Cells as dynamic, interactive, and self contained units of life have a need for molecular motors that can create physical forces to move cargoes within the cell. Cytoplasmic dynein 1 is one such molecular motor that has many functions in the cell. The number and variety of functions that involve cytoplasmic dynein 1 suggest that there are a number of different binding sites on dynein for different proteins. Cytoplasmic dynein 1 is a multiprotein complex made up of six different subunit families. The many different combinations of subunits that could be used to make up a cytoplasmic dynein 1 holocomplex provides the variety of different binding sites for cargoes that can be individually regulated. The following chapters flush out how light intermediate chain 1 (LIC1), a subunit of cytoplasmic dynein 1, is involved with multiple dynein functions involving the binding of different cargoes to the cytoplasmic dynein 1 holocomplex, and how the binding of these cargoes can be regulated. First, LIC1 is found to be involved in the spindle assembly checkpoint. LIC1 appears to facilitate the removal of Mad1-Mad2, a complex important in producing a wait anaphase signal, from kinetochores. Second, the involvement of LIC1 in the spindle assembly checkpoint requires the phosphorylation of LIC1 at a putative Cdk1 phosphorylation site. This site is located in a domain of LIC1 that binds various proteins suggesting that this phosphorylation could also regulate these interactions. Third, LIC1 is involved in the centrosomal assembly of pericentrin, an important centrosomal protein. From the data presented herein, LIC1 is shaping up as a multifunctional cargo binder for cytoplasmic dynein 1 that requires regulation of its various cargoes.

Summary Basic biological processes require gene expression. Tightly regulated molecules known as transcription factors mediate the expression of genes in development and disease. Signal transduction pathways, which respond to environmental cues or stressors are major regulators of the transcription factors. Use of macromolecular synthesis inhibitors in models of normal neurodevelopment and neurodegenerative cell death has led to the discovery that gene expression is required for these processes to occur (Martin et. al.,(1988), J Cell Biol 106p829). To date, however, the identities of very few of the genes required in these events have been revealed. Hence, the activation or requirement of specific signaling pathways leading to the expression of known apoptotic genes is not well established. Utilizing the neurothrophic factor deprivation and neurotoxin models of programmed cell death we address these gaps in our understanding of the molecular mechanism of apoptosis as it occurs in neuronal cell death. Nerve growth factor (NGF) withdrawal from PC12 cells leads to the activation of p38 and apoptosis. The functional significance of 38 activation in this paradigm of cell death is not known. To increase our understanding of apoptosis I examined the requirement for p38 activity in pro-apoptotic gene expression in PC12 cells. I performed a subtractive hybridization that led to the identification of the monoamine oxidase (MAG) gene as induced in response to NGF withdrawal. Using the p38 inhibitor PD169316 I showed that the NGF withdrawal stimulated induction of the MAG gene and apoptosis is blocked by inhibition of the p38 MAP kinase pathway. I also determined that the MAG inhibitor clorgyline blocked cell death indicating that MAG activity contributes to the cell death caused by NGF withdrawal. Together, these data indicate that the p38 MAP kinase pathway targets the MAG gene in response to apoptotic stimuli. To study the requirement for the JNK signaling pathway in neurodegeneration I stimulated primary cortical neurons with the neurotoxin arsenite. Arsenite treatment of primary neurons leads to both JNK and p38 activation and subsequently apoptosis. Utilizing transgenic mice lacking the JNK3 gene I demonstrated that JNK3 specifically contributes to the effects of arsenite in these cells. Ribonuclease protection assays were used to identify Fas ligand as a molecule whose arsenite-induced expression is dependent on the JNK3 signal transduction pathway. Furthermore, I have shown that neurons deficient in signaling mediated by the receptor for Fas ligand are resistant to cell death due to arsenite treatment. These results in total have established that the JNK3 mediated expression of Fas ligand contributes to the arsenite induced death of cortical neurons. In summary, the work presented in these studies identifies the JNK and p38 MAP kinase signal transduction pathways as mediators of apoptosis in neuronal cells. Importantly, I have provided evidence that these stress activated pathways are responsible for the expression of specific genes in apoptotic neuronal cells.

