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.     

Albumin evolution in polyploid species of the genusXenopus

Electrophoresis of serum from 21 Xenopus species and subspecies reveals variable numbers of albumin bands. The diploid X. tropicalis has one albumin, while the tetraploid species (laevis, borealis, muelleri, clivii, fraseri, epitropicalis) have two. The octoploid species (amieti, boumbaensis, wittei, vestitus, andrei) have two to three bands, and the dodecaploid X. ruwenzoriensis has three. The molecular weight of the Xenopus albumins varies from 68 kd (in the tropicalis group) to 74 kd. The subspecies of X. laevis possess two albumins of different molecular weights (70 and 74 kd), whereas most species have only 70-kd albumins. Peptide maps have been obtained from albumin electromorphs by limited proteolysis in sodium dodecyl sulfate (SDS) gels, using S. aureus V8 protease. The peptide patterns produced by electromorphs from the same tetraploid Xenopus species generally differ from each other, suggesting that the two albumin genes contain a substantial amount of structural differences. In addition, the peptide maps are diagnostic for most tetraploid species and for some subspecies of X. laevis as well. Proteolysis of albumins from most octoploid and dodecaploid species results in patterns which are very similar to the ones produced by the electromorphs from X. fraseri. The albumins of X. vestitus differ from those of the other octoploid species. X. andrei possesses two fraseri-type and one vestitus-type albumin, which indicates that it probably originated by allopolyploidy.     

Use of restrained molecular dynamics in water to determine three-dimensional protein structure: prediction of the three-dimensional structure of Ecballium elaterium trypsin inhibitor II

Refinement of distance geometry (DG) structures of EETI-II (Heitz et al.: Biochemistry 28:2392-2398, 1989), a member of the squash family trypsin inhibitor, have been carried out by restrained molecular dynamics (RMD) in water. The resulting models show better side chain apolar/polar surface ratio and estimated solvation free energy than structures refined "in vacuo." The consistent lower values of residual NMR constraint violations, apolar/polar surface ratio, and solvation free energy for one of these refined structures allowed prediction of the 3D folding and disulfide connectivity of EETI-II. Except for the few first residues for which no NMR constraints were available, this computer model fully agreed with X-ray structures of CMTI-I (Bode et al.: FEBS Lett. 242:285-292, 1989) and EETI-II complexed with trypsin that appeared after the RMD simulation was completed. Restrained molecular dynamics in water is thus proved to be highly valuable for refinement of DG structures. Also, the successful use of apolar/polar surface ratio and of solvation free energy reinforce the analysis of Novotny et al. (Proteins 4:19-30, 1988) and shows that these criteria are useful indicators of correct versus misfolded models.     

Systemic GDF11 stimulates the secretion of adiponectin and induces a calorie restriction‐like phenotype in aged mice

Aging is a negative regulator of general homeostasis, tissue function, and regeneration. Changes in organismal energy levels and physiology, through systemic manipulations such as calorie restriction and young blood infusion, can regenerate tissue activity and increase lifespan in aged mice. However, whether these two systemic manipulations could be linked has never been investigated. Here, we report that systemic GDF11 triggers a calorie restriction‐like phenotype without affecting appetite or GDF15 levels in the blood, restores the insulin/IGF‐1 signaling pathway, and stimulates adiponectin secretion from white adipose tissue by direct action on adipocytes, while repairing neurogenesis in the aged brain. These findings suggest that GDF11 has a pleiotropic effect on an organismal level and that it could be a linking mechanism of rejuvenation between heterochronic parabiosis and calorie restriction. As such, GDF11 could be considered as an important therapeutic candidate for age‐related neurodegenerative and metabolic disorders.     

Unexpected Negative Effect of Available Water Capacity Detected on Recent Conifer Forest Growth Trends Across Wide Environmental Gradients

Ecosystems - National Forest Inventories (NFIs) perform systematic forest surveys across space and time. They are hence powerful tools to understand climate controls on forest growth at wide...     

Human nongastric H+-K+-ATPase: transport properties of ATP1al1 assembled with different beta-subunits

To investigate whether nongastric H+-K+-ATPases transport Na+ in exchange for K+ and whether different beta-isoforms influence their transport properties, we compared the functional properties of the catalytic subunit of human nongastric H+-K+-ATPase, ATP1al1 (AL1), and of the Na+-K+-ATPase alpha1-subunit (alpha1) expressed in Xenopus oocytes, with different beta-subunits. Our results show that betaHK and beta1-NK can produce functional AL1/beta complexes at the oocyte cell surface that, in contrast to alpha1/beta1 NK and alpha1/betaHK complexes, exhibit a similar apparent K+ affinity. Similar to Na+-K+-ATPase, AL1/beta complexes are able to decrease intracellular Na+ concentrations in Na+-loaded oocytes, and their K+ transport depends on intra- and extracellular Na+ concentrations. Finally, controlled trypsinolysis reveals that beta-isoforms influence the protease sensitivity of AL1 and alpha1 and that AL1/beta complexes, similar to the Na+-K+-ATPase, can undergo distinct K+-Na+- and ouabain-dependent conformational changes. These results provide new evidence that the human nongastric H+-K+-ATPase interacts with and transports Na+ in exchange for K+ and that beta-isoforms have a distinct effect on the overall structural integrity of AL1 but influence its transport properties less than those of the Na+-K+-ATPase alpha-subunit.     

