Interleukin-12 but not interleukin-18 is required for immunity to Trypanosoma cruzi in mice

Protective immunity to the parasite Trypanosoma cruzi in mice depends on a pro-inflammatory T cell response involving the production of interferon-gamma (IFN-gamma). In conjunction with interleukin-12 (IL-12), IL-18 promotes the synthesis of IFN-gamma and a T helper type 1 immune response. We investigated the requirements of IL-12 and IL-18 in murine T. cruzi infection by use of C57BL/6 mice genetically deficient in either cytokine. IL-12p40(-/-) mice succumbed to infection at doses of 100 parasites, whereas IL-18(-/-) and wild-type mice resisted infectious doses up to 1000 parasites to the same extent. Levels of parasitemia were comparable between the latter groups, as were tissue parasite burdens according to quantitative real-time PCR. In contrast, IL-12p40(-/-) mice displayed vastly increased levels of parasites both in blood and in tissue. IFN-gamma concentrations in the serum of infected mice and in supernatants of splenocytes stimulated in vitro were decreased in IL-18(-/-) mice, whereas in IL-12p40(-/-) mice, IFN-gamma was undetectable in the serum and drastically reduced in cell supernatants. Levels of IL-12 production were generally comparable between wild-type and IL-18(-/-) mice, as were levels of IL-4, IL-2 and nitric oxide. Thus, the requirement for endogenous pro-inflammatory cytokines for a protective murine immune response against T. cruzi is satisfied by the expression of IL-12, while IL-18 is dispensable.     

The electrochemical proton potential generated by the sulphur respiration of Wolinella succinogenes

Wolinella succinogenes grown on formate and elemental sulphur was found to use the polysulphide derivatives 2,2-tetrathiobispropionate (R₂S₄) or pentathionate (S₅O ₆ ⁼ ) as acceptors for formate oxidation. The specific activities of formate oxidation with these acceptors were similar to those with elemental sulphur. The main reaction products of R₂S₄ reduction were 2,2-dithiobispropionate (R₂S₂) and sulphide. Pentathionate was converted to thiosulphate and some elemental sulphur. The electrochemical proton potential across the cytoplasmic membrane of the bacterium was measured in the steady state of electron transport from formate to R₂S₄. The electrical proportion () of the determined through the distribution of labeled tetraphenylphosphonium cation was obtained as 0.17 Volt. The was zero, when a protonophore was present. The pH-difference across the membrane was negligible. Thus the generated by sulphur respiration is close to that measured earlier with fumarate as the terminal acceptor of electron transport.Abbreviations DMO 5,5-dimethyloxazolidine-2,4-dione - R₂S_n (n=2–5) 2,2-polythiobispropionate - TTFB 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazol - TPP tetraphenylphosphonium cation     

The hepatitis C virus RNA 3'-untranslated region strongly enhances translation directed by the internal ribosome entry site

The positive-strand RNA genome of the hepatitis C virus (HCV) is flanked by 5'- and 3'-untranslated regions (UTRs). Translation of the viral RNA is directed by the internal ribosome entry site (IRES) in the 5'-UTR, and subsequent viral RNA replication requires sequences in the 3'-UTR and in the 5'-UTR. Addressing previous conflicting reports on a possible function of the 3'-UTR for RNA translation in this study, we found that reporter construct design is an important parameter in experiments testing 3'-UTR function. A translation enhancer function of the HCV 3'-UTR was detected only after transfection of monocistronic reporter RNAs or complete RNA genomes having a 3'-UTR with a precise 3' terminus. The 3'-UTR strongly stimulates HCV IRES-dependent translation in human hepatoma cell lines but only weakly in nonliver cell lines. The variable region, the poly(U . C) tract, and the most 3' terminal stem-loop 1 of the highly conserved 3' X region contribute significantly to translation enhancement, whereas stem-loops 2 and 3 of the 3' X region are involved only to a minor extent. Thus, the signals for translation enhancement and for the initiation of RNA minus-strand synthesis in the HCV 3'-UTR partially overlap, supporting the idea that these sequences along with viral and possibly also cellular factors may be involved in an RNA 3'-5' end interaction and a switch between translation and RNA replication.     

