The Th1/Th2/Th17 cytokine profile of HIV-infected individuals: A multivariate cytokinomics approach

HIV infection causes the dysregulation of cytokine production. A cytokinomics approach employing cytometric bead array (CBA) technology, flow cytometry and multivariate analysis was applied to the investigation of HIV-induced T helper cell type 1 (Th1), Th2 and Th17 cytokine changes in the sera of treatment naive individuals. Stepwise linear discriminant analysis (LDA) and logistic regression identified interleukin (IL)-6 to be discriminatory for HIV infection with 74.6% and 71.2% of the cases correctly classified. Analysis of variance (ANOVA) confirmed IL-6 and IL-10 concentrations to be significantly (p = 0.001 and p = 0.025) different between the groups. A scatter plot of the log IL-6 and IL-10 concentrations for the groups largely overlapped, with improved differentiation where patients were advancing to the acquired immunodeficiency syndrome (AIDS). IL-17A levels were higher than other cytokines but did not significantly distinguish the groups suggesting that the HIV? and HIV+ individuals had similar immune profiles. This possibility was supported by other clinical indicators. Taken together, the measured cytokines (IL-6, 10 and 17) have potential prognostic value.     

Adenovirus core protein pVII is translocated into the nucleus by multiple import receptor pathways

Adenoviruses are nonenveloped viruses with an approximately 36-kb double-stranded DNA genome that replicate in the nucleus. Protein VII, an abundant structural component of the adenovirus core that is strongly associated with adenovirus DNA, is imported into the nucleus contemporaneously with the adenovirus genome shortly after virus infection and may promote DNA import. In this study, we evaluated whether protein VII uses specific receptor-mediated mechanisms for import into the nucleus. We found that it contains potent nuclear localization signal (NLS) activity by transfection of cultured cells with protein VII fusion constructs and by microinjection of cells with recombinant protein VII fusions. We identified three NLS-containing regions in protein VII by deletion mapping and determined important NLS residues by site-specific mutagenesis. We found that recombinant protein VII and its NLS-containing domains strongly and specifically bind to importin alpha, importin beta, importin 7, and transportin, which are among the most abundant cellular nuclear import receptors. Moreover, these receptors can mediate the nuclear import of protein VII fusions in vitro in permeabilized cells. Considered together, these data support the hypothesis that protein VII is a major NLS-containing adaptor for receptor-mediated import of adenovirus DNA and that multiple import pathways are utilized to promote efficient nuclear entry of the viral genome.     

Nitrogen source effects on the growth and toxicity of two strains of the ciguatera-causing dinoflagellate Gambierdiscus toxicus

Two Caribbean strains (1651 and 1655) of the ciguatera-causing dinoflagellate Gambierdiscus toxicus were grown in xenic, batch culture under defined, measured nutrient conditions with nitrate, ammonium, urea, a mix of free amino acids (FAA), or putrescine as the nitrogen source. Cultures were maintained at 27 °C, salinity 35, 110 μmol m⁻² s⁻¹ (12 h:12 h light:dark cycle) on L2 medium at an initial nitrogen concentration of 50 μM N. Toxicity was determined using a ouabain/veratridine-dependent cytotoxicity assay (N2A assay) standardized to a ciguatoxin standard. Nitrate, ammonium, FAA, and putrescine supported growth, but urea did not. The appearance of ammonium in the organic nitrogen cultures indicated that G. toxicus and/or associated bacteria remineralized the available organic nitrogen. Both strains were exposed to nitrogen-limiting conditions as evidenced by chlorophyll a content per cell, nitrogen content, and nitrogen (N) to phosphorus (P) (N:P) ratio significantly declining once nitrogen was no longer available in the medium and cells entered stationary phase. Strain 1651 grew significantly faster than strain 1655 when nitrate, FAA, and putrescine was the nitrogen source, but not ammonium. Nitrogen source had no effect on growth rate (0.14 d⁻¹) in strain 1651. The growth rate of strain 1655 (0.10–0.13 d⁻¹) was significantly faster on ammonium than the other nitrogen sources. Strain 1655 was significantly more toxic (10-fold) than strain 1651 except when growing on ammonium at exponential phase. Toxicity ranged from 1.3 to 8.7 fg C-CTX1-Eq cell⁻¹ in strain 1651 and from 30.7 to 54.3 fg C-CTX1-Eq cell⁻¹ in strain 1655. Nitrogen source had no significant affect on toxicity. Toxicity was greater in stationary versus exponential phase cells for strain 1651 when grown on nitrate and strain 1655 regardless of nitrogen source. The difference in toxicity between growth phases may result from an increase in ciguatoxin and/or maitotoxin. Our results suggest that some strains of G. toxicus when associated with bacteria are able to take advantage of organic as well as inorganic nitrogen sources on short time scales to support future growth. The uncoupling of total nitrogen and phosphorus pools from conditions in the water column suggest that instantaneous growth rates can be supported by nutrients acquired hours to days earlier.     

