Heparin oligosaccharide sequence and size essential for inhibition of pulmonary artery smooth muscle cell proliferation

Heparin has a wide range of important biological activities including inhibition of pulmonary artery smooth muscle cell proliferation. To determine the minimum size of the heparin glycosaminoglycan chain essential for antiproliferative activity, porcine intestinal mucosal heparin was partially depolymerized with heparinase and fractionated to give oligosaccharides of different sizes. The structure of these oligosaccharides was fully characterized by 1D and 2D 1H NMR spectroscopy. These oligosaccharides were assayed for antiproliferative effects on cultured bovine pulmonary artery smooth muscle cells (PASMCs). The tetrasaccharide (4-mer) exhibited no heparin-like activity. Decasaccharides (10-mers) and dodecasaccharides (12-mers) displayed a reduced level of activity when compared to full-length heparin. Little effect on activity was observed in deca- and dodecasaccharides with one less 2-O-sulfo group. The 14-, 16-, and 18-mers showed comparable growth-inhibition effects on PAMSC as porcine intestinal mucosal heparin. These data suggest that a 14-mer is the minimum size of oligosaccharide that is essential for full heparin-like antiproliferative activity. Since the 14- to 18-mers have no 3-O-sulfo groups in their glucosamine residues, their full activity confirms that these 3-O-sulfonated glucosamine residues, which are required for heparin's anticoagulant activity, are not an essential requirement for antiproliferative activity.     

Effect of disinfectants on stability and transmissibility of R-plasmid in E. coli isolated from drinking water

Drug resistant enteropathogenic E. coli (EPEC, 086 serotype) isolated from contaminated piped drinking water supply (Fecal coliform 160/100 ml) was studied for effect of disinfectants (chlorine and UVB) on stability and transmissibility of R-plasmid. The strain was resistant to streptomycin and bacitracin and tolerant to multiple metal ions of Cd, Cr, Co, As, Ni, Zn and Hg. A plasmid of molecular size of 3.7 Kb was detected in the organism. After exposure to sublethal doses of disinfectants, complete elimination of resistances to streptomycin and Cr was observed. Partial loss of resistance to Hg due to chlorine was detected. Although UVB did not effected the pattern of transmissibility effect on frequency of transfer was observed. Surprisingly, in UVB irradiated cells, significantly enhanced rate of transfer was noted.     

Biosorption and elution of chromium from immobilized Bacillus coagulans biomass

Bacillus coagulans, a tannery wastewater isolate, previously shown to bind dissolved Cr(VI), retained its ability to biosorb Cr(VI) in different matrices. Polymeric materials like agar, agarose, calcium alginate and polyacrylamide were screened. Agarose emerged as the suitable candidate for biomass immobilization mainly due to its higher stability and integrity in acidic pH. Aptness of agarose as the matrix for B. coagulans biomass was revealed during Cr(VI) biosorption from natural wastewater.     

Increased detection of structural templates using alignments of designed sequences

Protein structure prediction by comparative modeling benefits greatly from the use of multiple sequence alignment information to improve the accuracy of structural template identification and the alignment of target sequences to structural templates. Unfortunately, this benefit is limited to those protein sequences for which at least several natural sequence homologues exist. We show here that the use of large diverse alignments of computationally designed protein sequences confers many of the same benefits as natural sequences in identifying structural templates for comparative modeling targets. A large-scale massively parallelized application of an all-atom protein design algorithm, including a simple model of peptide backbone flexibility, has allowed us to generate 500 diverse, non-native, high-quality sequences for each of 264 protein structures in our test set. PSI-BLAST searches using the sequence profiles generated from the designed sequences ("reverse" BLAST searches) give near-perfect accuracy in identifying true structural homologues of the parent structure, with 54% coverage. In 41 of 49 genomes scanned using reverse BLAST searches, at least one novel structural template (not found by the standard method of PSI-BLAST against PDB) is identified. Further improvements in coverage, through optimizing the scoring function used to design sequences and continued application to new protein structures beyond the test set, will allow this method to mature into a useful strategy for identifying distantly related structural templates.     

Intracellular segregation of phosphatidylinositol-3,4,5-trisphosphate by insulin-dependent actin remodeling in L6 skeletal muscle cells

Insulin stimulates glucose uptake by recruiting glucose transporter 4 (GLUT4) from an intracellular pool to the cell surface through a mechanism that is dependent on phosphatidylinositol (PI) 3-kinase (PI3-K) and cortical actin remodeling. Here we test the hypothesis that insulin-dependent actin filament remodeling determines the location of insulin signaling molecules. It has been shown previously that insulin treatment of L6 myotubes leads to a rapid rearrangement of actin filaments into submembrane structures where the p85 regulatory subunit of PI3-K and organelles containing GLUT4, VAMP2, and the insulin-regulated aminopeptidase (IRAP) colocalize. We now report that insulin receptor substrate-1 and the p110alpha catalytic subunit of PI3-K (but not p110beta) also colocalize with the actin structures. Akt-1 was also found in the remodeled actin structures, unlike another PI3-K effector, atypical protein kinase C lambda. Transiently transfected green fluorescent protein (GFP)-tagged pleckstrin homology (PH) domains of general receptor for phosphoinositides-1 (GRP1) or Akt (ligands of phosphatidylinositol-3,4,5-trisphosphate [PI-3,4,5-P(3)]) migrated to the periphery of the live cells; in fixed cells, they were detected in the insulin-induced actin structures. These results suggest that PI-3,4,5-P(3) is generated on membranes located within the actin mesh. Actin remodeling and GLUT4 externalization were blocked in cells highly expressing GFP-PH-GRP1, suggesting that PI-3,4,5-P(3) is required for both phenomena. We propose that PI-3,4,5-P(3) leads to actin remodeling, which in turn segregates p85alpha and p110alpha, thus localizing PI-3,4,5-P(3) production on membranes trapped by the actin mesh. Insulin-stimulated actin remodeling may spatially coordinate the localized generation of PI-3,4,5-P(3) and recruitment of Akt, ultimately leading to GLUT4 insertion at the plasma membrane.     