The Mitogen-Activated Protein Kinase Kinase/Extracellular Signal-Regulated Kinase Cascade Activation Is a Key Signalling Pathway Involved in the Regulation of G1 Phase Progression in Proliferating Hepatocytes

In this study, activation of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signalling pathway was analyzed in proliferating rat hepatocytes both in vivo after partial hepatectomy and in vitro following epidermal growth factor (EGF)-pyruvate stimulation. First, a biphasic MEK/ERK activation was evidenced in G(1) phase of hepatocytes from regenerating liver but not from sham-operated control animals. One occurred in early G(1) (30 min to 4 h), and the other occurred in mid-late G(1), peaking at around 10.5 h. Interestingly, the mid-late G(1) activation peak was located just before cyclin D1 induction in both in vivo and in vitro models. Second, the biological role of the MEK/ERK cascade activation in hepatocyte progression through the G(1)/S transition was assessed by adding a MEK inhibitor (PD 98059) to EGF-pyruvate-stimulated hepatocytes in primary culture. In the presence of MEK inhibitor, cyclin D1 mRNA accumulation was inhibited, DNA replication was totally abolished, and the MEK1 isoform was preferentially targeted by this inhibition. This effect was dose dependent and completely reversed by removing the MEK inhibitor. Furthermore, transient transfection of hepatocytes with activated MEK1 construct resulted in increased cyclin D1 mRNA accumulation. Third, a correlation between the mid-late G(1) MEK/ERK activation in hepatocytes in vivo after partial hepatectomy and the mitogen-independent proliferation capacity of these cells in vitro was established. Among hepatocytes isolated either 5, 7, 9, 12 or 15 h after partial hepatectomy, only those isolated from 12- and 15-h regenerating livers were able to replicate DNA without additional growth stimulation in vitro. In addition, PD 98059 intravenous administration in vivo, before MEK activation, was able to inhibit DNA replication in hepatocytes from regenerating livers. Taken together, these results show that (i) early induction of the MEK/ERK cascade is restricted to hepatocytes from hepatectomized animals, allowing an early distinction of primed hepatocytes from those returning to quiescence, and (ii) mid-late G(1) MEK/ERK activation is mainly associated with cyclin D1 accumulation which leads to mitogen-independent progression of hepatocytes to S phase. These results allow us to point to a growth factor dependency in mid-late G(1) phase of proliferating hepatocytes in vivo as observed in vitro in proliferating hepatocytes and argue for a crucial role of the MEK/ERK cascade signalling pathway.     

Biosynthesis of glycoconjugates in mitochondrial outer membranes

The results reported in this paper show two distinct ways for the incorporation ofN-acetylglucosamine into mitochondrial outer membranes. The first one is the glycosylation of dolichol acceptors, which is indicated by the inhibition of the synthesis of these products by the inhibitors of the dolichol intermediates (tunicamycin and GDP). The second one is the incorporation ofN-acetylglucosamine into protein acceptors directly from UDP-N-acetylglucosamine. This second way of glycosylation is only localized in mitochondria outer membranes.The existence of a direct route forN-glycoprotein biosynthesis has been based on the following evidence. First, the synthesis of theN-acetylglucosaminylated protein acceptors was not inhibited by tunicamycin or GDP. Second, the addition of exogenous dolichol-phosphate did not change the rate of biosynthesis of glycosylated protein material. Third, the sequential incorporation ofN-acetylglucosamine and mannose from their nucleotide derivatives in the presence of GDP and tunicamycin led to the synthesis of glycosylated protein material which entirely bound to Concanavalin A-Sepharose. The oligosaccharide moiety of the glycosylated protein material resulting from the direct transfer of sugars from their nucleotide derivatives to the protein acceptor is of theN-glycan type. On sodium dodecylsulphate polyacrylamide gel electrophoresis, this glycosylated material migrated as a marker protein with a molecular weight between 45 000 and 63 000. HPLC chromatofocusing analysis revealed that the fraction studied was anionic. The oligosaccharide moiety of the glycoprotein material can only be elongated by the incorporation ofN-acetylglucosamine and galactose from their nucleotide derivatives.     

Identification and molecular cytogenetic characterization of a novel complex Y chromosome rearrangement in a boy with disorder of sexual development

Ambiguous genitalia or disorder of the sexual development is a birth defect where the external genitals do not have the typical appearance of either a male or female. Here we report a boy with ambiguous genitalia and short stature. The cytogenetic analysis by G-banding revealed a small Y chromosome and an additional material on the 15p arm. Further, molecular cytogenetic analysis by Fluorescence in situ hybridization (FISH) using whole chromosome paint probes showed the presence of Y sequences on the 15p arm, confirming that it is a Y;15 translocation. Subsequent, FISH with centromere probe Y showed two signals depicting the presence of two centromeres and differing with a balanced translocation. The dicentric nature of the derivative 15 chromosome was confirmed by FISH with both 15 and Y centromeric probes. Further, the delineation of the Y chromosomal DNA was also done by quantitative real time PCR. Additional Y-short tandem repeat typing was performed to find out the extent of deletion on small Y chromosome. Fine mapping was carried out with 8 Y specific BAC clones which helped in defining the breakpoint regions. MLPA was performed to check the presence or absence of subtelomeric regions and SHOX regions on Y. Finally array CGH helped us in confirming the breakpoint regions. In our study we identified and characterized a novel complex Y chromosomal rearrangement with a complete deletion of the Yq region and duplication of the Yp region with one copy being translocated onto the15p arm. This is the first report of novel and unique Y complex rearrangement showing a deletion, duplication and a translocation in the same patient. The possible mechanism of the rearrangement and the phenotype–genotype correlation are discussed.     