Cell migration involves complex mechanical interactions between cells or between cells and the underlying substrate. Using a newly developed technique, "traction force microscopy", I have been able to visualize the dynamic characteristics of mechanical forces exerted by migrating fibroblasts such as magnitude, direction, and shear. For NIH 3T3 fibroblasts, I found that the lamellipodium provides nearly all of the force necessary for cell migration. A high shear zone separates the lamellipodium from the remainder of the cell body, suggesting that they are mechanically distinct entities. The timing of the tractions at the leading edge, as well as the spatial distribution, bears no apparent relationship to concurrent local protrusive activities, yet changes in traction force patterns often precede changes in migration direction. In H-ras transformed cells I found isolated regions of weak, transient traction forces in pseudopods all along the cell that appeared to act against one another. The resulting shear pattern suggested that there were multiple disorganized mechanical domains. These results support a frontal towing model for cell migration where the dynamic traction forces at the leading edge served to actively pull the cell body forward. In H-ras transformed cells, the weak poorly coordinated traction forces coupled with weak cell substrate-adhesions were likely responsible for the abnormal motile behavior of these cells. To probe the mechanical interactions beneath various regions of migrating fibroblasts, a cell substrate inhibitor (GRGDTP peptide) was locally applied while imaging stress distribution on the substrate utilizing traction force microscopy. I found that both spontaneous and GRGDTP induced detachment of the trailing edge resulted in extensive cell shortening with no change in overall traction force magnitude or cell migration. Conversely, leading edge disruption resulted in a dramatic global loss of traction forces pnor to any significant cell shortening. These results suggested that fibroblasts transmit their contractile forces to the substrate through two distinct types of adhesions. Leading edge adhesions were unique in their ability to transmit active propulsive forces whereas trailing end adhesions created passive resistance during cell migration and readily redistributed their loads upon detachment. I have also investigated how fibroblasts regulate traction forces based on mechanical input. My results showed that stretching forces applied through the flexible substrate induced increases in both intracellular calcium concentration and traction forces in fibroblasts. Treatment with gadolinium, a well known stretch-activated ion channel inhibitor, was found to inhibit both traction forces and cell migration without inhibiting cellular spread morphology or protrusive activities. Gadolinium treatment also caused a pronounced decrease in vinculin and phosphotyrosine concentrations from focal adhesions. Local application of gadolinium to the trailing region had no detectable effect on overall traction forces or cell migration, whereas local application to the leading edge caused a global inhibition of traction forces and an inhibition of migration. These observations suggest that stretch activated entry of calcium ions in the frontal region serves to regulate the organization of focal adhesions and the output of mechanical forces. Together my experiments elucidate how fibroblasts exert mechanical forces to propel their movements, and how fibroblasts utilize mechanical input to regulate their movements.

