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Call number: LD2668 .T4 1966 Q1 / Master of Science

Methoprene is a Juvenile Hormone (JH) analogue commonly used for the control of mosquito larvae. When applied to a mosquito breeding site, methoprene enters the haemolymph, where it mimics the function of JH and interferes with normal metamorphosis, resulting in larval mortality. Methoprene is commonly used for the control of larvae and has not been used as an adulticide, due to an absence of acute effects. This study evaluated possible chronic effects caused by the exposure of adult Aedes mosquitoes to methoprene. Methoprene was applied, in both technical grade and the commercially available Altosid®, topically to adults through droplet application on the abdomen and as a spray application. Mosquitoes were examined for treatment effects on ovary development, adult male and female mortality, and fecundity. The results demonstrate that relatively high doses are required to affect adult survivorship. In contrast, significant impacts on both fecundity and egg hatch were observed for females treated at the lower dosages. I discuss the results in relation to autocidal strategies for mosquito control in which the release of fecund females is to be avoided.

The start of the menopausal transition involves the introduction of various somatic, urogenital, and psychological symptoms; of the symptoms, irritability is one of the main complaints reported by women. The use of bioidentical hormone replacement therapy has become more prevalent in society, specifically treating the somatic and urogenital symptoms of the menopausal transition. This study aims to determine the effects of bioidentical hormone replacement therapy (BHRT) on irritability in menopausal women. To test the hypotheses, an online survey was distributed to women via social media and word-of-mouth. Participants were asked to respond to various questions, which were then analyzed based on BHRT use. An independent samples t-test was used to analyze the data. The results exemplified no significant relationship between BHRT and irritability; using BHRT does not significantly reduce irritability scores.

Stress promotes the use of methamphetamine and other recreational substances and is often implicated in relapse to stimulant use. Thus, it is of critical importance to examine the consequences of the co-occurance of stress and methamphetamine use. Activity of the glutamatergic N-methyl D-aspartate (NMDA) receptor system appears to be involved in the neurotoxic effects of both chronic stress and methamphetamine exposure. The current studies investigated the hypothesis that chronic pre-exposure to the stress hormone corticosterone (CORT) results in an increase of NMDA receptor activity and that this will potentiate the neurotoxic effects of methamphetamine (METH). Co-cultures of the ventral tegmental area, nucleus accumbens, and medial prefrontal cortex were pre-exposed to CORT (1 μM) for 5 days prior to co-exposure to METH (100 μM) for 24 hours to investigate the combined effects on neurotoxicity and protein density of NMDA receptor subunits. The combination of CORT and METH resulted in significant neurotoxicity within the medial prefrontal cortex compared to either CORT or METH alone. The CORT+METH-induced toxicity was attenuated by co-exposure to the NMDA receptor antagonist (2R)-amino-5-phosphonovaleric acid (APV; 50 μM) during the 24 hour CORT and METH co-exposure. Although CORT alone did not significantly alter the density of the NR1 and NR2B subunits of the NMDA receptor, METH exposure for 24 hours resulted in a significant loss of the polyamine sensitive NR2B subunit. Co-exposure to CORT and METH also resulted in decreased extracellular glutamate while not significantly altering extracellular dopamine. These results suggest an enhancement of NMDA receptor systems or downstream effectors in areas of the mesolimbic reward pathway following chronic pre-exposure to CORT, which leads to enhanced neuronal vulnerability to future excitotoxic insults. This may be of critical importance as use of psychostimulants such as METH and other drugs of abuse may produce excitotoxic events in these areas, thus further compromising neuronal viability.

