Absence of post-phosphoryl modification in dystroglycanopathy mouse models and wild-type tissues expressing non-laminin binding form of α-dystroglycan

α-Dystroglycan (α-DG) is a membrane-associated glycoprotein that interacts with several extracellular matrix proteins, including laminin and agrin. Aberrant glycosylation of α-DG disrupts its interaction with ligands and causes a certain type of muscular dystrophy commonly referred to as dystroglycanopathy. It has been reported that a unique O-mannosyl tetrasaccharide (Neu5Ac-α2,3-Gal-β1,4-GlcNAc-β1,2-Man) and a phosphodiester-linked modification on O-mannose play important roles in the laminin binding activity of α-DG. In this study, we use several dystroglycanopathy mouse models to demonstrate that, in addition to fukutin and LARGE, FKRP (fukutin-related protein) is also involved in the post-phosphoryl modification of O-mannose on α-DG. Furthermore, we have found that the glycosylation status of α-DG in lung and testis is minimally affected by defects in fukutin, LARGE, or FKRP. α-DG prepared from wild-type lung- or testis-derived cells lacks the post-phosphoryl moiety and shows little laminin-binding activity. These results show that FKRP is involved in post-phosphoryl modification rather than in O-mannosyl tetrasaccharide synthesis. Our data also demonstrate that post-phosphoryl modification not only plays critical roles in the pathogenesis of dystroglycanopathy but also is a key determinant of α-DG functional expression as a laminin receptor in normal tissues and cells.     

Hypoxia-reoxygenation-induced mitochondrial damage and apoptosis in human endothelial cells are inhibited by vitamin C

Hypoxia and hypoxia-reperfusion (H-R) play important roles in human pathophysiology because they occur in clinical conditions such as circulatory shock, myocardial ischemia, stroke, and organ transplantation. Reintroduction of oxygen to hypoxic cells during reperfusion causes an increase in generation of reactive oxygen species (ROS), which can alter cell signaling, and cause damage to lipids, proteins, and DNA leading to ischemia-reperfusion injury. Since vitamin C is a potent antioxidant and quenches ROS, we investigated the role of intracellular ascorbic acid (iAA) in endothelial cells undergoing hypoxia-reperfusion. Intracellular AA protected human endothelial cells from H-R-induced apoptosis. Intracellular AA also prevents loss of mitochondrial membrane potential and the release of cytochrome C and activation of caspase-9 and caspase-3 during H-R. Additionally, inhibition of caspase-9 activation prevented H-R-induced apoptosis, suggesting a mitochondrial site of initiation of apoptosis. We found that H-R induced an increase in ROS in endothelial cells that was abrogated in the presence of iAA. Our results indicate that vitamin C prevents hypoxia and H-R-induced damage to human endothelium.     

Bacteroides fragilis interferes with iNOS activity and leads to pore formation in macrophage surface

Bacteroides fragilis is the anaerobe most commonly recoverable from clinical specimens. The wide genetic diversity of this bacterium related with virulence potential is still an open question. In this study, we analyzed the morphological aspects and microbicide action of M? during interactions with B. fragilis. A filamentous cytoplasm content release and a different actin organization colocalized with iNOS were detected. It was also possible to observe the reduction of NO production in the same conditions. The scanning electron microscopy showed the formation of pore-like structures in the surface of macrophages in the bacterial presence and by transmission electron microscopy we could observe the extrusion of cytoplasm contents as well as the condensation of chromatin in the nucleus periphery. These data suggest the existence of an inhibitory mechanism developed by B. fragilis strains for one of the macrophage microbicide actions.     

Muscular dystrophy and neuronal migration disorder caused by mutations in a glycosyltransferase, POMGnT1.

Muscle-eye-brain disease (MEB) is an autosomal recessive disorder characterized by congenital muscular dystrophy, ocular abnormalities, and lissencephaly. Mammalian O-mannosyl glycosylation is a rare type of protein modification that is observed in a limited number of glycoproteins of brain, nerve, and skeletal muscle. Here we isolated a human cDNA for protein O-mannose beta-1,2-N-acetylglucosaminyltransferase (POMGnT1), which participates in O-mannosyl glycan synthesis. We also identified six independent mutations of the POMGnT1 gene in six patients with MEB. Expression of most frequent mutation revealed a great loss of the enzymatic activity. These findings suggest that interference in O-mannosyl glycosylation is a new pathomechanism for muscular dystrophy as well as neuronal migration disorder.     

Host-soluble galectin-1 promotes HIV-1 replication through a direct interaction with glycans of viral gp120 and host CD4

Sexual transmission of HIV-1 requires virus adsorption to a target cell, typically a CD4(+) T lymphocyte residing in the lamina propria, beneath the epithelium. To escape the mucosal clearance system and reach its target cells, HIV-1 has evolved strategies to circumvent deleterious host factors. Galectin-1, a soluble lectin found in the underlayers of the epithelium, increases HIV-1 infectivity by accelerating its binding to susceptible cells. By comparison, galectin-3, a family member expressed by epithelial cells and part of the mucosal clearance system, does not perform similarly. We show here that galectin-1 directly binds to HIV-1 in a β-galactoside-dependent fashion through recognition of clusters of N-linked glycans on the viral envelope gp120. Unexpectedly, this preferential binding of galectin-1 does not rely on the primary sequence of any particular glycans. Instead, glycan clustering arising from the tertiary structure of gp120 hinders its binding by galectin-3. Increased polyvalency of a specific ligand epitope is a common strategy for glycans to increase their avidity for lectins. In this peculiar occurrence, glycan clustering is instead exploited to prevent binding of gp120 by galectin-3, which would lead to a biological dead-end for the virus. Our data also suggest that galectin-1 binds preferentially to CD4, the host receptor for gp120. Together, these results suggest that HIV-1 exploits galectin-1 to enhance gp120-CD4 interactions, thereby promoting virus attachment and infection events. Since viral adhesion is a rate-limiting step for HIV-1 entry, modulation of the gp120 interaction with galectin-1 could thus represent a novel approach for the prevention of HIV-1 transmission.     

