Association of homocysteine and methylene tetrahydrofolate reductase (MTHFR C677T) gene polymorphism with coronary artery disease (CAD) in the population of North India

The implications of the methylene tetrahydrofolate reductase (MTHFR) gene and the level of homocysteine in the pathogenesis of coronary artery disease (CAD) have been extensively studied in various ethnic groups. Our aim was to discover the association of MTHFR (C677T) polymorphism and homocysteine level with CAD in north Indian subjects. The study group consisted of 329 angiographically proven CAD patients, and 331 age and sex matched healthy individuals as controls. MTHFR (C677T) gene polymorphism was detected based on the polymerase chain reaction and restriction digestion with HinfI. Total homocysteine plasma concentration was measured using immunoassay. T allele frequency was found to be significantly higher in patients than in the control group. We found significantly elevated levels of mean homocysteine in the patient group when compared to the control group (p = 0.00). Traditional risk factors such as diabetes, hypertension, smoking habits, a positive family history and lipid profiles (triglyceride, total cholesterol, HDL-cholesterol, LDL-cholesterol, VLDL-cholesterol), were found significantly associated through univariate analysis. Furthermore, multivariable logistics regression analysis revealed that CAD is significantly and variably associated with diabetes, hypertension, smoking, triglycerides and HDL-cholesterol. Our findings showed that MTHFR C677T polymorphism and homocysteine levels were associated with coronary artery disease in the selected population.     

Effects of larval mosquitoes (Aedes triseriatus) and stemflow on microbial community dynamics in container habitats.

The dynamics of the microbial food sources for Aedes triseriatus larvae in microcosms were found to be strongly influenced by larval presence. The total abundance of bacteria in water samples generally increased in response to larvae, including populations of cultivable, facultatively anaerobic bacteria. Additionally, a portion of the community shifted from Pseudomonaceae to Enterobacteriaceae. Bacterial abundance on leaf material was significantly reduced in the presence of actively feeding larvae. Principle-component analysis of whole community fatty acid methyl ester (FAME) profiles showed that larvae changed the microbial community structure in both the water column and the leaf material. Cyclopropyl FAMEs, typically associated with bacteria, were reduced in microcosms containing larvae; however, other bacterial fatty acids showed no consistent response. Long-chain polyunsaturated fatty acids characteristic of microeukaryotes (protozoans and meiofauna) declined in abundance when larvae were present, indicating that larval feeding reduced the densities of these microorganisms. However, presumed fungal lipid markers either increased or were unchanged in response to larvae. Larval presence also affected microbial nitrogen metabolism through modification of the physiochemical conditions or by grazing on populations of bacteria involved in nitrification-denitrification. Stemflow primarily influenced inorganic ion and organic compound concentrations in the microcosms and had less-pronounced effects on microbial community parameters than did larval presence. Stemflow treatments diluted concentrations of all inorganic ions (chloride, sulfate, and ammonium) and organic compounds (total dissolved organic carbon, soluble carbohydrates, and total protein) measured, with the exceptions of nitrite and nitrate. Stemflow addition did not measurably affect larval biomass in the microcosms but did enhance development rates and early emergence patterns of adults.     

Affinity purification of fibrinogen using a ligand from a peptide library.

An affinity resin containing the peptide ligand Phe-Leu-Leu-Val-Pro-Leu (FLLVPL) has been developed for the purification of fibrinogen. The ligand was identified by screening a solid-phase combinatorial peptide library using an immunostaining technique. The specific binding of fibrinogen to the ligand has been characterized by isothermal calorimetry and adsorption isotherms and is dominated by both hydrophobic interactions and ionic interactions with the N-terminal free amino group. The effective association constant of fibrinogen was substantially higher when the peptide was immobilized on the resin than in solution; moreover, it increased with increasing peptide density, suggesting a cooperative binding effect. A low ionic strength buffer at pH 4 was used successfully to elute adsorbed fibrinogen from the column with high purity, retention of factor XIII crosslinking activity, and minimal, if any, loss of biological function. This general approach to ligand selection and characterization can be used to develop peptide ligands for the affinity purification of diverse proteins on a large scale.     

