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.     

Porcine D-amino acid oxidase: production of the biologically active enzyme in Escherichia coli

DNA molecules coding either for mature porcine D-amino acid oxidase or for truncated forms of the enzyme have been obtained by stepwise addition of synthetic oligonucleotides to a partial cDNA. Under the control of the lambda PL thermoregulatable promoter, these DNAs were respectively expressed in Escherichia coli as 36, 28 and 25 kilodalton polypeptides, specifically recognised by antibodies raised against the natural enzyme. None of the truncated proteins were biologically active whereas the mature recombinant species was able to hydrolyze D-alanine in vitro as efficiently as the natural product.     

Rearing, reproductive behaviour and gamma sterilization of fruit fly, Dacus zonatus (Diptera: Tephritidae)

Investigations were made on rearing, reproductive behaviour and gamma sterilization of one-day old male adults of Dacus zonatus. The larvae were successfully reared on an artificial diet based on wheat shorts. Adult emergence ranged from 89–99% with a sex ratio of about 1:1. Mating occurred at dusk and its duration ranged from 8–13 hours. Males mated a second time with the same female but preferred mating if the already mated female was replaced with a sexually mature virgin female. The optimum dosage for inducing sterility amongst one-day old male adults was 12 kR.

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Zusammenfassung Zucht und Fortpflanzungsverhalten von Dacus zonatus (Saunders) wurde untersucht. Die Larven wurden vier Generationen lang an einer Diät aus Weizenkleie, Bierhefe, granuliertem Zucker, Agar, Nipagin, Salzsäure und Wasser gehalten. Die Arbeit gibt Daten über Verpuppungsprozentsatz (69,3%), Puppengewicht, Dauer der Ei + Larvenperiode, Schlüpfen der Adulten, Präovipositionszeit, Fruchtbarkeit, Fertilität und Lebensdauer der Adulten. Die Schlüpfrate der Adulten betrug 89–99%, das Geschlechtsverhältnis lag etwa bei 1:1. Die Kopulation findet während der Abenddämmerung statt, sie dauert 8–13 Stunden. Maximum der Kopulationen zwischen dem 10. und 15. Tag nach dem Schlüpfen. Männchen paarten sich ein zweites Mal mit dem gleichen Weibchen, bevorzugten jedoch geschlechtsreife jung-fräuliche Weibchen. Die Eiablage begann am 2.–7. Tag nach der Paarung, die Eizahl betrug bei gepaarten Weibchen 91–564.Die optimale Dosis zur Erzeugung von 99,3% Sterilität bei Bestrahlung von einem Tag alten Männchen war 12 kR. Die Lebensdauer der Bestrahlten war vermindert.

     

Guanine deaminase inhibitor from rat liver. Isolation and characterization

1. An inhibitor of cytoplasmic guanine deaminase of rat liver was isolated from liver ;heavy mitochondrial' fraction after freezing and thawing and treatment with Triton X-100. 2. Submitochondrial fractionation revealed that the inhibitor was localized in the outer-membrane fraction. 3. The method of purification of inhibitor, involving precipitation with (NH(4))(2)SO(4) and chromatography on DEAE-cellulose, its precipitability by trichloroacetic acid and the pattern of absorption in the u.v. indicated that the inhibitor was a protein. In confirmation, tryptic digestion of the isolated material resulted in destruction of the inhibitor activity. The inhibitor was stable to acid, but labile to heat. 4. The isolated inhibitor required phosphatidylcholine (lecithin) for activity. Phosphatidylcholine also partially protected the inhibitor against heat inactivation. 5. When detergent treatment was omitted, the inhibitor activity of frozen mitochondria was precipitated by (NH(4))(2)SO(4) in a fully active form without supplementation with phosphatidylcholine, indicating that Triton X-100 ruptured the linkage between inhibitor and lipid. 6. A reconstituted sample of inhibitor-phosphatidylcholine complex was precipitated in a fully active form by dialysis against 2-mercaptoethanol, but treatment of the precipitate with NaCl yielded an extract which was inactive unless supplemented with fresh phosphatidylcholine. 7. We interpret the results as evidence that the inhibitor was present in vivo as a lipoprotein and that once the complex was dissociated by the action of detergent and the protein precipitated, there was an absolute need for exogenous phosphatidylcholine for its activity. The manner in which inhibitor associated with the outer membrane of rat liver mitochondria might regulate the activity of the enzyme in the supernatant has been suggested.     

