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PubMed2004

Detection of Aspergillus fumigatus mycotoxins: immunogen synthesis and immunoassay development.

Immunological detection of secreted low molecular weight toxins represents a potentially novel means of diagnosing infection by the fungus Aspergillus fumigatus. Two such metabolites, gliotoxin and helvolic acid, were selected and conjugated to thyroglobulin for antisera generation in rabbits. Gliotoxin was initially activated using N-[p-maleimidophenyl] isocyanate (PMPI) and subsequently conjugated to S-acetyl thioglycolic acid N-hydroxysuccinimide-activated thyroglobulin, whereas helvolic acid was activated with N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) in the presence of thyroglobulin prior to immunisation. To facilitate subsequent antisera evaluation, both toxins were similarly conjugated to bovine serum albumin (BSA). Matrix-Assisted Laser Desorption Ionisation-Time Of Flight (MALDI-TOF) mass spectrometry and SDS-PAGE analysis confirmed covalent attachment of toxins to BSA in the ratios of 15 and 2.4 mol per mol BSA for gliotoxin and helvolic acid, respectively. Resultant high titer antisera were capable of detecting both BSA-conjugated toxins (inhibitory concentration (IC)(50): 4-5 microg/ml). Free toxins were also detectable by competitive immunoassay, whereby 10 microg/ml free gliotoxin (30 microM) and helvolic acid (17 microM), respectively, inhibited antibody binding to cognate toxin-BSA previously immobilised on microwells. This work confirms that sensitive and specific antisera can be raised against fungal toxins and may have an application in diagnosing fungal infection.

Animals↗

PubMed1992

Absorption and Raman spectroscopy study of cyt c-thiol complexes in acidic solutions.

Absorption UV-VIS and pre-resonance Raman spectra of acidic cyt c solutions with a series of thiols added (thiophenol, n-propanethiol, isopropanethiol, L-cysteine, dithiothreitol, 2-mercaptoethanol, N-acetyl-L-cysteine, p-acetamidothiophenol, 2-mercaptoethanamine, thioglycolic acid and mercaptopropionic acid), are presented. Interactions of cyt c molecule with the thiols were studied with the aim to identify binding of the thiols with the cyt c heme as its iron axial ligands. Absorption and Raman spectra showed some correlation between maxima of 700 nm region absorption band (typical for Fe-S axial bond in cyt c heme) and also wave numbers of spin state marker and axial ligand sensitive Raman bands on one, and pKa constant values of appropriate thiols on the other hand. These results imply thiol replacement of Met-80 from axial bond with heme iron and suggest that the force of Fe-L-cysteine axial bond is very close to the native axial bond (Fe-Met) for cyt c in neutral solution.

Cytochrome c Group↗

PubMedDate not supplied

Amino acid analysis for meat protein evaluation.

The Food Safety and Inspection Service procedure for determination of essential amino acid content of mechanically processed products from red meat animals and poultry is based on hydrolysis of a powder prepared by blending samples in acetone-chloroform. The hydrolysis procedure incorporates thioglycolic acid to prevent loss of tryptophan. Aliquots of prepared hydrolysates are injected into a liquid chromatographic system, using gradient elution on an ion-exchange column for separation. The system also uses post-column hypochlorite oxidation coupled with orthophthalaldehyde reagent and fluorescence detection. Modification of the elution program allows concurrent determination of tryptophan with minimal added cost. Chromatograms from beef, pork, and poultry products show adequate separation and quantitation of beta-alanine, 1-methyl-histidine, and 3-methyl-histidine, indicating that the procedure could be used to estimate muscle content of products. A colorimetric procedure for assay of hydroxyproline was introduced and validated as an adjunct method for protein quality estimation.

Amino Acids↗

PubMed1997

A novel white laccase from Pleurotus ostreatus.

