Influence of oxidatively modified LDL on monocyte-macrophage differentiation

Cellular cytoskeletal remodeling reflects alterations in local biochemical and mechanical changes in terms of stress that manifests relocation of signaling molecules within and across the cell. Although stretching due to load and chemical changes by high homocysteine (HHcy) causes cytoskeletal re-arrangement, the synergism between stretch and HHcy is unclear. We investigated the contribution of HHcy in cyclic stretch-induced focal adhesion (FA) protein redistribution leading to cytoskeletal re-arrangement in mouse aortic endothelial cells (MAEC). MAEC were subjected to cyclic stretch (CS) and HHcy alone or in combination. The redistribution of FA protein, and small GTPases were determined by Confocal microscopy and Western blot techniques in membrane and cytosolic compartments. We found that each treatment induces focal adhesion kinase (FAK) phosphorylation and cytoskeletal actin polymerization. In addition, CS activates and membrane translocates small GTPases RhoA with minimal effect on Rac1, whereas HHcy alone is ineffective in both GTPases translocation. However, the combined effect of CS and HHcy activates and membrane translocates both GTPases. Free radical scavenger NAC (N-Acetyl-Cysteine) inhibits CS and HHcy-mediated FAK phosphorylation and actin stress fiber formation. Interestingly, CS also activates and membrane translocates another FA protein, paxillin in HHcy condition. Cytochalasin D, an actin polymerization blocker and PI3-kinase inhibitor Wortmannin inhibited FAK phosphorylation and membrane translocation of paxillin suggesting the involvement of PI3K pathway. Together our results suggest that CS- and HHcy-induced oxidative stress synergistically contribute to small GTPase membrane translocation and focal adhesion protein redistribution leading to endothelial remodeling.     

Parallel changes in fibronectin and alpha5beta1 integrin in articular cartilage in type II collagen-induced arthritis

Interactions of cells with extracellular matrix (ECM) are mediated through specific cell surface receptors, belonging to the integrin family of transmembrane proteins. Integrins have been shown to be involved in chondrocyte-matrix interactions in the cartilage. In this study, the status of a matrix glycoprotein fibronectin (FN) and its receptor alpha5beta1 integrin in the articular cartilage in collagen type II-induced experimental arthritis in rats, as well as in synovial fluid from osteoarthritic patients was investigated. Experimental arthritis was induced by intradermal injection of type-II collagen (300 microg/100 g body wt) and Freund's complete adjuvant. Saline-treated animals served as control. Clinical severity was indicated by increase in paw volume. Significant increase in the activities of lysosomal enzymes beta-glucuronidase and beta-hexosaminidase was observed in synovial effusate, serum and cartilage of arthritic animals, when compared to untreated control, indicating dysfunction of cartilage. Changes in FN and alpha5beta1 integrin were studied by ELISA. A progressive increase was observed in the FN level in synovial effusate and cartilage of arthritic animals, when compared to untreated controls. FN levels were also significantly high in synovial fluid of osteoarthritic patients. A significant increase in the levels of alpha5beta1 integrin was found in cartilage of arthritic rats. Parallel changes in FN and alpha5beta1 integrin indicated that alterations in FN and alpha5beta1 integrin in chondrocytes constituted one of the molecular mechanisms during progression of arthritis.     

Modulation of angiogenic factors by ursolic acid

Investigations were carried out to understand the molecular basis of the effect of ursolic acid on angiogenesis by analysing its effects on the expression of modulators of angiogenesis by HUVECs in culture. Treatment with ursolic acid increased the expression of adhesion molecules such as E-selectin, CD-31 and I-CAM, upregulated angiogenic growth factors such as VEGF and FGF-2 and their receptors and caused increase in the ratio of PGE₂ to PGD₂. Reversal of the effect of ursolic acid by inhibition of PI3K-Akt pathway and increase in the level of phospho Akt suggest that the ursolic acid effect is mediated through PI3K-Akt pathway.     