Thesis (M.A.)--University of Hawaii at Manoa, 2005. / Includes bibliographical references (leaves 34-45). / 52 leaves, bound ill. 29 cm

The large conductance Ca++-activated K+ channel (BKCa) regulates neuronal excitability through the efflux of K+, in response to membrane depolarization and increases in intracellular Ca++. The activity of the BKCa channel is increased by acute exposure to ethanol (EtOH), which is thought to underlie, in part, the influence of the drug on peptide hormone release from neurohypophysial nerve terminals (Dopico et al., 1996, 1998). Moreover, chronic EtOH exposure attenuates acute drug action on hormone release, and reduces the sensitivity of BKCa channels to acute EtOH exposure (Knott et al., 2002). The factors regulating EtOH action on BKCa channels are not well understood. Several lines of evidence suggest, however, that the lipid composition of the plasma membrane may influence channel sensitivity to the drug. The plasma membrane is highly complex in its organization (Welti and Glaser, 1994; Brown and London, 1998). There is a growing body of literature indicating that the local lipid composition of the membrane can influence the function of ion channels, including BKCa (Chang et al., 1995a, b; Moczydlowski et al., 1985; Park et al., 2003; Turnheim et al., 1999). Interestingly, chronic exposure to EtOH in animal models results in alterations in the composition of synaptic plasma membranes, including changes in the amount and distribution of membrane cholesterol (CHS) (Chin et al., 1978; Chin et al., 1979; Wood et al., 1989). The significance of these alterations is unclear. Here, we set out to determine the ability of membrane lipids to modulate BKCa channel activity and EtOH sensitivity. To address this, we implement the planar lipid bilayer technique, allowing control of both the protein and lipid components of the membrane. Native BKCa channels retain EtOH sensitivity in this reductionist preparation (Chu et al., 1998), and we extend the study here to examine cloned human brain (hslo) BKCachannels. We show here that hslo channels maintain their characteristic large conductance, voltage and Ca++-dependent gating, and sensitivity to 50 mM EtOH in bilayers cast from a 3:1 mixture of 1-pamiltoyl-2-oleoyl-phosphatidylethanolamine (POPE) and 1-pamiltoyl-2-oleoyl-phosphatidylserine (POPS). The addition of CHS to the bilayer decreases both the basal activity and EtOH sensitivity of the channels, in a concentration-dependent manner. This lends support to the notion that alterations in plasma membrane CHS levels following chronic EtOH exposure may reflect adaptations to the acute actions of the drug on ion channels. Furthermore, the EtOH sensitivity and CHS modulation of these reconstituted hslo channels are greatly reduced in the absence of negatively charged POPS in the bilayer (pure POPE). Based on these findings, we look to gain mechanistic insight into the lipid headgroup and acyl chain properties that may regulate BKCa channel modulation by EtOH and CHS. When POPS is replaced with the uncharged lipid 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), the hslo response to EtOH and CHS is restored, suggesting that the loss of negative surface charge or PS headgroup structure itself cannot explain the lack of channel modulation by these agents in POPE bilayers. Moreover, increases in the proportion of unsaturated acyl chains in the bilayer cannot significantly influence the hslo response to EtOH. The loss of EtOH sensitivity in pure POPE and CHS-containing bilayers may, therefore, reflect the propensity of POPE and CHS to form nonlamellar (nonbilayer) structures. Regarding the basal activity of the channel, we demonstrate that decreases in negative surface charge, increases in the proportion of unsaturated acyl chains, and increases in the complexity of head group interactions can all influence the steady-state activity of reconstituted hslochannels, relative to control POPE/POPS (3:1) bilayers. Overall, these data further suggest the ability of the local lipid environment to regulate the basal function and EtOH sensitivity of an ion channel protein. Parts of this dissertation have appeared in separate publications: Treistman, S.N., O'Connell, R.J., and Crowley, J.J. (2002). Artificial Bilayer Techniques in Ion Channel Study. In Methods in Alcohol-Related Neuroscience Research, D. Lovinger and Y. Liu, eds. (Boca Raton, Florida: CRC Press) Crowley, J.J., Treistman, S.N., and Dopico, A.M. (2003). Cholesterol antagonizes ethanol potentiation of human BKCA channels in binary phospholipid bilayers. Mol. Pharma. 64(2):364-372.

