Major Histocompatibility Complex (MHC) Class II molecules bind peptides derived from endocytosed protein and the MHC/peptide complexes are expressed at the surface of immune cells. When the endocytosed protein is foreign, such as a bacterial toxin, the MHC/peptide complex may be recognized by a T lymphocyte, intiating a cascade of events that constitute an immune response. HLA-DM is an intracellular MHC II-like protein that regulates the loading of peptides onto MHC Class II molecules. We are using mouse/human hybrid DM molecules and mutant DM molecules to map the regions of the the DM molecule involved in its function. The student will assist in generating and studying the effects of the hybrid proteins on peptide loading in an in vitro system. Numerous molecular biology techniques will be employed including PCR, sequencing, subcloning, as well as biochemical analysis of the hybrid molecules by immunoprecipitation and western blotting.
Lucy Rasmussen, Ph. D.
A Research Assistant is needed to participate in several ongoing projects aimed at understanding the human immune response to the viral glycoproteins of human cytomegalovirus (CMV). Duties to include: Participation in the design and coordination of the research protocols. Evaluation of scientific literature pertinent to project. Specific specialized techniques include generation and characterization of monoclonal antibodies to CMV envelope glycoproteins, immunization of small animals with purified glycoproteins; radiolabeling CMV infected cells; analysis of antibodies by immunoprecipitation using SDS-PAGE and Western blots; cloning and expression of viral glycoprotein genes in mammalian systems; performing both polymerase chain reaction (PCR) assays and DNA gels for analysis of plasmids; ELISA assays for determination of human antibody reactivity to viral glycoproteins; trouble shoot and solve technical problems associated with development of specialized techniques. Background will include experience in standard molecular biological techniques such as cloning as well experience with bacterial and cell culture methods. Must have initiative, be able to work independently with general direction, establish priorities, be neat and organized with excellent laboratory skills, and possess the degree of flexibility that is necessary for developmental research work. MacIntosh skills necessary and familiarity with scientific graphing, spreadsheet, and word processing programs.
Peter Jackson, Ph. D.
Assistant Professor of Pathology and Microbiology &
I study how cyclin-dependent kinases and other biochemical factors control the initiation of DNA replication. Much of the work uses extracts from Xenopus eggs. These extracts recapitulate major events in the cell cycle including the controls that program the initiation of DNA replication, that ensure its fidelity, and that limit replication to once per cell cycle. Using biochemical tests and novel microscopy-based assays for subreactions of DNA replication, my group dissects the biochemical connections between the cyclin-dependent kinases, other regulators, and the replication machinery itself. Our goals include: (1) identifying the components of a 450 kD complex containing the cyclin E-dependent protein kinase; (2) establishing which components of the initiation complex are regulated by the cyclin E/Cdk2 kinase; and (3) understanding how nuclear structure and the nuclear membrane contribute to establishing a replication-competent nucleus.
We are also interested in how ubiquitin-dependent proteolysis
gates specific steps in DNA replication and the role of small
molecules in controlling the cell cycle.
1. Jackson, P. K., S. Chevalier, M. Phillipe, and M. W. Kirschner (1995). Early events in DNA replication require cyclin E and are blocked by p21CIP1. J. Cell Biol. 130, 755-769.
2. J. Chen, P. K. Jackson, M. W. Kirschner, and A. Dutta (1995). Inhibition of cdk2 kinase, but not PCNA, is essential for the growth suppression activity of p21. Nature 374, 386-388.
3. R. W. King, Jackson, P. K., and M. W. Kirschner (1994). Mitosis in Transition.
Cell, 79, 563-571.
4. Jackson, P. K., D. Baltimore, and D. Picard (1993). Hormone-conditional transformation by fusion proteins of c-Abl and its transforming variants. EMBO J., 12, 2809-2819.
