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Prolonging the clinical effectiveness of β-lactams, which remain first-line antibiotics for many infections, is an important part of efforts to address antimicrobial resistance. We report here that inactivation of the predicted d-cycloserine (DCS) transporter gene cycA resensitized methicillin-resistant Staphylococcus aureus (MRSA) to β-lactam antibiotics. The cycA mutation also resulted in hypersusceptibility to DCS, an alanine analogue antibiotic that inhibits alanine racemase and d-alanine ligase required for d-alanine incorporation into cell wall peptidoglycan. Alanine transport was impaired in the cycA mutant, and this correlated with increased susceptibility to oxacillin and DCS. The cycA mutation or exposure to DCS were both associated with the accumulation of muropeptides with tripeptide stems lacking the terminal d-ala-d-ala and reduced peptidoglycan cross-linking, prompting us to investigate synergism between β-lactams and DCS. DCS resensitized MRSA to β-lactams in vitro and significantly enhanced MRSA eradication by oxacillin in a mouse bacteremia model. These findings reveal alanine transport as a new therapeutic target to enhance the susceptibility of MRSA to β-lactam antibiotics., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Metabolic feedback from the periphery to the brain results from a dynamic physiologic fluctuation of nutrients and hormones, including glucose and fatty acids, ghrelin, leptin, and insulin. The specific interactions between humoral factors and how they influence feeding is largely unknown. We hypothesized that acute glucose availability may alter how the brain responds to ghrelin, a hormonal signal of energy availability. Acute glucose administration suppressed a range of ghrelin-induced behaviors as well as gene expression changes in hypothalamic neuropeptide Y (NPY) and agouti-related peptide (AgRP) neurons after ghrelin administration. Knockdown of the energy-sensing molecule AMP-activated protein kinase (AMPK) in AgRP neurons resulted in loss of the glucose effect, and mice responded as though pretreated with saline. Conversely, 2-deoxyglucose (2-DG), which decreases glucose availability, potentiated ghrelin-induced feeding and increased hypothalamic NPY mRNA levels. AMPK knockdown did not alter the additive effect of 2-DG and ghrelin on feeding. Our findings support the idea that computation of energy status is dynamic, is informed by multiple signals, and responds to acute fluctuations in metabolic state. These observations are broadly relevant to the investigation of neuroendocrine control of feeding and highlight the underappreciated complexity of control within these systems.

Context: Estrogens affect the incidence and progression of colorectal cancer (CRC), although the precise molecular mechanisms remain ill-defined., Objective: The present study investigated prereceptor estrogen metabolism through steroid sulphatase (STS) and 17β-hydroxysteroid dehydrogenase activity and subsequent nongenomic estrogen signaling in human CRC tissue, in The Cancer Genome Atlas colon adenocarcinoma data set, and in in vitro and in vivo CRC models. We aimed to define and therapeutically target pathways through which estrogens alter CRC proliferation and progression., Design, Setting, Patients, and Interventions: Human CRC samples with normal tissue-matched controls were collected from postmenopausal female and age-matched male patients. Estrogen metabolism enzymes and nongenomic downstream signaling pathways were determined. CRC cell lines were transfected with STS and cultured for in vitro and in vivo analysis. Estrogen metabolism was determined using an ultra-performance liquid chromatography-tandem mass spectrometry method., Primary Outcome Measure: The proliferative effects of estrogen metabolism were evaluated using 5-bromo-2'-deoxyuridine assays and CRC mouse xenograft studies., Results: Human CRC exhibits dysregulated estrogen metabolism, favoring estradiol synthesis. The activity of STS, the fundamental enzyme that activates conjugated estrogens, is significantly (P < 0.001) elevated in human CRC compared with matched controls. STS overexpression accelerates CRC proliferation in in vitro and in vivo models, with STS inhibition an effective treatment. We defined a G-protein-coupled estrogen receptor (GPER) proproliferative pathway potentially through increased expression of connective tissue growth factor in CRC., Conclusion: Human CRC favors estradiol synthesis to augment proliferation via GPER stimulation. Further research is required regarding whether estrogen replacement therapy should be used with caution in patients at high risk of developing CRC., (Copyright © 2017 Endocrine Society)

ERG, an ETS family transcription factor frequently overexpressed in human leukemia, has been implicated as a key regulator of hematopoietic stem cells. However, how ERG controls normal hematopoiesis, particularly at the stem and progenitor cell level, and how it contributes to leukemogenesis remain incompletely understood. Using homologous recombination, we generated an Erg knockdown allele (Erg kd ) in which Erg expression can be conditionally restored by Cre recombinase. Erg kd/kd animals die at E10.5-E11.5 due to defects in endothelial and hematopoietic cells, but can be completely rescued by Tie2-Cre-mediated restoration of Erg in these cells. In Erg kd/+ mice, ∼40% reduction in Erg dosage perturbs both fetal liver and bone marrow hematopoiesis by reducing the numbers of Lin - Sca-1 + c-Kit + (LSK) hematopoietic stem and progenitor cells (HSPCs) and megakaryocytic progenitors. By genetic mosaic analysis, we find that Erg-restored HSPCs outcompete Erg kd/+ HSPCs for contribution to adult hematopoiesis in vivo. This defect is in part due to increased apoptosis of HSPCs with reduced Erg dosage, a phenotype that becomes more drastic during 5-FU-induced stress hematopoiesis. Expression analysis reveals that reduced Erg expression leads to changes in expression of a subset of ERG target genes involved in regulating survival of HSPCs, including increased expression of a pro-apoptotic regulator Bcl2l11 (Bim) and reduced expression of Jun. Collectively, our data demonstrate that ERG controls survival of HSPCs, a property that may be used by leukemic cells. Stem Cells 2017;35:1773-1785., (© 2017 AlphaMed Press.)

Background: Deprivation of tumor bioenergetics by inhibition of multiple energy pathways has been suggested as an effective therapeutic approach for various human tumors. However, this idea has not been evaluated in glioblastoma (GBM). We hypothesized that dual inhibition of glycolysis and oxidative phosphorylation could effectively suppress GBM tumorspheres (TS)., Methods: Effects of 2-deoxyglucose (2DG) and metformin, alone and in combination, on GBM-TS were evaluated. Viability, cellular energy metabolism status, stemness, invasive properties, and GBM-TS transcriptomes were examined. In vivo efficacy was tested in a mouse orthotopic xenograft model., Results: GBM-TS viability was decreased by the combination of 2DG and metformin. ATP assay and PET showed that cellular energy metabolism was also decreased by this combination. Sphere formation, expression of stemness-related proteins, and invasive capacity of GBM-TS were also significantly suppressed by combined treatment with 2DG and metformin. A transcriptome analysis showed that the expression levels of stemness- and epithelial mesenchymal transition-related genes were also significantly downregulated by combination of 2DG and metformin. Combination treatment also prolonged survival of tumor-bearing mice and decreased invasiveness of GBM-TS., Conclusion: The combination of 2DG and metformin effectively decreased the stemness and invasive properties of GBM-TS and showed a potential survival benefit in a mouse orthotopic xenograft model. Our findings suggest that targeting TS-forming cells by this dual inhibition of cellular bioenergetics warrants expedited clinical evaluation for the treatment of GBM., (© The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com)

Leucocytes respond rapidly to pathogenic and other insults, with responses ranging from cytokine production to migration and phagocytosis. These are bioenergetically expensive, and increased glycolytic flux provides adenosine triphosphate (ATP) rapidly to support these essential functions. However, much of this work is from animal studies. To understand more clearly the relative role of glycolysis and oxidative phosphorylation in human leucocytes, especially their utility in a translational research setting, we undertook a study of human peripheral blood mononuclear cells (MNCs) bioenergetics. Glycolysis was essential during lipopolysaccharide (LPS)-mediated interleukin (IL)-1β, IL-6 and tumour necrosis factor (TNF)-α production, as 2-deoxy-D-glucose decreased significantly the output of all three cytokines. After optimizing cell numbers and the concentrations of all activators and inhibitors, oxidative phosphorylation and glycolysis profiles of fresh and cryopreserved/resuscitated MNCs were determined to explore the utility of MNCs for determining the bioenergetics health profile in multiple clinical settings. While the LPS-induced cytokine response did not differ significantly between fresh and resuscitated cells from the same donors, cryopreservation/resuscitation significantly affected mainly some measures of oxidative phosphorylation, but also glycolysis. Bioenergetics analysis of human MNCs provides a quick, effective means to measure the bioenergetics health index of many individuals, but cryopreserved cells are not suitable for such an analysis. The translational utility of this approach was tested by comparing MNCs of pregnant and non-pregnant women to reveal increased bioenergetics health index with pregnancy but significantly reduced basal glycolysis and glycolytic capacity. More detailed analysis of discrete leucocyte populations would be required to understand the relative roles of glycolysis and oxidative phosphorylation during inflammation and other immune responses., (© 2015 British Society for Immunology.)

