Retreat 2006 M.D./Ph.D. Student Abstracts
Acculturation and Health Risks in Mexican-American Adolescents
PSTP Student: Teresa Barcellos
Mentor: Dr. Marc B. Schenker
Every year, California agriculture employs more than 1.1 million farm workers, most of whom are Mexican-origin or Mexican American. Moreover, Mexican-Americans are the fastest growing ethnic group in the United States and make up more than 25% of new immigrants. Many immigrate as families, and children, whether born in the US or Mexico, face many of the same increased health risks as their parents, often arising from poor access to care and lack of primary care and preventative health services. Nevertheless, many indicators of adverse health effects, such as low birth weights, smoking habits, and sexually transmitted infection rates are better among Mexican- and Latin American-origin individuals than their socioeconomically-similar counterparts, a phenomenon known as the Latino paradox. Acculturation is believed to play a significant role in this trend and changes in acculturation have been linked to changes in health status in previous studies. However, little work has been done on the effects of acculturation in rural immigrants or their children. It is likely that at least part of the effects of acculturation occur through the probability of engaging in high-risk behaviors, such as smoking, alcohol consumption, and early initiation of sexual activity. Therefore, we propose a cohort study of adolescent children of Mexican-origin farm workers that will examine the association between acculturation and participation in health-risk behaviors. In general, we expect more highly acculturated adolescents to participate more frequently than their less-acculturated peers.
A Mechanism For Agrin in Synaptic Development in the Superior Cervical Ganglion: A Preliminary Study
PSTP Student: Jolene M. Chang
Mentor: Dr. Michael Ferns
Synaptogenesis is important for formation, regeneration, and plasticity of the nervous system, and is regulated by bidirectional signalling between pre and postsynaptic neurons. Synaptogenesis has been studied extensively in the neuromuscular junction, where an extracellular matrix protein called agrin is deposited by the presynaptic motoneuron into the basal lamina of the muscle cell, where it signals through MuSK receptor tyrosine kinase to cluster acetylcholine receptors at the postsynaptic site. Outside the neuromuscular junction, agrin has also been studied in the superior cervical ganglion (SCG). Interneuronal synapses in the SCG system shares some similarities to the neuromuscular junction – it is also a fast cholinergic synapse. Agrin knockout mice have fewer synapses in SCG and impaired synaptic transmission. Thus, agrin regulates synaptic differentiation in the peripheral nervous system as well as at the neuromuscular junction. The mechanism through which agrin acts to stabilize and maintain synapses in the PNS is unknown. Most importantly, the receptor for agrin in the SCG has not yet been identified. My project is to characterize and identify the agrin receptor in neurons. One approach is to use recombinant agrin fragments to to map agrin’s interaction domain with the receptor, in order to find an existing or novel receptor using biochemistry and/or expression cloning. Another approach is to take a candidate receptor, MuSK, and examine for similar synaptic defects to agrin knockouts. Preliminary results show that there is a 40% decrease in PDZ staining on SCG neurons, which is similar to agrin knockout cultures, which suggests that MuSK is possibly the receptor for agrin in the SCG. Future studies will have to localize MuSK to see if it is synaptically localized, further characterize the MuSK knockouts using different synaptic markers and to examine synaptic defects in vivo, and to continue to map agrin’s receptor interaction domain.
