Modulation of glycolysis induction in primary cultures of rabbit kidney proximal tubule cells: the role of shaking,glucose and insulin.

We have assessed the impact of increasing oxygen availability on cellular phenotype expression of rabbit proximal tubule cells in primary culture developed with variable glucose and/or insulin contents. To mitigate hypoxia at the cell/medium interface, cells were shaken for the whole culture duration and their expressed phenotype was compared with those expressed by static cultures. O₂ and CO₂ tensions were kept constant in the incubator atmosphere. Glycolysis and gluconeogenesis pathways, detoxication system, and mitochondrial, apical and basolateral membrane marker enzyme activities were assessed. This study showed that the induction of glycolysis which appear in primary cultures of proximal tubule cells may be partially prevented by continuously shaking the cultures. This effect was more marked in the presence of glucose, suggesting better substrate oxidation in shaken cultures.     

Productive infection maintains a dynamic steady state of residual viremia in human immunodeficiency virus type 1-infected persons treated with suppressive antiretroviral therapy for five years

To provide insight into the dynamics and source of residual viremia in human immunodeficiency virus (HIV) patients successfully treated with antiretroviral therapy, 14 intensely monitored patients treated with indinavir and efavirenz sustaining HIV RNA at <50 copies/ml for >5 years were studied. Abacavir was added to the regimen of eight patients at year 5. After the first 9 months of therapy, HIV RNA levels had reached a plateau ("residual viremia") that persisted for over 5 years. Levels of residual viremia differed among patients and ranged from 3.2 to 23 HIV RNA copies/ml. Baseline HIV DNA was the only significant pretreatment predictor of residual viremia in regression models including baseline HIV RNA, CD4 count, and patient age. In the four of five patients with detectable viremia who added abacavir to their regimen after 5 years, HIV RNA levels declined rapidly. The estimated half-life of infected cells was 6.7 days. Decrease in activated memory cells and a reduction in gamma interferon production to HIV Gag and p24 antigen in ELISpot assays were observed, consistent with a decrease in HIV replication. Thus, in patients treated with efavirenz plus indinavir, levels of residual viremia were established by 9 months, were predicted by baseline proviral DNA, and remained constant for 5 years. Even after years of highly suppressive therapy, HIV RNA levels declined rapidly after the addition of abacavir, suggesting that productive infection contributes to residual ongoing viremia and can be inhibited with therapy intensification.     

Regulation of the mitogen-activated protein kinase signaling pathway by SHP2.

Gab1-SHP2 association is required for Erk mitogen-activated protein kinase activation by several growth factors. Gab1-SHP2 interaction activates SHP2. However, an activated SHP2 still needs to associate with Gab1 to mediate Erk activation. It was unclear whether SHP2 is required to dephosphorylate a negative phosphorylation site on Gab1 or whether SHP2 needs the Gab1 pleckstrin homology (PH) domain to target it to the plasma membrane. We found that expression of a fusion protein consisting of the Gab1 PH domain and an active SHP2 (Gab1PH-SHP2DeltaN) induced constitutive Mek1 and Erk2 activation. Linking the active SHP2DeltaN to the PDK1 PH domain or the FRS2beta myristoylation sequence also induced Mek1 activation. Mek1 activation by Gab1PH-SHP2DeltaN was inhibited by an Src inhibitor and by Csk. Significantly, Gab1PH-SHP2DeltaN induced Src activation. Gab1PH-SHP2DeltaN expression activated Ras, and the Gab1PH-SHP2DeltaN-induced Mek1 activation was blocked by RasN17. These findings suggest that Gab1PH-SHP2DeltaN activated a signaling step upstream of Src and Ras. The SHP2 tyrosine phosphatase activity is essential for the function of the fusion protein. Together, these data show that the Gab1 sequence, besides the PH domain and SHP2 binding sites, is dispensable for Erk activation, suggesting that the primary role of Gab1 association with an activated SHP2 is to target it to the membrane.     

