cAMP Treatment Research Articles

Conditions for Tumor-free and Dopamine Neuron–enriched Grafts After Transplanting Human ES Cell–derived Neural Precursor Cells

We have previously demonstrated derivation of neural precursor (NP) cells of a midbrain-type from human embryonic stem (hES) cells to yield an enriched population of dopamine (DA) neurons. These hES-derived NPs can be expanded in vitro through multiple passages without altering their DA neurogenic potential. Here, we studied two aspects of these hES-NP cells that are critical issues in cell therapeutic approaches for Parkinson's disease (PD): cell survival and tumorigenic potential. Neuroepithelial rosettes, a potentially tumorigenic structure, disappeared during hES-NP cell expansion in vitro. Although a minor population of cells positive for Oct3/4, a marker specific for undifferentiated hES cells, persisted in culture during hES-NP cell expansion, they could be completely eliminated by subculturing hES-NPs under differentiation-inducing conditions. Consistently, no tumors/teratomas are formed in rats grafted with multipassaged hES-NPs. However, extensively expanded hES-NP cells easily underwent cell death during differentiation in vitro and after transplantation in vivo. Transgenic expression of Bcl-XL and sonic hedgehog (SHH) completely overcame the cell survival problems without increasing tumor formation. These findings indicate that hES-NP cell expansion in conjunction with Bcl-XL+SHH transgene expression may provide a renewable and safe source of DA neurons for transplantation in PD.

Liver Receptor Homolog-1 Regulates the Transcription of Steroidogenic Enzymes and Induces the Differentiation of Mesenchymal Stem Cells into Steroidogenic Cells

Steroidogenic factor-1 (SF-1, also known as Ad4BP) has been demonstrated to be a primary transcriptional regulator of steroidogenic-related genes. However, mRNA for liver receptor homolog-1 (LRH-1), which together with SF-1, belongs to the NR5A nuclear receptor family, is expressed at much higher levels than SF-1 mRNA in the human gonad. In our previous studies, we demonstrated that SF-1 induced the differentiation of bone marrow-derived mesenchymal stem cells (MSCs) into steroidogenic cells such as Leydig or adrenocortical cells. The introduction of LRH-1 into human MSCs (hMSCs) with the aid of cAMP also induced the expression of steroidogenic enzymes, including CYP17, and their differentiation into steroid hormone-producing cells. Promoter analysis, EMSA, and chromatin immunoprecipitation assay using LRH-1-transduced hMSCs indicated that three LRH-1 binding sites were responsible for CYP17 transactivation. Immunohistochemical studies showed that LRH-1 protein was expressed in human Leydig cells. The CYP17 promoter region was highly methylated in hMSCs, whereas it was demethylated by the introduction of LRH-1 and cAMP treatment. These results indicate that LRH-1 could represent another key regulator of the steroidogenic lineage in MSCs and play a vital role in steroid hormone production in human Leydig cells.

Activation of TR4 orphan nuclear receptor gene promoter by cAMP/PKA and C/EBP signaling

In earlier studies, we had suggested that the fasting signal induces TR4 orphan nuclear receptor expression in vivo. The detailed mechanism(s), however, remain unclear. In this study, we found that cAMP/PKA, the mediator of fasting and glucagon signals, could induce TR4 gene expression that in turn modulates gluconeogenesis. Mechanistic dissection by in vitro studies in hepatocytes demonstrated that cAMP/PKA might trigger C/EBP alpha and beta binding to the selective cAMP response element, which is located at the TR4 promoter, thus inducing TR4 transcription. We also demonstrated that the binding activity of C/EBPs to the TR4 promoter is increased in response to cAMP treatment. Together, our data identified a new signaling pathway from the fasting signal --> cAMP/PKA --> C/EBP alpha and beta --> TR4 --> gluconeogenesis in hepatocytes; and suggested that TR4 could be an important regulator to control glucose homeostasis. The identification of activator(s)/inhibitor(s) or ligand(s) of TR4 may provide us an alternative way to control gluconeogenesis.

