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.     

Effect of tubulozole, a new synthetic microtubule inhibitor, on the induction of casein gene expression by prolactin

Colchicine and related drugs are known to inhibit milk secretion. They are also able to prevent stimulation of casein and DNA synthesis by prolactin in the mammary gland. The present report reports data obtained with tubulozole, a new antimitotic drug. Tubulozole C added to culture medium of isolated rabbit epithelial mammary cells strongly inhibited their multiplication. Simultaneously, at a concentration of 1 microM, it prevented almost completely the induction of beta-casein mRNA. Induced cells were rapidly deinduced by addition of the drug to the medium. A similar inhibition was observed when the induction was obtained with prolactin alone or with its two stimulators insulin and glucocorticoids. Tubulozole T, an isomer of tubulozole C which is known to be ineffective in disrupting microtubules, did not alter prolactin actions. These data and those obtained with other tubulin-binding drugs strongly suggest that the integrity of microtubules is required for prolactin to deliver its message to the mammary cell.     

Pyridoxal 5'phosphate binding site of Escherichia coli beta cystathionase and cystathionine gamma synthase comparison of their sequences

The phosphopyridoxyl peptides of beta cystathionase and cystathionine gamma synthase of Escherichia Coli were identified after reduction, carboxymethylation and proteolysis of the holoenzymes. Their comparison with those obtained from rat liver gamma cystathionase (Fearon, C.W., Rodkey, J.A. and Abeles R.H. 1982. Biochemistry 21 3790-3794.) showed a high degree of homology between the three PLP binding sites with the presence of the tripeptide sequence: Thr-Lys(Pxy)-Tyr in their structure. This homology suggests that these enzymes of methionine metabolism have probably the same origin.     

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.     

Gene amplification at a locus encoding a putative Na+/H+ antiporter confers sodium and lithium tolerance in fission yeast.

We have identified a new locus, sodium 2 (sod2) based on selection for increased LiCl tolerance in fission yeast, Schizosaccharomyces pombe. Tolerant strains have enhanced pH-dependent Na+ export capacity and sodium transport experiments suggest that the gene encodes an Na+/H+ antiport. The predicted sod2 gene product can be placed in the broad class of transporters which possess 12 hydrophobic transmembrane domains. The protein shows some sequence similarity to the human and bacterial Na+/H+ antiporters. Overexpression of sod2 increased Na+ export capacity and conferred sodium tolerance. Osmotolerance was not affected and sod2 cells were unaffected for growth in K+. In a sod2 disruption strain cells were incapable of exporting sodium. They were hypersensitive to Na+ or Li+ and could not grow under conditions that approximate pH7. The sod2 gene amplification could be selected stepwise and the degree of such amplification correlated with the level of Na+ or Li+ tolerance.     

Brain somatostatin receptors in spontaneously hypertensive rats: an autoradiographic study.

The apparent densities of brain somatostatin (SRIF) receptor sites were compared in adult spontaneously hypertensive rats (SH) and their normotensive genetic counterparts (Wistar-Kyoto; WKY) using quantitative receptor autoradiography. Globally, the distribution of brain [125I][Tyr0, D-Trp8]SRIF14 binding sites was very similar in both strains. However, apparent densities of specific labeling were either higher (subfornical organ, 3.2 x; locus coeruleus, 1.9 x; lateroanterior hypothalamic nucleus, 1.3 x) or lower (basolateral amygdaloid nucleus, 0.8 x; spinal trigeminal sensory nucleus, 0.6 x) in SH than WKY rats in areas especially relevant to CNS cardiovascular integration. This provides further evidence for the possible involvement of brain SRIF neurons in cardiovascular regulation.     

Kinetic studies of aminoglycoside acetyltransferase and phosphotransferase from Staphylococcus aureus RPAL. Relationship between the two activities

In the Staphylococcus aureus strain harbouring the plasmid RPAL, the resistance to aminoglycoside antibiotics results from two inactivating reactions catalyzed by a 6'-N-aminoglycoside acetyltransferase and a 2"-O-amino-glycoside phosphotransferase. These enzymes are copurified with a constant ratio between the two activities, the purification process consisting in affinity chromatography, native electrophoresis and gel exclusion chromatography. The kinetic mechanisms of each activity have been determined from studies of initial velocities, as well as product and dead-end inhibitions. Both activities follow a random rapid equilibrium mechanism. The substrates and cofactors of one reaction have been tested as effectors of the other reaction. No interaction between the two activities has been observed. However, the GTP cofactor of phosphotransferase protects, at weak concentrations, the acetyltransferase against thermal inactivation, which suggests that the two activities may be associated.     

Effect of mifepristone (RU 486) on concentrations of prostaglandin E-2 binding sites in the rat endometrium

Progesterone implants in ovariectomized rats increased endometrial concentrations of PGE-2 receptors. The increase was completely inhibited by simultaneous daily injection (7.5 mg/kg) of mifepristone (RU 486). A single injection of mifepristone on the morning of Day 1 of pseudopregnancy (day of oestrus) decreased the amount of PGE-2 receptors found in the endometrium on Day 5 by 64%. This inhibitory effect probably resulted from the antiprogesterone activity of this compound since it was not counteracted by simultaneous treatment with dexamethasone, shown to reverse totally the antiglucocorticoid action of mifepristone. The inhibition by mifepristone lasted only for 1 day; endometrial PGE-2 receptor levels on Day 6 of pseudopregnancy returned to the high values present in controls. Under these conditions, administration of the mifepristone did not affect the plasma oestradiol and progesterone concentrations during the 1st week of pseudopregnancy. The administration of mifepristone on Days 2 and 3 of pseudopregnancy kept the endometrial PGE-2 receptor levels low, even by 4 days after the end of treatment. We therefore concluded that, in the rat, progesterone priming leading to uterine receptivity can be delayed, at least by 1 day. In contrast, interruption of the progesterone action for a longer period later during the early pseudopregnant period resulted in an altered subsequent evolution of the endometrium, in terms of acquisition of the PGE-2 binding sites.     

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.