The purpose of this study was to investigate the mechanism of action of lipolysis by growth hormone in rat adipocytes. GH-induced lipolysis, in contrast to that of isoproterenol (ISO), is slow in onset (lag time >1h), small in magnitude (~2X basal). and requires corticosteroid. Evidence for direct coupling between GH receptors and adenylyl cyclase or G-proteins is lacking, and although we could detect no measurable change in cAMP content after treatment with GH + dexamethasone (Dex), it is likely that cAMP activation of protein kinase A is a central event in GH-induced lipolysis. Rp-cAMPS, a competitive antagonist of cAMP was equally effective in decreasing lipolysis in tissues treated with GH/Dex or a comparably lipolytic dose of ISO. Incorporation of 32P from γ-32P-ATP into kemptide, a synthetic oligopeptide substrate for protein kinase A, was increased in homogenates of GH/Dex-treated tissue. This increase was correlated with increased lipolysis. Earlier estimates based upon 32P-ribosylation of Gi catalysed by pertussis toxin (PTx) suggested that the abundance of Gi in adipocyte membranes was decreased 4h after treatment of hypophysectomized rats with GH. We therefore examined the possibility that changes in amount or distribution of G-proteins in adipocyte membranes might account for the lipolytic action of GH. Homogenates of GH/Dex-treated and control adipocytes were subjected to differential centrifugation and the abundance of G-proteins in low speed, l6k x g (16k), pellets and high speed, 100k x g (100k), pellets were determined by quantitative Western analysis with densitometry. A 35% loss of Giα2 from the l6k pellet compared from tissues treated with GH/Dex was associated with a 70% increase of Giα2 in the 100k pellet. No change in Gsα was observed in the l6k pellet but a 35% loss of Gsα was seen in the 100k pellet. The G proteins in the l6k pellet were fractionated on a continuous sucrose gradient followed by quantitation with Western analysis or autoradiography after 32P-NAD ribosylation. Giα2 was consistently shifted from heavier to lighter fractions of the l6k pellet after treatment with GH/Dex. Similar shifts of Gsα were not seen. The distribution of 32P-labelled proteins was comparably altered after incubation of homogenates of control and GH/Dex treated adipocytes with PTx and 32P-NAD. These shifts were blocked by treatment of adipocytes with 100μM colchicine which also blocked the lipolytic action of GH/Dex. We propose that an action of GH/Dex on the cytoskeleton of fat cells may change the cellular distribution of G-proteins in a manner that produces a relative decrease in the tonic inhibitory influence of Gi on adenylyl cyclase.

Prostate cancer increases its incidence with age after men in their fifth decade as the ratio of estrogen to androgen rises. Epidemiological studies indicated that high levels of estrogens are associated with the high-risk ethnic groups for prostate cancer. Therefore, estrogens may be involved in prostatic carcinogenesis. It is widely believed that the actions of estrogens are mediated by estrogen receptors. However, expression of estrogen receptor in normal prostate and lesions of the gland was controversial. With the recent discovery of second estrogen receptor (ER-β), this issue became more complicated and it needs to be readdressed. In addition, the biological involvement of ER-β in human prostate remains to be investigated. In this study, we demonstrated that human normal prostate epithelial cells express ER-β but not ER-α, suggesting that estrogens act directly on these epithelial cells via ER-β. Using RT-PCR analysis, the transcripts of ER-β were detected in our primary human prostatic epithelial cell cultures that were derived from the ultrasound-guided peripheral zone biopsies and the cells express two estrogen-regulated genes such as progesterone receptor (PR) and