ARR19 (Androgen Receptor Corepressor of 19 kDa), an Antisteroidogenic Factor, Is Regulated by GATA-1 in Testicular Leydig Cells

ARR19 (androgen receptor corepressor of 19 kDa), which encodes for a leucine-rich protein, is expressed abundantly in the testis. Further analyses revealed that ARR19 was expressed in Leydig cells, and its expression was differentially regulated during Leydig cell development. Adenovirus-mediated overexpression of ARR19 in Leydig cells inhibited testicular steroidogenesis, down-regulating the expression of steroidogenic enzymes, which suggests that ARR19 is an antisteroidogenic factor. Interestingly, cAMP/luteinizing hormone attenuated ARR19 expression in a fashion similar to that of GATA-1, which was previously reported to be down-regulated by cAMP. Sequence analysis of the Arr19 promoter revealed the presence of two putative GATA-1 binding motifs. Further analyses with 5' deletion and point mutants of putative GATA-1 binding motifs showed that these GATA-1 binding sites were critical for high promoter activity. CREB-binding protein coactivated GATA-1 and markedly increased the activity of the Arr19 promoter. Both GATA-1 and CREB-binding proteins occupied the GATA-1 motifs within the Arr19 promoter, which was repressed by cAMP treatment. Altogether, these findings demonstrate that ARR19 is the target gene of GATA-1 and suggest that ARR19 gene expression in testicular Leydig cells is regulated by luteinizing hormone/cAMP signaling via the control of GATA-1 expression, resulting in the control of testicular steroidogenesis.

Catecholamine mediated S49 T lymphoma apoptosis is regulated by p38 MAPK (136.15)

Abstract p38 MAPK is an important intracellular signaling mediator for a variety of cellular and environmental stressors and plays a role in cell survival, apoptosis, and inflammation. Previously, our lab has shown that norepinephrine (NE) treatment of S49 T lymphoma cells leads to increased activity of p38 MAPK by the classical βAR/AC/cAMP/PKA pathway. Exogenous cAMP or βAR stimulation of S49 cells leads to apoptosis. To study the relationship between p38 MAPK activity and apoptosis, S49 cells were incubated in the presence and absence of a cAMP analogue (8-bromo) with or without the p38 MAPK inhibitor SB203580. After 72 hours of cAMP treatment, S49 cell viability decreased by 50%, yet in the presence of SB203580 cell viability decreased by 35%. The steady state levels of FasL mRNA (an early biochemical marker of apoptosis) were analyzed after two hours of S49 cell treatment with 8-bromo cAMP in the absence or presence of SB203580. FasL mRNA levels increased 9.8 fold with 8-bromo cAMP treatment for 2 hours and was reversed 50% by p38 MAPK inhibition. These results suggest that activation of p38 MAPK by NE or cAMP is associated with T lymphoma cell apoptosis. Supported by NIH

Low‐energy laser irradiation promotes synovial fibroblast proliferation by modulating p15 subcellular localization

Low-energy laser irradiation (low-level laser therapy) (LELI/LLLT/photobiomodulation) has been found to modulate various biological effects, especially those involved in promoting cell proliferation. Synovial fibroblasts are important in maintaining the homeostasis of articular joints and have strong chondrogenetic capacity. Here, we investigated the effect and molecular basis of LELI on synovial fibroblast proliferation. HIG-82 rabbit synovial fibroblasts were cultured, and laser irradiation (660 nm) was applied at the power density of 40 mW/cm(2) for 2 minutes, corresponding to laser fluence of 4.8 J/cm(2). The effect of LELI on cell proliferation, cell cycle progression, and expression of cyclin-dependent kinase inhibitors (CKIs) were investigated. We also examined whether the effects of LELI on HIG-82 cell proliferation were affected by cAMP content, which is known to influence the cell cycle via inducing CKIs. LELI promoted HIG-82 synovial fibroblast proliferation and induced cytoplasmic localization of cyclin-dependent kinase inhibitor p15 (INK4B/CDKN2B). Moreover, the proliferation of HIG-82 synovial fibroblasts was reduced by cAMP, while cAMP inhibitor, SQ22536, induced p15 cytoplasmic localization and as a result, elevated synovial fibroblast proliferation was observed. In addition, the promotive effect of LELI-induced HIG-82 synovial fibroblast proliferation was abolished by cAMP treatment. Our findings suggest that cAMP may be involved in the effect of LELI on synovial fibroblast proliferation. We revealed the effect and molecular link involved in synovial fibroblast proliferation induced by 660-nm LELI. Our study provides new insights into the mechanisms by which LELI has biological effects on synovial fibroblast proliferation. These insights may contribute to further investigation on biological effects and application of LELI in regenerative medicine.