FXYD7 is a brain-specific regulator of Na,K-ATPase alpha 1-beta isozymes

Recently, corticosteroid hormone-induced factor (CHIF) and the gamma-subunit, two members of the FXYD family of small proteins, have been identified as regulators of renal Na,K-ATPase. In this study, we have investigated the tissue distribution and the structural and functional properties of FXYD7, another family member which has not yet been characterized. Expressed exclusively in the brain, FXYD7 is a type I membrane protein bearing N-terminal, post-translationally added modifications on threonine residues, most probably O-glycosylations that are important for protein stabilization. Expressed in Xenopus oocytes, FXYD7 can interact with Na,K-ATPase alpha 1-beta 1, alpha 2-beta 1 and alpha 3-beta 1 but not with alpha-beta 2 isozymes, whereas, in brain, it is only associated with alpha 1-beta isozymes. FXYD7 decreases the apparent K(+) affinity of alpha 1-beta 1 and alpha 2-beta 1, but not of alpha 3-beta1 isozymes. These data suggest that FXYD7 is a novel, tissue- and isoform-specific Na,K-ATPase regulator which could play an important role in neuronal excitability.     

Na self inhibition of human epithelial Na channel: temperature dependence and effect of extracellular proteases

The regulation of the open probability of the epithelial Na(+) channel (ENaC) by the extracellular concentration of Na(+), a phenomenon called "Na(+) self inhibition," has been well described in several natural tight epithelia, but its molecular mechanism is not known. We have studied the kinetics of Na(+) self inhibition on human ENaC expressed in Xenopus oocytes. Rapid removal of amiloride or rapid increase in the extracellular Na(+) concentration from 1 to 100 mM resulted in a peak inward current followed by a decline to a lower quasi-steady-state current. The rate of current decline and the steady-state level were temperature dependent and the current transient could be well explained by a two-state (active-inactive) model with a weakly temperature-dependent (Q(10)act = 1.5) activation rate and a strongly temperature-dependant (Q(10)inact = 8.0) inactivation rate. The steep temperature dependence of the inactivation rate resulted in the paradoxical decrease in the steady-state amiloride-sensitive current at high temperature. Na(+) self inhibition depended only on the extracellular Na(+) concentration but not on the amplitude of the inward current, and it was observed as a decrease of the conductance at the reversal potential for Na(+) as well as a reduction of Na(+) outward current. Self inhibition could be prevented by exposure to extracellular protease, a treatment known to activate ENaC or by treatment with p-CMB. After protease treatment, the amiloride-sensitive current displayed the expected increase with rising temperature. These results indicate that Na(+) self inhibition is an intrinsic property of sodium channels resulting from the expression of the alpha, beta, and gamma subunits of human ENaC in Xenopus oocyte. The extracellular Na(+)-dependent inactivation has a large energy of activation and can be abolished by treatment with extracellular proteases.     

Comparative vaccine studies in HLA-A2.1-transgenic mice reveal a clustered organization of epitopes presented in hepatitis C virus natural infection

A polyepitopic CD8(+)-T-cell response is thought to be critical for control of hepatitis C virus (HCV) infection. Using transgenic mice, we analyzed the immunogenicity and dominance of most known HLA-A2.1 epitopes presented during infection by using vaccines that carry the potential to enter clinical trials: peptides, DNA, and recombinant adenoviruses. The vaccines capacity to induce specific cytotoxic T lymphocytes and interferon gamma-producing cells revealed that immunogenic epitopes are clustered in specific antigens. For two key antigens, flanking regions were shown to greatly enhance the scope of epitope recognition, whereas a DNA-adenovirus prime-boost vaccination strategy augmented epitope immunogenicity, even that of subdominant ones. The present study reveals a clustered organization of HCV immunogenic HLA.A2.1 epitopes and strategies to modulate their dominance.     

Selected contribution: limiting Na(+) transport rate in airway epithelia from alpha-ENaC transgenic mice: a model for pulmonary edema.

The amiloride-sensitive epithelial Na(+) channel (ENaC) is essential for fluid clearance from the airways. An experimental animal model with a reduced expression of ENaC, the alpha-ENaC transgenic rescue mouse, is prone to develop edema under hypoxia exposure. This strongly suggests an involvement of ENaC in the pathogenesis of pulmonary edema. To investigate the pathogenesis of this type of edema, primary cultures of tracheal cells from these mice were studied in vitro. An ~60% reduction in baseline amiloride-sensitive Na(+) transport was observed, but the pharmacological characteristics and physiological regulation of the channel were similar to those observed in cells from wild-type mice. Aprotinin, an inhibitor of serine proteases, blocked 50-60% of the basal transepithelial current, hypoxia induced downregulation of Na(+) transport, and beta-adrenergic stimulation was effective to stimulate Na(+) transport after the hypoxia-induced decrease. When downregulation of ENaC activity (such as observed under hypoxia) is added to a low "constitutive" ENaC expression, the resulting reduced Na(+) transport rate may be insufficient for airway fluid clearance and favor pulmonary edema.