Engineered CD4- and CXCR4-using simian immunodeficiency virus from African green monkeys is neutralization sensitive and replicates in nonstimulated lymphocytes

During human immunodeficiency virus type 1 (HIV-1) infection, disease progression correlates with the occurrence of variants using the coreceptor CXCR4 for cell entry. In contrast, apathogenic simian immunodeficiency virus (SIV) from African green monkeys (SIVagm), specifically the molecular virus clone SIVagm3mc, uses CCR5, Bob, and Bonzo as coreceptors throughout the course of infection. The influence of an altered coreceptor usage on SIVagm3mc replication was studied in vitro and in vivo. The putative coreceptor binding domain, the V3 region of the surface envelope (SU) glycoprotein, was replaced by the V3 loop of a CD4- and CXCR4-tropic HIV-1 strain. The resulting virus, termed SIVagm3-X4mc, exclusively used CD4 and CXCR4 for cell entry. Consequently, its in vitro replication was inhibited by SDF-1, the natural ligand of CXCR4. Surprisingly, SIVagm3-X4mc was able to replicate in vitro not only in interleukin-2- and phytohemagglutinin-stimulated but also in nonstimulated peripheral blood mononuclear cells (PBMCs) from nonhuman primates. After experimental infection of two pig-tailed macaques with either SIVagm3-X4mc or SIVagm3mc, the coreceptor usage was maintained during in vivo replication. Cell-associated and plasma viral loads, as well as viral DNA copy numbers, were found to be comparable between SIVagm3mc and SIVagm 3-X4mc infections, and no pathological changes were observed up to 14 months postinfection. Interestingly, the V3 loop exchange rendered SIVagm3-X4mc susceptible to neutralizing antibodies present in the sera of SIVagm3-X4mc- and SIVagm3mc-infected pig-tailed macaques. Our study describes for the first time a successful exchange of a V3 loop in nonpathogenic SIVagm resulting in CD4 and CXCR4 usage and modulation of virus replication in nonstimulated PBMCs as well as sensitivity toward neutralization.     

Structural investigation of the binding of a herpesviral protein to the SH3 domain of tyrosine kinase Lck

Herpesvirus saimiri codes for a tyrosine kinase interacting protein (Tip) that interacts with both the SH3 domain and the kinase domain of the T-cell-specific tyrosine kinase Lck via two separate motifs. The activation of Lck by Tip is considered as a key event in the transformation of human T-lymphocytes during herpesviral infection. We investigated the interaction of proline-rich Tip peptides with the LckSH3 domain starting with the structural characterization of the unbound interaction partners. The solution structure of the LckSH3 was determined by heteronuclear multidimensional nuclear magnetic resonance (NMR) spectroscopy using 44 residual dipolar couplings in addition to the conventional experimental restraints. Circular dichroism spectroscopy proved that the polyproline helix of Tip is already formed prior to SH3 binding and is conformationally stable. NMR titration experiments point out three major regions of the Tip-Lck interaction comprising the RT loop, the n-src loop, and a helical turn preceding the last strand of the beta-sheet. Further changes of the chemical shifts were observed for the N- and C-terminal beta-strands of the SH3 domain, indicating additional contacts outside the proline-rich segment or subtle structural rearrangements transmitted from the binding site of the proline helix. Fluorescence spectroscopy shows that Tip binds to the SH3 domains of several Src kinases (Lck, Hck, Lyn, Src, Fyn, Yes), exhibiting the highest affinities for Lyn, Hck, and Lck.     

The plastidic pentose phosphate translocator represents a link between the cytosolic and the plastidic pentose phosphate pathways in plants

Plastids are the site of the reductive and the oxidative pentose phosphate pathways, which both generate pentose phosphates as intermediates. A plastidic transporter from Arabidopsis has been identified that is able to transport, in exchange with inorganic phosphate or triose phosphates, xylulose 5-phosphate (Xul-5-P) and, to a lesser extent, also ribulose 5-phosphate, but does not accept ribose 5-phosphate or hexose phosphates as substrates. Under physiological conditions, Xul-5-P would be the preferred substrate. Therefore, the translocator was named Xul-5-P/phosphate translocator (XPT). The XPT shares only approximately 35% to 40% sequence identity with members of both the triose phosphate translocator and the phosphoenolpyruvate/phosphate translocator classes, but a higher identity of approximately 50% to glucose 6-phosphate/phosphate translocators. Therefore, it represents a fourth group of plastidic phosphate translocators. Database analysis revealed that plant cells contain, in addition to enzymes of the oxidative branch of the oxidative pentose phosphate pathway, ribose 5-phosphate isomerase and ribulose 5-phosphate epimerase in both the cytosol and the plastids, whereas the transketolase and transaldolase converting the produced pentose phosphates to triose phosphates and hexose phosphates are probably solely confined to plastids. It is assumed that the XPT function is to provide the plastidic pentose phosphate pathways with cytosolic carbon skeletons in the form of Xul-5-P, especially under conditions of a high demand for intermediates of the cycles.     

