligence’s  Scientific Journal http://pharmaceuticalintelligence.com, acting as Curator, Co-Curator, and e-Publishing Article Architecture Designer and, chiefly, Editor-in-Chief of a Five e-Series in BioMed,

https://pharmaceuticalintelligence.com/biomed-e-books/

Forward to Volume Two

Volume Two: Cardiovascular Original Research: Cases in Methodology Design for Content Co-Curation

Curation is explained by it being contrasted with the Art of Scientific Creation, both are expored below by examples.

Part 1: The Scientific Creation

I shall try to identify the important features and criteria that contribute to scientific curation of medical, biological, and pharmaceutical research, including structural and functional content from the sciences of anatomy, physiology, physics and chemistry.

The principles that I seek to realized is a foundation in the body of knowledge that precedes the discovery or innovation.  Is the discovery essential, but unnoticed because of unlinkings to prior established concepts.  This is extremely difficult to cull out, but it has had a recurrent history.  It might be easiest to refer to examples in physics, such as, the unique Nobel Prize discovery of pseudo-crystals that has had an impact on materials science. But actually, in the history of mathematics, astronomy, and physics, and later in anatomy and physiology, we have an “audit trail” in writings from the Hellenistic period, interrupted by the dark ages and the Bubonic Plague, and a reawakening in the period preceding and through the enlightenment and reformation. This carried significant risks for great thinkers in a society that changes slowly, and with repeated interruptions throughout all periods by wars.  One might say that this has no relevance to curation, but repeatedly, libraries and museums preserved discovery that could be re-examined later. Thus, we can’t discard the brilliance of Hipparchos, whose influence on Ptolemy is known, and who discovered the centrality of the Sun to our universe, even though the extent to which he accepted societal belief in astrology is at best limited.  The work of Copernicus later was under great duress, but gave precedence to Galileo and Newton.  The Hellenistic period also gave us Euclid and Archimedes, which was critical for the development of mathematics and measurement, and El Gibr’ gave us algebra. In his time, Archimedes found no-one who he could share his ideas with other than Conon, who died too early, but he was later read by Omar Kayyam,  Leonardo da Vinci, Galileo and Newton.  The Greek diagrams used by Archimedes of Syracuse were a major contribution to cognition and inference.  The Archimedes Palimpses, which were given to us as by the priest-scribe, Ioannes Myronas in 1229, is historically a major contribution revealing Archimedes work in the Method. There is the center of gravity of a triangle, and the treatise on Balancing Planes, from which he deduces that if you place two objects on a balance on which the distances are movable from the fulcrum, the distance of the lighter object is five times the distance of the heavier object.  The rule is that weights balance when they are reciprocal to their distance. Then there is Fermat’s Last Theorem, unsolved problem for centuries since the seventeenth century.The theorem state that while the square of a whole number can can be broken down into two other squares of whole numbers the same cannot be done for cubes or any higher power. The theorem took seven years to write, with a ynother year to edit.The principle was incorporated into the Pythagorean Theorem, and in 1955 two japanese mathematicians made a far reaching conjecture that paved the way to the solution by Andrew Wiles at Princeton in 1995.

Notably, the great mathematician, Gauss, who published Disquisition on Mathematics in 1801, on  number theory at age 24, refused to engage in the solution, but his work in complex analysis, based on earlier work by Euler involving imaginary numbers was crucial to the 20th century understanding.Perhaps another apt example is Einstein’s general theory of relativity, the prediction of gravitational radiation bringing a new attention to the tiny ripples in space-time that has opened our eyes to modern cosmology. Finally, we find that a small piece of our universe is viewed as a chunk of Hilbert space, developing as a nest of interacting probability waves. The waves of Hilbert space are actually the waves Schroedinger derived before we had the tools to observe their behavior.The mathematics of entanglement identifies the high-probability areas of a joint-Hilbert space developed from the interaction having consistent histories. This has led to the description of Schroedinger’s principle, the things that we consider to be real are stable persistent patterns. This gives rise to debate about many worlds.

We leave the seemingly esoteric world of problems in mathematics and theoretic physics and return to the world of biochemistry, molecular biology, genomics, proteomics and allied medical sciences.