Background: Systemic sclerosis (SSc) is a complex autoimmune disease that involves dysregulation of immune homeostasis. The failure of impaired regulatory T cells (Tregs) to maintain immune homeostasis plays a major role in the development of SSc. Transcriptional silencing of the forkhead box protein 3 gene (FOXP3) via hypermethylation of regulatory regions has been identified as a hallmark of committed Tregs and several autoimmune disorders., Objectives: To investigate whether aberrant expression and methylation of FOXP3 occurs in CD4+ T cells of patients with SSc and their roles in the pathogenesis of SSc., Methods: FOXP3 expression in CD4+ T cells was measured by real-time quantitative reverse-itranscriptase polymerase chain reaction and western blot. Bisulfite sequencing was performed to determine the methylation status of the FOXP3 proximal promoter sequence. The percentage of Treg cells was estimated by flow cytometry., Results: Decreased FOXP3 expression was observed in CD4+ T cells from patients with SSc. The methylation levels of the FOXP3 regulatory sequences were elevated and inversely correlated with FOXP3 mRNA expression in patients with SSc. The number of Tregs was significantly reduced in patients with SSc. Treatment of SSc CD4+ T cells with a DNA methylation inhibitor, 5-azacytidine, reduced the mean methylation levels, and enhanced FOXP3 expression and Treg generation. The promoter methylation status and expression level of FOXP3 are significantly associated with disease activity., Conclusions: The contribution of the hypermethylation of the FOXP3 promoter to decreased FOXP3 expression and the subsequent quantitative defects of Tregs may mediate the immune dysfunction in SSc., (© 2014 British Association of Dermatologists.)

Background: Antitumour necrosis factor (anti-TNF)-α therapy has made a significant impact on the treatment of psoriasis. Despite these agents being designed to neutralize TNF-α activity, their mechanism of action in the resolution of psoriasis remains unclear., Objectives: To understand better the mechanism of action of etanercept by examining very early changes in the lesional skin of patients with psoriasis responding to etanercept., Methods: Twenty patients with chronic plaque psoriasis were enrolled and received etanercept 50 mg twice weekly. Skin biopsies were obtained before treatment and on days 1, 3, 7 and 14 post-treatment. Skin mRNA expression was analysed by quantitative reverse-transcription polymerase chain reaction and microarray; cytokine and phosphoprotein levels were assessed using multiplexed bead arrays., Results: In etanercept responders, we observed no significant changes in interleukin (IL)-17A, IL-22 or interferon-γ mRNA or protein in the first week of treatment; however, there was a 2·5-fold downregulation of IL-17 receptor C (IL-17RC) mRNA (P < 0·05) after day 1, accompanied by decreased extracellular signal-regulated kinase-1/2 phosphorylation. Transcriptional analysis revealed that genes suppressed by etanercept significantly overlapped with IL-17A-induced genes, and a marked overlap was also observed between the genes suppressed by etanercept and by the anti-IL-17A agent ixekizumab. Finally we show that TNF-α enhances the expression of IL-17RC, and short hairpin RNA inhibition of IL-17R expression abrogates synergistic gene induction by TNF and IL-17A., Conclusions: These results suggest that the early responses of psoriasis plaques to etanercept may be due to decreased tissue responsiveness to IL-17A due to suppressed IL-17RC expression in keratinocytes, blunting the strong synergy between TNF-α and IL-17, which contributes to the maintenance of psoriasis lesions., (© 2014 British Association of Dermatologists.)

Megakaryocytes associate with the bone marrow vasculature where they convert their cytoplasm into proplatelets that protrude through the vascular endothelium into the lumen and release platelets. The extracellular matrix (ECM) microenvironment plays a critical role in regulating these processes. In this work we demonstrate that, among bone marrow ECM components, fibronectin, type IV collagen, and laminin are the most abundant around bone marrow sinusoids and constitute a pericellular matrix surrounding megakaryocytes. Most importantly, we report, for the first time, that megakaryocytes express components of the basement membrane and that these molecules contribute to the regulation of megakaryocyte development and bone marrow ECM homeostasis both in vitro and in vivo. In vitro, fibronectin induced a threefold increase in the proliferation rate of mouse hematopoietic stem cells leading to higher megakaryocyte output with respect to cells treated only with thrombopoietin or other matrices. However, megakaryocyte ploidy level in fibronectin-treated cultures was significantly reduced. Stimulation with type IV collagen resulted in a 1.4-fold increase in megakaryocyte output, while all tested matrices supported proplatelet formation to a similar extent in megakaryocytes derived from fetal liver progenitor cells. In vivo, megakaryocyte expression of fibronectin and basement membrane components was upregulated during bone marrow reconstitution upon 5-fluorouracil induced myelosuppression, while only type IV collagen resulted upregulated upon induced thrombocytopenia. In conclusion, this work demonstrates that ECM components impact megakaryocyte behavior differently during their differentiation and highlights a new role for megakaryocyte as ECM-producing cells for the establishment of cell niches during bone marrow regeneration., (© AlphaMed Press.)

Gene-environment interactions influence an individual's risk of disease development. A common human 8-oxoguanine DNA glycosylase 1 (OGG1) variant, Cys326-hOGG1, has been associated with increased cancer risk. Evidence suggests that this is due to reduced repair ability, particularly under oxidising conditions but the underlying mechanism is poorly understood. Oxidising conditions may arise due to internal cellular processes, such as inflammation or external chemical or radiation exposure. To investigate wild-type and variant OGG1 regulation and activity under oxidising conditions, we generated mOgg1 (-/-) null mouse embryonic fibroblasts cells stably expressing Ser326- and Cys326-hOGG1 and measured activity, gene expression, protein expression and localisation following treatment with the glutathione-depleting compound L-buthionine-S-sulfoximine (BSO). Assessment of OGG1 activity using a 7,8-dihydro-8-oxodeoxyguanine (8-oxo dG) containing molecular beacon demonstrated that the activity of both Ser326- and Cys326-hOGG1 was increased following oxidative treatment but with different kinetics. Peak activity of Ser326-hOGG1 occurred 12 h prior to that of Cys326-hOGG1. In both variants, the increased activity was not associated with any gene expression or protein increase or change in protein localisation. These findings suggest that up-regulation of OGG1 activity in response to BSO-induced oxidative stress is via post-transcriptional regulation and provide further evidence for impaired Cys326-hOGG1 repair ability under conditions of oxidative stress. This may have important implications for increased mutation frequency resulting from increased oxidative stress in individuals homozygous for the Cys326 hOGG1 allele.

The use of natural compounds as inhibitory agents for virulence factor production is a new approach to overcome increased antimicrobial resistance in pathogenic bacteria. In this study, we examined whether red chilli (Capsicum annuum) contains any such compound(s) that can repress the cholera toxin (CT) production in Vibrio cholerae. We found that the methanol extract of red chilli could inhibit CT production in recently emerged V. cholerae O1 El Tor variant strains without affecting their viability. Interestingly, capsaicin, a well-studied active component of red chilli, also drastically inhibited CT production in V. cholerae strains belonging to various serogroups including variants. Real-time quantitative reverse transcription-PCR assay revealed that capsaicin effectively repressed the transcription of ctxA, tcpA and toxT genes, but not of toxR and toxS genes. On the contrary, capsaicin significantly enhanced the transcription of the hns gene, the product of which is known to regulate negatively the transcription of ctxAB, tcpA and toxT genes. These results suggest that capsaicin might act as a potent repressor for CT production possibly by enhancing the transcription of hns.

