Journal article summaries!

Robert Moss Mossre at wofford.edu
Mon Aug 17 23:31:56 EST 1998


        Are you keeping up with the literature as much as you would like to? 
Modern biology moves so quickly, few of us can keep up.   I can help, with a
new service aimed at helping keep YOU up to date. 
        I will be reviewing many of the major and popular journals, and
sending
out succinct summaries of some of the most interesting articles via email.  In
just a few minutes you will be able to read over each summary and learn about
the most interesting, cutting edge science.  Just read my summaries, or use
them to lead you to the full articles in your library.  Either way, these
summaries will help you sort through the literature and learn about what is
going on in the world of biology.
        The summaries are written using fairly non-technical language, and are
accompanied by background explanations.  They should be appropriate for
educators at both the high school and college level, physicians, students of
biology, and scientists as well.  Each issue will summarize at least 10
articles, plus one reviewing or explaining some of the science behind one of 
the articles.   Issues will be sent every other Monday, at a cost of less than
46 cents each!
        I hold a PhD in Cell and Developmental Biology from Harvard
University,
and teach at Wofford College in the fields of genetics, molecular biology,
development, immunology, and cancer biology.  You may have read articles I
have
authored in The Scientist, The Journal of College Science Teaching, The
American
Biology Teacher, Cancer, and Technology Review among others.  You can expect
interesting and readable summaries every week!
        You will find a sample issue below.

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BIOLOGY UPDATES   -   Volume 1, Issue #1  - August 17, 1998

1.  Gene therapy: Ribozyme mediated repair of sickle beta-globin
2.  Human genome project: Shotgun sequencing of the human genome
3.  Left-right body plan determination in mice
4.  Ethical and social aspects of testing for Alzheimer Disease
5.  Biological based treatments for breast cancer
6.  Gamma-delta T cells in the immune system    
7.  Tissue microarrays for molecular profiling of tumor specimens
8.  CFTR mutation providing resistance to typhoid
9.  Transplant of embryonic neurons to treat Huntingtons Disease
10. Defeating AIDS: Reviews
11. Background: Knock-out mice

1.  Lan et al: RIBOZYME-MEDIATED REPAIR OF SICKLE BETA-GLOBIN mRNAs IN
ERYTHROCYTE PRECURSORS: Science, v. 280, p. 1593: June 5, 1998
        Red blood cell precursors from patients with sickle cell anemia were
transfected with a trans-splicing ribozyme.   Ribozymes were able to
convert 8%
of the sickle cell transcripts into mRNAs encoding the anti-sickling protein
gamma-globin.  The half-time of the reaction was approximately 60 minutes.
        The authors state that sickle cell patients expressing gamma globin at
10-20% of the level of sickle cell globin usually have greatly improved
clinical prognosis, so that even the fairly small percentage of globin
converted here could be clinically relevant.
        This paper is quite interesting, as it involves correcting a genetic
defect by altering the RNA, not the gene!
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2. Venter et al: SHOTGUN SEQUENCING OF THE HUMAN GENOME: Science, v. 280, p.
1540: June 5, 1998
        The Human Genome Project is scheduled to complete its sequence of the
three billion base pairs of human genomic DNA in 2005, for an estimated
cost of
$3 billion.  Although the project has been ahead of schedule in mapping DNA
markers, actual sequencing has been fairly slow going.    Current sequencing
technology involves flourescent labeling of DNA fragments generated by the
Sanger dideoxy chain termination method, and automated sequencing.   The
limiting step in sequencing is building “sequence ready” maps covering large
areas of the genome.  
        A number of smaller genomes have been sequenced using a different
approach, a whole-genome “shotgun” method.   Unordered DNA fragments are
randomly cloned and sequenced, and the order of the fragments is determined by
overlapping the completed sequences.
        In this paper, the authors describe a new commercial joint venture
between The Institute for Genomic Research and Perkin-Elmer Applied
Biosystems.  The latter company has announced a new fully automated sequencer,
capable of processing 1,000 samples per day with only 15 minutes hands-on
operator time.  This compares with 8 hours for the previous model.  
        The authors propose to complete the human sequence in only 3 years,
at a
cost of $200-$250 million, including the computational and laboratory
infrastructure required for the task.  The lab will contain 230 automated
sequencers, with a combined daily sequencing capacity of 100 million bases.
        Their approach involves randomly breaking genomic DNA into segments of
various sizes, and cloning into multiple vectors.  A plasmid library
containing
inserts of approximately 2 kb will be supplemented by a library of plasmids
with 10 kb inserts.  This will allow multiple overlapping sequences, and
reduce
the number of sequences that are unclonable or under-represented in one of the
libraries.  The goal is to generate 70 million sequences totaling 35 billion
base pairs, covering the entire genome ten times.
        To make ordering the sequences easier, and check the reliability of
their results, the sequences will be compared to 30,000 known Sequence Tagged
Site (STS) markers, as well as Expressed Sequence Tags (EST).
        Once the project is completed, the group intends to make the actual
sequence data available free of charge, without restrictions.  They expect to
earn online access fees, and market the database system to pharmaceutical and
biotechnology companies as well.

