Antidiarrheal effect of sodium hydrosulfide in diabetic rats: In vitro and in vivo studies.

BACKGROUND: The inhibitory effects of H2 S on spontaneous contractions of smooth muscles of small, and large intestines well-established but its role in the pathophysiology of diarrhea has not been identified. Therefore, this study evaluated the role of exogenous H2 S (NaHS) on diabetic-induced diarrhea and determined mRNA expression of cystathionine ß-lyase (CSE) and cystathionine γ-synthase (CBS) in diabetic rats. METHODS: In order to evaluate antidiarrheal effect of H2 S, normal and diabetic rats received NaHS and L-Cysteine and the total number of fecal pellets (FP) determined. The effect of NaHS on intestinal transit ratio (ITR) was also evaluated in diabetic rats. The level of mRNA expressions of CBS and CSE determined in smooth muscles of jejunum, ileum, and colon in normal, and diabetic rats. The effect of NaHS on frequency and tension of spontaneous contractions of smooth muscle strips of colon, ileum, and jejunum were investigated. KEY RESULTS: NaHS decreased ITR, total number of FP, frequency and tension of spontaneous contractions of colon, ileum, and jejunum muscle strips in diabetic rats. The level of mRNA expression of CSE and CBS in diabetic rats were lower than in normal rats. NaHS, and L-Cysteine decreased the number of FP in normal rats. CONCLUSIONS & INFERENCES: These findings showed NaHS effectively controlled diarrhea in diabetic rats through decreasing the frequency, and tension of spontaneous contraction of smooth muscles of large, and small intestines. The increased frequency and tension of spontaneous contractions of smooth muscles in diabetic rats may be due to down-regulation of H2 S biosynthesis enzymes.

Improved immunodetection of nuclear antigens after sodium dodecyl sulfate treatment of formaldehyde-fixed cells.

Immunostaining techniques are commonly employed to determine the temporal and spatial patterns of cellular components in in situ preparations of cells and tissues. Usually, cells are formalin-fixed, permeabilized with nonionic detergents, and probed with specific antibodies. The incorporation of a sodium dodecyl sulfate (SDS) treatment after chemical crosslinking has been shown to improve the immunodetection of some cytosolic and cell surface antigens. By incorporating an SDS treatment after crosslinking, we report a significant improvement in the detection of two nuclear antigens (i.e.) the DNA binding proteins apurinic/apyrimidinic endonuclease and DNA polymerase-beta) and bromodeoxyuridine-tagged DNA by indirect immunofluorescence of whole cells. In bromodeoxyuridine-tagged DNA, the improvement in detection after an SDS treatment was observed only after long incorporation protocols (>48 hr) and, interestingly, it was more pronounced in cultured human foreskin keratinocytes than in bovine aorta endothelial cells. In addition, the SDS treatment proved in these studies to be superior to the standard Triton X-100 permeabilization. SDS thus provides a potential means to visualize previously undetectable or poorly detectable nuclear antigens.

Intragenic suppression of an active site mutation in the human apurinic/apyrimidinic endonuclease.

The apurinic/apyrimidinic endonucleases (APE) contain several highly conserved sequence motifs. The glutamic acid residue in a consensus motif, LQE96TK98 in human APE (hAPE-1), is crucial because of its role in coordinating Mg2+, an essential cofactor. Random mutagenesis of the inactive E96A mutant cDNA, followed by phenotypic screening in Escherichia coli, led to isolation of an intragenic suppressor with a second site mutation, K98R. Although the Km of the suppressor mutant was about sixfold higher than that of the wild-type enzyme, their kcat values were similar for AP endonuclease activity. These results suggest that the E96A mutation affects only the DNA-binding step, but not the catalytic step of the enzyme. The 3' DNA phosphoesterase activities of the wild-type and the suppressor mutant were also comparable. No global change of the protein conformation is induced by the single or double mutations, but a local perturbation in the structural environment of tryptophan residues may be induced by the K98R mutation. The wild-type and suppressor mutant proteins have similar Mg2+ requirement for activity. These results suggest a minor perturbation in conformation of the suppressor mutant enabling an unidentified Asp or Glu residue to substitute for Glu96 in positioning Mg2+ during catalysis. The possibility that Asp70 is such a residue, based on its observed proximity to the metal-binding site in the wild-type protein, was excluded by site-specific mutation studies. It thus appears that another acidic residue coordinates with Mg2+ in the mutant protein. These results suggest a rather flexible conformation of the region surrounding the metal binding site in hAPE-1 which is not obvious from the X-ray crystallographic structure.

Neuroprotective effects of an antioxidant in cortical cerebral ischemia: prevention of early reduction of the apurinic/apyrimidinic endonuclease DNA repair enzyme.

We examined the effects of the free radical scavenger, 21-aminosteroid, on apurinic/apyrimidinic endonuclease (APE/Ref-1) protein expression and subsequent infarction volume after photothrombotic cortical cerebral ischemia in mice. Immunohistochemistry and Western blot analysis showed a significant reduction in APE/Ref-1 expression 6 and 24 h after ischemia in untreated animals, whereas in drug-treated animals the reduction was much less at the same time points. The administration of 21-aminosteroid significantly decreased subsequent infarction volume 3 days after ischemia. These data suggest that 21-aminosteroid prevents the early decrease of APE/Ref-1 expression, thereby reducing cortical infarction after photothrombotic cerebral ischemia.