Genomic organization and chromosomal localization of three members of the UDP-N-acetylgalactosamine: polypeptide N- acetylgalactosaminyltransferase family

A homologous family of UDP- N -acetylgalactosamine: polypeptide N - acetylgalactosaminyltransferases (GalNAc-transferases) initiate O- glycosylation. These transferases share overall amino acid sequence similarities of approximately 45-50%, but segments with higher similarities of approximately 80% are found in the putative catalytic domain. Here we have characterized the genomic organization of the coding regions of three GalNAc-transferase genes and determined their chromosomal localization. The coding regions of GALNT1 , -T2 , and -T3 were found to span 11, 16, and 10 exons, respectively. Several intron/exon boundaries were conserved within the three genes. One conserved boundary was shared in a homologous C. elegans GalNAc- transferase gene. Fluorescence in situ hybridization showed that GALNT1 , -T2 , and -T3 are localized at chromosomes 18q12-q21, 1q41-q42, and 2q24-q31, respectively. These results suggest that the members of the polypeptide GalNAc-transferase family diverged early in evolution from a common ancestral gene through gene duplication.      

Dissociation between rate of hepatic lipoprotein secretion and hepatocyte microtubule content

The fact that colchicines inhibits hepatic secretion of very low density lipoprotein (VLDL) particles has been interpreted to mean that microtubules are involved in hepatic VLDL secretion. To further define this relationship, we have attempted to see if changes in hepatic VLDL secretion are associated with changes in hepatocyte microtubule or tubulin content. Accordingly, hepatic secretion of VLDL was increased in rats, and the hepatocyte content of both microtubules (using quantitative morphometric methods) and tubulin (using a time-decay colchicine binding assay) was determined. In acute experiments, VLDL secretion was increased by perfusion of isolated rat livers for 2 h with varying concentrations of free fatty acids (FFA). Results indicate that hepatic VLDL triglyceride (TG) secretion at perfusate FFA levels of 0.7 μEq/ml is threefold greater (P < 0.01) than when livers are perfused without added FFA. However, no differences are observed in the content of microtubules in these livers: specifically, microtubules occupy 0.029 percent of hepatocyte cytoplasm in livers perfused without FFA and 0.030 percent of cytoplasm in livers perfused with FFA. In chronic experiments, rats were fed for 1 wk with either standard rat chow or a hyperlipidemic (sucrose/lard) diet. With the experimental diet, plasma triglyceride levels increase threefold over controls, and liver VLDL-TG production, as determined by [(3)H]glycerol turnover studies, is 55 percent greater (P < 0.01) than controls. However, microtubules occupy 0.027 percent of the cytoplasm of hepatocyte cytoplasm whether rats are on standard or hyperlipidemic diets. Furthermore, the tubulin content of isolated hepatocytes does change, and represents 1 percent of hepatocyte soluble protein, irrespective of diet. These results suggest that increases in hepatic VLDL secretion can occur without any demonstrable change in hepatocyte assembled microtubule or tubulin content, and raise questions as to the role played by microtubules in hepatic VLDL secretion.     

Manganese catalyzed auto-oxidation of dopamine to 6-hydroxydopamine in vitro

Manganese (Mn) is an essential trace element which, upon excessive exposure, produces a neurological syndrome similar to chronic Parkinson's disease in animals and humans. Previous work demonstrated that Mn was more potent than other transition metals in stimulating dopamine (DA) auto-oxidation. In these experiments, DA was incubated under physiological conditions in the presence and absence of Mn for up to 60 min. 6-Hydroxydopamine (6-OHDA) was produced in the presence of Mn, while the incubation mixture without Mn showed no DA oxidation. 6-Hydroxydopamine is a neurotoxicant which exerts its effects by destroying DA nerve terminals in the CNS. Therefore, this work suggests that the Mn catalyzed increase in DA auto-oxidation could be linked mechanistically to the appearance of Mn-induced neurotoxic effects.     

