What regulates placental steroidogenesis in 90-day pregnant ewes?

By day-90, the placenta secretes half of the circulating progesterone and 85% of the circulating estradiol-17beta [Weems YS, Vincent D, Tanaka Y, et al. Effects of prostaglandin F(2alpha) on sources of progesterone and pregnancy in intact, ovariectomized, and hysterectomized 90-100 day pregnant ewes. Prostaglandins 1992;43:203-22; Weems YS, Vincent DL, Nusser K, et al. Effects of prostaglandin F(2alpha) (PGF(2alpha)) on secretion of estradiol-17beta and cortisol in 90-100 day hysterectomized, intact, or ovariectomized pregnant ewes. Prostaglandins 1994;48:139-57]. Ovariectomy (OVX) or prostaglandin (PG) F(2alpha) (PGF(2alpha)) does not abort intact or OVX 90-day pregnant ewes and PGF(2alpha) regresses the corpus luteum, but does not affect placental progesterone secretion in vivo [Weems YS, Vincent D, Tanaka Y, et al. Effects of prostaglandin F(2alpha) on sources of progesterone and pregnancy in intact, ovariectomized, and hysterectomized 90-100 day pregnant ewes. Prostaglandins 1992;43:203-22]. Luteal progesterone secretion in vitro at day-90 of pregnancy in ewes is regulated by PGE(1)and/or PGE(2), not by ovine luteinizing hormone (LH; 3). Concentrations of PGE in uterine or ovarian venous plasma averaged 6 ng/ml at 90-100 days of pregnancy in ewes [Weems YS, Vincent DL, Tanaka Y, Nusser K, Ledgerwood KS, Weems CW. Effect of prostaglandin F(2alpha) on uterine or ovarian secretion of prostaglandins E and F(2alpha) (PGE; PGF(2alpha)) in vivo in 90-100 day hysterectomized, intact or ovariectomized pregnant ewes. Prostaglandins. 1993;46:277-96]. Ovine placental PGE secretion is regulated by LH up to day-50 and by pregnancy specific protein B (PSPB) after day-50 of pregnancy [Weems YS, Kim L, Humphreys V, Tsuda V, Weems CW. Effect of luteinizing hormone (LH), pregnancy specific protein B (PSPB), or arachidonic acid (AA) on ovine endometrium of the estrous cycle or placental secretion of prostaglandins E(2) (PGE(2)) and F(2alpha) (PGF(2alpha)), and progesterone in vitro. Prostaglandins Other Lipid Mediators 2003;71:55-73]. Indomethacin (INDO), a prostaglandin synthesis inhibitor [Lands WEM. The biosynthesis and metabolism of prostaglandins. Annu Rev Physiol 1979;41:633-46], lowers jugular venous progesterone [Bridges PJ, Weems YS, Kim L, et al. Effect of prostaglandin F(2alpha) (PGF(2alpha)), indomethacin, tamoxifen or estradiol-17beta on pregnancy, progesterone and pregnancy specific protein B (PSPB) secretion in 88-90 day pregnant ewes. Prostaglandins Other Lipid Mediators 1999;58:113-24] and inferior vena cava PGE of pregnant ewes with ovaries by half at day-90 [Bridges PJ, Weems YS, Kim L, LeaMaster BR, Vincent DL, Weems CW. Effect of prostaglandin F(2alpha) (PGF(2alpha)), indomethacin, tamoxifen or estradiol-17beta on prostaglandin E (PGE), PGF(2alpha) and estradiol-17beta secretion in 88-90 day pregnant sheep. Prostaglandins Other Lipid Mediators 1999;58:167-78]. In addition, treatment of 90 day ovine diced placental slices with androstenedione in vitro increased placental estradiol-17beta, but treatment with PGF(2alpha)in vitro did not decrease placental progesterone secretion, which indicates that ovine placenta progesterone secretion is resistant to the luteolytic action of PGF(2alpha) [Weems YS, Bridges PJ, LeaMaster BR, Sasser RG, Vincent DL, Weems CW. Secretion of progesterone, estradiol-17beta, prostaglandins (PG) E (PGE), F(2alpha) (PGF(2alpha)), and pregnancy specific protein B (PSPB) by day 90 intact or ovariectomized pregnant ewes. Prostaglandins Other Lipid Mediators 1999;58:139-48]. This also explains why ovine uterine secretion of decreased around day-50 [Weems YS, Kim L, Humphreys V, Tsuda V, Weems CW. Effect of luteinizing hormone (LH), pregnancy specific protein B (PSPB), or arachidonic acid (AA) on ovine endometrium of the estrous cycle or placental secretion of prostaglandins E(2) (PGE(2)) and F(2alpha) (PGF(2alpha)), and progesterone in vitro. Prostaglandins Other Lipid Mediators 2003;71:55-73], when placental estradiol-17beta secretion is increasing [Weems C, Weems Y, Vincent D. Maternal recognition of pregnancy and maintenance of gestation in sheep. In: Reproduction and animal breeding: advances and strategies. Enne G, Greppi G, Lauria A, editors, Elsevier Pub., Amsterdam 1995. p. 277-93]. Treatment of 90 day pregnant ewes with estradiol-17beta+ PGF(2alpha), but not either treatment alone, caused a linear increase in both estradiol-17beta and PGF(2alpha) and ewes were aborting [Bridges PJ, Weems YS, Kim L, Sasser RG, LeaMaster BR, Vincent DL, Weems CW. Effect of prostaglandin F(2alpha) (PGF(2alpha)), indomethacin, tamoxifen or estradiol-17beta on pregnancy, progesterone and pregnancy specific protein B (PSPB) secretion in 88-90 day pregnant ewes. Prostaglandins Other Lipid Mediators 1999;58:113-24; Bridges PJ, Weems YS, Kim L, LeaMaster BR, Vincent DL, Weems CW. Effect of prostaglandin F(2alpha) (PGF(2alpha)), indomethacin, tamoxifen or estradiol-17beta on prostaglandin E (PGE), PGF(2alpha) and estradiol-17beta secretion in 88-90 day pregnant sheep. Prostaglandins Other Lipid Mediators 1999;58:167-78]. Pregnant ewes OVX on day 83 of pregnancy and placental slices cultured in vitro secretes 2-3-fold more estradiol-17beta, PSPB, PGE, and progesterone than placental slices from 90 day intact pregnant ewes, but placental PGF(2alpha) secretion by placental slices from intact or OVX ewes did not change [Denamur R, Kann G, Short R V. How does the corpus luteum of the sheep know that there is an embryo in the uterus? In: Pierrepont G, editor. Endocrinology of pregnancy and parturition, vol. 2. Cardiff, Wales, UK: Alpha Omega Pub Co.; 1973. p. 4-38]. The objective of these experiments was to determine what regulates ovine placental progesterone and estradiol-17beta secretion at day-90 of pregnancy, since the hypophysis [Casida LE, Warwick J. The necessity of the corpus luteum for maintenance of pregnancy in the ewe. J Anim Sci 1945;4:34-9] or ovaries [Weems CW, Weems YS, Randel RD. Prostaglandins and reproduction in female farm animals. Vet J 2006;171:206-28] are not necessary after day-55 to maintain pregnancy. In Experiment 1, diced placental slices from day-90 intact or OVX pregnant ewes that were ovariectomized or laparotomized and ovaries were not removed on day 83 were collected on day-90 and incubated in vitro in M-199 with Vehicle, ovine luteinizing hormone (oLH), ovine follicle stimulating hormone (oFSH), ovine placental lactogen (oPL), PGE(l), PGE(2), PGD(2), PGI(2), insulin-like growth factor (IGF) 1 or 2 (IGF(l); IGF(2)), leukotriene C(4) (LTC(4)), platelet activating factor (PAF) 16 or 18 (PAF-16; PAF-18) at doses of 0, 1, 10, or 100ng/ml for 4h. In Experiment 2, placental slices from day-90 intact and OVX (intact or OVX laporotomized 7 days earlier) pregnant ewes were incubated in vitro with vehicle, INDO, Meclofenamate (MECLO), PGE(l), PGE(2), INDO+PGE(1), MECLO+PGE(l), INDO+PGE(2), or MECLO+PGE(2) for 4h. Media were analyzed for progesterone, estradiol-17beta, PGE, or PGF(2alpha) by RIA. Hormone data in media were analyzed in Experiment 1 by a 2x3x13 and in Experiment 2 by a 2x9 Factorial Design for ANOVA. In Experiment 1, placental progesterone, PGE, or estradiol-17beta secretion were increased (P< or =0.05) two-fold by OVX. Progesterone was not increased (P> or =0.05) by any treatment other than OVX and only FSH increased (P< or =0.05) estradiol-17beta secretion by placental slices in both OVX and intact ewes 90-day pregnant ewes. In Experiment 2, INDO or MECLO decreased (P< or =0.05) placental progesterone secretion by 88% but did not decrease (P> or =0.05) placental estradiol-17beta secretion from intact or OVX ewes. PGE(l) or PGE(2) increased (P< or =0.05) progesterone secretion only in ewes treated with INDO or MECLO. It is concluded that FSH probably regulates day-90 ovine placental estradiol-17beta secretion, while PGE(l) or PGE(2) regulates day-90 placental progesterone secretion.     