The effects of human immunodeficiency virus recombinant envelope glycoprotein on immune cell functions in vitro

The effect of human immunodeficiency virus (HIV) recombinant envelope glycoprotein 120 (rgp 120) on the functions of peripheral blood mononuclear cells (PBMC) in vitro was investigated. The results demonstrate that rgp 120 used at concentrations less than 1 microgram/ml has no significant effects on PBMC function in vitro. However, the addition of 1-20 micrograms/ml of rgp 120 significantly inhibits the tetanus toxoid-induced PBMC proliferative response in a dose-related manner as determined by [3H]thymidine incorporation. The data also show that rgp 120 (5 micrograms/ml) causes up to 70% reduction in the number of immunoglobulin G-secreting cells in pokeweed mitogen-stimulated PBMC cultures. Further, rgp 120 can selectively interact with the CD4a epitope of the CD4 helper cell membrane receptor. These results indicate that microgram per milliliter levels of rgp 120 can depress certain immune functions in vitro. The significance of these findings to the pathogenesis of immunodeficiency in HIV infection remains to be determined.     

Homocysteine induces programmed cell death in human vascular endothelial cells through activation of the unfolded protein response.

Severe hyperhomocysteinemia is associated with endothelial cell injury that may contribute to an increased incidence of thromboembolic disease. In this study, homocysteine induced programmed cell death in human umbilical vein endothelial cells as measured by TdT-mediated dUTP nick end labeling assay, DNA ladder formation, induction of caspase 3-like activity, and cleavage of procaspase 3. Homocysteine-induced cell death was specific to homocysteine, was not mediated by oxidative stress, and was mimicked by inducers of the unfolded protein response (UPR), a signal transduction pathway activated by the accumulation of unfolded proteins in the lumen of the endoplasmic reticulum. Dominant negative forms of the endoplasmic reticulum-resident protein kinases IRE1alpha and -beta, which function as signal transducers of the UPR, prevented the activation of glucose-regulated protein 78/immunoglobulin chain-binding protein and C/EBP homologous protein/growth arrest and DNA damage-inducible protein 153 in response to homocysteine. Furthermore, overexpression of the point mutants of IRE1 with defective RNase more effectively suppressed the cell death than the kinase-defective mutant. These results indicate that homocysteine induces apoptosis in human umbilical vein endothelial cells by activation of the UPR and is signaled through IRE1. The studies implicate that the UPR may cause endothelial cell injury associated with severe hyperhomocysteinemia.     

Identification of Mycobacterium tuberculosis clinical isolates with altered phagocytosis by human macrophages due to a truncated lipoarabinomannan

Phenotypically distinct clinical isolates of Mycobacterium tuberculosis are capable of altering the balance that exists between the pathogen and human host and ultimately the outcome of infection. This study has identified two M. tuberculosis strains (i.e. HN885 and HN1554) among a bank of clinical isolates with a striking defect in phagocytosis by primary human macrophages when compared with strain Erdman, a commonly used laboratory strain for studies of pathogenesis. Mass spectrometry in conjunction with NMR studies unequivocally confirmed that both HN885 and HN1554 contain truncated and more branched forms of mannose-capped lipoarabinomannan (ManLAM) with a marked reduction of their linear arabinan (corresponding mainly to the inner Araf-alpha(1-->5)-Araf unit) and mannan (with fewer 6-Manp residues and more substitutions in the linear Manp-alpha(1-->6)-Manp unit) domains. The truncation in the ManLAM molecules produced by strains HN885 and HN1554 led to a significant reduction in their surface availability. In addition, there was a marked reduction of higher order phosphatidyl-myo-inositol mannosides and the presence of dimycocerosates, triglycerides, and phenolic glycolipid in their cell envelope. Less exposed ManLAM and reduced higher order phosphatidyl-myo-inositol mannosides in strains HN885 and HN1554 resulted in their low association with the macrophage mannose receptor. Despite reduced phagocytosis, ingested bacilli replicated at a fast rate following serum opsonization. Our results provide evidence that the clinical spectrum of tuberculosis may be dictated not only by the host but also by the amounts and ratios of surface exposed mycobacterial adherence factors defined by strain genotype.     

Mechanochemical regulation of oscillatory follicle cell dynamics in the developing Drosophila egg chamber

During tissue elongation from stage 9 to stage 10 in Drosophila oogenesis, the egg chamber increases in length by ∼1.7-fold while increasing in volume by eightfold. During these stages, spontaneous oscillations in the contraction of cell basal surfaces develop in a subset of follicle cells. This patterned activity is required for elongation of the egg chamber; however, the mechanisms generating the spatiotemporal pattern have been unclear. Here we use a combination of quantitative modeling and experimental perturbation to show that mechanochemical interactions are sufficient to generate oscillations of myosin contractile activity in the observed spatiotemporal pattern. We propose that follicle cells in the epithelial layer contract against pressure in the expanding egg chamber. As tension in the epithelial layer increases, Rho kinase signaling activates myosin assembly and contraction. The activation process is cooperative, leading to a limit cycle in the myosin dynamics. Our model produces asynchronous oscillations in follicle cell area and myosin content, consistent with experimental observations. In addition, we test the prediction that removal of the basal lamina will increase the average oscillation period. The model demonstrates that in principle, mechanochemical interactions are sufficient to drive patterning and morphogenesis, independent of patterned gene expression.     