3. Meno et al: LEFTY-1 IS REQUIRED FOR LEFT-RIGHT DETERMINATION AS A REGULATOR
OF LEFTY-2 AND NODAL: Cell, v. 94, p. 287.  August 7, 1998

        Certainly one of the most interesting questions in biology is that of
embryonic development: How can a single cell grow into such a complex
organism,
with such complex patterns?  How do cells “know” where to go and what to
become
to make a complex body plan?  Developmental biologists have come a long way
towards understanding the basic genetic processes in a number of experimental
organisms, most notably the fruit fly Drosophila melanogaster.  But until
recently, they have lacked the tools to address pattern formation in mammals.
        Just over a decade ago, a new technology called “knock-out mice” was
developed.  Using this technology [see “BACKGROUND” section in this issue],
scientists can create mice lacking any gene of interest, and study the
effects.  A number of genes involved in body plan in mice have been identified
in this way.
        This paper reports the results of an experiment eliminating the
“lefty-1" gene in mice.  Lefty-1, lefty-2, and nodal are all expressed on the
left side of developing mouse embryos, and are implicated in left-right
determination.  Mice lacking the lefty-1 gene showed a number of positional
defects in internal organs, including transformations of one side of the heart
and lung.  The most common feature of these mice was left isomerism.  Since
lefty-1 is expressed in the left side, it was expected that eliminating the
gene would cause right isomerism.  The mice lacking lefty-1 now expressed
lefty-2 and nodal on BOTH sides, suggesting the normal role of lefty-1 is to
limit the expressoin of lefty-2 and nodal to only the left side.

4.  McConnell et al: GENETIC TESTING AND ALZHEIMER DISEASE: HAS THE TIME COME?
Nature Medicine, v. 4, p. 757 (July, 1998)
        When the Human Genome Project was first funded, the bill mandated that
5% of the three billion dollars going to the project be spent studying the
“Ethical, Legal, and Social Implications” (ELSI) of the technology.  In this
paper, Stanford University’s ELSI group presents their recommendations
regarding genetic testing for Alzheimer disease (AD).
        There are a number of genes associated with AD.  Three, APP, PS1, and
PS2 are dominant, and lead to early onset AD.  These are often found in
“familial” cases of AD, that is, when the disease seems to “run in the
family”.  Another gene, APOE, may be predictive of AD.  Every person has two
alleles for APOE.  A person having one APOE4 allele is at higher risk for AD
later in life; someone having two APOE4 alleles has a higher risk still.  
        A number of commercial gene tests have been developed as
diagnostics and
predictive tests.  The authors claim that these tests should not be used as
screening tools or predictive tests, except in certain cases where there is a
strong familial association with AD.  They argue that the tests are not good
enough; they only predict higher risk, not certainty of getting the disease. 
So the tests would have many “false positives”, many people would be burdened
with the prediction that they will get the disease who never will develop
symptoms.  Since there is not yet any good treatment or prevention for the
disease, there does not seem to be significant benefit in testing.
        Other recommendations made by the group include:
        - No type of genetic testing for AD should be done for children,
fetuses
or embryos at this time.
        - Skilled genetic counseling is essential whenever offering AD genetic
testing.  The counselors must assess the decision-making capacity of the
person
interested in testing, educate him or her about the implications for both the
patient and untested family members, discuss the limits of current knowledge
regarding diagnosis, treatment, and prevention of AD, and make referrals for
sources of ongoing education and psychological support.
        - Expanded genomics education is essential both for health
professionals, and for the public in general.

