Nitric oxide-induced S-glutathionylation and inactivation of glyceraldehyde-3-phosphate dehydrogenase.
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S-Nitrosylation of protein thiol groups by nitric oxide (NO) is a widely recognized protein modification. In this study we show that nitrosonium tetrafluoroborate (BF4NO), a NO+ donor, modified the thiol groups of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by S-nitrosylation and caused enzyme inhibition. The resultant protein-S-nitrosothiol was found to be unstable and to decompose spontaneously, thereby restoring enzyme activity. In contrast, the NO-releasing compound S-nitrosoglutathione (GSNO) promoted S-glutathionylation of a thiol group of GAPDH both in vitro and under cellular conditions. The GSH-mixed protein disulfide formed led to a permanent enzyme inhibition, but upon dithiothreitol addition a functional active GAPDH was recovered. This S-glutathionylation is specific for GSNO because GSH itself was unable to produce protein-mixed disulfides. During cellular nitrosative stress, the production of intracellular GSNO might channel signaling responses to form protein-mixed disulfide that can regulate intracellular function. (+info)
Arsenic-related Bowen's disease, palmar keratosis, and skin cancer.
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Chronic arsenical intoxication can still be found in environmental and industrial settings. Symptoms of chronic arsenic intoxication include general pigmentation or focal "raindrop" pigmentation of the skin and the appearance of hyperkeratosis of the palms of the hands and soles of the feet. In addition to arsenic-related skin diseases including keratosis, Bowen's disease, basal-cell-carcinoma, and squamous-cell carcinoma, there is also an increased risk of some internal malignancies. Arsenic-related diseases are common in areas of the world where the drinking water has a high arsenic content. In this paper, we describe a 35-year-old male patient who had arsenic-related keratosis, squamous-cell carcinoma in the palmar area of his left hand, and Bowen's disease on his left thigh. The patient worked in a borax mine for 15 years, so he was exposed to arsenic in drinking water, airborne arsenic in his workplace, and had direct contact. The patient was treated for 11 months for arsenic-related keratosis until an axillary lymph node metastasis occurred; the lesion was excised and diagnosed to be malignant. Bowen's disease was detected when the patient was being treated for cancer. No other malignancy was found. The patient is still receiving regular follow-up care. (+info)
Chloride channels of glycine and GABA receptors with blockers: Monte Carlo minimization and structure-activity relationships.
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GABA and glycine receptors (GlyRs) are pentameric ligand-gated ion channels that respond to the inhibitory neurotransmitters by opening a chloride-selective central pore lined with five M2 segments homologous to those of alpha(1) GlyR/ ARVG(2')LGIT(6')TVLTMTTQSSGSR. The activity of cyanotriphenylborate (CTB) and picrotoxinin (PTX), the best-studied blockers of the Cl(-) pores, depends essentially on the subunit composition of the receptors, in particular, on residues in positions 2' and 6' that form the pore-facing rings R(2') and R(6'). Thus, CTB blocks alpha(1) and alpha(1)/beta, but not alpha(2) GlyRs (Rundstrom, N., V. Schmieden, H. Betz, J. Bormann, and D. Langosch. 1994. Proc. Natl. Acad. Sci. U.S.A. 91:8950-8954). PTX blocks homomeric receptors (alpha(1) GlyR and rat rho(1) GABAR), but weakly antagonizes heteromeric receptors (alpha(1)/beta GlyR and rho(1)/rho(2) GABAR) (Pribilla, I., T. Takagi, D. Langosch, J. Bormann, and H. Betz. 1992. EMBO J. 11:4305-4311; Zhang D., Z. H. Pan, X. Zhang, A. D. Brideau, and S. A. Lipton. 1995. Proc. Natl. Acad. Sci. U.S.A. 92:11756-11760). Using as a template the kinked-helices model of the nicotinic acetylcholine receptor in the open state (Tikhonov, D. B., and B. S. Zhorov. 1998. Biophys. J. 74:242-255), we have built homology models of GlyRs and GABARs and calculated Monte Carlo-minimized energy profiles for the blockers pulled through the pore. The profiles have shallow minima at the wide extracellular half of the pore, a barrier at ring R(6'), and a deep minimum between rings R(6') and R(2') where the blockers interact with five M2s simultaneously. The star-like CTB swings necessarily on its way through ring R(6') and its activity inversely correlates with the barrier at R(6'): Thr(6')s and Ala(2')s in alpha(2) GlyR confine the swinging by increasing the barrier, while Gly(2')s in alpha(1) GlyR and Phe(6')s in beta GlyR shrink the barrier. PTX has an egg-like shape with an isopropenyl group at the elongated end and the rounded end trimmed by ether and carbonyl oxygens. In the optimal binding mode to alpha(1) GlyR and rho(1) GABAR, the rounded end of PTX accepts several H-bonds from Thr(6')s, while the elongated end enters ring R(2'). The lack of H-bond donors on the side chains of Phe(6')s (beta GlyR) and Met(6')s (rho(2) GABAR) deteriorates the binding. The hydrophilic elongated end of picrotin does not fit the hydrophobic ring of Pro(2')s/Ala(2')s in GABARs, but fit a more hydrophilic ring with Gly(2')s in GlyRs. This analysis provides explanations for structure-activity relationships of noncompetitive agonists and predicts a narrow pore of LGICs in agreement with experimental data on the permeation of organic cations. (+info)
Recognition control of the nucleic acid model through conformational switching of nucleobase induced by borate ester formation of cis-2',3'-diol.
