Cycle and duration of the seminiferous epithelium in puma (Puma concolor)

Puma or sussuarana (Puma concolor) is the second largest feline in the American continent and has an ample latitudinal distribution in very diverse habitats. In relation to its conservation status, the puma is considered an extinction-threatened species. The study of the testis morphology and the spermatogenic process in a species is fundamental for establishing the physiologic patterns that will make possible the selection of the protocols for assisted reproduction. A number of peculiarities associated with the reproductive biology of specific species such as the duration of spermatogenic process can be used to determine the frequency of sperm collection. Nine adult male pumas maintained in captivity were used to determine the relative frequency of stages in the seminiferous epithelium cycle. Three of them received intra-testicular injections of 0.1ml tritiated thymidine to determine the duration of the seminiferous epithelium cycle, and were subjected to biopsy 7 days later. The cycle of the seminiferous epithelium in puma was didactically described into eight stages by the tubular morphology method. The total duration of one seminiferous epithelium cycle in puma was calculated to be 9.89 days, and approximately 44.5 days are required for development of spermatozoon from spermatogonia. The duration of spermiogenesis, prophase and other events of meiosis were 14.08, 15.20 and 1.79 days, respectively. The relative frequency of the pre-meiotic, meiotic and post-meiotic phases were 3.98, 1.79 and 4.12 days, respectively.     

UDPglucose pyrophosphorylase from Xanthomonas spp. Characterization of the enzyme kinetics, structure and inactivation related to oligomeric dissociation

The genes encoding for UDPglucose pyrophosphorylase in two Xanthomonas spp. were cloned and overexpressed in Escherichia coli. After purification to electrophoretic homogeneity, the recombinant proteins were characterized, and both exhibited similar structural and kinetic properties. They were identified as dimeric proteins of molecular mass 60kDa, exhibiting relatively high specific activity ( approximately 80Units/mg) for UDPglucose synthesis. Both enzymes utilized UTP or TTP as substrate with similar affinity. The purified Xanthomonas enzyme was inactivated after dilution into the assay medium. Studies of crosslinking with the bifunctional lysyl reagent bisuberate suggest that inactivation occurs by enzyme dissociation to monomers. UTP effectively protects the enzyme against inactivation, from which a dissociation constant of 15microM was calculated for the interaction substrate-enzyme. The UTP binding to the enzyme would induce conformational changes in the protein, favoring the subunits interaction to form an active dimer. This view was reinforced by protein modeling of the Xanthomonas enzyme on the basis of the prokaryotic UDPglucose pyrophosphorylase crystallographic structure. The in silico approach pointed out two main critical regions in the enzyme involved in subunit-subunit interaction: the region surrounding the catalytic-substrate binding site and the C-term.     

A multi-layer method to study genome-scale positions of nucleosomes

The basic unit of eukaryotic chromatin is the nucleosome, consisting of about 150 bp of DNA wrapped around a protein core made of histone proteins. Nucleosomes position is modulated in vivo to regulate fundamental nuclear processes. To measure nucleosome positions on a genomic scale both theoretical and experimental approaches have been recently reported. We have developed a new method, Multi-Layer Model (MLM), for the analysis of nucleosome position data obtained with microarray-based approach. The MLM is a feature extraction method in which the input data is processed by a classifier to distinguish between several kinds of patterns. We applied our method to simulated-synthetic and experimental nucleosome position data and found that besides a high nucleosome recognition and a strong agreement with standard statistical methods, the MLM can identify distinct classes of nucleosomes, making it an important tool for the genome wide analysis of nucleosome position and function. In conclusion, the MLM allows a better representation of nucleosome position data and a significant reduction in computational time.     

A Preliminary Study of the Temporal and Spatial Biomass Patterns of Herbaceous Vegetation Consumed by Mountain Gorillas in an Afromontane Rain Forest

Although many animal species consume herbaceous vegetation found in African tropical forests, little is known of the temporal and spatial availability of these plants. From September 2004 to August 2005 we conducted a study that quantified the temporal and spatial biomass availability of 20 species of herbs frequently consumed by endangered mountain gorillas at two locations (Buhoma and Ruhija) in Bwindi Impenetrable National Park, Uganda. In general, the biomass of herbs varied over the study period, but these changes were relatively small. For 12 of 18 and nine of 11 species in Buhoma and Ruhija, respectively, herb biomass differed significantly among habitat types. Of the nine species found in both locations, seven species had a higher biomass at Ruhija, one species had a higher biomass at Buhoma, and one species showed no difference. These results demonstrate that herb biomass varied little temporally but spatial differences in herb biomass were more pronounced. Future studies should investigate the variables that may influence herb phenological patterns such as rainfall, light, soil quality, previous disturbance regimes, and animal foraging and trampling damage.     