Changes in the Treatment Responses to Artesunate-Mefloquine on the Northwestern Border of Thailand during 13 Years of Continuous Deployment

BackgroundArtemisinin combination treatments (ACT) are recommended as first line treatment for falciparum malaria throughout the malaria affected world. We reviewed the efficacy of a 3-day regimen of mefloquine and artesunate regimen (MAS3), over a 13 year period of continuous deployment as first-line treatment in camps for displaced persons and in clinics for migrant population along the Thai-Myanmar border.Methods and Findings3,264 patients were enrolled in prospective treatment trials between 1995 and 2007 and treated with MAS3. The proportion of patients with parasitaemia persisting on day-2 increased significantly from 4.5% before 2001 to 21.9% since 2002 (p<0.001). Delayed parasite clearance was associated with increased risk of developing gametocytaemia (AOR = 2.29; 95% CI, 2.00–2.69, p = 0.002). Gametocytaemia on admission and carriage also increased over the years (p = 0.001, test for trend, for both). MAS3 efficacy has declined slightly but significantly (Hazards ratio 1.13; 95% CI, 1.07–1.19, p<0.001), although efficacy in 2007 remained well within acceptable limits: 96.5% (95% CI, 91.0–98.7). The in vitro susceptibility of P. falciparum to artesunate increased significantly until 2002, but thereafter declined to levels close to those of 13 years ago (geometric mean in 2007: 4.2 nM/l; 95% CI, 3.2–5.5). The proportion of infections caused by parasites with increased pfmdr1 copy number rose from 30% (12/40) in 1996 to 53% (24/45) in 2006 (p = 0.012, test for trend).ConclusionArtesunate-mefloquine remains a highly efficacious antimalarial treatment in this area despite 13 years of widespread intense deployment, but there is evidence of a modest increase in resistance. Of particular concern is the slowing of parasitological response to artesunate and the associated increase in gametocyte carriage.

Induction of senescence by progesterone receptor-B activation in response to cAMP in ovarian cancer cells

Objective Progesterone receptor (PR) expression is a favorable prognostic marker in ovarian cancer. We previously demonstrated that the induction of PR-B by treatment with cAMP was associated with G0/G1 arrest of the cell cycle and growth inhibition in NIH 3T3 cells. In this study, we examined the effect of cAMP treatment on cell growth in Ras-transformed NIH3T3 cells and ovarian cancer cells. Methods 1) The levels of PR-B and cell cycle associated proteins (p21, p27 and Rb) following treatment with cAMP in the Ras-transformed NIH3T3 cells (K12V) and ovarian cancer cell lines (SKOV cells) were investigated by Western blots. 2) The effects of PR overexpression following treatment with cAMP or after infection of an adenovirus expressing PR-B on cell growth and tumorigenicity in a soft agar culture were examined. Results 1) Treatment with cAMP increased PR-B and p27 levels in K12V cells and inhibited cell growth by inducing premature senescence. Induction of senescence was specific to the transformed cells. 2) In SKOV cells, treatment with cAMP induced PR-B, p27 and p21 expression, reduced the level of phosphorylated Rb, caused accumulation of cells in the G0/G1 fraction of the cell cycle, and induced senescence. 3) Both anchorage-dependent and -independent SKOV cell growths were inhibited by cAMP treatment. 4) Induction of both the expression and transcriptional activity of PR-B is critical for the induction of senescence and suppression of tumorigenicity. Conclusion Treatment of cAMP, through activation of PR-B, induced senescence and suppressed tumorigenicity in ovarian cancer cells.