5. Nass et al: BREAST CANCER BIOLOGY BLOSSOMS IN THE CLINIC: Nature
Medicine, v.
4 p. 761 (July, 1998)
        This short “News and Views” article reviews some of the recent
clinical
work relating to biological therapies for breast cancer.
        A humanized monoclonal antibody against the HER-2 protein, which is
over-expressed in 30% of breast tumors, is being tested, and shows promise. 
Monoclonal antibodies are normally made in mice, and are cleared from patients
fairly quickly by the immune system, since they are recognized as foreign.  By
making these proteins look more like human antibodies, researchers can
increase
the life of these proteins in the body, and thus increase their effectiveness.
        Estrogen is required for the growth of a majority of breast cancers,
those that express the estrogen receptor.  Analysis of 55 randomized clinical
trials has shown that tamoxifen, and anti-estrogen, substantially improves the
10 year survival of women with early stage breast cancers, regardless of
age or
menopausal status.  Only tumors expressing the estrogen receptor are affected.
        This success has prompted clinical trials to investigate the
ability of
tamoxifen to prevent breast cancer in high risk women.  Early results show a
significant, perhaps more than 40% reduction of breast cancer in all age
groups.
        Tamoxifen and raloxifene, another antiestrogen, are selective, in that
they enhance estrogen in some tissues, and block it in others.  These drugs
may
be able to prevent breast cancer while promoting other beneficial effects of
estrogen such as maintenance of bone density, and lowering of cholesterol
levels.  There are rare side effects though, including uterine cancer and
embolisms due to clots.
        The authors suggest that these studies will stimulate the
development of
new and superior drugs, as well as monoclonal antibodies, and vaccines.

6. Mak and Ferrick: THE GAMMA-DELTA T CELL BRIDGE: LINKING INNATE AND ACQUIRED
IMMUNITY: Nature Medicine, v. 4, p. 764 (1998)
        The immune system has two major divisions: the “humoral” immune
system,
consisting of antibodies, and the “cellular” immune system, consisting of
the T
cells, responsible for fighting intracellular parasites like viruses, killing
cancer cells, and regulating the entire immune system.  Most studies of T
cells
to date have been of the more common, “alpha-beta” T cells.  They have a T
cell
receptor made up of two chains, and alpha and a beta.  Until recently, the
function of the “gamma-delta” T cells, with T cell receptors containing a
gamma
and a delta chain, has remained a mystery.
        This short “news and views” article summarizes recent discoveries
regarding the gamma-delta cells.
        Gamma-delta T cells were first discovered due to the structural
similarity between their T cell receptors and those of the alpha-beta cells.
Fewer than ten percent of circulating T cells carry the gamma delta receptor. 
New discoveries show that unlike the more common T cells, gamma-delta cells
are
not antigen specific, and do not bind to the polymorphic MHC-peptide
complexes. 
Instead, they bind to “stress-associated antigens”, presented by epithelial
cells when damaged or infected.  The chemokines and cytokines they release
upon
activation recruit inflammatory cells, and mediate both the innate and
acquired
immune response.
        Studies show that the gamma-delta cells enter the intestinal mucosa
directly from the bone marrow, bypassing the thymus.  They recognize MHC class
I-related molecules MICA and MICB, which are induced by stress from injury or
infection.  The recognition is independent of antigen processing, showing
these
gamma-delta cells are not antigen specific.  The authors conclude that these
gamma-delta cells may be an evolutionary link between non-specific cells such
as macrophages, and the very specific alpha-beta T cells.  

7.  Kononen et al: TISSUE MICROARRAYS FOR HIGH-THROUGHPUT MOLECULAR PROFILING
OF TUMOR SPECIMENS: Nature Medicine, v. 4, p. 844, with commentary on p. 767
(July, 1998).
        Many genes are involved in cancer development.  Many studies have
looked
at individual genes and their roles in tumor formation; the current challenge
is to examine the combinations of gene mutations and their roles and effect on
prognosis.  Scientists also need to examine large numbers of tumors, of many
stages and types.  The authors of this article have designed an array-based
technique, placing as many as 1,000 cylindrical tissue biopsies from tumors on
a 45 x 20 mm filter, for in-situ detection of DNA, RNA, or protein. 
Consecutive sections through the tumor array allow the use of a number of
different probes on the same 1,000 tissue samples.  They used the technique to
detect six gene amplifications as well as p53 and estrogen receptor expression
in breast cancers.  