Fibrogenic Potential of Human Multipotent Mesenchymal Stromal Cells in Injured Liver

Multipotent mesenchymal stromal cells (MSC) are currently investigated clinically as cellular therapy for a variety of diseases. Differentiation of MSC toward endodermal lineages, including hepatocytes and their therapeutic effect on fibrosis has been described but remains controversial. Recent evidence attributed a fibrotic potential to MSC. As differentiation potential might be dependent of donor age, we studied MSC derived from adult and pediatric human bone marrow and their potential to differentiate into hepatocytes or myofibroblasts in vitro and in vivo. Following characterization, expanded adult and pediatric MSC were co-cultured with a human hepatoma cell line, Huh-7, in a hepatogenic differentiation medium containing Hepatocyte growth factor, Fibroblast growth factor 4 and oncostatin M. In vivo, MSC were transplanted into spleen or liver of NOD/SCID mice undergoing partial hepatectomy and retrorsine treatment. Expression of mesenchymal and hepatic markers was analyzed by RT-PCR, Western blot and immunohistochemistry. In vitro, adult and pediatric MSC expressed characteristic surface antigens of MSC. Expansion capacity of pediatric MSC was significantly higher when compared to adult MSC. In co-culture with Huh-7 cells in hepatogenic differentiation medium, albumin expression was more frequently detected in pediatric MSC (5/8 experiments) when compared to adult MSC (2/10 experiments). However, in such condition pediatric MSC expressed alpha smooth muscle more strongly than adult MSC. Stable engraftment in the liver was not achieved after intrasplenic injection of pediatric or adult MSC. After intrahepatic injection, MSC permanently remained in liver tissue, kept a mesenchymal morphology and expressed vimentin and alpha smooth muscle actin, but no hepatic markers. Further, MSC localization merges with collagen deposition in transplanted liver and no difference was observed using adult or pediatric MSC. In conclusion, when transplanted into an injured or regenerating liver, MSC differentiated into myofibroblasts with development of fibrous tissue, regardless of donor age. These results indicate that MSC in certain circumstances might be harmful due to their fibrogenic potential and this should be considered before potential use of MSC for cell therapy.     

<Emphasis Type="Italic">In vitro</Emphasis> antioxidant activity of the prostatic secretory granules in rabbit semen after exposure to organic peroxides

Background  

The prostate gland of rabbits produces numerous granules, which are specifically implicated in the inhibition of sperm capacitation during the first hours after mating. These granules are rich in vitamin E, but their role in the antioxidant protection of rabbit sperm has not been studied.     

Human Bone Marrow Mesenchymal Stem Cells Display Anti-Cancer Activity in SCID Mice Bearing Disseminated Non-Hodgkin's Lymphoma Xenografts

Background

Although multimodality treatment can induce high rate of remission in many subtypes of non-Hodgkin''s lymphoma (NHL), significant proportions of patients relapse with incurable disease. The effect of human bone marrow (BM) mesenchymal stem cells (MSC) on tumor cell growth is controversial, and no specific information is available on the effect of BM-MSC on NHL.

Methodology/Principal Findings

The effect of BM-MSC was analyzed in two in vivo models of disseminated non-Hodgkin''s lymphomas with an indolent (EBV⁻ Burkitt-type BJAB, median survival = 46 days) and an aggressive (EBV⁺ B lymphoblastoid SKW6.4, median survival = 27 days) behavior in nude-SCID mice. Intra-peritoneal (i.p.) injection of MSC (4 days after i.p. injection of lymphoma cells) significantly increased the overall survival at an optimal MSC∶lymphoma ratio of 1∶10 in both xenograft models (BJAB+MSC, median survival = 58.5 days; SKW6.4+MSC, median survival = 40 days). Upon MSC injection, i.p. tumor masses developed more slowly and, at the histopathological observation, exhibited a massive stromal infiltration coupled to extensive intra-tumor necrosis. In in vitro experiments, we found that: i) MSC/lymphoma co-cultures modestly affected lymphoma cell survival and were characterized by increased release of pro-angiogenic cytokines with respect to the MSC, or lymphoma, cultures; ii) MSC induce the migration of endothelial cells in transwell assays, but promoted endothelial cell apoptosis in direct MSC/endothelial cell co-cultures.