The vestibular evoked myogenic potential (VEMP) has been shown to be clinically useful in providing diagnostic information regarding the function of the otolith receptors, inferior vestibular nerve, and vestibulospinal pathways. The VEMP is a biphasic response elicited by loud clicks or tone bursts and recorded from the tonically contracted sternocleidomastoid (SCM) muscle. Because the VEMP is an inhibitory response, it is important to investigate stimulus and parameter characteristics in order to determine the optimal test protocol and maximize clinical usefulness. The aims of this study were 1) to evaluate the effects of 4 different methods of SCM muscle activation and the effect of visual biofeedback on VEMP latency, amplitude, asymmetry ratio, mean rectified EMG level, and difficulty ratings, and 2) to determine the influence of SCM muscle activation pattern and visual biofeedback level on test-retest reliability. Forty-eight healthy volunteers between the ages of 18 and 50 underwent VEMP testing using each of the following muscle activation patterns: supine with the head raised (SE), supine with the head turned away from the test ear (SR), supine with the head raised and turned away from the test ear (SER), and sitting with the head turned away from the test ear (SitR). Testing subjects with the SER method yielded the most robust amplitude response and sternocleidomastoid EMG activity. No statistically significant differences were found in interaural asymmetry ratios among the 4 methods of SCM activation. Subjects rated the SE and SER methods as more difficult than the SE and SitR methods at each of the 3 target levels. Test-retest reliability was high for P1/N1 amplitude and mean rectified EMG levels when subjects were provided visual biofeedback to monitor the level of tonic SCM muscle activity. The study demonstrates the importance of providing patients a means of monitoring and maintaining the amplitude of the rectified EMG at a constant target level during SCM muscle activation. Although no evidence to reject or strongly favor a specific method was found, monaural-ipsilateral recording with the SitR method was found to be advantageous for individuals with weakness or decreased endurance for sustained muscle contraction.

Attention uses sensory inputs and goals to select information from our environment. Monkey electrophysiological literature demonstrates that visuo-tactile bimodal neurons (respond to visual and tactile stimuli presented on/near the hand) facilitate multisensory integration. Human behavioral studies show that hand position/function bias visual attention. Event-related potentials (ERPs) reveal the cortical dynamics coordinating visual inputs, body position, and action goals. Early, sensory ERPs (N1) indicate multisensory integration. Later, cognitive ERPs (P3) reflect task-related processing. Study 1 investigates a discrepancy between monkey and human literatures. Monkey studies demonstrate bimodal neuron responses equidistantly around the whole hand, but human studies demonstrate attentional bias for grasping space. In a visual detection paradigm, participants positioned their hand so target and non-target stimuli appeared near the palm or back of the hand; ERPs were measured. N1 components indicated no amplitude differences between Palm vs. Back conditions, but P3 components revealed greater target vs. non-target differentiation for Palm conditions. Results suggest cortical timing underlies grasping vs. whole hand bias differences: early processing does not differentiate using hand function, but cognitive processing does when stimuli are discriminated for action. Study 2 investigates whether proprioceptive inputs facilitate visual processing. In a visual detection paradigm, participants viewed stimuli presented between occluders blocking view of a hand positioned either near or far from the stimuli. N1 amplitudes were similar for near and far conditions, but P3 amplitudes for target/non-target differences were accentuated for near conditions. Proprioceptive effects emerge later in processing. ERP reveals the cortical dynamics underlying hand position effects on vision.

Voltage-gated K+ channels associate with multiple regulatory proteins to form complexes with diverse gating properties and pharmacological sensitivities. Small molecules which activate or inhibit channel function are valuable tools for dissecting the assembly and function of these macromolecular complexes. My thesis focuses on the discovery and use of small molecules to probe the structure and function of the KCNQ family of voltage-gated K+ channels. One protein that obligatorily assembles with KCNQ channels to mediate proper assembly, trafficking, and gating is the calcium sensor, calmodulin. Although resolution of the crystal structures of calmodulin associated with isolated peptide fragments from other ion channels has provided some insight into how calmodulin interacts with and modulates KCNQ channels, structural information for calmodulin bound to a fully folded ion channel in the membrane is unknown. In Chapter II, I developed an intracellular tethered blocker approach to determine the location of calmodulin binding with respect to the KCNQ ion-conducting pathway. Using distance restraints from a panel of these intracellular tethered blockers we then generated models of the KCNQ-calmodulin complex. Our model places calmodulin close to the gate of KCNQ channels, providing structural insight into how CaM is able to communicate changes in intracellular calcium levels to KCNQ channel complexes. In addition to pore blockers, chemical modification of ion channels has been used to probe ion channel function. During my initial attempt to chemically activate KCNQ channels, I discovered that some boronates modulate KCNQ complexes. In Chapter III, the activating derivative, phenylboronic acid, is characterized. Characterization of activation by phenylboronic acid showed that it targeted the ion conduction pathway of KCNQ channels with some specificity over other voltage-gated K+ channels. The commercial availability of thousands of boronic acid derivatives provides a large class of compounds with which to systematically dissect the mechanisms of KCNQ gating and may lead to the discovery of a potent activator of KCNQ complexes for the treatment of channelopathies. All of the electrophysiological studies presented in this thesis were conducted in Xenopus oocytes. Unexpectedly, during the studies described above, the quality of our Xenopus oocytes declined. The afflicted oocytes developed black foci on their membranes, had negligible electric resting potentials, and poor viability. Culturing the compromised oocytes determined that they were infected with multi-drug resistant Stenotrophomonas maltophilia, Pseudomonas fluorescens and Pseudomonas putida. Antibiotic testing showed that all three species of bacteria were susceptible to amikacin and ciprofloxacin, which when included in the oocyte storage media prevented the appearance of black foci and resulted in oocytes that were usable for electrophysiological recordings. This study provides a solution to a common issue that plagues many electrophysiologists who use Xenopus oocytes. Taken together, these findings provide new insights into activation of KCNQ channel complexes and provide new tools to study the structure-function relationship of voltage-gated K+ channels.

Neonatal mice have immature immune systems with defects in several components of inflammatory, innate, and specific immune responses and develop a preferential T helper type 2 (Th2) response following immunization with many vaccine antigens. Although maternal antibody is the major form of protection from disease in early life when the neonatal immune system is still immature, the presence of maternal antibody also interferes with active immunization, placing infants at risk for severe bacterial and viral infection. Recent studies have suggested that immunizing with DNA plasmids encoding the vaccine antigen of interest is highly efficacious in a variety of adult animal models. However, similar extensive studies have not been conducted in infants. In this dissertation, we examine both the quantitative and qualitative differences between neonatal and adult humoral and cell-mediated immune responses in the presence or absence of maternal antibody. First, we wished to determine if one-day-old neonatal mice immunized with plasmid DNA expressing influenza A/PR/8/34 hemagglutinin (HA) by either intramuscular (i.m.) or gene gun (g.g.) inoculation were capable of generating humoral responses comparable to those in mice immunized as adults. We found that newborn mice developed stable, long-lived, protective anti-HA-specific IgG responses similar in titer to those of adult DNA-immunized mice. However, unlike the adult i.m. and g.g. DNA immunizations, which develop polarized IgG2a and IgG1 responses, respectively, mice immunized as neonates developed a variety of IgG1-, IgG2a-, and mixed IgG1/IgG2a responses regardless of the inoculation method. Boosting increased, but did not change these antibody profiles. We also found that, in contrast to the DNA immunizations, inoculations of newborn mice with an A/PR/8/34 viral protein subunit preparation failed to elicit an antibody response. Further, temporal studies revealed that both responsiveness to protein vaccination and development of polarized patterns of T help following DNA immunization appeared by 2 weeks of age. To determine if the disparity of polarized IgG responses between neonatal and adult DNA vaccinated mice was due to deficiencies in Th1 promoting cytokines, we addressed the ability of DNA encoding Th1 cytokines to bias the isotype of antibody raised by neonatal DNA immunization. We found that neonatal mice coimmunized with HA and either IL-12 or IFNγ-expressing DNAs developed IgG2a-biased immune responses, regardless of inoculation method, whereas these DNAs had no effect on IgG subtype patterns in adult DNA immunized mice. Consistent with the Th1-promoting effects of these cytokines, we also observed that codelivery of IL-12 or IFNγ DNAs raised T helper responses toward Th1 in mice immunized both as neonates or adults. Thus, codelivery of cytokine DNAs may be effective at tailoring immune responses depending on the required correlates of protection for a given pathogen. Finally, we addressed the effect of maternal antibody on the elicitation of humoral and cell-mediated immune responses. We tested the ability of i.m. and g.g. immunization with DNA expressing influenza HA and/or nucleoprotein (NP) to raise protective humoral and cellular responses in the presence and absence of maternal antibody. We found that neonatal mice born to influenza-immune mothers raised full antibody responses to NP but failed to generate antibody responses to HA. In contrast, the presence