Distinct Roles of Molecular Chaperones HSP90α and HSP90β in the Biogenesis of KCNQ4 Channels

Loss-of-function mutations in the KCNQ4 channel cause DFNA2, a subtype of autosomal dominant non-syndromic deafness that is characterized by progressive sensorineural hearing loss. Previous studies have demonstrated that the majority of the pathogenic KCNQ4 mutations lead to trafficking deficiency and loss of KCNQ4 currents. Over the last two decades, various strategies have been developed to rescue trafficking deficiency of pathogenic mutants; the most exciting advances have been made by manipulating activities of molecular chaperones involved in the biogenesis and quality control of the target protein. However, such strategies have not been established for KCNQ4 mutants and little is known about the molecular chaperones governing the KCNQ4 biogenesis. To identify KCNQ4-associated molecular chaperones, a proteomic approach was used in this study. As a result, two major molecular chaperones, HSP70 and HSP90, were identified and then confirmed by reciprocal co-immunoprecipitation assays, suggesting that the HSP90 chaperone pathway might be involved in the KCNQ4 biogenesis. Manipulating chaperone expression further revealed that two different isoforms of HSP90, the inducible HSP90α and the constitutive HSP90β, had opposite effects on the cellular level of the KCNQ4 channel; that HSP40, HSP70, and HOP, three key components of the HSP90 chaperone pathway, were crucial in facilitating KCNQ4 biogenesis. In contrast, CHIP, a major E3 ubiquitin ligase, had an opposite effect. Collectively, our data suggest that HSP90α and HSP90β play key roles in controlling KCNQ4 homeostasis via the HSP40-HSP70-HOP-HSP90 chaperone pathway and the ubiquitin-proteasome pathway. Most importantly, we found that over-expression of HSP90β significantly improved cell surface expression of the trafficking-deficient, pathogenic KCNQ4 mutants L274H and W276S. KCNQ4 surface expression was restored by HSP90β in cells mimicking heterozygous conditions of the DFNA2 patients, even though it was not sufficient to rescue the function of KCNQ4 channels.     

Asymmetrical radical formation in D- and L-alanines irradiated with yttrium-90β-rays

Radical formation in^{9 0}Y--irradiated D- and L-alanines was studied using ESR. It was observed that the relative radical concentration by -irradiation was distinguishably (13.9–21.5%) more in D-alanine than in L-alanine. Discussion was made on the possible mechanisms for the observed results.     

