Differential desensitization and internalization of three different bullfrog gonadotropin-releasing hormone receptors

We previously demonstrated the presence of three distinct types of the gonadotropin-releasing hormone receptor (GnRHR) in a bullfrog (denoted bfGnRHR-1, bfGnRHR-2, and bfGnRHR-3). The bfGnRHRs exhibited differential tissue distribution and ligand selectivity. In the present study, we demonstrated the desensitization and internalization kinetics of these receptors in both transiently-transfected HEK293 cells and retrovirus-mediated stable cells. The time-course accumulation of the inositol phosphate in response to GnRH revealed that bfGnRHR-1 and -2 were rapidly desensitized, whereas bfGnRHR-3 was slowly desensitized. A comparison of the internalization kinetics revealed the most rapid rate and highest extent of internalization of bfGnRHR-2 among the three receptors. Interestingly, the mechanisms that underlie the receptor internalization appear to differ from each other. Internalization of bfGnRHR-1 was dependent on both dynamin and beta-arrestin, whereas those of bfGnRHR-2 and -3 were dependent on dynamin, but not on arrestin. These results, therefore, suggest that differential regulatory mechanisms for desensitization and internalization of the GnRHR are involved in diverse cellular and physiological responses to GnRH stimulation.     

Lithium action on adrenomedullary and adrenocortical functions and serum ionic balance in different age-groups of albino rats

The aim of the current study was to investigate lithium action on adrenomedullary and adrenocortical functions and on serum ionic balance in rats. Three age-groups of male rats (juvenile: 30 days, adult: 100 days and aged: 3 years) were used. Each age-group of animal was exposed to short- (10 days) and long-term (25 days) treatments with lithium. Each age-group of rat received lithium at a dose 2mEq/kg body weight daily for 10 and 25 days. Each daily dose (2mEq) was divided equally into half (1 mEq) and each half was injected intraperitoneally twice (at 9 am and 9 pm) for both the durations of experiments. Control animals received physiological saline for similar duration of experiments. Thirty animals were used for each age-group and they were divided equally into 6 groups with 5 each. After termination of all the experiments rats were sacrificed and, adrenal glands were quickly dissected out and processed for epinephrine, norepinephrine and corticosterone estimations and, 3 beta-hydroxysteroid dehydrogenase (3 beta-HSDH) activity of the adrenal gland. Blood was drawn from the heart of each rat and, serum was collected and stored at -20 degrees C until assayed for lithium, calcium, sodium, potassium and corticosterone concentrations. The findings revealed that lithium in both short- and long-term treatments was maintained well within the therapeutic range (0.3-0.8 mEq/l) in all the age-groups of rats. This alkali metal caused depletions of both epinephrine and norepinephrine concentrations from adrenal glands, and elevations of corticosterone in both adrenal and blood serum of each age-group of rat (juvenile, adult and aged). Additionally adrenal 3beta-HSDH activity was also increased in all the age-groups of rats irrespective of duration of the treatments. Short-term treatment of lithium elevated only serum K+ level in juvenile and adult rats and, Ca+ level only in adult animals. Significant elevations of serum K+ and Ca+ levels were observed following long-term treatments of lithium in all the age group of rats. No significant change in serum Na+ level was recorded after lithium treatment, irrespective of duration of treatments, in any age-group of rats. The findings suggest that lithium action, in respect of adrenomedullary and adrenocortical functions and, serum ionic balance, may not be largely related to the age-group of rats and that, lithium acts on adrenomedullary activity probably by stimulating the release mechanism of epinephrine and norepinephrine from the adrenal gland of rats, but stimulates adrenocortical activity by stimulating both synthesis (including 3 beta-HSDH activity) and release of corticorterone. Simultaneously, lithium disturbs normal ionic balance by elevating K+ and Ca+ levels in all the age-group of rats. Thus, the antimanic drug certainly disturbs both adrenomedullary and adrenocortical functions and, serum ionic balance in all the age-group of rats.     

