AP180P Sigma-AldrichDonkey Anti-Goat IgG Antibody, HRP conjugate, Species Adsorbed

In the mammalian testis, the ubiquitin-proteasome system plays important roles in the process that promotes the formation of mature sperm. We recently identified zygote-specific proteasome assembly chaperone (ZPAC), which is specifically expressed in the mouse gonads and zygote. ZPAC mediates a unique proteasome assembly pathway in the zygote, but the expression profile and function of ZPAC in the testis is not fully understood. In this study, we investigated the possible role of ZPAC during mouse spermatogenesis. First, we analyzed the expression of ZPAC and 20S proteasome subunit α4/PSMA7 in the adult mouse testis. ZPAC and α4 were expressed in spermatogonia, spermatocytes, and round spermatids. In elongating spermatids, ZPAC was expressed until step 10, whereas expression of α4 persisted until step 12. We then examined the expression profile of ZPAC and α4 in a mouse model of experimental unilateral cryptorchidism. Consistent with appearance of morphologically impaired germ cells following cryptorchidism, the ZPAC protein level was significantly decreased at 4 days post induction of experimental cryptorchidism (D4) compared with the intact testis, although the amount of α4 protein persisted at least until D10. Moreover, intense ZPAC staining was co-localized with staining of annexin V, an early indicator of apoptosis in mammalian cells, in germ cells of cryptorchid testis, but ZPAC was also expressed in germ cells showing no detectable expression of annexin V. These results suggest that ZPAC plays a role during spermatogenesis and raises the possibility that 20S proteasome mediated by ZPAC may be involved in the regulation of germ cell survival during spermatogenesis.

Severe muscle wasting is a debilitating condition in critically ill intensive care unit (ICU) patients, characterized by general muscle weakness and dysfunction, resulting in a prolonged mobilization, delayed weaning from the ventilator, and a decreased quality of life post-ICU. The mechanisms underlying limb muscle weakness in ICU patients are complex and involve the impact of primary disease, but also factors common to critically ill ICU patients such as sepsis, mechanical ventilation (MV), immobilization, and systemic administration of corticosteroids (CS). These factors may have additive negative effects on skeletal muscle structure and function, but their respective role alone remain unknown. The primary aim of this study was to examine how CS administration potentiates ventilator and immobilization-related limb muscle dysfunction at the gene level. Comparing biceps femoris gene expression in pigs exposed to MV and CS for 5 days with only MV pigs for the same duration of time showed a distinct deregulation of 186 genes according to microarray. Surprisingly, the decreased force-generation capacity at the single muscle fiber reported in response to the addition of CS administration in mechanically ventilated and immobilized pigs was not associated with an additional upregulation of proteolytic pathways. On the other hand, an altered expression of genes regulating kinase activity, cell cycle, transcription, channel regulation, oxidative stress response, cytoskeletal, sarcomeric, and heat shock protein, as well as protein synthesis at the translational level, appears to play an additive deleterious role for the limb muscle weakness in immobilized ICU patients.

Degradation of maternally stored mRNAs after fertilization is an essential process for mammalian embryogenesis. Maternal mRNA degradation depending on deadenylases in mammalian early embryos has been mostly speculated, rather than directly demonstrated. Previously, we found that gene expression of nocturnin, which functions as a circadian clock-controlled deadenylase in mammalian cells, was clearly changed during the maternal-to-zygotic transition (MZT). Here, we investigated the possible role of nocturnin during mouse MZT. First, we examined the expression profile and localization of nocturnin in mouse oocytes and early embryos. The abundance of Nocturnin mRNA level was significantly decreased from the MII to 4-cell stages and slightly increased from the 8-cell to blastocyst stages, whereas the Nocturnin protein level was almost stable in all examined cells including GV and MII oocytes and early embryos. Nocturnin was localized in both the cytoplasm and the nucleus of all examined cells. We then examined the effect of loss or gain of Nocturnin function on early embryonic development. Knockdown of Nocturnin by injection of Nocturnin antisense expression vector into 1-cell embryos resulted in the delay of early embryonic development to the early blastocyst stage. Moreover, Nocturnin-overexpressed embryos by injection of Nocturnin expression vector impaired their development from the 1-cell to 2-cell or 4-cell stages. These results suggest that precise expression of nocturnin is critical to proper development of early mouse embryos. Functional analysis of nocturnin may contribute to the understanding of the possible role of the deadenylase at mouse MZT.

