Cloning, expression, and characterization of a glycoside hydrolase family 50 beta-agarase from a marine Agarivorans isolate.

The gene for a thermostable beta-agarase from Agarivorans sp. JA-1 was cloned and sequenced. It comprised an open reading frame of 2,988 base pairs, which encode a protein of 109,450 daltons consisting of 995 amino acid residues. A comparison of the entire sequence showed that the enzyme has 98.8% sequence similarities to beta-agarase from Vibrio sp. JT1070, indicating that it belongs to the family glycoside hydrolase (GH)-50. The gene corresponding to a mature protein of 976 amino acids was inserted and expressed in Escherichia coli. The recombinant beta-agarase was purified to homogeneity. It had maximal activity at 40 degrees C and pH 8.0 in the presence of 1 mM NaCl and 1 mM CaCl(2). The enzyme hydrolyzed agarose as well as neoagarohexaose and neoagarotetraose to yield neoagarobiose as the main product. Thus, the enzyme would be useful for the industrial production of neoagarobiose.

Increased expression of phospholipase D in the heart with experimental autoimmune myocarditis in Lewis rats.

The expression of phospholipase D (PLD) in the hearts of rats with experimental autoimmune myocarditis (EAM) was studied to elucidate the functional role of PLD in the pathogenesis of EAM. Western blot analysis showed that the level of the PLD1 isoform was significantly increased in the hearts of rats with EAM on days 14, 17 and 21 postimmunization (pi) (P < 0.01; control vs EAM at 14 pi, 17 pi and 21 pi). The phenotypes of cells exhibiting increased PLD1 expression were primarily inflammatory cells, including ED1 positive macrophages, in the inflammatory EAM lesions. Some cardiomyocytes also showed increased PLD1 immunoreaction in and around EAM lesions. Some PLD1-positive cells were also positive for proliferating cell nuclear antigen in some cardiomyocytes or terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling in some macrophages, suggesting that PLD1 positive cells have a capacity for proliferation or apoptosis depending on cell types in the target organ. Thus, it is postulated that increased expression of PLD1 in EAM may support an early inflammatory response in proliferating inflammatory cells, and its expression in cardiomyocytes may help them to survive by activation of survival factors in hearts with EAM.

Sodium salicylate-induced amelioration of experimental autoimmune encephalomyelitis in Lewis rats is associated with the suppression of inducible nitric oxide synthase and cyclooxygenases.

We studied the effects of oral administration of sodium salicylate on the expression of the pro-inflammatory mediators, nitric oxide synthase (iNOS) and cyclooxygenase- (COX-) 1 and 2, in rats with experimental autoimmune encephalomyelitis (EAE). Sodium salicylate (200 mg/kg) was administered orally for 13 days after the induction of EAE by immunization with guinea pig myelin basic protein and complete Freund's adjuvant. The onset (P<0.0001) and severity (P<0.05) of EAE paralysis in salicylate-treated animals were delayed and suppressed significantly compared with vehicle-treated controls. Western blot analysis showed that expression of COX-2 and iNOS, but not COX-1, decreased significantly in the spinal cords of salicylate-treated rats compared with vehicle-treated controls (P<0.05) and this finding was paralleled by immunohistochemical observations. These results suggest that the amelioration by salicylate of paralysis in rats with EAE is mediated in part by the suppression of COX and iNOS.