of maternal antibody did not affect the generation of long-lived CD4+ and CD8+ T cell responses to both HA and NP. Thus, maternal antibody did not affect cell-mediated responses, but rather it limited humoral responses, with the ability to limit the antibody response correlating with whether the DNA-expressed immunogen was localized in the plasma membrane or within the cell. We further observed that protection from influenza virus challenge was dependent on the presence of anti-HA IgG and was independent of the presence T cell responses. Taken together with other published studies, the data presented in this dissertation help better characterize the responses elicited by DNA vaccines at birth. This dissertation presents several novel observations including the temporal development of polarized IgG subtype responses, the ability of codelivered Th1 cytokine DNA to affect both antibody and T cell responses in the neonate, and the ability to generate humoral responses to intracellular, but not plasma membrane proteins, in the presence of maternal antibody. Furthermore, the data provides rationale for further development of DNA vaccines in the neonate.

It has been well documented that mRNA is associated with the cytoskeleton, and that this relationship is involved in translation and mRNA sorting. The molecular components involved in the attachment of mRNA to the cytoskeleton are only poorly understood. The objective of this thesis was to directly visualize the interaction of mRNA with the cytoskeleton, with sufficient resolution to identify the filament systems and structures involved. This work required the development of novel in situ hybridization methods for use with electron microscopy. This allowed resolution to visualize single mRNA molecules and individual filaments. The development of a silver enhancement methodology for both the light and electron microscopic detection of biotinated oligo-dT probes permitted a synoptic view of the intracellular distribution of poly(A) mRNA. At the light microscope, the distribution of poly(A) mRNA did not resemble the individual distribution patterns of microfilaments, intermediate filaments or microtubules. Ultrastructural examination revealed that poly(A) mRNA was not uniformly distributed along cytoskeletal filaments, but clustered at their intersections. The composition of these mRNA containing structures was investigated by both morphologic and in situ hybridization analysis using antibodies to cytoskeletal proteins. In thin sections, polysomes were observed attached to both microfilaments and intermediate filaments. To permit the simultaneous detection of oligo-dT hybridization and specific cytoskeletal proteins, a double labelling method using colloidal gold conjugated antibodies was developed. The majority of poly(A) mRNA was associated with the actin cytoskeleton, with 72% of the hybridization localized within 5nm of a labelled microfilament. Within the actin cytoskeleton, poly(A) mRNA was localized to intersections of orthogonal networks. Greater than 50% of poly(A) colocalized with the actin crosslinking proteins, filamin and α-actinin, but not vinculin. A significant amount of poly(A) mRNA was found to be associated with intermediate filaments. The double label gold analysis demonstrated that 33% of the hybridization signal localized within 5nm of labelled vimentin filaments. Prior disorganization of the actin cytoskeleton using cytochalasin did not disrupt the association of mRNA with vimentin. These observations are consistent with our morphologic results of polysome-intermediate filament associations, and indicate that microfilaments are not the only filament system to which mRNA is bound. Furthermore, a small amount of hybridization signal (12%) consistently was observed along microtubules, providing an additional cytoskeletal network to distribute mRNA. To further characterize the spatial organization of mRNA within the cytoskeleton, ultrastructural methods were developed to directly visualize individual mRNA molecules. First, oligonucleotide probes chemically modified with a single hapten and directly conjugated primary reagents were used to permit detection of an individual hybridized probe molecule by a single gold particle. Second, biotin and digoxigenin labelled oligonucleotide probes were used to simultaneously visualize the intermolecular and intramolecular relationships of two nucleic acid sequences. Third, reverse transcriptase was used to extend hybridized primers in situ which permitted visualization of the poly(A) sequence concomittant with the conformation of an mRNA molecule. These methods have permitted analysis of how single mRNA molecules may be positioned with respect to each other within the cytoskeleton. The ultrastructural visualization of mRNA within its structural environment has demonstrated heterogeneous interactions with the cytoskeleton. Future work will be needed to further characterize the mechanism of mRNA attachment. The proteins which bridge nucleic acid sequences to specific intersections can be identified. It will be interesting to learn how the identified mRNA-cytoskeletal interactions might be involved in the regulation of both mRNA translation and intracellular location. Lastly, and perhaps the most challenging goal, is to investigate whether the identified mRNA-cytoskeletal interactions are used by the cell to influence its own shape, polarity and architecture.