Cell surface heparan sulfate proteoglycans are involved in the binding of Hsp90α and Hsp90β to the cell plasma membrane

Extracellular membrane-bound and secreted heat shock protein 90 (Hsp90) is known to be involved in cell motility and invasion. The mechanism of Hsp90 anchoring to the plasma membrane remains obscure. We showed that treatment of human glioblastoma A-172 and fibrosarcoma HT1080 cells with sodium chlorate, heparinase, and heparin causes a prominent loss of 2 Hsp90 cytosolic isoforms, Hsp90α and Hsp90β, from the cell surface and strongly inhibits the binding of exogenous Hsp90 to cells. We revealed that Hsp90α and Hsp90β are partly colocalized with heparan sulfate proteoglycans (HSPGs) on the cell surface and that this colocalization was sensitive to heparin. The results demonstrate that cell surface HSPGs are involved in the binding/anchoring of Hsp90α and Hsp90β to the plasma membrane.     

Stat1 mediates an auto-regulation of hsp90β gene in heat shock response

We have reported earlier that a heat shock element in the first intron of human hsp90β gene (iHSE) acts as an intronic enhancer to bind the heat shock factor (HSF1) and activates hsp90β gene under heat shock. Here, we show that, in addition to the HSF1, Stat1 phosphorylation is indispensable in the event. We show that Jak2, a Janus kinase specifically associated with the β subunit of IFNγ receptor, and PKCε? an isoform of the atypical PKC family, are the two dominant kinases responsible for the heat shock induced phosphorylation on Y701 and S727 of Stat1. However, the activation of these kinases under heat shock requires the association of chaperone proteins of the Hsp90 family, in particular, the Hsp90β under heat shock. Furthermore, Brg1, an ATPase subunit of the SWI/SNF chromatin remodeling complex is likely recruited by HSF1 and Stat1 at the iHSE under heat shock. Brg1 further confers an open chromatin conformation at the promoter region that is pivotal to the heat shock induced fully activation of the hsp90β gene in Jurkat cells. This is a novel example of how multiple activation steps occur under heat shock, first on the kinases and then the Stat1 and the SWI/SNF chromatin remodeling complex that follows to conduct an auto-regulation based fully activation of the gene.     

The role of secreted heat shock protein-90 (Hsp90) in wound healing - how could it shape future therapeutics?

Introduction: Defects in tissue repair or wound healing pose a clinical, economic and social problem worldwide. Despite decades of studies, there have been few effective therapeutic treatments.

Areas covered: We discuss the possible reasons for why growth factor therapy did not succeed. We point out the lack of human disorder-relevant animal models as another blockade for therapeutic development. We summarize the recent discovery of secreted heat shock protein-90 (Hsp90) as a novel wound healing agent.

Expert commentary: Wound healing is a highly complex and multistep process that requires participations of many cell types, extracellular matrices and soluble molecules to work together in a spatial and temporal fashion within the wound microenvironment. The time that wounds remain open directly correlates with the clinical mortality associated with wounds. This time urgency makes the healing process impossible to regenerate back to the unwounded stage, rather forces it to take many shortcuts in order to protect life. Therefore, for therapeutic purpose, it is crucial to identify so-called ‘driver genes’ for the life-saving phase of wound closure. Keratinocyte-secreted Hsp90α was discovered in 2007 and has shown the promise by overcoming several key hurdles that have blocked the effectiveness of growth factors during wound healing.     

Role of Hsp90 in Biogenesis of the β-Cell ATP-sensitive Potassium Channel Complex