Mesothelial cells in suspension expose an enriched integrin repertoire capable of capturing soluble fibronectin and laminin

Pleural cavities are lined by a polarized monolayer of mesothelial cells (MC). During pleuritis, MC are shed into effusions, and pleural obstruction may occur. Integrins are cell surface receptors mediating interactions with extracellular matrix (ECM) proteins. The distribution of beta 1-, beta 3-, beta 4-integrins and fibronectin and laminin in normal and chronically inflamed pleura and in/on MC from pleural effusions was examined by immunomorphology and flow cytometry. Adhesion assays of MC to fibronectin and laminin were performed. In situ, resting MC expressed beta 1-, beta 3-, and beta 4-, and alpha v-subunits. Activated MC were beta 1- and alpha v-positive and also expressed alpha 3 and alpha 6; beta 4 was confined to the basal surface of MC; beta 3 was absent. Floating MC from effusions neoexpressed alpha 5 and reexpressed beta 3. In vitro, MC surface expressed beta 1, beta 3, alpha 3, alpha 5, alpha 6, alpha v, and also alpha 1 and alpha 2. In normal pleura, fibronectin and laminin were components of the basement membrane. In pleuritis, the basement membrane was desintegrated. Instead, newly formed fibronectin/laminin containing fibrils extended into the submesothelial connective tissue. Floating MC freshly isolated from effusions carried fibronectin and laminin on their surface and showed specific binding to these ECM proteins. Binding was blocked by anti-beta 1 or anti-alpha 5 and anti-alpha 6 antibodies, respectively. MC incubated with fibronectin showed a clear shift to the S phase, while laminin had no effect. In conclusion, activated and detached MC progressively enrich their integrin repertoire. By capturing soluble fibronectin and laminin and by matrix-mediated bridging, readhering MC may contribute to pleural obstruction. Further, soluble fibronectin bound to alpha 5 beta 1 might be life-sustaining for floating MC by driving cells into cell cycle.     

Genetic engineering of brewing yeast to reduce the content of ethanol in beer

The GPD1 gene encoding the glycerol-3-phosphate dehydrogenase was overexpressed in an industrial lager brewing yeast (Saccharomyces cerevisiae ssp. carlsbergensis) to reduce the content of ethanol in beer. The amount of glycerol produced by the GPD1-overexpressing yeast in fermentation experiments simulating brewing conditions was increased 5.6 times and ethanol was decreased by 18% when compared to the wild-type. Overexpression of GPD1 does not affect the consumption of wort sugars. Only minor changes in the concentration of higher alcohols, esters and fatty acids could be observed in beer produced by the GPD1-overexpressing brewing yeast. However, the concentrations of several other by-products, particularly acetoin, diacetyl and acetaldehyde, were considerably increased.     

Overexpression of Hsp27 affects the metastatic phenotype of human melanoma cells in vitro

Overexpression of the small heat shock protein Hsp27 has been shown by us to inhibit the in vitro proliferation rate and to delay tumor development of a human melanoma cell line (A375) in nude mice. We hypothesized that Hsp27 may influence the neoplastic phenotype. In the present study Hsp27 transfectants from this cell line were analyzed for various cellular aspects associated with the metastatic process. We found that Hsp27-overexpressing clones exhibited an altered cellular morphology as compared with control transfected cells. The Hsp27-positive cells tended to develop an epithelial-like phenotype growing in clusters and were characterized by a loss of transcytoplasmic stressfibers. In parallel, Hsp27-expressing cells lost the ability to form colonies in soft agar. The invasive potential was studied in vitro by the use of a reconstituted extracellular matrix-coated filter (Matrigel). Compared with controls, Hsp27-overexpressing cells showed decreased cell invasiveness through Matrigel. A correlation between invasion and activation of matrix metalloproteinases (MMPs) has been shown in several cell models. Secretion of MMPs (MMP-2 and MMP-9) was studied by gelatin-substrate zymogram analysis, as well as by a sensitive gelatinase activity assay. The Hsp27-transfected A375 melanoma cell line showed decreased secretion of MMP-2 and MMP-9 as compared with the control transfected cells. Integrins are adhesion receptors and function in cell invasion by mediating cell movement on matrix molecules and by regulating the expression of MMPs. Both fluorescence-activated cell sorter analysis and immunofluorescence analysis revealed a loss of alpha(v)beta3 integrin in Hsp27-transfected cell colonies. Our results demonstrate that Hsp27 overexpression has a profound impact on several parameters regulating the invasive and metastatic potential of melanoma cells in vitro.     

