The Renin prosequence enhances constitutive secretion of Renin and optimizes Renin activity.

Renin is cleaved from its precursor prorenin into mature renin. We investigated the impact of the renin proregion on the generation and secretion of enzymatically active renin. We compared the effects of the following sequences of human prorenin with those of wild type prorenin[1-383]: prosequence [1-43], hinge sequence [1-62], Des[1-43]prorenin ("renin"), Des[1-62]prorenin and prorenin[N260]. These sequences were individually expressed in CV1 cells (constitutive pathway model) and AtT20 cells (regulated and constitutive pathways model), and Des[1-43]prorenin was also coexpressed together with the different prosequences. Renin concentration and activity were measured in cell extracts and culture media. Deletion of the prosequence reduces renin activity in both cell types, but it leaves (total) renin concentration unchanged. Coexpression of the prosequence with renin enhances renin secretion in both cell types: Constitutively secreted renin is enhanced by coexpression of renin together with any of the prosequence containing molecules [1-43], [1-62] or prorenin[N260]. Immunofluorescence in AtT20 cells shows lysosomal typical labeling of prorenin and Des[1-43]prorenin. In AtT20 cells expressing prorenin[1-383], stimulation of regulated secretion increases prorenin but not renin release. The renin prosequence [1-43] optimizes renin activity possibly through appropriate protein folding and it enhances the constitutive secretion of (pro)renin. The major part of generated renin may be targeted to lysosomes.

Molecular mechanisms of insulin exocytosis role of small monomeric GTP-ASES

SUMMARYInsulin secretion from pancreatic beta-cells is a fundamental condition for the maintenance of blood glucose levels. During the last decades, important components of the molecular machinery controlling hormone release have been characterized. My PhD thesis was dedicated to the study of new signaling pathways regulating insulin exocytosis and in particular to the role of small monomelic guanine triphosphatase or GTPases controlling the last events of hormone release.The first part of my thesis focused on Ras-like (Ral) RalA and RalB proteins. We investigated the mechanisms leading to activation of Ral proteins in pancreatic beta-cells and analyzed their impact on different steps of the insulin-secretory process. Our results have shown that RalA is the predominant isoform expressed in pancreatic islets and insulin-secreting cell lines. Silencing of this GTPase in INS-IE cells by RNA interference led to a decrease in secretagogue-induced hormone release. The activation of the GTPase, followed by FRET imaging, is triggered by increases in intracellular Ca and cAMP. Defective insulin release in cells lacking RalA is associated with a decrease in the secretory granules docked at the plasma membrane, detected by TIRF microscopy and with strong impairment in PLD1 activation in response to secretagogues. RalA was found to be activated by the exchange factor RalGDS, which regulates hormone secretion induced by secretagogues and the docking step of insulin-containing granules at the plasma membrane. In the second part of this work we have shown that a member of the Rab family, Rab37, is present on insulin-containing secretory granules of pancreatic beta-cells. In addition, our experiments have suggested that Rab37 is required to obtain an optimal insulin secretory response induced by secretogogues and is important for the docking step of insulin-containing granules at the plasma membrane.

Adapting the Process Writing Approach to English Language Learners with Special Needs: Using Visuals

The available literature on the writing characteristics and best practices to teach writing to English Language Learners who also present some disability is scarce. In order to understand and provide some insight on the developments in this field, I propose an adaptation of the Process Writing Approach based on a literature review of the existing bibliography about the writing characteristics of English Language Learners, Special Needs Learners, and English Language Learners with Special Needs’ writing, the effects of the Process Writing Approach in teaching writing to these groups, and the use of visuals in writing instruction. The main assumptions of this study are: a) The Process Writing Approach provides an opportunity to differentiate instruction to ELLs with special needs and gives them additional opportunities to bring their funds of knowledge to the classroom, improving their writing, and b) By allowing students to rely on visuals in different phases of the writing process teachers will be addressing the needs of both visual and verbal learners, therefore allowing students more options to develop writing skills. The main pedagogical implication is that by dividing writing in recursive stages and inserting visuals as scaffolding throughout the entire writing process, teachers will provide an alternative approach to writing instruction that may be more effective to English Language Learners with Special Needs.

Use of NIR spectroscopy and multivariate process spectra calibration methodology for pharmaceutical solid samples analysis

Accomplish high quality of final products in pharmaceutical industry is a challenge that requires the control and supervision of all the manufacturing steps. This request created the necessity of developing fast and accurate analytical methods. Near infrared spectroscopy together with chemometrics, fulfill this growing demand. The high speed providing relevant information and the versatility of its application to different types of samples lead these combined techniques as one of the most appropriated. This study is focused on the development of a calibration model able to determine amounts of API from industrial granulates using NIR, chemometrics and process spectra methodology.

