The follicle is the compartment of the ovary responsible for estrogen production. In the pig follicular estrogen production is accomplished by a concerted cooperation between the two cell types of the follicle, the granulosa cells, and the thecal cells, under the control of the pituitary gonadotropins Follicle Stimulating Hormone (FSH) and Luteinizing Hormone (LH). Under the influence of LH stimulation, the thecal cells increase their production of the androgen, androstenedione. FSH stimulation of the enzyme aromatase, increases granulosa cell estradiol production from androstenedione provided by the thecal cells. Granulosa cells from large preovulatory follicles luteinize in vitro when placed in culture. This process is characterized by a decline in estrogen production and by increased progesterone production. To better define the steroid metabolic activity of granulosa cells during this period of in vitro luteinization, studies were performed to assess the metabolism of (A-dione) by granulosa cells in culture. The results of these studies demonstrated that aromatase activity of the granulosa cells declined substantially between 12 and 48 hours of culture resulting in the loss of estrogen production. In addition to this finding, an alternative pathway of A-dione metabolism was discovered. This pathway involves the production of the novel acidic steroid 19-oic-androstenedione (19-oic-A), and two additional novel metabolites, the C{dollar}\sb{lcub}18{rcub}{dollar} neutral steroids 5(10)estrene-3{dollar}\beta,\ 17\beta{dollar}-diol (estrenediol) and 19-nor-testosterone (19-nor-T). Evaluation of the time course of A-dione metabolism suggested that aromatase was responsible for the production of 19-oicA from A-dione. The data also suggested that changes in metabolism associated with luteinization of the granulosa cells resulted in the metabolism of 19-oic-A to estrenediol and 19-nor-T. Further investigation confirmed that aromatase was the enzyme responsible for the production of 19-oic-A from A-dione. The metabolism of 19-oic-A by granulosa cells was assessed, and the results demonstrated that this steroid was indeed the metabolic precursor of the two C{dollar}\sb{lcub}18{rcub}{dollar} neutral steroids, estrenediol and 19-nor-T. Isolation of 19-oic-A, estrenediol, and 19-nor-T in pig ovarian follicular fluid provided evidence that the metabolic pathways observed in culture were reflective of in vivo follicular steroidogenesis. The effects of metabolites of A-dione were tested for their ability to modulate parameters associated with follicular development and maturation. Granulosa cells isolated from prepubertal pigs were cultured with various combinations of FSH and steroids to assess the effects of these hormone combinations on the induction of LH receptors and aromatase activity. 19-nor-T significantly augmented the ability of FSH to induce LH receptors on granulosa cells, while estradiol and estrenediol were without effect. Likewise, the androgens A-dione, testosterone(T), 19-nor-T and all significantly enhanced the FSH stimulation of granulosa cell aromatase activity, while estradiol was without effect.;To determine if the regulation of LH receptor and aromatase induction by androgens, but not estrogens could be explained by the expression of steroid hormone receptors, immunohistochemical studies were performed to localize androgen and estrogen receptors in the pig ovary. Granulosa cells from small preantral and antral follicles expressed androgen receptors, but were devoid of estrogen receptors. As follicles increased in size as a result of follicular development and maturation, the expression of androgen receptors decreased. Weak expression of estrogen receptors was observed only in mature follicles and the developing corpus luteum. Collectively, these results were interpreted to indicate that androgens are potentially important modulators of FSH action during follicular development and maturation.

