Studies on toll-like receptor stimuli responsiveness in HIV-1 and HIV-2 infections

Background: HIV-1 and HIV-2 are two related viruses with distinct clinical outcomes, where HIV-1 is more pathogenic and transmissible than HIV-2. The pathogenesis of both infections is influenced by the dysregulation and deterioration of the adaptive immune system. However, their effects on the responsiveness of innate immunity are less well known. Here, we report on toll-like receptor (TLR) stimuli responsiveness in HIV-1 or HIV-2 infections. Methods: Whole blood from 235 individuals living in Guinea-Bissau who were uninfected, infected with HIV-1, infected with HIV-2, and/or infected with HTLV-I, was stimulated with TLR7/8 and TLR9 agonists, R-848 and unmethylated CpG DNA. After TLR7/8 and TLR9 stimuli, the expression levels of IL-12 and IFN-α were related to gender, age, infection status, CD4⁺ T cell counts, and plasma viral load. Results: Defective TLR9 responsiveness was observed in the advanced disease stage, along with CD4⁺ T cell loss in both HIV-1 and HIV-2 infections. Moreover, TLR7/8 responsiveness was reduced in HIV-1 infected individuals compared with uninfected controls. Conclusions: Innate immunity responsiveness can be monitored by whole blood stimulation. Both advanced HIV-1 and HIV-2 infections may cause innate immunity dysregulation.     

Enhanced dopaminergic differentiation of human neural stem cells by synergistic effect of Bcl-xL and reduced oxygen tension

Neural stem cells constitute a promising source of cells for transplantation in Parkinson's disease, but a protocol for controlled dopaminergic differentiation is not yet available. Here we investigated the effect of the anti-apoptotic protein Bcl-x_L and oxygen tension on dopaminergic differentiation and survival of a human ventral mesencephalic stem cell line (hVM1). hVM1 cells and a Bcl-x_L over-expressing subline (hVMbcl-x_L) were differentiated by sequential treatment with fibroblast growth factor-8, forskolin, sonic hedgehog, and glial cell line-derived neurotrophic factor. After 10 days at 20% oxygen, hVMbcl-x_L cultures contained proportionally more tyrosine hydroxylase(TH)-positive cells than hVM1 control cultures. This difference was significantly potentiated from 11 ± 0.8% to 17.2 ± 0.2% of total cells when the oxygen tension was lowered to 3%. Immunocytochemistry and Q-PCR-analysis revealed expression of several dopaminergic markers besides of TH just as dopamine was detected in the culture medium by HPLC analysis. Although Bcl-x_L-over-expression reduced cell death in the cultures, it did not alter the relative content of GABAergic, neurons, while the content of astroglial cells was reduced in hVMbcl-x_L cell cultures compared with control. We conclude that Bcl-x_L and lowered oxygen tension act in concert to enhance dopaminergic differentiation and survival of human neural stem cells.     

Designed Human Serum Hyaluronidase 1 Variant, HYAL1��L, Exhibits Activity up to pH 5.9

