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PubMed1989

A systematic evaluation of bathophenanthroline, ferrozine and ferene in an ICSH-based method for the measurement of serum iron.

The chromogenic substrates ferrozine and ferene were compared to bathophenanthroline disulphonic acid for the measurement of iron concentrations in aqueous and serum samples in an assay based on that of the Iron Panel of the International Committee for Standardisation in Haematology. Ferrozine and ferene were more sensitive than bathophenanthroline. Copper at physiological concentrations in plasma caused only minimal positive interference with all three chromogenic substrates when thioglycollic acid was used as the reducing agent, but when ascorbic acid was used significant positive interference occurred with ferrozine and ferene. Interference due to contaminating haem was comparable with all agents. Bilirubin and carotene produced no interference. Profound reductions in colour development were noted with EDTA plasma.

Chromogenic Compounds↗

PubMed1999

Measurement of thiols in human plasma using liquid chromatography with precolumn derivatization and fluorescence detection.

A liquid chromatography (LC) method for the simultaneous measurement of the main low molecular mass thiols (i.e., cysteine, cysteinylglycine, homocysteine, and glutathione) in human plasma is described. The sample treatment consists of the reduction of disulfide bounds with tri-n-butylphosphine and protein precipitation with trichloroacetic acid followed by precolumn derivatization with a thiol-selective fluorogenic reagent (7-fluoro-2,1,3-benzoxadiazole-4-sulfonamide). The structure of thiol derivatives is assessed using electrospray ionization-mass spectrometry (MS). The stability of resulting adducts in acidic medium (24 h at 10 degrees C) allows the automation of the technique and a high throughput of samples (approximately 50 per day). Separation is complete within 12 min using isocratic reversed-phase mode, and detection is operated by spectrofluorimetry (lambda ex = 385 nm and lambda em = 515 nm). Quantitation is performed by an internal standardization mode using thioglycolic acid. The LC method is fully validated, and homocysteine concentrations obtained in plasma samples are compared with values measured using either fluorescence polarization immunoassay or capillary gas chromatography-MS; a good correlation is observed between LC and both methods. The method has been applied in daily use to a large-scale study in a human healthy population, and some resulting data are discussed.

Adolescent↗

PubMed1989

Oxidative inactivation of rhodanese by hydrogen peroxide produces states that show differential reactivation.

Controlled conditions have been found that give complete reactivation and long term stabilization of rhodanese (EC 2.8.1.1) after oxidative inactivation by hydrogen peroxide. Inactivated rhodanese was completely reactivated by reductants such as thioglycolic acid (TGA) (100 mM) and dithiothreitol (DTT) (100 mM) or the substrate thiosulfate (100 mM) if these reagents were added soon after inactivation. Reactivability fell in a biphasic first order process. At pH 7.5, in the presence of DTT inactive rhodanese lost 40% of its reactivability in less than 5 min, and the remaining 60% was lost more gradually (t 1/2 = 3.5 h). TGA reactivated better than DTT, and the rapid phase was much less prominent. If excess reagents were removed by gel filtration immediately after inactivation, there was time-independent and complete reactivability with TGA for at least 24 h, and the resulting samples were stable. Reactivable enzyme was resistant to proteolysis and had a fluorescence maximum at 335 nm, just as the native protein. Oxidized rhodanese, Partially reactivated by DTT, was unstable and lost activity upon further incubation. This inactive enzyme was fully reactivated by 200 mM TGA. Also, the enzyme could be reactivated by arsenite and high concentrations of cyanide. Addition of hydrogen peroxide (40-fold molar excess) to inactive rhodanese after column chromatography initiated a time-dependent loss of reactivability. This inactivation was a single first order process (t 1/2 = 25 min). Sulfhydryl titers showed that enzyme could be fully reactivated after the loss of either one or two sulfhydryl groups. Irreversibly inactivated enzyme showed the loss of one sulfhydryl group even after extensive reduction with TGA. The results are consistent with a two-stage oxidation of rhodanese. In the first stage there can form sulfenyl and/or disulfide derivative(s) at the active site sulfhydryl that are reducible by thioglycolate. A second stage could give alternate or additional oxidation states that are not easily reducible by reagents tried to date.

