Enhanced cardiovascular function and energy level by a novel chromium (III)-supplement

The impetus for the novel Energy Formula (EF) which combines the niacin-bound chromium (III) (0.45%) (NBC), standardized extract of Withania somnifera extracts (10.71%), caffeine (22.76%), D-ribose (10.71%) and selected amino acids such as phenylalanine, taurine and glutamine (55.37%) was based on the knowledge of the cardioprotective potentials of the Withania somnifera extract, caffeine and D-ribose as well as their abilities to increase energy levels and the abilities of amino acids to increase the muscle mass and energy levels. The effect of oral supplementation of EF on the safety, myocardial energy levels and cardioprotective ability were investigated in an ischemic-reperfused myocardium model in both male and female Sprague-Dawley rats over 90 days trial period. At the completion of 90 days, the EF-treated male and female rats gained 9.4% and 3.1% less body weights, respectively, as compared to their corresponding control groups. No significant difference was found in the levels of lipid peroxidation and activities of hepatic Aspartate transaminase, Alanine transaminase and Alkaline phosphatase in EF treatment when compared with control animals. The male and female rat hearts were subjected to 30 min of global ischemia followed by 2 h of reperfusion at 30 and 90 days of EF treatment. Cardiovascular functions including heart rate, coronary flow, aortic flow, dp/dt(max), left ventricular developed pressure (LVDP) and infarct size were monitored. The levels of myocardial adenosine triphosphate (ATP), creatine phosphate (CP), phospho-adenosine monophosphate kinase (p-AMPK) levels, were analyzed at the end of 30 and 90 days of treatment. Significant improvement was observed in all parameters in the EF treatment groups as compared to their corresponding controls. Thus the niacin-bound chromium (III) based energy formula is safe and effective supplement to boost energy levels and cardioprotection.     

Whole-genome analysis of Oryza sativa reveals similar architecture of two-component signaling machinery with Arabidopsis

The two-component system (TCS), which works on the principle of histidine-aspartate phosphorelay signaling, is known to play an important role in diverse physiological processes in lower organisms and has recently emerged as an important signaling system in plants. Employing the tools of bioinformatics, we have characterized TCS signaling candidate genes in the genome of Oryza sativa L. subsp. japonica. We present a complete overview of TCS gene families in O. sativa, including gene structures, conserved motifs, chromosome locations, and phylogeny. Our analysis indicates a total of 51 genes encoding 73 putative TCS proteins. Fourteen genes encode 22 putative histidine kinases with a conserved histidine and other typical histidine kinase signature sequences, five phosphotransfer genes encoding seven phosphotransfer proteins, and 32 response regulator genes encoding 44 proteins. The variations seen between gene and protein numbers are assumed to result from alternative splicing. These putative proteins have high homology with TCS members that have been shown experimentally to participate in several important physiological phenomena in plants, such as ethylene and cytokinin signaling and phytochrome-mediated responses to light. We conclude that the overall architecture of the TCS machinery in O. sativa and Arabidopsis thaliana is similar, and our analysis provides insights into the conservation and divergence of this important signaling machinery in higher plants.     

Secretory factors of human neuroblastoma (IMR-32) and human glioblastoma (U87MG) cell lines induce neurite outgrowths in PC12 cells

Neurite outgrowth is essential for the communication of the nervous system. The rat Pheochromocytoma (PC12) cells are commonly used in the neuronal cell study. It is well known that exogenous stimuli such as Nerve Growth Factor (NGF) induce neurite outgrowth. In the present study it has been investigated whether or not the conditioned medium from human neuroblastoma cell line (IMR-32) and human glioblastoma cell line (U87MG) may augment neurite outgrowth in PC12 cells. PC12 were cultured with and without conditioned media of IMR-32 and U87MG. The result showed that both the conditioned media induce neurite outgrowth within 48 hr and stops further proliferation of PC12 cells. However no outgrowth was noted in PC12 cells incubated without conditioned medium. In conclusion, it is shown that both the conditioned media (IMR-32 and U87MG) have the potential to induce the neurite outgrowth in the PC12 cells.     