Oviductal sperm storage structure and their changes during the seasonal (dissociated) reproductive cycle in the soft-shelled turtle Lissemys punctata punctata

The oviduct of the Indian fresh water soft-shelled turtle Lissemys punctata punctata was examined throughout the year under light and scanning electron microscopes to determine the location, histomorphological characteristics, and function of sperm storage structure, as well as their changes at different phases of the seasonal reproductive cycle. Sperm storage structures in the form of tubules were observed in the wall of isthmus throughout the year. These tubules developed either by folding or fusion of the oviductal mucosal folds and were lined by both ciliated and nonciliated epithelial cells. The height and secretory activities of the epithelia were markedly high during the breeding phase (August to September) but low in the nonbreeding phase (October to June). A few short tubules lined by cuboidal epithelium appear in the wall of infundibulum only during the breeding phase. Following mating (May), inseminated sperm were stored within the tubules of isthmus up to the pre-ovulatory stage (August). Thereafter, sperm associated with PAS-positive materials secreted from the epithelium (referred to as a carrier matrix) moved forward to the infundibulum and were stored within the storage tubules of the infundibulum for a short time. Subsequently, sperm evacuated the storage tubules and entered the oviductal lumen to fertilize the subsequently ovulated eggs during or prior to ovulation. The isthmus-tubules become shorter and narrower in the regressive phase (October to November) and remained so until the early preparatory phase (April). Sperm release might have been stimulated by estrogen secreted from the ovarian follicles of pre-ovulatory turtles. Stored sperm not utilized for fertilization remained viable not less than six months in the present turtle species.     

Isolation and functional characterization of a temperature-sensitive mutant of the yeast Saccharomyces cerevisiae in translation initiation factor eIF5: an eIF5-dependent cell-free translation system

Eukaryotic translation initiation factor 5 (eIF5) interacts with the 40S ribosomal initiation complex (40S.eIF3.AUG.Met-tRNA(f).eIF2.GTP) to promote the hydrolysis of bound GTP. In Saccharomyces cerevisiae, eIF5, a protein of 45346Da, is encoded by a single-copy essential gene, TIF5. In this paper, we have isolated a temperature-sensitive S. cerevisiae strain, TMY5-1, by replacing the wild-type chromosomal copy of TIF5 with one mutagenized in vitro. The mutant yeast cells rapidly cease protein synthesis when grown under non-permissive conditions, lose polyribosomes and accumulate free 80S ribosomes. Further characterization of mutant eIF5 showed that the mutant protein, expressed in Escherichia coli, is defective both in its interaction with eIF2 as well as in mediating the hydrolysis of GTP bound to the 40S initiation complex and consequently in the formation of the 80S initiation complex. Additionally, the availability of a yeast strain containing temperature-sensitive mutation in the eIF5 gene allowed us to construct a cell-free translation system that was dependent on exogenously added eIF5 for translation of mRNAs in vitro.     

Induction of male and female sterility in isabgol (<Emphasis Type="Italic">Plantago ovata</Emphasis>) due to floral infection of downy mildew (<Emphasis Type="Italic">Pernospora plantaginis</Emphasis>)

Downy mildew (Peronospora plantaginis) caused two different types of infection in the floral parts of isabgol (Plantago ovata). Systemic infection resulted in long spikes bearing weak and sterile florets, which later turned black due to saprophytic growth. Localised infection produced various symptoms ranging between normal flower opening and failure to bloom. Different parts of infected flowers such as sepal, petal, filament and anther were reduced in size compared to healthy flowers. However, gynoecium was elongated in localised infection. P. plantaginis induced gradual sterility of isabgol flowers. Androecium was affected more than the gynoecium was. Pollen number, pollen viability and germination reduced drastically due to localised infection. On the contrary, there were no significant differences between healthy and locally infected flowers in terms of stigma receptivity. In systemically infected spikes, bud development was arrested leading to sterility. When localised disease severity was high, secondary systemic infection caused similar symptoms. Microscopic observations showed presence of the pathogen in different parts of the flowers. Downy mildew adversely affected seed yield and quality; producing seeds, which were smaller and lighter than the healthy ones and later, became black. Seed yield was reduced by as much as 73.45 percent. Husk content per unit seed mass increased relatively as the total surface area of infected seeds increased.     

Linear Mixed Model Selection for False Discovery Rate Control in Microarray Data Analysis

Summary In a microarray experiment, one experimental design is used to obtain expression measures for all genes. One popular analysis method involves fitting the same linear mixed model for each gene, obtaining gene‐specific p‐values for tests of interest involving fixed effects, and then choosing a threshold for significance that is intended to control false discovery rate (FDR) at a desired level. When one or more random factors have zero variance components for some genes, the standard practice of fitting the same full linear mixed model for all genes can result in failure to control FDR. We propose a new method that combines results from the fit of full and selected linear mixed models to identify differentially expressed genes and provide FDR control at target levels when the true underlying random effects structure varies across genes.     