pS2. Moreover, we had developed an ER-β antibody with fully characterizations and used it for immunohistochemistry. Results indicated that ER-p protein is expressed in the basal compartment of prostatic epithelium of the gland. Our findings lead to a new hypothesis that estrogens directly act on human prostatic epithelial cells to modulate its biological functions. To investigate expression of ERs in prostate cancer, RT-PCR analysis was used. We found that all three human prostate metastatic cancer cell lines, DU145, PC-3 and LNCaP, express ER-β transcripts while ER-α mRNA expression only in PC-3 cells. Expressions of PR and pS2 in these cell lines are various. LNCaP cells express both PR and pS2 mRNAs but DU145 cells with only PR and PC-3 cells with only pS2. Our immunohistochemical results on prostatic lesions revealed down-regulation of ER-β expression in high-grade of dysplasia and carcinoma of peripheral zone of the prostate compared to their low-grade lesions. This down-regulation in high-grade carcinoma was verified in transcriptional level by RT-PCR analysis on micro dissected normal epithelium and lesion samples of the gland. In the metastasis, ER-β was found to be reactivated as we observed ER-β mRNA expression in prostate cancer cell lines. Recent evidence suggests that ER-β may be antiproliferative factor for a protective effect against the mitogenic activity of estrogens in breast and androgens in prostate. Activation of the receptor may exhibit cell growth inhibition. We demonstrated that antiestrogens [ICI-182,780 (ICI) and 4-hydroxytamoxifen], raloxifene and phytoestrogen (resveratrol), but not estrogens (17β-estradiol and diethylstilbestrol), inhibit growth of DU145 cells which express only ER-β while PC-3 cells with both ERs showed growth inhibition in response to estrogen and antiestrogen treatments. In DU145 cells, the ICI-induced cell growth inhibition was prevented by blockade of ER-β expression using antisense oligonucleotide. It indicated that the inhibition is mediated via ER-p associated pathway. Using flow cytometry, we found that ICI-treatment could induce accumulation of cells at GO-G1 phase of cell cycle. Similarly, this GO-G1 cell accumulation was also induced by raloxifene in DU145 cells. For resveratrol, the treatment exhibited dual effects on cell cycle distribution in DU145 cells. In the early treatment, resveratrol induced cell cycle arrests at GO-G1phase. The prolonged treatment leads to S-phase cell cycle arrest. To study the molecular mechanism of this ER-p associated cell growth inhibition, real-time RT-PCR analysis was used to semi-quantitate the transcript levels of tentative ER-β regulated genes such as telomerase reverse transcriptase (TERT), survivin and thymidylate synthase (TS) in the treated cells compared to those in control. Results demonstrated that the treatment of ICI could down-regulate TERT and survivin mRNA expressions with dose-dependent fashion. As the ICI-treatment, resveratrol downregulated expression levels of TERT, survivin and TS in DU145 cells. Down-regulation of TS may be related to the S-phase cell cycle arrest observed in the prolonged treatment of resveratrol. Taken together, our findings support the concept that ER-β participates in cell cycle regulation in normal and malignant prostatic epithelial cells. Presence of ER-β in basal cells of the prostate acini indicates that the direct actions of estrogens may be involved in the normal physiology of the gland. Loss of this receptor in primary prostate cancer and its re-expression in metastasis suggests the roles of ER-β in the cancer progression. Activation of the receptor by antiestrogen and phytoestrogen induced cell growth inhibition in prostate cancer cells. The mechanism may be mediated by reduction of cell survival factors and eventually decrease in cell viability and induction of cell cycle arrests.

