Biology 610: Current Topics in Cell and Molecular
Biology
Fall Semester 2003 Molecular Biology 610: Molecular and Cell Biology of Disease
(Please note meeting room, date and time of lectures)
Instructor:
Roger Davis, LS307, rdavis@sunstroke.sdsu.edu;
619-594-7936).
The goal of this course is to provide students with
insights into how modern cell and molecular biology is used to gain and
understanding of disease processes and/or the development of therapies. Each week the course will concentrate
on one related topic. On Mondays
(except November 18- see below) outside experts will come a give the first
lecture (didactic and up to 1.5 hours).
This lecture will provide students with both a review of the current
literature as well as the presenter’s own view of where the field is headed.
The second class meeting will be a class discussion in
which two research publications (provided via the class website in the form of
a PDF file) are discussed by the students and led by the instructor. Each student is responsible for having
read the paper and being prepared to describe to the class the salient points
of the paper, which include:
1)
understanding all data
presented in figures and tables
2)
understanding what
conclusions were offered by the authors
3)
understanding how these
conclusions are (or are not) supported by the data
4)
being prepared to
propose alternative conclusions and experiments to further examine the major issues
of the papers
Grades will be calculated as 50% class participation
and 50% final exam. Each person
can only miss one class meeting!
Oct 22
Sandy Bernstein will finish discussion of first part of class. Class should begin studying papers for
week of Oct 27.
Oct 27 Stanley
Maloy, Professor of Biology, SDSU; Topic: “Infectious
bacteria and human disease”
Nov 3 Linda
Curtiss, Member, Scripps Research Institute; Topic:
“Biology of bone marrow transplantation and its therapeutic importance”
Nov 10
Simon Hui, Research Assistant Professor of Biology, SDSU; Topic:
“Nuclear receptors”
TUESDAY, Nov 18 (LS101) Chris Glass, Professor of Cell
and Molecular Medicine, UCSD; Topic: “PPARs and metabolic diseases”
Nov 24
Mark Sussman, Professor of Biology, SDSU; Topic:“Cardiac myocyte
development and regeneration”
Dec
1 Jim Paterniti, Senior Director,
Pharmacology, Amylin Pharmaceuticals, Inc.; Topic: “Diabetes, obesity and energy metabolism”
Dec
8 Joseph Witztum, Professor
of Medicine, UCSD; Topic:“innate immunity and atherosclerosis”
Dec 10 Final examination
___________________________________________________________
a. Host
immunity
b. Other
host functions
a. What
is a pathogen? Microbe vs host
b. What
is a virulence gene? Molecular Koch’s postulates
c. Evolution
of pathogenesis
d. How
can you identify bacterial genes
required for virulence
Two
papers that emphasize some of these concepts are attached
How
did they demonstrate that the observed host response was elicited by
Bacteroides?
Why
did they focus on Bacteroides? Do all commensal bacteria stimulate a similar
response?
How
might bacterial cells promote this host response?
How
did they visualize the microvasculature development in the small intestine?
Are
there better ways this could be done?
Why
did they focus on Paneth cells?
Not
as simple a question as it sounds ... see Nat Immunol. 2003 4: 269]
Why
is this a “symbiosis”?
What
is the evidence that internalin is specifically involved in the transit of
Listeria across the gut?
Does
internalin affect the systemic infection?
What
is the evidence that E-cadherin is the host receptor?
What
is the difference in E-cadherin between humans, guinea pigs, and mice?hat is
the proposed role for inlB and why might this be required
Nuclear
Receptors Review 1 and Co-activators/repressors
Pick
the BEST answer in each
1) LXR binds to a DR4 site on the CYP7A1
promoter. Which of the following
sequences are may describe the LXR binding site?
A) AGGTCATAGAAGGTCA
B) AGGTCATAGAGGTCA
C)
AGGTCATAGATGACCT
D)
AGGTCAXTGACCT
E)
AGGTCATAGAAGTTC
2) Which of the following site on a
nuclear receptor is responsible for binding to NCOR?
A) AF1
B) AF2
C) AF3
D)
H3
E)
A/B
3) Which portion of a PPARg is likely to
be similar to that of PPARa?
A) The ligand binding domain
B) The AF2 domain
C) The DNA binding domain
D)
A and B
E)
B and C
4) What best describes the LXXLL domain?