Heparin inhibits SMC growth in the presence of human and fetal bovine serum

Heparin (HP) has antiproliferative as well as anticoagulant properties, but not all HP preparations are equally antiproliferative. A recent report found that HP lost its total antiproliferative activity when fetal bovine serum (FBS) was replaced with human serum (HS) in culture media. This observation led to the investigation of our most potent antiproliferative Upjohn HP preparation effects on bovine pulmonary artery smooth muscle cells (PASMC) and systemic SMC growth stimulated in the presence of either FBS or HS. Bovine PASMC, human PASMC, and bovine aortic SMC were treated with 10 microg/ml Upjohn HP in either 15% FBS or 15% HS and the cell number was determined by a Coulter counter. We found that Upjohn HP significantly inhibited bovine PASMC and systemic SMC proliferation in both HS and FBS. The antiproliferative activity of the above HP preparation in HS may lead to an effective treatment of pulmonary vascular and systemic remodeling.     

An adaptive strategy for single- and multi-cluster gene assignment

Strict assignment of genes to one class, dimensionality reduction, a priori specification of the number of classes, the need for a training set, nonunique solution, and complex learning mechanisms are some of the inadequacies of current clustering algorithms. Existing algorithms cluster genes on the basis of high positive correlations between their expression patterns. However, genes with strong negative correlations can also have similar functions and are most likely to have a role in the same pathways. To address some of these issues, we propose the adaptive centroid algorithm (ACA), which employs an analysis of variance (ANOVA)-based performance criterion. The ACA also uses Euclidian distances, the center-of-mass principle for heterogeneously distributed mass elements, and the given data set to give unique solutions. The proposed approach involves three stages. In the first stage a two-way ANOVA of the gene expression matrix is performed. The two factors in the ANOVA are gene expression and experimental condition. The residual mean squared error (MSE) from the ANOVA is used as a performance criterion in the ACA. Finally, correlated clusters are found based on the Pearson correlation coefficients. To validate the proposed approach, a two-way ANOVA is again performed on the discovered clusters. The results from this last step indicate that MSEs of the clusters are significantly lower compared to that of the fibroblast-serum gene expression matrix. The ACA is employed in this study for single- as well as multi-cluster gene assignments.     

Effect of alternaria pathotoxin(s) on expression of p53-like apoptotic protein in calli and leaves of Brassica campestris

Possible involvement of apoptosis was investigated in pathotoxin-treated and nutritionally-depleted in vitro cultured calli by comparing levels of p53-like protein. Antibodies raised against human p53 were used to detect and quantify p53 in B. campestris. Expression of p53-like protein increased from proliferating to static growth stage and reached to constant level at decaying stage. Both ELISA and dot immuno-binding assay showed that p53-like protein was over expressed in toxin treated and nutritionally depleted calli. Almost similar changes were seen in senescent damage in Brassica species indicating involvement of p53 dependent pathways.     

Role of antioxidants in chronic fatigue syndrome in mice

The present study was carried out using mice model of chronic fatigue syndrome (CFS) in which mice were forced to swim everyday for 7 days for a 6 min session. There was a significant increase in despair behavior (immobility period) in saline treated mice on successive days. Treatment with potent antioxidants carvedilol (5 mg/kg, i.p.) and melatonin (10 mg/kg, i.p.) produced a significant reduction in immobility period. Similar results were observed with herbal products St. John's Wort (Hypericum perforatum L) (10 mg/kg, p.o.) and GS-02 (20 mg/kg, p.o.). Fluoxetine, a selective serotonin reuptake inhibitor produced a significant effect only on first and second day of its treatment. Biochemical analysis revealed that chronic swim test significantly increased lipid peroxidation and catalase levels in whole brains of mice. There was a decrease in the levels of super oxide dismutase (SOD) and glutathione reductase (GSH) in the brain. Administration of carvedilol, melatonin, GS-02 and St. John's Wort restored the levels of lipid peroxidation and glutathione. The enzymes SOD and catalase were also restored. Fluoxetine affected the biochemical variables not to the same extent as other treatments. The findings of the present study suggest that oxidative stress might play a significant role in the pathophysiology of CFS. Thus antioxidants and herbal products like St. Johns wort and GS-02 could be useful in the treatment of CFS.