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Thiols as peroxidase substrates.

The abilities of haem peroxidases to catalyse the oxidation of various thiols were studied using the spin-trapping electron spin resonance (ESR) technique. Myeloperoxidase, a neutrophil and monocyte enzyme, catalysed the oxidation of cysteamine, cysteine methyl, and ethyl ester and to some extent 2-mercaptoethanol and thioglycollic acid. This peroxidase poorly catalysed the oxidation of cysteine, N-acetylcysteine, penicillamine, and glutathione under the same conditions. The dependence on pH of peroxidase-catalysed thiol oxidation may indicate that the thiolate anion form is the actual peroxidase substrate. Another leucocyte peroxidase, eosinophil peroxidase, had similar catalytic properties toward thiols as myeloperoxidase. Lactoperoxidase (found in milk, saliva, and tears) and the plant horseradish peroxidase were, however, different from the aforementioned leucocyte peroxidases in their abilities to catalyse the oxidation of thiols.

Catalysis↗

Thioether adducts of a new imine reactive intermediate of the pneumotoxin 3-methylindole.

Cytochrome P450 enzymes can potentially oxygenate 3-methylindole to form 2,3-epoxy-3-methylindoline which could rearrange to the stable metabolite 3-methyloxindole or open to form 3-hydroxy-3-methylindolenine, a putative electrophilic imine. The purpose of the current work was to determine if the imine was formed, and to characterize it via its adducts with thiol nucleophiles. Thiols were added to incubations of goat lung microsomes with 3-methylindole and deuterated analogues of 3-methylindole to trap the imine intermediate as its thioether conjugates. The N-acetylcysteine conjugate of 3-hydroxy-3-methylindolenine was detectable by LC/MS, but a molecular ion was not observed because the adduct rapidly dehydrated to form the 2-substituted indole. However, the imine was S-alkylated, and the intermediate carbinol was intramolecularly trapped using thioglycolic acid as a trapping agent that induced cyclocondensation to a lactone. The retention of one atom of deuterium from [2-2H]-3-methylindole and three from 3-[2H3-methyl]indole substantiated the mechanism in which the lactone adduct was produced by sulfur addition to either 3-hydroxy-3-methylindolenine or the epoxide. Tandem mass spectrometry of the lactone adduct produced a daughter ion spectrum consistent with this adduct. These studies demonstrated the existence of a new reactive intermediate of 3-methylindole, 3-hydroxy-3-methylindolenine, which may play a role in the pneumotoxicity of this chemical.

Animals↗

Specific inhibition of sclerotium formation by 2-mercaptoethanol and related sulfhydryl compounds in Sclerotium rolfsii.

Sclerotium formation in Sclerotium rolfsii was completely inhibited by 2-mercaptoethanol at a concentration of 2-4 mM without any adverse effect on mycelial growth. Concentrations lower than 2 mM had no effect on mycelial growth and sclerotium formation, whereas both were inhibited at concentrations higher than 4 mM. Complete inhibition of sclerotium formation with no effect on mycelial growth was also obtained by propyl mercaptan, 1-butyl mercaptan and 2-butyl mercaptan at a concentration of 0.10 mM. Sclerotium formation was also inhibited by benzyl mercaptan and thioglycolic acid at 0.15 mM and 2-4 mM concentration respectively, whereas it was only partially inhibited by L-cysteine and glutathione at 20 mM. Mycelium grown for 21 days in nutrient medium supplemented with mercaptoethanol at a concentration of 3 mM, when transferred into fresh medium without the chemical, grew normally and produced abundant mature sclerotia. Mercaptoethanol inhibited the initiation as well as the further development of young, unpigmented sclerotia. The mechanism of sclerotium formation was arrested completely when mercaptoethanol was added to the growth medium at any time between inoculation and the appearance of sclerotia of the "development" stage. It is suggested that the specific inhibitory action of mercaptoethanol could be used to study the mechanism of sclerotium formation

Cysteine↗

A novel oxytocin-like and vasotocin-like peptide in human plasma after administration of estrogen.

