Matrix metalloproteinase-9 in homocysteine-induced intestinal microvascular endothelial paracellular and transcellular permeability

Although elevated levels of homocysteine (Hcy), known as hyperhomocysteinemia (HHcy), is associated with inflammatory bowel disease (IBD), the mechanism of Hcy action is unclear. In the present study, we tested the hypothesis that HHcy activates matrix metalloproteinase-9 (MMP-9), which in turn enhances permeability of human intestinal microvascular endothelial cell (HIMEC) layer by decreasing expression of endothelial junction proteins and increasing caveolae formation. HIMECs were grown in Transwells and treated with 500 μM Hcy in the presence or absence of MMP-9 activity inhibitor. Hcy-induced permeability to FITC-conjugated bovine serum albumin (FITC-BSA) was assessed by measuring fluorescence intensity of solutes in the Transwells' lower chambers. The cell-cell interaction and cell barrier function was estimated by measuring trans-endothelial electrical impedance. Confocal microscopy and flow cytometry were used to study cell junction protein expressions. Hcy-induced changes in transcellular transport of HIMECs were estimated by observing formation of functional caveolae defined as caveolae labeled by cholera toxin and antibody against caveolin-1 and one that have taken up FITC-BSA. Hcy instigated HIMEC monolayer permeability through activation of MMP-9. The increased paracellular permeability was associated with degradation of vascular endothelial cadherin and zona occludin-1 and transcellular permeability through increased caveolae formation in HIMECs. Elevation of Hcy content increases permeability of HIMEC layer affecting both paracellular and transcellular transport pathways, and this increased permeability was alleviated by inhibition of MMP-9 activity. These findings contribute to clarification of mechanisms of IBD development.     

Vitamin E biosynthesis genes in rice: Molecular characterization, expression profiling and comparative phylogenetic analysis

Vitamin E comprises four tocopherols and four tocotrienols, collectively termed tocochromanols that play an essential role as antioxidants in humans, animals and photosynthetic organisms and are also believed to play a role in modulation of signal transduction and gene expression pathways. In rice and Populus genome, we have identified 7 and 11 tocochromanol biosynthesis genes, respectively. A detailed study of domain organization and phylogenetic analysis of these genes in rice, Arabidopsis and other plants has revealed the presence of homologous genes. Expression profiling of rice and Populus genes has been done by full-length cDNA and EST-based analysis. In rice, real-time PCR analysis was done to reveal the light-regulated expression pattern. Microarray-based expression analysis in different rice tissues and developmental stages revealed expression of these genes in almost all plant tissues/organs. Under abiotic stress conditions, expression of gene coding for HPPD enzyme, that regulates pathway flux, was also found to be increased. This information is expected to be helpful for further functional characterization of tocochromanol biosynthesis genes in different plant tissues under diverse growth conditions.     

MicroRNAs as a therapeutic target for cardiovascular diseases

MicroRNAs (miRNAs) are tiny, endogenous, conserved, non-coding RNAs that negatively modulate gene expression by either promoting the degradation of mRNA or down-regulating the protein production by translational repression. They maintain optimal dose of cellular proteins and thus play a crucial role in the regulation of biological functions. Recent discovery of miRNAs in the heart and their differential expressions in pathological conditions provide glimpses of undiscovered regulatory mechanisms underlying cardiovascular diseases. Nearly 50 miRNAs are overexpressed in mouse heart. The implication of several miRNAs in cardiovascular diseases has been well documented such as miRNA-1 in arrhythmia, miRNA-29 in cardiac fibrosis, miRNA-126 in angiogenesis and miRNA-133 in cardiac hypertrophy. Aberrant expression of Dicer (an enzyme required for maturation of all miRNAs) during heart failure indicates its direct involvement in the regulation of cardiac diseases. MiRNAs and Dicer provide a particular layer of network of precise gene regulation in heart and vascular tissues in a spatiotemporal manner suggesting their implications as a powerful intervention tool for therapy. The combined strategy of manipulating miRNAs in stem cells for their target directed differentiation and optimizing the mode of delivery of miRNAs to the desired cells would determine the future potential of miRNAs to treat a disease. This review embodies the recent progress made in microRNomics of cardiovascular diseases and the future of miRNAs as a potential therapeutic target - the putative challenges and the approaches to deal with it.     

Bioconjugates of curcumin display improved protection against glutathione depletion mediated oxidative stress in a dopaminergic neuronal cell line: Implications for Parkinson’s disease

Oxidative stress is implicated in mitochondrial dysfunction associated with neurodegeneration in Parkinson’s disease (PD). Depletion of the cellular antioxidant glutathione (GSH) resulting in oxidative stress is considered as an early event in neurodegeneration. We previously showed that curcumin, a dietary polyphenol from turmeric induced GSH synthesis in experimental models and protected against oxidative stress. Here we tested the effect of three bioconjugates of curcumin (involving diesters of demethylenated piperic acid, valine and glutamic acid) against GSH depletion mediated oxidative stress in dopaminergic neuronal cells and found that the glutamic acid derivative displayed improved neuroprotection compared to curcumin.     

