Dopamine receptor agonist bromocriptine restrains the follicular development,hatchling success and puberty in Gambusia affinis

The present investigation was conducted to determine the influence of dopamine (DA) receptor agonist bromocriptine (BRO) on reproduction and onset of puberty in the viviparous fish Gambusia affinis. In the first experiment, the mean number of stage I and II follicles (previtellogenic) in 0.8 or 5 mg BRO treated fish did not show significant difference compared to those of experimental controls, whereas the mean number of stage III follicles were significantly lower in 5 mg BRO treated fish compared to experimental controls. However, treatment of 0.8 or 5 mg BRO resulted in significantly lower numbers of stage IV (early vitellogenic) and V (late vitellogenic) follicles compared to those of experimental controls. There was decrease in the percent occurrence of pregnancy and different stages of embryos in BRO treated fish compared with the experimental controls. Concomitant with this, sparsely distributed gonadotropin releasing hormone immunoreactive (GnRH‐ir) fibres were observed in the proximal pars distalis (PPD) region of the pituitary gland in BRO treated fish compared to those of dense accumulations of these fibres in the PPD region of the pituitary gland in experimental controls. In the second experiment, exposure of juveniles (25 DPH) to same doses of BRO for 45 days resulted in complete absence of vitellogenic follicles and presence of few GnRH‐ir fibres in 5 mg BRO treated juvenile in contrast to presence of vitellogenic follicles and dense aggregation of GnRH fibres in treatment controls. Overall, the results of the present investigation suggest that DA affects ovarian follicular and embryonic development and onset of puberty in viviparous species.     

Platelet-Derived Growth Factor CC-Mediated Neuroprotection against HIV Tat Involves TRPC-Mediated Inactivation of GSK 3beta

Platelet-derived growth factor-CC (PDGF-CC) is the third member of the PDGF family, and has been implicated both in embryogenesis and development of the CNS. The biological function of this isoform however, remains largely unexplored in the context of HIV-associated dementia (HAD). In the present study, we demonstrate that exposure of human neuroblastoma cells SH-SY5Y to HIV transactivator protein Tat resulted in decreased intrinsic expression of PDGF-CC as evidenced by RT-PCR and western blot assays. Reciprocally, pretreatment of SH-SY5Y cells with PDGF-CC abrogated Tat-mediated neurotoxicity by mitigating apoptosis and neurite & MAP-2 loss. Using pharmacological and loss of function approaches we identified the role of phosphatidylinositol 3-kinase (PI3K)/Akt signaling in PDGF-CC-mediated neuroprotection. We report herein a novel role about the involvement of transient receptor potential canonical (TRPC) channel 1 in modulation of calcium transients in PDGF-CC-mediated neuroprotection. Furthermore we also demonstrated PDGF-CC-mediated inactivation of the downstream mediator - glycogen synthase kinase 3β (GSK3β) evidenced by its phosphorylation at Ser-9. This was further validated by gain and loss of function studies using cells transfected with either the wild type or mutant GSK3β constructs. Intriguingly, pretreatment of cells with either the PI3K inhibitor or TRPC blocker resulted in failure of PDGF-CC to inactivate GSK3β, thereby suggesting the intersection of PI3K and TRPC signaling at GSK3β. Taken together our findings lead to the suggestion that PDGF-CC could be developed as a therapeutic target to reverse Tat-mediated neurotoxicity with implications for HAD.     

Engineering of deglycosylated and plasmin resistant variants of recombinant streptokinase in Pichia pastoris

Applied Microbiology and Biotechnology - Streptokinase, a therapeutically important thrombolytic agent, is prone to C-terminal degradation and plasmin-mediated proteolytic processing. Since the...     

Cellular function of elastase in Pseudomonas aeruginosa: role in the cleavage of nucleoside diphosphate kinase and in alginate synthesis

Elastase is a major virulence factor in Pseudomonas aeruginosa that is believed to cause extensive tissue damage during infection in the human host. Elastase is secreted in non-mucoid P. aeruginosa. It is known that secretion of most virulence factors such as elastase, lipase, exotoxin A, etc., in P. aeruginosa is greatly reduced in alginate-secreting mucoid cells isolated from the lungs of cystic fibrosis (CF) patients. We have previously reported that in mucoid P. aeruginosaan intracellular protease cleaves the 16 kDa form of nucleoside diphosphate kinase (Ndk) to a truncated 12 kDa form. This smaller form is membrane associated and has been observed to form complexes with specific proteins to predominantly generate GTP, an important molecule in alginate synthesis. The main aim of this study was to purify and characterize this protease. The protease was purified by hydrophobic interaction chromatography of the crude extract of mucoid P. aeruginosa 8821, a CF isolate. Further analysis using a gelatin containing SDS–polyacrylamide gel detected the presence of a 103 kDa protease, which when boiled, migrated as a 33 kDa protein on a SDS–polyacrylamide gel. The first 10 amino acids from the N-terminus of the 33 kDa protease showed 100% identity to the mature form of elastase. An elastase-negative lasB ::Cm knock-out mutant in the mucoid 8821 background was constructed, and it showed a non-mucoid phenotype. This mutant showed the presence of only the 16 kDa form of Ndk both in the cytoplasm and membrane fractions. We present evidence for the retention of active elastase in the periplasm of mucoid P. aeruginosa and its role in the generation of the 12 kDa form of Ndk. Finally, we demonstrate that elastase, when overproduced in both mucoid and non-mucoid cells, stimulates alginate synthesis. This suggests that the genetic rearrangements that trigger mucoidy in P. aeruginosa also allow retention of elastase in the periplasm in an active oligomeric form that facilitates cleavage of 16 kDa Ndk to its 12 kDa form for the generation of GTP, required for alginate synthesis.     