Survey of Src Activity and Src-related Growth and Migration in Prostate Cancer Lines
PSTP Student: Yu-Ming Chang
Mentors: Dr. Hsing-Jien Kung, Dr. Christopher P. Evans
Prostate cancer (CaP) often develops androgen independence (AI) and proliferates uninhibited despite anti-androgen therapy. Src kinase is a non-receptor tyrosine kinase that mediates a variety of cellular functions. Previous studies have demonstrated that the neuropeptide bombesin and IL-8 can activate src kinase and stimulate AI growth in CaP cells. However, a role for src in prostate cancer is not yet defined. We evaluated src activation and expression levels in the most commonly used CaP cell lines. We also examined the effect of src kinase inhibition on the proliferation and migration in CaP cell lines. Lastly, we identified src downstream factors through its inhibition. Src activation and basal src expression levels were profiled in CaP cell lines CWR22Rv1, LNCaP, PC3, DU145, and LAPC-4 in two-day charcoal-stripped serum with minimal androgen and small molecules (CS) and regular serum (RS) conditions. CaP cell lines and immortalized prostate epithelial cells RWPE-1 and PZ-HPV-7 were treated with 62.5nM to 16µM of AZD0530, a novel Src kinase inhibitor in RS and its effects on cell growth were examined. PC3 and DU145 cell migration were assayed in Boyden Chamber in the presence of 62.5nM to 4µM AZD0530 with fibronectin (FN) as chemoattractant. In the scratch migration assay, cells were seeded, a cell-free zone was made by “scratching” cells in a linear fashion with a pipette tip followed by washes, and cells were allowed to migrate for two to four days. Src expression level was found to vary in the CaP cell lines examined with androgen-responsive cell lines LNCaP, LAPC-4, and CWR22Rv1 expressing the higher level of src than androgen-independent lines PC3 and DU145. Src activation does not vary between CS and RS conditions in all cell lines. LNCaP has the highest level of src activation, followed by DU145 and CWR22Rv1 while LAPC-4 and PC3 have the lowest level of src activation. All CaP cells treated with AZD0530 demonstrated dose-dependent inhibition of cell growth in MTT proliferation and migration in Boyden chamber and scrach migration assays. IC50s for growth in LNCaP, LAPC-4, CWR22Rv1, DU145, PC3, RWPE-1, PZ-HPV-7 were 3 to 6µM, greater than 16µM, 6 to 9µM, 3 to 6µM, 0.5 to 3µM, 8 to 16µM, and 8 to 16µM respectively. IC50 for PC3 and DU145 in Boyden chamber migration were 62.5nM and 500nM respectively. Scratch migration results were in accordance with the Boyden chamber migration results. Src inhibition in LNCaP resulted in inactivation of ERK1/2, STAT3, and p38 while src inhibition in DU145 resulted in inactivation of STAT3 and p38 but not ERK1/2.
We therefore conclude that src may be important mediator of cell growth, migration, and antiapoptosis in CaP. Rationale exists for the inhibition of Src in the treatment of CaP.
Monoenergetic X-ray Cancer Phototherapy and the Compton Light Source
PSTP Student: Bill Frederick
Mentor: Dr. Jonathan P. Heritage
X-ray phototherapy is the delivery of monochromatic x-ray radiation at and above the K-edge of various heavy metals bonded to substrates within neoplastic cells. Such a dose enhancing treatment should be more effective that conventional radiation therapy for killing neoplastic cells. In vitro Experiments to verify this conjecture are underway at several synchrotron facilities including the Stanford Synchrotron Radiation Laboratory (SSRL), the Advanced Light Source (ALS), and the Advanced Photon Source (APS). While radiosensitization may be demonstrated using synchrotron light, a more compact source is need to allow such treatment in a hospital setting. This program is part of the Compton Light Source (CLS) project at the Stanford Linear Accelerator Center (SLAC) Klystron Department to develop a tunable, compact source of quasi-monoenergetic x-rays for use in cancer detection and treatment. The proposed work will use synchrotron beamlines to irradiate established cell lines labeled with high-Z atoms. Dose enhancement will be assessed through dose-response curves generated both by traditional colony counting and by flow cytometry. Candidates for radiosensitizing agents include Iodine, Gadolinium, Indium, Platinum, and Gold. Samples will be irradiated at energies 0-30 keV above the K-edges of these elements. Although synchrotron beamlines produce relatively low fluxes at these energies, cell culture geometry can be adjusted to the small beam size, and samples may be irradiated for long times (minutes) to reach higher integrated photon intensities. Broader bandwidths (up to 1 keV) are permitted in these experiments as a trade-off for reaching higher fluxes. Initial experiments have shown dose enhancement using iodine in prostate cancer cells (PC3 line) comparable to predictions by Monte Carlo simulation.