Potential use of chitosan nanoparticles for oral delivery of DNA vaccine in Asian sea bass (Lates calcarifer) to protect from Vibrio (Listonella) anguillarum

In recent years, attention has been focused on the possibility of utilizing DNA vaccines in fish aquaculture. A successful regime for intramuscular injection of naked DNA into fish has been developed and novel methods to deliver this DNA to fish are under investigation. The potential of chitosan as a polycationic gene carrier for oral administration has been explored since 1990s. The present study examines the potential efficacy of DNA vaccine against Vibrio anguillarum through oral route using chitosan nanoparticles encapsulation. The porin gene of V. anguillarum was used to construct DNA vaccine using pcDNA 3.1, a eukaryotic expression vector and the construct was named as pVAOMP38. The chitosan nanoparticles were used to deliver the constructed plasmid. In vitro and in vivo expression of porin gene was observed in sea bass kidney cell line (SISK) and in fish, respectively by fluorescent microscopy. The cytotoxicity of chitosan encapsulated DNA vaccine construct was analyzed by MTT assay and it was found that the cytotoxicity of pVAOMP38/chitosan was quite low. Distribution of gene in different tissues was studied in fish fed with the DNA (pVAOMP38) encapsulated in chitosan by using immunohistochemistry. The results indicate that DNA vaccine can be easily delivered into fish by feeding with chitosan nanoparticles. After oral vaccination Asian sea bass were challenged with Vibrio anguillarum by intramuscular injection. A relative percent survival (RPS) rate of 46% was recorded. The results indicate that Sea bass (Lates calcarifer) orally vaccinated with chitosan-DNA (pVAOMP38) complex showed moderate protection against experimental V. anguillarum infection.     

High throughput lipidomic profiling of schizophrenia and bipolar disorder brain tissue reveals alterations of free fatty acids, phosphatidylcholines, and ceramides

A mass spectrometry based high throughput approach was employed to profile white and gray matter lipid levels in the prefrontal cortex (Brodmann area 9) of 45 subjects including 15 schizophrenia and 15 bipolar disorder patients as well as 15 controls samples. We found statistically significant alterations in levels of free fatty acids and phosphatidylcholine in gray and white matter of both schizophrenia and bipolar disorder samples compared to controls. Also, ceramides were identified to be significantly increased in white matter of both neuropsychiatric disorders as compared to control levels. The patient cohort investigated in this study includes a number of drug naive as well as untreated patients, allowing the assessment of drug effects on lipid levels. Our findings indicate that while gray matter phosphatidylcholine levels were influenced by antipsychotic medication, this was not the case for phosphatidylcholine levels in white matter. Changes in free fatty acids or ceramides in either white or gray matter also did not appear to be influenced by antipsychotic treatment. To assess lipid profiles in the living patient, we also profiled lipids of 40 red blood cell samples, including 7 samples from drug naive first onset patients. We found significant alterations in the concentrations of free fatty acids as well as ceramide. Overall, our findings suggest that lipid abnormalities may be a disease intrinsic feature of both schizophrenia and bipolar disorder reflected by significant changes in the central nervous system as well as peripheral tissues.     

Evaluating genomic DNA extraction methods from human whole blood using endpoint and real-time PCR assays

The extraction of genomic DNA is the crucial first step in large-scale epidemiological studies. Though there are many popular DNA isolation methods from human whole blood, only a few reports have compared their efficiencies using both end-point and real-time PCR assays. Genomic DNA was extracted from coronary artery disease patients using solution-based conventional protocols such as the phenol–chloroform/proteinase-K method and a non-phenolic non-enzymatic Rapid-Method, which were evaluated and compared vis-a-vis a commercially available silica column-based Blood DNA isolation kit. The appropriate method for efficiently extracting relatively pure DNA was assessed based on the total DNA yield, concentration, purity ratios (A₂₆₀/A₂₈₀ and A₂₆₀/A₂₃₀), spectral profile and agarose gel electrophoresis analysis. The quality of the isolated DNA was further analysed for PCR inhibition using a murine specific ATP1A3 qPCR assay and mtDNA/Y-chromosome ratio determination assay. The suitability of the extracted DNA for downstream applications such as end-point SNP genotyping, was tested using PCR-RFLP analysis of the AGTR1-1166A>C variant, a mirSNP having pharmacogenetic relevance in cardiovascular diseases. Compared to the traditional phenol–chloroform/proteinase-K method, our results indicated the Rapid-Method to be a more suitable protocol for genomic DNA extraction from human whole blood in terms of DNA quantity, quality, safety, processing time and cost. The Rapid-Method, which is based on a simple salting-out procedure, is not only safe and cost-effective, but also has the added advantage of being scaled up to process variable sample volumes, thus enabling it to be applied in large-scale epidemiological studies.     