Hyaluronidases from diverse species and sources have different pH optima. Distinct mechanisms with regard to dynamic structural changes, which control hyaluronidase activity at varying pH, are unknown. Human serum hyaluronidase 1 (HYAL1) is active solely below pH 5.1. Here we report the design of a HYAL1 variant that degrades hyaluronan up to pH 5.9. Besides highly conserved residues in close proximity of the active site of most hyaluronidases, we identified a bulky loop formation located at the end of the substrate binding crevice of HYAL1 to be crucial for substrate hydrolysis. The stretch between cysteine residues 207 and 221, which normally contains 13 amino acids, could be replaced by a tetrapeptide sequence of alternating glycine serine residues, thereby yielding an active enzyme with an extended binding cleft. This variant exhibited hyaluronan degradation at elevated pH. This is indicative for appropriate substrate binding and proper positioning being decisively affected by sites far off from the active center.Hyaluronan (HA),³ a linear polysaccharide found in the extracellular matrix of most tissues and body fluids of vertebrates, is enzymatically degraded by hyaluronidases (1). Mammalian-type hyaluronidases are grouped into EC 3.2.1.35 (2, 3) or the glycoside hydrolase family 56 (4). Members of this enzyme family hydrolyze the 1,4-β-glycosidic linkage between N-acetyl-d-glucosamine and d-glucuronate within HA polymers (5). In mammalians, hyaluronidases have been found in testis, liver lysosomes, and serum. They are involved in controlling HA levels and are thus implicated in various diseases related to defects of HA metabolism (6).The crystal structures of hyaluronidase from bee (7), wasp (8), and only recently that of human serum hyaluronidase 1 (HYAL1) (9) have been deciphered. In addition to the N-terminal catalytic domain of the insect enzymes, which resembles a distorted (β/α)₈ barrel, HYAL1 contains yet another domain. HA hydrolysis is achieved by a pair of acidic amino acids via a retaining double displacement mechanism and a substrate-assisted catalysis, in which the carbonyl oxygen of the N-acetyl group of the cleaved HA subunit acts as the catalytic nucleophile (7).Mammalian-type hyaluronidases display different pH optima. HYAL1 (10) and hyaluronidase 2 (HYAL2) (11) exhibit highest activities at acidic conditions, whereas the hyaluronidase found in Xenopus laevis kidney is only active at neutral pH (12). Bee venom hyaluronidase (13), as well as sperm hyaluronidase, PH20 (SPAM1) (14), are capable of degrading HA over a broad pH range. Up to three PH20 isoforms with greatly different pH optima could be found in protein preparations from bovine testis (15). Extensive analysis of hyaluronidase structures did not bring forward any insights as to what residues or regions of the enzymes specify a specific pH optimum.Profiles of pH-dependent activities can be assigned by computing the electrostatic interactions of the enzyme, which are primarily determined by the ionization states of its amino acid side chains. The pK_a values of titratable groups of the enzyme reflect pH-dependent properties such as stability, enzymatic interaction, and substrate interactions (16). Here we present computational and experimental data on the replacement of a loop region located at the end of the substrate binding groove yielding a variant hyaluronidase with an altered pH profile.     

Structural determinants for selective recognition of peptide ligands for endothelin receptor subtypes ETA and ETB

The molecular basis for recognition of peptide ligands endothelin‐1, ‐2 and ‐3 in endothelin receptors is poorly understood. Especially the origin of ligand selectivity for ET_A or ET_B is not clearly resolved. We derived sequence‐structure‐function relationships of peptides and receptors from mutational data and homology modeling. Our major findings are the dissection of peptide ligands into four epitopes and the delineation of four complementary structural portions on receptor side explaining ligand recognition in both endothelin receptor subtypes. In addition, structural determinants for ligand selectivity could be described. As a result, we could improve the selectivity of BQ3020 about 10‐fold by a single amino acid substitution, validating our hypothesis for ligand selectivity caused by different entrances to the receptors' transmembrane binding sites. A narrow tunnel shape in ET_A is restrictive for a selected group of peptide ligands' N‐termini, whereas a broad funnel‐shaped entrance in ET_B accepts a variety of different shapes and properties of ligands. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Hyperbaric oxygen induces apoptosis via a mitochondrial mechanism

During therapeutic hyperbaric oxygenation lymphocytes are exposed to high partial pressures of oxygen. This study aimed to analyze the mechanism of apoptosis induction by hyperbaric oxygen. For intervals of 0.5–4 h Jurkat-T-cells were exposed to ambient air or oxygen atmospheres at 1–3 absolute atmospheres. Apoptosis was analyzed by phosphatidylserine externalization, caspase-3 activation and DNA-fragmentation using flow cytometry. Apoptosis was already induced after 30 min of hyperbaric oxygenation (HBO, P < 0.05). The death receptor Fas was downregulated. Inhibition of caspase-9 but not caspase-8 blocked apoptosis induction by HBO. Hyperbaric oxygen caused a loss of mitochondrial membrane potential and caspase-9 induction. The mitochondrial pro-survival protein Bcl-2 was upregulated, and antagonizing Bcl-2 function potentiated apoptosis induction by HBO. In conclusion, a single exposure to hyperbaric oxygenation induces lymphocyte apoptosis by a mitochondrial and not a Fas-related mechanism. Regulation of Fas and Bcl-2 may be regarded as protective measures of the cell in response to hyperbaric oxygen.     

Serotonin transporter oligomerization documented in RN46A cells and neurons by sensitized acceptor emission FRET and fluorescence lifetime imaging microscopy

The serotonin transporter is a member of the monoamine transporter family that also includes transporters of dopamine and norepinephrine. We have used sensitized acceptor emission fluorescence resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM) to study the oligomerization of SERT in HEK-MSR-239 cells, RN46A cells and in cultured hippocampal neurons. We were able to show identical FRET efficiencies in cell lines as well as in primary cultured hippocampal neurons, demonstrating that the oligomerization is cell type independent. The results obtained with both FRET approaches are very similar and furthermore, in agreement with previous results obtained by donor bleaching FRET microscopy.     