A) It's the charged part of the AF2 domain
responsible for binding a co-activator
B) It's the hydrophobic part of a co-activator
responsible for binding to the AF2 domain.
C)
It's the hydrophobic part of a co-activator responsible for binding to DNA
D)
It's the hydrophilic part of a co-activator responsible for binding to the AF1
domain.
E)
It's the portion of the DNA binding domain of a nuclear receptor.
5) Deacetylated histones tend to
A)
Be associated with active transcription
B)
Be associated with less active transcription
C)
Be associated with less accessible DNA
D)
A and C
E)
B and C
6) Which of the following proteins binds
to the TATA box with high affinity (<10-7 M?
A)
TBP
B)
Polymerase III
C)
TFIID
D)
Polymerase II
E)
B and D
7) Which of the following factors
represses transcription but has no DNA binding site?
A)
HNF4
B)
LXR
C)
FXR
D)
SHP
E)
CAR
8) FOX and JUN bind to which DNA element?
A)
IR1
B)
DR7
C)
AP1
D)
IR
E)
all of the above
Topic 3 presented by Chris Glass Professor of Cell and Molecular Medicine, UCSD on TUESDAY, Nov 18 (LS101)
“PPARs and metabolic diseases”
Paper 1 Li PPARg and athero.pdf and paper 2 PPARd
and athero.pdf
Questions for discussion:
Discussion questions for Li,
et al., 2000 and Lee, et al., 2003
1. What are the major physiological roles of each of the PPARs?
(PPARa is highly
expressed in liver and heart and plays an important role in fatty acid
metabolism and energy homeostasis.
PPARa is the molecular target of the fibrate class of drugs that raise
HDL and lower VLDL in the circulation.
PPARg is most highly expressed in adipose tissue and is required for fat
cell development. PPARg also
regulated glucose and fatty acid metabolism and is the target of the TZD class
of drugs that improve insulin resistance in patients with type 2 diabetes mellitus. The physiologic roles of PPARd have not
as yet been clearly established, but recent studies suggest roles in energy homeostasis. Specific, high affinity ligands for
PPARd have recently been developed and should allow the pharmacology of PPARd
to be explored.)
2. What is the potential significance of PPARg expression in
macrophages with respect to treatment of diabetes and the development of
atherosclerosis?
(The discovery that PPARg
is expressed in macrophages raised the possibility that it might have
additional functions beyond regulation of adipogenesis and regulation of
glucose homeostasis. The goals of
treatment of type 2 diabetes are to lower glucose levels and symptoms of
hyperglycemia in the short term and inhibit the development of complication in
the long term. Diabetic patients are
at markedly increased risk of development of atherosclerosis and its clinical
complications. If the activation
of PPARg in macrophages by TZDs had an adverse effect on atherosclerosis, it
would not be a desirable drug for use in diabetics despite its ability to lower
circulating glucose levels.
Conversely, if activation of PPARg inhibited the development of
atherosclerosis, it would be predicted to decrease atherosclerotic
complications in diabetic patients.)
3. Why might activation of PPARg promote the development of
atherosclerosis?
(Some of the initial studies
of PPARg action in macrophages led to the discovery that it can induce the
expression of CD36, a macrophage scavenger receptor that can
mediate the uptake of atherogenic forms of LDL. These observations raised the question of whether PPARg might activate other atherogenic
genes.)
4. Why might activation of PPARg inhibit the development of
atherosclerosis?
(First, TZDs are insulin
sensitizers and improve glucose homeostasis. This in itself may be anti-atherogenic. Second, studies of PPARg action in the
macrophage indicate that it can inhibit inflammatory response genes, including
inducible nitric oxide synthase and matrix metalloproteinase 9 (MMP9). Atherosclerosis is a chronic inflammatory
disease, in which the expression of genes that promote influx and activation of
monocyte/macrophages and lymphocytes is thought to play a critical role in
disease progression. Inhibition of
the expression of these genes by PPARg agonists would be predicted to be
antiatherogenic.
Third, recent studies have suggested that PPARa and PPARg can induce the
expression of the liver X receptor alpha (LXRa). LXRa is a nuclear receptor that is activated by oxysterols
and acts in a feed-forward regulatory pathway to lower cellular cholesterol
levels. LXRa activates genes such
as the ABCA1 transporter that mediates transfer of cellular cholesterol to
extracellular acceptors.
