Dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN)-mediated enhancement of dengue virus infection is independent of DC-SIGN internalization signals

Dengue virus (DV) is a mosquito-borne flavivirus that causes hemorrhagic fever in humans. In the natural infection, DV is introduced into human skin by an infected mosquito vector where it is believed to target immature dendritic cells (DCs) and Langerhans cells (LCs). We found that DV productively infects DCs but not LCs. We show here that the interactions between DV E protein, the sole mannosylated glycoprotein present on DV particles, and the C-type lectin dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN) are essential for DV infection of DCs. Binding of mannosylated N-glycans on DV E protein to DC-SIGN triggers a rapid and efficient internalization of the viral glycoprotein. However, we observed that endocytosis-defective DC-SIGN molecules allow efficient DV replication, indicating that DC-SIGN endocytosis is dispensable for the internalization step in DV entry. Together, these results argue in favor of a mechanism by which DC-SIGN enhances DV entry and infection in cis. We propose that DC-SIGN concentrates mosquito-derived DV particles at the cell surface to allow efficient interaction with an as yet unidentified entry factor that is ultimately responsible for DV internalization and pH-dependent fusion into DCs.     

DC-SIGN and L-SIGN are high affinity binding receptors for hepatitis C virus glycoprotein E2

The hepatitis C virus (HCV) genome codes for highly mannosylated envelope proteins, which are naturally retained in the endoplasmic reticulum. We found that the HCV envelope glycoprotein E2 binds the dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) and the related liver endothelial cell lectin L-SIGN through high-mannose N-glycans. Competing ligands such as mannan and an antibody directed against the carbohydrate recognition domains (CRD) abrogated binding. While no E2 interaction with distant monomeric CRDs on biosensor chips could be detected, binding is observed if CRDs are closely seeded (Kd = 48 nm) and if the CRD is part of the oligomeric-soluble extracellular domain of DC-SIGN (Kd = 30 nm). The highest affinity is seen for plasma membrane-expressed DC-SIGN and L-SIGN (Kd = 3 and 6 nm, respectively). These results indicate that several high-mannose N-glycans in a structurally defined cluster on E2 bind to several subunits of the oligomeric lectin CRD. High affinity interaction of viral glycoproteins with oligomeric lectins might represent a strategy by which HCV targets to and concentrates in the liver and infects dendritic cells.     

Physiological and biochemical parameters: new tools to screen barley root exudate allelopathic potential (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L. subsp. <Emphasis Type="Italic">vulgare</Emphasis>)

Morphological markers/traits are often used in the detection of allelopathic stress, but optical signals including chlorophyll a fluorescence emission could be useful in developing new screening techniques. In this context, the allelopathic effect of barley (Hordeum vulgare subsp. vulgare) root exudates (three modern varieties and three landraces) were assessed on the morphological (root and shoot length, biomass accumulation), physiological (F_v/F_m and F₀), and biochemical (chlorophyll and protein contents) variables of great brome (Bromus diandrus Roth., syn. Bromus rigidus Roth. subsp. gussonii Parl.). All the measured traits were affected when great brome was grown in a soil substrate in which barley plants had previously developed for 30 days before being removed. The response of receiver plants was affected by treatment with activated charcoal, dependent on barley genotype and on the nature of the growing substrate. The inhibitory effect was lower with the addition of the activated charcoal suggesting the release of putative allelochemicals from barley roots into the soil. The barley landraces were more toxic than modern varieties and their effect was more pronounced in sandy substrate than in silty clay sand substrate. In our investigation, the chlorophyll content and F_v/F_m were the most correlated variables with barley allelopathic potential. These two parameters might be considered as effective tools to quantify susceptibility to allelochemical inhibitors in higher plants.     

