Modulation of cyclooxygenase in endothelial cells by fibronectin: relevance to angiogenesis

Cyclooxygenases (COX), which catalyze the formation of prostaglandins (PGs), have been implicated in angiogenesis. Adhesion of endothelial cells (ECs) to extracellular matrix (ECM) induces the expression of COX-2 and PG production. The present study was carried out to analyze the influence of the adhesive ECM protein, fibronectin (FN), in modulating COX expression and its implications to angiogenesis using in vitro cultures of human umbilical vein ECs. RT-PCR analysis showed that the level of COX-2 mRNA was significantly high while that of COX-1 decreased in ECs maintained on FN. On treatment with p38 MAPK inhibitor and anti-alpha(5)beta(1) integrin antibody, FN dependent effect on COX expression was not observed. Analysis by ELISA and immunoblotting confirmed FN-dependent upregulation of COX-2 protein. The ratio of PG E(2):PG D(2) was significantly high in cells maintained on FN and on treatment with p38 MAPK inhibitor, the relative level of PG D(2) increased and that of PG E(2) decreased. Concomitant with the modulation of COX-2 and changes in PGs, ECs maintained on FN showed angiogenic response in an alpha(5)beta(1) integrin/p38 MAPK dependent manner as evidenced by the expression of angiogenic markers, CD 31 and E-selectin. These results suggest a FN-alpha(5)beta(1)/FAK/p38 MAPK dependent upregulation of COX-2 causing a shift in the relative levels of PGs in HUVECs which contributes to the angiogenic effect of FN.     

Production of deuterated β-carotene by metabolic labelling of Spirulina platensis

Method for production of deuterated -carotene for the bioavailability studies of vitamin A has been developed using Spirulina platensis in culture. Suspension cultures of Spirulina in heavy water (99.4% D₂O) medium produced maximum biomass and -carotene in 28 to 30 days. Of the total carotenoids, lutein constituted 30 to 35% while -carotene was about 24%. MS showed that 60 to 65% H atoms in -carotene were deuterated. 100% replacement of H atom with deuterium was achieved by preventing exchange with atmospheric moisture. The medium could be used in several cycles for metabolic labelling of carotenoids whereby the cost of production is reduced.     

Monocyte-macrophage differentiation in three dimensional collagen lattice

Human peripheral blood mononuclear cells (PBMC) upon transendothelial migration interact with subendothelial matrix components and differentiate into macrophages. In order to study whether the shape of the cells as dictated by the extracellular matrix can influence monocyte-macrophage (mo-m(phi)) differentiation, human PBMC were maintained in vitro on a three dimensional collagen I (COL I) lattice and studied for various macrophage specific functions, viz. endocytosis of [(125)I]acetyl bovine serum albumin (BSA), expression of specific cell surface antigens and expression of matrix metalloproteinases (MMPs). The cells maintained in three dimensional COL gel exhibited a higher rate of endocytosis of [(125)I]acetyl BSA than those on COL-coated plastic. FACS analysis showed that the mean fluorescence intensity (MFI) corresponding to monocyte specific LPS receptor CD14 was significantly decreased while MFI corresponding to macrophage specific transferrin receptor CD71 was significantly increased in cells maintained in vitro on three dimensional COL gel compared to two dimensional COL substrata. Expression of macrophage specific MMPs (gelatinase A and gelatinase B) was significantly high in cells maintained on COL gel than on COL I-coated plastic. Appearance of 67 kDa gelatinase in the COL gel suggested that induction as well as activation of MMPs occur when cells are maintained in a three dimensional environment. These results indicate that monocytes undergo a rapid rate of differentiation when maintained in vitro on three dimensional COL I lattice suggesting that apart from the chemical nature of the matrix, the shape of the cells as provided by the matrix also influences mo-m(phi) differentiation.     