Dual Role of the Oligopeptide Permease Opp3 during Growth of Staphylococcus aureus in Milk

Staphylococcus aureus RN6390 presents a diauxic growth in milk, due to amino acid limitation. Inactivation of the oligopeptide permease Opp3 (dedicated to the nitrogen nutrition of the strain) not only affects the growth of the strain but also results in reduced expression levels of three major extracellular proteases.Staphylococcus aureus is a ubiquitous gram-positive pathogen commonly found in the environment. Some S. aureus strains are able to produce enterotoxins that cause food poisoning (6). In France, contaminated dairy products are the main source of staphylococcal food-borne disease outbreaks (3). Thus, the development of this bacterium in milk is a major concern for the safety of dairy products. Our objective was to evaluate the growth of S. aureus in milk, with special attention focused on the role of the peptide transport systems.     

Kinetic characterization of recombinant mouse retinal dehydrogenase types 3 and 4 for retinal substrates

Background

Retinal dehydrogenases (RALDHs) catalyze the dehydrogenation of retinal into retinoic acids (RAs), which are required for embryogenesis and tissue differentiation. This study sought to determine the detailed kinetic properties of 2 mouse RALDHs, namely RALDH3 and 4, for retinal isomer substrates, to better define their specificities in RA isomer synthesis.

Methods

RALDH3 and 4 were expressed in Escherichia coli as His-tagged proteins and affinity-purified. Enzyme kinetics were performed with retinal isomer substrates. The enzymatic products were analyzed by high pressure liquid chromatography.

Results

RALDH3 oxidized all-trans retinal with high catalytic efficiency (V_max/K_m = 77.9) but did not show activity for either 9-cis or 13-cis retinal substrates. On the other hand, RALDH4 was inactive for all-trans retinal substrate, exhibited high activity for 9-cis retinal oxidation (V_max/K_m = 27.4), and oxidized 13-cis retinal with lower catalytic efficiency (V_max/K_m = 8.24). β-ionone, a potent inhibitor of RALDH4 activity, suppressed 9-cis and 13-cis retinal oxidation competitively with inhibition constants of 0.60 and 0.32, respectively, but had no effect on RALDH3 activity. The divalent cation MgCl₂ activated 13-cis retinal oxidation by RALDH4 by 3-fold, did not significantly influence 9-cis retinal oxidation, and slightly activated RALDH3 activity.

Conclusions

These data extend the kinetic characterization of RALDH3 and 4, providing their specificities for retinal isomer substrates.

General significance

The kinetic characterization of RALDHs should give useful information in determining amino acid residues that are involved in the specificity for retinal isomers and on the role of these enzymes in the synthesis of RAs in specific tissues.     

Dissecting the Unique Nucleotide Specificity of Mimivirus Nucleoside Diphosphate Kinase