TWO IN VIVO SURGICAL APPROACHES FOR LUMBAR CORPECTOMY USING ALLOGRAFT AND A METALLIC IMPLANT: A CONTROLLED CLINICAL AND BIOMECHANICAL STUDY
PSTP Student: Phil Huang
Mentor: Dr. Sarigul-Klijn
Collaborators: BS Gupta, S Hazelwood
Both bone graft and metallic implants have been used in combination with the necessary anterior rod or plate instrumentation to fill the voids left by vertebral body removal, with the ultimate goal of restoring stability. One type of device that has recently been introduced is an expandable titanium telescoping cage that is designed to be used as a strut implant to fill corpectomy defects. The use of these devices has met varying success. Acceptance by surgeons and spine biomechanicians has been limited by clinical failure with subsequent loss of reduction and increase in kyphosis. In order to further improve patient care, it is critical to evaluate the use of these implants through biomechanical as well as other modes of testing. The purpose of this study is to compare and contrast the spinal fusion outcome of using allograft bone versus the expandable vertebral body replacement titanium implant in a lumbar corpectomy procedure. A controlled biomechanical study of lumbar spine fusion was performed using bone graft and the expandable cage in an in vivo bovine model following a 4-month postoperative healing period. Calves aged 4-6 months with L3 removed were used to create a simulated lumbar corpectomy defect. The calves were randomly allocated to groups for corpectomy defect repair with 1) allograft metatarsal bone and Thoracolumbar Spine Locking Plate, n=6, or 2) Expandable vertebral body replacement device and Thoracolumbar Spine Locking Plate, n=6. Following a 4-month postoperative period, the animals were sacrificed and lumbar spines harvested for biomechanical testing. Strength of fusion was assessed by stiffness of ex vivo spine specimens in flexion-extension, lateral bending, and torsion obtained from biomechanical testing. Uniaxial strain at various positions on the surface of the anterior plate was measured during loading as an additional stability parameter. Loading tests were repeated following removal of the anterior instrumentation (plate and screws).At 4 months postoperative, the stiffness of the calf spines repaired by the bone graft and locking plate was significantly greater than that of the spines repaired by the expandable cage and locking plate. This finding was true for all three directions of loading. Greater strain values were observed from the gauges on the locking plate of the spines utilizing the expandable cage than the spines utilizing allograft bone. This finding held for all gauge positions (anterior edge, anterior face, posterior edge, and posterior face of the plate). Following locking plate removal and a repeat of the loading tests, a decrease in stiffness of the construct was observed for both the allograft and cage groups. These data may indicate that the use of allograft bone for corpectomy defect repair in the lumbar spine contributes to a stiffer and perhaps more stable spine segment compared to using the expandable cage device for such a repair following a 4-month healing period in this in vivo calf model.
SMRT AND N-COR COREPRESSORS ARE REGULATED BY DISTINCT KINASE SIGNALING PATHWAYS AND BY ALTERNATIVE SPLICING
PSTP Student: Brian A. Jonas
Mentor: Dr. Martin L. Privalsky
N-CoR and SMRT are corepressor paralogs that partner with and mediate transcriptional repression by a wide variety of metazoan transcription factors, including nuclear hormone receptors. Repression plays a particularly critical role in the functions of these nuclear receptors, including development, differentiation, reproduction, and homeostasis. Although encoded by distinct genetic loci, N-CoR and SMRT share substantial sequence interrelatedness and form analogous assemblies with histone deacetylases and auxiliary factors. In addition, N-CoR and SMRT can interact with overlapping sets of transcription factor partners and exert overlapping functions in cells. Our lab previously reported that SMRT is subject to negative regulation by MAPK signaling pathways operating downstream of growth factor and stress signaling pathways. As a result of our studies, we report that whereas activation of MEKK1 leads to phosphorylation of SMRT, its dissociation from its transcription factor partners in vivo and in vitro, and its redistribution from the cell nucleus to a cytoplasmic compartment, N-CoR is refractory to all these forms of regulation. In contrast to this MAPK cascade, other signal transduction pathways operating downstream of growth factor/cytokine receptors appear able to affect both corepressor paralogs. We also report that SMRT is expressed in cells by alternative mRNA splicing to yield two distinct variants or isoforms. We designate these isoforms SMRTalpha and SMRTtau and demonstrate that these isoforms have significantly different affinities for different nuclear receptors. These isoforms of SMRT are evolutionarily conserved and are expressed in a tissue-specific manner. N-CoR, on the other hand, appears to be less extensively regulated by alternative splicing. These results suggest that differential mRNA splicing serves to customize corepressor function in different cells, allowing the transcriptional properties of nuclear receptors to be adapted to different contexts. Taken together, our results indicate that SMRT and N-CoR are embedded in distinct regulatory networks and that the two corepressors interpret growth factor, cytokine, differentiation, and prosurvival signals differently.