The scientific underpinnings of biology and medicine transitioned from a largely observational and descriptive phase in the 19th century with the scientifc leadership of Rudolph Virchow, Louis Pasteur, Robert Koch, John Hunter, Edward Jennings, Walter Reed, Karl Landsteiner, and Thomas Hunt Morgan.  Pasteur, Koch, Landsteiner and Morgan were outstanding experimentalists.  The latter two were to receive Nobel Prizes that began in 2001.  The idea of a more fundamental basis for biological sciences was concerned with studying the chemical structures and processes of biological phenomena that involve the basic units of life, and it developed out of the related fields of biochemistrygenetics, and biophysics. The primary focus became the study of proteins and nucleic acids—i.e., the macromolecules that are essential to life processes. A great impetus was provided by enabling the three-dimensional structure of these macromolecules through such techniques as X-ray diffraction and electron microscopy. In seeking to understand the molecular basis of genetic processes; molecular biologists map the location of genes on specific chromosomes, associate these genes with particular characters of an organism, and use recombinant DNA technology to isolate, sequence, and modify specific genes.

The above is tied to a dominance of Western scientific discovery, as seen in the recipients of the Nobel Prize, but it is only a two dimensional view. Here another type of graphical display would be more informative, and it has been developed. I might consider a separation by type for physics, chemistry and medicine, leaving out the others, and then, in combination. I would bet that there are interactions.

For instance – 2001 – Roentgen, Physics; Pierre and Marie Curie, Physics; E.O. Lawrence, Chemistry, Berkeley Radiation Lab; Max Planck, following on Boltzmann and on Josiah Willard Gibbs (pre-Nobel) work. Then you have radiology and radioisotope chemistry and photosynthesis, Martin Kamen. Of course, modern physiology and metabolism traces back to the work on oxygen, carcon dioxide, and heat, adiabatic systems, and leads to the calorimeter, the Warburg apparatus, which credits Pasteur’s work 60 years earlier. The fruit fly genetics was an impetus for cracking the genetic code, but the impetus for that was both from Gregor Mendel and Charles Darwin, and then the mathematical work of Pearson and of Fischer. The work on the chemical bond by Linus Pauling really opened up a foundation for understanding organic and inorganic reactions based on atomic orbital theory that was essential for pursuit of the double helix. This was so important that it unlocked the structure of polymeric proteins through the disulfide bond, and also metalloprotein complexes (heme, …). Wouldn’t it be incredible to map the Nobel work to seminal work done in the 100 years before the Prize with different colored arrows to show stromg and weaker associations? This is in a strong sense, a method of CURATION (as opposed to creation), that is very important for a fundamental grasp of the growth of and ties in the development of the knowledgebase.

Wouldn’t it be incredible to map the Nobel work to seminal work done in the 100 years before the Prize with different colored arrows to show stromg and weaker associations? This is in a strong sense, a method of CURATION (as opposed to creation), that is very important for a fundamental grasp of the growth of and ties in the development of the knowledge-base.

Such a discussion in depth is the curation that is intended for https://pharmaceuticalintelligence.com/biomed-e-books/series-e-titles-in the strategic-plan-for-2014-1015/2014-milestones-in-physiology-discoveries-in-medicine

Part 2: Scientifc Results – The Art of Curation

Dr. Lev-Ari continued her work, beyond Volume Two, above, on Curation as a Methodology for Critique of the Scientific Frontier and the most effective method for synthesis of scientific milestones in the following selective list of articles:

e-Recognition via Friction-free Collaboration over the Internet: “Open Access to Curation of Scientific Research by Aviva Lev-Ari, PhD, RN

Digital Publishing Promotes Science and Popularizes it by Access to Scientific Discourse by Aviva Lev-Ari, PhD, RN

Synthetic Biology: On Advanced Genome Interpretation for Gene Variants and Pathways: What is the Genetic Base of Atherosclerosis and Loss of Arterial Elasticity with Aging

The Heart: Vasculature Protection – A Concept-based Pharmacological Therapy including THYMOSIN

Paradigm Shift in Human Genomics – Predictive Biomarkers and Personalized Medicine – Part 1

The Fatal Self Distraction of the Academic Publishing Industry: The Solution of the Open Access Online Scientific Journals