New neurons in the adult brain transiently express molecules related to neuronal development, such as the polysialylated form of neural cell adhesion molecule, or doublecortin (DCX). These molecules are also expressed by a cell population in the rat paleocortex layer II, whose origin, phenotype, and function are not clearly understood. We have classified most of these cells as a new cell type termed tangled cell. Some cells with the morphology of semilunar-pyramidal transitional neurons were also found among this population, as well as some scarce cells resembling semilunar, pyramidal. and fusiform neurons. We have found that none of these cells in layer II express markers of glial cells, mature, inhibitory, or principal neurons. They appear to be in a prolonged immature state, confirmed by the coexpression of DCX, TOAD/Ulip/CRMP-4, A3 subunit of the cyclic nucleotide-gated channel, or phosphorylated cyclic adenosine monophosphate response element-binding protein. Moreover, most of them lack synaptic contacts, are covered by astroglial lamellae, and fail to express cellular activity markers, such as c-Fos or Arc, and N-methyl-d-aspartate or glucocorticoid receptors. We have found that none of these cells appear to be generated during adulthood or early youth and that most of them have been generated during embryonic development, mainly in E15.5.

Several adverse health effects, including irritant and allergic contact dermatitis, have been reported among workers who are occupationally exposed to chromium-containing compounds. Human dermal fibroblasts were used as an in vitro experimental model to study the potential mechanisms underlying hexavalent chromium [Cr(VI)]-induced dermal toxicity. Exposure of the fibroblasts to 5 microM Cr(VI) (LC50 for a 24-h exposure period) followed by microarray analysis of the gene expression profile revealed overexpression of several genes including those involved in cell stress response. The cellular level of glutathione, the major antioxidant molecule present in the cells, was significantly lower in the Cr(VI)-treated cells compared to the corresponding control cells. The Cr(VI)-induced overexpression of heme-oxygenase 1 messenger RNA (HO-1) in the fibroblasts was significantly blocked by actinomycin D and by inhibitors of MAP kinase pathways. The Cr(VI)-induced cytotoxicity and the overexpression of the HO-1 gene were dependent on the glutathione level of the fibroblasts. Buthionine sulfoximine-mediated GSH depletion resulted in enhanced Cr(VI) cytotoxicity and further overexpression of the HO-1 gene. On the other hand, elevated cellular levels of glutathione resulting from pretreating the cells with GSH significantly protected the cells against the Cr(VI)-induced cytotoxicity and blocked the HO-1 gene's overexpression. Pretreating the fibroblasts with N-acetyl cysteine also significantly reduced the Cr(VI)-induced cytotoxicity and overexpression of the HO-1 gene. In conclusion, depletion of GSH leading to cellular stress is a major mechanism responsible for Cr(VI)-induced cytotoxicity. Furthermore, the expression level of HO-1 gene is a marker for Cr(VI)-induced cell stress leading to cytotoxicity.

Changes in the oxidant/antioxidant environment of aging Leydig cells have been shown to be correlated with the reduced ability of these cells to produce testosterone. With this in mind, we hypothesized that the experimental depletion of glutathione (GSH), an abundant Leydig cell intracellular antioxidant, might result in reduced testosterone production. Incubation of Leydig cells isolated from the testes of adult Brown Norway rats with buthionine sulfoximine (BSO) reduced GSH content by more than 70% and testosterone production by about 40%. The antioxidants vitamin E, N-tert-butyl-alpha-phenylnitrone and Trolox countered BSO's effect on steroidogenesis but not on GSH depletion. Together, BSO and glutathione ethyl ester maintained intracellular GSH and also testosterone production, whereas 1,2-dithiole-3-thione, which increases intracellular GSH, increased testosterone production. In vivo studies also were conducted. Young (4 month old) and old (24 month old) rats were injected with BSO twice a day for 7 d, after which Leydig cells were isolated and analyzed in vitro. BSO treatment reduced Leydig cell GSH content by 70% and the ability of the Leydig cells to produce testosterone by more than 50%. As with aging, decreases were seen in LH-stimulated cAMP production, steroidogenic acute regulatory protein, cholesterol side-chain cleavage, 3beta-hydroxysteroid dehydrogenase, and 17alpha-hydroxylase/17,20-lyase. The results of these studies, taken together, are consistent with the hypothesis that alteration in the oxidant/antioxidant environment may play a significant, causative role in the age-related reduced ability of Leydig cells to produce testosterone.

Saccharomyces cerevisiae cells (strain W303-1A) treated with 5-fluorouracil and grown in 2% (fermentative conditions) or in 0.1% glucose (oxidative conditions) accumulated two types of 5-fluoro-UDP-sugars (FUDP-sugars): FUDP-N-acetylglucosamine and FUDP-glucose. No difference was observed in both conditions of culture. The viability of yeast cells on treatment with 5-fluorouracil was also followed. Both FUDP-sugars were partially purified by column chromatography (on Hypersil ODS and Mono Q columns) and characterized by: (i) treatment with alkaline phosphatase (EC 3.1.3.1), snake venom phosphodiesterase (EC 3.1.4.1) and UDP-glucose dehydrogenase (EC 1.1.1.22); (ii) UV spectra; and (iii) matrix-assisted laser desorption/ionization-time of flight mass analysis and 1H-nuclear magnetic resonance spectrometry. The syntheses of both FUDP-sugars were inversely related to the concentration of uracil and directly related to the concentration of 5-fluorouracil in the culture medium. The strain W303-1A, requiring uracil for growth, was useful as a tool to analyze the effect of 5-fluorouracil on nucleotide metabolism.

To study the possible influence of cell-cycle delay on cells reaching mitosis during conventional radiation-induced chromatid break experiments using colcemid as a blocking agent, we have compared the chromatid break kinetics following a single dose of gamma rays (0.75 Gy) in metaphase CHO cells using calyculin-induced premature chromosome condensation (PCC), with those using colcemid block. Calyculin-induced PCC causes very rapid condensation of G2 cell chromosomes without the need for a cell to progress to mitosis, hence eliminating any effect of cell-cycle checkpoint on chromatid break frequency. We found that the kinetics of the exponential first-order decrease in chromatid breaks with time after irradiation was similar (not significantly different) between the two methods of chromosome condensation. However, use of the calyculin-PCC technique resulted in a slightly increased rate of disappearance of chromatid breaks and thus higher frequencies of breaks at 1.5 and 2.5 h following irradiation. We also report on the effect of the nucleoside analogue ara A on chromatid break kinetics using the two chromosome condensation techniques. Ara A treatment of cells abrogated the decrease in chromatid breaks with time, both using the calyculin-PCC and colcemid methods. We conclude that cell-cycle delay may be a factor determining the absolute frequency of chromatid breaks at various times following irradiation of cells in G2 phase but that the first-order disappearance of chromatid breaks with time and its abrogation by ara A are not significantly influenced by the G2 checkpoint.

The anticancer drug cyclophosphamide induces granulosa cell apoptosis and is detoxified by glutathione (GSH) conjugation. We previously showed that both cyclophosphamide treatment and GSH depletion induced granulosa cell apoptosis in rats, but the role of GSH in apoptosis in human ovarian cells has not been studied. Using the COV434 human granulosa cell line, we tested the hypotheses that (1) GSH depletion or treatment with 4-hydroperoxycyclophosphamide (4HC), a preactivated form of cyclophosphamide, induces apoptosis, (2) GSH depletion potentiates 4HC-induced apoptosis, and (3) 4HC-induced apoptosis is mediated by GSH depletion and oxidative stress. Cells were treated with buthionine sulfoximine (BSO), a specific inhibitor of GSH synthesis, with or without follicle stimulating hormone (FSH) or serum. A significant increase in the number of apoptotic cells, assessed by terminal deoxynucleotidyl transferase-mediated deoxy-uridine triphosphate nick-end labeling (TUNEL) and Hoechst 33342 staining, occurred with BSO treatment. Treatment with 4HC dose-dependently induced apoptosis by TUNEL, Hoechst staining, and caspase 3 activation. Treatment with 4HC caused an increase in reactive oxygen species generation, measured by dichlorofluorescein fluorescence, oxidative DNA damage, measured by 8-hydroxyguanosine immunostaining, and an oxidation of the redox potential for the oxidized glutathione/reduced glutathione couple. Total intracellular GSH declined after 4HC treatment, preceding the onset of cell death. Treatment with antioxidants inhibited 4HC-induced apoptosis. Combined treatment with BSO and 4HC caused greater induction of apoptosis than either treatment alone. These findings are consistent with roles for oxidative stress and GSH depletion in mediating the induction of apoptosis in COV434 cells by cyclophosphamide.

Ectopic genes transferred to cells are temporally expressed, although this phenomenon has not yet been well characterized. We found that 5-bromodeoxyuridine dramatically increased transient expression of ectopic genes in human cells. This effect was elicited by adding 5-bromodeoxyuridine prior to or after transfection. No promoter specificity was observed. Real time PCR analysis showed an approximately 2-fold increase in mRNA levels. Since 5-bromodeoxyuridine decondenses heterochromatin and changes the nuclear envelope, these changes might affect transcriptional and post-transcriptional events in the gene expression of plasmids.

