Semen characteristics of the captive Indian white-backed vulture (Gyps bengalensis)

The present paper describes, to our knowledge for the first time, the successful collection and evaluation of semen from the Indian white-backed vulture (Gyps bengalensis), a critically endangered bird. Over a period of 2 yr, semen was collected using the manual massage method and evaluated for semen volume, semen pH, sperm concentration, percentage normal/abnormal spermatozoa, and percentage motile spermatozoa. It appears that the concentration of spermatozoa and percentage motile spermatozoa in the Indian white-backed vultures are low compared to those in other birds. Tyrode medium supplemented with albumin, lactate, and pyruvate (TALP) proved to be the best semen extender compared to two others (Beltsville Poultry Semen Extender and Lake diluent). Furthermore, TALP with 20% egg yolk and supplemented with 8% dimethyl sulfoxide maintained 50% of the initial percentage of motile spermatozoa following cryopreservation and thawing. A computer-aided semen analysis indicated that the spermatozoa of the Indian white-backed vulture are extremely active and swim in linear trajectories for up to 5 h following dilution in TALP. The trajectories were linear with time, but we noticed a decrease in the velocity parameters (average path velocity, curvilinear velocity, and progressive velocity). Thus, the present study provides baseline data on semen characteristics of the highly endangered Indian white-backed vulture, and these data could be of immense importance to reproductive and conservation biologists attempting to breed these animals in captivity, which to date has not been achieved.     

Temporal and spatial control of nucleophosmin by the Ran-Crm1 complex in centrosome duplication

Centrosome duplication is tightly controlled during faithful cell division, and unnecessary reduplication can lead to supernumerary centrosomes and multipolar spindles that are associated with most human cancer cells. In addition to nucleocytoplasmic transport, the Ran-Crm1 network is involved in regulating centrosome duplication to ensure the formation of a bipolar spindle. Here, we discover that nucleophosmin (NPM) may be a Ran-Crm1 substrate that controls centrosome duplication. NPM contains a functional nuclear export signal (NES) that is responsible for both its nucleocytoplasmic shuttling and its association with centrosomes, which are Ran-Crm1-dependent as they are sensitive to Crm1-specific nuclear export inhibition, either by leptomycin B (LMB) or by the expression of a Ran-binding protein, RanBP1. Notably, LMB treatment induces premature centrosome duplication in quiescent cells, which coincides with NPM dissociation from centrosomes. Moreover, deficiency of NPM by RNA interference results in supernumerary centrosomes, which can be reversed by reintroducing wild-type but not NES-mutated NPM. Mutation of a potential proline-dependent kinase phosphorylation site at residue 95, from threonine to aspartic acid (T95D) within the NES motif, abolishes NPM association and inhibition of centrosome duplication. Our results are consistent with the hypothesis that the Ran-Crm1 complex may promote a local enrichment of NPM on centrosomes, thereby preventing centrosome reduplication.     

Direct one-pot conversion of acylated carbohydrates into their alkylated derivatives under heterogeneous reaction conditions using solid NaOH and a phase transfer catalyst

A convenient one-pot protocol for the direct conversion of acyl-protected carbohydrates into their alkylated counterparts has been developed by using alkyl halides in the presence of solid sodium hydroxide and a phase transfer catalyst. These economically convenient, mild, two-phase reaction conditions allow the preparation of a variety of monosaccharide intermediates for use in the synthesis of complex oligosaccharides.     

Primary non-Hodgkin's lymphoma of the breast: a case report

Extranodal non-Hodgkin's lymphoma (NHL) of the breast is a rare entity. It represents 0.04-1.1% of malignant tumors of the breast. 1.7-2.2% of extranodal lymphomas and 0.7% of all NHL. However, primary NHL (PNHL) is the most frequent hematopojetic tumor of the breast. CASE: A 23-year-old woman presented with a mass in the left breast for 3 months followed by enlarged left axillary lymph nodes. Mammography showed a diffuse increase in the density of the left breast. Other investigations were unremarkable. Both fine needle aspiration cytology (FNAC) and histopathology were diagnostic of NHL. Immunohistochemistry was confirmatory of NHL, diffuse large cell type, of B-cell lineage. CONCLUSION: Primary and secondary lymphomas of the breast, though rare, should be considered in the differential diagnosis of breast malignancies. PNHL of the breast is usually right sided, but this patient had left breast involvement. Diagnosis by FNAC was successful as the cytologic picture is similar to that of any other lymphoid or extranodal NHL. When histopathology and immunohistopathology are conclusive, FNAC, supplemented by immunocytochemistry, can be applied as a simple, reliable and cost-effective tool in the early diagnosis of breast lymphomas.     