Glycogen synthase kinase 3beta phosphorylates Alzheimer's disease-specific Ser396 of microtubule-associated protein tau by a sequential mechanism

Phosphorylation of tau on S(396) was suggested to be a key step in the development of neurofibrillary pathology in Alzheimer's disease brain [Bramblett, G. T., Goedert, M., Jacks, R., Merrick, S. E., Trojanowski, J. Q., and Lee, V. M.-Y. (1993) Neuron 10, 1089-1099]. GSK3beta phosphorylates Ser(396) of tau in the brain by a mechanism which is not clear. In this study, when HEK-293 cells were cotransfected with tau and GSK3beta, GSK3beta co-immunoprecipitated with tau and phosphorylated tau on S(202), T(231), S(396), and S(400) but not on S(262), S(235), and S(404). Blocking phosphorylation on T(231), S(235), S(396), S(400), or S(404) did not prevent the subsequent phosphorylation on S(202) by GSK3beta. These data suggest that GSK3beta directly phosphorylates tau on S(202) (without requiring prephosphorylation). However, preventing phosphorylation on S(235), S(400), and S(404) prevented GSK3beta-dependent phosphorylation of T(231), S(396), and S(400), respectively. This indicates that phosphorylation of T(231), S(396), and S(400) by GSK3beta depends on a previous phosphorylation of S(235), S(400), and S(404), respectively. To examine S(396) phosphorylation, we analyzed phosphorylation of S(396), S(400), and S(404). Blocking phosphorylation of S(404) prevented the subsequent GSK3beta-dependent phosphorylation of both S(400) and S(396). When phosphorylation of S(404) was allowed but S(400) blocked, GSK3beta failed to phosphorylate S(396). Thus, GSK3beta phosphorylates S(396) by a two-step mechanism. In the first step, GSK3beta phosphorylates S(400) of previously S(404)-phosphorylated tau. This event primes tau for second-step phosphorylation of S(396) by GSK3beta. We conclude that GSK3beta phosphorylates tau directly at S(202) but requires the previous phosphorylation on S(235) to phosphorylate T(231). Phosphorylation of S(396), on the other hand, occurs sequentially. Once a priming kinase phosphorylates S(404), GSK3beta sequentially phosphorylates S(400) and then S(396).     

Cyclin-dependent protein kinase 5 primes microtubule-associated protein tau site-specifically for glycogen synthase kinase 3beta

In the preceding paper, we showed that GSK3beta phosphorylates tau at S(202), T(231), S(396), and S(400) in vivo. Phosphorylation of S(202) occurs without priming. Phosphorylation of T(231), on the other hand, requires priming phosphorylation of S(235). Similarly, priming phosphorylation of S(404) is essential for the sequential phosphorylation of S(400) and S(396) by GSK3beta. The priming kinase that phosphorylates tau at S(235) and S(404) in the brain is not known. In this study, we find that in HEK-293 cells cotransfected with tau, GSK3beta, and Cdk5, Cdk5 phosphorylates tau at S(202), S(235), and S(404). S(235) phosphorylation enhances GSK3beta-catalyzed T(231) phosphorylation. Similarly, Cdk5 by phosphorylating S(404) stimulates phosphorylation of S(400) and S(396) by GSK3beta. These data indicate that Cdk5 primes tau for GSK3beta in intact cells. To evaluate if Cdk5 primes tau for GSK3beta in mammalian brain, we examined localizations of Cdk5, tau, and GSK3beta in rat brain. We also analyzed the interaction of Cdk5 with tau and GSK3beta in brain microtubules. We found that Cdk5, GSK3beta, and tau are virtually colocalized in rat brain cortex. When bovine brain microtubules are analyzed by FPLC gel filtration, Cdk5, GSK3beta, and tau coelute within an approximately 450 kDa complex. From the fractions containing the approximately 450 kDa complex, tau, Cdk5, and GSK3beta co-immunoprecipitate with each other. In HEK-293 cells transfected with tau, Cdk5, and GSK3beta in different combinations, tau binds to Cdk5 in a manner independent of GSK3beta and to GSK3beta in a manner independent of Cdk5. However, Cdk5 and GSK3beta bind to each other only in the presence of tau, suggesting that tau connects Cdk5 and GSK3beta. Our results suggest that in the brain, tau, Cdk5, and GSK3beta are components of an approximately 450 kDa complex. Within the complex, Cdk5 phosphorylates tau at S(235) and primes it for phosphorylation of T(231) by GSK3beta. Similarly, Cdk5 by phosphorylating tau at S(404) primes tau for a sequential phosphorylation of S(400) and S(396) by GSK3beta.     