Increased survival in sepsis by in vivo adenovirus-induced expression of IL-10 in dendritic cells

The dendritic cell (DC) is the most potent APC of the immune system, capable of stimulating naive T cells to proliferate and differentiate into effector T cells. Recombinant adenovirus (Adv) readily transduces DCs in vitro allowing directed delivery of transgenes that modify DC function and immune responses. In this study we demonstrate that footpad injection of a recombinant Adv readily targets transduction of myeloid and lymphoid DCs in the draining popliteal lymph node, but not in other lymphoid organs. Popliteal DCs transduced with an empty recombinant Adv undergo maturation, as determined by high MHC class II and CD86 expression. However, transduction with vectors expressing human IL-10 limit DC maturation and associated T cell activation in the draining lymph node. The extent of IL-10 expression is dose dependent; transduction with low particle numbers (10(5)) yields only local expression, while transduction with higher particle numbers (10(7) and 10(10)) leads additionally to IL-10 appearance in the circulation. Furthermore, local DC expression of human IL-10 following in vivo transduction with low particle numbers (10(5)) significantly improves survival following cecal ligation and puncture, suggesting that compartmental modulation of DC function profoundly alters the sepsis-induced immune response.     

Evidence for the mechanism of the irreversible inhibition of oestrone sulphatase (ES) by aminosulphonate based compounds

In our search for the mechanism of the enzyme oestrone sulphatase (ES) we have synthesised and evaluated a number of compounds that were predicted to possess some inhibitory activity. Some of these compounds were indeed found to be inhibitors of ES, whilst other compounds were not. From a consideration of the structure–activity relationship (SAR) of the inhibitors and non-inhibitors of this enzyme, we discovered a factor which we now believe is the main inhibitory moiety within the aminosulphonated inhibitors. We therefore report the results of our study into a series of phenyl and alkyl sulphamated compounds as inhibitors of ES. The results of the study show that the substituted phenyl sulphamates are potent inhibitors, whereas the alkyl compounds are, in general, non-inhibitors. Using the results of our SAR study, we postulate the probable mechanism for the irreversible and reversible inhibition of ES, and rationalise the role of the different physicochemical factors in the inhibition of this crucial enzyme.     

The Field of Art Production and Western Desert Acrylics1

Following the logic of Bourdieu's notion of ‘field’, acrylic art production in the Western Desert of Australia is positioned as a sub‐field in the field of Western art production. The significance of the cultural identity of the producer subordinates Western Desert acrylic art within the field and the shift in the criteria of legitimation constitutes it as a sub‐field. The capital at stake in the sub‐field of Western Desert acrylic art is differentiated and contingent upon legitimation of authenticity as defined by the Western field of art production. This capital is constituted through the positioning of Western Desert acrylics within the field of art production and is a manifestation of the struggle to maintain domination within the field.     

Knockdown resistance (kdr) to DDT and pyrethroid insecticides maps to a sodium channel gene locus in the housefly (Musca domestica)

The voltage-sensitive sodium channel is generally regarded as the primary target site of dichlorodiphenyl-trichloro-ethane (DDT) and pyrethroid insecticides, and has been implicated in the widely reported mechanism of nerve insensitivity to these compounds. This phenomenon is expressed as knockdown resistance (kdr) and has been best characterised in the housefly where several putative alleles, including the more potent super-kdr factor, have been identified. We report the isolation of cDNA clones containing part of a housefly sodium channel gene, designated Msc, which show close homology to the para sodium channel of Drosophila (99% amino acid identity within the region of overlap). Using Southern blots of insect DNA, restriction fragment length polymorphisms (RFLPs) at the Msc locus were identified in susceptible, kdr and super-kdr housefly strains. These RFLPs showed tight linkage to resistance in controlled crosses involving these strains, thus providing clear genetic evidence that kdr, and hence pyrethroid mode of action, is closely associated with the voltage-sensitive sodium channel.