Synapse formation involves multiple coordinated events between the presynaptic and the postsynaptic nerve that ultimately results in the expression of the appropriate neurotransmitters at the presynaptic nerve terminal and the matching neurotransmitter receptors on the opposing postsynaptic membrane. For my thesis research, I investigate different aspects of neurotransmitter receptor gene expression, an early step in the process of synaptogenesis. Specifically, I focus on two different neurotransmitter-gated ion channels that are expressed on neonatal rat peripheral neurons: the serotonin 5-HT3 receptor (5-HT3R) and the neuronal nicotinic acetylcholine receptor (nAChR). / The 5-HT3R is expressed on both nodose sensory neurons and sympathetic neurons. However, little is known about 5-HT3R expression during neonatal development or about the factors that regulate its expression. To investigate 5-HT3R gene expression, first I examined 5-HT 3R mRNA levels in nodose and sympathetic neurons as they develop in vivo and in culture. My results show that 5-HT 3R gene expression is differentially regulated in these two populations of neurons. In addition, I demonstrate that 5-HT3R gene expression in nodose neurons depends on target innervation and can be modulated by neurotrophins. / Neonatal sympathetic neurons express five different neuronal nAChR subunit genes. One unresolved issue is the contribution of these five subunits to nAChR function. To investigate this issue, I altered the expression of one nAChR subunit gene, alpha3, using transient transfection procedures. To do this, first I modified and optimized gene transfer procedures for sympathetic neurons, based on recombinant adenovirus vectors. sing this approach, I overexpressed sense and antisense alpha3 mRNA and investigated how the changes in alpha3 subunit expression affect ACh-evoked currents on cultured sympathetic neurons. I show that changes in alpha3 mRNA levels alter the magnitude of ACh-evoked current densities. My results indicate that alpha3 gene expression is rate-limiting for the assembly and insertion of nAChRs on sympathetic neurons. / Together, my results show that multiple mechanisms influence the expression neurotransmitter-gated ion channel genes on peripheral neurons.

For many years, the fact that the primary transcript of human immunodeficiency virus type 1 (HIV-1) is inefficiently spliced was largely taken for granted and the underlying mechanism remained unexplored. We set out to define the cis-acting elements involved in splicing regulation of the HIV-1 tat/rev intron. This task was facilitated by the use of a plasmid designed for transient expression in COS-7 cells of a synthetic gene that contained the first intron of human beta-globin. By exchanging either the 5' splice site (5' ss) or the 3' splice site (3' ss) of beta-globin with various portions of the HIV-1 genome spanning the tat/ rev splice sites, we could draw several conclusions. / By trimming away flanking sequences, we demonstrated that the tat/rev 3' ss is intrinsically inefficient. Mutagenesis studies revealed that both the branchpoint and polypyrimidine tract elements were suboptimal. The tat/rev 5' ss, on the other hand, was shown to be utilized efficiently. The efficiency of the tat/rev 5' ss was not affected by inclusion of the tat/ rev intron sequences. Therefore, we concluded that the only requirement for inefficient splicing of the tat/rev intron is the suboptimal nature of the branchpoint and polypyrimidine tract elements. / Deletion studies revealed that two cis-acting elements within the downstream exon modulate splicing of the tat/ rev intron. These elements include a positive element, the exon splicing enhancer (ESE), and an adjacent negative element, the exon splicing silencer (ESS). The ESE was mapped to a 10 nt purine-rich element that is functionally interchangeable with other purine-rich ESEs. The ESS exhibited context dependence in that its extent of splicing inhibition was sensitive to mutations that improve the branchpoint or polypyrimidine tract. / During the course of our investigation of the HIV-1 ESS in a heterologous context, we made the unexpected discovery of novel splicing regulatory elements within the EDA exon of the human fibronectin gene. As in HIV-1, these elements include adjacent positive and negative elements. Although the fibronectin ESE is not purine-rich, we demonstrated that it is functionally analogous to the ESE of HIV-1. The fibronectin ESS is relatively large. Evolutionary alignment and mutagenesis studies implicate secondary structures as determinants of splicing inhibition. / Finally, using chimeric beta-globin/HIV-1 introns as above, we tested a model for nuclear retention of unspliced pre-mRNA which proposes that the RNA becomes trapped in "handicapped" splicing complexes as a consequence of the pairing of a strong splice site with a weak one. We present evidence that the inefficient tat/rev 3' ss is insufficient to cause nuclear retention; additional tat/rev intron sequences are required to prevent the cytoplasmic accumulation of unspliced RNA.