CAMP and Pyk2 interact to regulate prostate cells proliferation and function

In cultured prostate cancer cells cAMP blocks proliferation and induces neuroendocrine differentiation. Pyk2 expression inversely correlates with malignancy of prostate cancer. The aim of this study was to investigate the interaction between cAMP and Pyk2 in the prostate. EPN cells, a line derived from human normal prostate expressing Pyk2, and EPN-PKM3 cells, an EPN clone bearing a Pyk2 kinase-negative mutant, were adopted as model system. cAMP inhibited cell growth in both prostate cell lines, and activated Pyk2, but not ERK1/2, in EPN cells. cAMP treatment, abolished the activation of AKT1, an important component of the pro-survival pathway, in the EPN cells but not in EPN-PKM3 cells. Finally, upon cAMP treatment, EPN and EPN-PKM3 cells exhibited different expression patterns of HOX genes, an important network controlling cell identity. These data demonstrated for the first time that Pyk2 and cAMP interact in regulating prostate cell functions and in "keeping" prostate identity.

Identification of secreted proteins regulated by cAMP in glioblastoma cells using glycopeptide capture and label‐free quantification

Exposure of glioblastoma U87MG cells to a cAMP analog leads to a decrease in proliferation, invasion, and angiogenic potential. Here, we apply a label-free MS-based approach to identify formerly N-linked glycopeptides that change in abundance upon cAMP treatment. Over 150 unique glycopeptides in three biological repetitions were quantified, leading to the identification of 14 upregulated proteins and 21 downregulated proteins due to cAMP treatment. Of these, eight have been validated, either through comparison with microarray data or by Western blot. We estimate our ability to identify differentially expressed peptides at greater than 85% in a single biological repetition, while the analysis of multiple biological repetitions lowers the false positive rate to approximately 2%. Many of the proteins identified in this study are involved in cell signaling and some, such as Tenascin C, Cathepsin L, Neuroblastoma suppressor of tumorigenicity, and AXL/UFO tyrosine-protein kinase receptor, have been previously shown to be involved in glioblastoma progression. We also identify several semitryptic peptides that increase in abundance upon cAMP treatment, suggesting that cAMP regulates protease activity in these cells. Overall, these results demonstrate the benefits of using a highly specific enrichment method for quantitative proteomic experiments.

Timing Is Critical for Effective Glucocorticoid Receptor Mediated Repression of the cAMP-Induced CRH Gene

Glucocorticoid negative feedback of the hypothalamus-pituitary-adrenal axis is mediated in part by direct repression of gene transcription in glucocorticoid receptor (GR) expressing cells. We have investigated the cross talk between the two main signaling pathways involved in activation and repression of corticotrophin releasing hormone (CRH) mRNA expression: cyclic AMP (cAMP) and GR. We report that in the At-T20 cell-line the glucocorticoid-mediated repression of the cAMP-induced human CRH proximal promoter activity depends on the relative timing of activation of both signaling pathways. Activation of the GR prior to or in conjunction with cAMP signaling results in an effective repression of the cAMP-induced transcription of the CRH gene. In contrast, activation of the GR 10 minutes after onset of cAMP treatment, results in a significant loss of GR-mediated repression. In addition, translocation of ligand-activated GR to the nucleus was found as early as 10 minutes after glucocorticoid treatment. Interestingly, while both signaling cascades counteract each other on the CRH proximal promoter, they synergize on a synthetic promoter containing ‘positive’ response elements. Since the order of activation of both signaling pathways may vary considerably in vivo, we conclude that a critical time-window exists for effective repression of the CRH gene by glucocorticoids.

Effect of follicle size and dibutyryl cAMP on the cAMP content and gap junctional communication of porcine prepubertal cumulus - oocyte complexes during IVM