Folic acid supplementation can prevent many cases of neural tube defects (NTDs), whereas suboptimal maternal folate status is a risk factor, suggesting that folate metabolism is a key determinant of susceptibility to NTDs. Despite extensive genetic analysis of folate cycle enzymes, and quantification of metabolites in maternal blood, neither the protective mechanism nor the relationship between maternal folate status and susceptibility are understood in most cases. In order to investigate potential abnormalities in folate metabolism in the embryo itself, we derived primary fibroblastic cell lines from foetuses affected by NTDs and subjected them to the dU suppression test, a sensitive metabolic test of folate metabolism. Significantly, a subset of NTD cases exhibited low scores in this test, indicative of abnormalities in folate cycling that may be causally linked to the defect. Susceptibility to NTDs may be increased by suppression of the methylation cycle, which is interlinked with the folate cycle. However, reduced efficacy in the dU suppression test was not associated with altered abundance of the methylation cycle intermediates, s-adenosylmethionine and s-adenosylhomocysteine, suggesting that a methylation cycle defect is unlikely to be responsible for the observed abnormality of folate metabolism. Genotyping of samples for known polymorphisms in genes encoding folate-associated enzymes did not reveal any correlation between specific genotypes and the observed abnormalities in folate metabolism. These data suggest that as yet unrecognized genetic variants result in embryonic abnormalities of folate cycling that may be causally related to NTDs.

A stable supramolecular cluster in T cells at the contact site of APCs, the immunological synapse (IS), is essential for full T cell activation. Failure of IS maturation, as determined by defective relocalization of the TCR/CD3 complex at the T cell/APC contact site, is linked with T cell hyporesponsiveness. The effects of clinically used immunosuppressants on these critical events, however, are undefined. Here, we show that treatment of T cells with cyclosporin A, FK506, and dexamethasone, which are known to inhibit calcineurin and NF-kappaB, respectively, but not rapamycin, the inhibitor of mammalian target of rapamycin, selectively prevented TCR/CD3 relocalization into the IS, while relocalization of adhesion and cytoskeletal proteins as well as T cell/APC conjugate formation remained unaltered. The involvement of calcineurin and NF-kappaB in IS maturation was confirmed by using specific inhibitors of these molecules (FR901725, gossypol, SN50). FK778, as an inhibitor of DNA replication and also TCR/CD3-activated tyrosine kinases, globally abrogated cytoskeletal, adhesion, and signaling molecule relocalization, thereby preventing formation of an IS at an earlier, immature stage along with impaired, antigen-specific T cell/APC conjugate formation. Collectively, blocking IS formation at distinct stages may mediate effects on T cell activation of currently used immunosuppressants, apart from their capacity to block gene transcription, cytokine signaling, and DNA replication. Furthermore, these data imply novel functions of calcineurin and NF-kappaB for successful IS maturation.

RNA polymerase II (RNAPII) in eukaryotic cells drives transcription of most messenger RNAs. RNAPII core enzyme is composed of 12 polypeptides where Rpb1 is the largest subunit. To further understand the mechanisms of RNAPII transcription, we isolated and characterized novel point mutants of RPB1 that are sensitive to the nucleotide-depleting drug 6-azauracil (6AU). In this work we reisolated the rpo21-24/rpb1-E1230K allele, which reduces the interaction of RNAPII-TFIIS, and identified five new point mutations in RPB1 that cause hypersensitivity to 6AU. The novel mutants affect highly conserved residues of Rpb1 and have differential genetic and biochemical effects. Three of the mutations affect the "lid" and "rudder," two small loops suggested by structural studies to play a central role in the separation of the RNA-DNA hybrids. Most interestingly, two mutations affecting the catalytic center (rpb1-N488D) and the homology box G (rpb1-E1103G) have strong opposite effects on the intrinsic in vitro polymerization rate of RNAPII. Moreover, the synthetic interactions of these mutants with soh1, spt4, and dst1 suggest differential in vivo effects.

This study examined the effects of three different cellular stresses on oocyte maturation in meiotically arrested mouse oocytes. Cumulus-cell enclosed oocytes (CEO) or denuded oocytes (DO) from immature, eCG-primed mice were cultured for 17-18 h in dbcAMP-containing medium plus increasing concentrations of the metabolic poison, sodium arsenite, or the free radical-generating agent, menadione. Alternatively, oocytes were exposed to osmotic stress by pulsing with sorbitol and returned to control inhibitory conditions for the duration of culture. Arsenite and menadione each dose-dependently induced germinal vesicle breakdown (GVB) in both DO and CEO. DO, but not CEO, pulsed for 60 min with 500 mM sorbitol were stimulated to resume maturation. The lack of effect in CEO suggests that the cumulus cells may be playing a protective role in osmotic stress-induced GVB. The AMP-activated protein kinase (PRKA; formerly known as AMPK) inhibitors, compound C and araA, completely blocked the meiosis-stimulating effects of all the tested stresses. Western blots showed that acetyl-CoA carboxylase, an important substrate of PRKA, was phosphorylated before GVB, supporting a role for PRKA in stress-induced maturation. Together, these data show that a variety of stresses stimulate GVB in meiotically arrested mouse oocytes in vitro and suggest that this effect is mediated through activation of PRKA.

The nematode Caenorhabditis elegans yields a substance(s) inducing the larval diapause, called dauer-inducing pheromone. We discovered that the crude pheromone extract extends the adult lifespan in the animal. This extension does not occur in the mutant animal, in which expansion of the lifespan caused by other mutations reducing insulin signaling is suppressed. This is the first description concerning the relevancy of the pheromone to the longevity in the animal.

We have isolated 14 different Schizosaccharomyces pombe mutants that synthesize invertase enzyme constitutively. Analyses of invertase activities revealed that the degrees of resistance to glucose repression were not similar among different complementation groups. One of the complementation groups appeared to be associated with functional and/or regulatory defects in hexose transport. Another complementation group appeared to be specific for the regulation of the inv1 gene alone, implying that these mutations might be associated with different genes acting on the glucose sensing and signaling pathway. In addition, we found that the wild-type level glucose uptake is essential for the full-level repression of inv1 expression.

Background: We investigated the expression of the hepatocyte growth factor (HGF) gene and protein by the stromal cells derived from women with or without endometriosis and its regulation by interleukin-6 (IL-6) and tumour necrosis factor alpha (TNFalpha)., Methods: Stromal cells immunoreactive to vimentin were isolated from the eutopic and ectopic endometrium of 18 infertile women with endometriosis and 12 women without endometriosis. The production of HGF in the culture media of basal and IL-6- or TNFalpha-stimulated stromal cells was examined with an enzyme-linked immunosorbent assay. The mRNA expression of HGF and its receptor c-Met in the stroma was investigated by RT-PCR. The localization of HGF and c-Met in isolated stromal cells and in intact tissue was examined by immunohistochemistry. The effect of HGF on the growth of stromal cells alone or in combination with IL-6 or TNFalpha was examined in a bromodeoxyuridine (BrdU) incorporation study., Results: The production of HGF in the culture medium of stromal cells was significantly increased after single or combined treatment with either IL-6 or TNFalpha when compared with non-treated cells. The production of HGF by stromal cells derived from the eutopic endometrium of women with endometriosis was significantly higher than that of cells from women without endometriosis. This effect was paralleled by increased expression of HGF and c-Met mRNA, as demonstrated by RT-PCR. The BrdU incorporation study indicated that the addition of HGF enhanced the growth of endometrial and endometriotic stroma alone or in combination with IL-6 or TNFalpha., Conclusion: IL-6 and TNFalpha are involved in the production of HGF by endometrial stromal cells and may be involved in the growth of endometriosis by an autocrine mechanism.

Neocortical projection neurons arise from a pseudostratified ventricular epithelium (PVE) from embryonic day 11 (E11) to E17 in mice. The sequence of neuron origin is systematically related to mechanisms that specify neuronal class properties including laminar fate destination. Thus, the neurons to be assembled into the deeper layers are the earliest generated, while those to be assembled into superficial layers are the later generated neurons. The sequence of neuron origin also correlates with the probability of cell cycle exit (Q) and the duration of G1-phase of the cell cycle (T(G1)) in the PVE. Both Q and T(G1) increase as neuronogenesis proceeds. We test the hypothesis that mechanisms regulating specification of neuronal laminar destination, Q and T(G1) are coordinately regulated. We find that overexpression of p27(Kip1) in the PVE from E12 to E14 increases Q but not T(G1) and that the increased Q is associated with a commensurate increase in the proportion of exiting cells that is directed to superficial layers. We conclude that mechanisms that govern specification of neocortical neuronal laminar destination are coordinately regulated with mechanisms that regulate Q and are independent of mechanisms regulatory to cell cycle duration. Moreover, they operate prior to postproliferative mechanisms necessary to neocortical laminar assembly.