The pancreatic β-cell ATP-sensitive potassium (K_ATP) channel is a multimeric protein complex composed of four inwardly rectifying potassium channel (Kir6.2) and four sulfonylurea receptor 1 (SUR1) subunits. K_ATP channels play a key role in glucose-stimulated insulin secretion by linking glucose metabolism to membrane excitability. Many SUR1 and Kir6.2 mutations reduce channel function by disrupting channel biogenesis and processing, resulting in insulin secretion disease. To better understand the mechanisms governing K_ATP channel biogenesis, a proteomics approach was used to identify chaperone proteins associated with K_ATP channels. We report that chaperone proteins heat-shock protein (Hsp)90, heat-shock cognate protein (Hsc)70, and Hsp40 are associated with β-cell K_ATP channels. Pharmacologic inhibition of Hsp90 function by geldanamycin reduces, whereas overexpression of Hsp90 increases surface expression of wild-type K_ATP channels. Coimmunoprecipitation data indicate that channel association with the Hsp90 complex is mediated through SUR1. Accordingly, manipulation of Hsp90 protein expression or function has significant effects on the biogenesis efficiency of SUR1, but not Kir6.2, expressed alone. Interestingly, overexpression of Hsp90 selectively improved surface expression of mutant channels harboring a subset of disease-causing SUR1 processing mutations. Our study demonstrates that Hsp90 regulates biogenesis efficiency of heteromeric K_ATP channels via SUR1, thereby affecting functional expression of the channel in β-cell membrane.     

Cell surface heparan sulfate proteoglycans are involved in the binding of Hsp90α and Hsp90β to the cell plasma membrane

Extracellular membrane-bound and secreted heat shock protein 90 (Hsp90) is known to be involved in cell motility and invasion. The mechanism of Hsp90 anchoring to the plasma membrane remains obscure. We showed that treatment of human glioblastoma A-172 and fibrosarcoma HT1080 cells with sodium chlorate, heparinase, and heparin causes a prominent loss of 2 Hsp90 cytosolic isoforms, Hsp90α and Hsp90β, from the cell surface and strongly inhibits the binding of exogenous Hsp90 to cells. We revealed that Hsp90α and Hsp90β are partly colocalized with heparan sulfate proteoglycans (HSPGs) on the cell surface and that this colocalization was sensitive to heparin. The results demonstrate that cell surface HSPGs are involved in the binding/anchoring of Hsp90α and Hsp90β to the plasma membrane.     

Role for Heat Shock Protein 90α in the Proliferation and Migration of HaCaT Cells and in the Deep Second-Degree Burn Wound Healing in Mice

Inflammation, proliferation, and tissue remodeling are essential steps for wound healing. The hypoxic wound microenvironment promotes cell migration through a hypoxia—heat shock protein 90 alpha (Hsp90α)—low density lipoprotein receptor-related protein-1 (LRP-1) autocrine loop. To elucidate the role of this autocrine loop on burn wound healing, we investigated the expression profile of Hsp90α at the edge of burn wounds and found a transient increase in both mRNA and protein levels. Experiments performed with a human keratinocyte cell line—HaCaT also confirmed above results. 17-dimethylaminoethylamino-17demethoxygeldanamycin hydrochloride (17-DMAG), an Hsp90α inhibitor, was used to further evaluate the function of Hsp90α in wound healing. Consistently, topical application of Hsp90α in the early stage of deep second-degree burn wounds led to reduced inflammation and increased tissue granulation, with a concomitant reduction in the size of the wound at each time point tested (p<0.05). Consequently, epidermal cells at the wound margin progressed more rapidly causing an expedited healing process. In conclusion, these results provided a rationale for the therapeutic effect of Hsp90α on the burn wound management.     

Hsp90 modulates PPARγ activity in a mouse model of nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) is a highly prevalent complication of obesity, yet cellular mechanisms that lead to its development are not well defined. Previously, we have documented hepatic steatosis in mice carrying a mutation in the Sec61a1 gene. Here we examined the mechanism behind NAFLD in Sec61a1 mutant mice. Livers of mutant mice exhibited upregulation of Pparg and its target genes Cd36, Cidec, and Lpl, correlating with increased uptake of fatty acid. Interestingly, these mice also displayed activation of the heat shock response (HSR), with elevated levels of heat shock protein (Hsp) 70, Hsp90, and heat shock factor 1. In cell lines, inhibition of Hsp90 function reduced Pparγ signaling and protein levels. Conversely, overexpression of Hsp90 increased Pparγ signaling and protein levels by reducing degradation. This may occur via a physical interaction as Hsp90 and Pparγ coimmunoprecipitated in vivo. Furthermore, inhibition of Hsp90 in Sec61a1 mutant hepatocytes also reduced Pparγ protein levels and signaling. Finally, overexpression of Hsp90 in liver cell lines increased neutral lipid accumulation, and this accumulation was blocked by Hsp90 inhibition. Our results show that the HSR and Hsp90 play an important role in the development of NAFLD, opening new avenues for the prevention and treatment of this highly prevalent disease.     