For a complete list of her Curations, go to

REFERENCES

1. George Sarton. A History of Science: Hellenistic Science and Culture in the last three centuries B.C. 1959. Harvard University Press. Cambridge, MA, USA.
2. Reviel Netz & William Noel. The Archimedes Codex: How a medieval prayer book is revealing the true genius of antiquity’s greatest scientist. 2007. Da Capo Press.
Perseus Books Group, Philadelphia, PA, USA.
3. Amir D Aczel. Fermat’s last theorem: Unlocking the secret of an ancient methematical problem.  Four Walls Eight Windows. 1996. New York, NY, USA.
4. Colin Bruce. Schroedinger’s Rabbits: the many worlds of quantum.  2004. Joseph Henry Press. Washington, DC, USA.
5. Marcia Bartusiak. Einstein’s Unfinished Symphony: listening to the sounds of spac^2 E-time.  The Berkley Publishing Group, New York, NY, USA.

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

The amazing history of the Nobel Prize, told in maps and charts
https://pharmaceuticalintelligence.com/2013/12/31/the-amazing-history-of-the-nobel-prize-told-in-maps-and-charts/

Quantum Biology And Computational Medicine
Curator: Larry H. Bernstein, MD, FCAP
https://pharmaceuticalintelligence.com/2013/04/03/quantum-biology-and-computational-medicine/

Metabolite Identification Combining Genetic and Metabolic Information: Genetic association links unknown metabolites to functionally related genes
Reporter: Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2012/10/22/metabolite-identification-combining-genetic-and-metabolic-information-genetic-association-links-unknown-metabolites-to-functionally-related-genes/

Breast Cancer, drug resistance, and biopharmaceutical targets
Reporter: Larry H Bernstein, MD
https://pharmaceuticalintelligence.com/2012/09/18/breast-cancer-drug-resistance-and-biopharmaceutical-targets/

The Initiation and Growth of Molecular Biology and Genomics – Part I
Curator: Larry H Bernstein, MD, FCAP
https://pharmaceuticalintelligence.com/2013/02/08/the-initiation-and-growth-of-molecular-biology-and-genomics/

Nitric Oxide and Sepsis, Hemodynamic Collapse, and the Search for Therapeutic Options
Curator, Reporter, EAW: Larry H Bernstein, MD, FCAP
https://pharmaceuticalintelligence.com/2012/10/20/nitric-oxide-and-sepsis-hemodynamic-collapse-and-the-search-for-therapeutic-options/

Sepsis, Multi-organ Dysfunction Syndrome, and Septic Shock: A Conundrum of Signaling Pathways Cascading Out of Control
Curator and Author: Larry H Bernstein, MD, FCAP
https://pharmaceuticalintelligence.com/2012/10/13/sepsis-multi-organ-dysfunction-syndrome-and-septic-shock-a-conundrum-of-signaling-pathways-cascading-out-of-control/

How Methionine Imbalance with Sulfur-Insufficiency Leads to Hyperhomocysteinemia
Curator: Larry H Bernstein, MD, FACP
https://pharmaceuticalintelligence.com/2013/04/04/sulfur-deficiency-leads_to_hyperhomocysteinemia/

Vegan Diet is Sulfur Deficient and Heart Unhealthy
Larry H. Bernstein, MD, FCAP, Curator
https://pharmaceuticalintelligence.com/2013/11/17/vegan-diet-is-sulfur-deficient-and-heart-unhealthy/

Portrait of a great scientist and mentor: Nathan Oram Kaplan
Author: Larry H. Bernstein, MD
https://pharmaceuticalintelligence.com/2013/01/26/portrait-of-a-great-scientist-and-mentor-nathan-oram-kaplan/

Read Full Post »


Reporter: Aviva Lev-Ari, PhD, RN

Gene found that regenerates heart tissue

DALLAS – April 17, 2013 – Researchers at UT Southwestern Medical Center have identified a specific gene that regulates the heart’s ability to regenerate after injuries.

Scientists led by Dr. Hesham Sadek have demonstrated that the gene Meis1 regulates the regenerative capability of newborn hearts.

Scientists led by Dr. Hesham Sadek have demonstrated that the gene Meis1 regulates the regenerative capability of newborn hearts.

The function of the gene, called Meis1, in the heart was not known previously. The findings of the UTSW investigation are available online in Nature.