Objectives: Candidiasis can be associated with the formation of biofilms on bioprosthetic surfaces and the intrinsic resistance of Candida albicans biofilms to the most commonly used antifungal agents has been demonstrated. In this study, we report on the antifungal activity of caspofungin at two different concentrations, on C. albicans and Candida parapsilosis biofilms with different ages of maturation., Methods: Fifteen strains of C. albicans (10 strains susceptible to fluconazole in vitro and five strains resistant to this antifungal agent) and six strains of C. parapsilosis (all were susceptible to fluconazole in vitro) were studied. The antifungal activity of caspofungin was assessed by looking for a significant inhibition of the metabolic activity of yeasts within biofilms. Biofilms of Candida were produced in vitro, on silicone catheters., Results: Caspofungin used at MIC did not modify the metabolic activity of C. albicans, whatever the maturation age of the biofilms. The same concentration of caspofungin significantly reduced the metabolism (P

We investigated the hypothesis that glutathione (GSH) in reproductive tract secretions (RTS) protects the preimplantation embryo from endogenous reactive oxygen species and is important for normal development during the embryo's sensitive period when it is incapable of synthesizing GSH de novo. Mice were administered buthionine sulfoximine (BSO) to inhibit GSH synthesis and decrease GSH concentration in RTS. Embryos were then allowed to develop either in vivo or in vitro in the presence of RTS and the GSH concentration of the embryos was quantified by HPLC and embryonic development was recorded. GSH concentration in RTS did not differ over the phases of the estrous cycle, but there were significant decreases in GSH concentration on Day 2 of gestation and due to BSO treatment. Embryos allowed to develop in vivo and in vitro in RTS with decreased GSH concentration did not exhibit decreased development or GSH concentration. Oocytes exposed to BSO during maturation in vivo experienced a significant decrease in GSH concentration and an increase in percent of degenerate embryos when compared with control. These data suggest that most of the GSH in RTS does not play a critical role in normal preimplantation embryo development but that GSH stored in the oocyte during maturation has an important role in subsequent embryo development. Our studies do not exclude the possibility that GSH in RTS plays an important role in protection of the preimplantation embryo during exposure to some toxicants.

Ghrelin, an endogenous ligand for the GH secretagogue receptor, is a hormone expressed in stomach and other tissues, such as hypothalamus, testis, and placenta. This hormone acts at a central level to stimulate GH secretion and food intake. Little is known, however, about the molecular forms and physiological roles of ghrelin within the hypothalamus. In this report, we detail the molecular forms, mRNA expression patterns, and peptide contents of ghrelin within the rat hypothalamus. Using the combination of reverse-phase HPLC and ghrelin-specific RIA, we determined that the rat hypothalamus contains both n-octanoyl-modified and des-acyl ghrelins. Fasting for 24 and 48 h significantly decreased ghrelin mRNA expression in the hypothalamus to 24% and 28% of control values, respectively. Both n-octanoyl-modified and des-acyl ghrelin content in the hypothalamus decreased after 24 and 48 h of fasting. These results contrast the changes in gastric ghrelin after fasting, which decreased in content despite increased mRNA expression. Two hours after injection of 2-deoxy-d-glucose (2-DG), a selective blocker of carbohydrate metabolism, ghrelin peptide levels also decreased. Thus, induction of glucoprivic states, such as fasting and 2-DG treatment, decreased ghrelin gene expression and peptide content within the hypothalamus.

Neurogenesis occurs throughout adult life in the dentate gyrus of mammalian hippocampus and has been suggested to play an important role in cognitive function. Multiple trophic factors including IGF-I have been demonstrated to regulate hippocampal neurogenesis. Ames dwarf mice live considerably longer than normal animals and maintain physiological function at youthful levels, including cognitive function, despite a deficiency of circulating GH and IGF-I. Here we show an increase in numbers of newly generated cells [bromodeoxyuridine (BrdU) positive] and newborn neurons (neuronal nuclear antigen and BrdU positive) in the dentate gyrus of adult dwarf mice compared with normal mice using BrdU labeling. Despite the profound suppression of hippocampal GH expression, hippocampal IGF-I protein levels are up-regulated and the corresponding mRNAs are as high in Ames dwarf as in normal mice. Our results suggest that local/hippocampal IGF-I expression may have induced the increase in hippocampal neurogenesis, and increased neurogenesis might contribute to the maintenance of youthful levels of cognitive function during aging in these long-lived animals.

Puromycin N-acetyl transferase gene (pac), of which the gene product catalyzes antibiotic puromycin (an effective inhibitor of protein synthesis), has been widely used as a dominant selection marker in embryonic stem (ES) cell-mediated transgenesis. The present study is the first to report on the usefulness of puromycin for production of enhanced green fluorescent protein (EGFP) transgenic piglets after somatic cell cloning and embryo transfer. Somatic cells isolated from porcine fetuses at 73 days of gestation were immediately electroporated with a transgene (pCAG-EGFPac) carrying both EGFP cDNA and pac. This procedure aims to avoid aging effects thought to be generated during cell culture. The recombinant cells were selected with puromycin at a low concentration (2 microg/ml), cultured for 7 days, and then screened for EGFP expression before somatic cell cloning. The manipulated embryos were transplanted into the oviducts of 14 foster mother sows. Four of the foster sows became pregnant and nine piglets were delivered. Of the nine piglets, eight died shortly after birth and one grew healthy after weaning. Results indicate that puromycin can be used for the selection of recombinant cells from noncultured cells, and moreover, may confer the production of genetically engineered newborns via nuclear transfer techniques in pigs.

We recently found that estrogen deficiency leads to a lowering of thiol antioxidant defenses in rodent bone. Moreover, administration of agents that increase the concentration in bone of glutathione, the main intracellular antioxidant, prevented estrogen-deficiency bone loss, whereas depletion of glutathione by buthionine sulfoximine (BSO) administration provoked substantial bone loss. It has been shown that the estrogen-deficiency bone loss is dependent on TNFalpha signaling. Therefore, a model in which estrogen deficiency causes bone loss by lowering antioxidant defenses predicts that the osteopenia caused by lowering antioxidant defenses should similarly depend on TNFalpha signaling. We found that the loss of bone caused by either BSO administration or ovariectomy was inhibited by administration of soluble TNFalpha receptors and abrogated in mice deleted for TNFalpha gene expression. In both circumstances, lack of TNFalpha signaling prevented the increase in bone resorption and the deficit in bone formation that otherwise occurred. Thus, depletion of thiol antioxidants by BSO, like ovariectomy, causes bone loss through TNFalpha signaling. Furthermore, in ovariectomized mice treated with soluble TNFalpha receptors, thiol antioxidant defenses in bone remained low, despite inhibition of bone loss. This suggests that the low levels of antioxidants in bone seen after ovariectomy are the cause, rather than the effect, of the increased resorption. These experiments are consistent with a model for estrogen-deficiency bone loss in which estrogen deficiency lowers thiol antioxidant defenses in bone cells, thereby increasing reactive oxygen species levels, which in turn induce expression of TNFalpha, which causes loss of bone.

The mammalian sperm must be highly motile for a long time to fertilize a egg. It has been supposed that ATP required for sperm flagellar movement depends predominantly on mitochondrial respiration. We assessed the contribution of mitochondrial respiration to mouse sperm motility. Mouse sperm maintained vigorous motility with high beat frequency in an appropriate solution including a substrate such as glucose. The active sperm contained a large amount of ATP. When carbonyl cyanide m-chlorophenylhydrazone (CCCP) was applied to suppress the oxidative phosphorylation in mitochondria, the vigorous motility was maintained and the amount of ATP was kept at the equivalent level to that without CCCP. When pyruvate or lactate was provided instead of glucose, both sperm motility and the amount of ATP were high. However, they were drastically decreased when oxidative phosphorylation was suppressed by addition of CCCP. We also found that sperm motility could not be maintained in the presence of respiratory substrates when glycolysis was suppressed. 2-Deoxy-d-glucose (DOG) had no effect on mitochondrial respiration assessed by a fluorescent probe, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1), but, it inhibited motility and decreased ATP content when pyruvate or lactate were provided as substrates. The present results suggest that glycolysis has an unexpectedly important role in providing the ATP required for sperm motility throughout the length of the sperm flagellum.