Arginine Deprivation: A Novel Therapy for Prostate Cancer
PSTP Student: Randie Kim
Mentor: Dr. Hsing-Jien Kung
Amino acid deprivation is a treatment concept first described for leukemias and lymphomas almost 50 years ago. Altered metabolic pathways in cancer cells can create different nutritional requirements for tumors than for normal cells. The enzyme argininosuccinate synthetase (ASS) is involved in the two-step conversion of citrulline into arginine. Deficiencies in ASS in hepatocellular carcinomas and melanomas cause these cancer cells to be dependent on exogenous sources of arginine. The use of arginine deiminase (ADI) to degrade arginine into citrulline has shown to be effective in inhibiting cancer growth. It has been observed that some prostate cancer cell lines are also sensitive to arginine depletion, indicating that prostate cancer growth can be inhibited with ADI treatment. In this study, correlating ASS mRNA and protein expression levels in prostate cancer lines with survival curves after ADI treatment would reinforce the validity of amino acid deprivation as a novel and effective chemotherapy.
To determine the levels of ASS in prostate cancer and normal prostate lines, expression of ASS was examined on the transcript and protein level. Using RT-PCR, ASS mRNA levels were determined to be present in the three prostate cancer cell lines LNCaP, PC3, and DU145 as well as in normal prostate PZ-HPV-7. Although the expression of ASS mRNA has been confirmed in these cell lines, protein expression data still needs to be determined. The effect of PEG-ADI on prostate cancer cell lines were examined in a dose-dependent manner. After six days of PEG-ADI treatment, cells were assayed for cytotoxicity with methylthiotetrazole. Preliminary results suggest that PEG-ADI has an effect on the inhibition of growth of the prostate cancer lines. However, no conclusions about the relationship between ASS expression and ADI response in prostate cancer lines can be made at this time.
RGS EXPRESSION SETS THE DURATION OF SIGNALING IN ROD PHOTORECEPTORS
PSTP Student: Claudia M. Krispel
Mentor: Dr. Marie E. Burns
Signaling through heterotrimeric G proteins is a ubiquitous mechanism in cell biology. Despite the intense study of processes that regulate G protein pathways (e.g. G protein receptor kinases, arrestins, RGSs), it is unknown which regulatory steps determine the overall duration of the resulting intracellular signals. Even in the well-studied G protein cascade of vertebrate rod photoreceptors, the identity of the biochemical step that dictates the time course of the response under normal conditions has been unclear. In rods, timely recovery of the photoresponse requires efficient deactivation of both the G protein coupled receptor, rhodopsin, and the G protein/effector complex. The slowest of these two steps determines the overall rate of response recovery (Nikonov et al., 1998). While some experiments have suggested that rhodopsin lifetime is the slowest, or rate-limiting, step in recovery, other studies have argued that the rate-limiting step is deactivation of the G-protein, transducin.
A principal difficulty in settling this controversy has been that most experimental approaches that have investigated these mechanisms have abnormally prolonged deactivation, either by diluting endogenous regulators in biochemical experiments or by slowing the time course of physiological responses through pharmacological or genetic loss-of-function manipulations. Because lengthening the lifetime of rhodopsin or transducin/PDE does not address which of these two species normally rate-limit recovery, settlement of this controversy demands a manipulation to directly speed the rate-limiting step in intact cells.
We have addressed this question by utilizing mice which over-express (and thus have increased total activity of) rhodopsin kinase and RGS9-1, the regulators that control rhodopsin and transducin/PDE deactivation, respectively. Using suction electrodes, we recorded flash responses from rods of RGS9-over expressing mice and rods of RK-over expressing mice, and compared the time courses of their flash responses. We report that increasing the expression of RGS9-1, accelerates recovery of the light response in mammalian rods, while over expression of RK has no effect on the time course of flash responses. This resolves a long-standing controversy by demonstrating that deactivation of many G protein molecules, rather than the shutoff of long-lived photoexcited rhodopsin, determines the duration of the rod's response to light. Furthermore, these results indicate that RGSs are not merely negative regulators of G protein activity, but can precisely tune the duration of cellular responses.
Nikonov S, Engheta N, Pugh EN Jr. (1998). Kinetics of recovery of the dark-adapted salamander rod response. J Gen Physiol 111(1):7-37.