Impaired integrin‐mediated adhesion contributes to reduced barrier properties in VASP‐deficient microvascular endothelium

Recent studies point to a significant role of vasodilator‐stimulated phosphoprotein (VASP) in the maintenance of endothelial barrier functions in vivo and in vitro. Moreover, it has been reported that VASP is required for activation of the small GTPase Rac 1. However, little is known whether VASP is involved in the regulation of cell adhesion molecules that are critical for maintenance of the endothelial barrier. Here we demonstrate that impaired barrier properties in VASP‐deficient (VASP?/?) microvascular myocardial endothelial cells (MyEnd) correlated with both impaired integrin‐mediated adhesion as revealed by laser tweezer trapping and reduced integrin‐dependent cell migration. This was paralleled by reduction of focal adhesions at the cell periphery as well as of β₁‐integrin and VE‐cadherin cytoskeletal anchorage. Incubation of MyEnd VASP wt with RGD peptide to block interaction of integrins with extracellular matrix (ECM) reduced barrier properties and Rac 1 activity in wt endothelial monolayers mimicking the situation in VASP (?/?) cells under resting conditions. Moreover, cAMP‐mediated Rac 1 activation was reduced under conditions of impaired integrin‐mediated adhesion in wt cells and cAMP‐induced increase in VE‐cadherin cytoskeletal anchorage was abolished in VASP (?/?) endothelium. In summary, these data indicate that VASP is required for integrin‐mediated adhesion which stabilizes endothelial barrier properties at least in part by facilitating Rac 1 activation. J. Cell. Physiol. 220: 357–366, 2009. © 2009 Wiley‐Liss, Inc.     

Experimentally reduced hip abductor function during walking: Implications for knee joint loads

Hip and knee functions are intimately connected and reduced hip abductor function might play a role in development of knee osteoarthritis (OA) by increasing the external knee adduction moment during walking. The purpose of this study was to test the hypothesis that reduced function of the gluteus medius (GM) muscle would lead to increased external knee adduction moment during level walking in healthy subjects. Reduced GM muscle function was induced experimentally, by means of intramuscular injections of hypertonic saline that produced an intense short-term muscle pain and reduced muscle function. Isotonic saline injections were used as non-painful control. Fifteen healthy subjects performed walking trials at their self-selected walking speed before and immediately after injections, and again after 20 min of rest, to ensure pain recovery. Standard gait analyses were used to calculate three-dimensional trunk and lower extremity joint kinematics and kinetics. Surface electromyography (EMG) of the glutei, quadriceps, and hamstring muscles were also measured. The peak GM EMG activity had temporal concurrence with peaks in frontal plane moments at both hip and knee joints. The EMG activity in the GM muscle was significantly reduced by pain (?39.6%). All other muscles were unaffected. Peaks in the frontal plane hip and knee joint moments were significantly reduced during pain (?6.4% and ?4.2%, respectively). Lateral trunk lean angles and midstance hip joint adduction and knee joint extension angles were reduced by ?1°. Thus, the gait changes were primarily caused by reduced GM function. Walking with impaired GM muscle function due to pain significantly reduced the external knee adduction moment. This study challenge the notion that reduced GM function due to pain would lead to increased loads at the knee joint during level walking.     

Tryptophan is a marker of human postmortem brain tissue quality

Postmortem human brain tissue is widely used in neuroscience research, but use of tissue originating from different brain bank centers is considered inaccurate because of possible heterogeneity in sample quality. There is thus a need for well-characterized markers to assess the quality of postmortem brain tissue. Toward this aim, we determined tryptophan (TRP) concentrations, phosphofructokinase-1 and glutamate decarboxylase activities in 119 brain tissue samples. These neurochemical parameters were tested in samples from autopsied individuals, including control and pathological cases provided by 10 different brain bank centers. Parameters were assessed for correlation with agonal state, postmortem interval, age and gender, brain region, preservation and freezing methods, storage conditions and storage time, RNA integrity, and tissue pH value. TRP concentrations were elevated significantly ( p  = 0.045) with increased postmortem interval; which might indicate increased protein degradation. Therefore, TRP concentration might be one useful and convenient marker for estimating the quality of human postmortem brain tissue.