Activation of LXRa expression by PPARa and PPARg agonists is not
consistently observed, however, and the importance of this pathway remains to
be established.)
5. How do you interpret the differential effects of PPARg
agonists on the development of
atherosclerosis in male and female mice?
(These observations
suggest that there is a sexual dimorphism that alters the balance of
atherogenic and antiatherogenic effects of PPARg agonists in male and female
animals. The basis for this effect has not been established. It does not appear to translate to
human subjects, as women are slightly more responsive to TZDs with respect to
effects on glucose levels than are men.
The findings in mice are of interest because they suggest that there are
regulatory mechanisms that can act to inhibit or counteract PPARg activity.)
6. To what extent do the findings in Li et al. support local as
opposed to metabolic roles of PPARg action in inhibiting the development of
atherosclerosis in male mice?
Mice fed the high
fat/high cholesterol diet became moderately obese and insulin resistant. Treatment with the PPARg agonists
improved insulin resistance and resulted in decreased levels of inflammatory
gene expression in the artery wall.
Thus, the observed results could be due to either local or metabolic
actions of PPARg agonists, or both.
The findings in Li et al were subsequently reproduced by three other
laboratories. In one study, a
hypercholesterolemic diet was used that did not cause obesity or insulin
resistance, suggesting antiatherogenic activities independent of glucose
lowering.
7. How could you directly test whether activation of PPARg expression in macrophages is essential for
antiatherogenic activities of TZDs?
The general approach to
this type of question is to lethally irradiate an atherosclerosis-prone strain
of mice (e.g., LDLR KO) and reconstitute with wild type or PPARg knockout bone
marrow progenitor cells. The
resulting mice can then be fed an atherosclerotic diet with or without drug and
the extent of atherosclerosis measured.
This type of experiment has been done and mice reconstituted with PPARg
KO bone marrow get more atherosclerosis than mice reconstituted with wild type
bone marrow. These studies
demonstrate that PPARg plays an antiatherogenic role in macrophages.
8. How might these findings be translated to humans?
9. The studies reported in Lee et al suggest that PPARd and
PPARg regulate different sets of target genes. The DNA binding domains of PPARd and PPARg are very
similar. How might different
target gene regulation be achieved?
One potential
explanation for this would be differential recruitment of coactivators and
corepressors. In the case of
MCP-1, PPARd appears to be able to selectively inhibit its expression due to its selective
ability to act as a sink for Bcl6.
The binding of agonists to PPARd are proposed to cause displacement of
Bcl6 and its redistribution to the MCP-1 promoter.
10. How might the redistribution model of
inhibition of MCP-1 be tested
further?
One approach would be to
use Bcl6-deficient cells.
PPARd agonists should have no effect on MCP-1 expression in these cells.
Another approach would be
to perform chromatin immunoprecipitation studies of the MCP-1 promoter. Bcl6 should be observed on the promoter
following addition of a PPARd agonist.
11. Atherosclerosis was more severe in LDLR
KO animals transplanted with wild type bone marrow progenitor cells that
PPARd-KO bone marrow progenitor cells.
Given the effects of PPARd agonists on MCP-1 expression, what do these
results imply about the availability of endogenous ligands for PPARd in
lesions?
These results suggest
that PPARd is unliganded in atherosclerotic lesions, otherwise Bcl6 would have
redistributed to the MCP-1 reporter gene.
This is somewhat surprising, because PPARd has been shown to be activated
in macrophages by the lipoprotein lipase-dependent hydrolysis of triglycerides
in VLDL. This bears looking into
further.
12. How would you test the hypothesis that
atherosclerosis is decreased in LDLR-KO mice due to decreased expression of
MCP-1?
Not an easy
experiment. It would require
developing double knockouts of
MCP-1 and PPARd and using bone marrow cells from these animals for
transplantation studies. Mice
transplanted with MCP-1 KO cells should already be relatively resistant to
atherosclerosis, but adding the PPARd KO should no longer result in a further
decrease.
13. Explain how the present studies
demonstrate the concept that the phenotype resulting from the lack of a nuclear
receptor can be quite different than the phenotype resulting from lack of a
ligand.
14. Do you think that activation of PPARd by a synthetic
ligand will inhibit the development of atherosclerosis?