The analysis of the Acanthamoeba polyphaga mimivirus genome revealed the first virus-encoded nucleoside diphosphate kinase (NDK), an enzyme that is central to the synthesis of RNA and DNA, ubiquitous in cellular organisms, and well conserved among the three domains of life. In contrast with the broad specificity of cellular NDKs for all types of ribo- and deoxyribonucleotides, the mimivirus enzyme exhibits a strongly preferential affinity for deoxypyrimidines. In order to elucidate the molecular basis of this unique substrate specificity, we determined the three-dimensional (3D) structure of the Acanthamoeba polyphaga mimivirus NDK alone and in complex with various nucleotides. As predicted from a sequence comparison with cellular NDKs, the 3D structure of the mimivirus enzyme exhibits a shorter Kpn loop, previously recognized as a main feature of the NDK active site. The structure of the viral enzyme in complex with various nucleotides also pinpointed two residue changes, both located near the active site and specific to the viral NDK, which could explain its stronger affinity for deoxynucleotides and pyrimidine nucleotides. The role of these residues was explored by building a set of viral NDK variants, assaying their enzymatic activities, and determining their 3D structures in complex with various nucleotides. A total of 26 crystallographic structures were determined at resolutions ranging from 2.8 Å to 1.5 Å. Our results suggest that the mimivirus enzyme progressively evolved from an ancestral NDK under the constraints of optimizing its efficiency for the replication of an AT-rich (73%) viral genome in a thymidine-limited host environment.Mimivirus, a DNA virus infecting Acanthamoeba, is the largest and most complex virus isolated to date (8, 37). It is the first representative and prototype member of the Mimiviridae, the latest addition to the large nucleocytoplasmic DNA viruses, including the poxviruses, the phycodnaviruses, (infecting algae), the iridoviruses (infecting invertebrates and fishes), and asfarvirus (the agent of a swine fever in Africa) (18). The mimivirus''s record genome size (1.2 Mb) and gene content (911 encoded proteins), as well as the presence of genes previously thought to be specific to cellular organisms (such as aminoacyl-tRNA synthetases [3]), revived the debate about the evolutionary origin of DNA viruses and their putative role in the emergence of the eukaryote nucleus (reviewed in reference 7) or in the advent of DNA genomes (13).In this peculiar context, we found the discovery of the first virus-encoded nucleoside diphosphate kinase (NDK) within the mimivirus genome of great interest and warranting a detailed study of the structural and biochemical properties of this unique viral enzyme. Ubiquitous in cellular organisms, NDKs are responsible for the last step of 2′-deoxynucleoside triphosphate (dNTP) pathways and as such play an essential role in the replication of DNA by providing the basic precursors for its synthesis. Acting indiscriminately on ribonucleotides and deoxyribonucleotides, the cellular NDKs are also responsible for supplying energy to various essential synthetic pathways, producing NTPs for RNA synthesis, CTP for lipid synthesis, UTP for polysaccharide synthesis, and GTP for protein synthesis elongation, signal transduction, and microtubules polymerization. Besides their direct role in the above metabolic pathways, cellular NDKs have been involved in the regulation of cell growth and differentiation in vertebrates (22).Cellular NDKs are small proteins of about 150 amino acids, the sequences of which are highly conserved among the three domains of life (>40% identity). They are most often hexameric enzymes, with a few occurrences of tetrameric and dimeric NDK structures in bacteria (19, 25, 26, 31, 38). They all catalyze the transfer of a phosphate group from an NTP onto a nucleotide diphosphate (NDP) through an Mg²⁺-dependent reaction. In vivo, the phosphate donor is usually the nonlimiting ATP nucleotide.In agreement with their implication in various metabolic pathways, cellular NDKs exhibit little substrate specificity and are equally able to act on purine and pyrimidine nucleotides, in their 2′ OH and deoxyribonucleotide forms. In clear contrast, our characterization of the mimivirus NDK revealed its enhanced affinity for deoxypyrimidine nucleotides (20). This marked difference between the viral and cellular NDKs offered a good opportunity to explore the sequence and structure features governing substrate specificity. For instance, cellular NDKs exhibit a conserved loop, the Kpn loop, involved both in substrate binding and in oligomerization of the enzyme (19). Interestingly, a sequence comparison predicted this loop to be shorter in the Acanthamoeba polyphaga mimivirus NDK (NDK_apm) sequence. However, many other single-residue changes could also be involved in modifying the enzyme properties. To explore these issues, we performed a detailed structure-function analysis of the NDK_apm protein in a variety of mutated forms and substrate-enzyme complexes. Despite its markedly different sequence, the three-dimensional structure of the mimivirus NDK was found to be very similar to that of cellular enzymes. Its peculiar substrate specificity is not attributable to a single sequence feature but rather appears to result from the conjunction of several factors, suggesting the progressive optimization of an ancestral enzyme for the replication of an AT-rich (73%) genome in a thymidine-limited host environment.     

Phosphoinositides and the endocytic pathway

Phosphorylation of the phosphatidylinositol headgroup generates seven varieties of phosphoinositide of which PtdIns(4,5)P₂, PtdIns3P and PtdIns (3,5)P₂ have established roles on the endocytic pathway. In this review, we discuss the enzymes responsible for generation and turnover of these lipids, which are keys to determining compartmental identity and the flux of material through the endocytic system. The enzymatic generation of lipids serves as an amplification mechanism through which a wide variety of effector molecules can be recruited.     

A structural model of 20S immunoproteasomes: effect of LMP2 codon 60 polymorphism on expression, activity, intracellular localisation and insight into the regulatory mechanisms

The immunoproteasome subunit low molecular weight protein 2 (LMP2) codon 60 polymorphism has been associated with autoimmune diseases. It has also been demonstrated to influence susceptibility to TNF-alpha-induced apoptosis in blood cells and proteasome activity in aged human brain. In the present study, an in silico model of immunoproteasome was used to examine the effect of the R60H polymorphism in the LMP2 subunit. The investigation of immunoproteasome expression, activity and intracellular localisation in an in vitro cellular model, namely lymphoblastoid cell lines, showed no major variations in functionality and amount, while a significant difference in antibody affinity was apparent. These data were integrated with previous results obtained in different tissues and combined with a structural model of the LMP2 polymorphism. Accordingly, we identified three prospective mechanisms that could explain the biological data for the polymorphism, such as modulation of the binding affinity of a putative non-catalytic modifier site on the external surface of the immunoproteasome core, or the modification of any channel between alpha and beta rings.