Proteus mirabilis is the most frequent cause of infection-induced bladder and kidney stones. P. mirabilis urease catalyzes the hydrolysis of urea thereby raising the pH of urine and initiating formation of urinary stones. The enzyme is critical for kidney colonization and the development of acute pyelonephritis. Urease is induced by urea and is not controlled by the nitrogen regulatory system (ntr) or catabolite repression. Genes necessary for urease activity, ureD,A,B,C,E,F, have previously been cloned and sequenced (Jones, B. D. and H. L. T. Mobley. 1989. J. Bacteriol. 171:6414-6422). Purified whole cell RNA from induced and uninduced cultures of P. mirabilis and Escherichia coli harboring cloned urease sequences probed with a 4.2 kb Bg/I fragment from within the urease operon revealed a 4.2-fold and 5.8-fold increase in urease-specific mRNA upon induction with urea. Northern blot analysis of whole-cell RNA from wild-type P. mirabilis HI4320, probed with the same 4.2 kb DNA segment revealed significantly higher amounts of mRNA transcripts of 2.8 and 3.6 kb when induced with urea which correspond to the predicted lengths of mRNA encoding transcripts of ureABCE and ureDABCE, respectively. However, the presence of putative promoters preceding both ureD and ureA makes this interpretation difficult. A 1.2 kb EcoRV-BamHI restriction fragment upstream of the urease gene sequences confers inducibility upon the operon in trans. Nucleotide sequencing of this fragment revealed a single open reading frame 400 bp upstream of ureD consisting of 882 nucleotides, designated ureR, which is transcribed in the opposite direction of the urease structural and accessory genes, and encodes a 293 amino acid polypeptide predicted to be 33,415 Da. Autoradiographs of SDS-polyacrylamide gels of ({dollar}\sp{lcub}35{rcub}{dollar}S) -methionine-labeled polypeptides obtained by in vitro transcription-translation of the PCR fragments carrying only ureR yielded a single band of 32 kDa apparent molecular size; fragments carrying an in-frame deletion within ureR synthesized a truncated product. The predicted UreR amino acid sequence shares amino acid similarity to a number of DNA binding proteins including E. coli regulatory proteins for acid phosphatase synthesis (AppY), porin synthesis (EnvY), and rhamnose utilization (RhaR). Subsequent analysis of this sequence revealed a potential helix-turn-helix motif and an AraC family signature (M. D. Island and H. L. T. Mobley, personal communication). These data suggest that UreR governs inducibility of P. mirabilis urease. To study the specificity of induction of urease, the ability of urea analogs and urease inhibitors to induce the enzyme was studied by constructing a fusion of ureA (urease subunit gene) and lacZ ({dollar}\beta{dollar}-galactosidase gene) within plasmid pMID1010 which encodes an inducible urease of P. mirabilis expressed in E. coli JM103 (Lac). The fusion protein, predicted to be 117 kDa, was induced by urea and detected on Western blots with anti-{dollar}\beta{dollar}-galactosidase antiserum. Induction was specific for urea as no structural analog of urea induced fusion protein activity. These data suggest that induction by UreR, the protein that governs regulation of the urease operon, is exquisitely specific for urea and does not respond to closely related structural analogs.

ATP utilization by P-type cation transport ATPases includes a phosphorylated intermediate which is formed by transfer of the ATP terminal phosphate onto an aspartyl residue at the catalytic site. The phosphorylation site and cation binding site of the ATPase molecule are separated by a fairly long distance of about 50 A. The coupling mechanism of these two functional sites is not yet fully understood and is currently under active investigation. Within the family of cation transport ATPases, the Ca2+-ATPase of sarcoplasmic reticulum (SR) provides an advantageous experimental system due to its abundance in the native membrane, and the availability of cDNA for expression of functional protein.;The sarcoplasmic reticulum Ca2+-ATPase segment extending from the phosphorylation site (Asp351) to the preceding transmembrane helix M4 (which is involved in Ca2+ binding in conjunction with transmembrane helices M5, M6 and M8), shares a marked sequence homology with the corresponding segments of other cation ATPases. We generated twenty six point mutations in this segment and expressed those mutant enzymes in COS-1 cells. We found that non-conservative mutations of residues which are homologous in various cation ATPases result in strong inhibition of catalytic and transport functions. Mutations of non-homologous residues to match the corresponding residues of other cation ATPases are not inhibitory, and in some cases produce higher activity. The inhibitory mutations specifically affect the phosphorylated intermediate turnover, which is associated with the vectorial translocation of bound Ca2+. The same mutations do not affect the kinetics of ATPase activation by Ca2+, which is required for enzyme phosphorylation by ATP. This indicates that activation of the phosphoryl transfer reaction by Ca2+ binding, and vectorial displacement of bound Ca2+ by enzyme phosphorylation, do not occur simply as the forward and reverse directions of the same process, but are linked to distinct structural features of the enzyme. The peptide segment extending from the phosphorylation site in the enzyme extramembranous headpiece, through the M4 helix in the membrane bound region, sustains a prominent role in transmission of the phosphorylation signal for displacement of bound Ca2+. A critical structural role of this segment is also demonstrated by the interference of specific mutations with membrane assembly of the expressed protein.