Measurement of the Attachment and Assembly of Small Amyloid-β Oligomers on Live Cell Membranes at Physiological Concentrations Using Single-Molecule Tools

It is thought that the pathological cascade in Alzheimer's disease is initiated by the formation of amyloid-β (Aβ) peptide complexes on cell membranes. However, there is considerable debate about the nature of these complexes and the type of solution-phase Aβ aggregates that may contribute to their formation. Also, it is yet to be shown that Aβ attaches strongly to living cell membranes, and that this can happen at low, physiologically relevant Aβ concentrations. Here, we simultaneously measure the aggregate size and fluorescence lifetime of fluorescently labeled Aβ_1-40 on and above the membrane of cultured PC12 cells at near-physiological concentrations. We find that at 350 nM Aβ concentration, large (>>10 nm average hydrodynamic radius) assemblies of codiffusing, membrane-attached Aβ molecules appear on the cell membrane together with a near-monomeric species. When the extracellular concentration is 150 nM, the membrane contains only the smaller species, but with a similar degree of attachment. At both concentrations, the extracellular solution contains only small (∼2.3 nm average hydrodynamic radius) Aβ oligomers or monomers. We conclude that at near-physiological concentrations only the small oligomeric Aβ species are relevant, they are capable of attaching to the cell membrane, and they assemble in situ to form much larger complexes.     

Evaluation of cyclooxygenase 2 derived endogenous prostacyclin in mouse preimplantation embryo development in vitro

Cyclooxygenase (COX) plays an important role in prostaglandin (PG) synthesis and has two isoforms, COX1 and COX2. PGI synthase (PGIS) catalyzes the isomeization of PGH(2) to prostacyclin (PGI(2)). It is reported that COX2 derived PGI2(2) plays a critical role in blastocyst implantation and decidualization and PGI2 mediates its function via PPARdelta receptor. It is also known that cyclooxygenase derived prostaglandins play an important role in mouse blastocyst hatching in vitro. In this study we hypothesized that COX2 derived PGI2 plays an important role in preimplantation embryonic development by increasing the cell number. To examine this hypothesis, 8-cell stage mouse embryos were cultured in the presence of selective inhibitors of COX1 (SC560), COX2 (NS398) and PGIS (U51605) respectively. COX2 and PGIS inhibitor significantly reduced the blastocyst development and presence of PGI2 analogue along with these inhibitors restored the blastocyst development by increasing the total number of embryonic cells. Our immunohistochemical analysis showed that COX1 is expressed at 2-cell, 8-cell, compaction and blastocyst stage whereas COX2 expression starts from eight cell stage embryos. PGIS and PPARdelta expression starts at 2-cell stage of development. Our results suggest that PGI(2) may affect blastomeres number via the so called hypothesis of PPARdelta nuclear receptor in autocrine manner.     

Thyroid responses to altered photoperiod in the soft-shelled turtle Lissemys punctata punctata bonnoterre

The aim of the current investigation was to investigate the effect of photoperiod on thyroid activity in soft-shelled turtles (Lissemys punctata punctata). Thirty days exposure of short photoperiod with 2L:22D increased relative weight, follicular epithelial height and peroxidase activity of the thyroid gland; whereas exposure of long photoperiod with 22L:2D for 30 days showed reversed changes to those of the short photoperiod in adult female turtles. These findings indicate that short photoperiod stimulates thyroid activity and long photoperiod inhibits its activity in soft-shelled turtles. It is suggested that photoperiod exerts its action on thyroid activity presumably via gonads and/or pineal-gonadal axis in turtles.     

Extracellular calcium is a direct effecter of VDR levels in proximal tubule epithelial cells that counter-balances effects of PTH on renal Vitamin D metabolism

In renal proximal tubules, VDR is transiently decreased by parathyroid hormone (PTH) during times of hypocalcemia and returns to normal levels with the rise in serum calcium (Ca). In this study we tested the hypothesis that elevated extracellular Ca induces VDR in a human renal proximal cell line (HK-2G) stably expressing PTH receptor type I. Exposure of HK-2G cells to increasing Ca concentration, up to 3 mM, induced the expression of VDR. The increase in VDR occurred within 1 h and was sustained over 24 h. The increase in VDR was also dose-dependently increased using 20–100 nM gadolinium, suggesting the induction of VDR is regulated via the extracellular Ca sensing receptor (CaSR) with is naturally expressed in HK-2G cells. In conclusion, an extracellular Ca concentration in the physiological range is capable of direct increase of renal proximal VDR expression, and the induction mechanism represents a strategy the body may use to counterbalance effects of PTH on renal Vitamin D metabolism.