Arsenic↗

PubMed1999

Hypoxia/aglycemia increases endothelial permeability: role of second messengers and cytoskeleton.

The effects of hypoxia/aglycemia on microvascular endothelial permeability were evaluated, and the second messenger systems and the cytoskeletal-junctional protein alterations in this response were also examined. Monolayers of human dermal microvascular endothelial cells on microcarrier beads were exposed to either thioglycolic acid (5 mM, an O(2) chelator), glucose-free medium, or both stresses together. Permeability measurements were performed over a 90-min time course. Although neither hypoxia alone nor aglycemia alone increased endothelial permeability significantly, the combination of both increased significantly as early as 15 min. Intracellular Ca(2+) measurements with fura 2-AM showed that hypoxia/aglycemia treatment increased Ca(2+) influx. To determine the second messengers involved in increased permeability, monolayers were incubated for 30 min with the cytosolic Ca(2+) scavenger 3,4, 5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8, 0.1 mM), a classical protein kinase C (PKC) blocker, Gö-6976 (10 nM), a cGMP-dependent protein kinase (PKG) antagonist, KT-5823 (0.5 microM), or the mitogen-activated protein (MAP) kinase inhibitor PD-98059 (20 microM). Hypoxia/aglycemia-mediated permeability changes were blocked by chelating cell Ca(2+), PKC blockade, PKG blockade, and by inhibiting p38 MAP kinase-1. Finally, changes in the binding of junctional proteins to the cytoskeleton under the same conditions were assessed. The concentrations of occludin and pan-reactive cadherin binding to the cytoskeleton were significantly decreased by only both stresses together. However, these effects were also blocked by pretreatment with TMB-8, Gö-6976, KT-5823 (not in occludin), and PD-98059. These data suggest that hypoxia/aglycemia-mediated endothelial permeability may occur through PKC, PKG, MAP kinase, and Ca(2+) related to dissociation of cadherin-actin and occludin-actin junctional bonds.

Alkaloids↗

PubMed1994

Defense Responses in Infected and Elicited Cucumber (Cucumis sativus L.) Hypocotyl Segments Exhibiting Acquired Resistance.

Segments from dark-grown cucumber (Cucumis sativus L.) hypocotyls were used to study defense reactions occurring upon fungal infection and induced by elicitors in the same tissue. The segments were rendered resistant to infection by Colletotrichum lagenarium either by growing the seedlings in the presence of dichloroisonicotinic acid (DCIA) or by preincubation of the cut segments with DCIA, salicylic acid (SA), or 5-chlorosalicylic acid (5CSA). This resistance appears to be due mainly to inhibition of fungal penetration into epidermal cells. In the resistant hypocotyl segments, the fungus induced, at the time of attempted penetration, an increased deposition of phenolics, which were visualized by autofluorescence. These phenolics were located mainly in the epidermal cell wall around and in the emerging papillae below appressoria and were quantified either as lignin-like polymers by the thioglycolic acid method or as 4-OH-benzaldehyde, 4-OH-benzoic, or 4-coumaric acid liberated upon treatment with alkali at room temperature. Pretreatment with DCIA, SA, and 5CSA induced little chitinase activity, but this activity greatly increased in resistant tissues upon subsequent infection. These observations indicate that resistance is associated with an improved perception of the pathogen stimulus resulting in the enhanced induction of diverse defense reactions. When the cut segments were pretreated with DCIA, SA, or 5CSA and then split and incubated with chitosan fragments, the deposition of cell wall phenolics was also enhanced. These pretreated and split segments also exhibited an increase in the rapid production of activated oxygen species induced by an elicitor preparation from Phytophthora megasperma f. sp. Glya. Pretreatment of the segments with methyl jasmonate neither induced resistance nor enhanced induction of cell wall phenolics upon fungal infection, although we observed in the corresponding split segments some increase in chitosan-induced cell wall phenolics and in elicitor-induced rapid production of activated oxygen species.