Effect of moisture and salt stress on selectedRhizobium phaseoli strains

Summary Studies in Rossmoyne silt loam by the plate count (PC) and fluorescent antibody (FA) techniques showed that flooding had no detrimental effects on survival and N₂-fixing capacity ofR. phaseoli strains. Short-term flooding apparently stimulated the activity of the bacteria, as indicated by the increase in nodulation and yield ofPhaseolus vulgaris. The strains also differed in their ability to survive desiccation, and a decline in the population of all strains occurred at moisture tensions greater than 15 bars. The inhibitory concentrations of salts to growth ofR. phaseoli in solution were 0.3% for Na and K chlorides and 1.0% for Ca and Mg chlorides. The effects of Na and K sulphates were similar to the Na and K chlorides at 0.3% except for strain C-05 which was inhibited by 0.1% Na sulphate. Comparative growth averaged over all strains grown with 0.1% salts showed that the degree of inhibition was the same for both chloride and sulphate salts but that Na salts showed the greatest, and Mg salts the least, inhibition. It was concluded that some strains ofR. phaseoli can survive soil flooding, desiccation, and salt stress in large enough numbers to initiate nodule formation in some soils.

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Resumen Estudios realizados en Rossmoyne, en suelos de tipo limoso, ricos en materia orgánica han mostrado mediante técnicas de conteo en placa (PC) y de anticuerpos fluorescentes (FA) que la inundación de campos no iba en detrimento de la supervivencia y de la capacidad de fijación de N₂ de algunas cepas deR. phaseoli. La actividad de las bacterias parece verse estimulada por inundaciones de corta duración según se deduce del incremento en la nodulación y en la cosecha dePhaseolus vulgaris. Las distintas cepas mostraron diferencias en su habilidad para sobrevivir a la sequía, observándose una disminución en la población de todas las cepas a tensiones superiores a 15 bars. Las sales en disolución inhibieron el crecimiento deR. phaseoli a las concentraciones siguientes: 0.3% para los cloruros de Na y K, y 0.1% para los cloruros de Ca y Mg. Los efectos de los sulfatos de Na y K a una concentración de 0.3% fueron similares a los observados para los cloruros, excepto para el caso de la cepa C-05 que fue inhibida por 0.1% de sulfato de Na. El crecimiento medio, comparativo, de todas las cepas en un medio con un 0.1% de sales mostró que cloruros y sulfatos tenían un mismo grado de inhibición y que las sales de Na tenían el mayor efecto inhibidor, teniendo las de Mg el menor.

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Résumé Des études effectuées sur la terre limoneuse de Rossmoyne par les techniques de numération sur gélose (PC) et d'anticorps fluorescents (FA) ont montré que l'inondation n'a pas d'effet défavorable sur la survie et la capacité à fixer l'azote de certaines souches deR. phaseoli. Une brève inondation du sol parait stimuler l'activité des bactéries, comme l'indique le fait que la nodulation et le rendement dePhaseolus vulgaris sont accrus. L'aptitude des souches à survivre à la dessication est variable. D'autre part, les populations de toutes les souches diminuent lorsque la tension d'humidité dépasse 15 bars. Les concentrations salines inhibant la croissance deR. phaseoli en milieu liquide sont de 0,3% pour le chlorure de Na et de K, et de 0,1% pour le chlorure de Ca et de Mg. Les sulfates de Na et de K sont, comme les chlorures, inhibiteurs à la concentration de 0,3%, sauf dans le cas de la souche C-05 qui est inhibée par 0,1% de sulfate de Na. A la concentration de 0,1%, les chlorures et les sulfates exercent le même degré d'inhibition sur toutes les souches, mais les sels de Na sont plus inhibiteurs que ceux de Mg. En conclusion, certaines souches deR. phaseoli peuvent survivre à l'inondation, à la dessication et à l'action des sels en nombres suffisants pour initier la formation de nodules.

     

Specific mRNA destabilization in Dictyostelium discoideum requires RNA synthesis.

We tested the effects of inhibitors of protein and RNA synthesis on the disaggregation-mediated destabilization of prespore mRNAs in Dictyostelium discoideum. Incubating disaggregated cells with daunomycin to inhibit RNA synthesis prevented the loss of prespore mRNAs, whereas the inhibitor decreased or did not affect levels of the common mRNAs CZ22 and actin. Protein synthesis inhibitors varied in their effects. Cycloheximide, which inhibited protein synthesis almost completely, prevented the loss of the prespore mRNAs, but puromycin, which inhibited protein synthesis less well, did not. These results indicate that the process of specific mRNA destabilization requires the synthesis of RNA and possibly of protein.     

A novel transforming growth factor-beta 1 responsive cytoplasmic trans-acting factor binds selectively to the 3'-untranslated region of mammalian ribonucleotide reductase R2 mRNA: role in message stability.

