Characterization of equine zona pellucida glycoproteins by polyacrylamide gel electrophoresis and immunological techniques.

This study was designed to explore the composition of the equine zona pellucida (EZP) by one- and two-dimensional polyacrylamide gel electrophoresis (1D- and 2D-PAGE), silver staining and immunoblotting techniques. Antral follicles palpable on frozen-thawed equine ovaries were aspirated with a needle and syringe, and the resultant follicular fluid, cellular material and oocytes were pooled. Oocytes were placed in Petri dishes, moved by narrow-bore pipette to droplets of phosphate-buffered saline (PBS) and mechanically cleaned of cumulus cells. The EZP from these collected oocytes was solubilized, and then analysed by 1D- and 2D-PAGE. Silver stained 2D-PAGE of the EZP revealed the presence of three EZP glycoprotein families of apparent molecular mass ranges of 93-120 kDa, 73-90 kDa and 45-80 kDa. Immunoblot analysis of EZP glycoproteins resolved by 2D-PAGE using rabbit antisera against pig zonae pellucidae (R alpha HSPZ) confirmed the presence of three EZP glycoprotein families and established the existence of common epitopes between equine and porcine ZP glycoproteins. Further immunodetection using 2D-PAGE-separated glycoproteins illustrated that the 45-80 kDa family is recognized by the monoclonal antibody R5, developed against the porcine ZP glycoprotein of molecular mass 55-120 kDa. Guinea-pig antiserum against endo-beta-galactosidase-treated rabbit ZP 55 kDa glycoprotein (R55K), which specifically recognizes the rabbit ZP glycoprotein with the lowest molecular mass, also recognized the EZP 45-80 kDa glycoprotein family. Guinea-pig polyclonal antisera developed against total heat-solubilized rabbit ZP (GP alpha HSRZ) recognized the 73-90 kDa EZP glycoprotein family exclusively. After heat solubilization and treatment of EZP with endo-beta-galactosidase to remove polylactosaminoglycans, silver stained 1D-PAGE again demonstrated the presence of three glycoproteins with apparent molecular masses of 60, 75 and 90 kDa. The partially deglycosylated 60 kDa equine glycoprotein is recognized on immunoblot by the monoclonal antibody R5; the 75 kDa EZP glycoprotein is recognized by GP alpha HSRZ; and all three EZP glycoproteins separated by 1D-PAGE are recognized by R alpha HSPZ. These data add further support to the concept of cross-species zona pellucida glycoprotein antigenicity.     

Repeated oral administration of capsaicin increases anxiety-like behaviours with prolonged stress-response in rats

This study was conducted to examine the psycho-emotional effects of repeated oral exposure to capsaicin, the principal active component of chili peppers. Each rat received 1 mL of 0.02% capsaicin into its oral cavity daily, and was subjected to behavioural tests following 10 daily administrations of capsaicin. Stereotypy counts and rostral grooming were significantly increased, and caudal grooming decreased, in capsaicin-treated rats during the ambulatory activity test. In elevated plus maze test, not only the time spent in open arms but also the percent arm entry into open arms was reduced in capsaicin-treated rats compared with control rats. In forced swim test, although swimming duration was decreased, struggling increased in the capsaicin group, immobility duration did not differ between the groups. Repeated oral capsaicin did not affect the basal levels of plasma corticosterone; however, the stress-induced elevation of plasma corticosterone was prolonged in capsaicin treated rats. Oral capsaicin exposure significantly increased c-Fos expression not only in the nucleus tractus of solitarius but also in the paraventricular nucleus. Results suggest that repeated oral exposure to capsaicin increases anxiety-like behaviours in rats, and dysfunction of the hypothalamic-pituitary-adrenal axis may play a role in its pathophysiology.     

Effect of weight loss and exercise on angiogenic factors in the circulation and in adipose tissue in obese subjects

Background:

Vascular growth is a prerequisite for adipose tissue (AT) development and expansion. Some AT cytokines and hormones have effects on vascular development, like vascular endothelial growth factor (VEGF‐A), angiopoietin (ANG‐1), ANG‐2 and angiopoietin‐like protein‐4 (ANGPTL‐4).