Streptonigrin is a antineoplastic antibiotic whose mode of action is dependent on formation of the hydroxyl radical. A streptonigrin resistance gene was cloned from a partial Sau3A digest of E. coli DNA. One to 10 Kilobase (Kb) fragments were ligated into the vector pUC18 and transformed into E. coli (DH5{dollar}\alpha{dollar}). Once prepared, the library was replica plated and then screened with differing streptonigrin concentrations in a top agar overlay. Clones exhibiting growth into the overlay at the highest STN concentrations (pH191, pH192) were rescued from the overlay and grown for plasmid isolation and for determination of theminimum inhibitory concentration (MIC) for streptonigrin in LB broth, 3 and 1 {dollar}\mu{dollar}g/ml respectively. The isolated plasmids were mapped by restriction endonuclease digestion followed by agarose gel electrophoresis. Restriction sites were assigned based on known location in the multicloning site of the vector, leading to the production of an approximate physical map of the insert in the plasmid. The resistance element (StnR) was localized by deletion studies on pH191. Sequencing and analysis of the deoxynucleotide sequence of pH191 led to the identification of an open reading frame (StnR) with a high homology (97.6%) to the first 185 residues of riboflavin synthase (RibC). The resistance gene was expressed in S. lividans on pIJ702 where it yielded streptonigrin resistance in excess of that found in the wild type producing species, S. flocculus (40 {dollar}\mu{dollar}g/ml). Expression of StnR and RibC in E. coli via the vector pET3C resulted in strains with elevated streptonigrin resistance as compared to the pET3C control in both solid and liquid media. A gene imparting resistance to the hydroxyl radical producing drug streptonigrin has been identified as the riboflavin synthase from E. coli.

The homeostatic mechanism for the respiratory system involves the tight control of expression of several gene products. One such gene is a member of the multi-substrate oxidase system called cytochrome P450 (P450). Pulmonary P450s may be involved in the tissue-specific detoxification, and/or bio-activation of chemical agents. Additionally, pulmonary cytochrome P450s, particularly the human iso-form 4B1, could play an important role in the development and homeostasis of the pulmonary tissue by removing androgens that cause deleterious effects on lung maturation. Human cytochrome P450 4B1 (CYP 4B1) is a predominant cytochrome P450 activity in the lung. CYP 4B1 protein is reported to bio-activate some pulmonary toxicants. Therefore it may mediate chemical-induced damage to that organ. However, the high expression levels of CYP 4B1 gene products in the lung (approximately 80% of the total P450 activity) may suggest the involvement of the iso-form in normal physiological functions for pulmonary tissue. On the other hand, the results of this and other laboratories have shown an apparent species-specific difference in the catalytic activity of 4B1 protein in man versus rodents. For instance, the rodent iso-form is potent at activating pro-carcinogens and devoid of androgen hydrolyzing activities towards androgen metabolism. The human 4B1 protein has higher 6-beta-testosterone hydroxylase activity while the rodent iso-form is devoid of this action. Furthermore, the manner in which the 4B1 gene is regulated appears to be species-specific. Therefore, my studies have been directed toward further defining the genetic mechanism of action that regulates the human 4B1 gene expression levels. My research objectives have been divided into three specific aims: to determine the presence or absence of cis-acting elements located in the 3.0 Kbp upstream fragment of the human 4B1 gene; to determine the DNA structure of the 4.5 Kbp fragment that overlaps and lies further upstream to the 3.0 Kbp regulatory region of the human 4B1 gene; and to determine the actions of a contiguous 6.5 Kbp fragment containing the entire putative upstream region of 4B1 gene on transcription of the luciferase reporter gene in human lung cells. The results of my studies show the presence of multiple promoter elements in addition to the TATA-box that are contained in the CYP 4B 13.0 Kbp upstream fragment. Nucleotide sequence analysis of the 4.5 Kbp upstream fragment that contains 1.0 Kbp overlapping fragment with the 3.0 Kbp fragment based on restriction mapping confirms that it is indeed part of the contiguous region of the CYP 4B1 gene. A number of consensus DNA regulatory motifs have been identified. (Abstract shortened by UMI.)

