Journal Articles Summaries!!

I used to have a life... now I have a computer. mossre at mail.wofford.edu
Tue Aug 18 20:01:58 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
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	I will be reviewing many of the major and popular journals, and sending out
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interesting, cutting edge science.  Just read my summaries, or use them to lead you
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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
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immunology, and cancer biology.  You may have read articles I have authored in The
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Cancer, and Technology Review among others.  You can expect interesting and readable
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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!
------------------------------------------------------------------------------------------------------------
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 ADVANTAGECAPITALIZING 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



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