Using three antisera to oxytocin (OT Pitt Ab-1, OT Pitt Ab-2, and TOR OT Ab), we found comparable levels of OT in response to infant suckling and during infusion of synthetic OT, and identical standard curves with biological and synthetic standards of OT. Pitt Ab-1, but not Pitt Ab-2 or TOR OT Ab, measured increased OT in response to estrogen. Using an arginine vasotocin RIA (TOR AVT Ab), we found an increase in AVT immunoreactivity after estrogen treatment. Mean basal OT levels measured with OT Pitt Ab-2 in plasma of men [0.75 +/- 0.06 (+/- SEM) microU/ml] and women (0.8 +/- 0.09 microU/ml) were lower than OT measured with Pitt Ab-1 (1.7 +/- 0.09 microU/ml in men and 1.7 +/- 0.07 microU/al in women; P less than 0.001). Mean OT measured with Pitt Ab-2 in the plasma of women given estrogen chronically (0.8 +/- 0.04 microU/ml) and acutely (0.6 +/- 0.15 microU/ml) were not significantly different from basal levels. OT levels measured with Pitt Ab-1 in the same samples were 4.6 +/- 0.5 and 4.3 +/- 0.5 microU/ml, respectively, both significantly increased from basal levels (P less than 0.001) and significantly higher than OT measured with Pitt Ab-2 (P less than 0.001). Mean OT measured with Pitt Ab-1 in the plasma of pregnant women was 8.6 +/- 1.02 microU/ml, significantly higher than OT measured with Pitt Ab-2 (1.0 +/- 0.3 microU/ml; P less than 0.001). Men given 25 mg diethylstilbestrol had significant increases in OT measured with Pitt Ab-1 and in AVT measured with TOR AVT (P less than 0.01), but not in OT measured with Pitt Ab-2. Plasma from a man given diethylstilbestrol was prepared for high performance liquid chromatography and applied to a C18 muBondapak reverse phase column. The plasma contained two peaks of immunoreactivity detected as OT with Pitt Ab-1 and as AVT using TOR AVT Ab. The material was not detected by Pitt Ab-2 or TOR OT Ab and did not coelute with standards of OT, AVT, or AVP. Pregnancy plasma, thioglycolic acid, chymotrypsin, and trypsin reduced Pitt Ab-1, Pitt Ab-2, and TOR OT immunoreactivity of synthetic OT. The percent recovery of OT immunoreactivity was not significantly different with Pitt Ab-1 vs. Pitt Ab-2. A novel peptide, which is increased in response to administered estrogen, is present in human plasma and is detected by some antisera to OT and AVT. The observation explains the wide variability in OT levels in the estrogen-primed state and provides a new mechanism to study estrogen-related physiology and pathophysiology.

Adult↗

The use of thioglycolic acd in hair-bearing skin inlay urethroplasty.

Thioglycolic acid, in commercially available spray, lotion or cream, was used to remove the hair from the scrotum, pubis and perineum in 45 patients, with minimal discomfort in only 11 patients and wound infection in only one. Eighty-five per cent of these patients said they preferred the depilatory cream to the routine pre-operative shave with a razor. In 3 of 4 patients where it was instilled intraurethrally to remove hairs from skin-inlay urethroplasties it was successful in removing hairs; in the fourth patient most of the hairs were removed. None of these patients had anything more than a short-lasting irritation afterwards.

Hair Removal↗

Synthesis and pharmacological activity of 5-substituted-s-triazole-3-thiols.

Two subgroups of 5-substituted triazoles were synthesized: derivatives of 3(3-substituted-amino-2-hydroxypropylthio)-5-substituted-4H-1,2,4- triazole and derivatives of N-substituted amides of 5-substituted-s-(1,2,4-triazole-3)thioglycolic acid. Selected members of the two subseries were tested pharmacologically. The blood pressure lowering effect in anaesthetized normotensive rats was weak to moderate. More pronounced was the inhibitory effect against adrenaline induced human blood platelet aggregation. In experiments on isolated rat heart atria and on isolated rat tail artery the activity of the agents was much weaker than in the case of known beta or alpha adrenoceptor antagonists. Generally, the chance to find a potential antihypertensive agent within the group studied seems to be low. Antiaggregatory activity of the compounds deserves further studies.

Animals↗

Cell column chromatography: a new research tool to quantify cerebral cell volume changes following chemically-induced anoxia/re-oxygenation.

OBJECTIVE: The roles of individual types of cerebral cells in contributing to brain edema are undefined. The objective of this study was to determine the role of cerebral cell-column chromatography in quantifying cell volumes of individual cerebral cell lines, under chemically-induced anoxia/re-oxygenation (A/R). METHODS: Cerebral endothelial cells (4 experiments) or type II astrocytes (4 experiments) were cultured to confluence on microcarrier beads. A chromatographic cell-column of 1.5 cm height was filled with non-treated cell-covered beads. The column was perfused at 1 ml/min with a balanced perfusate for one hour (Baseline). The perfusate was then switched to that containing 5 mM thioglycolic acid for one hour (Anoxia). Then the column was perfused with the normal perfusate for another two hours (Re-oxygenation). The total free space in the column, reversely reflecting cell volumes, was determined by averaged transit time (TTa) of a non-permeable flow tracer blue dextran. Decreased TTa means that cells swell, and vice versa. RESULTS: TTa in endothelial cell columns increased with a peak at 60 minutes of re-oxygenation. TTa in astrocyte columns decreased with a nadir at 30 minutes of re-oxygenation. CONCLUSION: Cell column chromatography can be used to determine the cerebral cell volume changes following chemically-induced anoxia/re-oxygenation.