Effect of glycosaminoglycans on matrix metalloproteinases in type II collagen-induced experimental arthritis

Matrix metalloproteinases (MMPs) are a family of neutral proteinases that are involved in tissue remodeling by mediating degradation of extracellular matrix components in both physiology and pathology. As MMPs appear to play a key role in the degradation of cartilage matrix in the progression of arthritic disease, MMPs are considered as potential therapeutic targets. The effect of chondroitin sulfate A (CSA) on MMPs in type II collagen-induced experimental arthritis was studied. The anti-arthritic effect of CSA was evidenced by a decrease in marker activities like lysosomal beta-hexosaminidase and beta-glucuronidase. Arthritic animals showed significantly higher activity of MMP2 and MMP9 and increased levels of other MMPs, including MMP3 and MT-1 MMP in cartilage and serum. Treatment with CSA significantly decreased the activity of MMPs, particularly MMP9 in serum and synovial effusate and cartilage. The effect of CSA was further studied by fragmenting CSA into low-molecular-weight oligosaccharides. The oligosaccharide-treated animals showed considerably lower MMP activity (particularly MMP9) compared with arthritic controls. The CSA (and the oligosaccharides derived from it) not only reduced the activity of MMPs but also decreased the protein level expression of MMPs, indicating that the production of MMPs is affected. These results indicate that the antiarthritic effect of CSA involves down-regulation of MMPs, which are critically involved in the progression of arthritic disease.     

Lycopene as a natural protector against gamma-radiation induced DNA damage, lipid peroxidation and antioxidant status in primary culture of isolated rat hepatocytes in vitro

The present study was designed to evaluate the radioprotective effect of lycopene, a naturally occurring dietary carotenoid, on gamma-radiation induced toxicity in cultured rat hepatocytes. The cellular changes were estimated using lipid peroxidative indices like thiobarbituric acid reactive substances (TBARS), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), reduced glutathione (GSH), ceruloplasmin, vitamins A, E, C and uric acid. The DNA damage was analysed by single cell gel electrophoresis (comet assay). The increase in the severity of DNA damage was observed with the increase in gamma-radiation dose (1, 2 and 4 Gy) in cultured rat hepatocytes. TBARS were increased significantly whereas the levels of GSH, vitamins C, E and A, ceruloplasmin, uric acid and antioxidant enzymes were significantly decreased in gamma-irradiated groups. The maximum damage to hepatocytes was observed at 4 Gy irradiation. Pretreatment with lycopene (1.86, 9.31 and 18.62 microM) showed a significant decrease in the levels of TBARS and DNA damage. The antioxidant enzymes increased significantly along with the levels of GSH, vitamins A, E, C, uric acid and ceruloplasmin. The maximum protection of hepatocytes was observed at 9.31 muM of lycopene pretreatment. Thus, our results show that pretreatment with lycopene offers protection against gamma-radiation induced cellular damage and can be developed as an effective radioprotector during radiotherapy.     

Structural and biochemical analyses of shikimate dehydrogenase AroE from Aquifex aeolicus: implications for the catalytic mechanism

The shikimate biosynthetic pathway is essential to microorganisms, plants, and parasites but absent from mammals. Therefore, shikimate dehydrogenase (SD) and other enzymes in the pathway are attractive targets for developing nontoxic antimicrobial agents, herbicides, and antiparasite drugs. SD catalyzes the fourth reaction in the pathway, the nicotinamide adenine dinucleotide phosphate- (NADP-) dependent reduction of 3-dehydroshikimic acid to shikimic acid (SA), as well as its reverse, by the transfer of a hydride. Previous structural studies reveal that the enzyme exists in two major conformations, an open and a closed form. For the reaction to occur, it is believed that the catalytic complex assumes the closed conformation. Nonetheless, the only structure containing both SA and NADP+ exhibits an open conformation (PDB entry 2EV9). Here, we present two crystal structures of Aquifex aeolicus SD, including a ternary complex with both SA and NADP+, which assumes the closed conformation and therefore contains a catalytically competent active site. On the basis of preexisting and novel structural and biochemical data, a catalytic mechanism is proposed.     