Apolipoprotein E and Low-Density Lipoprotein Binding and Internalization in Primary Cultures of Rat Astrocytes: Isoform-Specific Alterations

Abstract: Apolipoprotein (apo) E is likely involved in redistributing cholesterol and phospholipids during compensatory synaptogenesis in the injured CNS. Three common isoforms of apoE exist in human (E2, E3, and E4). The apoE4 allele frequency is markedly increased in both late-onset sporadic and familial Alzheimer's disease (AD). ApoE concentration in the brain of AD subjects follows a gradient: ApoE levels decrease as a function of E2 > E3 ? E4. It has been proposed that the poor reinnervation capacity reported in AD may be caused by impairment of the apoE/low-density lipoprotein (LDL) receptor activity. To understand further the role of this particular axis in lipid homeostasis in the CNS, we have characterized binding, internalization, and degradation of human ¹²⁵I-LDL to primary cultures of rat astrocytes. Specific binding was saturable, with a K_D of 1.8 nM and a B_max of 0.14 pmol/mg of proteins. Excess unlabeled human LDL or very LDL (VLDL) displaced 70% of total binding. Studies at 37°C confirmed that astrocytes bind, internalize, and degrade ¹²⁵I-LDL by a specific, saturable mechanism. Reconstituted apoE (E2, E3, and E4)-liposomes were labeled with ¹²⁵I and incubated with primary cultures of rat astrocytes and hippocampal neurons to examine specific binding. Human LDL and VLDL displaced binding and internalization of all apoE isoforms similarly in both astrocytes and neurons. ¹²⁵I-ApoE2 binding was significantly lower than that of the other ¹²⁵I-apoE isoforms in both cell types. ¹²⁵I-ApoE4 binding was similar to that of ¹²⁵I-apoE3 in both astrocytes and neurons. On the other hand, ¹²⁵I-apoE3 binding was significantly higher in neurons than in astrocytes. These isoform-specific alterations in apoE-lipoprotein pathway could explain some of the differences reported in the pathophysiology of AD subjects carrying different apoE alleles.     

The effects of soil warming on plant recruitment

We demonstrate, using a simple field experiment, that soil warming is sufficient, independent of other cues, to potentially alter the recruitment of Betula pendula seedlings. This study suggests that changing temperature, and correlated changes in edaphic factors, can alter patterns in plant recruitment via differential effects on seed dormancy. Increased soil temperatures (of less than 5 °C) throughout the year led to a significant increase in the number of birch seedlings that emerged from soil samples collected in the spring. No concomitant changes in the emergence of other species was observed. Such temperature induced changes in seedbank dynamics and their subsequent effects on the competitive interactions within a plant community suggest far reaching ecological consequences of current increases in global temperature.     

How Many Nucleotides Are Required to Resolve a Phylogenetic Problem? The Use of a New Statistical Method Applicable to Available Sequences

The evolution of bootstrap proportions (BP) with sequence length was studied using a 28S ribosomal RNA data set. For different sequence lengths, informative sites were jackknifed several times. Bootstrapping was subsequently performed on each of these subsamples. For each node, BPs so obtained were plotted against sequence length, showing the evolution of the robustness with increasing number of informative sites. For robust nodes (BP of 100%), the pattern of BPs is unvarying and is described by a simple function BP = 100(1− e^{−b(x − x′)}), where x is the number of informative sites and b and x′ are two parameters estimated using a nonlinear regression procedure. When a node has a BP <100% and the pattern of BPs fits this function, it is possible to estimate the number of informative sites required to obtain a given average BP. The method also identifies nonrobust nodes (nonascending clusters of BP dots), for which it seems to be more cost effective and fruitful to turn to other species and/or genes rather than to continue sequencing longer gene lengths from the same species to reach a BP of 95%.     

Evidence for linkage between K88ab, K88ac intestinal receptors to Escherichia coli and transferrin loci in pigs

Segregation at the loci coding for the K88ab and K88ac small intestinal receptors to E. coli adhesins (K88abR, K88acR) and at the transferrin (TF) locus was studied in 38 pig families including 273 piglets. The TF locus showed a segregation deviation towards the B variant while each of the K88 receptors behaved as a single autosomal dominant gene. Recombinants between K88abR and K88acR provide evidence that they are under the control of two different loci. Thirty-two triple backcross families were selected to test linkage and estimate recombination rates (θ). Our results demonstrate that the two K88 receptor loci are closely linked (θ= 0.02) with a maximum lod score value (Z_m) of 46.0. In addition, they are linked to the TF locus, θ= 0.14, Z_m= 19.6 for the K88abR locus and θ= 0.16, Z_m= 17.9 for the K88acR locus. The estimated recombination rates, smaller in males than in females, are consistent with the order TF-K88abR-K88acR. This linkage thus localizes the K88 loci, as the TF locus, on chromosome 13.