Journal Article↗

PubMed1987

Permanent waving and straightening of hair.

A significant number of consumers use products to curl or straighten their hair. These products function by chemically breaking and reforming keratin disulfide bonds. The most popular waving products contain salts of thioglycolic acid to break the bonds and hydrogen peroxide to reform them. Straighteners generally rely on high alkalinity to break the bonds and reform them with acid. Problems with permanent waving are usually due to overtreatment or undertreatment of the hair rather than allergy or irritation. Straighteners can be irritating because of their high alkalinity and can seriously damage hair if used excessively.

Dermatitis, Contact↗

PubMed1965

The use of gas-liquid chromatography in the analysis of neutral monosaccharides in hydrolysates of gastric mucopolysaccharides.

1. Conditions are described for the separation and estimation of the neutral monosaccharides obtained on acidic hydrolysis of human gastric mucopolysaccharides. 2. The technique involves the formation of the trimethylsilyl derivatives of the sugars and the analysis of these by gas-liquid chromatography. 3. The monosaccharides estimated in gastric mucopolysaccharides by this technique were l-fucose, d-mannose, d-galactose and d-glucose. 4. The analytical values for glucose and fucose obtained by this method agreed well with values obtained by the glucose oxidase and thioglycollic acid methods respectively. 5. Evidence is presented which clearly indicates that gas-liquid chromatography is a faster, more sensitive and more convenient technique for the measurement of these compounds than any other in use at present.

Chromatography, Gas↗

PubMed1975

[Microeletrophoresis in continuous polyacrylamide gel gradients. IV. The effect of reducing agents on the electrophoresis of proteins in sodium dodecylsulfate buffer systems (author's transl)].

The migration of reducing agents (e.g. 2-mercaptoethanol, dithiothreitol and thioglycolic acid) was analysed in various electrophoretic buffer systems containing sodium dodecylsulfate. It is shown that proteins loaded with dodecylsulfate and previously not reduced can be reduced during the electrophoretic separation.

Bence Jones Protein↗

PubMed1982

[Colorimetric determination of urinary iron, chelated with deferoxamine B, using a single reagent].

A new colorimetric method for the determination of urinary iron chelated with desferrioxamine B is proposed. A single reagent (ferrozine plus thioglycolic acid 1%) is utilized. Analytical range reached about 25 mg of iron/l, while the detection limit was 0.2 p.p.m. The evaluation of imprecision and inaccuracy were within acceptable limits. Moreover, there is good correlation with the atomic absorption spectrophotometric technique. This test shows good reliability.

Colorimetry↗

PubMed2001

Oxyluciferin, a luminescence product of firefly luciferase, is enzymatically regenerated into luciferin.

The activity regenerating luciferin from the luminescent product oxyluciferin was found in the protein fraction of a lantern extract from Photinus pyralis. The protein, luciferin-regenerating enzyme (LRE), was purified to homogeneity by ammonium sulfate precipitation followed by successive column chromatography on Ultrogel AcA34, S-Sepharose FF, Q-Sepharose FF, TSKgel super Q 5pw and TSKgel G3000 SW(XL). This enzyme was a single polypeptide with a molecular mass of 38 kDa. LRE converted oxyluciferin to 2-cyano-6-hydroxybenzothiazole and thioglycolic acid. In the presence of d-cysteine, 2-cyano-6-hydroxybenzothiazole was turned over into luciferin. The same activities were detected in the extracts from two Japanese fireflies, Luciola cruciata and Luciola lateralis. We have cloned a cDNA encoding LRE from poly(A)+ RNA of the lantern of P. pyralis using reverse transcription-polymerase chain reaction, 5'-RACE (rapid amplification of cDNA ends) and 3'-RACE. The primary structure of LRE from P. pyralis deduced from the nucleotide sequence was shown to consist of 308 amino acids with a molecular weight of 33,619. The cDNA was successfully expressed under the control of the tac promoter in Escherichia coli.