Molecular biology of β-lactam acylases

-Lactam acylases such as penicillin G acylases, penicillin V acylases and glutaryl 7-aminocephalosporanic acid acylases are used in the manufacture of 6-aminopenicillanic acid, 7-aminodesacetoxycephalosporanic acid and 7-aminocephalosporanic acid (7-ACA). Genetically-engineered strains producing 1050 U/g, 3200 U/g and 7000 to 10,000 U/I of penicillin G acylase, penicillin V acylase and glutaryl-7-ACA acylase, respectively, have been developed. The penicillin G acylase studied to date and the glutaryl-7-ACA acylase from Pseudomonas sp. share some common features: the active enzyme molecules are composed of two dissimilar subunits that are generated from respective precursor polypeptide; the proteolytic processing is a post-translational modification which is regulated by temperature; and the Ser residue at the N-terminus of the -sub-unit (Ser²⁹⁰; penicillin G acylase numbering) is implicated as the active site residue. Protein engineering, to generate penicillin G acylase molecules and their precursors with altered sequences, and the structure-function correlation of the engineered molecules are discussed.The authors are with Research and Development, Hindustan Antibiotics Ltd, Pimpri, Pune 411 018, India;     

Deciphering the DNA repair protein, Rad23 from kuruma shrimp Marsupenaeus japonicus: full-length cDNA cloning and characterization

Aims: Lesions of DNA are removed by nucleotide excision repair (NER) process in the living systems. NER process‐related host factors are believed to aid recovery steps during viral integration. Here, we report identification and characterization of a DNA repair molecule Rad23 from kuruma shrimp Marsupenaeus japonicus. Methods and Results: The full‐length cDNA of M. japonicus Rad23 gene (MjRad23) has 1149 bp coding for a putative protein of 382 amino acids with a 5′ untranslated region (UTR) of 92 bp and 3′ UTR region of 1116 bp. Quantitative expression analysis revealed MjRad23 is constitutively expressed in all the organs of healthy shrimp, whereas with high level in muscle tissue. Although MjRad23 expression is observed in every haemolymph samplings to post‐white spot syndrome virus infection, high expression is recorded at 2 h post infection (h.p.i.). MjRad23 consists of putative functional domains including one ubiquitin domain (UBQ), two ubiquitin‐associated domains (UBA) and one heat‐shock chaperonin‐binding motif (STI1). Multiple alignment of MjRad23 with Rad23 of other species showed highly significant identity ranging from 37 to 53%; however, high homology is observed with Rad23 of Bombyx mori (BmRad23). UBQ domain region alignment revealed maximum of 66% homology with Rad23 of Apis melifera (AmRad23). MjRad23 clustered with invertebrate sector along with insect species in evolution analysis. Three‐dimensional structural analyses demonstrated the highest identity between MjRad23 and human Rad23A (hHR23A). Conclusions: The present work revealed the presence of MjRad23 gene, which is essential in DNA repair process. Further studies are required to clarify the involvement of MjRad23 in NER process. Significance and Impact of the Study: This is the first report on identification and characterization of DNA repair protein in crustaceans, which will lead to further investigation to explore the molecular mechanisms behind the NER process.     

Modulation of anti-inflammatory response in lipopolysaccharide stimulated human THP-1 cell line and mouse model at gene expression level with indigenous putative probiotic lactobacilli

The anti-inflammatory potential of eight indigenous probiotic Lactobacillus isolates was evaluated in vitro in terms of modulating the expression of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) in human acute monocytic leukemia (THP-1) cells under inflammatory conditions. Amongst these, Lactobacillus plantarum Lp91 was the most potent anti-inflammatory strain as it evoked a significant (P < 0.001) down-regulation of TNF-α by −1.45-fold relative to the control in THP-1 cells. However, in terms of IL-6 expression, all the strains could up-regulate its expression considerably at different levels. Hence, based on in vitro expression of TNF-α, Lp91 was selected for in vivo study in lipopolysaccharide (LPS)-induced mouse model to look at the expression of TNF-α, IL-6, monocyte chemotactic protein-1 (MCP-1), vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule (ICAM-1) and E-selectin in mouse aorta. In LPS challenged (2 h) mice group fed with Lp91 for 10 days, TNF-α, IL-6, MCP-1, VCAM-1, ICAM-1 and E-selectin expressions were significantly down-regulated by 3.10-, 10.02-, 4.22-, −3.14-, 2.28- and 5.71-fold relative to control conditions. In conclusion, Lp91 could serve as a candidate probiotic strain to explore it as a possible biotherapeutic anti-inflammatory agent against inflammatory diseases including cardiovascular disease.

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