Down-regulated expression of exocytotic proteins in pancreatic islets of diabetic GK rats

Exocytosis is regulated by exocytotic proteins, which are present in insulin-secreting beta-cells and play regulatory roles in insulin secretion. Non-insulin dependent diabetes mellitus (type 2 diabetes) is a disease characterized by impaired insulin secretion and insulin resistance. Exocytotic protein immunoreactivities were studied in pancreatic islets of type 2 diabetic Goto-Kakizaki (GK) rats using immunofluorescence histochemistry. The immunoreactivities for vesicle-associated membrane protein-2 (VAMP-2), synaptotagmin III, cysteine string protein (CSP), mammalian homologue of the unc-18 gene (Munc-18), alpha-soluble N-ethylmaleimide-sensitive attachment protein (alpha-SNAP), N-ethylmaleimide-sensitive factor (NSF) and synaptosomal-associated protein of 25 kDa (SNAP-25) exhibited weaker immunofluorescence intensity in islets of GK rats as compared to control Wistar rats. Insulin immunoreactivity was also decreased in GK rat beta-cells, whereas no detectable alterations in the expression of actin immunoreactivity could be detected. The data suggest that reduced expression of exocytotic proteins and decreased insulin content may contribute to the diabetic syndrome in the GK rat.     

The ductal origin of structural and functional heterogeneity between pancreatic islets

Islets form in the pancreas after the first endocrine cells have arisen as either single cells or small cell clusters in the epithelial cords. These cords constitute the developing pancreas in one of its earliest recognizable stages. Islet formation begins at the time the cords transform into a branching ductal system, continues while the ductal system expands, and finally stops before the exocrine tissue of ducts and acini reaches its final expansion. Thus, islets continuously arise from founder cells located in the branching and ramifying ducts. Islets arising from proximal duct cells locate between the exocrine lobules, develop strong autonomic and sensory innervations, and pass their blood to efferent veins (insulo-venous efferent system). Islets arising from cells of more distal ducts locate within the exocrine lobules, respond to nerve impulses ending at neighbouring blood vessels, and pass their blood to the surrounding acini (insulo-acinar portal system). Consequently, the section of the ductal system from which an islet arises determines to a large extent its future neighbouring tissue, architecture, properties, and functions. We note that islets interlobular in position are frequently found in rodents (rats and mice), whereas intralobularly-located, peripheral duct islets prevail in humans and cattle. Also, we expound on bovine foetal Laguesse islets as a prominent foetal type of type 1 interlobular neuro-insular complexes, similar to neuro-insular associations frequently found in rodents. Finally, we consider the probable physiological and pathophysiological implications of the different islet positions within and between species.     

Rat olfactory bulb mitral cells receive sparse glomerular inputs

Center-surround receptive fields are a fundamental unit of brain organization. It has been proposed that olfactory bulb mitral cells exhibit this functional circuitry, with excitation from one glomerulus and inhibition from a broad field of glomeruli within reach of the lateral dendrites. We investigated this hypothesis using a combination of in vivo intrinsic imaging, single-unit recording, and a large panel of odors. Assuming a broad inhibitory field, a mitral cell would be influenced by >100 contiguous glomeruli and should respond to many odors. Instead, the observed response rate was an order of magnitude lower. A quantitative model indicates that mitral cell responses can be explained by just a handful of glomeruli. These glomeruli are spatially dispersed on the bulb and represent a broad range of odor sensitivities. We conclude that mitral cells do not have center-surround receptive fields. Instead, each mitral cell performs a specific computation combining a small and diverse set of glomerular inputs.     