Amino Acid Sequence↗

PubMed1996

Metal binding and ferritin immunoreactivity in a high molecular weight fraction from rat brain.

A high molecular weight protein fraction (PI) from rat brain that exhibited heat- and acid-resistant properties like ferritin was characterized with respect to its reactivity with spleen ferritin antiserum, iron (Fe) content and Fe and aluminum (Al) binding. This fraction eluted on Sepharose 4B and Sepharose 6B columns with an apparent Mr of 2 100 000. A second Fe-rich fraction (PII) with an Mr of 500 000 showed lower anti-ferritin activity. The PI and PII fractions from liver, when prepared without the heat and acid steps, were similar to those found in brain. In both tissues, the PI fraction had a low Fe/ferritin ratio (0.28 +/- 0.08 microgram/microgram for brain and 0.22 +/- 0.05 microgram/microgram for liver). Similarly the PII fractions had high Fe/ferritin ratios (1.48 +/- 0.44 microgram/microgram for brain and 1.34 +/- 0.10 microgram/microgram for liver). Analysis of Fe binding to brain PI revealed one high affinity site (Kd = 157 +/- 30.0 X 10(-6) M; Bmax = 1.42 +/- 0.11 micromol Fe/mg protein) and one low affinity site (Kd = 890 +/- 100 X 10(-6) M; Bmax = 5.24 +/- 0.96 micromol Fe/mg protein). Removal of Fe from brain PI with 1% thioglycollic acid solution prior to Fe binding, increased binding affinity at both sites, while total binding remained unchanged. A1 bound to brain PI with high affinity (Kd = 59.7 +/- 13.6 X 10(-6) M; Bmax = 0.24 +/- 0.02 micromol A1/mg protein). These results demonstrate the presence of two ferritin-like, metal-binding (MB) proteins in rat brain.

Aluminum↗

PubMed1975

Determination of some in vitro growth requirements of Bacteroides nodosus.

Physical and nutritional factors required for growth of Bacteroides nodosus isolates from ovine foot-rot lesions were examined. Simplified anaerobic culture techniques were devised utilizing a fully soluble, autoclavable, liquid medium (TAS) which contained proteose-peptone, yeast and meat extracts and certain other essential compounds required to promote prompt and serially transferrable growth of cultures from small inocula. The latter included Trypticase, arginine, a reducing agent (most suitably thioglycollic acid) and CO2; serine and Mg2+ markedly increased growth yields. Trypticase could not be replaced by a commercial preparation of acid-hydrolysed casein; other forms of hydrolysed protein gave delayed and inconsistent growth. Maximum growth of cultures required concentrations of 0-02 to 0-35 M-arginine, which could not be replaced by glutamic acid, citrulline or ornithine. Exogenous carbohydrate compounds were not required. The temperature range for optimum growth of cultures was 37 to 39 degrees C, and anaerobic culture conditions were essential for growth and the production of B. nodosus organisms of normal morphology. Solidified TAS media for the isolation and maintenance of B. nodosus cultures were also devised.

Anaerobiosis↗

PubMed2000

Modified lignin in tobacco and poplar plants over-expressing the Arabidopsis gene encoding ferulate 5-hydroxylase.

Ferulate 5-hydroxylase (F5H) is a cytochrome P450-dependent monooxygenase that catalyses the hydroxylation of ferulic acid, coniferaldehyde and coniferyl alcohol in the pathways leading to sinapic acid and syringyl lignin biosynthesis. Earlier studies in Arabidopsis have demonstrated that F5H over-expression increases lignin syringyl monomer content and abolishes the tissue-specificity of its deposition. To determine whether this enzyme has a similar regulatory role in plants that undergo secondary growth, we over-expressed the F5H gene in tobacco and poplar. In tobacco, over-expression of F5H under the control of the cauliflower mosaic virus 35S promoter increased lignin syringyl monomer content in petioles, but had no detectable effect on lignification in stems. By contrast, when the cinnamate 4-hydroxylase (C4H) promoter was used to drive F5H expression, there was a significant increase in stem lignin syringyl monomer content. Yields of thioglycolic acid and Klason lignin in C4H-F5H lines were lower than in the wild-type, suggesting that F5H over-expression leads to a reduced deposition or an altered extractability of lignin in the transgenic plants. Histochemical analysis suggested that the novel lignin in C4H-F5H transgenic lines was altered in its content of hydroxycinnamyl aldehydes. Transgenic poplar trees carrying the C4H-F5H transgene also displayed enhanced lignin syringyl monomer content. Taken together, these data show that hydroxylation of guaiacyl-substituted lignin precursors controls lignin monomer composition in woody plants, and that F5H over-expression is a viable metabolic engineering strategy for modifying lignin biosynthesis in forest species.