To investigate the role played by the orexigenic peptide, neuropeptide Y (NPY), in adaptive responses to insulin-induced hypoglycemia, we measured hypothalamic, feeding, and hormonal responses to this stimulus in both wild-type (Npy+/+) and NPY-deficient (Npy-/-) mice. After administration of insulin at a dose (60 mU ip) sufficient to cause moderate hypoglycemia (plasma glucose levels, 40 +/- 3 and 37 +/- 2 mg/dl for Npy+/+ and Npy-/- mice, respectively; P = not significant), 4-h food intake was increased 2.5-fold in Npy+/+ mice relative to saline-injected controls. By comparison, the increase of intake in Npy-/- mice was far smaller (45%) and did not achieve statistical significance (P = 0.08). Hyperphagic feeding in response to insulin-induced hypoglycemia was therefore markedly attenuated in mice lacking NPY, and a similar feeding deficit was detected in these animals after neuroglucopenia induced by 2-deoxyglucose (500 mg/kg ip). A role for NPY in glucoprivic feeding is further supported by our finding that Npy mRNA content (measured by real-time PCR) increased 2.4-fold in the hypothalamus of Npy+/+ mice by 7 h after insulin injection. Unlike the feeding deficits observed in mice lacking NPY, the effect of hypoglycemia to increase plasma glucagon and corticosterone levels was fully intact in these animals, as were both the nadir glucose value and time to recovery of euglycemia after insulin injection (P = not significant). We conclude that NPY signaling is required for hyperphagic feeding, but not neuroendocrine responses to moderate hypoglycemia.

The neuropeptide galanin is expressed in sympathetic nerve terminals that surround islet cells and inhibits insulin secretion. To explore its role for islet function, we studied mice with a loss-of-function mutation in the galanin gene [galanin knockout (KO) mice]. Intravenous 2-deoxy-glucose, which activates both the sympathetic and parasympathetic branches of the autonomic nervous system, caused an initial (1-5 min) inhibition of insulin secretion that was impaired in galanin KO mice (P = 0.027), followed by a subsequent stimulation of insulin secretion that was augmented in galanin KO mice (P < 0.01). Similar effects were seen after chemical sympathectomy by 6-hydroxydopamine. In contrast, galanin KO mice had a reduced insulin response to glucose, both in vivo (P < 0.001) and in isolated islets (P < 0.001), and to arginine, both in vivo (P = 0.012) and in vitro (P = 0.018). During an iv glucose tolerance test, galanin KO mice had impaired glucose disposal (P = 0.005) due to a reduced insulin response (P < 0.001) and a reduced insulin-independent glucose elimination (glucose effectiveness; P = 0.040). Insulin sensitivity, as judged by a euglycemic, hyperinsulinemic clamp technique, was slightly increased in galanin KO mice (P = 0.032). We conclude that 1) galanin may contribute to sympathetic influences inhibiting insulin secretion in mice, and 2) galanin KO mice have a reduced glucose-induced insulin secretion.

The present study examined the relative importance of blood glucose vs. free fatty acids as a metabolic signal regulating GnRH release as measured electrophysiologically by multiple-unit activity (MUA) in the arcuate nucleus/median eminence region in ovariectomized, estradiol-treated goats. MUA was recorded before, during, and after: 1) cellular glucoprivation by peripheral infusion of 2-deoxy-d-glucose (2DG; 25, 50, and 75 mg/kg.h, iv); 2) peripheral hypoglycemia in response to various doses (15-195 mU/kg.h, iv) of insulin infusion; and 3) cellular lipoprivation induced by peripheral infusion of sodium mercaptoacetate (MA; 2.4 mg/kg.h alone or combined with 25 mg/kg.h of 2DG, iv), and effects on the interval of characteristic increases in MUA (MUA volleys) were examined. Infusion of the highest dose of 2DG increased the mean interval between MUA volleys, whereas the lower doses of 2DG had no effect on volley interval. The MUA volley intervals lengthened as insulin-induced hypoglycemia became profound. There was a negative correlation between MUA volley intervals and blood glucose concentrations during insulin infusion, and coinfusion of glucose with insulin returned the MUA volley interval to a normal frequency. Infusion of MA alone or MA with 2DG did not increase MUA volley intervals. These findings demonstrate that glucose availability, but not fatty acids, regulates the GnRH pulse generator activity in the ruminant. Glucose is considered a key metabolic regulator that fine-tunes pulsatile GnRH release.

CRH neurons within the medial parvicellular part of the hypothalamic paraventricular nucleus (PVHmp) can respond to afferent inputs encoding stress-related information by initiating peptide synthesis (signaling cascades, transcription, and translation) and/or peptide release. However, understanding these cellular events is hampered by three outstanding issues: 1) neural inputs that activate CRH neurons remain incompletely identified; 2) the identity and temporal dynamics of signaling pathways within CRH neurons are poorly understood; and 3) the precise coupling of the first two issues has not been established. Here, we report that the phosphorylated forms of p44/p42 MAPKs (pERK1/2) are rapidly detected in PVHmp cells after i.v. infusion of the antimetabolite, 2-deoxy-D-glucose (2-DG). Combined immunocytochemistry and in situ hybridization revealed that pERK1/2 immunoreactivity is detectable 10 min after 2-DG infusion not only within most PVHmp neurons containing CRH mRNA (78.6% of mean total CRH cells counted) but also in many non-CRH neurons (45.5% of mean total sampled cells). In contrast, Fos protein in the PVHmp was not detected within this time period, consistent with the known time course for its translation. Stress associated with halothane exposure also robustly elevated pERK1/2 levels in PVHmp neurons approximately 10 min after exposure. Our results implicate pERK1/2 in stress-induced activation of CRH neurosecretory cells and underscore their utility as indices of rapid cellular activation. Because 2-DG-induced activation of CRH gene transcription in these neurons requires a catecholaminergic input, our data also suggest that pERK1/2 could couple afferent catecholaminergic signals with CRH gene expression in these neurons.

Study Objectives: Besides regulating hormone secretion, steroids also modulate sleep architecture in specific ways. To simulate a state of altered glucocorticoid- and mineralocorticoid-receptor occupation, we administered metyrapone, an 11-beta-hydroxylase inhibitor, that blocks adrenal cortisol synthesis and inhibits the 11-beta-hydroxysteroiddehydrogenase type-1 enzyme in the central nervous system and investigated endocrine changes and the sleep electroencephalogram., Design: Each volunteer spent 4 nights in the sleep laboratory, the first of which served to habituate the subject to the conditions in the laboratory. The volunteers underwent 3 trial conditions in random order and in a single-blind design receiving 2 doses of metyrapone (4.5 g or 6.0 g) or placebo on the day before the sleep electroencephalogram recordings., Setting: Sleep laboratory., Participants: 8 healthy male volunteers., Measurements and Results: The corticotropin secretion was significantly enhanced by metyrapone, while the cortisol secretion remained largely unchanged. Growth hormone, progesterone, and dehydroepiandrosterone concentrations were also significantly increased by both doses. Metyrapone induced a pronounced reduction in slow-wave sleep and had slight but nonlinear effects upon rapid eye movement sleep. Parameters of sleep quality were not different between groups., Conclusions: Metyrapone induces pronounced effects on hormonal secretion and the sleep electroencephalogram. These results are in part comparable to those from experiments with the administration of corticotropin-releasing hormone and with experiments that deplete mineralocorticoid receptors. The findings may be explained by altered steroid synthesis proximal to the enzyme block. Metyrapone exerts not only effects upon adrenocortical steroid synthesis, but also important extra-adrenal effects on central corticosteroid metabolism.