Role of omega-3 polyunsaturated fatty acids on cyclooxygenase-2 metabolism in brain-metastatic melanoma

Cyclooxygenase-2 (COX-2) is important in the progression of epithelial tumors. Evidence indicates that omega-6 PUFAs such as arachidonic acid (AA) promote the growth of tumor cells; however, omega-3 fatty acids [eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)] inhibit tumor cell proliferation. We investigated the effects of omega-3 PUFA on the expression and function of COX-2 in 70W, a human melanoma cell line that metastasizes to the brain in nude mice. We show that 1) tumor necrosis factor-alpha upregulates the expression of both COX-2 mRNA and prostaglandin E2 (PGE2) production, and 2) omega-3 and omega-6 PUFA regulate COX-2 mRNA expression and PGE2 production. AA increased COX-2 mRNA expression and prostaglandin production in omega-6-stimulated 70W cells. Conversely, COX-2 mRNA expression decreased in cells incubated with EPA or DHA. AA increased Matrigel invasion 2.4-fold, whereas EPA or DHA did not. Additionally, PGE2 increased in vitro invasion 2.5-fold, whereas exposure to PGE3 significantly decreased invasion. Our results demonstrate that incubation of 70W cells with either AA or PGE2 increased invasiveness, whereas incubation with EPA or DHA downregulated both COX-2 mRNA and protein expression, with a subsequent decrease in Matrigel invasion. Taken together, these results indicate that omega-3 PUFA regulate COX-2-mediated invasion in brain-metastatic melanoma.     

Expression and sub cellular localization of the sodium hydrogen exchanger isoform-1 in rat tissues: A possible functional relevance

In an attempt to understand the mechanism underlying the tissue-dependent function, the expression of NHE-1 protein and its sub cellular localization was examined in the rat GI-tract and other tissues. Rat NHE-1 polyclonal antibodies were raised in rabbits using a NHE-1 fusion protein antigen. The antibodies recognized a 110 kD protein in rats and mice, but not in human or rabbit RBCs. Colon, stomach, brain, spleen and kidney expressed NHE-1 protein abundantly, whereas the skeletal muscle the least abundant. Ouabain-sensitive-K⁺-stimulated p-nitrophenylphosphatase (PNPPase), the partial activity of the sodium pump and alkaline phosphatase (Apase) were used as the markers of the basolateral and apical membranes. NHE-1 was detected predominantly in the PNPPase enriched membrane fractions, but was also detected in the apical membrane enriched fractions in the kidney cortex, jejunum and colon at a lower level. NHE-1 was detected in the plasma membrane enriched fractions from the skeletal muscle and ventricle. Immunofluorescence data showed a similar localization pattern of NHE-1 in the colon and kidney sections. These findings suggest that NHE-1 is localized both on the apical and basolateral membrane. In view of its similar sub cellular localization in the GI-tract and kidney, but a different level of expression, might suggest that the level of protein, but not the sub cellular distribution is important to regulate its tissue-dependent function.     

Vascular Dysfunction in Obstructive Sleep Apnea and Type 2 Diabetes Mellitus

Despite the high prevalence of obstructive sleep apnea (OSA) in type 2 diabetes mellitus (DM), the attributable vascular risk from each condition is unknown. We hypothesize that OSA may have a similar effect on vascular function as type 2 diabetes does. Healthy normal‐weight subjects, healthy obese subjects, subjects with type 2 diabetes, and obese subjects with OSA were enrolled. Vascular function was assessed with brachial artery ultrasound for flow‐mediated dilatation (FMD) and in skin microcirculation by laser Doppler flowmetry. One hundred fifty‐three subjects were studied: healthy normal‐weight controls (NCs) (n = 14), healthy obese controls (OCs) (n = 33), subjects with DM (n = 68), and obese subjects with OSA (n = 38). The DM group did not undergo sleep study and thus may have had subclinical OSA. The OSA and type 2 diabetes groups had impaired FMD as compared to both the normal‐weight and OC groups (5.8 ± 3.8%, 5.4 ± 1.6% vs. 9.1 ± 2.5%, 8.3 ± 5.1%, respectively, P < 0.001, post hoc Fischer test). When referenced to the NC group, a multiple linear regression model adjusting for covariates found that baseline brachial artery diameter (β = ?3.75, P < 0.001), OSA (β = ?2.45, P = 0.02) and type 2 diabetes status (β = ?2.31, P = 0.02), negatively predicted % FMD. OSA status did not seem to affect nitroglycerin‐induced vasodilation (endothelium‐independent) of the brachial artery or vascular function in the skin microcirculation. OSA impairs endothelial function in the brachial artery to a similar degree as type 2 diabetes does. OSA, however, does not appear to affect brachial endothelium‐independent vasodilation or skin microcirculatory function. Treatment of OSA in patients with concomitant type 2 diabetes, therefore, may be a potential therapeutic option to improve macro‐, but not microvascular outcomes.