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A novel nucleic acid model that possessed 5'-amino-5'-deoxyuridine at alpha- and gamma-position of L-glutamic acid through amide linkage using 5'-amino group was synthesized and the conformation and the hybridization properties were studied. The complex of alpha-PRNA with complementary DNA/RNA was more stable than the corresponding natural duplex in the absence of borate. Its recognition ability was however lost when borax was added to the solution. (+info)
Identification of functional arginine residues in ribonuclease A and lysozyme.
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A specific color reaction has been developed for the detection of N-7, N-8-(1,2-dihydroxycyclohex-1,2-ylene)-L-arginine-containing peptides. The reaction is based on the fact that hydroxylamine converts the blocking group to cyclohexanedione dioxime, which forms a red nickel complex. N-7, N-8-(1,2-dihydroxycyclohex-1,2-ylene)-L-arginine-containing peptides can also be detected by diagonal electrophoresis from the change of electrophoretic mobility of these peptides on interaction of the blocking group with borate. Since the modified arginine residues are resistant to tryptic cleavate, changes in tryptic peptide patterns can also be utilized to identify the presence of modified arginine residues. A combination of these approaches was used to identify the arginine residues modified by cyclohexanedione treatment. Bovine panctreatic RNase A loses approximately 90% of its activity on cyclohexanedione treatment with the modification of 2 to 3 arginine residues. Arginine-39 reacts most rapidly and its modification contributes most to inactivation of the enzyme. Arginine-85 also reacts rapidly with cyclohexanedione. Arginine-10 reacts slowly and no reaction was observed with arginine-33. Removal of the blocking groups by hydroxylamine treatment resulted in complete recovery of enzyme activity in samples where arginine-39 and arginine-85 had been modified, whereas 80% of activity was regained from samples where arginine-10 had also been modified. With egg white lysozyme, all 11 arginine residues react with cyclohexanedione, resulting in partial inactivation of the enzyme. The fully modified enzyme retains 35% of its activity. Since arginine residues are important for electrostatic interaction between the enzyme and the negatively charges cell surface, even the modified, basic residues can provide the necessary positive charges. In the presence of borate, activity is almost completely abolished, since the modified arginine-borate complex has a reduced net positive charge. Upon removal of the blocking groups by hydroxylamine, even the fully modified lysozyme regains complete activity. With the exception of the most reactive arginine (residue 5), modification of all other arginine residues contributes equally to inactivation of the enzyme. The possible reason for the importance of arginine-5 in maintaining activity is discussed. Advantages of the present method for the selective reversible modification of arginine residues of proteins and for the identification of reactive arginine residues are evaluated. (+info)
The amino acid profiles of the whole plant and of four plant residues from temperate and tropical forages.