Interferons (IFNs) are polypeptides that protect the body from microbial infection by the activation of genes that inhibit the replication of such pathogens. STAT1 is a transcription factor that is activated by IFNs and virus infection, and propagates signals to the nucleus by binding DNA enhancer elements to induce the expression of IFN-stimulated genes (ISGs). One such gene product is the double-stranded RNA (dsRNA)dependent protein kinase, PKR. PKR is an important determinant of host resistance since it has the ability to bind dsRNA, an intermediate produced during viral replication, and to phosphorylate the eukaryotic translation initiation factor-2alpha (eIF-2alpha), a modification that inhibits of protein synthesis. Additionally, recent findings have shown that PKR can modulate various signaling pathways. The objective of this research was to understand the involvement of PKR in the IFN signaling pathways. We observed that the catalytic domain of PKR interacts with the DNA-binding region of STAT1 in vitro and in vivo. Overexpression of catalytic mutants of PKR blocked STAT1 DNA-binding and ISG expression upon IFN or dsRNA treatment. The capacity of catalytic mutants of PKR to negatively regulate STAT1 DNA-binding is attributed to its ability to interact with STAT1 since in cells lacking PKR or expressing an RNA-binding mutant of PKR, unable to associate with STAT1, STAT1 displays augmented activity. A mutant of STAT1 (TM) unable to interact with PKR displayed elevated biochemical and biological activities; findings consistent with the role of PKR in regulating STAT1 function. Alternatively, the interaction between PKR and STAT1 has the ability to inhibit PKR activity in vitro and in vivo; a phenomenon not observed with STAT1 TM. / In parallel, PKR was also found to interact with TYK2, the upstream activator of STAT1. IFN treatment promoted the dissociation of PKR from TYK2 in a manner dependent on TYK2 kinase activity. Furthermore, we observed that TYK2 phosphorylates the amino terminus of PKR in vitro and in vivo on tyrosine. Taken together, PKR appears to function as an important scaffolding protein for the IFN signaling pathways. Alternatively, PKR activity itself is also regulated at multiple steps along this pathway. Thus, it appears that there is an intimate cross-talk between components of the JAK/STAT pathway and translational machinery.

Voltage-gated potassium (K) channels are important in controlling a neuron's excitability. In this thesis I show that neonatal rat nodose and superior cervical ganglion (SCG) neurons express three voltage-gated K currents: a non-inactivating delayed rectifier type current (IK); a rapidly inactivating A-current (IAf), and; a slowly inactivating A-current (IAs). The channels that underlie IAf and IAs differ in their voltage-dependent, kinetic and pharmacological properties, but share the same single channel conductance, suggesting that rapidly and slowly inactivating A-channels are distinct subtypes of the same basic channel. My preliminary molecular biology experiments establish an approach for identifying the genes that encode for IAf, IAs and IK channels on SCG neurons. By studying the expression of IAf, IAs and IK on peripheral neurons during the first 2 postnatal weeks, I showed that there is no change in the expression of the 3 currents on nodose neurons, whereas the outward current on SCG neurons changes significantly from one dominated by IAs to one dominated by IAf. These results indicate that the pattern of developmental expression of similar types of K channels can differ for each neuron type. Next, I investigated various factors involved in the postnatal control of expression of K channels on SCG neurons. I demonstrated that neither preganglionic nor target factors influence postnatal changes in K currents. However, I observed that neonatal SCG neurons that develop in culture without other cell types lose their expression of IAf and IAs, suggesting that extrinsic factor(s) are involved in the regulation of the expression of these currents. I showed that this loss of A-currents is due, in part, to the absence of a soluble factor provided by nonneuronal cells. In addition, my analysis of the different patterns of expression of voltage-gated K currents suggests that peripheral neurons use intrinsic mechanisms to coordinate their expression of IAf, IAs and

