VEGF-induced mobilization of caveolae and increase in permeability of endothelial cells

Glomerular epithelial cells (GEC) are aknown site of vascular endothelial growth factor (VEGF) production. Weestablished immortalized rat GEC, which retained the ability to produceVEGF. The isoforms expressed by GEC were defined as VEGF-205, -188, -120, and -164. The electrical resistance of endothelial cells culturedon GEC-conditioned matrix, an indicator of the permeability ofmonolayers to solutes, was significantly increased by the treatment with the neutralizing polyclonal antibodies to VEGF and decreased byVEGF-165. Transfection of endothelial cells with green fluorescence protein-caveolin construct and intravital confocal microscopy showedthat VEGF results in a rapid appearance of transcellular elongatedstructures decorated with caveolin. Transmission electron microscopy ofendothelial cells showed that caveolae undergo rapid internalizationand fusion 30 min after application of VEGF-165. Later (36 h),endothelial cells pretreated with VEGF developed fenestrae and showed adecrease in electrical resistance. Immunoelectron microscopy ofglomeruli confirmed VEGF localization to podocytes and in the basementmembrane. In summary, immortalized GEC retain the ability to synthesizeVEGF. Matrix-deposited and soluble VEGF leads to the enhancement ofcaveolae expression, their fission and fusion, formation of elongatedcaveolin-decorated structures, and eventual formation of fenestrae,both responsible for the increase in endothelial permeability.

     

Efficacité et innocuité du prélèvement percutané de sperme testiculaire dans la prise en charge de l’infertilité masculine

Objective

To assess the efficacy and safety of percutaneous testicular biopsy to provide sperm cells for ICSI in male patients with azoospermia not amenable to surgical treatment.

Materials and methods

From October 1995 to December 2001, 175 biopsies were performed in men with azoospermia to provide material for intracytoplasmic sperm injection. Azoospermia was obstructive (OA) in 41 cases and non-obstructive (NOA) in 134 cases. Open biopsy was performed in the first 15 patients in the series and percutaneous biopsy was performed on an outpatient basis, under local anesthesia, with a Biopty Gun® (14G needle), in the subsequent patients as the first step in management. Open surgical biopsies were performed in another 15 patients following a sperm cell-negative percutaneous biopsy.

Results

All biopsies performed for OA were positive, but only 51/134 biopsies (38%) were positive in the NOA group. The material provided by percutaneous biopsy, when positive for sperm cells, was always sufficient to perform ICSI. When percutaneous biopsy was negative, open surgical biopsy failed to give better results. Five men developed minor complications (acute hematocele) following percutaneous biopsies requiring reoperation for hemostasis (3.12%). No major complications were observed. Results were comparable in terms of fertilization and pregnancy rates whether fresh or frozen-thawed sperm was used.

Conclusion

Percutaneous testicular sperm extraction is a safe, well-tolerated and cost-effective procedure in the management of male-factor infertility related to azoospermia.     

Unseen proteome: mining below the tip of the iceberg to find low abundance and membrane proteins

Abundant and hydrophilic nonmembrane proteins with isoelectric points below pH 8 are the predominant proteins identified in most proteomics projects. In yeast, however, low-abundance proteins make up 80% of the predicted proteome, approximately 50% have pl's above pH 8 and 30% of the yeast ORFs are predicted to encode membrane proteins with at least 1 trans-membrane span. By applying highly solubilizing reagents and isoelectric fractionation to a membrane fraction of yeast we have a purified and identified 780 protein isoforms, representing 323 gene products, including 28% low abundance proteins and 49% membrane or membrane associated proteins. More importantly, considering the frequency and importance of co- and post-translational modifications, the separation of protein isoforms is essential and two-dimensional electrophoresis remains the only technique which offers sufficient resolution to address this at a proteomic level.     

Roles of hnRNP A1, SR proteins,and p68 helicase in c-H-ras alternative splicing regulation

Human ras genes play central roles in coupling extracellular signals with complex intracellular networks controlling proliferation, differentiation, and apoptosis, among others processes. c-H-ras pre-mRNA can be alternatively processed into two mRNAs due to the inclusion or exclusion of the alternative exon IDX; this renders two proteins, p21H-Ras and p19H-RasIDX, which differ only at the carboxy terminus. Here, we have characterized some of the cis-acting sequences and trans-acting factors regulating IDX splicing. A downstream intronic silencer sequence (rasISS1), acting in concert with IDX, negatively regulates upstream intron splicing. This effect is mediated, at least in part, by the binding of hnRNP A1. Depletion and add-back experiments in nuclear extracts have confirmed hnRNP A1's inhibitory role in IDX splicing. Moreover, the addition of two SR proteins, SC35 and SRp40, can counteract this inhibition by strongly promoting the splicing of the upstream intron both in vivo and in vitro. Further, the RNA-dependent helicase p68 is also associated with both IDX and rasISS1 RNA, and suppression of p68 expression in HeLa cells by RNAi experiments results in a marked increase of IDX inclusion in the endogenous mRNA, suggesting a role for this protein in alternative splicing regulation.     