Sequence analysis of the grey mouse lemur (<Emphasis Type="Italic">Microcebus murinus</Emphasis>) <Emphasis Type="Italic">MHC class II DQ</Emphasis> and <Emphasis Type="Italic">DR</Emphasis> region

We here report the genomic organisation of the grey mouse lemur (Microcebus murinus) MHC class II DQ and DR region based on BAC clone analysis. The sequenced Mimu-MHC haplotype spans 343 kb and encompasses the genes TAP2, DOB, DQB, DQA, DRB, DRA, BTNL2 and a further BTNL gene. The DQ and DR genes of this haplotype are not duplicated. Mimu-DOB is not transcribed and represents a pseudogene due to deletions and premature stop codons. Analysis of BAC clone DNA, a cDNA sample and eight genomic DNA samples suggests that Mimu-DRB, Mimu-DQA and Mimu-DQB are highly polymorphic with the majority of peptide-binding residues being affected by polymorphisms. In contrast, Mimu-DRA is moderately polymorphic, and the variable amino acid positions are not part of the peptide-binding region. Phylogenetic analysis of Mimu-DQA and Mimu-DQB and other primate DQA and DQB genes indicates that duplication of DQA and DQB loci occurred in Anthropoidea after the split from Strepsirrhini.     

Blood monocytes: distinct subsets, how they relate to dendritic cells, and their possible roles in the regulation of T-cell responses

Monocytes can have important effects on the polarization and expansion of lymphocytes and may contribute to shaping primary and memory T-cell responses in humans and mice. However, their precise contribution in terms of cellular subsets and the molecular mechanisms involved remains to be determined. Mouse monocytes originate from a bone marrow progenitor, the macrophage and DC precursor (MDP), which also gives rise to conventional dendritic cells through a separate differentiation pathway. Mouse monocytes may be grouped in different functional subsets. The CD115(+) Gr1(+) 'inflammatory' monocyte subset can give rise not only to immunostimulatory 'TipDCs' in infected mice but also to immunosuppressive 'myeloid-derived suppressor cells' in tumor-bearing mice. CD115(+) Gr1(+) monocytes can also contribute to the renewal of several resident subsets of macrophages and DCs, such as microglia and Langerhans cells, in inflammatory conditions. The CD115(+) Gr1(-) 'resident' monocyte subset patrols blood vessels in the steady state and extravasates during infection with Listeria monocytogenes or in the healing myocardium. CD115(+) Gr1(-) monocytes are responsible for an early and transient inflammatory burst during Lm infection, which may play a role in the recruitment of other effector cells and subsequently differentiate toward 'M2'-like macrophages that may be involved in wound healing. More research will no doubt confirm the existence of more functional subsets, the developmental relationship between mouse subsets as well as the correspondence between mouse subsets and human subsets of monocytes. We will discuss here the potential roles of monocytes in the immune response, the existence of functional subsets and their relationship with other myeloid cells, including dendritic cells.     

Worldwide distribution of transposable element copy number in natural populations of Drosophila simulans

Abstract.— Transposable elements (TEs), which promote various kinds of mutations, constitute a large fraction of the genome. How they invade natural populations and species is therefore of fundamental importance for understanding the dynamics of genetic diversity and genome composition. On the basis of 85 samples of natural populations of Drosophila simulans , we report the distributions of the genome insertion site numbers of nine TEs that were chosen because they have a low average number of sites. Most populations were found to have 0–3 insertion sites, but some of them had a significantly higher number of sites for a given TE. The populations located in regions outside Africa had the highest number of sites for all elements except HMS Beagle and Coral , suggesting a recent increase in the activity of some TEs associated with the colonization patterns of Drosophila simulans . The element Tirant had a very distinctive pattern of distribution: it was identified mainly in populations from East Africa and some islands in the Indian Ocean, and its insertion site number was low in all these populations. The data suggest that the genome of the entire species of Drosophila simulans may be being invaded by TEs from populations in which they are present in high copy number.     

Misfolded plant virus proteins: elicitors and targets of ubiquitylation

Mutant tobacco mosaic virus (TMV) coat proteins (CPs) with known amino acid replacements provide well defined examples of destabilized tertiary structures. Here we show that misfolded TMV CPs, but not functional wild-type CPs, induce massive ubiquitylation in tobacco cells and that denatured, insoluble CP subunits are the main substrates of ubiquitin conjugation. As TMV CPs can be easily manipulated they are unique tools to study the molecular basis of the plant cell's response to aberrant protein structures and the associated intracellular stress reactions.