The lower ability of oocytes from prepubertal pigs to yield viable embryos than those from adult pigs appears, in part, a consequence of their reduced ability to accumulate cAMP during IVM. The present study examined the cAMP content of oocytes and cumulus-oocyte complexes (COCs), cumulus expansion and gap junctional communication (GJC) in COCs from 3- and 5-8-mm follicles during IVM. The effect of 1 mm dibutyryl cAMP (db-cAMP) treatment for the first 22 h of IVM was also examined for both follicle size classes. The cAMP concentration of oocytes from 5-8-mm follicles was threefold greater than that in oocytes from 3-mm follicles following 11 h of IVM (11.9 +/- 5.9 v. 3.6 +/- 1.8 fmol, respectively; P < 0.05). In the presence of db-cAMP, the cAMP content of oocytes from 3- and 5-8-mm follicles was no longer significantly different at 11 h IVM. The cAMP concentration of intact COCs from 5-8-mm follicles was significantly higher than that in COCs from 3-mm follicles at 11 h (1110.6 +/- 318.0 v. 116.9 +/- 55.7 fmol, respectively; P < 0.05). Despite maturation with db-cAMP, the cAMP content in COCs from 3- and 5-8-mm follicles at 11 h of IVM remained significantly different (15.1 +/- 4.9 v. 196.2 +/- 33.3 fmol, respectively; P < 0.05). The COCs from 3-mm follicles displayed lower cumulus expansion than did COCs from 5-8-mm follicles at both 11 h (cumulus expansion index (CEI) 1.0 +/- 0.1 v. 1.8 +/- 0.1, respectively; P < 0.01) and 22 h (CEI 1.9 +/- 0.3 v. 2.9 +/- 0.2, respectively; P < 0.05) of IVM. The level of cumulus cell-oocyte GJC decreased during IVM, with the number of GJC significantly greater in COCs from 3-mm compared with 5-8-mm follicles at both 6 h (613 +/- 55 v. 304 +/- 44 fluorescence intensity (FI), respectively; P < 0.05) and 11 h (644 +/- 99 v. 337 +/- 38 FI, respectively; P < 0.05) of IVM. By 22 h of IVM, the GJC of COCs from 3-mm follicles had decreased (227 +/- 18 FI) and was no longer significantly different to that of COCs from 5-8-mm follicles (139 +/- 15 FI; P > 0.05). Dibutyryl cAMP had no effect on the cAMP content, cumulus expansion or GJC of the whole COC. In conclusion, the results of the present study demonstrate that COCs from 3-mm follicles accumulate less intraoocyte and inter-COC cAMP, display lower cumulus expansion and maintain their cumulus cell-oocyte GJC for longer during IVM than do COCs from 5-8-mm follicles.

235 A COMPARISON OF PARTHENOGENETIC DEVELOPMENT OF PORCINE OOCYTES SYNCHRONISED DURING MATURATION BY CYCLOHEXIMIDE OR cAMP

In vitro maturation of porcine oocytes is characterized by a high level of asynchrony between oocytes. Previous studies reported that cycloheximide (CHX) and 3′, 5′-cyclic AMP (cAMP) synchronize porcine oocytes and improve development to blastocyst stage following IVF or have been used for somatic cell nuclear transfer (SCNT) (Ye et al. 2005 Biol. Reprod. 72(2), 399–406; Betthauser et al. 2000 Nat. Biotechnol. 18(10), 1055–1059). We previously reported that cAMP was more effective than CHX in synchronizing porcine oocyte maturation, producing MII oocytes in a shorter time window and providing a more homogenous population for future SCNT studies (Chen et al. 2008 SRF conference, 2008 abst, p34). Here we compared parthenogenetic development of porcine oocytes synchronized by these two treatments. Selected cumulus–oocyte complexes (COC) obtained from slaughtered gilts were randomly divided into three groups and cultured at 39°C, 5% CO2 in air in modified NCSU-23 medium (with 1 μm glutathione, 1 mm cysteine, 5 mg L–1 insulin, 10 ng mL–1 epidermal growth factor, 10% (v/v) porcine follicular fluid, 1% essential and 0.5% nonessential amino acids) ± hormones (10 IU mL–1 PMSG and 10 IU mL–1 hCG): (1) with hormones for the first 22 h and then without hormones until 44 h; (2) with hormones and 5 μg mL–1 CHX for 12 h, and then with hormones but no CHX until 44 h; (3) with hormones and 1 mm cAMP for 22 h, and then without hormones and cAMP until 44 h. Parthenogenetic development of cycloheximide and cAMP treated oocytes was compared by cleavage rate at 48 h postactivation (hpa) and blastocyst formation at 168 hpa. No significant differences were observed in the frequency of cleavage (96.7 ± 2.1% v. 81.4 ± 11.6% v. 84.5 ± 5.7%), development to blastocyst (28.3 ± 11.4% v. 27.1 ± 5.7% v. 32.8 ± 5.3%) between control, CHX or cAMP treated oocytes, respectively (chi-square test, P &gt; 0.05). However, total cell number was significantly higher in the CHX group than cAMP group (42.7 ± 4.1 v. 31.8 ± 2.0, respectively; t-test, P &lt; 0.05). The results demonstrate that synchronization of porcine oocytes by treatment with CHX or cAMP does not affect subsequent parthenogenetic development if judged by the blastocyst formation, although the meaning of the difference of total cell numbers between CHX and cAMP treatments is still unclear.

