Hydrogen sulfide attenuates homocysteine-induced osteoblast dysfunction by inhibiting mitochondrial toxicity

Homocysteine (Hcy) is detrimental to bone health in a mouse model of diet-induced hyperhomocysteinemia (HHcy). However, little is known about Hcy-mediated osteoblast dysfunction via mitochondrial oxidative damage. Hydrogen sulfide (H₂S) has potent antioxidant, anti-inflammatory, and antiapoptotic effects. In this study, we hypothesized that the H₂S mediated recovery of osteoblast dysfunction by maintaining mitochondrial biogenesis in Hcy-treated osteoblast cultures in vitro. MC3T3-E1 osteoblastic cells were exposed to Hcy treatment in the presence or absence of an H₂S donor (NaHS). Cell viability, osteogenic differentiation, reactive oxygen species (ROS) production were determined. Mitochondrial DNA copy number, adenosine triphosphate (ATP) production, and oxygen consumption were also measured. Our results demonstrated that administration of Hcy increases the intracellular Hcy level and decreases intracellular H₂S level and expression of the cystathionine β-synthase/Cystathionine γ-lyase system, thereby inhibiting osteogenic differentiation. Pretreatment with NaHS attenuated Hcy-induced mitochondrial toxicity (production of total ROS and mito-ROS, ratio of mitochondrial fission (DRP-1)/fusion (Mfn-2)) and restored ATP production and mitochondrial DNA copy numbers as well as oxygen consumption in the osteoblast as compared with the control, indicating its protective effects against Hcy-induced mitochondrial toxicity. In addition, NaHS also decreased the release of cytochrome c from the mitochondria to the cytosol, which induces cell apoptosis. Finally, flow cytometry confirmed that NaHS can rescue cells from apoptosis induced by Hcy. Our studies strongly suggest that NaHS has beneficial effects on mitochondrial toxicity, and could be developed as a potential therapeutic agent against HHcy-induced mitochondrial dysfunction in cultured osteoblasts in vitro.     

Variability in ionization state,stoichiometry and aggregation in histidine complexes with formic acid

The crystal structures of the complexes of L and DL histidine with formic acid have been determined as part of an effort to define biologically and evolutionarily important interactions and aggregation patterns. In terms of ionization state and stoichiometry they may be described as L-histidine formate formic acid and DL-histidine formate monohydrate respectively. In the L-histidine complex, amino acid molecules arranged in head-to-tail sequences centred around 2₁ screw axes are interconnected by formic acid molecules and formate ions. Histidine-formate interactions in the structure gives rise to a characteristic interaction pattern involving a linear array of alternating imidazole groups and formate ions. In DL-histidine formale monohydrate, head-to-tail sequences involving glide related molecules are interconnected through main chain-side chain interactions leading to amino acid layers. The layers are held together by formate ions and water molecules arranged in strings along which the ion and the molecule alternate. The patterns of amino acid aggregation in histidine complexes exhibit considerably higher variability than those in complexes involving arginine and lysine do. X-ray studies on crystalline complexes involving amino and peptides Part XXIX.     

Rapid and Efficient Synthesis of Peptides Containing α,α-dialkylamino acids employing KOBt

Summary Several Fmoc-α,α-dialkylamino acids and their acid chlorides have been prepared, isolated and characterised. The synthesis of peptides containing sterically hindered dialkylamino acids has been accomplished using acid chloride/KOBt in dichloromethane. The yields as well as the purity of the peptides were satistactory.     

Production of interleukin-1 and tumour necrosis factor in non-Hodgkin's lymphoma patients

Summary Peripheral blood monocytes from non-Hodgkin's lymphoma (NHL) patients were assessed for the monocyte functions with respect to their ability to secrete interleukin-1 and tumour necrosis factor (TNF) and their cytotoxic potential to tumour target WEHI 164 clone 13. Our results indicate comparable levels of interleukin-1 and TNF production by NHL patients. The cytotoxic potential by monocytes was also not depressed in these patients. The data obtained suggest normal monocyte functions in NHL patients.     

Clinical evaluation of a new two-site assay for CA125 antigen

As appropriate surgery and chemotherapy can improve both quality of life and survival of patients with ovarian adenocarcinoma, there has been a pressing need for "serodiagnostic" assays to enable close patient monitoring. CA 125 antigen has previously been described as a useful tumor marker of ovarian cancer. This is the first clinical evaluation of a radioimmunoassay using two new monoclonal antibodies, B27.1 and B43.13, that react with separate sites on the glycoprotein marker CA 125. Using the new assay, the majority of patients with clinically or radiologically detectable disease had serum CA 125 antigen levels well above the upper limit seen with random apparently healthy donors, while only three patients who were believed free of disease had elevated levels. Disease progression was associated with increasing values of serum CA 125 antigen, while response to therapy was associated with a steady decline in serum CA 125 antigen levels. Seven patients had steadily rising serum CA 125 antigen levels after initially having normal levels. The mean lead time between rise above normal and clinical or radiological evidence of relapse was 5 months (range 2 to 12 months). The merits of further surgical intervention are illustrated by the serial values of two patients followed after chemotherapy. The assay appears to have value in monitoring response to therapy and in detecting disease relapse at a time when appropriate therapeutic intervention is still possible or likely to be beneficial. Furthermore, monitoring CA 125 antigen was shown to be of benefit in assessing response to chemotherapy in a few patients with metastatic adenocarcinoma of unknown primary, and may be useful in this group of patients in determining those likely to benefit from aggressive chemotherapy.     