After fertilization, the sperm and oocyte genomes undergo extensive epigenetic reprogramming to form a totipotent zygote. The dynamic epigenetic changes during early embryo development primarily involve DNA methylation and demethylation. We have previously identified Gse (gonad-specific expression gene) to be expressed specifically in germ cells and early embryos. Its encoded protein GSE is predominantly localized in the nuclei of cells from the zygote to blastocyst stages, suggesting possible roles in the epigenetic changes occurring during early embryo development. Here, we report the involvement of GSE in epigenetic reprogramming of the paternal genome during mouse zygote development. Preferential binding of GSE to the paternal chromatin was observed from pronuclear stage 2 (PN2) onward. A knockdown of GSE by antisense RNA in oocytes produced no apparent effect on the first and second cell cycles in preimplantation embryos, but caused a significant reduction in the loss of 5-methylcytosine (5mC) and the accumulation of 5-hydroxymethylcytosine (5hmC) in the paternal pronucleus. Furthermore, DNA methylation levels in CpG sites of LINE1 transposable elements, Lemd1, Nanog and the upstream regulatory region of the Oct4 (also known as Pou5f1) gene were clearly increased in GSE-knockdown zygotes at mid-pronuclear stages (PN3-4), but the imprinted H19-differential methylated region was not affected. Importantly, DNA immunoprecipitation of 5mC and 5hmC also indicates that knockdown of GSE in zygotes resulted in a significant reduction of the conversion of 5mC to 5hmC on LINE1. Therefore, our results suggest an important role of maternal GSE for mediating active DNA demethylation in the zygote.

Exchange mechanisms across the blood-cerebrospinal fluid (CSF) barrier in the choroid plexuses within the cerebral ventricles control access of molecules to the central nervous system, especially in early development when the brain is poorly vascularised. However, little is known about their molecular or developmental characteristics. We examined the transcriptome of lateral ventricular choroid plexus in embryonic day 15 (E15) and adult mice. Numerous genes identified in the adult were expressed at similar levels at E15, indicating substantial plexus maturity early in development. Some genes coding for key functions (intercellular/tight junctions, influx/efflux transporters) changed expression during development and their expression patterns are discussed in the context of available physiological/permeability results in the developing brain. Three genes: Secreted protein acidic and rich in cysteine (Sparc), Glycophorin A (Gypa) and C (Gypc), were identified as those whose gene products are candidates to target plasma proteins to choroid plexus cells. These were investigated using quantitative- and single-cell-PCR on plexus epithelial cells that were albumin- or total plasma protein-immunopositive. Results showed a significant degree of concordance between plasma protein/albumin immunoreactivity and expression of the putative transporters. Immunohistochemistry identified SPARC and GYPA in choroid plexus epithelial cells in the embryo with a subcellular distribution that was consistent with transport of albumin from blood to cerebrospinal fluid. In adult plexus this pattern of immunostaining was absent. We propose a model of the cellular mechanism in which SPARC and GYPA, together with identified vesicle-associated membrane proteins (VAMPs) may act as receptors/transporters in developmentally regulated transfer of plasma proteins at the blood-CSF interface.

The melatonin system in preeclamptic pregnancies has been largely overlooked, especially in the placenta. We have previously documented melatonin production and expression of its receptors in normal human placentas. In addition, we and others have shown a beneficial role of melatonin in placental and fetal functions. In line with this, decreased maternal blood levels of melatonin are found in preeclamptic compared with normotensive pregnancies. However, melatonin production and expression of its receptors in preeclamptic compared with normotensive pregnancy placentas has never been examined. This study compares (i) melatonin-synthesizing enzyme expression and activity, (ii) melatonin and serotonin, melatonin's immediate precursor, levels and (iii) expression of MT1 and MT2 melatonin receptors in placentas from preeclamptic and normotensive pregnancies. Protein and mRNA expression of aralkylamine N-acetyltransferase (AANAT) and hydroxyindole O-methyltransferase (HIOMT), the melatonin-synthesizing enzymes, as well as MT1 and MT2 receptors were determined by RT-qPCR and Western blot, respectively. The activities of melatonin-synthesizing enzymes were assessed by radiometric assays while melatonin levels were determined by LC-MS/MS. There is a significant inhibition of AANAT, melatonin's rate-limiting enzyme, expression and activity in preeclamptic placentas, correlating with decreased melatonin levels. Likewise, MT1 and MT2 expression is significantly reduced in preeclamptic compared with normotensive pregnancy placentas. We propose that reduced maternal plasma melatonin levels may be an early diagnostic tool to identify pregnancies complicated by preeclampsia. This study indicates a clinical utility of melatonin as a potential treatment for preeclampsia in women where reduced maternal plasma levels have been identified.

ATP-sensitive K(+) (K(ATP)) channel subunits were investigated in rat submandibular gland (SMG). RT-PCR detected the presence of mRNA transcripts of the Kir6.1, Kir6.2, SUR2A, and SUR2B in the SMG, whereas SUR1 mRNA was barely detected. Western blot analysis provided the evidence that these four K(ATP) channel subunits are expressed in rat SMG. Immunostaining detected that these four K(ATP) channel subunits are widely distributed, with different intensities, in myoepithelial cells, epithelial cells of intercalated ducts, granular convoluted tubules, striated ducts, and excretory ducts. Immunofluorescence double staining showed that Kir6.1 and Kir6.2 colocalized with SUR2A in the myoepithelial cells, granular convoluted tubules, striated ducts, and excretory ducts. Kir6.1 and Kir6.2 also colocalized with SUR2B, mainly in the duct system, e.g., the granular convoluted tubules, striated ducts, and excretory ducts. Taken together, these results indicate that the K(ATP) channels in SMG may consist of Kir6.1, Kir6.2, SUR2A, and SUR2B, with various combinations of colocalization with each other, and may play important roles in rat SMG during salivary secretion.