The large subunit of ribonucleotide reductase (RR1) encoded by Herpes Simplex Virus Type 1 and 2 (ICP6 and ICP10, respectively) is a chimeric protein consisting of a Ser/Threonine protein kinase (PK) domain at the amino terminus and a ribonucleotide reductase (RR) domain at the carboxy terminus. The findings that the PK domain is present only in HSV RR1 proteins, it is dispensable for ribonucleotide reduction and it functions as immediate-early (IE) protein during HSV infection, suggest that the PK activity plays a significant role in virus pathogenesis. The present work was initiated to confirm the intrinsic nature of ICP10 PK activity and to elucidate its role in HSV-2 infection. In stably transfected eukaryotic cells, ICP10 PK activity was eliminated by deletion of the conserved PK catalytic motifs or of the transmembrane (TM) segment and it was significantly impaired by mutation of the invariant Lys (Lys{dollar}\sp{lcub}176{rcub}{dollar}). Loss of PK activity by Lys{dollar}\sp{lcub}176{rcub}{dollar} mutation resulted in the failure to bind ATP. A truncated ICP10 PK expressed in bacteria (pp29{dollar}\rm\sp{lcub}la1{rcub}{dollar}) retained auto- and transphosphorylating activity (for calmodulin) after purification to apparent homogeneity. In cells infected with laboratory and patient isolates of HSV, RR1 had auto- and transphosphorylating activity for the small subunit of HSV ribonucleotide reductase (RR2) and Immunoglobulin G (IgG). Two HSV-2 (G) mutants deleted in the protein kinase or ribonucleotide reductase domains of ICP10 (ICP10{dollar}\Delta{dollar}PK and ICP10{dollar}\Delta{dollar}RR, respectively) were constructed by marker transfer. ICP10{dollar}\Delta{dollar}PK virus lost its intrinsic PK activity but retained its RR activity; ICP10{dollar}\Delta{dollar}RR virus retained its PK activity but lost its RR activity. ICP10{dollar}\Delta{dollar}PK virus does not replicate during the first 10 hrs postinfection (p.i.). However, its titers catch up with those of the wild type virus by 24 hrs p.i.; ICP10{dollar}\Delta{dollar}RR virus replicates as well as the wild type virus in exponentially growing cells but it is significantly impaired for growth in growth-restricted cells. HSV-2 and ICP10{dollar}\Delta{dollar}RR virus produce similar clear plaques but ICP10{dollar}\Delta{dollar}PK virus produces hazy plaques, which under magnification consist of a mixture of lysed and unlysed cells. The studies suggest that (i) ICP10 has intrinsic auto- and transphosphorylating PK activity, (ii) ICP10 PK and RR are functionally dissociable in virus infected cells, (iii) ICP10 PK is required for virus replication during the first 10 hrs p.i. and (iv) ICP10 PK may be involved in cell death in virus infected cells.

Chromosomal DNA replication in animal cells is a complex process. A complete understanding of this process will require information on the organization and the control of the DNA synthetic machinery. To date several mammalian enzymes and protein factors have been shown to be required for DNA replication in vitro. A variety of mechanisms describing how these proteins might function at the mammalian cell replication fork have also been proposed. However, the manner in which these proteins, and those as yet to be identified, associate with one another, as well as their ability to act in concert allowing for the efficient replication of DNA have not been well defined. Over the years evidence for these proteins organizing into a large macromolecule complex to mediate DNA replication has emerged. In order to pursue the hypothesis of multiprotein complex playing a role in mammalian DNA replication, this study was directed towards identifying and characterizing such DNA replication multiprotein complex from murine cells. By using a series of differential centrifugation, polyethylene glycol precipitation, and ion-exchange chromatography steps, a multiprotein DNA replication complex was isolated and purified from mouse mammary carcinoma cell line (FM3A). A number of key enzymes and protein factors for DNA replication were found to associate with this multiprotein complex including RPA, DNA topoisomerase I and II, DNA polymerase alpha, DNA primase, DNA polymerase delta, PCNA, RF-C, DNA ligase I and DNA helicase. The complex has a 17S sedimentation coefficient as determined by sucrose density gradient analysis. The integrity of the complex is maintained after treatment with detergents, salt, RNase and DNase, suggesting that the association of the proteins with one another is independent of nonspecific interaction with other cellular macromolecule components. Most importantly, it was demonstrated that this complex of proteins is fully competent to replicate polyomavirus (PyV) DNA in vitro. Based on the fractionation and chromatographic profiles of the individual proteins found to co-purify with the complex, a model is proposed to represent the mammalian Multiprotein DNA Replication Complex (MRC). It was also shown that the in vitro PyV DNA replication reaction catalyzed by the Q-Sepharose column purified mouse cell MRC is dependent on polyoma virus DNA replication origin, Py large T-antigen, Mg++, the energy regenerating system, and an exogenously added PyV origin containing DNA molecule. Kinetics analysis of the initiation of in vitro PyV DNA replication mediated by the purified MRC demonstrated that the lag time for initiation of the DNA replication was reduced significantly compared with what has been reported using human cell crude extracts in the SV40 DNA replication system. A study of the cell cycle-dependent regulation of the MRC in terms of its activity and integrity showed that a fully assembled MRC is maintained in both quiescent and proliferating stages of cells. However, the DNA replication-active form of the MRC can only be found in the S-phase cells.