HIV Controller CD4+ T Cells Respond to Minimal Amounts of Gag Antigen Due to High TCR Avidity

HIV controllers are rare individuals who spontaneously control HIV replication in the absence of antiretroviral treatment. Emerging evidence indicates that HIV control is mediated through very active cellular immune responses, though how such responses can persist over time without immune exhaustion is not yet understood. To investigate the nature of memory CD4+ T cells responsible for long-term anti-HIV responses, we characterized the growth kinetics, Vβ repertoire, and avidity for antigen of patient-derived primary CD4+ T cell lines. Specific cell lines were obtained at a high rate for both HIV controllers (16/17) and efficiently treated patients (19/20) in response to the immunodominant Gag293 peptide. However, lines from controllers showed faster growth kinetics than those of treated patients. After normalizing for growth rates, IFN-γ responses directed against the immunodominant Gag293 peptide showed higher functional avidity in HIV controllers, indicating differentiation into highly efficient effector cells. In contrast, responses to Gag161, Gag263, or CMV peptides did not differ between groups. Gag293-specific CD4+ T cells were characterized by a diverse Vβ repertoire, suggesting that multiple clones contributed to the high avidity CD4+ T cell population in controllers. The high functional avidity of the Gag293-specific response could be explained by a high avidity interaction between the TCR and the peptide-MHC complex, as demonstrated by MHC class II tetramer binding. Thus, HIV controllers harbor a pool of memory CD4+ T cells with the intrinsic ability to recognize minimal amounts of Gag antigen, which may explain how they maintain an active antiviral response in the face of very low viremia.     

CD4 dynamics over a 15 year-period among HIV controllers enrolled in the ANRS French observatory

Background

There are few large published studies of HIV controllers with long-term undetectable viral load (VL). We describe the characteristics and outcomes of 81 French HIV controllers.

Methods and Results

HIV controllers were defined as asymptomatic, antiretroviral-naïve persons infected ≥10 years previously, with HIV-RNA <400 copies/mL in >90% of plasma samples. All available CD4 and VL values were collected at enrolment. Mixed-effect linear models were used to analyze CD4 cell count slopes since diagnosis. HIV controllers represented 0.31% of all patients managed in French hospitals. Patients infected through intravenous drug use were overrepresented (31%) and homosexual men were underrepresented (26% of men) relative to the ANRS SEROCO cohort of subjects diagnosed during the same period. HIV controllers whose VL values were always below the detection limit of the assays were compared with those who had rare “blips” (<50% of VL values above the detection limit) or frequent blips (>50% of VL values above the detection limit). Estimated CD4 cell counts at HIV diagnosis were similar in the three groups. CD4 cell counts remained stable after HIV diagnosis in the “no blip” group, while they fell significantly in the two other groups (−0.26√CD4 and −0.28√CD4/mm³/year in the rare and frequent blip groups, respectively). No clinical, immunological or virological progression was observed in the no blip group, while 3 immunological and/or virological events and 4 cancers were observed in the blip subgroups.

Conclusions

Viral blips in HIV controllers are associated with a significant decline in CD4 T cells and may be associated with an increased risk of pathological events, possibly owing to chronic inflammation/immune activation.     

Visualization of elusive structures using intracardiac echocardiography: Insights from electrophysiology

Echocardiography has the ability to noninvasively explore hemodynamic variables during pharmacologic or exercise stress test in patients with heart failure. In this review, we detail some important potential applications of stress echocardiography in patients with heart failure. In patients with coronary artery disease and chronic LV dysfunction, dobutamine stress echocardiography is able to distinguish between viable and fibrotic tissue to make adequate clinical decisions. Exercise testing, in combination with echocardiographic monitoring, is a method of obtaining accurate information in the assessment of functional capacity and prognosis. Functional mitral regurgitation is a common finding in patients with dilated and ischaemic cardiomyopathy and stress echocardiography in the form of exercise or pharmacologic protocols can be useful to evaluate the behaviour of mitral regurgitation. It is clinical useful to search the presence of contractile reserve in non ischemic dilated cardiomyopathy such as to screen or monitor the presence of latent myocardial dysfunction in patients who had exposure to cardiotoxic agents. Moreover, in patients with suspected diastolic heart failure and normal systolic function, exercise echocardiography could be able to demonstrate the existence of such dysfunction and determine that it is sufficient to limit exercise tolerance. Finally, in the aortic stenosis dobutamine echocardiography can distinguish severe from non-severe stenosis in patients with low transvalvular gradients and depressed left ventricular function.