“We found that the activity of the Meis1 gene increases significantly in heart cells soon after birth, right around the time heart muscle cells stop dividing. Based on this observation we asked a simple question: If the Meis1 gene is deleted from the heart, will heart cells continue to divide through adulthood? The answer is ‘yes’,” said Dr. Hesham Sadek, assistant professor of internal medicine in the division of cardiology, and senior author of the study.

In 2011, Dr. Sadek’s laboratory showed that the newborn mammalian heart is capable of a vigorous, regenerative response to injury through division of its own cells. As the newborn develops, the heart rapidly loses the ability to regenerate and to repair injuries such as heart attacks.

The research team demonstrated that deletion of Meis1 extended the proliferation period in the hearts of newborn mice, and also re-activated the regenerative process in the adult mouse heart without harmful effect on cardiac functions. This new finding demonstrates that Meis1 is a key factor in the regeneration process, and the understanding of the gene’s function may lead to new therapeutic options for adult heart regeneration. The findings also provide a possible alternative to current adult heart regeneration research, which focuses on the use of stem cells to replace damaged heart cells.

Meis1 is a transcription factor, which acts like a software program that has the ability to control the function of other genes. In this case, we found that Meis1 controls several genes that normally act as brakes on cell division,” Dr. Sadek said. “As such, Meis1 could possibly be used as an on/off switch for making adult heart cells divide. If done successfully, this ability could introduce a new era in treatment for heart failure.”

According to the American Heart Association, almost 6 million people in the U.S. have heart failure, which occurs when the heart cannot pump enough blood and oxygen to support other organs. Heart disease is the leading cause of death for both men and women in the country, according to the Centers for Disease Control and Prevention.

The study received support from the American Heart Association, the Gilead Research Scholars Program in Cardiovascular Disease, the Foundation for Heart Failure Research, and the National Institutes of Health.

The co-first authors of the study are Dr. Ahmed I. Mahmoud, who is now a postdoctoral fellow at Harvard University; Dr. Fatih Kocabas, who is now a postdoctoral fellow at North American College; and Dr. Shalini A. Muralidhar, a postdoctoral research fellow II of internal medicine. Other researchers at UT Southwestern involved in the study are Wataru Kimura, a visiting senior researcher of internal medicine; Ahmed Koura, now a medical student at Ain Shams University in Egypt; Dr. Enzo Porrello, research fellow and faculty member at the University of Queensland in Australia; and Suwannee Thet, a research associate of internal medicine.

About UT Southwestern Medical Center
UT Southwestern, one of the premier academic medical centers in the nation, integrates pioneering biomedical research with exceptional clinical care and education. The institution’s faculty includes many distinguished members, including five who have been awarded Nobel Prizes since 1985. Numbering more than 2,700, the faculty is responsible for groundbreaking medical advances and is committed to translating science-driven research quickly to new clinical treatments. UT Southwestern physicians provide medical care in 40 specialties to nearly 100,000 hospitalized patients and oversee more than 2.1 million outpatient visits a year.

Media Contact: Remekca Owens
214-648-9344
remekca.owens@utsouthwestern.edu

 http://www.utsouthwestern.edu/newsroom/news-releases/year-2013/april/heart-sadek.html?goback=.gde_4842427_member_233989854

Genetics: A gene of rare effect

A mutation that gives people rock-bottom cholesterol levels has led geneticists to what could be the next blockbuster heart drug.

09 April 2013
ADAPTED FROM: PETER DAZELEY/GETTY

When Sharlayne Tracy showed up at the clinical suite in the University of Texas (UT) Southwestern Medical Center in Dallas last January, the bandage wrapped around her left wrist was the only sign of anything medically amiss. The bandage covered a minor injury from a cheerleading practice led by Tracy, a 40-year-old African American who is an aerobics instructor, a mother of two and a college student pursuing a degree in business. “I feel like I’m healthy as a horse,” she said.

Indeed, Tracy’s well-being has been inspiring to doctors, geneticists and now pharmaceutical companies precisely because she is so normal. Using every tool in the modern diagnostic arsenal — from brain scans and kidney sonograms to 24-hour blood-pressure monitors and cognitive tests — researchers at the Texas medical centre have diagnostically sliced and diced Tracy to make sure that the two highly unusual genetic mutations she has carried for her entire life have produced nothing more startling than an incredibly low level of cholesterol in her blood. At a time when the target for low-density lipoprotein (LDL) cholesterol, more commonly called ‘bad cholesterol’, in Americans’ blood is less than 100 milligrams per decilitre (a level many people fail to achieve), Tracy’s level is just 14.