Regulation of P450 oxidoreductase by gonadotropins in rat ovary and its effect on estrogen production

BackgroundP450 oxidoreductase (POR) catalyzes electron transfer to microsomal P450 enzymes. Its deficiency causes Antley-Bixler syndrome (ABS), and about half the patients with ABS have ambiguous genitalia and/or impaired steroidogenesis. POR mRNA expression is up-regulated when mesenchymal stem cells (MSCs) differentiate into steroidogenic cells, suggesting that the regulation of POR gene expression is important for steroidogenesis. In this context we examined the regulation of POR expression in ovarian granulosa cells by gonadotropins, and its possible role in steroidogenesis.MethodsChanges in gene expression in MSCs during differentiation into steroidogenic cells were examined by DNA microarray analysis. Changes in mRNA and protein expression of POR in the rat ovary or in granulosa cells induced by gonadotropin treatment were examined by reverse transcription-polymerase chain reaction and western blotting. Effects of transient expression of wild-type or mutant (R457H or V492E) POR proteins on the production of estrone in COS-7 cells were examined in vitro. Effects of POR knockdown were also examined in estrogen producing cell-line, KGN cells.ResultsPOR mRNA was induced in MSCs following transduction with the SF-1 retrovirus, and was further increased by cAMP treatment. Expression of POR mRNA, as well as Cyp19 mRNA, in the rat ovary were induced by equine chorionic gonadotropin and human chorionic gonadotropin. POR mRNA and protein were also induced by follicle stimulating hormone in primary cultured rat granulosa cells, and the induction pattern was similar to that for aromatase. Transient expression of POR in COS-7 cells, which expressed a constant amount of aromatase protein, greatly increased the rate of conversion of androstenedione to estrone, in a dose-dependent manner. The expression of mutant POR proteins (R457H or V492E), such as those found in ABS patients, had much less effect on aromatase activity than expression of wild-type POR proteins. Knockdown of endogenous POR protein in KGN human granulosa cells led to reduced estrone production, indicating that endogenous POR affected aromatase activity.ConclusionWe demonstrated that the expression of POR, together with that of aromatase, was regulated by gonadotropins, and that its induction could up-regulate aromatase activity in the ovary, resulting in a coordinated increase in estrogen production.

Regulation of Cyclin D1 and Wnt10b Gene Expression by cAMP-responsive Element-binding Protein during Early Adipogenesis Involves Differential Promoter Methylation

Cyclin D1 expression is elevated and Wnt10b is repressed by cAMP during the first few hours of adipogenesis. cAMP-responsive element-binding protein (CREB) is a primary target for cAMP signaling, and we have shown that activation of CREB promotes adipogenesis and adipocyte survival. Here we tested the impact of CREB on expression of cyclin D1 and wingless-related mouse mammary tumor virus integration site 10b (Wnt10b) in 3T3-L1 cells. Forced depletion of CREB blocked Bt(2)cAMP-stimulated cyclin D1 expression and basal Wnt10b gene expression. Two CREB-binding sites were identified in the Wnt10b promoter region. Ablation of either site partially blocked promoter activity, while mutation of both sites completely suppressed promoter activity. These results suggest that CREB activates transcription from both the cyclin D1 and Wnt10b gene promoters. What accounts for the differential regulation of cyclin D1 and Wnt10b genes by cAMP? Chromatin immunoprecipitation revealed CREB bound to the Wnt10b promoter in untreated preadipocytes but not following treatment with Bt(2)cAMP. CREB binding to the cyclin D1 promoter was detected in untreated cells and post-Bt(2)cAMP. Differences between CREB binding to the two genes correlated with increasing methylation of the Wnt10b promoter following Bt(2)cAMP treatment, whereas no methylation of the cyclin D1 promoter was observed. Treatment of cells with the methylase inhibitor 5-azacytidine restored CREB binding to the Wnt10b gene promoter and prevented the inhibition of Wnt10b RNA expression by Bt(2)cAMP. We conclude that cAMP stimulates phosphorylation and binding of CREB to the cyclin D1 gene promoter. Simultaneously, hypermethylation of the Wnt10b gene promoter suppresses binding of CREB, allowing adipogenesis to proceed.

