Simian immunodeficiency virus infection in neonatal macaques

Children with human immunodeficiency virus infection often have higher viral loads and progress to AIDS more rapidly than adults. Since the intestinal tract is a major site of early viral replication and CD4(+) T-cell depletion in adults, we examined the effects of simian immunodeficiency virus (SIV) on both peripheral and intestinal lymphocytes from 13 neonatal macaques infected with SIVmac239. Normal neonates had more CD4(+) T cells and fewer CD8(+) T cells in all tissues than adults. Surprisingly, neonates had substantial percentages of CD4(+) T cells with an activated, memory phenotype (effector CD4(+) T cells) in the lamina propria of the intestine compared to peripheral lymphoid tissues, even when examined on the day of birth. Moreover, profound and selective depletion of jejunum lamina propria CD4(+) T cells occurred in neonatal macaques within 21 days of infection, which was preceded by large numbers of SIV-infected cells in this compartment. Furthermore, neonates with less CD4(+) T-cell depletion in tissues tended to have higher viral loads. The persistence of intestinal lamina propria CD4(+) T cells in some neonates with high viral loads suggests that increased turnover and/or resistance to CD4(+) T-cell loss may contribute to the higher viral loads and increased severity of disease in neonatal hosts.     

Superoxide reacts with hydroethidine but forms a fluorescent product that is distinctly different from ethidium: potential implications in intracellular fluorescence detection of superoxide

Hydroethidine (HE) or dihydroethidium (DHE), a redox-sensitive probe, has been widely used to detect intracellular superoxide anion. It is a common assumption that the reaction between superoxide and HE results in the formation of a two-electron oxidized product, ethidium (E+), which binds to DNA and leads to the enhancement of fluorescence (excitation, 500-530 nm; emission, 590-620 nm). However, the mechanism of oxidation of HE by the superoxide anion still remains unclear. In the present study, we show that superoxide generated in several enzymatic or chemical systems (e.g., xanthine/xanthine oxidase, endothelial nitric oxide synthase, or potassium superoxide) oxidizes HE to a fluorescent product (excitation, 480 nm; emission, 567 nm) that is totally different from E+. HPLC measurements revealed that the HE/superoxide reaction product elutes differently from E+. This new product exhibited an increase in fluorescence in the presence of DNA. Mass spectral data indicated that the molecular weight of the HE/superoxide reaction product is 330, while ethidium has a molecular weight of 314. We conclude that the reaction between superoxide and HE forms a fluorescent marker product that is different from ethidium. Potential implications of this finding in intracellular detection and imaging of superoxide are discussed.     

Control of mitochondrial respiration by NO*, effects of low oxygen and respiratory state

Nitric oxide (NO.) inhibits mitochondrial respiration by binding to the binuclear heme a3/CuB center in cytochrome c oxidase. However, the significance of this reaction at physiological O2 levels (5-10 microM) and the effects of respiratory state are unknown. In this study mitochondrial respiration, absorption spectra, [O2], and [NO.] were measured simultaneously at physiological O2 levels with constant O2 delivery, to model in vivo respiratory dynamics. Under these conditions NO. inhibited mitochondrial respiration with an IC50 of 0.14 +/- 0.01 microm in state 3 versus 0.31 +/- 0.04 microM in state 4. Spectral data indicate that the higher sensitivity of state 3 respiration to NO. is due to greater control over respiration by an NO.-dependent spectral species in the respiratory chain in this state. These results are discussed in the context of regulation of respiration by NO. in vivo and its implications for the control of vessel-parenchymal O2 gradients.     

