Cross-Reactions between the Cytotoxic T-Lymphocyte Responses of Human Immunodeficiency Virus-Infected African and European Patients

The great variability of protein sequences from human immunodeficiency virus (HIV) type 1 (HIV-1) isolates represents a major obstacle to the development of an effective vaccine against this virus. The surface protein (Env), which is the predominant target of neutralizing antibodies, is particularly variable. Here we examine the impact of variability among different HIV-1 subtypes (clades) on cytotoxic T-lymphocyte (CTL) activities, the other major component of the antiviral immune response. CTLs are produced not only against Env but also against other structural proteins, as well as some regulatory proteins. The genetic subtypes of HIV-1 were determined for Env and Gag from several patients infected either in France or in Africa. The cross-reactivities of the CTLs were tested with target cells expressing selected proteins from HIV-1 isolates of clade A or B or from HIV type 2 isolates. All African patients were infected with viruses belonging to clade A for Env and for Gag, except for one patient who was infected with a clade A Env-clade G Gag recombinant virus. All patients infected in France were infected with clade B viruses. The CTL responses obtained from all the African and all the French individuals tested showed frequent cross-reactions with proteins of the heterologous clade. Epitopes conserved between the viruses of clades A and B appeared especially frequent in Gag p24, Gag p18, integrase, and the central region of Nef. Cross-reactivity also existed among Gag epitopes of clades A, B, and G, as shown by the results for the patient infected with the clade A Env-clade G Gag recombinant virus. These results show that CTLs raised against viral antigens from different clades are able to cross-react, emphasizing the possibility of obtaining cross-immunizations for this part of the immune response in vaccinated individuals.     

Gene expression during CAM induction under salt stress in Mesembryanthemum: cDNA library and increased levels of mRNA for phosphoenolpyruvate carboxylase and pyruvate orthosphosphate dikinase

Mesembryanthemum crystallinum responds to high salinity in the soil by shifting the mode of carbon assimilation from the C3 mode to Crassulacean acid metabolism (CAM). Several enzymes of carbon metabolism have increased apparent activities in the CAM mode, including phosphoenolpyruvate carboxylase (PEPcase) and pyruvate orthophosphate dikinase (PPDK). We have identified cDNA clones for PEPcase and PPDK by immunological screening of a cDNA library constructed in the protein expression vector lambda gt11. The clones were characterized by immunoblotting and RNA blotting techniques. RNA blotting showed that during CAM induction the steady-state level of mRNAs for both PEP case and PPDK increased.Abbreviations IPTG isopropyl thiogalactoside - PEP phosphoenolpyruvate - PEPcase phosphoenolpyruvate carboxylase - PPDK pyruvate orthophosphate dikinase - Xgal-5 bromo-4-chloro-3-indolyl-beta-D-galactopyranoside     

Model experiments on squid axons and NG108-15 mouse neuroblastoma × rat glioma hybrid cells

Three types of ionic current essentially determine the firing pattern of nerve cells: the persistent Na⁺ current, the M current and the low-voltage-activated Ca²⁺ current. The present article summarizes recent experiments concerned with the basic properties of these currents. Keynes and Meves (Proc R Soc Lond B (1993) 253, 61–68) studied the persistent or steady-state Na⁺ current on dialysed squid axons and measured the probability of channel opening both for the peak and the steady-state Na⁺ current (PF_peak and PF_ss) as a function of voltage. Whereas PF_peak starts to rise at −50 mV and reaches a maximum at +40 to +50 mV, PF_ss only begins to rise appreciably at around 0 mV and is still increasing at +100 mV. This differs from observations on vertebrate excitable tissues where the persistent Na⁺ current turns on in the threshold region and saturates at around 0 mV. Schmitt and Meves (Pflügers Arch (1993) 425, 134–139) recorded M current, a non-inactivating K⁺ current, from NG108-15 neuroblastoma × glioma hybrid cells, voltage-clamped in the whole-cell mode, and studied the effects of phorbol 12,13-dibutyrate (PDB), an activator of protein kinase C (PKC), and arachidonic acid (AA). PDB and AA both decreased I_M, the effective concentrations being 0.1–1 μM and 5–25 μM, respectively; while the PDB effect was regularly observed, the M current depression by AA was highly variable from cell to cell. The PKC 19–31 peptide, an effective inhibitor of PKC, in a concentration of 1 μM almost totally prevented the effects of PDB and AA on M current, suggesting that both are mediated by PKC. Schmitt and Meves (Pflügers Arch (1994a) 426, Suppl R 59) measured low-voltage-activated (l-v-a) and high-voltage-activated (h-v-a) Ca²⁺ currents on NG108-15 cells and investigated the effect of AA and PDB on both types of current. At pulse potentials > −20 mV, AA (25–100 μM) decreased l-v-a and h-v-a I_Ca. The decrease was accompanied by a small negative shift and a slight flattening of the activation and inactivation curves of the l-v-a I_Ca. The AA effect was not prevented by 50 μM eicosa-5,8,11,14-tetraynoic acid (ETYA), an inhibitor of AA metabolism, or PKC 19–31 peptide and not mimicked by 0.1–1 μM PDB. Probably, AA acts directly on the channel protein or its lipid environment. The physiological relevance of these three sets of observations is briefly discussed.     

miR-155 Inhibition Sensitizes CD4+ Th Cells for TREG Mediated Suppression

Background

In humans and mice naturally occurring CD4⁺CD25⁺ regulatory T cells (nTregs) are a thymus-derived subset of T cells, crucial for the maintenance of peripheral tolerance by controlling not only potentially autoreactive T cells but virtually all cells of the adaptive and innate immune system. Recent work using Dicer-deficient mice irrevocably demonstrated the importance of miRNAs for nTreg cell-mediated tolerance.