Synthesis and bioevaluation of glycosyl ureas as alpha-glucosidase inhibitors and their effect on mycobacterium

Glycosyl amino esters (2-13) on reaction with different isocyanates resulted in quantitative conversion to glycosyl ureas (14--32). Few of the selected ureas (15-20, 22-28, 30 and 32) on cyclative amidation with DBU/TBAB/4 A MS gave respective dihydropyrimidinones in fair to good yields (33-47). The compounds were screened for alpha-glucosidase inhibitory activity and two (19 and 23) of them showed strong inhibition against rat intestinal alpha-glucosidase. The compounds were also screened against Mycobacterium aurum, however, only one (19) of them exhibited marginal antitubercular activity.     

Synthesis of glycosylated beta-amino hydroxamates as new class of antimalarials

Glycosylated β-amino acids (3–18, 38, 39), obtained by hydrolysis of glycosylated β-amino esters on reaction with hydroxylamine hydrochloride in presence of DIC/DCC afforded glycosyl β-amino hydroxamates (19–34, 40, 41) in fair to good yields. Compounds (19–34, 40, 41) were screened against human malarial parasite Plasmodium falciparum in vitro for their schizontocidal activity. Compounds (19, 24, 26, 28, 40 and 41) exhibited good activity at 2 μg/mL concentrations.     

Monoubiquitination and endocytosis direct gamma-secretase cleavage of activated Notch receptor

Activation of mammalian Notch receptor by its ligands induces TNFalpha-converting enzyme-dependent ectodomain shedding, followed by intramembrane proteolysis due to presenilin (PS)-dependent gamma-secretase activity. Here, we demonstrate that a new modification, a monoubiquitination, as well as clathrin-dependent endocytosis, is required for gamma-secretase processing of a constitutively active Notch derivative, DeltaE, which mimics the TNFalpha-converting enzyme-processing product. PS interacts with this modified form of DeltaE, DeltaEu. We identified the lysine residue targeted by the monoubiquitination event and confirmed its importance for activation of Notch receptor by its ligand, Delta-like 1. We propose a new model where monoubiquitination and endocytosis of Notch are a prerequisite for its PS-dependent cleavage, and discuss its relevance for other gamma-secretase substrates.     

Novel human prostate-specific cDNA: molecular cloning,expression, and immunobiology of the recombinant protein

The differential display-polymerase chain reaction technique was employed to obtain a prostate-specific approximately 300-bp cDNA fragment. On screening the human prostate-lambdagt10 library with this fragment, a full-length approximately 1.5-kb cDNA encoding for a prostate antigen, designated as human novel prostate-specific antigen (hNPSA), was found. Extensive database searches revealed that the hNPSA cDNA is a novel sequence. It has an open reading frame (ORF) of 735-bp encoding for 245 amino acids (aa), with a calculated molecular mass of approximately 27kDa. Hydrophilicity analysis of the deduced aa sequence indicated that hNPSA is a membrane-anchored peptide. Analysis for tissue-specificity by Northern blot and RT-PCR-Southern blot procedures indicated that hNPSA is specifically expressed only in human prostate. The hNPSA (ORF) was subcloned into pET22b(+) vector and expressed using the histidine-tagged gene fusion system. The recombinant (r) protein of approximately 27kDa was purified and antibodies (Ab) were raised in rabbits. The rhNPSA Ab recognized a specific protein band of approximately 35kDa in solubilized human prostate tissue and not in any of the other 10 human tissues tested in the Western blot procedure. The hNPSA expression is upregulated 2.5- to 3-fold, both at the mRNA and protein levels in androgen-dependent LNCaP cells, as compared to normal whole prostate tissue. Antisense, but not the sense, phosphothiorate-conjugated oligonucleotides based on the hNPSA cDNA sequence significantly (p<0.001) inhibited proliferation of LNCaP cells in a concentration-dependent manner. Thus, the novel hNPSA, which has prostate-specific expression and seems to be involved in carcinogenesis, may have applications in the specific diagnosis and treatment of prostate cancer.     

Aerobic microbial manufacture of nanoscale selenium: exploiting nature’s bio-nanomineralization potential

The potential of the environment to yield organisms that can produce functional bionanominerals is demonstrated by selenium-tolerant, aerobic bacteria isolated from a seleniferous rhizosphere soil. An isolate, NS3, was identified as a Bacillus species (EU573774.1) based on morphological and 16S rRNA characterization. This strain reduced Se(IV) under aerobic conditions to produce amorphous α Se(0) nanospheres. A room-temperature washing treatment was then employed to remove the biomass and resulted in the production of clusters of hexagonal Se(0) nano-rods. The Se(0) nanominerals were analyzed using electron microscopy and X-ray diffraction techniques. This Bacillus isolate has the potential to be used both in the neutralizing of toxic Se(IV) anions in the environment and in the environmentally friendly manufacture of nanomaterials.