Neuronal nicotinic acetylcholine receptors (nAChRs) in the brain, especially in the hippocampus, are thought to play a crucial role in the physiology of learning and memory. Characterization of the neuronal nAChRs by molecular, immunological and physiological techniques has revealed that their molecular structures, physiology and pharmacology are highly diverse and different from those of the muscle nAChR. However, little is known at this time about noncompetitive antagonism of these nAChRs. The purpose of this study was to characterize the interaction of bupivacaine, an open channel blocker of muscle nAChR, with the neuronal nAChRs in cultured hippocampal neurons. Using whole-cell and single-channel modes of the patch-clamp technique, the interactions of bupivacaine with the neuronal nAChRs were studied under different experimental conditions. Whole-cell currents with two different types of decay kinetics could be induced by ACh (300 muM). Currents that were fast-decaying were highly sensitive to blockade by the competitive nicotinic antagonist methyllycaconitine (MLA) at 10 nM. Currents having a slow decay were only partially blocked by MLA. Both the fast and slowly decaying ACh-induced whole-cell currents were blocked by bupivacaine, but with different sensitivity. Open-channel blockade by the drug was evidenced by a substantial decrease in the decay time constants of slowly decaying whole-cell currents and was confirmed by a shortening of the lifetime of ACh-activated single channels. The weak voltage dependence of the reduction of the decay time constant of the slowly decaying current suggests that the bupivacaine-binding site(s) responsible for open-channel blockade is(are) probably located near the orifice of the receptor ion channel. The non-linear relationship of this blockade with increasing concentration of bupivacaine indicated two distinct blocking rates, and suggested a closed-channel interaction of the drug at higher concentration, which was supported by the bupivacaine-induced decrease in the probability of a channel being opened. The blockade of the slowly decaying current by bupivacaine at high concentration was use dependent. Bupivacaine decreased the peak amplitude but did not change the decay time constant of the fast decaying ACh-induced currents, suggesting that a closed-channel blockade, but not open-channel blockade was taking place. This closed-channel blockade was time dependent and was noncompetitive. The neuronal nAChRs were more sensitive than NMDA, kainate, quisqualate, GABA, or glycine receptors to the action of bupivacaine.