Stereotyped Axon Pruning via Plexin Signaling is Associated with Synaptic Complex Elimination in the Hippocampus
PSTP Student: Lawrence Low
Mentor: Dr. Hwai-Jong Cheng
The nervous systems of vertebrates are characterized by a complex array of intricate connections. However, early in development these connections are in excess and are less precise. Proper nervous system development requires the removal of inappropriate connections while functionally relevant ones are retained. One mechanism for removing excess connections is through the process of stereotyped axon pruning. In stereotyped axon pruning, transient long axon collaterals are predictably pruned away from their targets. We have previously addressed the importance of plexin signaling on the stereotyped pruning of long axon collaterals in the vertebrate CNS; however, a cellular basis for plexins on stereotyped pruning has not been determined. Using quantitative electron microscopy and immunocytochemistry, we found that infrapyramidal mossy fiber axon collaterals form transient synaptic complexes with basal dendrites of CA3 pyramidal cells in the early postnatal mouse hippocampus. At later postnatal ages, these synaptic complexes stop maturing and are removed before stereotyped pruning by a mechanism that does not involve axon degeneration and glial cell engulfment. In knock-out mice that lack plexin-A3 signaling, the synaptic complexes continue to mature, and, as a result, the collaterals are not pruned. Thus, our results suggest that intact plexin-A3 signaling contributes to synaptic complex elimination, which is associated with stereotyped axon pruning.
Investigating the Cancer Stem Cell Hypothesis in Human Glioblastomas: Co-localization of ATF5 and CD133 Identifies a Subpopulation of Progenitor-like Cells
PSTP Student: Joyce H. Ma
Mentor: Dr. David Pleasure
The cancer stem cell hypothesis suggests that cells with progenitor qualities may play a role in the origin and maintenance of neoplastic clones. The ependymal cells lining the ventricles, as well as the subependymal area have been identified as a source of neurocortical progenitor cells which supply the normal brain with additional differentiated cells. The cancer stem cell hypothesis explores the possibility that mutations in progenitor cells may possibly be the origin of tumorigenesis. ATF5 is a transcription factor that plays an important role in suppressing differentiation while stimulating proliferation of neuroprogenitor cells into three main possible terminal cell fates, and thus is a general mediator of progenitor cell cycle control. Downregulation of ATF5 is required for differentiation of progenitor cells into neurons, astrocytes, and oligodendrocytes. Our studies focus on the identification of a subpopulation of progenitor cells in human glioblastoma. To study the cancer stem cell hypothesis in human brain glioblastomas, immunohistochemical staining of tumor sections were performed to determine the presence and distribution of adult stem cell markers CD133, GFAP, and their colocalization with ATF5. CD133+ cells were localized to the ventricular zone in patient tumor tissues and in non-tumor contralateral tissues at coronal tumor section. Absence of CD133+ cells were noted in sections more rostral to the tumor section, while robust staining of CD133+ cells were identified in ipsilateral tumor sections. In comparison, non-tumor contralateral tissue at the same coronal section demonstrated minimal staining for CD133. At sections more rostral to the tumor, ATF5 staining is minimal compared to tumor ventricular zone. Our preliminary results in human tissue support previous findings demonstrating the presence of a progenitor cell population in the ventricular area in murine models, and may be the first to demonstrate the co-existence of a putative stem cell population in human glioblastoma pathogenesis. Our study is also the first to confirm the specificity of CD133+ as a useful marker in identifying cancer stem cells in human glioblastoma. Further studies proving or disproving whether gliomas originate from subjacent stems cells remain to be investigated.