A compact woman with wide-eyed energy, Tracy (not her real name) is one of a handful of African Americans whose genetics have enabled scientists to uncover one of the most promising compounds for controlling cholesterol since the first statin drug was approved by the US Food and Drug Administration in 1987. Seven years ago, researchers Helen Hobbs and Jonathan Cohen at UT-Southwestern reported1 that Tracy had inherited two mutations, one from her father and the other from her mother, in a gene called PCSK9, effectively eliminating a protein in the blood that has a fundamental role in controlling the levels of LDL cholesterol. African Americans with similar mutations have a nearly 90% reduced risk of heart disease. “She’s our girl, our main girl,” says Barbara Gilbert, a nurse who has drawn some 8,000 blood samples as part of Cohen and Hobbs’ project to find genes important to cholesterol metabolism.

Of all the intriguing DNA sequences spat out by the Human Genome Project and its ancillary studies, perhaps none is a more promising candidate to have a rapid, large-scale impact on human health than PCSK9. Elias Zerhouni, former director of the US National Institutes of Health (NIH) in Bethesda, Maryland, calls PCSK9 an “iconic example” of translational medicine in the genomics era. Preliminary clinical trials have already shown that drugs that inhibit the PCSK9 protein — used with or without statins — produce dramatic reductions in LDL cholesterol (more than 70% in some patients). Half-a-dozen pharmaceutical companies — all aiming for a share of the global market for cholesterol-reducing drugs that could reach US$25 billion in the next five years according to some estimates — are racing to the market with drugs that mimic the effect of Tracy’s paired mutations.

Free interview

Stephen Hall talks about Sharlayne’s unusual condition and whether similar cases might lead to a new line of drugs.

Zerhouni, now an in-house champion of this class of drug as an executive at drug firm Sanofi, headquartered in Paris, calls the discovery and development of PCSK9 a “beautiful story” in which researchers combined detailed physical information about patients with shrewd genetics to identify a medically important gene that has made “super-fast” progress to the clinic. “Once you have it, boy, everything just lines up,” he says. And although the end of the PCSK9 story has yet to be written — the advanced clinical trials now under way could still be derailed by unexpected side effects — it holds a valuable lesson for genomic research. The key discovery about PCSK9‘s medical potential was made by researchers working not only apart from the prevailing scientific strategy of genome research over the past decade, but with an almost entirely different approach.

As for Tracy, who lives in the southern part of Dallas County, the implications of her special genetic status have become clear. “I really didn’t understand at first,” she admits. “But now I’m watching ads on TV [for cholesterol-lowering drugs], and it’s like, ‘Wow, I don’t have that problem’.”

A heart problem

Cardiovascular disease is — and will be for the foreseeable future, according to the World Health Organization — the leading cause of death in the world, and its development is intimately linked to elevated levels of cholesterol in the blood. Since their introduction, statin drugs have been widely used to lower cholesterol levels. But Jan Breslow, a physician and geneticist at Rockefeller University in New York, points out that up to 20% of patients cannot tolerate statins’ side effects, which include muscle pain and even forgetfulness. And in many others, the drugs simply don’t control cholesterol levels well enough.

The search for better treatments for heart disease gained fresh impetus after scientists published the draft sequence of the human genome in 2001. In an effort to identify the genetic basis of common ailments such as heart disease and diabetes, geneticists settled on a strategy based on the ‘common variant hypothesis’. The idea was that a handful of disease-related versions (or variants) of genes for each disease would be common enough — at a frequency of roughly 5% or so — to be detected by powerful analyses of the whole genome. Massive surveys known as genome-wide association studies compared the genomes of thousands of people with heart disease, for example, with those of healthy controls. By 2009, however, many scientists were lamenting the fact that although the strategy had identified many common variants, each made only a small contribution to the disease. The results for cardiovascular disease have been “pretty disappointing”, says Daniel Steinberg, a lipoprotein expert at the University of California, San Diego.