Conclusions/Significance

Our data demonstrate that BM-MSC exhibit anti-lymphoma activity in two distinct xenograft SCID mouse models of disseminated NHL.     

Resveratrol potentiates glucose-stimulated insulin secretion in INS-1E beta-cells and human islets through a SIRT1-dependent mechanism

Resveratrol, a polyphenol compound, is known for its effects on energy homeostasis. With properties of energy sensors mediating effects of calorie restriction, sirtuins are targets of resveratrol. The mammalian sirtuin homolog SIRT1 is a protein deacetylase playing a role in glucose metabolism and islet function. Here, we investigated the effects of resveratrol and possible link with SIRT1 in β-cells. Insulinoma INS-1E cells and human islets were cultured with resveratrol before analyzing their physiological responses. Treatment of INS-1E cells for 24 h with 25 μM resveratrol resulted in marked potentiation of glucose-stimulated insulin secretion. This effect was associated with elevated glycolytic flux, resulting in increased glucose oxidation, ATP generation, and mitochondrial oxygen consumption. Such changes correlated with up-regulation of key genes for β-cell function, i.e. Glut2, glucokinase, Pdx-1, Hnf-1α, and Tfam. In human islets, chronic resveratrol treatment similarly increased both the glucose secretory response and expression of the same set of genes, eventually restoring the glucose response in islets obtained from one type 2 diabetic donor. Overexpression of Sirt1 in INS-1E cells potentiated resveratrol effects on insulin secretion. Conversely, inhibition of SIRT1 achieved either by expression of an inactive mutant or by using the EX-527 inhibitor, both abolished resveratrol effects on glucose responses. Treatment of INS-1E cells with EX-527 also prevented resveratrol-induced up-regulation of Glut2, glucokinase, Pdx-1, and Tfam. Resveratrol markedly enhanced the glucose response of INS-1E cells and human islets, even after removal of the compound from the medium. These effects were mediated by and fully dependent on active SIRT1, defining a new role for SIRT1 in the regulation of insulin secretion.     

Prospective Randomized Trial Comparing Hepatic Venous Outflow and Renal Function after Conventional versus Piggyback Liver Transplantation

Background

This randomized prospective clinical trial compared the hepatic venous outflow drainage and renal function after conventional with venovenous bypass (n = 15) or piggyback (n = 17) liver transplantation.

Methods

Free hepatic vein pressure (FHVP) and central venous pressure (CVP) measurements were performed after graft reperfusion. Postoperative serum creatinine (Cr) was measured daily on the first week and on the 14^th, 21^st and 28^th postoperative days (PO). The prevalence of acute renal failure (ARF) up to the 28th PO was analyzed by RIFLE-AKIN criteria. A Generalized Estimating Equation (GEE) approach was used for comparison of longitudinal measurements of renal function.

Results

FHVP-CVP gradient > 3 mm Hg was observed in 26.7% (4/15) of the patients in the conventional group and in 17.6% (3/17) in the piggyback group (p = 0.68). Median FHVP-CVP gradient was 2 mm Hg (0–8 mmHg) vs. 3 mm Hg (0–7 mm Hg) in conventional and piggyback groups, respectively (p = 0.73). There is no statistically significant difference between the conventional (1/15) and the piggyback (2/17) groups regarding massive ascites development (p = 1.00). GEE estimated marginal mean for Cr was significantly higher in conventional than in piggyback group (2.14 ± 0.26 vs. 1.47 ± 0.15 mg/dL; p = 0.02). The conventional method presented a higher prevalence of severe ARF during the first 28 PO days (OR = 3.207; 95% CI, 1.010 to 10.179; p = 0.048).

Conclusion

Patients submitted to liver transplantation using conventional or piggyback methods present similar results regarding venous outflow drainage of the graft. Conventional with venovenous bypass technique significantly increases the harm of postoperative renal dysfunction.

Trial Registration

ClinicalTrials.gov https://clinicaltrials.gov/ct2/show/NCT01707810