Brain Edema↗

Solubilization and stabilization of an anti-HIV thiocarbamate, NSC 629243, for parenteral delivery, using extemporaneous emulsions.

The O-alkyl-N-aryl thiocarbamate, I, (2-chloro-5-[[(1-methyl-ethoxy)thioxomethyl]amino]benzoic acid, 1-methylethylester, NSC 629243, also known as Uniroyal Jr.) is an experimental anti-HIV drug with very low water solubility (1.5 micrograms/mL). Early clinical studies required an injectable solution at approximately 15 mg/mL, representing a solubility increase of approximately 10(4)-fold. Adequate solubilization of this hydrophobic drug was achieved in 20% lipid emulsions. Extemporaneous emulsions were prepared by adding a concentrated drug solution to a commercially available parenteral emulsion. Various methods of preparation to minimize drug precipitation during its addition and enhance redissolution of precipitated drug were evaluated. The stability and mechanism(s) of decomposition of NSC 629243 in both 20% lipid emulsions and in natural oil vehicles were examined. In lipid emulsions, the shelf life at 25 degrees C varied from 1 to > 10 weeks, depending on the extent to which air was excluded from the preparation. The shelf life of 50 mg/mL solutions in natural oils at 25 degrees C varied from < 1 to > 100 days depending on the oil and its supplier. A qualitative correlation was found between the initial rate of oxidation and the peroxide concentration in the oil. The primary degradation product in both systems was shown to be a disulfide dimer, II, formed via oxidation. Oxidation was inhibited by vacuum-sealing of emulsion formulations or incorporation of an oil-soluble thiol, thioglycolic acid (TGA), into oil formulations. TGA may inhibit oxidation by consuming free radicals or peroxide initiators or by reacting with the disulfide, II, to regenerate the starting drug.

Antioxidants↗

Modified aca method for determination of iron chelated by deferoxamine and other chelators.

Iron in serum and urine specimens containing deferoxamine (a chelator used in treating acute iron intoxication) can be measured by the DuPont aca discrete-analyzer method for iron if 0.5 mL of a 100 mL/L solution of thioglycolic acid (mercaptoacetic acid) is added to the IRN-1 and IRN-2 test packs before their use. This reagent releases the unreactive chelated iron, allowing reliable colorimetry. Because about 50% of hemoglobin iron is measured by the modified method, hemolysed samples should not be used (or results should be corrected for hemoglobin iron). The modification allows emergency determination to total serum iron and assessment of urinary iron during chelation therapy, as well as measurement of plasma iron in samples treated with sodium citrate or oxalate plus fluoride (the unmodified aca iron method gives falsely low iron values in the presence of these chelators). About 60% of iron present as the dextran complex (Imferon) is measured by the modified aca method, as compared with <5% by the unmodified aca technique.

Anticoagulants↗

Cell differentiation, secondary cell-wall formation and transformation of callus tissue of Pinus radiata D. Don.

Tracheid and sclereid differentiation was induced in callus cultures of Pinus radiata D. Don by culturing on a basal medium containing activated charcoal but no phytohormones; sclereids differentiated in callus derived from xylem strips, but not in callus derived from hypocotyl segments. The tracheids differentiated in hypocotyl-derived callus had helical, scalariform, reticulated or pitted secondary cell-wall patterns, but those differentiated in xylem-derived callus had a reticulate or pitted pattern. The thickened tracheid and sclereid walls contained lignin as indicated by the red colour reaction given with phloroglucinol-HCl. The presence of lignin in the cell walls of differentiated callus was confirmed using pyrolysis gas chromatography-mass spectrometry by the detection of phenylpropanoid components derived from lignin. Lignin was also detected using solid-state (13)C cross-polarisation/magic-angle spinning nuclear magnetic resonance spectroscopy and quantified as thioglycolic acid lignin. Monosaccharide analyses of the cell walls isolated from differentiated and undifferentiated calli showed that the cell walls of the differentiated calli contained higher proportions of glucose and mannose, consistent with the presence of greater proportions of gluco- and/or galactogluco-mannans in the secondary cell walls of the differentiated cells. A protocol for the stable transformation of undifferentiated, xylem-derived cultures was successfully developed. Transgenic cell lines were established following Biolistic particle bombardment with a plasmid containing the coding region of the nptII gene and the coding region of the cad gene from P. radiata. Expression of the nptII gene in transgenic lines was confirmed by an NPTII-enzyme-linked immunosorbent assay. The overexpression of cad in the transgenic lines resulted in a down-regulation of cinnamyl alcohol dehydrogenase (EC 1.1.1.195) expression.