Coreceptor tropism in human immunodeficiency virus type 1 subtype D: high prevalence of CXCR4 tropism and heterogeneous composition of viral populations

In human immunodeficiency virus type 1 (HIV-1) subtype B, CXCR4 coreceptor use ranges from approximately 20% in early infection to approximately 50% in advanced disease. Coreceptor use by non-subtype B HIV is less well characterized. We studied coreceptor tropism of subtype A and D HIV-1 collected from 68 pregnant, antiretroviral drug-naive Ugandan women (HIVNET 012 trial). None of 33 subtype A or 10 A/D-recombinant viruses used the CXCR4 coreceptor. In contrast, nine (36%) of 25 subtype D viruses used both CXCR4 and CCR5 coreceptors. Clonal analyses of the nine subtype D samples with dual or mixed tropism revealed heterogeneous viral populations comprised of X4-, R5-, and dual-tropic HIV-1 variants. In five of the six samples with dual-tropic strains, V3 loop sequences of dual-tropic clones were identical to those of cocirculating R5-tropic clones, indicating the presence of CXCR4 tropism determinants outside of the V3 loop. These dual-tropic variants with R5-tropic-like V3 loops, which we designated "dual-R," use CCR5 much more efficiently than CXCR4, in contrast to dual-tropic clones with X4-tropic-like V3 loops ("dual-X"). These observations have implications for pathogenesis and treatment of subtype D-infected individuals, for the association between V3 sequence and coreceptor tropism phenotype, and for understanding potential mechanisms of evolution from exclusive CCR5 use to efficient CXCR4 use by subtype D HIV-1.     

Isolation of intact RNA from cytometrically sorted Saccharomyces cerevisiae for the analysis of intrapopulation diversity of gene expression

Characterizing and understanding the functional heterogeneity in a given population on the cellular and molecular level is a great challenge in microbiology. Each microorganism contributes differently to the overall performance of the community and responds differently to changing microenvironmental conditions. Here, we present a method for isolation of intact RNA out of small subpopulations of live Saccharomyces cerevisiae cells for differential gene expression analysis. The protocol includes fluorescence staining, flow cytometric analysis and sorting of live yeast cells, subsequent isolation of RNA from the resulting subpopulations and finally RNA quantification and integrity check. The isolated RNA can be transcribed into cDNA and successfully used for microarray analysis. This aids in relating molecular regulation processes within subpopulations with the dynamics and functioning of the entire population. The procedure can be accomplished in 2 d.     

Rap1 promotes endothelial mechanosensing complex formation,NO release and normal endothelial function

Decreased nitric oxide (NO) bioavailability underlies a number of cardiovascular pathologies, including hypertension. The shear stress exerted by flowing blood is the main determinant of NO release. Rap1 promotes integrin‐ and cadherin‐mediated signaling. Here, we show that Rap1 is a critical regulator of NO production and endothelial function. Rap1 deficiency in murine endothelium attenuates NO production and diminishes NO‐dependent vasodilation, leading to endothelial dysfunction and hypertension, without deleterious effects on vessel integrity. Mechanistically, Rap1 is activated by shear stress, promotes the formation of the endothelial mechanosensing complex—comprised of PECAM‐1, VE‐cadherin and VEGFR2‐ and downstream signaling to NO production. Our study establishes a novel paradigm for Rap1 as a regulator of mechanotransduction.     

The flare gene, which encodes the AIP1 protein of Drosophila, functions to regulate F-actin disassembly in pupal epidermal cells

Adult Drosophila are decorated with several types of polarized cuticular structures, such as hairs and bristles. The morphogenesis of these takes place in pupal cells and is mediated by the actin and microtubule cytoskeletons. Mutations in flare (flr) result in grossly abnormal epidermal hairs. We report here that flr encodes the Drosophila actin interacting protein 1 (AIP1). In other systems this protein has been found to promote cofilin-mediated F-actin disassembly. In Drosophila cofilin is encoded by twinstar (tsr). We show that flr mutations result in increased levels of F-actin accumulation and increased F-actin stability in vivo. Further, flr is essential for cell proliferation and viability and for the function of the frizzled planar cell polarity system. All of these phenotypes are similar to those seen for tsr mutations. This differs from the situation in yeast where cofilin is essential while aip1 mutations result in only subtle defects in the actin cytoskeleton. Surprisingly, we found that mutations in flr and tsr also result in greatly increased tubulin staining, suggesting a tight linkage between the actin and microtubule cytoskeleton in these cells.