Chronic moderate ethanol (ETOH) consumption prevents the development of the age-dependent increase in blood pressure (BP) in both humans and experimental animals. In the present studies, we proposed that the natriuretic peptide family may be partially responsible for this antihypertensive effect of ETOH. The natriuretic system consists of the atrial natriuretic peptide (ANP), the brain natriuretic peptide (BNP), the C-type natriuretic peptide (CNP) and the natriuretic peptide receptors (NPRs). The major function of the natriuretic system is to decrease BP. Thus, the main objective of the present studies was to investigate the interactions of acute and chronic ETOH administration with various components of the natriuretic system. / Acute studies. The injection of 1 and 2 g of ETOH/kg B.W. in Sprague-Dawley rats and 0.25 and 0.50 g of ETOH/kg B.W. in humans resulted in a rapid transient increase of circulating ANP levels. In the rats, this increase in plasma ANP levels was associated with a rapid decrease of atrial ANP content and with a delayed increase in ventricular ANP levels. / Chronic studies. A 20% v/v solution of alcohol was given to spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats for 8 months. In both SHR and WKY rats, chronic ETOH treatment decreased the BP. This lower BP in ETOH-treated animals was associated with lower circulating ANP and BNP levels, whereas ETOH treatment increased atrial ANP and BNP tissue contents, but not ANP and BNP mRNA. Furthermore, chronic ETOH treatment reduced heart ventricular ANP content and ANP mRNA while it increased ventricular BNP content and BNP mRNA in SHR, but not in WKY rats. Chronic ETOH reduced total natriuretic peptide binding sites (NPR-A and NPR-C) in the renal glomeruli. Quantification of the receptor subtypes demonstrated that this decrease was due to the down-regulation of NPR-C. In the renal papilla, chronic ETOH treatment increased natriuretic peptide binding sites (NPR-A). In addition to its effects on the peripheral natriuretic system, chronic ETOH treatment altered the activity of the natriuretic system at the level of the brain. Thus, chronic ETOH increased ANP and CNP levels in the hypothalamus, pons and medulla of SHR rats. In WKY rats, ETOH had no effect on brain ANP levels, but enhanced the concentration of CNP in the hypothalamus and medulla. Chronic ETOH treatment also decreased the affinity of NPR-C in some of the circumventricular organs of the brain, in the subfornical organ and choroid plexus, but not in the area postrema. / These results demonstrate that both acute and chronic moderate ETOH administration alters the activity of various components of the natriuretic system. Therefore, these ETOH-induced modifications in cardiac, renal and brain natriuretic peptides and receptors may contribute to the antihypertensive effect of chronic moderate ETOH drinking.

The present thesis has examined in detail the dynamics of adrenomedullary glucocorticoid (GC) receptors at various concentrations of steroid, the regulation of levels of receptor following various treatments, and the regulation of phenylethanolamine N-methyltransferase (PNMT) activity following acute exposure to GCs and various time delays, providing evidence that GC regulation of adrenomedullary catecholamine biosynthesis is more dynamic than was classically thought. We report that adrenomedullary GC receptors are translocated, both in response to nM concentrations of GCs, and in response to higher concentrations of GCs encountered by the glands during stress. We show that long-term increases in cyclic nucleotide second messengers are able to decrease GC receptor binding in adrenal medullary cells, via a mechanism independent of released cortisol, and provide the first evidence that changes in adrenomedullary GC receptor levels are reflected in an alteration in a GC-mediated function, i.e. induction of PNMT. We also provide novel in vitro evidence for the regulation of adrenomedullary PNMT activity, following a necessary lag period, by acute changes in both cortisol and nicotine. In addition, our in vitro studies are supported by our in vivo findings which show increases in adrenal tyrosine hydroxylase and PNMT activity 18h following a single episode of mild acute stress (20 min restraint) in rats.

Hypercholesterolemia, a major risk factor for atherosclerosis, is present in many patients with chronic renal failure (CRF). The present study was carried out in order to determine the mechanisms which underlie this increase in serum cholesterol levels and to test the feasibility of using lovastatin, a HMG-CoA reductase inhibitor in its treatment. / A mouse model of surgically-induced CRF was employed in the experiments. Five weeks after the onset of renal failure, the mice were characterized with regard to various biochemical and hematological parameters. At this time, treatment with lovastatin was initiated. The drug (50, 100 and 200 mg/kg BW/day) was incorporated in powdered diet and was given fresh daily for four weeks. Upon sacrifice, blood was collected for the estimation of blood urea nitrogen and serum lipids and livers were excised for the measurement of hepatic HMG-CoA reductase activity. / The mice exhibited the major manifestations of CRF--retention of nitrogenous wastes, elevated levels of alkaline phosphatase, suggesting the presence of bone disease, and severe anemia. CRF mice also had elevated serum total cholesterol levels with a concomitant, but not significantly correlated, increase in hepatic HMG-CoA reductase activity. Furthermore, their serum lipoprotein profiles were abnormal. Treatment with lovastatin resulted in a dose-dependent reduction in serum total cholesterol levels and correction of the serum lipoprotein profile. However, hepatic HMG-CoA reductase activity was unchanged. / These results indicate that the hypercholesterolemia observed in CRF mice is probably due to an increase in de novo synthesis of cholesterol in both the liver and extranepatic tissues. Lovastatin may decrease serum total cholesterol levels in CRF mice by inhibiting peripheral, rather than hepatic, HMG-CoA reductase activity.