8.  Prince, Alice: THE CFTR ADVANTAGE–CAPITALIZING ON A QUIRK OF FATE: Nature
Medicine, v. 4, p. 663 (June, 1998).
        Scientists have been looking for an explanation for the high frequency
of the cystic fibrosis gene mutation for years.  Since people who receive two
copies of the CFTR gene have generally died from CF before passing on the
gene,
one might expect the gene frequency to have decreased over time.  A similar
situation exists for the sickle cell anemia gene: Its frequency is much higher
than expected.  In the case of sickle cell, there is a good explanation -
having one copy of the sickle cell gene protects the carrier from infection
with malaria.  So in areas where malaria is common, the advantage of having
one
mutant sickle cell gene outweighs the less common disadvantage of having two
mutant genes.
        There has been much speculation about a possible advantage of
having one
mutant CFTR gene.  This short News and Views paper describes experimental
evidence that the intestinal CFTR protein serves as a receptor for the S.
typhi
bacterium, which causes typhoid.  The bacteria must bind to this protein to
enter the body.  Thus it is possible that people having one mutant copy of the
CFTR gene might be resistant to typhoid.  However, there is not yet any
clinical evidence to back this hypothesis up.

9.  Kendall et al: FUNCTIONAL INTEGRATION OF STRIATAL ALLOGRAFTS IN A PRIMATE
MODEL OF HUNTINGTONS DISEASE: Nature Medicine, v. 4, p. 727, with
commentary on
p. 669 (June, 1998)
        Huntingtons Disease (HD) is a progressive neurodegenerative disorder
characterized by involuntary movements (chorea), as well as cognitive and
personality defects.   Studies in mice have already shown that transplant of
embryonic brain cells expressing the normal huntingtin protein can improve the
motor defects.  These authors have extended those studies to primates, using
marmosets.  Striatal neurons from “P-zones” and non-striatal cells from
“NP-zones” were mixed, and implanted.   All six of the transplant recipients
showed improved motor skills.  All six maintained their improvement for at
least nine months, and some for 12 months, even without suppressing their
immune systems.  All animals had graft tissue surviving in the brain at
time of
autopsy, 10-12 months following transplantation.  The factor with the
strongest
correlation to degree of improvement was placement of the graft, with the best
site being the lateral edge of the globus pallidus. These studies might clear
the way for clinical trials of either primate or human embryonic tissue
transplants for the treatment of HD.

10. DEFEATING AIDS: WHAT WILL IT TAKE?  Scientific American, July, 1998
        This issue has a 25 page “special report” on defeating AIDS.  It
contains 9 articles, titled “HIV 1998: The Global Picture”, “Improving HIV
Therapy”, “How Drug Resistance Arises”, “Viral Load Tests Provide Valuable
Answers”, “When Children Harbor HIV”, “Preventing HIV Infection”, “HIV
Vaccines: Prospects and Challenges”, “Avoiding Infection after HIV Exposure”,
and “Coping with HIVs Ethical Dilemmas”.
        Since this entire section is too long to summarize here, and since you
all have access to Scientific American and will want to read some of these for
yourselves, I will summarize only the longest one: “IMPROVING HIV THERAPY”. 
This article gives an overview of HIV infection, which I will not repeat
here. 
Much of the article discusses drug therapy for HIV infection.
        The new “cocktails”, containing drugs to block reverse transcriptase
plus protease inhibitors, have reduced deaths due to AIDS by 44% between the
first half of 1996 and the first half of 1997. The treatment is burdensome and
costly though, and not avialable to most HIV infected people, in developing
nations.  No one knows how long these gains will be sustained, until the virus
can mutate to create strains resistant to these coctails. 
        How the infection progresses is often determined by the strength of
the
patient’s own immune response to the virus.  The stronger the CD8 T cell
response (“killer” T cells, which eliminate virally infected cells), the lower
the amount of virus found in the patient, and the more slowly the progression
to full-blown AIDS.  At any stage, viral levels correlate with prognosis.  The
antiviral cocktails lower the viral levels dramatically, often to undetectable
levels.  Clinics report that approximately 50% of patients given triple drug
cocktails reach the goal of less than 500 copies of virus per milliliter of
blood (currently considered “undetectable”) between six and 52 weeks after
initiating treatment.  Most of the other half of patients show more limited
results.  Although far from perfect, these results have brought the number of
hospitalizations due to major AIDS related problems down by 50-80%.
        However, even if the virus is not detectable in blood, virus
remains in
the body, and patients will have to continue taking the drugs for years;
probably indefinitely, or the virus will re-emerge.
        The current therapy of choice contains two reverse transcriptase
inhibitors, plus a protease inhibitor.  The drugs run $10,000-$12,000 per
year.  And the therapy is difficult to follow: patients must swallow at least
8, often 16 or more, anti-HIV pills each day, in addition to others to control
opportunistic infections and pain.  And they must keep track of which can be
taken with or without food, etc.   The drugs have many toxic side effects,
causing some patients to simply stop taking some of the drugs.  And many
patients are unable to follow the regimen, particularly the homeless,
demented,
or drug users.  Incomplete adherence to treatment plans accounts for about
half
of treatment failures.
        Most experts agree that the best time to begin treatment is early
on in
the acute phase of infection, before patients’ immune systems have suffered
much damage.  Yet it is often difficult to convince patients who are feeling
well, who do not yet have symptoms of the disease, to take such a toxic
regimen, which makes them feel ill.  Most patients with high T cell counts opt
to wait until they become ill, or until better treatments are available,
before
beginning.
        A number of new treatments are under investigation, including
“antisense
RNAs” to inactivate certain HIV genes, and viruses engineered to infect only
HIV infected cells and then activate “suicide genes”.  A particularly
promising
approach involves isolating blood-forming stem cells from an HIV patient.  The
stem cells do not yet have CD4 protein on their surface, so they are not
infected by the virus.  These stem cells are grown, transfected with a gene to
protect them from HIV when they later differentiate into T cells, and then
reinjected into the patient.  