Two laccase isoenzymes (POXA1 and POXA2) produced by Pleurotus ostreatus were purified and fully characterized. POXA1 and POXA2 are monomeric glycoproteins with 3 and 9% carbohydrate content, molecular masses of about 61 and 67 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis, of about 54 and 59 kDa by gel filtration in native conditions, and of 61 kDa by matrix-assisted laser desorption ionization mass spectrometry (only for POXA1) and pI values of 6.7 and 4.0, respectively. The N terminus and three tryptic peptides of POXA1 have been sequenced, revealing clear homology with laccases from other microorganisms, whereas POXA2 showed a blocked N terminus. The stability of POXA2 as a function of temperature was particularly low, whereas POXA1 showed remarkable high stability with respect to both pH and temperature. Both enzymes oxidize syringaldazine and ABTS (2, 2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) together with a variety of different substituted phenols and aromatic amines with the concomitant reduction of oxygen, but POXA1 is unable to oxidize guaiacol. Both enzymes were strongly inhibited by sodium azide and thioglycolic acid but not by EDTA. UV/visible absorption spectra, atomic adsorption, and polarographic data indicated the presence of 4 copper atoms/mol of POXA2 but only one copper, two zinc, and one iron atoms were found/mol of POXA1. The neutral pI and the anomalous metal content of POXA1 laccase render this enzyme unique in its structural characteristics. The lack of typical absorbance at 600 nm allows its classification as a "white" laccase.

Amino Acid Sequence↗

PubMed1972

Lumenal plasma membrane of the urinary bladder. II. Isolation and structure of membrane components.

A technique has been devised for isolation of lumenal plasma membranes from transitional epithelial cells lining the urinary bladder in rabbits and for subsequent separation of particle-bearing plaque regions from particle-free areas of the membranes. The success of the procedures employed and their effects on the isolates were assessed by electron microscopy of conventional plastic sections, negatively stained preparations, and freeze-etch replicas. When bladders are distended with a solution of 0.01 M thioglycolic acid, which reduces sulfhydryl bridges, cytoplasmic filaments are disrupted, and large segments of the lumenal membranes rupture and float free into the lumen. A centrifugation procedure was developed for isolating a fraction enriched with the large fragments. A comparison of membranes isolated in the presence of thioglycolate with those isolated from epithelial cells homogenized in sucrose medium indicates that thioglycolate has little effect on their fine structure except for the removal of filaments which are normally associated with their cytoplasmic surface. The curved plaques of hexagonally arrayed particles and the particle-free interplaque regions, both characteristic of membranes before exposure to thioglycolate, are well preserved. Subsequent treatment of thioglycolate-isolated lumenal membranes with 1% sodium desoxycholate (DOC) severs many of the interplaque regions, releasing individual plaques in which the particles are more clearly visible than before exposure to desoxycholate. Presumably, DOC acts by disrupting the hydrophobic bonds within the membrane; therefore, this type of cohesive force probably is a major factor maintaining the structural integrity of interplaque regions. This conclusion is consistent with the observation that interplaque regions undergo freeze-cleaving like simple bilayers with a plane of hydrophobic bonding.

Animals↗

PubMed1992

Sensitive, direct procedures for simultaneous determinations of iron and copper in serum, with use of 2-(5-nitro-2-pyridylazo)-5-(N-propyl-N-sulfopropylamino)phenol (nitro-PAPS) as ligand.

We developed a direct, simple, and sensitive procedure for the simultaneous colorimetric assay of iron and copper in serum, using sodium dodecyl sulfate-ascorbic acid to dissociate iron and copper from transferrin and ceruloplasmin, respectively. We also use a new water-soluble reagent, 2-(5-nitro-2-pyridylazo)-5-(N-propyl-N-sulfopropylamino)phenol disodium salt (nitro-PAPS) and thioglycolic acid to eliminate interference from copper in the measurement of iron. Within- and between-run precisions of the present method were 2.5-2.8% for iron and 1.8-4.6% for copper. The proposed method is susceptible to interference by hemoglobin and lipemia, especially for the iron assay. Linear-regression analyses of results of the proposed method with those of the bathophenanthroline method for iron and of the atomic absorption spectroscopic method for copper correlated well (r = 0.996, Sy/x = 0.73 and r = 0.959, Sy/x = 1.11, respectively).

Azo Compounds↗

PubMed2002

Metabolism of caffeic acid by isolated rat hepatocytes and subcellular fractions.