Detection of 65 kD heat shock protein in cerebrospinal fluid of tuberculous meningitis patients

Background  

Diagnosis of tuberculous meningitis (TBM) is difficult. Rapid confirmatory diagnosis is essential to initiate required therapy. There are very few published reports about the diagnostic significance of 65 kD heat shock protein (hsp) in TBM patients, which is present in a wide range of Mycobacterium tuberculosis species and elicits a cellular and humoral immune response. In the present study we have conducted a prospective evaluation for the demonstration of 65 kD hsp antigen in cerebrospinal fluid (CSF) of TBM patients, by indirect ELISA method using monoclonal antibodies (mAb) against the 65 kD hsp antigen, for the diagnosis of TBM.     

An Interleukin-10 Gene Promoter Polymorphism (?592A/C) Associated with Type 2 Diabetes: A North Indian Study

In this first report on the association of an IL-10 promoter polymorphism with type 2 diabetes mellitus in a North Indian population, the -592A/C SNP (rs1800872) was genotyped by PCR-RFLP and the IL-10 level measured using ELISA. Although no significant difference was observed in the genotypic frequencies (P = 0.657), diabetes patients carried a significantly higher number of A alleles at the -592 position, 25.6% (P < 0.001, odds ratio 0.887, 95% CI 0.670-1.184). Significant correlations were detected in postprandial glucose levels of CC-genotype patients and controls (P = 0.025), age and waist-hip ratio of CA patients and controls (P ≤ 0.001), and fasting glucose (P = 0.045) and low-density lipoprotein (P = 0.049) in all patients and controls. The serum IL-10 level was significantly higher in patients than in controls (P = 0.033). The polymorphism was significantly associated with disease incidence and its biochemical manifestations.     

Comparative structural modeling of a monothiol GRX from chickpea: insight in iron-sulfur cluster assembly

Glutaredoxins (GRXs) are small, ubiquitous, multifunctional, heat-stable and glutathione-dependent thiol-disulphide oxidoreductases, classified under thioredoxin-fold superfamily. In the green lineage, GRXs constitute a complex family of proteins. Based on their active site, GRXs are classified into two subfamilies: dithiol and monothiol. Monothiol GRXs contain 'CGFS' as a redox active motif and assist in maintaining redox state and iron homeostasis within the cell. Using RACE strategy, a full length cDNA of chickpea (Cicer arietinum) glutaredoxin 3 (CarGRX3) was cloned and sequenced. The cDNA contains open reading frame of 537 bp encoding 178 amino acids and exhibits features of other known 'CGFS' type GRXs. Based on the multiple sequence alignment among CarGRX3 and monothiol GRXs of other photosynthetic organisms, the characteristic motif (KGX4PXCGFSX([29/30/32])KX4WPTXPQX4GX3GGXDI) with 18 invariant residues was observed. The proposed structure of CarGRX3 was compared with structurally resolved monothiol GRXs of other organisms. The CarGRX3 and nearest Arabidopsis homolog (AtGRXcp) shares 76% sequence identity which was reflected by their 3D-structure conservation. The structure of chickpea monothiol GRX (CarGRX3) coordinates glutathione ligated [2Fe-2S] cluster in a homodimeric form, highlighting the structural basis for iron-sulfur cluster (ISC) assembly and delivery to acceptor proteins. The present study on CarGRX3 model highlighted the utility of the theoretical approaches to understand complex biological phenomena such as glutathione docking and incorporation of GSH-ligated [2Fe-2S] cluster.     

Defining the boundaries of species specificity for the Saccharomyces cerevisiae glycosylphosphatidylinositol transamidase using a quantitative in?vivo assay

In eukaryotes, GPI (glycosylphosphatidylinositol) lipid anchoring of proteins is an abundant post-translational modification. The attachment of the GPI anchor is mediated by GPI-T (GPI transamidase), a multimeric, membrane-bound enzyme located in the ER (endoplasmic reticulum). Upon modification, GPI-anchored proteins enter the secretory pathway and ultimately become tethered to the cell surface by association with the plasma membrane and, in yeast, by covalent attachment to the outer glucan layer. This work demonstrates a novel in vivo assay for GPI-T. Saccharomyces cerevisiae INV (invertase), a soluble secreted protein, was converted into a substrate for GPI-T by appending the C-terminal 21 amino acid GPI-T signal sequence from the S. cerevisiae Yapsin 2 [Mkc7p (Y21)] on to the C-terminus of INV. Using a colorimetric assay and biochemical partitioning, extracellular presentation of GPI-anchored INV was shown. Two human GPI-T signal sequences were also tested and each showed diminished extracellular INV activity, consistent with lower levels of GPI anchoring and species specificity. Human/fungal chimaeric signal sequences identified a small region of five amino acids that was predominantly responsible for this species specificity.