Potential role of kinins in the effects of taurine in high-fructose-fed rats

The present work investigates the involvement of kinins in the effects of taurine in fructose-fed hypertensive rats. The effects of taurine on blood pressure, plasma glucose, insulin, and the insulin sensitivity index were determined. Angiotensin-converting enzyme (ACE) activity and nitrite content in plasma, plasma and tissue kallikrein activity, and taurine content were also investigated. The blood pressure changes in response to the coadministration of inhibitors of the synthesis of nitric oxide (NO), prostaglandins (PGs), or a kinin receptor blocker along with taurine was also evaluated. Fructose-fed rats had higher blood pressure and elevated plasma levels of glucose and insulin. Kallikrein activity, taurine, and nitrite contents were significantly lower in fructose-fed rats as compared with controls. The increases in systolic blood pressure, hyperglycemia, and hyperinsulinemia were controlled by taurine administration in fructose-fed rats. ACE activity was lower, while nitrite and taurine content and kallikrein activity were higher, in taurine-supplemented rats as compared with fructose-fed rats. A significant increase in blood pressure was observed in rats cotreated with the inhibitors Hoe 140 (a kinin receptor blocker), L-NAME (a NO synthase inhibitor), or indomethacin (a PG synthesis inhibitor) with taurine for 1 week as compared with taurine-treated fructose-fed rats. This suggests that the antihypertensive effect of taurine in fructose-fed rats was blocked by the inhibitors. Augmented kallikrein activity and, hence, increased kinin availability may be implicated in the effects of taurine in fructose-fed hypertensive rats.     

Ceramide-induced intracellular oxidant formation, iron signaling, and apoptosis in endothelial cells: protective role of endogenous nitric oxide

Sphingolipid ceramide (N-acetylsphingosine), a bioactive second messenger lipid, was shown to activate reactive oxygen species (ROS), mitochondrial oxidative damage, and apoptosis in neuronal and vascular cells. The proapoptotic effects of tumor necrosis factor-alpha, hypoxia, and chemotherapeutic drugs were attributed to increased ceramide formation. Here we investigated the protective role of nitric oxide (.NO) during hydrogen peroxide (H(2)O(2))-mediated transferrin receptor (TfR)-dependent iron signaling and apoptosis in C(2)-ceramide (C(2)-cer)-treated bovine aortic endothelial cells (BAECs). Addition of C(2)-cer (5-20 microm) to BAECs enhanced .NO generation. However, at higher concentrations of C(2)-cer (> or =20 microm), .NO generation did not increase proportionately. C(2)-cer (20-50 microm) also resulted in H(2)O(2)-mediated dichlorodihydrofluorescein oxidation, reduced glutathione depletion, aconitase inactivation, TfR overexpression, TfR-dependent uptake of (55)Fe, release of cytochrome c from mitochondria into cytosol, caspase-3 activation, and DNA fragmentation. N(w)-Nitro-l-arginine methyl ester (l-NAME), a nonspecific inhibitor of nitricoxide synthases, augmented these effects in BAECs at much lower (i.e. nonapoptotic) concentrations of C(2)-cer. The 26 S proteasomal activity in BAECs was slightly elevated at lower concentrations of C(2)-cer (< or =10 microm) but was greatly suppressed at higher concentrations (>10 microm). Intracellular scavengers of H(2)O(2), cell-permeable iron chelators, anti-TfR receptor antibody, or mitochondria-targeted antioxidant greatly abrogated C(2)-cer- and/or l-NAME-induced oxidative damage, iron signaling, and apoptosis. We conclude that C(2)-cer-induced H(2)O(2) and TfR-dependent iron signaling are responsible for its prooxidant and proapoptotic effects and that .NO exerts an antioxidative and cytoprotective role.     

Structural, biochemical, and functional characterization of the calcium sensor neurocalcin delta in the inner retinal neurons and its linkage with the rod outer segment membrane guanylate cyclase transduction system

This study documents the detailed biochemical, structural, and functional identity of a novel Ca(2+)-modulated membrane guanylate cyclase transduction system in the inner retinal neurons. The guanylate cyclase is the previously characterized ROS-GC1 from the photoreceptor outer segments (PROS), and its new modulator is neurocalcin delta. At the membrane, the myristoylated form of neurocalcin delta senses submicromolar increments in free Ca(2+), binds to its specific ROS-GC1 domain, and stimulates the cyclase. Neurocalcin delta is not present in PROS, indicating the absence of the pathway in the outer segments and the dissociation of its linkage with phototransduction. Thus, the pathway is linked specifically with the visual transduction machinery in the secondary neurons of the retina. With the inclusion of this pathway, the findings broaden the understanding of the existing mechanisms showing how ROS-GC1 is able to receive and transduce diverse Ca(2+) signals into the cell-specific generation of second-messenger cyclic GMP in the retinal neurons.     