Alcohol Oxidoreductases↗

Analysis of collagen type III by uninterrupted sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting: changes in collagen type III polymorphism in aging rats.

A new method of type III collagen analysis by uninterrupted sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) combined with immunoblotting was developed. The electrophoresis was carried out with gels containing 4 M urea. A negatively charged reducing agent, thioglycolic acid, was added to the running buffer of the cathodic reservoir between 15 and 20 min after Bromphenol Blue (BPB) migrated to the top of the separating gel, to reduce interchain disulfide binding of the collagen. The polymorphic type III collagens, i.e., an alpha-chain derived from a trimer [alpha 1(III)]3 with interchain disulfide bonds but without covalent cross-links, alpha 1(III), a beta-chain with covalent cross-links, beta(III), or an alpha-chain released from a trimer without reduction of the disulfide bonds, alpha*1(III), were identified by immunostaining and quantified by densitometry. Using this method, changes in collagen type III polymorphism with aging were examined in the aorta, brachial artery, and skin of rats. The total quantity of collagen type III decreased with aging in all tissues. beta(III) was the major component in the aorta and brachial artery, but alpha 1(III) was the major component in the skin. With increasing age from 3 to 60 weeks, the ratio of beta(III) to alpha 1(III), which is correlated with the extent of covalent cross-linking, showed a steep increase in the aorta but only a slight increase in the skin and it remained almost constant in the brachial artery.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗

Thiolated chitosan/DNA nanocomplexes exhibit enhanced and sustained gene delivery.

PURPOSE: Thiolated chitosan appears to possess enhanced mucoadhesiveness and cell penetration properties, however, its potential in gene-drug delivery remains unknown. Herein, we report on a highly effective gene delivery system utilizing a 33-kDa thiol-modified chitosan derivative. METHODS: Thiolated chitosan was prepared by the reaction with thioglycolic acid. Nanocomplexes of unmodified chitosan or thiolated chitosan with plasmid DNA encoding green fluorescenct protein (GFP) were characterized for their size, zeta potential, their ability to bind and protect plasmid DNA from degradation. The transfection efficiency of thiolated chitosan and sustained gene expression were evaluated in various cell lines in vitro and in Balb/c mice in vivo. RESULTS: Thiolated chitosan-DNA nanocomplexes ranged in size from 75 to 120 nm in diameter and from +2.3 to 19.7 mV in zeta potential, depending on the weight ratio of chitosan to DNA. Thiolated chitosan, CSH360, exhibited effective physical stability and protection against DNase I digestion at a weight ratio>or=2.5:1. CSH360/DNA nanocomplexes induced significantly (P<0.01) higher GFP expression in HEK293, MDCK and Hep-2 cell lines than unmodified chitosan. Nanocomplexes of disulphide-crosslinked CSH360/DNA showed a sustained DNA release and continuous expression in cultured cells lasting up to 60 h post transfection. Also, intranasal administration of crosslinked CSH360/DNA nanocomplexes to mice yielded gene expression that lasted for at least 14 days. CONCLUSIONS: Thiolated chitosans condense pDNA to form nanocomplexes, which exhibit a significantly higher gene transfer potential and sustained gene expression upon crosslinking, indicating their great potential for gene therapy and tissue engineering.

Adhesives↗

[Enantioselective biotransformation of a new theophylline derivative].

Reductive amination of 4 with (RS)-2 is an improved method to obtain 7-((RS)2-((RS)-1-Methyl-2-phenyl-ethylamino)propyl)-theophylline (3) (ratio of diastereomers 1:1) compared with SN-displacement of proxyphyllintosylate with (RS)-2 (ratio of diastereomers 9:1). Due to steric hindrance 3 is no noteworthy prodrug for the intermediate formation of (RS)-2 in rats. The enantioselectivity of 2 after a single oral dose of 3 is determined in 24-h urine by HPLC on a Cyclobond-column using the isoindole derivatives, formed from o-phthalaldehyde, thioglycolic acid, and 2. The R(-)-amphetamine isomer is formed to a lesser amount (34.8%, range 32-37%) than the S(+)-isomer (65.2%, range 63-68%).