Intravenous, intraperitoneal, and subcutaneous routes of administering 64Cu-DOTA-HB22.7 display equivalent tumor targeting ability
PSTP Student: Shiloh Martin
Mentor: Dr. Joseph Tuscano
B lymphocyte signaling via surface molecule CD22 has been shown to modulate B cell receptor (BCR)-mediated signals. HB22.7 is a mouse monoclonal antibody that binds to the ligand binding domain of CD22 and blocks CD22 ligand binding. Previous studies demonstrated that HB22.7 has pro-apoptotic effects on B cell lymphoma cell lines and lymphomacidil properties in vivo. We have developed a 64Cu-DOTA-HB22.7 antibody, for in vivo imaging of lymphoma xenografts. 64Cu with its multiple emissions has applications both in imaging and in therapy. DOTA was conjugated to HB22.7 in varying ratios and all conjugated antibodies were determined to retain CD22 targeting via flow cytometry. The 64Cu-DOTA-HB22.7 antibody was injected into lymphoma xenografted mice either intravenously, intraperitoneally, or subcutaneously and biodistribution assessed using microPET. Imaging was performed at 3, 24, and 48 hours post-injection. 64Cu-DOTA-HB22.7 demonstrated highly specific tumor targeting by 48 hours. Tumor targeting was equivalent regardless of route of administration. Additionally, 64Cu-DOTA-HB22.7 was easily cleared from the circulation over time after injection. These findings establish 64Cu-DOTA-HB22.7 as a potential radioimmunotherapeutic or lymphoma-specific imaging agent. Furthermore, these findings provide evidence that more accessible routes of administration can achieve equivalent results in terms of biodistribution, and may lead to more efficient and reproducible administration of antibody-based therapeutics in mice.
Gaze Patterns Distinguish Infants At-Risk for Autism from Control Infants at 6 months
PSTP Student: Noah Merin
Mentor: Dr. Sally Rogers
Autism is a neurodevelopmental disorder that profoundly affects social development and language. Genetic factors are thought to play an important role in the risk of developing the disorder; twin-concordance studies have estimated heritability at 60-90%, one of the highest for any psychiatric disorder. It is presently diagnosed using a structured clinical observation of young toddlers who show signs of abnormal development. This clinical observation assesses behaviors that do not emerge until the second year of life (such as language, peer interaction, pointing, etc.). Therefore, autism cannot currently be diagnosed until after these behaviors typically appear. If we could identify the presence of behavioral markers of the disorder at an earlier age, there would be more time to intervene before the onset of symptoms. Knowing the age of onset also has theoretical importance—it would help in the search for environmental triggers and focus attention on specific neurodevelopmental processes. Infant gaze behavior, particularly during reciprocal social interaction, may be a useful behavioral marker for autism in young infants. In the present study, we tested 26 six-month-old infants at elevated risk for developing autism, and 24 typically-developing controls. We recorded eye tracking data during a live reciprocal interaction between the infant and the mother. In the at-risk group, we found seven infants who made poor eye contact, and focused visual attention predominantly on the mother’s mouth. Only one infant in the control group showed this pattern of gaze. Ongoing longitudinal work will determine if the subgroup of at-risk infants who do not look at their mother’s eyes later develop autism or autism-related disorders. Of the seven infants in this category, so far only one has returned to the lab for evaluation at 24 months of age—this infant has received a diagnosis of autism.
The Role of Chloride Channels in Inner Ear Homeostasis
PSTP Student: Karen J. Mu
Mentor: Dr. Ebenezer N. Yamoah
The endocochlear potential (EP) (> 80 mV), which is required for normal sound transduction is generated in part by the high K+ throughput across the apical membrane of basal/intermediate cells (BC/ICs) of the stria vascularis (StV). This is conferred by the K+ transporter, NKCC1, K+ pumps, Na+/K+ATPase, and K+ and Cl- channels. Although the sources for EP generation are known, the detailed mechanisms remain uncertain. To ensure electroneutrality, the flux of K+ is accompanied by Cl-, handled mainly by NKCC1 and Cl- channels. The importance of Cl- channels in the inner ear is demonstrated by the fact that mutations in barttin, an auxillary subunit of Cl- channels in the inner ear, results in Bartter’s Syndrome Type IV of which deafness is a component. To understand underlying cellular mechanism of Cl- channels in the inner ear, we cloned ClC-K1 and barttin from the lateral wall of the inner ear. We expressed the channels in the pNLR expression vector containing the 5'- and 3'-untranslated regions of the Xenopus ß-globin gene. Co-injection of RNAs of ClC-K1 and barttin yielded robust Cl- current. Measurements were taken in solutions with differing CaCl2 concentrations, pH, and anion types. The currents were elicited at voltages more positive than +40 mV and negative to -100 mV. Traces were recorded by applying step potentials from +60 mV to -120 mV and then to +60 mV from a holding potential of -30 mV. The current amplitude increased upon extracellular alkalinization to pH 8.0, was decreased by two-fold at pH 7.5 and was very small at more acidic pH. Replacement of 100 mM extracellular chloride with other anions indicated a permeability sequence of Br- > Cl- > NO3- > I- > Glutamate. Of particular interest, we observed very minimal inward current with glutamate substitution. While we expected the outward current to decrease as extracellular chloride was removed, the small inward current implies that there may be an extracellular chloride-dependent gating mechanism. Wild type ClC-K1 current amplitude increased by nearly four-fold upon elevation of extracellular calcium levels from 2 mM CaCl2 solution to 10 mM CaCl2. We are examining the effect of Ca2+ on gating in the ClC-K1 channel with the use of site-directed mutagenesis techniques. We have identified three areas along the ion permeation pathway that are important for pore function: K149, K165, and V166. To this end, we have constructed the following ClC-K1 mutants: K149R, K149A, K149E, K165C, V166E, and V166C. Upon initial examination, the ClC-K1 V166E mutant tail currents display dose-dependent, Ca2+ sensitivity not exhibited in the wild type channel. We are performing detailed characterization of these ClC-K1 mutants and their modulation by Ca2+. This should shed more light on the contribution of ClC-K1 toward the generation of endocochlear potential.