11.  BACKGROUND: KNOCK OUT MICE
        One of the most important tools in developmental biology is
mutagenesis.  Scientists need to be able to create mutations in genes of
interest, and then observe the effect on development.  In haploid organisms,
such as bacteria and haploid yeast, this has been relatively easy.  But in
diploid organisms, it is much more difficult.  Early in the 1980's, scientists
developed the ability to create “transgenic” animals, that is, animals
containing an extra, foreign gene.  As it turns out, adding an extra gene is
much easier than altering or removing an existing gene.
        In 1989 Mario Capecchi (Science v. 244 p. 1288) announced he had
learned
how to “knock out” a gene in mice.  Knock-out technology is widely used today,
and has allowed scientists to unlock many secrets about the function of many
different kinds of genes in mammals.
        Here is how it works.  You start with mouse embryos, at the blastocyst
stage.  At this stage, the cells in the center, called the “Inner Cell Mass”,
are “totipotent”... that is, the cells have not differentiated, and each cell
can form an entire mouse under the proper conditions.  These cells are
removed,
and grown in culture.  They are now called “embryonic stem cells” [ES cells]. 
In culture, the cells can easily be transfected with foreign DNA.  The
trick is
to use the proper genetic markers to be able to select for the rare cell in
which the foreign DNA enters the genome not at random, but by homologous
recombination.  In this rare cell, the foreign copy of the gene replaces the
original gene in the mouse.  If the foreign copy was altered so that it is
nonfunctional, the cell now has lost a copy of the normal gene.
        By injecting these particular cells back into a blastocyst, you can
create a whole mouse from these cells.  This mouse now has one normal, and one
mutated copy of the gene.  By breeding this mouse and then inbreeding the
offspring, you can create a new mouse lacking BOTH copies of the gene in two
generations.
        This is a very powerful technique.  “Knock outs” have been made
lacking
certain oncogenes, tumor suppressor genes, genes critical to the immune
system,
and genes critical for development, among others.  The technology has been
vital to working out the function of many important biological pathways in
mice.




- Please feel free to distribute or forward this particular issue.      
- If you would like to contribute summaries of articles you find particularly
interesting, please email them to me.  The summary will retain your name as
contributor.  And if I use your summary, I will tack on a month to your
subscription as a small “thank you”.
- Please let me know if there are topics you would like me to cover in the
“background” section, or questions you would like addressed about any of these
articles or topics.

Email questions or comments to Bob Moss, MOSSRE at WOFFORD.EDU 
-----------------------------------------------
Bob Moss
Associate Professor of Biology
Wofford College
429 N. Church Street
Spartanburg, SC  29303
(864)597-4623; fax -4620
email MOSSRE at WOFFORD.EDU
http://www.wofford.edu/~mossre/
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