Animals↗

Synthesis and biological investigations of some new thiazolylbenzimidazoles and benzimidazolylthiazolo[3,2-a]pyridines.

2-[(4-Oxo-4,5-dihydrothiazol-2-yl)methyl]-1H-benzimidazole (2) was prepared through the reaction of 2-cyanomethyl-1H-benzimidazole (1) with thioglycolic acid. The syntheses of its arylidene 3 and diazo-coupled compounds 5 and the cyclization of the aforementioned thiazolylbenzimidazole to benzimidazolylthiazolo[3,2-a]pyridines 8 were also performed. The prepared compounds were screened for their in-vitro antibacterial, antifungal, anti-HIV, and anticancer activities: they show promising antifungal activity.

Anti-Infective Agents↗

Novel carbostyril anchored heterocycles.

The behaviour of 3-acetylcarbostyril derivative 1, towards the Mannich reaction and the reactivity of the products formed in their reactions with aniline, phenylhydrazine and benzenesulphonyl chloride were studied. Furthermore, the reaction of 1 with thioglycolic acid was also investigated.

Aniline Compounds↗

Cytofluorometric determination of protein-bound thiols and DNA in cell nuclei.

The aim of the present study was to establish a cytofluorometric method for the simultaneous determination of protein-bound sulfhydryl-groups (PSH) and DNA in isolated cell nuclei. DNA was stained with ethidiumbromide and PSH with N-iodoacetyl-N(5-sulfo-1-naphthyl) ethylendiamine (AEDANS). Disulfide groups of nuclear proteins were determined by the same method after reduction with sodium borohydride or thioglycollic acid. The method was established by using nuclei of human lymphocytes, which then served as a biological standard for further investigations of the nuclei of different mammalian cell types: nuclei from mouse liver cells and nuclei from the cells of two human melanoma cell lines. For non-proliferating lymphocytes distinct DNA- and PSH-values could be measured. The PSH-values detected in the nuclei of the other cell types were higher by comparison and varied within the cell cycle; i.e., PSH increased during the S-phase and was almost doubled during the cell generation cycle from G1- to G2-phase. Cell line and cell cycle-dependent variations of nuclear disulfides could also be detected. These results are discussed with respect to their radiobiological implications. In conclusion, thiol groups may represent one factor determining the radiosensitivity of cells, but they are not the only decisive one.

Animals↗

A new micellar electrokinetic capillary chromatography method for separation of the components of the aminoglycoside antibiotics.

Aminoglycoside antibiotics are always a mixture of structurally related amino sugars, which do not have a chromophore or fluorophore. The aim of the study was to find one method for evaluation of the components and impurities of the antibiotics. Derivatization with o-phthaldialdehyde and thioglycolic acid is found to be appropriate for all antibiotics. The components of gentamicin (GM), sisomicin, netilmicin, kanamycin, amikacin, and tobramycin were tried to separate by means of micellar electrokinetic chromatography. The background electrolyte was composed of sodium tetraborate (100 mM, pH 10.0), sodium deoxycholate (20 mM), and beta-cyclodextrin (15 mM). This method is valid for evaluation of GM, kanamycin, and tobramycin. It has to be improved for amikacin and netilmicin. In addition, 46 bulk samples of GM of different manufacturer or pharmaceutical companies were investigated. Many samples were found to contain many minor products and different amounts. Beside different patterns of the main compounds GM C1, GM C1a, GM C2a, and GM C2, many lots were found consisting of a substantial number of minor products. The appearance of a high number of minor products is always associated with the existence of sisomicin, which is not found in "pure" samples. However, almost all samples met the requirements of the European Pharmacopoeia (EP) and United States Pharmacopoeia (USP).

Amikacin↗

Molecular and Supramolecular Architecture of the Salmo gairdneri Proteinaceous Eggshell during Development

A detailed developmental study of eggshell architecture of the fish Salmo gairdneri (rainbow trout) was performed using transmission and scanning electron microscopy. Thioglycollic acid treatment and freeze-fracturing reveal that fibrils ca. 5-10 nm in diameter constitute each lamella of the helicoidal eggshell. Freeze-fracturing also permits a direct visualization of the helicoidal architecture. Laser-Raman studies of the eggshell indicate abundant antiparallel beta-pleated-sheet conformation in the eggshell proteins of S. gairdneri during all developmental stages. Apparently, this conformation dictates formation of the helicoidal structure. Disulfide bonds, together with isopeptide bonds, cross-link S. gairdneri eggshell proteins throughout development.

Journal Article↗