There are four serotypes of dengue virus designated dengue 1, 2, 3 and 4 (D1, D2, D3 and D4) and epidemiological studies indicate that a severe complication of dengue virus infection - dengue hemorrhagic fever (DHF) is more likely to occur following a secondary infection. DHF is hypothesized to be immunologically mediated and may be triggered by virus-specific T cells. It is also likely that dengue virus-specific cytotoxic T lymphocytes (CTLs) are important for recovery from dengue virus infections. An analysis of the immune response during acute illness and when the patient has recovered from the infection (immune state) is therefore important as it will provide insights into the immunopathological nature of the disease. This thesis initially examines the CD8+CTL responses in volunteers who have received live attenuated dengue vaccines and then investigates acute and immune T cell responses in children following natural infection with dengue. When this project was initiated, there was little available information on the human CD8+ T cell responses to dengue viruses. PBMC from one donor had generated memory CD8+CTL to the nonstructural protein NS3 of dengue virus. Memory CD8+CTL responses were therefore analyzed to determine the diversity of the T cell response to dengue virus and to identify immunodominant proteins using PBMC from eight healthy adult American volunteers who had received monovalent live-attenuated candidate vaccines of the 4 dengue serotypes. All the donors had specific T cell proliferation to dengue viruses and to other flaviviruses that we tested. CTLs were generated from the stimulated PBMC of all donors and in the seven donors tested, dengue virus-specific CD8+CTL activity was demonstrated. The nonstructural proteins NS3 and NS1.2a and the structural protein E were recognized by CD8+CTLs from six, five and three donors respectively. All donors recognized either NS3 or NS 1.2a. In a donor who received a dengue 4 vaccine, CTL killing was seen in bulk culture against the premembrane protein (prM). This is the first demonstration of a CTL response against the prM protein. The CTL responses using PBMC of two donors were serotype-specific whereas all other donors had serotype-cross reactive responses. For one donor, CTLs specific for E, NSl.2a and NS3 proteins were all HLA-B44 restricted. For the three other donors tested the potential restricting alleles for recognition of NS3 were HLA-B38, A24 and/or B62 and B35. These results indicate that the CD8+CTL responses of humans after immunization with a single serotype of dengue virus are diverse and directed against a variety of proteins. The nonstructural proteins NS3 and NSl.2a appear to be immunodominant and should be considered when designing subunit vaccines for dengue. Previously T cell responses had not been examined in people who have had natural infections with dengue. The HLA diversity between North American Caucasians and populations where dengue is a serious health problem, calls for the analysis of immune responses in people who have been infected with natural circulating strains of the virus. We examined the memory cytotoxic T lymphocytic (CTL) responses of peripheral blood mononuclear cells (PBMC) obtained from patients in Thailand 12 months after natural symptomatic secondary dengue infections. In all four patients analyzed, CTLs were detected in bulk culture PBMC against nonstructural dengue proteins. Numerous CD4+ and CD8+ CTL lines were generated from the bulk cultures of two patients, KPP94-037 and KPP94-024, which were specific for the NSl.2a and NS3 proteins respectively. All CTL lines derived from both patients were crossreactive with other serotypes of dengue virus. The CD8+ NS1.2a specific lines from patient KPP94-037 were HLA-B57 restricted and the CD8+ NS3 specific lines from patient KPP94-024 were HLA-B7 restricted. The CD4+ CTL lines from patient KPP94-037 were HLA-DR7 restricted. A majority of the CD8+CTLs isolated from patient KPP94-024 were found to recognize a.a. 221-232 on NS3. These results demonstrate that after symptomatic secondary natural dengue infections in Thai patients, CTLs are mainly directed against nonstructural proteins and are broadly crossreactive. The data correlate