Caffeic acid (CA) is found in a wide variety of foods such as vegetables, fruits, tea, coffee, and wine. However, enzymes involved in its metabolism have not been identified. In the following, caffeic (CA), chlorogenic (CGA), and dihydrocaffeic (DHCA) acids were incubated with hepatocytes and shown to undergo metabolism by cytochrome P450, catechol-O-methyltransferase (COMT), and beta-oxidation enzymes. Ferulic (FA) or dihydroferulic (DHFA) acids, formed as the result of CA- or DHCA-O-methylation by COMT, were also O-demethylated by CYP1A1/2 but not CYP2E1. DHCA or DHFA also underwent side chain dehydrogenation to form CA and FA, respectively, which was prevented by thioglycolic acid, an inhibitor of the beta-oxidation enzyme acyl CoA dehydrogenase. The rates of glutathione conjugate formation catalyzed by NADPH/microsomes (CYP2E1) in decreasing order DHCA>CA>CGA trend which was in reverse order to the rates of their O-methylation by COMT. The CA- and DHCA-o-quinones formed by NADPH/P450 likely inhibited COMT but can readily form glutathione conjugates. CA, DHCA and DHFA were inter-metabolized to each other and to FA by isolated rat hepatocytes whereas FA was metabolized only to CA but not to DHCA or DHFA. CA, DHCA, FA, DHFA and CGA showed a dose-dependent hepatocyte toxicity and the LD(50) (2 h), determined were in decreasing order of effectiveness DHCA>CA>DHFA>CGA>FA. In summary, evidence has been provided that O-methylation, GSH conjugation, hydrogenation and dehydrogenation are involved in the hepatic metabolism of CA and DHCA. The O-methylation pathway for CA and DHCA is a detoxification route whereas o-quinones formation catalyzed by P450 is the toxification route.

Animals↗

PubMed1976

[Newer antimycotics. II. Arylhydrazones of derivatives of mesoxalic acid (author's transl)].

The author produced a number of aryl-hydrazone-derivatives of the esters and amides of mesoxalic-acid-seminitril and of mesoxalic-acid-diethylester, investigated their fungistatic efficiency in vitro, and discussed the association of chemical structure with the fungistatic effect in the series of these compounds. From the author's results it can be concluded that some compounds from the series of the aryl-hydrazones of mesoxalic-acid-seminitril-esters exert a strong fungistatic effect on Trichophyton gyps. (Tabl. 1); namely those, whose ester-alkyl-group is relatively short and in that there are the substituents of benzolring Cl-atom, CH3- or NO2- groups. On the other hand the majority of the compounds of this series is totally ineffective (Tab. 2). Each aryl-hydrazones of the amides of mesoxalic-acid-seminitril ant that ones of the mesoxalic-acid-diethylester are ineffective on Trichophyton gyps. (Tab. 2, 4, 5). Not a compound of the investigated series exerts any fungistatic effect on fungi-strains other then Trichophyton gyps. Those aryl-hydrazones of the mesoxalic-acid-seminitrilesters, which proved to be most effective on Trichophyton gyps. are on the mouse only negligible toxic and they do not irritate the skin and mucous membranes of the experimental animals; their fungistatic effect is not influenced by cystein, cysteamin or thioglycolic-acid. On the basis of the intensive fungistatic efficiency and small toxicity of some aryl-hydrazones of the mesoxalic-acid-seminitril-esters, it seems that a number of representatives of these types of chemical compounds could be given a role in the chemotherapy of superficial human and animal dermatomycoses.

Animals↗

PubMed1997

Indolyl nicotinic hydrazides and their neurological studies.

2-Substituted-indole-3-carboxaldehyde I was cyclised with nicotinic acid hydrazide II with few drops of glacial acetic acid which resulted in 2-substituted-2-methyl-3 (2-substituted-3-indole-3-yl-methylene imino) indolyl aldehyde III. Catalytic reduction of III by Pd/C and hydrazine hydrate (99%) in N,N'-dimethyl formamide (DMF) yielded compound IV. III further underwent reaction with thioglycolic acid and anhydrous ZnCl2 to give VI. Compound VIII were synthesised from IV by the reaction of substituted arylaldehyde in the presence of anhydrous sodium acetate and VII with 2-chloropropionic acid in the presence of Et3N respectively. III underwent cyclisation with chloroacetyl chloride and Et3N in DMF to yield compound V. Diazotisation of III with various substituted arylamines afforded IX. The compounds were evaluated for their C.N.S., anti-parkinsonian and MAO inhibitory activities.

Animals↗

PubMed1984

Cysteine: a potential source of error in amino acid analysis of mercaptoethane sulfonic or hydrochloric acid hydrolysates of proteins and peptides.

Hydrolysis of proteins and peptides with mercaptoethane sulfonic acid is liable to produce overestimation of the proline content owing to the production of ninhydrin-positive material (probably cysteine) which coelutes with proline on many ion-exchange analytical systems. A similar error occurs with HCl hydrolysis (especially in the presence of mercaptoethanol or thioglycollic acid) if care is not taken to oxidize cysteine during reconstitution of the hydrolysate before amino acid analysis.