ARFGAP1 Is Dynamically Associated with Lipid Droplets in Hepatocytes

The ARF GTPase Activating Protein 1 (ARFGAP1) associates mainly with the cytosolic side of Golgi cisternal membranes where it participates in the formation of both COPI and clathrin-coated vesicles. In this study, we show that ARFGAP1 associates transiently with lipid droplets upon addition of oleate in cultured cells. Also, that addition of cyclic AMP shifts ARFGAP1 from lipid droplets to the Golgi apparatus and that overexpression and knockdown of ARFGAP1 affect lipid droplet formation. Examination of human liver tissue reveals that ARFGAP1 is found associated with lipid droplets at steady state in some but not all hepatocytes.     

Interaction of the disintegrin and cysteine-rich domains of ADAM12 with integrin alpha7beta1

We describe a novel interaction between the disintegrin and cysteine-rich (DC) domains of ADAM12 and the integrin alpha7beta1. Integrin alpha7beta1 extracted from human embryonic kidney 293 cells transfected with alpha7 cDNA was retained on an affinity column containing immobilized DC domain of ADAM12. 293 cells stably transfected with alpha7 cDNA adhered to DC-coated wells, and this adhesion was partially inhibited by 6A11 integrin alpha7 function-blocking antibody. The X1 and the X2 extracellular splice variants of integrin alpha7 supported equally well adhesion to the DC protein. Integrin alpha7beta1-mediated cell adhesion to DC had different requirements for Mn2+ than adhesion to laminin. Furthermore, integrin alpha7beta1-mediated cell adhesion to laminin, but not to DC, resulted in efficient cell spreading and phosphorylation of focal adhesion kinase (FAK) at Tyr397. We also show that adhesion of L6 myoblasts to DC is mediated in part by the endogenous integrin alpha7beta1 expressed in these cells. Since integrin alpha7 plays an important role in muscle cell growth, stability, and survival, and since ADAM12 has been implicated in muscle development and regeneration, we postulate that the interaction between ADAM12 and integrin alpha7beta1 may be relevant to muscle development, function, and disease. We also conclude that laminin and the DC domain of ADAM12 represent two functional ligands for integrin alpha7beta1, and adhesion to each of these two ligands via integrin alpha7beta1 triggers different cellular responses.     

Acetaminophen: antipyretic or hypothermic in mice? In either case, PGHS-1b (COX-3) is irrelevant

Acetaminophen (AC) reduces the core temperatures (T(c)) of febrile and non-febrile mice alike. Evidence has been adduced that the selectively AC-sensitive PGHS isoform, PGHS-1b (COX-3), mediates these effects. PGHS-1b, however, has no catalytic potency in mice. To resolve this contradiction, AC was injected intravenously (i.v.) into conscious PGHS-1 gene-sufficient (wild-type (WT)) and -deficient (PGHS-1(-/-)) mice 60 min before or after pyrogen-free saline (PFS) or E. coli LPS (10 microg/kg) i.v. T(c) was monitored continuously; brain and plasma PGE(2) levels were determined hourly. AC at <160 mg/kg did not affect T(c) when given before PFS or LPS; at 160 mg/kg, it caused a approximately 2.5 degrees C T(c) fall in 60 min. LPS given after AC (all doses) induced a approximately 1 degrees C fever, not different from that in AC-untreated mice. But this rise was insufficient to overcome the hypothermia of the 160 mg/kg-treated mice; their T(c) culminated 1 degrees C below baseline. LPS given before AC similarly elevated T(c) approximately 1 degrees C. This rise was reduced to baseline in 30 min by 80 mg AC/kg; T(c) rebounded to its febrile level over the next 30 min. At 160 mg/kg, AC reduced T(c) to 4 degrees C below baseline in 60 min, where it remained until the end of the experiment. WT and PGHS-1(-/-) mice responded similarly to all the treatments. The basal brain and plasma PGE(2) levels of PFS mice and the elevated plasma levels of LPS mice were unchanged by AC at 160 mg/kg; but the latter's brain levels were reduced at 1h, then recovered. Thus, AC could exert an anti-PGHS-2 effect when this enzyme is upregulated in the brain of febrile mice. The hypothermia it induces in non-febrile mice, therefore, is due to another mechanism. PGHS-1b is not involved in either case.