with our observations that nonstructural proteins are immunodominant proteins in volunteers who received dengue vaccines. We were interested in examining CTL responses in children during their acute illness and comparing them to memory CTLs obtained from the same children a year or more after the infection. A detailed analysis on samples from nine patients during their acute illness failed to generate any dengue virus-specific CTL responses. We therefore decided to determine if cell mediated responses are altered during acute dengue infection. Decreased proliferative responses to mitogens and recall antigens have been observed in PBMC obtained during several acute human viral infections. All responses of PBMC during acute illness were compared to the same patients PBMC obtained at least 6 months after their infection. Proliferative responses to PHA, anti-CD3, tetanus toxoid and dengue antigens were significantly decreased in PBMC obtained during the acute infection. The proliferative responses to PHA were restored by the addition of gamma-irradiated autologous immune or allogeneic PBMC. Cell contact with the irradiated PBMC was necessary to restore proliferation. Non-T cells from the acute PBMC of dengue patients did not support proliferation of T cells from control donors in response to PHA, but T cells from the PBMC of patients with acute dengue proliferated if accessory cells from a control donor were present. Addition of anti-CD28 antibodies restored anti-CD3-induced proliferation of the PBMC of some patients. The percentage of monocytes was reduced in the acute sample of PBMC of the dengue patients. Addition of IL-2 or IL-7, but not IL-4 or IL-12 also restored proliferation of acute PBMC stimulated with anti-CD3. The results demonstrate that both quantitative and qualitative defects in the accessory cell population during acute dengue illness result in a depression of in vitro T cell proliferation. The data generated from this project shed light on the nature of the immune responses during acute natural dengue infections. It strengthens the existing data on the human memory CD8+CTL responses to dengue viruses and validates the observations by examining memory CTL responses after natural dengue infection in patients from Thailand. In addition, we demonstrate a profound defect in lymphoproliferative responses during dengue illness.

Bile acid metabolism is integrally involved in cholesterol homeostasis in mammals because it is the major means by which cholesterol is eliminated from the body. We have undertaken an effort to study the molecular mechanisms underlying the regulation of bile acid metabolism by isolating and characterizing the cDNA and gene for an enzyme that hydroxylates lithocholic acid (LCA) at position 6β, lithocholic acid 6β-hydroxylase; the first bile acid-induced gene reported. LCA is a very hydrophobic, toxic bile acid formed from chenodeoxycholic acid in the gut lumen upon reduction of the 7α-hydroxy group by microbial enzymes. The proper elimination of LCA is essential for maintenance of the bile acid pool and for prevention of cholestasis which results from LCA precipitating in the cannaculi of the liver when its concentration is high. The LCA 6β-hydroxylase cDNA was isolated by differential hybridization of hamster liver libraries prepared from animals fed either a cholic acid enriched diet or a cholestipol-rich chow and was named CYP3A10 based on its homology with other cytochrome P450s (P450) in family 3A. We found that CYP3A10 was essentially expressed only in males. A statistical analysis of RNA from young males fed with cholic acid and normal chow showed that the cholic acid induction was about 50% at the RNA level. We determined the biological nature of the protein encoded by CYP3A10 by expression of the cDNA in COS cells. Microsomes prepared from transfected cells were assayed with LCA as a substrate and found to hydroxylate LCA predominantly at position 6β. We examined whether CYP3A10 could hydroxylate other steroid compounds by assays with testosterone, progesterone and androstenedione and found that, although 6β-hydroxylase (as well as others) activity was observed with all three, LCA was the preferred substrate based on kinetic analysis. A