CD93 is required for maintenance of antibody secretion and persistence of plasma cells in the bone marrow niche.

Plasma cells represent the end stage of B-cell development and play a key role in providing an efficient antibody response, but they are also involved in numerous pathologies. Here we show that CD93, a receptor expressed during early B-cell development, is reinduced during plasma-cell differentiation. High CD93/CD138 expression was restricted to antibody-secreting cells both in T-dependent and T-independent responses as naive, memory, and germinal-center B cells remained CD93-negative. CD93 was expressed on (pre)plasmablasts/plasma cells, including long-lived plasma cells that showed decreased cell cycle activity, high levels of isotype-switched Ig secretion, and modification of the transcriptional network. T-independent and T-dependent stimuli led to re-expression of CD93 via 2 pathways, either before or after CD138 or Blimp-1 expression. Strikingly, while humoral immune responses initially proceeded normally, CD93-deficient mice were unable to maintain antibody secretion and bone-marrow plasma-cell numbers, demonstrating that CD93 is important for the maintenance of plasma cells in bone marrow niches.

In vitro functionality of isolated embryonic hypothalamic vasopressinergic and oxytocinergic neurons: modulatory effects of brain-derived neurotrophic factor and angiotensin II.

There are only a few studies on the ontogeny and differentiation process of the hypothalamic supraoptic-paraventriculo-neurohypophysial neurosecretory system. In vitro neuron survival improves if cells are of embryonic origin; however, surviving hypothalamic neurons in culture were found to express small and minimal amounts of arginine-vasopressin (AVP) and oxytocin (OT), respectively. The aim of this study was to develop a primary neuronal culture design applicable to the study of magnocellular hypothalamic system functionality. For this purpose, a primary neuronal culture was set up after mechanical dissociation of sterile hypothalamic blocks from 17-day-old Sprague-Dawley rat embryos (E17) of both sexes. Isolated hypothalamic cells were cultured with supplemented (B27)-NeuroBasal medium containing an agent inhibiting non-neuron cell proliferation. The neurosecretory process was characterized by detecting AVP and OT secreted into the medium on different days of culture. Data indicate that spontaneous AVP and OT release occurred in a culture day-dependent fashion, being maximal on day 13 for AVP, and on day 10 for OT. Interestingly, brain-derived neurotrophic factor (BDNF) and Angiotensin II (A II) were able to positively modulate neuropeptide output. Furthermore, on day 17 of culture, non-specific (high-KCl) and specific (Angiotensin II) stimuli were able to significantly (P < 0.05) enhance the secretion of both neuropeptides over respective baselines. This study suggests that our experimental design is useful for the study of AVP- and OT-ergic neuron functionality and that BDNF and A II are positive modulators of embryonic hypothalamic cell development.

Microsomal triglyceride transfer protein promotes the secretion of Xenopus laevis vitellogenin A1.

Vitellogenins (Vtg) are ancient lipid transport and storage proteins and members of the large lipid transfer protein (LLTP) gene family, which includes insect apolipophorin II/I, apolipoprotein B (apoB), and the microsomal triglyceride transfer protein (MTP). Lipidation of Vtg occurs at its site of synthesis in vertebrate liver, insect fat body, and nematode intestine; however, the mechanism of Vtg lipid acquisition is unknown. To explore whether Vtg biogenesis requires the apoB cofactor and LLTP family member, MTP, Vtg was expressed in COS cells with and without coexpression of the 97-kDa subunit of human MTP. Expression of Vtg alone gave rise to a approximately 220-kDa apoprotein, which was predominantly confined to an intracellular location. Coexpression of Vtg with human MTP enhanced Vtg secretion by 5-fold, without dramatically affecting its intracellular stability. A comparison of wild type and a triglyceride transfer-defective form of MTP revealed that both were capable of promoting Vtg secretion, whereas only wild type MTP could promote the secretion of apoB41 (amino-terminal 41% of apoB). These studies demonstrate that the biogenesis of Vtg is MTP-dependent and that MTP is the likely ancestral member of the LLTP gene family.

Regulation of insulin secretion by phosphatidylinositol-4,5-bisphosphate.