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This study compared the amino acid (AA) profile of five residues (original forage, borate-phosphate buffer residue (BPR), neutral detergent fiber residue with (NDF+) and without (NDF-) sodium sulfite, and acid detergent fiber residue (ADF). Fourteen grasses and legumes from tropical and temperate regions were used in this study. The use of sodium sulfite did not affect the NDF concentration, but the NDF insoluble protein was lower (P < 0.05) in the NDF+ than in the NDF- (3.9 vs 4.5% DM, respectively). For all of the amino acids tested, the amino acid content, expressed as a percentage of CP, was lower in the ADF residue than in the original forage. There were no differences in the amino acid concentrations of the NDF- and NDF+ extracts (P > 0.05). Only in the case of methionine was there a difference in the amount of amino acid when the original forage was compared with the BPR (1.84 vs 1.45 % CP). When the AA profile of each residue was corrected for the AA content of the ADF, no difference was observed between the AA profile of the original forage and of the BPR (P > 0.05). Similar to the result without correction for the amino acids in ADF, the AA profiles of the NDF+ and NDF- fractions were similar (P > 0.05). From this result, we infer that the sodium sulfite had similar effects on all AA in the NDF residue that we tested. There were differences in amino acid concentrations in the original forage and the NDF residues for several amino acids (Met, Cys, Lys, Thr, Arg, Ile, Leu, and Phe) (P < 0.05). When the amino acid values of the original forage and the BPR were used with animal data in the Cornell Net Carbohydrate and Protein System model, few differences in animal predicted performance were evident. These findings suggest that the AA profile of the original forage can be used to predict the AA profile of the undegraded intake protein instead of using the borate-phosphate buffer residue for amino acid analyses. This would simplify obtaining feed amino acid values for use in the Cornell Net Carbohydrate and Protein System. (+info)
Brief exposure to low-pH stress causes irreversible damage to the growing root in Arabidopsis thaliana: pectin-Ca interaction may play an important role in proton rhizotoxicity.
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The viability of Arabidopsis thaliana (strain Landsberg) roots exposed to a low pH (4.5 or 4.7) solution that contained 100 microM CaCl(2) was examined by staining with fluorescein diacetate-propidium iodide. The elongation zone of growing roots lost viability within 1-2 h following exposure to low pH, but non-growing roots showed no damage under the same treatment. Low-pH damage in growing roots was irreversible after 1 h incubation at pH 4.5 as judged by regrowth in growing medium at pH 5.6. Growing lateral roots also lost viability in the same treatment, whereas non-growing lateral roots remained viable during and after the treatment. The low-pH damage was ameliorated by the simultaneous application of calcium, indicating the involvement of a calcium-requiring process in overcoming proton toxicity. At pH 5.0, growing roots required 25 microM of calcium to maintain elongation, and at pH 4.8 and pH 4.5 more than 250 microM and 750 microM, respectively. The low-pH damage was ameliorated by divalent cations in the order of Ba2+, approximately Sr2+>/=Ca2+>Mg2+. The monovalent cation K+ showed no ameliorative effect, but borate showed a strong ameliorative effect with Ca2+. These results indicate that the primary target of proton toxicity may be linked to a disturbance of the stability in the pectic polysaccharide network, where calcium plays a key role in plant roots. (+info)
Inflammatory response, growth, and thyroid hormone concentrations are affected by long-term boron supplementation in gilts.
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An experiment was conducted to determine the long-term effects of dietary boron (B) on growth performance, immune function, and plasma and serum characteristics in gilts. Fifty weanling gilts were allotted to 10 pens based on weaning weight and litter origin. Pens were randomly assigned to receive one of two dietary treatments. Treatments consisted of a basal diet low in B (control) and the basal diet supplemented with 5 mg B/kg diet as sodium borate. Gilts remained on their respective experimental diets and with their penmates throughout the nursery, growing, and finishing phases. The B concentration of the basal diet was 0.98, 2.1, and 2.2 mg/kg diet during the nursery, growing, and finishing phases, respectively. At the end of each production phase, animals were weighed and feed consumption was determined to assess growth performance variables. In addition, blood samples were obtained from three randomly selected gilts per pen at the completion of each phase. Boron had no affect (P > 0.58) on growth performance during the nursery phase, but gilts receiving supplemental B had increased (P < 0.05) ADG at the end of the finishing phase and over the entire growing-finishing period. Serum concentrations of triiodothyronine (T3) tended (P < 0.07) to be reduced by dietary B at the end of the nursery phase, but serum thyroxine (T4) was not affected (P = 0.46) by B. At the completion of the growing phase, supplemental B decreased (P < 0.05) the concentrations of T3 and T4 in the serum. In addition, serum concentrations of total cholesterol and the activity of alkaline phosphatase were increased (P < 0.05) by dietary B at the end of the growing phase. Serum concentrations of urea N tended (P < 0.09) to be increased by B at the end of the growing phase. Beginning at d 95 of the experimental period, measures of immune function were assessed in randomly selected gilts. Boron decreased (P < 0.05) the inflammatory response to an intradermal injection of phytohemagglutinin. Boron did not affect (P > 0.30) the blastogenic response of isolated lymphocytes to mitogen stimulation or the humoral immune response against a sheep red blood cell suspension. Results indicate that B may affect serum thyroid hormone concentrations, the inflammatory response, and growth in pigs. (+info)