Effects of vasodilator stimulated phosphoprotein on intestinal barrier dysfunction after hemorrhagic shock in rats

Objective To observe the effects of vasedilator stimulated phosphoprotein (VASP) of small intestine mucous membrane on the intestinal barrier dysfunction during hemorrhagic shock (HS) in rats. Methods Forty Wistar rats were divided into normal group, HS 1 hour, HS 2 hours and HS 4 hours groups, as well as HS 2 hours + cyclic adenosine monophosphate (cAMP) treatment group. The activity of sermn diamine oxidase (DAO), content of hematoplasma D-lactic acid of each group were determined, and their relationship with the expression of VASP was analyzed. Results The expression of VASP was increased, serum DAO activity and hematoplasma D-lactic acid content were decreased by cAMP in rats at 2 hours after HS. There were differences upon the levels of VASP expression, DAO activity and hematoplasma D-lactic acid content between HS 2 hours group and HS 2 hours + cAMP treatment group ( t = 18.62, 9.28, 2.83, P < 0.05 ). The serum DAO activity increased while VASP expression decreased significantly after HS, which showed an obvious negative correlation between the two indexes (r=-0.95, P<0.05). Conclusions The decrease of VASP contributes to the intestinal barrier dysfunction after HS in rats, while the intestinal barrier dysfunction can be alleviated by cAMP. Key words: Shock; Intestinal barrier dysfunction; Vasodilator stimulated phosphoprotein

Reconsidering the impact of conflict on HIV infection among women in the era of antiretroviral treatment scale-up in sub-Saharan Africa: a gender lens

As increasing international and local efforts focus on scaling up access to antiretroviral treatment (ART) in sub-Saharan Africa the United Nations High Commissioner for Refugees (UNHCR) Medecins Sans Frontieres and the Joint United Nations Program on HIV/AIDS have increasingly advocated that the lack of essential medicines in conflict settings including ART is a violation of human rights. HIV infection and armed conflict are disproportionately represented in sub-Saharan Africa with women and children experiencing the largest burden of harms and adverse health outcomes due to both HIV infection risk and impact of conflict. Given emerging disparities in access to ART among HIV-positive women in conflict zones there is an urgent need to reconsider the impact of conflict on HIV infection. Importantly recent evidence suggests that there may be a protective effect of armed conflict on HIV prevalence as a result of reduced mobility and urbanization disruption of sexual networks and decreased casual consensual sex. At the same time substantial evidence suggests an increased vulnerability to HIV in conflict settings among women and children due to widespread human rights violations rape as a weapon of war forced occupational exposure the need for women to turn to sex work to support themselves and their families (i.e. survival sex) and reduced access to HIV prevention and reproductive health services. Regardless inequitable access to ART scale up in conflict zones suggests a need to re-examine the complex relationship between conflict and HIV infection. (excerpt)

Expression of stathmin, a microtubule regulatory protein, is associated with the migration and differentiation of cultured early trophoblasts

The microtubule-destabilizing protein stathmin is expressed by the villous cytotrophoblasts and invasive extravillous trophoblasts (EVTs) in the first-trimester human placenta. Here, we evaluated the significance of stathmin expression in terms of the functions of trophoblasts. We employed two choriocarcinoma cell lines (BeWo and JEG-3), an EVT cell line (HTR-8/SVneo) and isolated first-trimester trophoblast cells. The effects of small-interfering (si) RNA-mediated stathmin knockdown on trophoblast proliferation and migration were measured by WST-1 and Transwell assays, respectively. Trophoblast differentiation was induced by dibutyryl (db)-cAMP treatment and evaluated by measuring human chorionic gonadotrophin beta (hCGbeta) and syncytin expression and cell fusion. We examined the effect of knockdown and induced stathmin expression on db-cAMP-induced differentiation. siRNA-induced silencing of stathmin expression had a marked inhibitory effect on BeWo, JEG-3 and HTR-8/SVneo cell migration and also suppressed their proliferation, albeit to a lesser extent. db-cAMP-enhanced hCGbeta and syncytin expression and cell fusion in BeWo cells was inhibited by stathmin knockdown. However, induced expression of stathmin reversed the hCGbeta and syncytin expression and cell fusion in the Tet-On BeWo cells. Suppression of stathmin expression also inhibited the migration of and hCGbeta production by first-trimester trophoblasts. Stathmin expression may be closely associated with early trophoblast migration and differentiation into syncytiotrophoblasts during placentation.