Prospection and Evaluation of (Hemi) Cellulolytic Enzymes Using Untreated and Pretreated Biomasses in Two Argentinean Native Termites

Saccharum officinarum bagasse (common name: sugarcane bagasse) and Pennisetum purpureum (also known as Napier grass) are among the most promising feedstocks for bioethanol production in Argentina and Brazil. In this study, both biomasses were assessed before and after acid pretreatment and following hydrolysis with Nasutitermes aquilinus and Cortaritermes fulviceps termite gut digestome. The chemical composition analysis of the biomasses after diluted acid pretreatment showed that the hemicellulose fraction was partially removed. The (hemi) cellulolytic activities were evaluated in bacterial culture supernatants of termite gut homogenates grown in treated and untreated biomasses. In all cases, we detected significantly higher endoglucanase and xylanase activities using pretreated biomasses compared to untreated biomasses, carboxymethylcellulose and xylan. Several protein bands with (hemi) cellulolytic activity were detected in zymograms and two-dimensional gel electrophoresis. Some proteins of these bands or spots were identified as xylanolytic peptides by mass spectrometry. Finally, the diversity of cultured cellulolytic bacterial endosymbionts associated to both Argentinean native termite species was analyzed. This study describes, for the first time, bacterial endosymbionts and endogenous (hemi) cellulases of two Argentinean native termites as well as their potential application in degradation of lignocellulosic biomass for bioethanol production.     

A naringenin–tamoxifen combination impairs cell proliferation and survival of MCF-7 breast cancer cells

Since over 60% of breast cancers are estrogen receptor positive (ER+), many therapies have targeted the ER. The ER is activated by both estrogen binding and phosphorylation. While anti-estrogen therapies, such as tamoxifen (Tam) have been successful they do not target the growth factor promoting phosphorylation of the ER. Other proliferation pathways such as the phosphatidylinositol-3 kinase, (PI3K) and the mitogen-activated protein kinase (MAPK) pathways are activated in breast cancer cells and are associated with poor prognosis. Thus targeting multiple cellular proliferation and survival pathways at the onset of treatment is critical for the development of more effective therapies. The grapefruit flavanone naringenin (Nar) is an inhibitor of both the PI3K and MAPK pathways. Previous studies examining either Nar or Tam used charcoal-stripped serum which removed estrogen as well as other factors. We wanted to use serum containing medium in order to retain all the potential inducers of cell proliferation so as not to exclude any targets of Nar. Here we show that a Nar–Tam combination is more effective than either Tam alone or Nar alone in MCF-7 breast cancer cells. We demonstrate that a Nar–Tam combination impaired cellular proliferation and viability to a greater extent than either component alone in MCF-7 cells. Furthermore, the use of a Nar–Tam combination requires lower concentrations of both compounds to achieve the same effects on proliferation and viability. Nar may function by inhibiting both PI3K and MAPK pathways as well as localizing ERα to the cytoplasm in MCF-7 cells. Our results demonstrate that a Nar–Tam combination induces apoptosis and impairs proliferation signaling to a greater extent than either compound alone. These studies provide critical information for understanding the molecular mechanisms involved in cell proliferation and apoptosis in breast cancer cells.     

Sensitive period for a multimodal response in human visual motion area MT/MST

The middle temporal complex (MT/MST) is a brain region specialized for the perception of motion in the visual modality. However, this specialization is modified by visual experience: after long-standing blindness, MT/MST responds to sound. Recent evidence also suggests that the auditory response of MT/MST is selective for motion. The developmental time course of this plasticity is not known. To test for a sensitive period in MT/MST development, we used fMRI to compare MT/MST function in congenitally blind, late-blind, and sighted adults. MT/MST responded to sound in congenitally blind adults, but not in late-blind or sighted adults, and not in an individual who lost his vision between ages of 2 and 3 years. All blind adults had reduced functional connectivity between MT/MST and other visual regions. Functional connectivity was increased between MT/MST and lateral prefrontal areas in congenitally blind relative to sighted and late-blind adults. These data suggest that early blindness affects the function of feedback projections from prefrontal cortex to MT/MST. We conclude that there is a sensitive period for visual specialization in MT/MST. During typical development, early visual experience either maintains or creates a vision-dominated response. Once established, this response profile is not altered by long-standing blindness.     

Osteoblast Menin Regulates Bone Mass in Vivo

Menin, the product of the multiple endocrine neoplasia type 1 (Men1) tumor suppressor gene, mediates the cell proliferation and differentiation actions of transforming growth factor-β (TGF-β) ligand family members. In vitro, menin modulates osteoblastogenesis and osteoblast differentiation promoted and sustained by bone morphogenetic protein-2 (BMP-2) and TGF-β, respectively. To examine the in vivo function of menin in bone, we conditionally inactivated Men1 in mature osteoblasts by crossing osteocalcin (OC)-Cre mice with floxed Men1 (Men1^f/f) mice to generate mice lacking menin in differentiating osteoblasts (OC-Cre;Men1^f/f mice). These mice displayed significant reduction in bone mineral density, trabecular bone volume, and cortical bone thickness compared with control littermates. Osteoblast and osteoclast number as well as mineral apposition rate were significantly reduced, whereas osteocyte number was increased. Primary calvarial osteoblasts proliferated more quickly but had deficient mineral apposition and alkaline phosphatase activity. Although the mRNA expression of osteoblast marker and cyclin-dependent kinase inhibitor genes were all reduced, that of cyclin-dependent kinase, osteocyte marker, and pro-apoptotic genes were increased in isolated Men1 knock-out osteoblasts compared with controls. In contrast to the knock-out mice, transgenic mice overexpressing a human menin cDNA in osteoblasts driven by the 2.3-kb Col1a1 promoter, showed a gain of bone mass relative to control littermates. Osteoblast number and mineral apposition rate were significantly increased in the Col1a1-Menin-Tg mice. Therefore, osteoblast menin plays a key role in bone development, remodeling, and maintenance.