The role of PIP(2) in pancreatic beta cell function was examined here using the beta cell line MIN6B1. Blocking PIP(2) with PH-PLC-GFP or PIP5KIgamma RNAi did not impact on glucose-stimulated secretion although susceptibility to apoptosis was increased. Over-expression of PIP5KIgamma improved cell survival and inhibited secretion with accumulation of endocytic vacuoles containing F-actin, PIP(2), transferrin receptor, caveolin 1, Arf6 and the insulin granule membrane protein phogrin but not insulin. Expression of constitutively active Arf6 Q67L also resulted in vacuole formation and inhibition of secretion, which was reversed by PH-PLC-GFP co-expression. PIP(2) co-localized with gelsolin and F-actin, and gelsolin co-expression partially reversed the secretory defect of PIP5KIgamma-over-expressing cells. RhoA/ROCK inhibition increased actin depolymerization and secretion, which was prevented by over-expressing PIP5KIgamma, while blocking PIP(2) reduced constitutively active RhoA V14-induced F-actin polymerization. In conclusion, although PIP(2) plays a pro-survival role in MIN6B1 cells, excessive PIP(2) production because of PIP5KIgamma over-expression inhibits secretion because of both a defective Arf6/PIP5KIgamma-dependent endocytic recycling of secretory membrane and secretory membrane components such as phogrin and the RhoA/ROCK/PIP5KIgamma-dependent perturbation of F-actin cytoskeleton remodelling.

Regulation of glucagon secretion by glucose transporter type 2 (glut2) and astrocyte-dependent glucose sensors.

Ripglut1;glut2-/- mice have no endogenous glucose transporter type 2 (glut2) gene expression but rescue glucose-regulated insulin secretion. Control of glucagon plasma levels is, however, abnormal, with fed hyperglucagonemia and insensitivity to physiological hypo- or hyperglycemia, indicating that GLUT2-dependent sensors control glucagon secretion. Here, we evaluated whether these sensors were located centrally and whether GLUT2 was expressed in glial cells or in neurons. We showed that ripglut1;glut2-/- mice failed to increase plasma glucagon levels following glucoprivation induced either by i.p. or intracerebroventricular 2-deoxy-D-glucose injections. This was accompanied by failure of 2-deoxy-D-glucose injections to activate c-Fos-like immunoreactivity in the nucleus of the tractus solitarius and the dorsal motor nucleus of the vagus. When glut2 was expressed by transgenesis in glial cells but not in neurons of ripglut1;glut2-/- mice, stimulated glucagon secretion was restored as was c-Fos-like immunoreactive labeling in the brainstem. When ripglut1;glut2-/- mice were backcrossed into the C57BL/6 genetic background, fed plasma glucagon levels were also elevated due to abnormal autonomic input to the alpha cells; glucagon secretion was, however, stimulated by hypoglycemic stimuli to levels similar to those in control mice. These studies identify the existence of central glucose sensors requiring glut2 expression in glial cells and therefore functional coupling between glial cells and neurons. These sensors may be activated at different glycemic levels depending on the genetic background.

Análisis y mejora de los procesos excepcionales de una multinacional del sector de la electrónica utilizando la metodología Business Process Management

El presente documento ilustra la aplicación de la metodología Business Process Managementpara el caso de una empresa multinacional del sector de la electrónica. Para ello se han tomado los procesos excepcionales de Supply Chain Operations en el área EMEA (Europa, Oriente Medio y África). Se ha analizado la situación inicial, donde la aparición de incidencias de calidad en productos terminados y listos para entregar a clientes generaba una serie de acciones descoordinadas y con resultados insatisfactorios. Todos los departamentos implicados comprometían recursos, tiempo y esfuerzo, sin estar alineados entre sí. A partir de la aplicación sistemática de la metodología BPM definida en 10 fases, se ha desarrollado una solución completa para los procesos excepcionales. El documento describe con detalle en proceso de Reflash y la documentación necesaria para poner el proceso bajo control y en mejora continua.

Expression of an uncleavable N-terminal RasGAP fragment in insulin-secreting cells increases their resistance toward apoptotic stimuli without affecting their glucose-induced insulin secretion.

Apoptosis of pancreatic beta cells is implicated in the onset of type 1 and type 2 diabetes. Consequently, strategies aimed at increasing the resistance of beta cells toward apoptosis could be beneficial in the treatment of diabetes. RasGAP, a regulator of Ras and Rho GTPases, is an atypical caspase substrate, since it inhibits, rather than favors, apoptosis when it is partially cleaved by caspase-3 at position 455. The antiapoptotic signal generated by the partial processing of RasGAP is mediated by the N-terminal fragment (fragment N) in a Ras-phosphatidylinositol 3-kinase-Akt-dependent, but NF-kappaB-independent, manner. Further cleavage of fragment N at position 157 abrogates its antiapoptotic properties. Here we demonstrate that an uncleavable form of fragment N activates Akt, represses NF-kappaB activity, and protects the conditionally immortalized pancreatic insulinoma betaTC-tet cell line against various insults, including exposure to genotoxins, trophic support withdrawal, and incubation with inflammatory cytokines. Fragment N also induced Akt activity and protection against cytokine-induced apoptosis in primary pancreatic islet cells. Fragment N did not alter insulin cell content and insulin secretion in response to glucose. These data indicate that fragment N protects beta cells without affecting their function. The pathways regulated by fragment N are therefore promising targets for antidiabetogenic therapy.