Topic 4 presented by Mark Sussman, Ph.D. on November 24, 2003
1) Paper#1 (Cell, Vol.
114, 763–776, September 19, 2003, Adult Cardiac Stem Cells Are Multipotent and
Support Myocardial Regeneration-) and Paper
#2 (Nat Med. 2003 Sep;9(9):1195-201. Mesenchymal stem cells modified with Akt
prevent remodeling and restore performance of infarcted hearts. Mangi AA, Noiseux N, Kong D, He H,
Rezvani M, Ingwall JS, Dzau VJ.-What are stem cells? Are there differences
between a “cardiac stem cell” versus a “hematopoeitic” stem cell?
2) Where do stem cells live in the heart?
3) Is the heart a “terminally differentiated” organ? What
is the meaning of “self renewal”
4) What are the problems with stem cell transfer for
cardiac regeneration?
5) How would you select for and enrich the stem cell
population?
6) Can genetic manipulation be helpful for stem cell therapy?
7) How does Akt potentiate stem cell activity?
8) Are stem cells capable of regenerating all the tissues
necessary to rebuild the damaged heart?
9) Which is better: using donated stem cells or
recruiting endogenous ones for cardiac repair?
10) Why are the resident stem cells not capable of
repairing the heart after a heart attack?
11) Do stem cells have a finite lifespan? How many times
can they replicate?
12) Why do stem cells behave differently in vitro than in
vivo? How do these differences manifest themselves?
13) What are current limitations of stem cell treatment
that need to be overcome for this to be a clinically useful therapy for
treatment of heart disease?
14) Are you convinced that hearts can be repaired by stem
cell transfer?
Topic 5 presented by Linda K. Curtiss, Ph.D. on December 1, 2003
Bone marrow transplantation
There will be two papers discussed. Study questions will be available by the beginning of class. Paper 1 (JCI96.pdf) describes the transfer of apo E into mice using hematopoietic stem cells (HSC) and paper 2 (PON1 for 610.pdf) describes the delivery of a paraoxonase1 transgene into mice using HSCs.
Study questions
Topic 6 presented by Joseph Witztum, MD, Professor of
Medicine, UCSD, “Innate immunity and atherosclerosis”
Study questions for two papers (paper 1 immunization) and (paper 2, innate vs acquired immunity)
How does innate and acquired immunity differ?
What components mediate innate immunity?
What components mediate innate immunity?
Why should "immunity" be involved in a chronic disease
that essentially
represents the excess accumulation of cholesterol in the artery?
What components of immunity are found in the artery wall?
What is the evidence that manipulations of innate or acquired
immunity
affect atherogenesis?
Innate immunity is for the most part genetically determined, if
so, why
should this be invoved in atherogenesis, a disease that occurs
later in
life, eg beyound the reproductive period?
What are natural antibodies?
What are "pattern recognition receptors" (PRR) and what
are "pathogen
associated molecular patterns"(PAMP)?
What are some examples of PRRs and PAMPS?
Is it good or bad to have an immune response involved in
atherogenesis?
Could one develop a "vaccine" to prevent or delay the
development of atherosclerosis?
Topic 7 presented by Roger Davis, Ph.D., SREBPs, regulators
of energy metabolism Please read review
of SREBP-
1) Which of the following forms of SREBP is primarily
involved in regulating genes involved in fatty acid metabolism and also
contains an AF2 domain that has a lower affinity for co-activators?
A. SREBP1a
B. SREBP1b
C. SREBP1c
D. SREBP2a
E. SREBP2b
2) Which of the following is a trans-membrane protein
in the endoplasmic reticulum?
A) INSIG-1
B) SREBP-1c
C) SCAP
D) SREBP2
E) all of the above
3) A cell displaying cholesterol auxotrophy is most
likely to have mutations that block the function of which gene(s)?
A. S2P
B. SCAP
C. INSIG-1
D. All of the above
E. A and B
4) Mature (nuclear) SREBP1a is rapidly degraded in
cells by
A. Lysosomes
B. proteasomes
C. Trypsin
D. The mitochondria
E. The peroxisomes
5) Which
of the following genes display a transcriptional activation by SREBP?
A) glucokinase
B) Fatty acid synthase
C) CYP7A1
D) A and B
E) Band C
6) Which end of SREBP binds to the which end of SCAP?
A) N to N
B) N to C
C) C to C
D) None of the above