Single-minded: Helen Hobbs and Jonathan Cohen’s approach to heart-disease genetics yielded a target for drugs that could compete with statins.MISTY KEASLER/REDUX/EYEVINE

More than a decade earlier, in Texas, Hobbs and Cohen had taken the opposite tack. They had backgrounds in Mendelian, or single-gene, disorders, in which an extremely rare variant can have a big — often fatal — effect. They also knew that people with a particular Mendelian disorder didn’t share a single common mutation in the affected gene, but rather had a lot of different, rare mutations. They hypothesized that in complex disorders, many different rare variants were also likely to have a big effect, whereas common variants would have relatively minor effects (otherwise natural selection would have weeded them out). “Jonathan and I did not see any reason why it couldn’t be that rare variants cumulatively contribute to disease,” Hobbs says. To find these rare variants, the pair needed to compile detailed physiological profiles, or phenotypes, of a large general population. Cohen spoke of the need to “Mendelize” people — to compartmentalize them by physiological traits, such as extremely high or low cholesterol levels, and then look in the extreme groups for variations in candidate genes known to be related to the trait.

The pair make a scientific odd couple. Hobbs, who trained as an MD, is gregarious, voluble and driven. Cohen, a soft-spoken geneticist from South Africa, has a laid-back, droll manner and a knack for quantitative thinking. In 1999, they set out to design a population-based study that focused on physical measurements related to heart disease. Organized with Ronald Victor, an expert on high blood pressure also at UT Southwestern, and funded by the Donald W. Reynolds Foundation in Las Vegas, Nevada, the Dallas Heart Study assembled exquisitely detailed physiological profiles on a population of roughly 3,500 Dallas residents2. Crucially, around half of the participants in the study were African Americans, because the researchers wanted to probe racial differences in heart disease and high blood pressure. The team measured blood pressure, body mass index, heart physiology and body-fat distribution, along with a battery of blood factors related to cholesterol metabolism — triglycerides, high-density lipoprotein (HDL) cholesterol and LDL cholesterol. In the samples of blood, of course, they also had DNA from each and every participant.

As soon as the database was completed in 2002, Hobbs and Cohen tested their rare-variant theory by looking at levels of HDL cholesterol. They identified the people with the highest (95th percentile) and lowest (5th percentile) levels, and then sequenced the DNA of three genes known to be key to metabolism of HDL cholesterol. What they found, both in Dallas and in an independent population of Canadians, was that the number of mutations was five times higher in the low HDL group than in the high group3. This made sense, Cohen says, because most human mutations interfere with the function of genes, which would lead to the low HDL numbers. Published in 2004, the results confirmed that rare, medically important mutations could be found in a population subdivided into extreme phenotypes.

Armed with their extensive database of cardiovascular traits, Hobbs and Cohen could now dive back into the Dallas Heart Study whenever they had a new hypothesis about heart disease and, as Cohen put it, “interrogate the DNA”. It wasn’t long before they had an especially intriguing piece of DNA at which to look.

The missing link

In February 2003, Nabil Seidah, a biochemist at the Clinical Research Institute of Montreal in Canada, and his colleagues reported the discovery of an enigmatic protein4. Seidah had been working on a class of enzymes known collectively as proprotein convertases, and the researchers had identified what looked like a new member of the family, called NARC-1: neural apoptosis-regulated convertase 1.

“We didn’t know what it was doing, of course,” Seidah says. But the group established that the gene coding the enzyme showed activity in the liver, kidney and intestines as well as in the developing brain. The team also knew that in humans the gene mapped to a precise genetic neighbourhood on the short arm of chromosome 1.

That last bit of geographical information pointed Seidah to a group led by Catherine Boileau at the Necker Hospital in Paris. Her team had been following families with a genetic form of extremely high levels of LDL cholesterol known as familial hypercholesterolaemia, which leads to severe coronary artery disease and, often, premature death. Group member Marianne Abifadel had spent five fruitless years searching a region on the short arm of chromosome 1 for a gene linked to the condition. When Seidah contacted Boileau and told her that he thought NARC-1 might be the gene she was looking for, she told him, “You’re crazy”, Seidah recalls. Seidah bet her a bottle of champagne that he was correct; within two weeks, Boileau called back, saying: “I owe you three bottles.”

“The PCSK9 story is a terrific example of an up-and-coming pattern of translational research.”