developmental time course of CYP3A10 expression in males showed little expression before puberty, a striking induction of expression at puberty and a fourfold induction thereafter through adulthood. We then examined the male-specific expression of CYP3A10 in hamster liver. We disrupted the pattern of GH secretion in male hamsters by hypophysectomy, neonatal glutamate treatment and by continuous infusion of GH via osmotic minipumps (to mimic the female pattern of GH secretion) and found no significant effect on CYP3A10 expression. Conversely, in females, hypophysectomy and neonatal glutamate treatment significantly induced CYP3A10 expression 5- to 10-fold. Additionally, when females treated neonatally with glutamate were injected twice daily with GH as adults (to mimic the male pattern of GH secretion), the levels of CYP3A10 expression were not significantly different from those of normal males. These results led us to conclude that the pattern of GH secretion in males does not control the male-specific expression of CYP3A10 but that in females expression can be induced by altering the tonic secretion of GH. No significant effect on CYP3A10 expression was observed by castration of adult males, indicating that circulating androgens were not required for expression. We found that gonadal hormones (e.g. estrogen and progesterone) do not have a suppressive effect on CYP3A10 expression in females since ovariectomy did not induce expression. Many genes are "imprinted" neonatally by exposure to a given effector for developmental-, tissue- or sexually regulated expression. We investigated whether neonatal androgen exposure was required for male-specific expression of CYP3A10 by castrating hamsters neonatally and determining the level of CYP3A10 expression in adulthood. Our results indicate that androgens are required neonatally for CYP3A10 expression since no expression was observed in neonatally castrated hamsters. We were unable to induce expression in neonatally castrated hamsters by either GH or testosterone injections. These results suggest several notable points 1) that CYP3A10 expression is programmed neonatally by androgen exposure; 2) that androgens exert their effect directly on the liver and not via the hypothalamus; 3) that neither testosterone nor GH can restore CYP3A10 expression when males have not been exposed to androgens neonatally; and 4) that in experimental conditions, females can be induced to express CYP3A10, which indicates that there are two modes for regulating expression: by "imprinting" in males and by GH and testosterone in females. We are now studying the molecular mechanisms involved in the bile acid-mediated induction and the male-specific expression of CYP3A10. We have cloned approximately 8 kb of 5' flanking DNA from a hamster genomic library and sequenced about 1 kb of proximal DNA. Primer extension and S1 digestion analyses indicate that the mRNA for CYP3A10 has multiple transcription initiation sites clustered about 90 bp from the initiator methionine codon. We have also prepared CYP3A10 promoter/lacZ chimeric constructs to begin delineating the cis-acting elements controlling CYP3A10 expression and regulation. We used H2.35 cells as recipients because they are a mouse hepatocyte cell line that has been transformed with a temperature sensitive SV40. These cells can be grown at the permissive temperature and can be induced to behave like liver cells, the differentiated condition, by switching to a nonpermissive temperature. We have found that the construct with 1 kb of proximal CYP3A10 5' flanking DNA was able to express the reporter gene at higher levels under differentiated conditions, which were consistent with higher expression of an albumin promoter/lacZconstruct, upon switching the cells to the more liver phenotype. The system characterized and described here is ideally suited for dissecting the molecular details governing bile acid-mediated regulation and sexually dimorphic expression of liver genes. Very little is known about both these very important biological phenomena. Much could be learned about transcriptional regulation of liver genes by investigating the cis-elements and trans-acting factors mediating regulation of CYP3A10 expression.