Although a marked pubertal increase in Sertoli cell number is a hallmark of testicular development in the rhesus monkey, the ontogeny of this somatic cell type before puberty is less clear. To clarify this issue, groups (n = 4) of neonate (1-2 d old), infant (4-5 months old), juvenile (14-17 months old), and adult male rhesus monkeys were injected with 5-bromo-2'-deoxyuridine (BrdU) 2 h before castration. Tissue was fixed in Bouin's fluid, and the percentage of BrdU-labeled Sertoli cells at each developmental stage was calculated. In addition to the labeling index, Sertoli cell number per testis for the neonate and infant groups was enumerated using standard histomorphometry and compared with that previously reported by this laboratory for juvenile and adult rhesus monkeys. The number of Sertoli cells per testis in infants (156 +/- 49 x 10(6), mean +/- SD) was 4-fold greater than that in neonates (42 +/- 12 x 10(6)). The previously established value for this parameter in juvenile monkeys was 286 +/- 121 x 10(6). Incorporation of BrdU into nuclei of Sertoli cells indicated that these cells were mitotically active at all three stages of prepubertal development. The labeling index in the neonate and infant groups (1.33% in both cases), however, was significantly greater than that in juveniles (0.25%). From the foregoing results, we conclude that Sertoli cell proliferation during prepubertal development in the rhesus monkey occurs predominantly during infancy, when gonadotropin secretion is elevated, and to a lesser extent during the juvenile phase of development, when circulating gonadotropin concentrations are undetectable.

In this study, we investigated the effect of prostaglandin E(2) (PGE(2)) on MAPK ERK1/2 protein phosphorylation and on proliferation of epithelial cells of the human endometrium. Treatment of proliferative phase endometrium with PGE(2) induced rapid phosphorylation of ERK1/2 proteins in glandular epithelial and endothelial cells. Treatment of human endometrial tissue with PGE(2) for 24 h resulted in increased incorporation of 5-bromo-2'-deoxyuridine (a marker of cellular proliferation) in glandular epithelial cells. To investigate further the effect of PGE(2) on proliferation of epithelial cells, we used an endometrial epithelial cell line (HES). HES cells express functional EP4 (with absence of expression of EP1, EP2, and EP3) receptors and stimulate cAMP release and rapid phosphorylation of ERK1/2 proteins in response to PGE(2) or forskolin. Treatment of HES cells with PGE(2) or forskolin alone resulted in a significant increase in HES cell proliferation compared with control untreated cells (P < 0.05). Cotreatment of the cells with PGE(2) or forskolin and PD98059 abolished the increase in cellular proliferation. These data demonstrate ERK1/2 phosphorylation in response to PGE(2) in the human endometrium and suggest that PGE(2) via EP4 receptor may induce glandular epithelial cell proliferation in ERK1/2- dependent manner during the proliferative phase of the menstrual cycle.

An understanding of testicular physiology and pathology requires knowledge of the regulation of cell death. Previous observation of suppression of apoptosis by hypoxia suggested a role for ATP in germ cell death. However, the exact effects of ATP production on germ cell death and of apoptosis on the levels of ATP and other adenine nucleotides (ANs) have remained unclear. We investigated the levels of ANs during human testicular apoptosis (analyzed by HPLC) and the role of chemical anoxia in germ cell death (detected by Southern blot analysis of DNA fragmentation, in situ end labeling of DNA, and electron microscopy). Incubation of seminiferous tubule segments under serum-free conditions induced apoptosis and concomitantly decreased the levels of ANs. Chemical anoxia, induced with potassium cyanide (KCN), an inhibitor of mitochondrial respiration, dropped ATP levels further and suppressed apoptosis at 4 h. After 24 h, many of the testicular cells underwent delayed apoptosis despite ATP depletion. Some cells showed signs of necrosis or toxicity. The addition of 2-deoxyglucose, an antimetabolite of glycolysis, did not alter the results obtained with KCN alone, whereas a toxic concentration of hydrogen peroxide switched apoptosis to necrosis. In most of the testicular cells, mitochondrial respiration appears to play a crucial role in controlling primary cell death cascades. In the human testis, there seem to be secondary apoptotic pathways that do not require functional respiration (or ATP).

Objectives: To evaluate the penetration of ketolide cethromycin (ABT-773) into human polymorphonuclear leucocytes (PMNs) and its intracellular activity., Methods: The uptake of radiolabelled cethromycin by PMNs was determined by a velocity gradient centrifugation technique. The activity of cethromycin against intracellular Staphylococcus aureus ATCC 25923 in PMNs was also evaluated., Results: The cellular to extracellular concentration (C/E) ratio for cethromycin was >200 at an extracellular concentration of 2 mg/L. The uptake of cethromycin into PMNs was rapid and saturable. Cethromycin was slowly released from the loaded PMNs (cell associated drug>50% after 2 h of incubation). Intracellular penetration was significantly affected by the environmental temperature (C/E ratio at 4 degrees C and 37 degrees C: 13 +/- 6 and 226 +/- 31, respectively; P < 0.05), by cell viability (C/E ratio for dead and viable cells: 100 +/- 38 and 226 +/- 31, respectively; P < 0.05), by pH (C/E ratio was significantly increased at basic pH) and by the metabolic inhibitors 2,4-dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone. The intracellular accumulation of cethromycin also decreased significantly when cells were activated with phorbol myristate acetate or opsonized zymosan. These data indicate that a potentially active mechanism could be involved in the uptake of cethromycin by PMNs. At high extracellular concentrations of 5 and 10 mg/L, cethromycin showed significant intracellular activity against S. aureus., Conclusions: Cethromycin achieves high intracellular concentrations within human PMNs, remaining active intracellularly.

Propylthiouracil (PTU) is a thioamide drug used clinically to inhibit thyroid hormone production. However, PTU is associated with some side effects in different organs. In the present study, the acute and direct effects of PTU on testosterone production in rat Leydig cells were investigated. Leydig cells were isolated from rat testes, and an investigation was performed on the effects of PTU on basal and evoked-testosterone release, the functions of steroidogenic enzymes, including protein expression of cytochrome P450 side-chain cleavage enzyme (P450(scc)) and mRNA expression of the steroidogenic acute regulatory protein (StAR). Rat Leydig cells were challenged with hCG, forskolin, and 8-bromo-cAMP to stimulate testosterone release. PTU inhibited both basal and evoked-testosterone release. To study the effects of PTU on steroidogenesis, steroidogenic precursor-stimulated testosterone release was examined. PTU inhibited pregnenolone production (i.e., it diminished the function of P450(scc) in Leydig cells). In addition to inhibiting hormone secretion, PTU also regulated steroidogenesis by diminishing mRNA expression of StAR. These results suggest that PTU acts directly on rat Leydig cells to diminish testosterone production by inhibiting P450(scc) function and StAR expression.

Estrogens stimulate cell proliferation in typical estrogen-responsive tissues including the anterior pituitary gland. Here we report that 17-beta estradiol (E2) has estrogen receptor-mediated mitogenic and antimitogenic actions on rat lactotrophs in primary culture, depending on the cell context. E2 did not affect basal proliferation at 2 d after treatment, but it increased it at 4 d. Insulin markedly increased proliferative activity, which was inhibited by simultaneous treatment with E2, even after only 2 d of treatment. This antimitogenic action on insulin-induced proliferation was also observed with other estrogens but not with nonestrogenic steroids. Treatment with antiestrogens in combination with E2 antagonized both the mitogenic and antimitogenic actions of E2. Antiestrogen treatment alone inhibited basal proliferation, and it mimicked the inhibitory action of E2 on insulin-induced proliferation with less potency. In parallel with cell proliferation, an insulin-induced increase in the cell number of cyclin D1-immunoreactive lactotrophs was inhibited by E2 treatment. Although the antimitogenic action of E2 was seen with a wide range of doses of insulin or IGF-1, proliferation was stimulated rather than inhibited by E2 when cells were treated with serum or forskolin/isobutylmethylxanthine instead of insulin, indicating a mitogen-specific, but not proliferative activity-dependent, inhibition by E2. The results of estrogen-occupied estrogen receptors as negative regulators of proliferation suggest a novel interaction between estrogen and growth factors in the regulation of proliferation in estrogen-responsive cells.

Although cellular mitochondrial DNA (mtDNA) copy number varies widely among cell lines and tissues, little is known about the mechanism of mtDNA copy number control. Most nascent replication strands from the leading, heavy-strand origin (O(H)) are prematurely terminated, defining the 3' boundary of the displacement loop (D-loop). We have depleted mouse LA9 cell mtDNA to approximately 20% of normal levels by treating with 2',3'-dideoxycytidine (ddC) and subsequently allowed recovery to normal levels of mtDNA. A quantitative ligation-mediated PCR assay was used to determine the levels of both terminated and extended nascent O(H) strands during mtDNA depletion and repopulation. Depleting mtDNA leads to a release of replication termination until mtDNA copy number approaches a normal level. Detectable total nascent strands per mtDNA genome remain below normal. Therefore, it is likely that the level of replication termination plays a significant role in copy number regulation in this system. However, termination of D-loop strand synthesis is persistent, indicating formation of the D-loop structure has a purpose that is required under conditions of rapid recovery of depleted mtDNA.