5. S. Wen, P. Jackson, and R. A. Van Etten (1996). EMBO J. 15, 1583-1595.
6. P. Jackson. Cull and Destroy. Current Biology 6, 1209-1212, 1996.
__________________________________ Christopher H. Contag firstname.lastname@example.org Director Ph. 415-723-0707 or 725-8781 Molecular Biophotonics Laboratory Lab 415-498-7246 Neonatal and Developmental Medicine Fax 415-725-7724 Department of Pediatrics, S257 Stanford University School of Medicine Stanford, CA 94305-5119
Rationale: There has been tremendous progress in HIV drug therapy within the past year. However, the extraordinary clinical benefits reported have occurred nearly exclusively in previously untreated patients who start one of the rationally-desgned triple drug combinations. Most of the patients we know from our clinical trials have been on antiretrovirals for many years and many appear to have HIV strains that are resistant to all of the available nucleoside analog reverse transcriptase (RT) and protease inhibitors.
This is not surprising since even though there are 5 available nucleoside analog RT inhibitors and 3 available protease inhibitors, there is a lot of cross-resistance between these drugs. For example, certain combinations of 3-4 reverse transcriptase mutations may occur in HIV strains from patients receiving either AZT+ddI or AZT+3TC causing these strains to become resistant to all 5 nucleosides. In addition, HIV strains from patients who have received both saquinavir and indinavir (or ritonavir) appear to develop multidrug resistance within months.
Because of their previous "exposure" to several different antiretroviral drugs, our patients with multidrug-resistant HIV strains are generally not eligible for most clinical trials. Yet studying their HIV strains could provide extremely useful information. Specifically, these multidrug resistant strains need to be tested for susceptibility against some of the many experimental antiretrovirals. The results of such studies could help prioritize which of the experimental antiretrovirals should undergo rapid clinical development. Experimental antiretrovirals with activity against current multidrug-resistant strains could be developed to be used for (1) treatment of patients with multidrug-resistant virus, and for (2) inclusion in combination-therapy regimens in order to decrease the likelihood of the development of multidrug resistance.
Description of work: The project would be broken into two separate parts - each of which would be expected to lead to at least one publication. The first part would involve testing HIV strains resistant to multiple RT inhibitors: 2-3 strains would be tested against 3-5 drugs. The strains would include (1) a multinucleoside-strain with Q151M and its associated mutations (the mutations identified in our laboratory) and (2) 1-2 multinucleoside-resistant strains with M41L + M184V + T215Y - this is a very common pattern we have observed in persons who have done poorly on AZT+3TC. The experimental RT inhibitors would include PMEA, 1592U89 (a potent guanosine analog being developed by Glaxo-Wellcome), and foscarnet (since it is a prototypic pyrophosphate analog), and possibly others.
The second part would involve testing 2-3 strains resistant to each of the protease inhibitors. However, we have not yet chosen the genotypes to be tested because we are just beginning to see this form of drug resistance. The experimental drugs would include the protease inhibitors nelfinavir, the Vertex compound, and several others.
Methods: (1) Cell culture - peripheral blood mononuclear cells, CD4+ lymphocyte cell lines, HeLa CD4 cells (2) HIV culture (3) Drug susceptibility testing (4) RT and protease gene sequencing - automated dideoxyterminator sequencing
[Note: If one or both of the projects (RT and protease) are completed - logical follow-up work would involve passage experiments and site-directed mutagenesis experiments]
Advantages: If the work is completed, the results will definitely be useful and publishable. This would apply even if only some of the above strains and drugs are tested. The student would not be expected to have previous laboratory experience and would be taught the above methods over a period of 2 months. Most likely the student would be 2nd author on publications; a student who learned the methods rapidly without much assistance and who also prepared the manuscript without much assistance could conceivably be a first author. The student will develop a thorough knowledge and understanding of HIV therapy which may prove useful as soon as clinical rotations begin.
Disadvantages: The project does not address any fundamental biological questions (it is very much applied research) and it doesn't involve the use of any new forms of technology (the methods are very standard in clinical HIV virology). In addition, the work is somewhat tedious - For example, all the susceptibilities have to be done in duplicate and ideally, two different methods for susceptibility testing should be used.
Last modified: January 18, 1998