Metabolite control of angiogenesis: angiogenic effect of citrate

Endothelial cells respond to hypoxic changes with resultant accumulation of several metabolites and switch over to angiogenic phenotype. Although certain intermediates of glycolytic and oxidative metabolic pathways have been known to affect angiogenesis, the effect of citrate, which accumulates in certain tumors, on angiogenesis is not known. Therefore, the effect of citrate on angiogenesis was studied using different model systems. Increased vascularization in chorioallantoic membrane assay, increased endothelial sprouting in rat aortic rings, and increased expression of CD31, E-selectin in endothelial cells suggested a possible proangiogenic effect of citrate. Upregulation of angiogenic factors such as vascular endothelial growth factor and fibroblast growth factor suggested that the effect of citrate involves modulation of expression of angiogenic growth factors. LY 294002, an inhibitor of PI3K–Akt pathway, and wortmannin, an inhibitor of Akt pathway, reversed the effect of citrate in human umbilical vein endothelial cells. Citrate induced significant upregulation and activation of Akt in endothelial cells. Rapamycin, an inhibitor of mTOR, also reversed the effect of citrate in human umbilical vein endothelial cells and sprouting of aortic rings suggesting that the angiogenic effect of citrate involves activation of PI3K–Akt–mTOR pathway.     

Enhanced adhesion of human mononuclear cells to nonenzymatically glycosylated collagen I

Nonenzymatic glycosylation of extracellular matrix components may contribute to altered interaction of cells with the matrix. We have examined the interaction of mononuclear cells with early glycosylated collagen I. Significantly more cells attached to glycosylated collagen compared to normal collagen. Radioiodinated glycosylated collagen I specifically bound to mononuclear cells in a time and concentration dependent manner with a Kd of 2.45×10^–9 M. Maximum binding was observed in the presence of Mn⁺⁺ ions. The iodinated ligand bound to mononuclear cell membrane immobilized on nitrocellulose disks and the interaction was found to be saturable. These results suggested an alteration in the interaction of human blood mononuclear cells with collagen I, when it gets glycosylated non enzymatically and also indicate that early glycosylated collagen interacts with mononuclear cells through specific, high affinity cell surface molecules. (Mol Cell Biochem148: 115–121, 1995)     

Water stress-induced alterations in the proline metabolism of drought-susceptible and -tolerant cassava (Manihot esculenta) cultivars

The free proline levels and activities of ornithine aminotransferase (EC 2.6.1.13) and proline oxidase (EC 1.5.2.2), two of the enzymes involved in proline metabolism were studied during the induction of water stress in a drought susceptible (M-4) and a drought tolerant (S-1315) cultivar of cassava ( Manihot esculenta Crantz). Water stress induced by polyethylene glycol (MW 6000, osmotic potential — 1.65 MPa) caused a ca 25-fold increase in proline in young excised leaves of the susceptible cultivar (M-4) while the increase was about 9-fold in the tolerant cultivar (S-1315). The activity of ornithine aminotransferase (OAT), a key enzyme involved in the biosynthesis of proline, was found to increase 3-fold in water stressed leaves of M-4 and about 2-fold in those of S-1315. The activity of proline oxidase, which is involved in the degradation of proline to pyrroline-5-carboxylate, was reduced by 50% in M-4 and nearly 25% in S-1315 on water stress. Comparison of the kinetic properties of OAT showed that the enzyme from water-stressed leaves is more stable to heat inactivation compared to that of control. These results indicate that during water stress there are alterations in the metabolism of proline in cassava, and the extent of alteration varies between drought-susceptible and -tolerant cultivars.     

Beijerinckia indica var.penicillanicum penicillin V acylase: enhanced enzyme production by catabolite repression-resistant mutant and effect of solvents on enzyme activity

Summary Beijerinckia indica var.penicillanicum mutant UREMS-5, producing 168% more penicillin V acylase, was obtained by successive treatment with UV, -irradiation and ethylmethane sulfonate. Penicillin V acylase production by the mutant strain was resistant to catabolite repression by glucose. Incorporation of glucose, sodium glutamate and vegetable oils in the medium enhanced enzyme production. The maximum specific production of penicillin V acylase was 244 IU/g dry weight of cells. Effect of solvents on hydrolysis of penicillin V by soluble penicillin V acylase and whole cells was studied. Methylene chloride, chloroform and carbon tetrachloride significantly stimulated the rate of penicillin V hydrolysis by whole cells.