Walter I. Silva

Caveolin isoform expression during differentiation of C6 glioma cells.

Caveolae, a specialized form of lipid rafts, are cholesterol‐ and sphingolipid‐rich membrane microdomains implicated in potocytosis, endocytosis, transcytosis, and as platforms for signal transduction. One of the major constituents of caveolae are three highly homologous caveolin isoforms (caveolin‐1, caveolin‐2, and caveolin‐3). The present study expands the analysis of caveolin isoform expression in C6 glioma cells. Three complementary approaches were used to assess their differential expression during the dibutyryl‐cyclic AMP‐induced differentiation of C6 cells into an astrocyte‐like phenotype. Immunoblotting, conventional RT‐PCR, and real‐time RT‐PCR analysis established the expression of the caveolin‐3 isoform in C6 cells, in addition to caveolin‐1 and caveolin‐2. Similar to the other isoforms, caveolin‐3 was associated with light‐density, detergent‐insoluble caveolae membrane fractions obtained using sucrose‐density gradient centrifugation. The three caveolin isoforms display different temporal patterns of mRNA/protein expression during the differentiation of C6 cells. Western blot and real‐time RT‐PCR analysis demonstrate that caveolin‐1 and caveolin‐2 are up‐regulated during the late stages of the differentiation of C6 cells. Meanwhile, caveolin‐3 is gradually down‐regulated during the differentiation process. Indirect immunofluorescence analysis via laser‐scanning confocal microscopy reveals that the three caveolin isoforms display similar subcellular distribution patterns. In addition, co‐localization of caveolin‐1/caveolin‐2 and caveolin‐1/caveolin‐3 was detected in both C6 glioma phenotypes. The findings reveal a differential temporal pattern of caveolin gene expression during phenotypic differentiation of C6 glioma cells, with potential implications to developmental and degenerative events in the brain.

Numerical and experimental investigation of transient pipe flow

Two mathematical formulations for the computation of transient flow in piping systems are compared with experimental data. The formulations are: a four-equations fluid structure interaction model (FSI) that includes Poisson coupling, and a two-equations model for the fluid. Both models are solved numericaly using the method of characteristics. A partial-closure of a valve located at an intermediate point in a pipeline is used to create transient flow. The two-equations model computed the maximum pressure peak satisfactorily but the FSI model gave an overall better simulation. An unsteady-friction model, added to the FSI model, did not influence the final results significantly. The experimental procedures followed to obtain the valve characteristics and the pressure history along the pipeline are explained in detail. Excellent numerical results at the valve are obtained when experimental data is used to simulate the time-dependent boundary condition.

Caveolins in glial cell model systems: from detection to significance

Glial cells prevail in number and in diversity of cellular phenotypes in the nervous system. They have also gained prominence due to their multiple physiological and pathophysiological roles. Our current knowledge of the asymmetry and heterogeneity of the plasma membrane demands an in depth analysis of the diverse array of membrane microdomains postulated to exist in the context of glial cells. This review focuses and analyzes the studies reported to date on the detection of caveolae membrane rafts and the caveolin family members in glial cell model systems, the conditions leading to changes in their level of expression, and their functional and clinical significance. Outstanding in this work emerge the ubiquitous expression of caveolins, including the typically regarded ‘muscle‐specific’ cav3, in diverse glial cell model systems, their participation in reactive astrogliosis, cancer, and their key relevance to calcium signaling. The knowledge obtained to date demands incorporation of the caveolins and caveolae membrane rafts in our current models on the role of glial cells in heath and neurological disease.

The α7-nicotinic receptor is upregulated in immune cells from HIV-seropositive women: consequences to the cholinergic anti-inflammatory response.