Chronic somatostatin treatment affects pituitary gonadotrophs, ovaries and onset of puberty in rats

The effects of chronic somatostatin (SRIH-14) treatment on the pituitary gonadotrophs (FSH and LH cells) and ovaries of female Wistar rats were examined. Females were given 20 microg/100 g b.w. twice per day from the immature (23rd day) till the adult period of life (71st day). The onset of puberty was determined by daily examination for vaginal opening. The peroxidase-antiperoxidase immunocytochemical procedure was used to study the gonadotrophs. Changes in the number per unit area (mm2), cell volume and volume densities of LH- and FSH-immunoreactive cells were evaluated by morphometry and stereology. Ovaries were analysed by simple point counting of follicles and corpora lutea (CL). Follicles were divided by size according to the classification of Gaytán and Osman. The mitotic indexes of granulosa and theca cells in the follicles were estimated at all stages of folliculogenesis. The number, volume and the volume density of FSH- and LH-immunoreactive cells decreased after chronic SRIH-14 treatment, particularly the latter. In the ovary, SRIH-14 treatment decreased the number of healthy follicles at all phases of folliculogenesis, lowered the mitotic indexes of granulosa and theca cells but increased the number of atretic follicles. Healthy CL were fewer in number, while regressive CL were increased. Vaginal opening occurred at a later age in treated females. It can be concluded that chronic SRIH-14 treatment markedly inhibited LH cells and to a lesser extent FSH cells. In the ovary SRIH-14 inhibited folliculogenesis, enhanced atretic processes and lowered proliferative activity of granulosa and theca cells. It also delayed puberty onset.     

Determining functionally important amino acid residues of the E1 protein of Venezuelan equine encephalitis virus

A new method for predicting interacting residues in protein complexes, InterProSurf, was applied to the E1 envelope protein of Venezuelan equine encephalitis (VEEV). Monomeric and trimeric models of VEEV-E1 were constructed with our MPACK program, using the crystal structure of the E1 protein of Semliki forest virus as a template. An alignment of the E1 sequences from representative alphavirus sequences was used to determine physical chemical property motifs (likely functional areas) with our PCPMer program. Information on residue variability, propensity to be in protein interfaces, and surface exposure on the model was combined to predict surface clusters likely to interact with other viral or cellular proteins. Mutagenesis of these clusters indicated that the predictions accurately detected areas crucial for virus infection. In addition to the fusion peptide area in domain 2, at least two other surface areas play an important role in virus infection. We propose that these may be sites of interaction between the E1–E1 and E1–E2 subdomains of the envelope proteins that are required to assemble the functional unit. The InterProSurf method is, thus, an important new tool for predicting viral protein interactions. These results can aid in the design of new vaccines against alphaviruses and other viruses.     

Defective in cuticular ridges (DCR) of Arabidopsis thaliana, a gene associated with surface cutin formation, encodes a soluble diacylglycerol acyltransferase

A key step in the triacylglycerol (TAG) biosynthetic pathway is the final acylation of diacylglycerol (DAG) by DAG acyltransferase. In silico analysis has revealed that the DCR (defective in cuticular ridges) (At5g23940) gene has a typical HX(4)D acyltransferase motif at the N-terminal end and a lipid binding motif VX(2)GF at the middle of the sequence. To understand the biochemical function, the gene was overexpressed in Escherichia coli, and the purified recombinant protein was found to acylate DAG specifically in an acyl-CoA-dependent manner. Overexpression of At5g23940 in a Saccharomyces cerevisiae quadruple mutant deficient in DAG acyltransferases resulted in TAG accumulation. At5g23940 rescued the growth of this quadruple mutant in the oleate-containing medium, whereas empty vector control did not. Lipid particles were localized in the cytosol of At5g23940-transformed quadruple mutant cells, as observed by oil red O staining. There was an incorporation of 16-hydroxyhexadecanoic acid into TAG in At5g23940-transformed cells of quadruple mutant. Here we report a soluble acyl-CoA-dependent DAG acyltransferase from Arabidopsis thaliana. Taken together, these data suggest that a broad specific DAG acyltransferase may be involved in the cutin as well as in the TAG biosynthesis by supplying hydroxy fatty acid.