Low relative abundances of the mucolytic bacterium Akkermansia muciniphila and Bifidobacterium spp. in feces of children with autism

Gastrointestinal disturbance is frequently reported for individuals with autism. We used quantitative real-time PCR analysis to quantify fecal bacteria that could influence gastrointestinal health in children with and without autism. Lower relative abundances of Bifidobacteria species and the mucolytic bacterium Akkermansia muciniphila were found in children with autism, the latter suggesting mucus barrier changes.     

Rapidly progressive course of Trypanosoma cruzi infection in mice heterozygous for hexamethylene bis-acetamide inducible 1 (Hexim1) gene

Infection with Trypanosoma cruzi causes Chagas disease and results in myocardial inflammation and cardiomyopathy. Downregulated Hexim1 expression, as in Hexim1^+/? mice, reduces cardiac inflammation and fibrosis following ischemic stress. We asked whether reduced expression of Hexim1 would also afford protection against T. cruzi-induced cardiomyopathy. C57BL/6J (wild type – WT) and Hexim1^+/? mice were infected with sub-lethal doses of T. cruzi (Brazil strain), and cardiac function, serologic markers of inflammation and tissue pathology were examined. Infected Hexim1^+/? mice had compromised cardiac function, altered expression of both pro- and anti-inflammatory cytokines, and increased inflammation and fibrosis. Cardiac failure was evidenced by severely diminished heart rate, compensatory increase in respiratory rate, and abnormally high left ventricular mass with severe transmural inflammation. Lungs displayed intense peribronchial inflammation and fibrosis extending into the parenchyma. We also observed Smad3-serine²⁰⁸ phosphorylation in hearts and lungs of infected mice, suggesting increased TGF-β signaling pathway activity. This was more pronounced in Hexim1^+/? mice and correlated with increased fibrosis in these tissues. Conspicuous splenomegaly in the Hexim1^+/? mice most likely resulted from the observed extensive white pulp expansion. T. cruzi infection induced colonic dilatation and marked villous atrophy in both the WT and Hexim1^+/? mice but more so in the latter. The profound exacerbation of pathologic findings suggests a protective role for Hexim1 in T. cruzi infection.     

Sexual Behavior and Network Characteristics and Their Association with Bacterial Sexually Transmitted Infections among Black Men Who Have Sex with Men in the United States

Background

Black men who have sex with men (MSM) have a high prevalence of bacterial sexually transmitted infections (STIs), and individual risk behavior does not fully explain the higher prevalence when compared with other MSM. Using the social-ecological framework, we evaluated individual, social and sexual network, and structural factors and their association with prevalent STIs among Black MSM.

Methods

The HIV Prevention Trials Network 061 was a multi-site cohort study designed to determine the feasibility and acceptability of a multi-component intervention for Black MSM in six US cities. Baseline assessments included demographics, risk behavior, and social and sexual network questions collected information about the size, nature and connectedness of their sexual network. Logistic regression was used to estimate the odds of having any prevalent sexually transmitted infection (gonorrhea, chlamydia, or syphilis).

Results

A total of 1,553 Black MSM were enrolled in this study. In multivariate analysis, older age (aOR = 0.57; 95% CI 0.49–0.66, p<0.001) was associated with a lower odds of having a prevalent STI. Compared with reporting one male sexual partner, having 2–3 partners (aOR = 1.74; 95% CI 1.08–2.81, p<0.024) or more than 4 partners (aOR = 2.29; 95% CI 1.43–3.66, p<0.001) was associated with prevalent STIs. Having both Black and non-Black sexual partners (aOR = 0.67; 95% CI 0.45–0.99, p = 0.042) was the only sexual network factor associated with prevalent STIs.

Conclusions

Age and the number and racial composition of sexual partners were associated with prevalent STIs among Black MSM, while other sexual network factors were not. Further studies are needed to evaluate the effects of the individual, network, and structural factors on prevalent STIs among Black MSM to inform combination interventions to reduce STIs among these men.     

PI3-kinase activation by GM-CSF in endothelium is upstream of Jak/Stat pathway: role of alphaGMR

GM-CSF has been identified as a growth factor for endothelial cells. In this study, we investigated the role of PI3-kinase pathway in mediating GM-CSF induced angiogenesis. GM-CSF induced tube formation in human umbilical vein endothelial cells, as examined using Matrigel assay, was inhibited by specific inhibitors of PI3-kinase, wortmannin, and LY294002. The regulatory subunit of PI3-kinase (p85) interacted with alphaGMR via its C-SH2 domain in a GM-CSF-dependent fashion with concomitant phosphorylation of p85 and activation of PI3-kinase pathway. p85 binding site on the alphaGMR was essential to induce GM-CSF receptor-dependent Stat activation. Furthermore, inhibition of PI3-kinase activity also abrogated GM-CSF induced Stat activation. These studies underscore the significance of the GM-CSF mediated PI3-kinase activation and its role in angiogenesis.