GATA4 Reduction Enhances 3′,5′-Cyclic Adenosine 5′-Monophosphate-Stimulated Steroidogenic Acute Regulatory Protein Messenger Ribonucleic Acid and Progesterone Production in Luteinized Porcine Granulosa Cells

Previous studies with cultured granulosa cells implicated GATA4 in gonadotropin regulation of the steroidogenic acute regulatory protein (STAR) gene. Caveats to these prior studies exist. First, GATA4 levels are reduced in granulosa-luteal cells after the LH surge when GATA6 expression is relatively high. Second, STAR mRNA expression is negligible in granulosa cells until after the LH surge. Both exogenous GATA4 and GATA6 can transactivate STAR gene promoter constructs. We used an RNA interference (RNAi) approach to determine the contributions of GATA4 and GATA6 to cAMP analog regulation of the endogenous STAR gene in luteinizing granulosa cells. STAR mRNA was stimulated by cAMP under control RNAi conditions. Surprisingly, GATA4 reduction by its respective RNAi approximately doubled the cAMP induction of STAR mRNA. At 24 h cAMP treatment, this augmentation was abolished by co-down-regulation of GATA4+GATA6. GATA6 down-regulation by itself did not alter STAR mRNA levels. GATA4+GATA6 co-down-regulation elevated basal CYP11A mRNA at 24 h treatment but did not affect its induction by cAMP. Basal levels of HSD3B mRNA were reduced by GATA4 RNAi conditions leading to a greater fold induction of its mRNA by cAMP. Fold cAMP-stimulated progesterone production was enhanced by GATA4 down-regulation but not by GATA4+GATA6 co-down-regulation. These data implicate GATA6 as the facilitator in cAMP-stimulated STAR mRNA and downstream progesterone accumulation under reduced GATA4 conditions. Data also demonstrate that basal levels of GATA4/6 are not required for cAMP induction of the STAR gene. The altered ratio of GATA4 to GATA6 after ovulation may allow GATA6 to enhance STAR mRNA accumulation.

An analysis of the role of a retroendocytosis pathway in ABCA1-mediated cholesterol efflux from macrophages

The ATP binding cassette transporter A-1 (ABCA1) is critical for apolipoprotein-mediated cholesterol efflux, an important mechanism employed by macrophages to avoid becoming lipid-laden foam cells, the hallmark of early atherosclerotic lesions. It has been proposed that lipid-free apolipoprotein A-I (apoA-I) enters the cell and is resecreted as a lipidated particle via a retroendocytosis pathway during ABCA1-mediated cholesterol efflux from macrophages. To determine the functional importance of such a pathway, confocal microscopy was used to characterize the internalization of a fully functional apoA-I cysteine mutant containing a thiol-reactive fluorescent probe in cultured macrophages. ApoA-I was also endogenously labeled with (35)S-methionine to quantify cellular uptake and to determine the metabolic fate of the internalized protein. It was found that apoA-I was specifically taken inside macrophages and that a small amount of intact apoA-I was resecreted from the cells. However, a majority of the label that reappeared in the media was degraded. We estimate that the mass of apoA-I retroendocytosed is not sufficient to account for the HDL produced by the cholesterol efflux reaction. Furthermore, we have demonstrated that lipid-free apoA-I-mediated cholesterol efflux from macrophages can be pharmacologically uncoupled from apoA-I internalization into cells. On the basis these findings, we present a model in which the ABCA1-mediated lipid transfer process occurs primarily at the membrane surface in macrophages, but still accounts for the observed specific internalization of apoA-I.