Anemia is a hallmark of chronic renal failure (CRF) which, if left untreated, is a major contributing factor to the high morbidity and mortality of this condition. This characteristically hypoproliferative anemia is due primarily to decreased erythropoietin (EPO) production by the diseased kidney. Currently, correction of the anemia with recombinant human EPO (r-HuEPO) constitutes the mainstay of management in patients with end-stage renal disease (ESRD). / An alternative approach to the treatment of the anemia of CRF is the administration of androgens. / My project utilized a well characterized mouse model of surgically-induced renal failure to study the mechanism(s) of androgen effect on the anemia of CRF. Recent experiments in this model revealed a dose-response to r-HuEPO similar to that in humans, absent EPO gene expression in the liver and full correction of the anemia of CRF by the administration of a combination of subtherapeutic doses of r-HuEPO and insulin-like growth factor-I (IGF-I), a known regulator of erythropoiesis in the normal physiological state. / My hypothesis followed two lines of investigation: androgens may act to increase EPO production or they may increase production of IGF-I, thereby increasing extra-renal erythropoietic activity by either of the two hormones. (Abstract shortened by UMI.)

We review the salient clinical and laboratory features of cyclical neutropenia (CN) and other periodic hematological disorders, and the insight into these diseases afforded by mathematical modeling. Using Lomb periodogram analysis, we show that occurrence of significant cycling in the serial blood counts of neutropenic patients is very prevalent and the dynamics of all the cell lineages in CN can be modified by the administration of recombinant granulocyte colony stimulating factor (G-CSF). The analysis of the serial blood counts in the animal model of CN---the grey collie (GC), reveals a complex pattern of oscillations that we could reproduce with a model of hematopoiesis combining a peripheral control of granulopoiesis through G-CSF, together with an oscillatory input from the pluripotential stem cell. Both the human and GC data analysis suggests that CN results from a complex interaction between the stem cells, the mature cells and G-CSF, and that the regulation of the different blood cell lineages are strongly linked together.

Modern cancer therapy has explored the use of enzymatically acting toxins coupled to specific binding proteins in an attempt to destroy specific cell populations. In light of these new therapies we have synthesized two hormonotoxins (HTs): (i) composed of the ribosome inactivating plant protein gelonin conjugated to luteinizing hormone (LH), (ii) gelonin conjugated to human chlorionic gonadotropin (hCG), both through a disulfide bond. / Characterization of both conjugates was carried out by SDS-PAGE analysis, radioimmunoassay and Western blotting (using polyclonal antibodies against both the hormone and the toxin). In addition, bioactivity of the hormonotoxins was determined by their ability to bind LH receptors in testicular membrane preparations. MA-10 cells showed a significant reduction in protein synthesis following a 24 hour exposure to the cytotoxic conjugates. / In vivo studies were carried out using the LH-gelonin hormonotoxin. Intravenous injections of $ sp{125}$I-HT into 28-30 day-old pseudopregnant mice and determination of radioactivity in the ovaries, kidneys, liver, thyroid, brain, and blood helped to establish preliminary results on the uptake of hormonotoxin by these tissues. Study of the cytotoxic effects of the conjugate in vivo in normal male mice (following a three week period of injection), revealed a significant reduction in testosterone production in animals which received the highest concentrations of hormonotoxin. In addition, treatment of animals with the separate components of the conjugate (same pattern and duration of treatment), established specificity of the effect exerted by the hormonotoxin. Treatment with the LH-gelonin hormonotoxin caused production of antibodies to both components of the conjugate. Results obtained from these studies have helped to establish the groundwork for the effects of HT in vivo in normal mice. Overall, the research on these HTs had provided encouraging information for their potential use as therapeutic agents. (Abstract shortened by UMI.)