Transposon Tn5 as an identifiable marker in rhizobia: Survival and genetic stability of Tn5 mutant bean rhizobia under temperature stressed conditions in desert soils

Five transposon Tn5 insertion mutants of a beanRhizobium strain (Rhizobium leguminosarum b. v.phaseoli) were used in an ecological study to evaluate the extent to which transposon Tn5 was stable to serve as an identifiable marker in rhizobia under a high temperature stress condition in two Sonoran Desert soils. All the mutants possessed single chromosomal insertions of the transposon. In both soils, under the temperature stress conditions that were employed (40°C), both wild type and mutant populations possessing functional transposable elements declined rapidly. After 12 days, mutant cells, when screened using the Tn5 coded antibiotic resistance markers, were significantly less in number than when they were screened using only their intrinsic antibiotic resistance markers. There were no significant differences in numbers between the mutant cell population and the wild type when the mutant cells were screened using only the intrinsic antibiotic resistance markers. DNA-DNA hybridizations using a probe indicated neither deletion nor transposition of the transposable element. The results indicate that transposon DNA sequences are present within cells under high temperature stress conditions, but kanamycin/neomycin resistance is not expressed by some of these cells, suggesting that Tn5 undergoes a possible functional inactivation under these conditions. The possible implications of these findings are discussed.     

Shifts in protein metabolism in some organs of freshwater fish, Cyprinus carpio under mercury stress

Changes in the protein metabolism of gill, kidney and intestine of freshwater fish, Cyprinus carpio exposed to 1, 15 and 30 days to sublethal concentration (0.1 mg/l) of mercury were studied. The total, soluble and structural protein contents recorded the depletion followed by progressive increase in accumulation of free aminoacids. Concurrently, the activity of protease in the tissues was also increased. A steady enhancement in the activities of aspartate aminotransferase and alanine aminotransferase paralleled the elevation of glutamate dehydrogenase activity in the organs studied. Levels of ammonia and urea have also reported elevation. All these changes clearly documented the induction of severe proteolysis. The magnitude of these changes increased overtime. These changes were more in the gill at the initial periods of exposure (1 and 15 days), but as the period of exposure increased, these changes were more pronounced in the kidney at 30 days of exposure to sublethal concentration of mercury.     

Phospholipid methylation of kidney cortex brush border membranes. Effect on fluidity and transport

Exposure of intact brush border membrane vesicles of hog kidney cortex to cholesterol oxidase resulted in 24% oxidation of membrane cholesterol compared with more than 95% oxidation of cholesterol in lipids isolated from membranes, showing that cholesterol is asymmetrically distributed in membranes. Phospholipase C, hydrolyzed 76% of phosphatidylcholine and 10–12% phosphatidylethanolamine while phosphatidylserine was not hydrolyzed, thus indicating that majority of phosphatidylcholine is present on the outer surface of these vesicles while phosphatidylethanolamine and phosphatidylserine are present on the inner surface. Methylation of phospholipids in brush border membrane with $\text{S-}\text{adenosyl}\text{-[methyl-}{}^3\text{H}\text{]}\text{methionine}$ resulted in the formation of $\text{phosphatidyl}\text{-N-}\text{monomethylethanolamine}$, $\text{phosphatidyl}\text{-N,N-}\text{dimethylethanolamine}$ and phosphatidylcholine from endogenous phosphatidylethanolamine. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for $\text{S-}\text{adenosylmethionine}$ was 1·10^?4 M with an optimum pH 9.0 for the formation of all three methyl derivatives. Mg²⁺ was without any effect between pH 5 to 10. Addition of exogenous mono- and dimethylphosphatidylethanolamine derivatives enhanced methyl group incorporation by 4–5-fold as compared to the addition of phosphatidylethanolamine. The conversion of endogenous phosphatidylethanolamine to $\text{phosphatidyl}\text{-N-}\text{monomethylethanolamine}$ or addition of exogenous phosphatidylmonomethylethanolamine to brush border membrane did not result in a change in bulk membrane fluidity as determined by fluorescence polarization of diphenylhexatriene. Methylation of phosphatidylethanolamine in brush border membrane did not affect the Na⁺-dependent uptake of either d-glucose or phosphate, although the accessibility of cholesterol in membrane to cholesterol oxidase was diminished by 21%, presumably due to altered flip-flop movement of cholesterol in the membrane.     

γ-Glutamyl transpeptidase: catalytic,structural and functional aspects

Summary -Glutamyl transpeptidase catalyzes transfer of the -glutamyl moiety of glutathione to amino acids, dipeptides, and to glutathione itself; the enzyme also catalyzes the hydrolysis of glutathione to glutamate and cysteinyl-glycine. This review deals with the tissue distribution and localization of the enzyme in mammals, the catalytic properties of the enzyme (including its inhibition by reversible and irreversible inhibitors), structural studies on the enzyme, and new findings about its physiological function.