Competitive advantages of Caedibacter-infected Paramecia

Intracellular bacteria of the genus Caedibacter limit the reproduction of their host, the freshwater ciliate Paramecium. Reproduction rates of infected strains of paramecia were significantly lower than those of genetically identical strains that had lost their parasites after treatment with an antibiotic. Interference competition occurs when infected paramecia release a toxic form of the parasitic bacterium that kills uninfected paramecia. In mixed cultures of infected and uninfected strains of either P tetraurelia or of P novaurelia, the infected strains outcompeted the uninfected strains. Infection of new host paramecia seems to be rare. Infection of new hosts was not observed in either mixtures of infected with uninfected strains, or after incubation of paramecia with isolated parasites. The competitive advantages of the host paramecia, in combination with their vegetative reproduction, makes infection of new hosts by the bacterial parasites unnecessary, and could be responsible for the continued existence of "killer paramecia" in nature. Caedibacter parasites are not a defensive adaptation. Feeding rates and reproduction of the predators Didinium nasutum (Ciliophora) and Amoeba proteus (Amoebozoa, Gymnamoebia) were not influenced by whether or not their paramecia prey were infected. Infection of the predators frequently occurred when they preyed on infected paramecia. Caedibacter-infected predators may influence competition between Paramecium strains by release of toxic parasites into the environment that are harmful to uninfected strains.     

Retinol-binding protein-deficient mice: biochemical basis for impaired vision

We reported previously that mice lacking plasma retinol-binding protein (RBP) are phenotypically normal except that they display impaired vision at the time of weaning. This visual defect is associated with greatly diminished eyecup levels of retinaldehyde and is reversible if the mutants are maintained for several months on a vitamin A-sufficient diet. Here we provide a biochemical basis for the visual phenotype of RBP-deficient mice. This phenotype does not result from inadequate milk total retinol levels since these are not different for RBP-deficient and wild-type mice. The eye, unlike all other tissues that have been examined, takes up dietary retinol very poorly. Moreover, compared to other tissues, the eye displays a strong preference for retinol uptake when retinol is delivered bound to RBP. The poor uptake of dietary retinol by the eye coupled with its marked ability to take up retinol from RBP, we propose, provides a basis for the impaired vision observed in weanling RBP-deficient mice. Further study of the mutants suggests that the impaired vision is reversible because the eyes of mutant mice slowly acquire sufficient retinol from the low levels of retinol present in their circulation either bound to albumin or present in lipoprotein fractions. Thus, the eye is unlike other tissues in the body in that it shows a very marked preference for acquiring retinol needed to support vision from the retinol-RBP complex and is unable to meet adequately its retinol need through uptake of recently absorbed dietary retinol. This provides an explanation for the impaired vision phenotype of RBP-deficient mice.     

HtrA2/Omi, a sheep in wolf's clothing

Mammalian mitochondrial HtrA2/Omi was originally described as an apoptosis inducer, but rather than having extra cells, mice with mutant HtrA2/Omi suffer from a neurodegenerative disease due to progressive mitochondrial damage. This suggests that instead of promoting cell death by antagonizing inhibitor of apoptosis (IAP) proteins, the primary function of HtrA2/Omi is to handle misfolded proteins in the mitochondria.     

Heterotrophic bacterial growth efficiency and community structure at different natural organic carbon concentrations

Batch cultures of aquatic bacteria and dissolved organic matter were used to examine the impact of carbon source concentration on bacterial growth, biomass, growth efficiency, and community composition. An aged concentrate of dissolved organic matter from a humic lake was diluted with organic compound-free artificial lake water to obtain concentrations of dissolved organic carbon (DOC) ranging from 0.04 to 2.53 mM. The bacterial biomass produced in the cultures increased linearly with the DOC concentration, indicating that bacterial biomass production was limited by the supply of carbon. The bacterial growth rate in the exponential growth phase exhibited a hyperbolic response to the DOC concentration, suggesting that the maximum growth rate was constrained by the substrate concentration at low DOC concentrations. Likewise, the bacterial growth efficiency calculated from the production of biomass and CO(2) increased asymptotically from 0.4 to 10.4% with increasing DOC concentration. The compositions of the microbial communities that emerged in the cultures were assessed by separation of PCR-amplified 16S rRNA fragments by denaturing gradient gel electrophoresis. Nonmetric multidimensional scaling of the gel profiles showed that there was a gradual change in the community composition along the DOC gradient; members of the beta subclass of the class Proteobacteria and members of the Cytophaga-Flavobacterium group were well represented at all concentrations, whereas members of the alpha subclass of the Proteobacteria were found exclusively at the lowest carbon concentration. The shift in community composition along the DOC gradient was similar to the patterns of growth efficiency and growth rate. The results suggest that the bacterial growth efficiencies, the rates of bacterial growth, and the compositions of bacterial communities are not constrained by substrate concentrations in most natural waters, with the possible exception of the most oligotrophic environments.