Insertion of lipidated Ras proteins into lipid monolayers studied by infrared reflection absorption spectroscopy (IRRAS)

Ras proteins have to be associated with the inner leaflet of the plasma membrane to perform their signaling functions. This membrane targeting and binding is controlled by post-translational covalent attachment of farnesyl and palmitoyl chains to cysteines in the membrane anchor region of the N- and H-Ras isoforms. Two N-Ras lipoproteins were investigated, namely a farnesylated and hexadecylated protein, presenting the natural hydrophobic modifications and a doubly hexadecylated construct, respectively. The proteins are surface active and form a Gibbs monolayer at the air-D2O interface. The contours of the amide-I bands were analyzed using infrared reflection absorption spectroscopy (IRRAS). Langmuir monolayers of a mixture of POPC, brain sphingomyelin, and cholesterol were used as half of a model biomembrane to study the insertion of these N-Ras proteins. They insert with their hydrophobic anchors into lipid monolayers but at higher surface pressures (30 mN/m); the farnesylated and hexadecylated protein desorbs completely from the monolayer, whereas the doubly hexadecylated protein remains incorporated. During the insertion process, changes in the orientation of the protein secondary structure were detected by comparison with simulated IRRA spectra, based on the information on the relative orientation of the secondary structure elements from the protein crystal structure data.     

Immune Modulation Mediated by Cryptococcal Laccase Promotes Pulmonary Growth and Brain Dissemination of Virulent Cryptococcus neoformans in Mice

C. neoformans is a leading cause of fatal mycosis linked to CNS dissemination. Laccase, encoded by the LAC1 gene, is an important virulence factor implicated in brain dissemination yet little is known about the mechanism(s) accounting for this observation. Here, we investigated whether the presence or absence of laccase altered the local immune response in the lungs by comparing infections with the highly virulent strain, H99 (which expresses laccase) and mutant strain of H99 deficient in laccase (lac1Δ) in a mouse model of pulmonary infection. We found that LAC1 gene deletion decreased the pulmonary fungal burden and abolished CNS dissemination at weeks 2 and 3. Furthermore, LAC1 deletion lead to: 1) diminished pulmonary eosinophilia; 2) increased accumulation of CD4+ and CD8+ T cells; 3) increased Th1 and Th17 cytokines yet decreased Th2 cytokines; and 4) lung macrophage shifting of the lung macrophage phenotype from M2- towards M1-type activation. Next, we used adoptively transferred CD4+ T cells isolated from pulmonary lymph nodes of mice infected with either lac1Δ or H99 to evaluate the role of laccase-induced immunomodulation on CNS dissemination. We found that in comparison to PBS treated mice, adoptively transferred CD4+ T cells isolated from lac1Δ-infected mice decreased CNS dissemination, while those isolated from H99-infected mice increased CNS dissemination. Collectively, our findings reveal that immune modulation away from Th1/Th17 responses and towards Th2 responses represents a novel mechanism through which laccase can contribute to cryptococcal virulence. Furthermore, our data support the hypothesis that laccase-induced changes in polarization of CD4+ T cells contribute to CNS dissemination.     

Intercellular calcium waves in primary cultured rat mesenteric smooth muscle cells are mediated by connexin43

Intercellular Ca(2+) wave propagation between vascular smooth muscle cells (SMCs) is associated with the propagation of contraction along the vessel. Here, we characterize the involvement of gap junctions (GJs) in Ca(2+) wave propagation between SMCs at the cellular level. Gap junctional communication was assessed by the propagation of intercellular Ca(2+) waves and the transfer of Lucifer Yellow in A7r5 cells, primary rat mesenteric SMCs (pSMCs), and 6B5N cells, a clone of A7r5 cells expressing higher connexin43 (Cx43) to Cx40 ratio. Mechanical stimulation induced an intracellular Ca(2+) wave in pSMC and 6B5N cells that propagated to neighboring cells, whereas Ca(2+) waves in A7r5 cells failed to progress to neighboring cells. We demonstrate that Cx43 forms the functional GJs that are involved in mediating intercellular Ca(2+) waves and that co-expression of Cx40 with Cx43, depending on their expression ratio, may interfere with Cx43 GJ formation, thus altering junctional communication.