Methods:

In this study, the independent and combined effects of diet‐induced weight loss and exercise on AT gene expression and proteins levels of those angiogenic factors were investigated. Seventy‐nine obese males and females were randomized to: 1. Exercise‐only (EXO; 12‐weeks exercise without diet‐restriction), 2. Hypocaloric diet (DIO; 8‐weeks very low energy diet (VLED) + 4‐weeks weight maintenance diet) and 3. Hypocaloric diet and exercise (DEX; 8‐weeks VLED + 4‐weeks weight maintenance diet combined with exercise throughout the 12 weeks). Blood samples and fat biopsies were taken before and after the intervention.

Results:

Weight loss was 3.5 kg in the EXO group and 12.3 kg in the DIO and DEX groups. VEGF‐A protein was non‐significantly reduced in the weight loss groups. ANG‐1 protein levels were significantly reduced 22‐25% after all three interventions (P < 0.01). The ANG‐1/ANG‐2 ratio was also decreased in all three groups (P < 0.05) by 27‐38%. ANGPTL‐4 was increased in the EXO group (15%, P < 0.05) and 9% (P < 0.05) in the DIO group. VEGF‐A, ANG‐1, and ANGPTL‐4 were all expressed in human AT, but only ANGPTL‐4 was influenced by the interventions.

Conclusions:

Our data show that serum VEGF‐A, ANG‐1, ANG‐2, and ANGPTL‐4 levels are influenced by weight changes, indicating the involvement of these factors in the obese state. Moreover, it was found that weight loss generally was associated with a reduced angiogenic activity in the circulation.     

β-arrestin 2 attenuates cardiac dysfunction in polymicrobial sepsis through gp130 and p38

Sepsis is an exaggerated systemic inflammatory response to persistent bacteria infection with high morbidity and mortality rate clinically. β-arrestin 2 modulates cell survival and cell death in different systems. However, the effect of β-arrestin 2 on sepsis-induced cardiac dysfunction is not yet known. Here, we show that β-arrestin 2 overexpression significantly enhances animal survival following cecal ligation and puncture (CLP)-induced sepsis. Importantly, overexpression of β-arrestin 2 in mice prevents CLP-induced cardiac dysfunction. Also, β-arrestin 2 overexpression dramatically attenuates CLP-induced myocardial gp130 and p38 mitogen-activated protein kinase (MAPK) phosphorylation levels following CLP. Therefore, β-arrestin 2 prevents CLP-induced cardiac dysfunction through gp130 and p38. These results suggest that modulation of β-arrestin 2 might provide a novel therapeutic approach to prevent cardiac dysfunction in patients with sepsis.     

Ligand-induced dynamic membrane changes and cell deletion conferred by vanilloid receptor 1

The real time dynamics of vanilloid-induced cytotoxicity and the specific deletion of nociceptive neurons expressing the wild-type vanilloid receptor (VR1) were investigated. VR1 was C-terminally tagged with either the 27-kDa enhanced green fluorescent protein (eGFP) or a 12-amino acid epsilon-epitope. Upon exposure to resiniferatoxin, VR1eGFP- or VR1epsilon-expressing cells exhibited pharmacological responses similar to those of cells expressing the untagged VR1. Within seconds of vanilloid exposure, the intracellular free calcium ([Ca(2+)](i)) was elevated in cells expressing VR1. A functional pool of VR1 also was localized to the endoplasmic reticulum that, in the absence of extracellular calcium, also was capable of releasing calcium upon agonist treatment. Confocal imaging disclosed that resiniferatoxin treatment induced vesiculation of the mitochondria and the endoplasmic reticulum ( approximately 1 min), nuclear membrane disruption (5-10 min), and cell lysis (1-2 h). Nociceptive primary sensory neurons endogenously express VR1, and resiniferatoxin treatment induced a sudden increase in [Ca(2+)](i) and mitochondrial disruption which was cell-selective, as glia and non-VR1-expressing neurons were unaffected. Early hallmarks of cytotoxicity were followed by specific deletion of VR1-expressing cells. These data demonstrate that vanilloids disrupt vital organelles within the cell body and, if administered to sensory ganglia, may be employed to rapidly and selectively delete nociceptive neurons.     

