Mechanisms of HIV-1 evasion to the antiviral activity of chemokine CXCL12 indicate potential links with pathogenesis

HIV-1 infects CD4 T lymphocytes (CD4TL) through binding the chemokine receptors CCR5 or CXCR4. CXCR4-using viruses are considered more pathogenic, linked to accelerated depletion of CD4TL and progression to AIDS. However, counterexamples to this paradigm are common, suggesting heterogeneity in the virulence of CXCR4-using viruses. Here, we investigated the role of the CXCR4 chemokine CXCL12 as a driving force behind virus virulence. In vitro, CXCL12 prevents HIV-1 from binding CXCR4 and entering CD4TL, but its role in HIV-1 transmission and propagation remains speculative. Through analysis of thirty envelope glycoproteins (Envs) from patients at different stages of infection, mostly treatment-naïve, we first interrogated whether sensitivity of viruses to inhibition by CXCL12 varies over time in infection. Results show that Envs resistant (RES) to CXCL12 are frequent in patients experiencing low CD4TL levels, most often late in infection, only rarely at the time of primary infection. Sensitivity assays to soluble CD4 or broadly neutralizing antibodies further showed that RES Envs adopt a more closed conformation with distinct antigenicity, compared to CXCL12-sensitive (SENS) Envs. At the level of the host cell, our results suggest that resistance is not due to improved fusion or binding to CD4, but owes to viruses using particular CXCR4 molecules weakly accessible to CXCL12. We finally asked whether the low CD4TL levels in patients are related to increased pathogenicity of RES viruses. Resistance actually provides viruses with an enhanced capacity to enter naive CD4TL when surrounded by CXCL12, which mirrors their situation in lymphoid organs, and to deplete bystander activated effector memory cells. Therefore, RES viruses seem more likely to deregulate CD4TL homeostasis. This work improves our understanding of the pathophysiology and the transmission of HIV-1 and suggests that RES viruses’ receptors could represent new therapeutic targets to help prevent CD4TL depletion in HIV+ patients on cART.     

The response of alkaline phosphatase to osmoregulatory changes in the trout,Salmo gairdneri

Summary The relationship between alkaline phosphatase and environmental salinity was examined in the rainbow trout and the migratory rainbow (steelhead),Salmo gairdneri. The enzyme activity in tissues involved in osmoregulation was strongly correlated with the adaptation salinity and thus to the degree of salt and fluid transport in those tissues. After transfer from freshwater to seawater, the specific activity of the enzyme increased over 260% in the intestine, decreased by 50% in kidney, and was unchanged in the liver, an organ not directly involved in osmoregulation. The sea-run steelhead trout response was similar to the nonmigratory rainbow; although, the pre-migratory transformation (smoltification) had no effect on enzyme activity. Amino acid inhibitors of alkaline phosphatase significantly reduced fluid absorption in the isolated intestine of rainbow trout, reaffirming the relationship between the enzyme and fluid movement. Electrophoretic identification of trout alkaline phosphatase isozymes, clearly distinguishes the enzyme from different tissue origins. However, from the analysis of intestinal electrophoretic patterns, osmoregulatory adjustments are not associated with the induction of new alkaline phosphatase isozymes, or in the large scale preferential stimulation of one of the two existing intestinal isozymes over the other.     

Cloning of an Arabidopsis thaliana gene encoding 5-enolpyruvylshikimate-3-phosphate synthase: sequence analysis and manipulation to obtain glyphosate-tolerant plants

Summary 5-enolpyruvylshikimate-3-phosphate synthase (EPSPs), the target of the herbicide glyphosate, catalyzes an essential step in the shikimate pathway common to aromatic amino acid biosynthesis. We have cloned an EPSP synthase gene from Arabidopsis thaliana by hybridization with a petunia cDNA probe. The Arabidopsis gene is highly homologous to the petunia gene within the mature enzyme but is only 23% homologous in the chloroplast transit peptide portion. The Arabidopsis gene contains seven introns in exactly the same positions as those in the petunia gene. The introns are, however, significantly smaller in the Arabidopsis gene. This reduction accounts for the significantly smaller size of the gene as compared to the petunia gene. We have fused the gene to the cauliflower mosaic virus 35 S promoter and reintroduced the chimeric gene into Arabidopsis. The resultant overproduction of EPSPs leads to glyphosate tolerance in transformed callus and plants.     

Mutational analysis of the Bradyrhizobium japonicum common nod genes and further nod box-linked genomic DNA regions

Summary By insertional and deletional marker replacement mutagenesis the common nod region of Bradyrhizobium japonicum was examined for the presence of additional, essential nodulation genes. An open reading frame located in the 800 bp large intergenic region between nodD1 and nodA did not appear to be essential for nodulation of soybean. Furthermore, a strain with a deletion of the nodI- and nodJ-like genes downstream of nodC had a Nod⁺ phenotype. A mutant with a 1.7 kb deletion immediately downstream of nodD1 considerably delayed the onset of nodulation. This region carried a second copy of nodD (nodD2). A nodD1-nodD2 double mutant had a similar phenotype to the nodD2 mutant. Using a 22-mer oligonucleotide probe partially identical to the nod box sequence, a total of six hybridizing regions were identified in B. japonicum genomic DNA and isolated from a cosmid library. Sequencing of the hybridizing regions revealed that at least three of them represented true nod box sequences whereas the others showed considerable deviations from the consensus sequence. One of the three nod box sequences was the one known to be associated with nodA, whereas the other two were located 60 to 70 kb away from nif cluster I. A deletion of one of these two sequences plus adjacent DNA material mmutant 308) led to a reduced nodulation on Vigna radiata but not on soybean. Thus, this region is probably involved in the determination of host specificity.Dedicated to Prof. Giorgio Semenza on the occasion of his 60th birthday     

Scopolin, a Biochemical Marker for Resistance to Thielaviopsis basicola in Callus and Crown-Gall Tissue Cultures of Tobacco

Scopolin concentration increased in tissue cultures where Thielaviopsis basicola growth ceased (primary and established callus cultures of the resistant tobacco cultivar Ky 170) while it decreased in tissue cultures where fungal growth persisted (primary and established callus cultures of the susceptible tobacco cultivar Ky 151 and crown-gall cultures of Ky 170 and Ky 151). The concentration of chlorogenic acid, scopoletin, and two other unknown soluble phenols varied after inoculation and no, correlation with tissue culture resistance could be established. Incorporation of Agrobacterium tumefaciens T-DNA in the genome of both cultivars induced a drastic reduction of scopolin in inoculated tissue cultures. T-DNA incorporation had less, influence on uninoculated tissues. Scopolin at concentrations found in tissue cultures was not toxic to the fungus.     

Resistance of Tobacco to Thielaviopsis basicola under Tissue Culture Conditions and Increased Susceptibility after Transformation with Agrobacterium tumefaciens T-DNA

Tobacco callus cultures from a Thielaviopsis basicola (Berk. et Br.) Ferraris resistant cultivar were less severely colonized than callus cultures from susceptible cultivars by the pathogen at all concentrations of kinetin and α-indoleacetic acid tested. However, at concentrations where these substances influenced the morphology of the callus cultures they also influenced the degree of colonization by the fungus. Incorporation of T-DNA of Agrobacterium tumefaciens induced susceptibility in tissue culture of the resistant cultivar but did not significantly modify the reaction of the susceptible cultivar tested.