Arabidopsis↗

PubMed1980

T suppressor cells and suppressor factor which act at the efferent stage of the contact sensitivity skin reaction: their production by mice injected with water-soluble, chemically reactive derivatives of oxazolone and picryl chloride.

The water soluble, chemically reactive thioglycollic acid thioether derivatives of oxazolone and picryl chloride were synthesized and tested for their ability to prevent the development of contact sensitivity. Mice given two injections of these agents showed partial or complete unresponsiveness when subsequently sensitized and challenged with oxazolone and picryl chloride, respectively. This unresponsiveness was associated with T suppressor cells, Ts-eff(cs), which blocked the efferent stage of the contact sensitivity reaction, i.e. the passive transfer of contact sensitivity. These Ts-eff(cs) were entirely specific when tested with the corresponding antigen. However, the suppression which they caused had a non-specific final common pathway. Cell from mice injected with the oxazolone and picryl thioethers and painted with the corresponding contact sensitizer produced a suppressor factor in vitro. This factor specifically blocked passive transfer by immune cells incubated in it. It also armed macrophages which then caused suppression. These macrophages were most effective when injected intraperitoneally. The suppressor factor had a molecular weight between 30,000 and 100,000 and the alpha-oxazolone factor was absorbed by oxazolone-albumin Sepharose and could be eluted with oxazolone-epsilon-aminocaproic acid. It was also absorbed by concanavalin-A-sepharose and could be eluted with alpha-methylmannoside. It is proposed that the ability of water soluble, chemically reactive haptenes to evoke a Ts-eff(cs) population may be relevent to the rarity of severe drug reactions following the injection of chemically reactive drugs.

Absorption↗

PubMedDate not supplied

[Thiodiglycol metabolism in Alcaligenes xylosoxydans subsp. denitrificans].

The investigation of the degradation of thiodiglycol (the major product of mustard gas hydrolysis) by Alcaligenes xylosoxydans subsp. denitrificans strain TD2 showed that thiodiglycol is metabolized through the oxidation of its primary alcohol groups and the subsequent cleavage of C-S bonds in the intermediate products, thiodiglycolic and thioglycolic acids. The end products of these reactions are SO4(2-) ions and acetate, the latter being involved in the central metabolism of strain TD2. The oxidation of the sulfur atom gives rise to diglycolsulfoxide, which is recalcitrant to further microbial degradation. Based on the data obtained, a metabolic pathway of thiodiglycol transformation by A. xylosoxydans subsp. denitrificans strain TD2 is proposed.

Alcaligenes↗

PubMed1976

In vitro release of ACTH from dispersed rat pars intermedia cells. I. Effect of secretagogues.

Studies were made to test the responsiveness of dispersed pars intermedia (PI) cells to a number of secretagogues, that are known to alter ACTH release from the pars distalis (PD) in vitro. In summary, (a) incubation in high (K+), which will increase ACTH release from the PD, did not alter ACTH release from the PI; (b) a crude extract of rat hypothalamus (HE) increased ACTH release from PD and PI; (c) the effect of HE was not due to its vasopression content, since pretreatment of the extract with thioglycolic acid did not modify its ACTH-releasing activity and neither lysine nor arginine vasopressin stimulated ACTH release from the PI; and (d) a partially purified CRF preparation, which will stimulate ACTH release from the PD, did not alter ACTH release from the PI. We conclude that the hypothalamus contains a substance(s) that will stimulate ACTH release from the PI and that the 'secretagogue' is neither vasopressin nor the same CRF that will stimulate ACTH release from the PD.