Signal transduction and biological function of placenta growth factor in primary human trophoblast

Placenta growth factor (PlGF), a member of the vascular endothelial growth factor family of angiogenic factors, is prominently expressed by trophoblast. In addition to its role as a paracrine angiogenic factor within the placenta and endometrium, presence of its receptor, Flt-1, on trophoblast suggests that PlGF also may have an autocrine role(s) in regulating trophoblast function. To elucidate its role in trophoblast, we examined the signal transduction and functional responses of primary human trophoblast to PlGF. Exogenous PlGF induced specific activation of the stress-activated protein kinase (SAPK) pathways, c-Jun-N terminal kinase (JNK) and p38 kinase, in primary term trophoblast with little to no induction of the extracellular signal regulated kinase (ERK-1 and -2) pathways. In contrast, PlGF induced significant ERK-1 and -2 activity in human umbilical vein endothelial cells but did not induce JNK or p38 activity. PlGF-induced activation of the SAPK signaling pathways protected trophoblast from growth factor withdrawal-induced apoptosis, but it did not protect trophoblast from apoptosis induced by the pro-inflammatory cytokines, interferon gamma and tumor necrosis factor alpha. These results provide the first direct evidence of a biochemical and functional role for PlGF/Flt-1 in normal trophoblast and suggest that aberrant PlGF expression during pregnancy may impact upon trophoblast function as well as vascularity within the placental bed.     

Two-dimensional polyacrylamide gel electrophoresis of equine seminal plasma proteins and their correlation with fertility

The objectives of this study were to 1) identify proteins found in stallion seminal plasma utilizing two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) in conjunction with Western blot analysis; and 2) to determine if any of these individual proteins were correlated with stallion fertility utilizing regression analysis. Fertility was quantified by assigning a breeding score for each stallion. Each score was calculated by dividing the number of conceptions by the number of breedings for each stallion for four successive breeding seasons (1992-1995). Ejaculates from stallions of known fertility (n = 6) were collected with a Missouri-style artificial vagina. Immediately after collection, the semen sample was filtered and the gel fraction removed. The resultant sperm-rich fraction was centrifuged in a Beckman Microfuge E at 10,000 x g and the seminal plasma aspirated from the pelleted sperm cells. Two-dimensional PAGE of the seminal plasma was performed under denaturing conditions which revealed that 14 proteins were common in all stallions in the research population. Four of these proteins (SP-1, SP-2, SP-3, and SP-4) were found to be significantly (P < 0.05) correlated with the breeding score assigned for each stallion. Regression analysis of protein optical densities with breeding score indicated that SP-1 (72 kDa, pI 5.6) was positively correlated with fertility (P < 0.05, r2 = 0.706), while SP-2 (75 kDa, pI 6.0), SP-3 (18 kDa, pI 4.3), and SP-4 (16 kDa, pI 6.5) were found to be negatively correlated (P < 0.05, r2 = 0.762, 0.730, 0.775 respectively) with fertility. Western blot analysis of SP-1 indicated there was an antigenic homology with a bovine 55 kDa fertility-associated seminal plasma protein identified in a study by Killian et al. (19). This suggests that the two proteins may have a similar physiological role and therefore common biological properties. These results indicate that analysis of stallion seminal plasma proteins can be used as an indicator of fertilizing capacity. Identification of such proteins in stallion seminal plasma could lead to better insight into the nature of subfertility or infertility in the horse, as well as to indicate better cryopreservation strategies.     