Human respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infection in infants, immunocompromised patients, and the elderly. The RSV fusion (F) protein mediates fusion of the viral envelope with the target cell membrane during virus entry and is a primary target for antiviral drug and vaccine development. The F protein contains two heptad repeat regions, HR1 and HR2. Peptides corresponding to these regions form a six-helix bundle structure that is thought to play a critical role in membrane fusion. However, characterization of six-helix bundle formation in native RSV F protein has been hindered by the fact that a trigger for F protein conformational change has yet to be identified. Here we demonstrate that RSV F protein on the surface of infected cells undergoes a conformational change following exposure to elevated temperature, resulting in the formation of the six-helix bundle structure. We first generated and characterized six-helix bundle-specific antibodies raised against recombinant peptides modeling the RSV F protein six-helix bundle structure. We then used these antibodies as probes to monitor RSV F protein six-helix bundle formation in response to a diverse array of potential triggers of conformational changes. We found that exposure of 'membrane-anchored' RSV F protein to elevated temperature (45-55 degrees C) was sufficient to trigger six-helix bundle formation. Antibody binding to the six-helix bundle conformation was detected by both flow cytometry and cell-surface immunoprecipitation of the RSV F protein. None of the other treatments, including interaction with a number of potential receptors, resulted in significant binding by six-helix bundle-specific antibodies. We conclude that native, untriggered RSV F protein exists in a metastable state that can be converted in vitro to the more stable, fusogenic six-helix bundle conformation by an increase in thermal energy. These findings help to better define the mechanism of RSV F-mediated membrane fusion and have important implications for the identification of therapeutic strategies and vaccines targeting RSV F protein conformational changes.

Abstract The degree to which coral reef ecosystems will be impacted by global climate change depends on regional and local differences in corals' susceptibility and resilience to environmental stressors. Here, we present data from a reciprocal transplant experiment using the common reef building coral Porites lobata between a highly fluctuating back reef environment that reaches stressful daily extremes, and a more stable, neighbouring forereef. Protein biomarker analyses assessing physiological contributions to stress resistance showed evidence for both fixed and environmental influence on biomarker response. Fixed influences were strongest for ubiquitin-conjugated proteins with consistently higher levels found in back reef source colonies both pre and post-transplant when compared with their forereef conspecifics. Additionally, genetic comparisons of back reef and forereef populations revealed significant population structure of both the nuclear ribosomal and mitochondrial genomes of the coral host (F(ST) = 0.146 P < 0.0001, F(ST) = 0.335 P < 0.0001 for rDNA and mtDNA, respectively), whereas algal endosymbiont populations were genetically indistinguishable between the two sites. We propose that the genotype of the coral host may drive limitations to the physiological responses of these corals when faced with new environmental conditions. This result is important in understanding genotypic and environmental interactions in the coral algal symbiosis and how corals may respond to future environmental changes.

In mammals, circadian genes, Clock, Arntl (also known as Bmal1), Cry1, Cry2, Per1, Per2, and Per3, are rhythmically transcribed every 24 h in almost all organs and tissues to tick the circadian clock. However, their expression and function in oocytes and preimplantation embryos have not been investigated. In this study we found that the circadian clock may stop in mouse oocytes and preimplantation embryos. Real-time PCR analysis revealed the presence of transcripts of these genes in both oocytes and preimplantation embryos; however, their amounts did not oscillate every 24 h in one- to four-cell and blastocyst-stage embryos. Moreover, immunofluorescence analyses revealed that CLOCK, ARNTL, and CRY1 were localized similarly in the nuclei of germinal vesicle (GV) oocytes and one-cell- to four-cell-stage embryos. Because CRY1 is known to interact with the CLOCK-ARNTL complex to suppress transcription-promoting activity of the complex for genes such as Wee1, Cry2, Per1, Per2, and Per3 in cells having the ticking circadian clock, we hypothesized that if the circadian clock functions in GV oocytes and one-cell- to four-cell-stage embryos, CLOCK, ARNTL, and CRY1 might suppress the transcription of these genes in GV oocytes and one-cell- to 4-cell-stage embryos as well. As a result, knockdown of CRY1 in GV oocytes by RNA interference did not affect the transcription levels of Wee1, Cry2, Per1, Per2, and Per3, but it reduced maturation ability. Thus, it seems that circadian genes are not involved in circadian clock regulation in mouse oocytes and preimplantation embryos but are involved in physiologies, such as meiosis.