Adrenocorticotropic Hormone↗

PubMedDate not supplied

New indole, aminoindole and pyranoindole derivatives with anti-inflammatory activity.

6-Methoxy-1-methyl-2,3-diphenyl indol-5-carboxaldehyde (2) was demethylated to give the 6-hydroxy derivative (3) which was cyclized to the pyrano[3,2-f]indole derivatives (4a-d) by the action of ethyl acetoacetate, diethyl malonate, malononitrile, ethyl cyanoacetate. When 4c was boiled in acetic acid, it gave 4d. Reduction of 4c by sodium borohydride yielded the orthoaminonitrile (5). Friedel Craft's acetylation of 1b yielded the 5-acetyl derivative (6), which reacted with hydrazine hydrate, o-toluidine and o-aminophenol to afford (7a-c). Demethylation of (1b) yielded the hydroxyl derivative (8), which differs from compound (9) obtained by demethylation of 6-methoxy-2,3-diphenyl-indole (1a). Friedel Craft's acetylation of 9 gave the 7-acetyl compound (10) which yielded the hydrazone (11). The reaction of primary aromatic amines, (i.e. p-nitroaniline, p-anisidine and p-bromo aniline) with 6-methoxy-1-methyl-2,3-diphenyl-indol-5-carboxaldehyde (2) gave the Schiff bases (12a-c). The latter compounds were reduced by sodium borohydride to yield the corresponding Mannich bases (13a-c). Treatment of 12a-c with thioglycolic acid led to the thiazolidin-4-one-derivatives (14a-c). When (12a-c) reacted with cyanoacetamide, the amino group was replaced by the active methylene to form the cyano compound (15). The structure was confirmed by reacting the carboxaldehyde (2) with cyanoacetamide to yield (15). Pharmacological screening was has been carried out to test the anti-inflammatory activity, ulcerogenecity, effect on the isolated rabbit intestine and the antispasmodic activity.

Animals↗

PubMed2003

Application of chitin and chitosan derivatives in the pharmaceutical field.

Chitin and chitosan derivatives are used as excipients and drug carriers in the pharmaceutical field. Their derivatization contributed to expansion of application and decrease toxicity. Chitosan is used as an excipient in oral dosage form. Chitosan tablet can exhibit a sustained drug release compared to commercial products. Films prepared using chitin or chitosan have been developed as wound dressings, oral mucoadhesive and water-resisting adhesive by virtue of their release characteristics and adhesion. Intratumoral administration of gadopentetic acid-chitosan complex nanoparticles (approximately 430 nm in diameter) has been more effective for gadolinium neutron-capture therapy compared with a group treated with the solution. Compared to intragastrical feeding with diphtheria toxoid (DT) in PBS, a strong enhancement of the systemic (IgG) and local (IgA) immune responses against DT has been observed in mice fed with DT loaded chitosan microparticles (approximately 4.7 microm in size). When DNA-loaded chitosan microspheres (1.15 - 1.28 microm) were intramuscularly administrated into mice, high beta-galactosidase and luciferase productions were obtained even after a long post-transfection period (12 weeks). N-Succinyl-chitosan (Suc-Chi) has been studied for cancer chemotherapy as a drug carrier and the conjugates of mitomycin C with Suc-Chi exhibited good antitumor activities against various tumors. Furthermore, trimethyl-chitosan and monocarboxymethyl-chitosan has been shown to be effective as intestinal absorption enhancers due to their physiological properties. Chitosan-thioglycolic acid conjugates has been found to be a promising candidate as scaffold material in tissue engineering due to their physicochemical properties. This review summarizes the application of chitin and chitosan derivatives for hospital preparations and drug carriers.

Adjuvants, Pharmaceutic↗