Antiretroviral therapy partially restores the immune system and markedly increases life expectancy of HIV‐infected patients. However, antiretroviral therapy does not restore full health. These patients suffer from poorly understood chronic inflammation that causes a number of AIDS and non‐AIDS complications. Here we show that chronic inflammation in HIV+ patients may be due to the disruption of the cholinergic anti‐inflammatory pathway by HIV envelope protein gp120IIIB. Our results demonstrate that HIV gp120IIIB induces α7 nicotinic acetylcholine receptor (α7) upregulation and a paradoxical proinflammatory phenotype in macrophages, as activation of the upregulated α7 is no longer capable of inhibiting the release of proinflammatory cytokines. Our results demonstrate that disruption of the cholinergic‐mediated anti‐inflammatory response can result from an HIV protein. Collectively, these findings suggest that HIV tampering with a natural strategy to control inflammation could contribute to a crucial, unresolved problem of HIV infection: chronic inflammation.

SorLA in Glia: Shared Subcellular Distribution Patterns with Caveolin-1

SorLA is an established sorting and trafficking protein in neurons with demonstrated relevance to Alzheimer’s disease (AD). It shares these roles with the caveolins, markers of membrane rafts microdomains. To further our knowledge on sorLA’s expression and traffic, we studied sorLA expression in various cultured glia and its relation to caveolin-1 (cav-1), a caveolar microdomain marker. RT-PCR and immunoblots demonstrated sorLA expression in rat C6 glioma, primary cultures of rat astrocytes (PCRA), and human astrocytoma 1321N1 cells. PCRA were determined to express the highest levels of sorLA’s message. Induction of differentiation of C6 cells into an astrocyte-like phenotype led to a significant decrease in sorLA’s mRNA and protein expression. A set of complementary experimental approaches establish that sorLA and cav-1 directly or indirectly interact in glia: (1) co-fractionation in light-density membrane raft fractions of rat C6 glioma, PCRA, and human 1321N1 astrocytoma cells; (2) a subcellular co-localization distribution pattern in vesicular perinuclear compartments seen via confocal imaging in C6 and PCRA; (3) additional confocal analysis in C6 cells suggesting that the perinuclear compartments correspond to their co-localization in early endosomes and the trans-Golgi; and; (4) co-immunoprecipitation data strongly supporting their direct or indirect physical interaction. These findings further establish that sorLA is expressed in glia and that it shares its subcellular distribution pattern with cav-1. A direct or indirect cav-1/sorLA interaction could modify the trafficking and sorting functions of sorLA in glia and its proposed neuroprotective role in AD.

Caveolin-1 Regulates the P2Y2 Receptor Signaling in Human 1321N1 Astrocytoma Cells.

Damage to the CNS can cause a differential spatio-temporal release of multiple factors, such as nucleotides, ATP and UTP. The latter interact with neuronal and glial nucleotide receptors. The P2Y2 nucleotide receptor (P2Y2R) has gained prominence as a modulator of gliotic responses after CNS injury. Still, the molecular mechanisms underlying these responses in glia are not fully understood. Membrane-raft microdomains, such as caveolae, and their constituent caveolins, modulate receptor signaling in astrocytes; yet, their role in P2Y2R signaling has not been adequately explored. Hence, this study evaluated the role of caveolin-1 (Cav-1) in modulating P2Y2R subcellular distribution and signaling in human 1321N1 astrocytoma cells. Recombinant hP2Y2R expressed in 1321N1 cells and Cav-1 were found to co-fractionate in light-density membrane-raft fractions, co-localize via confocal microscopy, and co-immunoprecipitate. Raft localization was dependent on ATP stimulation and Cav-1 expression. This hP2Y2R/Cav-1 distribution and interaction was confirmed with various cell model systems differing in the expression of both P2Y2R and Cav-1, and shRNA knockdown of Cav-1 expression. Furthermore, shRNA knockdown of Cav-1 expression decreased nucleotide-induced increases in the intracellular Ca2+ concentration in 1321N1 and C6 glioma cells without altering TRAP-6 and carbachol Ca2+ responses. In addition, Cav-1 shRNA knockdown also decreased AKT phosphorylation and altered the kinetics of ERK1/2 activation in 1321N1 cells. Our findings strongly suggest that P2Y2R interaction with Cav-1 in membrane-raft caveolae of 1321N1 cells modulates receptor coupling to its downstream signaling machinery. Thus, P2Y2R/Cav-1 interactions represent a novel target for controlling P2Y2R function after CNS injury.