Nuclear hormone receptors (NHR) constitute a superfamily of ligand inducible transcriptional activators that enable an organism to regulate development and homeostasis through switching on or off target genes in response to stimuli reflecting changes in environment as well as endocrine. NHRs include classical steroid hormone receptors (GR, AR, ER and MR) and retinoid, thyroid hormone receptors. One long-term goal of our lab is to understand the molecular mechanisms through which the transcriptional activity of NHRs is regulated. Extensive studies in the past few years have revealed that in addition to the dependence on ligand availability, the transcriptional activity of NHRs is also regulated by two types of proteins: co activators and corepressors. In the absence of ligand, many NHRs, including TR and RAR can actively repress target gene transcription with the help of corepressors, proteins that physically interact with both NHRs and histone deacetylases (HDACs). Functional interactions between NHRs and corepressors therefore lead to tightly compact and transcriptionally non-permissive chromatin structures after the removal of obstructive acetyl groups from histone tails by HDACs. On the other hand, ligand binding stabilizes NHRs in a conformation that favors interaction with proteins other than corepressors; many of these proteins are able to potentiate the transcriptional activity of NHRs through various mechanisms, such as histone acetylation, chromatin remodeling and recruitment of basal transcription machinery and are collectively termed coactivators. Two highly related corepressors, SMRT (silencing mediator of retinoid and thyroid hormone receptors) and N-CoR (nuclear receptor corepressor), have been cloned. This research in corepressor SMRT started by a systematic study of its subcellular localization. We found that SMRT predominantly forms a specific nuclear punctuate structure that does not appear to overlap with any other well-known subnuclear domains/speckles. Although our searching for specific sequence signals that may determine the specific speckle localization of SMRT did not yield conclusive results, we discovered the colocalization of unliganded RAR and certain HDACs, including HDAC1, 3,4 and 5, in the SMRT nuclear speckles. Moreover, SMRT is likely to be the organizer of such speckles since it appears to be able to recruit other proteins into these speckles. The presence of HDAC1 in the SMRT speckles suggests a direct association between these two proteins, which has not been detected by previous biochemical analyses. Interestingly, HDAC1 point mutants that are completely defective in deacetylase activity failed to locate to SMRT nuclear speckles, while another partially active mutant maintained the colocalization. These discoveries may indicate SMRT nuclear speckles as novel nuclear domains involved in transcriptional repression. More physiologically relevant support for this hypothesis arises from study of HDAC4 and 5. HDAC4 and 5 are potent inhibitors of transcriptional activator MEF2C. Nuclear presence of HDAC4/5 can block the activation of MEF2C, which is required during muscle differentiation. Normally, HDAC4 is predominantly located in cytoplasm. However, we found that in the presence of SMRT overexpression, HDAC4 was found mostly in SMRT nuclear speckles. This accumulation enhanced HDAC4 mediated inhibition on MEF2C transcriptional activity in a transient transfection assay. SMRT overexpression also resulted in accumulation of HDAC5 in the SMRT nuclear speckles compared to the nuclear diffuse distribution in the absence of SMRT. Again, this accumulation of HDAC5 in nuclear speckles correlated with enhanced inhibition of MEF2C. Taken together, our study suggested that instead of being merely a corepressor for NHRs, SMRT might function as an organizer of a nuclear repression domain, which may be involved in a broad array of cellular processes. In contrast to the limited number of corepressors, numerous co activators have been identified; the SRC (or p160) family is relatively well studied. This family includes three highly related members, SRC-1, TIF2/GRIP1, RAC3/AIB1/ACTR/p/CIP. Similar domain structures are shared among these factors, with the most highly conserved region, the bHLH-PAS domain found within the N terminal ~400 amino acid residues. This study of RAC3 aims to identify the function of the highly conserved N terminal bHLH-PAS domain by isolating interacting proteins through yeast two-hybrid screening. One candidate gene isolated encodes the C terminal fragment of the human homologue of the yeast protein MMS19. Functional studies of this small fragment revealed that it specifically interacted with human estrogen receptors (ERs) and inhibited ligand induced transcriptional activity of ERs in the transient transfection assay. Then we cloned the full-length human MMS19 cDNA and characterized the hMMS19 as a weak coactivator for estrogen receptors in the transient transfection assay. Furthermore, when tested on separate AF-1 or AF-2 of ERs, hMMS19 specifically enhanced AF-1 but had no effect on AF-2. These results identified hMMS19 as a specific coactivator for ER AF-1.