Ultradian rhythmicity (circa 90 min) in physiologic processes ‐ evidence for multioscillatory phenomena

Abstract

Data accumulated in recent years have shown the existence of multiple about 90 min ultradian rhythms in gastric motility, urine flow and osmolality and physiologic indices of arousal. While these data support Kleitman's hypothesis that the REM‐NONREM sleep cycles are only fragments of a 24‐h rhythm (Basic Rest‐Activity Cycle) which is manifested in wakefulness in recurrent fluctuations in arousal, it further indicates that both the sleep and the waking rhythms are part of a more complex multioscillatory system with a dominant periodicity centered at 90 min.     

Non-Chinese Immigrants: Challenge Faced by Yunnan of China to Achieve the 90–90–90 Goals

正China’s AIDS epidemic started among people who inject drugs(PWID)in Ruili county-level city,a China–Myanmar border city of Yunnan Province,in 1989(Fig.1).Since then HIV has spread rapidly across the country(He and Detels 2005).China launched a‘‘Four Frees and One Care’’policy in 2003 to fight against HIV/AIDS.As one of     

新型清洗液--DECON90

所有实验室都会遇到清洗的问题，高标准的清洗是需要的，这就给实验室带来了比较困难的问题和繁重的工作。特别是随着科学技术的进步和发展，对清洗的要求和标准也有了更高的要求。迄今为止在中国许多实验室一直是在延续使用铬酸盐混合洗液或类似腐蚀剂如硫酸做清洗液，或家用洗衣粉，家用洗涤灵等清洗产品，这一过程的缺点是：     

Growth stimulating activity of heat shock protein 90α to lymphoid cell lines in serum-free medium

A growth stimulating factor was purified from the culture supernatant of human-human hybridoma SH-76 cells in serum-free RPMI 1640 medium by the serial use of DEAE anion and heparin affinity chromatographies. The factor was a 85 kDa protein on SDS-polyacrylamide gel electrophoresis. N-terminal amino acid sequence (PEETQTQDQPME) of the protein was coincident with that of heat shock protein 90α (HSP90α). The protein reacted with anti-HSP90 monoclonal antibody. These results suggest that the protein was a member of HSP90α family after taking other circumstantial evidence into account. The protein stimulated the growth of some lymphoid cell lines such as human B-lymphoblastoid cell line HO-323-3 hybridomas derived from HO-323, and several other lymphoid cell lines. There were several lymphoid cell lines which did not respond to the protein. Growth stimulating activity of the protein was heat-unstable and significantly decreased at above 60°C. These are the first data that describe growth-stimulating activity of heat shock protein 90α.     

真菌Hsp90研究进展

近年来,随着广谱抗生素、化疗以及器官移植技术的广泛应用,真菌感染日益严重,从分子水平揭示病原真菌的致病机制对真菌感染的防控、治疗至关重要。微生物适应宿主微环境压力的能力在其共生与感染过程中发挥着关键作用,heat shock protein 90（Hsp90）是真核生物参与压力应答响应的分子伴侣,它不仅参与胞内蛋白质的折叠,还与许多底物蛋白相互作用共同调节病原真菌的形态发育、生物被膜形成、有性生殖、毒力以及耐药性。本文从真菌Hsp90的活性调节、底物蛋白,以及Hsp90与病原真菌形态发生、有性生殖、耐药性调控等方面综述了近年来真菌Hsp90信号通路的研究进展。