High blood glucose and osmolality, but not high urinary glucose and osmolality, affect neuronal nitric oxide synthase expression in diabetic rat kidney.

We recently demonstrated that neuronal nitric oxide synthase (nNOS) messenger RNA (mRNA) is markedly increased in the kidneys of diabetic rats and water-deprived rats. It can be inferred that high plasma glucose and osmolality and high renal tubular glucose and osmolality are somehow involving in renal NOS synthesis in diabetic rats. Phlorizin, a competitive inhibitor of glucose transport in the proximal tubule, causes renal glycosuria in nondiabetic rats and reverses hyperglycemia in diabetic rats. To further investigate whether high plasma glucose and osmolality or high renal tubular glucose and osmolality influence renal NOS synthesis in diabetic rats, we measured nNOS mRNA levels in phlorizin-treated normal and diabetic rats. Neuronal NOS mRNA expression in the kidneys was not significantly different between normal rats and phlorizin-treated normal rats with high urinary glucose and osmolality. The phlorizin-treated diabetic rats showed a significant decrease in the ratio of nNOS to beta-actin mRNA compared with diabetic rats. On linear-regression analysis, plasma glucose was strongly positively correlated with nNOS mRNA expression in the cortex, outer medulla, and inner medulla (r(2) =.378, r(2) =.680, and r(2) =.445, respectively) of rat kidneys. Neither urine glucose concentration nor urine osmolality was correlated with nNOS mRNA expression in rat kidneys. In conclusion, our results indicate that nNOS mRNA expression in the kidneys of diabetic rats is directly affected by high blood glucose/osmolality but not by high urinary glucose or osmolality.

Enrichment of the antibodies against the C-terminus of Taiwan cobra cobrotoxin using dimeric glutaraldehyde-modified toxin as an immunogen.

The repertoire of antibodies producing by immunizing rabbits with cobrotoxin and dimeric glutaraldehyde-modified cobrotoxin (dGA-cobrotoxin) was analyzed by studying the immunoreactivity of the two antibody preparations toward cobrotoxin, GA-cobrotoxin and recombinant cobrotoxin. The results of enzyme-linked immunoassay revealed that the two antibody preparations exhibited a higher reactivity against their cognate antigen. Moreover, different behavior was observed for the reactivity of the two antibody preparations against GA-cobrotoxin and recombinant cobrotoxin. Notably, distortion of disulfide linkages at the C-terminus resulted in a reduced decrease in the antigenic activity of recombinant cobrotoxin toward anti-cobrotoxin antibodies compared to anti-dGA-cobrotoxin antibodies. Affinity purification of the antibodies against the C-terminus of cobrotoxin revealed that its amount represented 77% and 35.5% of the total anti-dGA-cobrotoxin antibodies and the total anti-cobrotoxin antibodies, respectively. These findings suggest that the antibody preparation elicited by dGA-cobrotoxin enriches the content of antibodies recognizes the C-terminal region of native cobrotoxin.

Characterization, primary structure and molecular evolution of anticoagulant protein from Agkistrodon actus venom.

An anticoagulant protein named AaACP was isolated from Agkistrodon actus (hundred-pace snake of Taiwan, Viperidae) venom. AaACP inhibited the factor Xa-induced plasma coagulation in a concentration-dependent manner. Thus, AaACP seems to bind to factor Xa in prothrombinase complex. AaACP was composed of A and B chains linked by disulphide bond(s). The amino acid sequences of A and B chains of AaACP were analysed with a few residues unidentified which were complemented from the nucleotide sequences of their cDNAs. The A chain consisted of 129 amino acid residues and the B chain 123 amino acid residues. Their amino acid sequences were highly similar to those of A and B chains of a series of anticoagulant proteins which had been purified from the venoms of some Viperidae snakes. The A and B chains structurally belong to C-type lectin-like protein family of snake venom origin. Construction of phylogenetic tree of C-type lectins and C-type lectin-like proteins based on their amino acid sequences indicated that their A and B chains diverged before speciation of snake species. The comparison of the nucleotide sequences of the cDNAs encoding A and B chains of AaACP and of Trimeresurus flavoviridis (Viperidae) venom-gland factors IX/X-binding protein and factor IX-binding protein showed that the mature protein-coding region is much more variable than the signal peptide-coding domain and the 5'- and 3'-untranslated regions, being in contrast to the case of the ordinary isoprotein genes. The ratios of the numbers of nucleotide substitutions per nonsynonymous site (K(A)) and per synonymous site (K(S)) in the mature protein-coding region in the cDNA pairs were about three times greater than those for the ordinary isoprotein genes, suggesting that these genes have been evolving in an accelerated manner. Taking account of the functional diversities of venom-gland C-type lectins and C-type lectin-like proteins including factors IX and/or X-binding proteins, it can be said that their functional diversities have been acquired by accelerated evolution.