Ribonucleotide reductase is a highly regulated enzyme that provides the four deoxyribonucleotides required for DNA synthesis. Our studies showed that TGF-beta 1 treatment of BALB/c 3T3 mouse fibroblasts markedly elevated ribonucleotide reductase R2 mRNA levels, and also increased the half-life of R2 message by 4-fold from 1.5 h in untreated cells to 6 h in treated cells. We describe a novel 75 Kd sequence-specific cytoplasmic factor (p75) that binds selectively to a 83-nucleotide 3'-untranslated region of R2 mRNA and did not bind to the 5'UTR, the coding region of the R2 message or to the 3'UTRs of other mRNAs (from c-myc, GM-CSF and the iron responsive element from the transferrin receptor mRNA), or to the homopolymer poly(A) sequence. p75-RNA binding activity, which requires new protein synthesis, is not present in untreated cells, but is induced following TGF-beta 1 stimulation. The in vivo kinetics of appearance of p75 binding activity paralleled the accumulation of R2 mRNA. Insertion of the 3'-untranslated region into the chloramphenicol acetyltransferase (CAT) message confers TGF-beta 1 induced stability of RNA in stably transfected cells, while the same insert carrying a deletion of the 83-nucleotide fragment had little affect on RNA levels. Furthermore, in vitro decay reactions that contained the 83-nucleotide RNA or deletion of this fragment caused a significant decrease in TGF-beta 1 stabilization of R2 message. A model is presented of R2 message regulation in which TGF-beta 1 mediated stabilization of R2 message involves a specific interaction of a p75-trans-acting factor with a cis-element(s) stability determinant within the 83-nucleotide sequence which is linked to a reduction in the rate of R2 mRNA degradation.     

Spontaneous Mutations in the env Gene of the Human Immunodeficiency Virus Type 1 NDK Isolate Are Associated with a CD4-Independent Entry Phenotype

Human immunodeficiency virus type 1 (HIV-1) entry into target cells is a multistep process initiated by envelope protein gp120 binding to cell surface CD4. The conformational changes induced by this interaction likely favor a second-step interaction between gp120 and a coreceptor such as CXCR4 or CCR5. Here, we report a spontaneous and stable CD4-independent entry phenotype for the HIV-1 NDK isolate. This mutant strain, which emerged from a population of chronically infected CD4-positive CEM cells, can replicate in CD4-negative human cell lines. The presence of CXCR4 alone renders cells susceptible to infection by the mutant NDK, and infection can be blocked by the CXCR4 natural ligand SDF-1. Furthermore, we have correlated the CD4-independent phenotype with seven mutations in the C2 and C3 regions and the V3 loop. We propose that the mutant gp120 spontaneously acquires a conformation allowing it to interact directly with CXCR4. This virus provides us with a powerful tool to study directly gp120-CXCR4 interactions.     

N-Linked Oligosaccharides Are Required for Cell Surface Expression of the Norepinephrine Transporter but Do Not Influence Substrate or Inhibitor Recognition

Abstract: The contribution of N-linked carbohydrates to the function of the human norepinephrine transporter (NET) was investigated using site-directed mutagenesis to inactivate the two most carboxy-terminal (NQQ mutant) or all three (QQQ mutant) sites for N -glycosylation within the extracellular loop between transmembrane domains 3 and 4. In HeLa cells transiently expressing the NET, two glycosylated forms of the transporter at 90 and 60 kDa are immunoprecipitated by NET antisera. A single 50-kDa species is observed in cells expressing the QQQ mutant, and it likely represents the NET core protein. Analyses of substrate transport kinetics showed rank order V _max of 19:9:1 for NET/NQQ/QQQ without a change in the apparent affinity of the wild-type and mutated carriers for either substrates or transport inhibitors. Cell surface biotinylation indicates that all NET, NQQ, and QQQ transporter species are detected at the plasma membrane but that glycosylated forms are selectively enriched. The transport activities exhibited by each of the carriers correlate well with cell surface content. Subcellular localization of transporters using immunofluorescence microscopy shows that reductions in surface expression and transport are associated with a corresponding increase in the intracellular retention of mutated carriers. Thus, N-linked glycosylation does not alter the apparent affinity of NET for either substrates or inhibitors of transport but, instead, appears to influence the abundance of carriers at the cell surface.