Niemann-Pick C-1 (NPC-1) protein is essential for trafficking of low density lipoprotein-derived cholesterol in mammalian cells. The low density lipoprotein pathway is a major route for supply of cholesterol for steroidogenesis in the adrenals and gonads of many species. We investigated the occurrence and regulation of NPC-1 in porcine tissues, with emphasis on the corpus luteum and on granulosa cells undergoing luteinization in vitro. The porcine open reading frame for NPC-1 predicted a protein of 1278 amino acids (aa). It displayed a domain structure consistent with the human protein, and overall homologies were 89% and 86% with the deduced human and mouse aa sequences, respectively. The mRNA for NPC-1 comprised two transcripts, migrating at 5.0 and 2.2 kb, respectively. Transcripts were detected in a variety of pig tissues and were in highest abundance in steroid-producing organs. NPC-1 mRNA abundance increased with the differentiation of the corpus luteum in vivo and with luteinization of granulosa cells in vitro. Actinomycin D blockade of transcription in luteinized granulosa cells resulted in reduced NPC-1 mRNA and provided a half-life estimate of 20 h. Cycloheximide treatment increased NPC-1 transcript abundance in excess of 5-fold over 24 h. Treatment of luteinized granulosa cells with 1 mM (Bu)(2)cAMP increased the abundance of the NPC-1 message by 2- to 4-fold. The 5'-flanking region of the pig sequence displayed consensus sequences for binding transcription factors, including specificity protein-1, cAMP response element-binding protein/activating transcription factor-1, activating protein-1, GATA, modified zinc finger protein-1, transcription factor-11 and a CpG island in the first 400 bp upstream of the ATG transcription initiation site. Transient transfection of 1.86 kb of the 5'-flanking region coupled to the luciferase reporter into three steroidogenic cell lines resulted in constitutive transcription. Treatment with (Bu)2cAMP for 24 h increased the luciferase signal in all three lines. Thus, three types of evidence indicate that cAMP regulates pig NPC-1 expression. These are the presence of consensus binding sites for cAMP-induced transcription factors (cAMP response element-binding protein/activating transcription factor-1) in the proximal 5'-flanking region of the gene, increases in transcription by the NPC-1 promoter, and increases in NPC-1 mRNA abundance induced by (Bu)2cAMP. We conclude that NPC-1 is expressed in the steroidogenic tissues of the pig and is regulated by the principal pathway of stimulation of steroidogenesis in the gonads and adrenal, the cAMP-PKA pathway.

The CCR4-NOT complex is involved in the regulation of gene expression both positively and negatively. The repressive effects of the complex appear to result in part from restricting TBP access to noncanonical TATAA binding sites presumably through interaction with multiple TAF proteins. We provide here genetic evidence that the CCR4-NOT complex also plays a role in transcriptional elongation. First, defects in CCR4-NOT components as well as overexpression of the NOT4 gene elicited 6-azauracil (6AU) and mycophenolic acid sensitivities, hallmarks of transcriptional elongation defects. A number of other transcription initiation factors known to interact with the CCR4-NOT complex did not elicit these phenotypes nor did defects in factors that reduced mRNA degradation and hence the recycling of NTPs. Second, deletion of ccr4 resulted in severe synthetic effects with mutations or deletions in the known elongation factors RPB2, TFIIS, and SPT16. Third, the ccr4 deletion displayed allele-specific interactions with rpb1 alleles that are thought to be important in the control of elongation. Finally, we found that a ccr4 deletion as well as overexpression of the NOT1 gene specifically suppressed the cold-sensitive phenotype associated with the spt5-242 allele. The only other known suppressors of this spt5-242 allele are factors involved in slowing transcriptional elongation. These genetic results are consistent with the model that the CCR4-NOT complex, in addition to its known effects on initiation, plays a role in aiding the elongation process.

Incubation behavior or broodiness in turkey hens is characterized by ovarian regression, hyperprolactinemia, and persistent nesting. Nest-deprivation of incubating turkey hens results in disruption of broodiness accompanied by a precipitous decline in plasma prolactin (PRL) concentrations. The objective of the present study is to examine cellular changes in the pituitary gland associated with nest-deprivation for 0, 1, 2, 3, 4, or 7 days. Bromodeoxyuridine (BrdU) was administered prior to kill to study proliferative activity. Pituitary tissue sections were immunostained using turkey growth hormone (GH) antibody, and/or chicken PRL peptide antibody, and BrdU antibody. Plasma PRL concentrations declined significantly following nest-deprivation for 1 or more days. The midsagittal pituitary area immunoreactive (ir) to GH was significantly increased while that of PRL was significantly decreased following nest-deprivation for 2 or more days. Terminal deoxy-UTP nick end labeling and PRL-immunostaining revealed an abundance of apoptotic nuclei in both cephalic and caudal lobes of the anterior pituitary gland, suggestive of programmed cellular death of lactotrophs in the pituitary gland of hens nest-deprived for 2 or more days. Mammosomatotrophs were abundant in hens nest-deprived on Day 0 but were absent in hens nest-deprived for 1 or more days. Proliferating (BrdU-ir) cells were significantly abundant in the pituitary cephalic and caudal lobes following nest-deprivation for 1 or more days but were absent on Day 0 or in laying hens. Dual-labeling studies indicated that most of the BrdU-ir nuclei in the caudal lobe were not colocalized in somatotrophs in hens nest-deprived for 1-4 days but did colocalize with GH following 7 days of nest-deprivation. In conclusion, nest-deprivation of incubating turkey hens results in 1) a precipitous decline in plasma PRL concentration, 2) programmed cell death of lactotrophs, 3) disappearance of mammosomatotrophs, 4) increased proliferative activity of pituitary cells, and 5) recruitment of somatotrophs arising primarily from mitosis of nonsomatotrophic cells.

Alteration of the redox potential has been proposed as a mechanism influencing gene expression. Reduced glutathione (GSH) is one of the cellular scavengers involved in the regulation of the redox potential. To test the role that GSH may play in thyroid cells, we cultured a differentiated rat thyroid cell strain (FRTL-5) in the presence of L-buthionine-(S,R)-sulfoximine (BSO). BSO affects GSH synthesis by irreversibly inhibiting gamma-glutamylcysteine synthetase (EC 6.3.2.2), a specific enzyme involved in GSH synthesis. BSO-treated FRTL-5 cells show a great decrease in the GSH level, whereas malondialdehyde increases in the cell culture medium as a sign of lipid peroxidation. In these conditions the activity of two thyroid-specific promoters, thyroglobulin (Tg) and thyroperoxidase (TPO), is strongly reduced in transient transfection experiments. As both Tg and TPO promoters depend upon the thyroid-specific transcription factors, thyroid-specific transcription factor-1 (TTF-1) and Pax-8 for full transcriptional activity, we tested whether reduction of GSH concentration impairs the activity of these transcription factors. After BSO treatment of FRTL-5 cells, both transcription factors fail to trans-activate the respective chimerical targets, C5 and B-cell specific activating protein promoters, containing, respectively, multimerized TTF-1- or Pax-8-binding sites only as well as the Tg and TPO natural promoters. Northern analysis revealed that endogenous Tg messenger RNA (mRNA) expression is also reduced by BSO treatment, whereas endogenous TPO expression is not modified. Furthermore, the Pax-8 mRNA steady state concentration does not change in BSO-treated cells, whereas TTF-1 mRNA slightly decreases. Immunoblotting analysis of FRTL-5 nuclear extracts does not show significant modification of the Pax-8 concentration in BSO-treated cells, whereas a decrease of 25% in TTF-1 protein is revealed. Furthermore, BSO treatment decreases the DNA-binding activity to the respective consensus sequence of both transcription factors. Finally, different mechanisms seem to act on TTF-1 and Pax-8 functional impairment in BSO-treated cells. Indeed, with a lowered GSH concentration, the overexpressed Pax-8 still activates transcription efficiently, whereas, on the contrary, the overexpressed TTF-1 does not recover its transactivation capability when the respective chimerical target sequences are used (C5 and BSAP). When the natural Tg and TPO promoter sequences are used, overexpression of Pax-8 parallels the effect on both promoters observed using the chimeric target sequences, whereas overexpression of TTF-1 increases TPO promoter transcriptional activity only.