Synthesis of a Modified Geminal Disulfone and Biological Evaluation of its Mode of Anti- HIV-1 Activity
PSTP Student: Ekama Onofiok
Mentor: Dr. Jacquelyn Gervay-Hague
HIV integrase (IN), a DNA nucleotidyl-transferase encoded by the HIV genome, is responsible for insertion of pro-viral DNA into the host genome in HIV-infected cells; thus critical for infectivity and replication of the virus. Previous investigative studies of a series of geminal disulfones (analogues of chicoric acids which are known to be potent antiviral IN inhibitors), revealed potency of a vinyl-trihydroxy-phenyl-substituted disulfone as both an inhibitor of HIV- IN in purified enzyme assays, and as an antiviral agent in cell-based assays. However, unlike other geminal disulfones in the study, purified enzyme inhibition by this compound had poor correlation with antiviral assays as evidenced by time-of-addition studies which indicated a cellular target early in the viral replication event, such as entry into the host cell. In an effort to evaluate its modes of activity as an HIV-IN and possibleentry inhibitor, functional-group modifications of the vinyl trihydroxy-phenyl disulfone compound, and subsequent comparisons of antiviral activities of the newly synthesized products were performed. The vinyl groups, hypothesized to confer some functionality to the compound were reduced, and thus, herein is reported the synthesis and biological evaluation of a hydrogenated trihydroxy-phenyl disulfone.
Microvascular changes during endotoxemia and treatment with polyethylene glycol-albumin in a rabbit endotoxemia model
PSTP Student: Patricia L. To
Mentor: Dr. Anthony Cheung
Sepsis, the systemic inflammatory response to infection, often progresses to vasodilatory shock and organ dysfunction. Increasing recognition of the role of microcirculatory dysfunction in sepsis and the development of multiple organ failure has led to microvascular resuscitation as a therapeutic goal in sepsis. Polyethylene glycol (PEG)-modified plasma expanders, in which PEG is covalently bound to proteins such as albumin to produce a high colloid osmotic pressure, may address the problem of resuscitation with the increased capillary leak that occurs during sepsis. Our laboratory is developing a rabbit endotoxemia model to study the effect of endotoxemia and treatment with PEGylated bovine serum albumin (PEG-BSA) on the microcirculation. Animals will be anesthetized and instrumented for monitoring of systemic function and oxygenation characteristics. Following an equilibration period, Escherichia coli endotoxin (lipopolysaccharide, E. coli 055:B5, Sigma Chemical) will be infused intravenously at a dose of 5 mg/kg over 30 minutes. After confirmation of a septic state, animals will be untreated (n=10) or treated with normal saline (n=10), hypertonic saline (n=10), BSA (n=10), or PEG-BSA (n=10). Computer-assisted intravital microscopy will be used to videotape and quantify microvascular changes in the conjunctival microcirculation during baseline, endotoxemia, and post-treatment phases of the study to correlate with systemic function and oxygenation changes. The results of this study will provide a better understanding of the changes that occur in the conjunctival microcirculation during endotoxemia and treatment with PEG-BSA, which has not yet been studied simultaneously with systemic function and oxygenation in the same animal.