In 2003, the Paris and Montreal groups reported that the French families with hypercholesterolaemia had one of two mutations in this newly discovered gene, and speculated that this might cause increased production of the enzyme5. Despite Seidah’s protests, the journal editors gave both the gene and its protein product a new name that fit with standard nomenclature: proprotein convertase subtilisin/kexin type 9, or PCSK9. At around the same time, Kara Maxwell in Breslow’s group at Rockefeller University6 and Jay Horton, a gastroenterologist at UT-Southwestern7 also independently identified the PCSK9 gene in mice and revealed its role in a previously unknown pathway regulating cholesterol8.

The dramatic phenotype of the French families told Hobbs that “this is an important gene”. She also realized that in genetics, mutations that knock out a function are much more common than ones that amplify function, as seemed to be the case with the French families. “So immediately I’m thinking, a loss-of-function mutation should manifest as a low LDL level,” she says. “Let’s go and see if that’s true.”

Going to extremes

Hobbs and Cohen had no further to look than in the extreme margins of people in the Dallas Heart Study. In quick order, they identified the highest and lowest LDL readings in four groups: black women, black men, white women and white men. They then resequenced the PCSK9 gene in the low-cholesterol groups, looking for mutations that changed the make-up of the protein.

They found seven African Americans with one of two distinct ‘nonsense’ mutations in PCSK9 — mutations that essentially aborted production of the protein. Then they went back and looked for the same mutations in the entire population. Just 2% of all black people in the Dallas study had either of the two PCSK9 mutations — and those mutations were each associated with a 40% reduction of LDL cholesterol in the blood9. (The team later detected a ‘missense mutation’ in 3% of white people, which impaired but did not entirely block production of the protein.) The frequency of the mutations was so low, Hobbs says, that they would never have shown up in a search for common variants.

When Hobbs and Cohen published their findings in 2005, they suggested that PCSK9 played a crucial part in regulating bad cholesterol, but said nothing about whether the mutations had any effect on heart disease. That evidence came later that year, when they teamed up with Eric Boerwinkle, a geneticist at the University of Texas Health Science Center in Houston, to look forPCSK9 mutations in the Atherosclerosis Risk in Communities (ARIC) study, a large prospective study of heart disease that had been running since 1987. To experts such as Steinberg, the results10 — published in early 2006 — were “mind-blowing”. African Americans in ARIC who had mutations in PCSK9 had 28% less LDL cholesterol and an 88% lower risk of developing heart disease than people without the mutations. White people with the less severe mutation in the gene had a 15% reduction in LDL and a 47% reduced risk of heart disease.

How did the gene exert such profound effects on LDL cholesterol levels? As researchers went on to determine11, the PCSK9 protein normally circulates in the bloodstream and binds to the LDL receptor, a protein on the surface of liver cells that captures LDL cholesterol and removes it from the blood. After binding with the receptor, PCSK9 escorts it into the interior of the cell, where it is eventually degraded. When there is a lot of PCSK9 (as in the French families), there are fewer LDL receptors remaining to trap and remove bad cholesterol from the blood. When there is little or no PCSK9 (as in the black people with mutations), there are more free LDL receptors, which in turn remove more LDL cholesterol.

“We didn’t understand why everybody wasn’t doing what we were doing.”

The UT-Southwestern group, meanwhile, went back into the community looking for family members who might carry additional PCSK9 mutations. In September 2004, Gilbert, the nurse known as ‘the cholesterol lady’ in south Dallas because of her frequent visits, knocked on the door of Sharlayne Tracy’s mother, an original member of the Dallas Heart Study. Gilbert tested Tracy, as well as her sister, brother and father. “They tested all of us, and I was the lowest,” Tracy says. Zahid Ahmad, a doctor working with Hobbs at UT-Southwestern, was one of the first to look at Tracy’s lab results. “Dr Zahid was in awe,” Tracy recalled. “He said, ‘You’re not supposed to be so healthy!’.”

It wasn’t just that her LDL cholesterol measured 14. As a person with two dysfunctional copies of the gene — including a new type of mutation — Tracy was effectively a human version of a knockout mouse. The gene had been functionally erased from her genome, and PCSK9 was undetectable in her blood without any obvious untoward effects. The genomics community might have been a little slow to understand the significance, Hobbs says, “but the pharmaceutical companies got it right away”.

The next statin?