Principal Findings

DNA-Microarray analyses of human as well as murine conventional CD4⁺ Th cells and nTregs revealed a strong up-regulation of mature miR-155 (microRNA-155) upon activation in both populations. Studying miR-155 expression in FoxP3-deficient scurfy mice and performing FoxP3 ChIP-Seq experiments using activated human T lymphocytes, we show that the expression and maturation of miR-155 seem to be not necessarily regulated by FoxP3. In order to address the functional relevance of elevated miR-155 levels, we transfected miR-155 inhibitors or mature miR-155 RNAs into freshly-isolated human and mouse primary CD4⁺ Th cells and nTregs and investigated the resulting phenotype in nTreg suppression assays. Whereas miR-155 inhibition in conventional CD4⁺ Th cells strengthened nTreg cell-mediated suppression, overexpression of mature miR-155 rendered these cells unresponsive to nTreg cell-mediated suppression.

Conclusion

Investigation of FoxP3 downstream targets, certainly of bound and regulated miRNAs revealed the associated function between the master regulator FoxP3 and miRNAs as regulators itself. miR-155 is shown to be crucially involved in nTreg cell mediated tolerance by regulating the susceptibility of conventional human as well as murine CD4⁺ Th cells to nTreg cell-mediated suppression.     

The mutagenic potential of high flash aromatic naphtha

Catalytic reforming is a refining process that converts naphthenes to aromatics by dehydrogenation to make higher octane gasoline blending components. A portion of this wide boiling range hydrocarbon stream can be separated by distillation and used for other purposes. One such application is a mixture of predominantly 9-carbon aromatic molecules (C9 aromatics, primarily isomers of ethyltoluene and trimethylbenzene), which is removed and used as a solvent — high-flash aromatic naphtha. A program was initiated to assess the toxicological properties of high-flash aromatic naphtha since there may be human exposure through inhalation or external body contact. The current study was conducted partly to assess the potential for mutagenic activity and also to assist in an assessment of carcinogenic potential. The specific tests utilized included the Salmonella/mammalian microsome mutagenicity assay, the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) forward mutation assay in CHO cells, in vitro chromosome aberration and sister chromatid exchange (SCE) assays in CHO cells, and an in vivo chromosome aberration assay in rat bone marrow.There was no evidence that high-flash aromatic naphtha was either a gene or chromosomal mutagen. Thus it is unlikely to be a genotoxic carcinogen.Abbreviations Brdu 5-Bromo-2-deoxyuridine - C9 Aromatic species with 9 carbons (i.e., ethyl toluene and trimethyl benzene isomers) - CE Cloning efficiency - CHO Chinese hamster embryo - CP Cyclophosphamide - DMSO Dimethyl sulfoxide - HGPRT Hypoxanthine-guanine phosphoribosyl transferase - HVAC Heating, Ventilation, Air Conditioning - 3MC 3 Methylcholanthrene - MMC Mitomycin C - MMS Methyl methanesulfonate - S9 S9 Mammalian microsomal enzyme activation mixture - SCE Sister chromatid exchange     

Effects of habitat fragmentation on the genetic structure of the monophagous butterfly Polyommatus coridon along its northern range margin

Population genetic patterns of species at their range margin have important implications for species conservation. We performed allozyme electrophoresis of 19 loci to investigate patterns of the genetic structure of 17 populations (538 individuals) of the butterfly Polyommatus coridon, a monophagous habitat specialist with a patchy distribution. The butterfly and its larval food plant Hippocrepis comosa reach their northern distribution margin in the study region (southern Lower Saxony, Germany). Butterfly population size increased with host plant population size. The genetic differentiation between populations was low but significant (FST = 0.013). No isolation-by-distance was found. Hierarchical F-statistics revealed significant differentiation between a western and an eastern subregion, separated by a river valley. The combination of genetic and ecological data sets revealed that the expected heterozygosity (mean: 18.5%) decreased with increasing distance to the nearest P. coridon population. The population size of P. coridon and the size of larval food plant population had no effect on the genetic diversity. The genetic diversity of edge populations of P. coridon was reduced compared to populations from the centre of its distribution. This might be explained by (i). an increasing habitat fragmentation towards the edge of the distribution range and/or (ii). a general reduction of genetic variability towards the northern edge of its distribution.     

Fish communities on staghorn coral: effects of habitat characteristics and resident farmerfishes

Branching corals, like many in the genus Acropora, provide structurally complex habitats for reef fishes and other organisms. Fluctuations in the abundance, distribution and characteristics of thicket-forming staghorn Acroporids may contribute to changes in the abundance and species composition of reef fishes due to changes in the availability of shelter habitat and food. Farming damselfishes of the genus Stegastes can occur in high abundances in staghorn corals and actively defend food and nest space against organisms that threaten these resources. Here we assess the value of staghorn as habitat for fishes in the central South Pacific, and how the presence of territorial farming damselfishes may influence the assemblage of fishes that associate with staghorn corals. Surveys of 185 Acropora pulchra patches located in the lagoons surrounding the island of Moorea, French Polynesia revealed 85 species of fish from 25 families. Total fish abundance and species richness values ranged from no fish on a patch to a high of 275 individuals and 26 species. Patch area was the most important characteristic in explaining variation in attributes of the fish assemblage, with other characteristics explaining little of the species composition or trophic structure. Behavioral observations revealed that farming damselfishes were most aggressive toward corallivores, herbivores, and egg predators, while they ignored most carnivores and omnivores. Despite this pattern, we observed positive covariance between Stegastes and the group of fishes that elicited the strongest aggressive response when the effect of patch area was removed, suggesting these fishes remain drawn to the resources produced or enhanced by Stegastes on A. pulchra.