The role of the putative sigma receptor in mediating neuroprotection against glutamate induced neuronal injury was examined in mature cultured rat cortical neurons. With the exception of the sigma1, selective ligand (+)-3-PPP, all of the sigma receptor ligands tested were neuroprotective, preventing glutamate induced morphological changes and increases in LDH release. When corrected for relative sigma versus PCP binding site affinity, it appears that a positive correlation exists between neuroprotective potency and sigma1, site affinity. None of the sigma ligands were neurotoxic when tested alone at concentrations at least 5-30 times their respective neuroprotective EC{dollar}\sb{lcub}50{rcub}{dollar} values. The effect of neuroprotective sigma ligands on the unique calcium responses evoked by glutamate, NMDA, potassium chloride (KCl) and trans-ACPD were investigated to elucidate the mechanism of sigma-mediated neuroprotection. In general, except for (+)-3-PPP all of the sigma ligands studied interfered with glutamate and NMDA induced (Ca{dollar}\sp{lcub}2+{rcub}\rbrack\sb{lcub}\rm i{rcub}{dollar} signaling, but, highly sigma{dollar}\sb1{dollar} selective ligands also lacking substantial PCP binding site affinity (i.e. carbetapentane, DTG and haloperidol) were much less effective in altering calcium influx induced by 80 {dollar}\mu{dollar}M glutamate. In contrast to glutamate, KCl (50 mM) produced changes in (Ca{dollar}\sp{lcub}2+{rcub}\rbrack\sb{lcub}\rm i{rcub}{dollar} which were not neurotoxic to the neurons as measured by LDH release. Sigma ligands which lack substantial PCP site afflinity were very effective in altering KCl induced calcium signaling while the sigma/PCP site ligand (+)-cyclazocine was ineffective or, in the case of (+)-SKF 10047, much less effective. Similar to the effects of sigma ligands on KCl induced calcium dynamics, the sigma selective ligands DTG, haloperidol, (+)-pentazocine, and carbetapentane were very effective in altering intracellular calcium dynamics evoked by trans-ACPD while the sigma/PCP ligand (+)-SKF 10047 was ineffective or, in the case of (+)-cyclazocine, much less effective. Importantly, (+)-3-PPP, a non-neuroprotective sigma selective ligand, actually potentiated the calcium response elicited by trans-ACPD. The ability of sigma ligands applied at maximal neuroprotective concentrations to attenuate receptor and/or voltage-gated changes in calcium dynamics suggests that modulation of neurotoxic (Ca{dollar}\sp{lcub}2+{rcub}\rbrack\sb{lcub}\rm i{rcub}{dollar} plays a significant role in sigma-mediated neuroprotection. The unique modulatory effects of sigma ligands on the buffering of neuronal (Ca{dollar}\sp{lcub}2+{rcub}\rbrack\sb{lcub}\rm i{rcub}{dollar} will likely have numerous therapeutic applications in the treatment of CNS injury and other neurodegenerative disorders.

Our laboratory have designed and synthesized a number of cytochrome P450 17alpha-hydroxylase/C17,20-lyase (P450c17) inhibitors with the aim of inhibiting androgen synthesis and as potential agents to treat prostate cancer. VN/63-1, VN/85-1, VN/87-1, VN/107-1, VN/108-1, and VN/109-1 inhibitor were chosen for study. To select the most potent inhibitors, a new method for evaluating inhibitors of the P450c17 enzyme was designed. Bacteria expressing the P450c17 were used as a source of the enzyme. HPLC was replaced by a rapid radiometric assay, acetic acid releasing assay. This new analytical system will be a powerful tool to accelerate the process of new compound selection. Utilizing this system, I showed that VN/85-1, VN/87-1, and VN/108-1 are the most potent inhibitors of P450c17. The effects of the new inhibitors on androgen dependent LNCaP and androgen independent PC-3 prostatic carcinoma cells proliferation were studied. I have shown that VN/85-1, VN/87-1, and VN/108-1 are the most potent inhibitors of LNCaP cell growth in vitro . VN/85-1 also has weak inhibitory effects against androgen independent PC-3 cells. The effects of azolyl steroids on LNCaP androgen receptor (AR) were determined. VN/63-1, VN/85-1, and VN/87-1 were full antagonists of LNCaP AR. Although, VN/107-1 , VN/108-1, and VN/109-1 were partial agonists of LNCaP AR, all of the evaluated inhibitors were antiandrogens against the wild-type AR. The new inhibitors were also evaluated in vivo. The male SCID mice bearing LNCaP tumor xenografts were utilized for new compounds evaluation. I showed that VN/85-1 and VN/87-1 have potent antitumor effects in this in vivo model. The ability of pregnenolone to stimulate LNCaP cell proliferation was discovered and investigated. I showed that this pregnenolone stimulation is mediated through the mutated AR and can be inhibited by our novel compounds, especially by VN/63-1, VN/85-1, and VN/87-1. This findings suggests that pregnenolone has a significant role in stimulating prostate cancer and that our new compounds by inhibiting not only androgens but also pregnenolone action may be more effective for treating prostate cancer than currently available agents.