Generation of highly selective VPAC2 receptor agonists by high throughput mutagenesis of vasoactive intestinal peptide and pituitary adenylate cyclase-activating peptide

Pituitary adenylate cyclase-activating peptide (PACAP) has a specific receptor PAC1 and shares two receptors VPAC1 and VPAC2 with vasoactive intestinal peptide (VIP). VPAC2 activation enhances glucose-induced insulin release while VPAC1 activation elevates glucose output. To generate a large pool of VPAC2 selective agonists for the treatment of type 2 diabetes, structure-activity relationship studies were performed on PACAP, VIP, and a VPAC2 selective VIP analog. Chemical modifications on this analog that prevent recombinant expression were sequentially removed to show that a recombinant peptide would retain VPAC2 selectivity. An efficient recombinant expression system was then developed to produce and screen hundreds of mutant peptides. The 11 mutations found on the VIP analog were systematically replaced with VIP or PACAP sequences. Three of these mutations, V19A, L27K, and N28K, were sufficient to provide most of the VPAC2 selectivity. C-terminal extension with the KRY sequence from PACAP38 led to potent VPAC2 agonists with improved selectivity (100-1000-fold). Saturation mutagenesis at positions 19, 27, 29, and 30 of VIP and charge-scanning mutagenesis of PACAP27 generated additional VPAC2 selective agonists. We have generated the first set of recombinant VPAC2 selective agonists described, which exhibit activity profiles that suggest therapeutic utility in the treatment of diabetes.     

The Role of Toll-Like Receptor 9 in Chronic Stress-Induced Apoptosis in Macrophage

Emerging evidence implied that chronic stress has been exerting detrimental impact on immune system functions in both humans and animals. Toll-like receptors (TLRs) have been shown to play an essential role in modulating immune responses and cell survival. We have recently shown that TLR9 deficiency protects against lymphocyte apoptosis induced by chronic stress. However, the exact role of TLR9 in stress-mediated change of macrophage function remains unclear. The results of the current study showed that when BALB/c mice were treated with restraint stress (12 h daily for 2 days), the number of macrophages recruited to the peritoneal cavity was obviously increased. Results also demonstrated that the sustained effects of stress elevated cytokine IL-1β, TNF-α and IL-10 production yet diminished IFN-γ production from macrophage, which led to apoptotic cell death. However, TLR9 deficiency prevented the chronic stress-mediated accumulation of macrophages. In addition, knocking out TLR9 significantly abolished the chronic stress-induced imbalance of cytokine levels and apoptosis in macrophage. TLR9 deficiency was also found to reverse elevation of plasma IL-1β, IL-10 and IL-17 levels and decrease of plasma IFN-γ level under the condition of chronic stress. These results indicated that TLR9-mediated macrophage responses were required for chronic stress-induced immunosuppression. Further exploration showed that TLR9 deficiency prevented the increment of p38 MAPK phosphorylation and reduction of Akt/Gsk-3β phosphorylation; TLR9 deficiency also attenuated the release of mitochondrial cytochrome c into cytoplasm, caused upregulation of Bcl-2/Bax protein ratio, downregulation of cleavage of caspase-3 and PARP, as well as decreased TUNEL-positive cells in macrophage of stressed mice. Collectively, our studies demonstrated that deficiency of TLR9 maintained macrophage function by modulating macrophage accumulation and attenuating macrophage apoptosis, thus preventing immunosuppression in restraint-stressed mice.     

Effect of chronic treatment with Rosiglitazone on Leydig cell steroidogenesis in rats: <Emphasis Type="Italic">In vivo</Emphasis> and <Emphasis Type="Italic">ex vivo</Emphasis> studies

Background  

The present study was designed to examine the effect of chronic treatment with rosiglitazone - thiazolidinedione used in the treatment of type 2 diabetes mellitus for its insulin sensitizing effects - on the Leydig cell steroidogenic capacity and expression of the steroidogenic acute regulatory protein (StAR) and cholesterol side-chain cleavage enzyme (P450scc) in normal adult rats.