This being biology, however, the road to the clinic was not completely smooth. The particular biology of PCSK9 has so far thwarted efforts to find a small molecule that would interrupt its interaction with the LDL receptor and that could be packaged in a pill. But the fact that the molecule operates outside cells means that it is vulnerable to attack by monoclonal antibodies — one of the most successful (albeit most expensive) forms of biological medicine.

The results of early clinical trials have caused a stir. Regeneron Pharmaceuticals of Tarrytown, New York, collaborating with Sanofi, published phase II clinical-trial results12 last October showing that patients with high LDL cholesterol levels who had injections every two weeks of an anti-PCSK9 monoclonal antibody paired with a high-dose statin saw their LDL cholesterol levels fall by 73%; by comparison, patients taking high-dose statins alone had a decrease of just 17%. Last November, Regeneron and Sanofi began to recruit 18,000 patients for phase III trials that will test the ability of their therapy to cut cardiovascular events, including heart attacks and stroke. Amgen of Thousand Oaks, California, has also launched several phase III trials of its own monoclonal antibody after it reported similarly promising results13. Among other companies working on PCSK9-based therapies are Pfizer headquartered in New York, Roche based in Basel, Switzerland, and Alnylam Pharmaceuticals of Cambridge, Massachusetts. (Hobbs previously consulted for Regeneron and Pfizer, and now sits on the corporate board of Pfizer.)

Not everyone is convinced that a huge market awaits this class of cholesterol-lowering drugs. Tony Butler, a financial analyst at Barclays Capital in New York, acknowledges the “beautiful biology” of the PCSK9 story, but wonders if the expense of monoclonal drugs — and a natural reluctance of both patients and doctors to use injectable medicines — will constrain potential sales. “I have no idea what the size of the market may be,” he says.

“Everything hinges on the phase III side effects,” says Steinberg. So far, the main side effects reported have been minor, such as reactions at the injection site, diarrhoea and headaches. But animal experiments have raised potential red flags: the Montreal lab reported in 2006 that knocking out the gene in zebrafish is lethal to embryos14. That is why the case of Tracy was “very, very helpful” to drug companies, says Hobbs. Although her twin mutations have essentially deprived her of PCSK9 throughout her life, doctors have found nothing abnormal about her.

That last point may revive a debate in the cardiology community: should drug therapy to lower cholesterol levels, including statins and the anti-PCSK9 medicines, if they pan out, be started much earlier in patients than their 40s or 50s? That was the message Steinberg took from the people withPCSK9 mutations in the ARIC study — once he got over his shock at the remarkable health effects. “My first reaction was, ‘This must be wrong. How could that be?’And then it hit me — these people had low LDL from the day they were born, and that makes all the difference.” Steinberg argues that cardiologists “should get off our bums” and reach a consensus about beginning people on cholesterol-lowering therapy in their early thirties. But Breslow, a former president of the American Heart Association, cautions against being too aggressive too soon. “Let’s start out with the high-risk individuals and see how they do,” he says.

Not long after Hobbs and Cohen published their paper in 2006, they began to get invited to give keynote talks at major cardiology meetings. Soon after, the genetics community began to acknowledge the strength of their approach. In autumn 2007, then-NIH director Zerhouni organized a discussion at the annual meeting of the institutes’ directors to raise the profile of the rare-variant approach and contrast it with genome-wide studies. “Obviously, the two approaches are opposed to each other, and the question was, what was the relative value of each?” says Zerhouni. “I thought the PCSK9 story was a terrific example of an up-and-coming pattern of translational research” — indeed, he adds, “a harbinger of things to come”.

Hobbs and Cohen might not have found their gene if they had not had a hunch about where to look, but improved sequencing technology and decreasing costs now allow genomicists to incorporate the rare variant approach and to mount large-scale sweeps in search of such variants. “Gene sequencing is getting cheap enough that if there’s another gene like PCSK9 out there, you could probably find it genome-wide,” says Jonathan Pritchard, a population biologist at the University of Chicago, Illinois.

“What was amazing to us,” says Hobbs, “was that the genome project was spending all this time, energy, effort sequencing people, and they weren’t phenotyped, so there was no potential for discovery. We didn’t understand, and couldn’t understand, why everybody wasn’t doing what we were doing. Particularly when we started making discoveries.”

Nature

 496,

152–155

(11 April 2013)

doi:10.1038/496152a

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