Amino Acids↗

PubMed1987

Studies on hydroperoxide-dependent folic acid degradation by hemin.

Hemin (ferric protoporphyrin IX chloride) in the presence of hydrogen peroxide or tert-butyl hydroperoxide was found to cleave folic acid at the C9-N10 bond. The ferrous form of hemin was not involved in hydroperoxide-dependent folic acid degradation, as indicated by the lack of inhibition by carbon monoxide. Molecular oxygen was not required for the degradation. GSH-Mn(II) or NAD(P)H in the presence of molecular oxygen did not support hemin-mediated folic acid degradation. The degradation increased as the temperature was elevated from 10 to 70 degrees C. Ascorbic acid and azide were potent inhibitors. Superoxide dismutase and hydroxyl radical quenchers, such as ethanol, mannitol, benzoate, and dimethyl sulfoxide did not inhibit the reaction. Catalase inhibited hydrogen peroxide-supported degradation but not the tert-butyl hydroperoxide-dependent one. Thiol compounds, such as thioglycolic acid, thiourea, glutathione, cysteine, and 2-mercaptoethanol, inhibited the hydrogen peroxide-dependent degradation but supported the tert-butyl hydroperoxide-mediated one. N5-formyl tetrahydrofolic acid, but not N10-formyl folic acid, was degraded by hemin in the presence of H2O2 or TBHP. The data obtained are suggestive of a mechanism similar to N-demethylation reactions catalyzed by cytochrome P-450 and some peroxidases.

Folic Acid↗

PubMed1992

Application and comparison of silver intensification methods for the diaminobenzidine and diaminobenzidine-nickel endproduct of the peroxidation reaction in immunohistochemistry and in situ hybridization.

Silver-intensification methods described in the literature for the diaminobenzidine (DAB) and diaminobenzidine-nickel (DAB/Ni) endproduct of the peroxidase reaction were compared in model systems after immunoperoxidase and in situ hybridization. First, these methods were compared in immunohistochemical model systems, using the demonstration of glial fibrillar acidic protein (GFAP) and prostate-specific antigen (PSA) in paraffin sections of human brain and prostate tissue, respectively. When DAB without Ni was used as substrate, tissue argyrophilia caused considerable background staining. Only when this tissue reactivity was quenched with, e.g., CuSO4 with H2O2 or thioglycolic acid, were the results acceptable. A considerable improvement in the signal-to-noise ratio could be obtained when nickel was included in the substrate mixture. The methods that proved to be best for demonstration of GFAP and PSA made use of acid developer solutions. Subsequently, these methods were compared with other sensitive immunostaining methods for demonstration of the gamma-delta T-cell receptor in frozen lymphoid tissue. In this model a considerable increase in the number of positive cells could be obtained using silver intensification. The different methods using DAB/Ni were also compared for use in DNA in situ hybridization (DISH). In this case two model systems were used: human papilloma virus type 11 (HPV-11) DNA in condyloma tissue (abundant target model) and Epstein-Barr virus (EBV) DNA in a mononucleosis lymph node (low target model). For demonstration of HPV-11, all methods gave more or less satisfactory results, which were best with the acid developer solutions. Moreover, for demonstration of EBV DNA, a signal could be obtained only with these developer solutions. Such a method also proved suitable in double immuno-hybrido stainings for the demonstration of EBV DNA in specific antigen-positive Reed-Sternberg cells in paraffin sections of Hodgkin lymph nodes.

3,3'-Diaminobenzidine↗

PubMed2001

Synthesis and evaluation of the analgesic and antiinflammatory activities of O-substituted salicylamides.

The present investigation deals with the synthesis of some new salicylamidoacetyl sulfonamides 3a,b, salicylamido ethylacetate 4, salicylamido acetic acid hydrazide 5, which is considered as the key intermediate for the synthesis of several series of new compounds such as salicylamido pyrazol 6 and pyrazolone 7. N-imido-derivatives 9, 10, 11, thiadiazole 13, oxadiazole 14, 15, Schiffs bases 16a-f. Cyclocondensation of Schiffs bases with thioglycolic acid gave thiazolidinone 18a-c while with acetylchloride afforded azitidinones 19a-c and with acetic anhydride gave 1,4-benzoxazepine-3,5-dione. Some of the compounds were tested for their analgesic and antiinflammatory activities as well as ulcerogenic effects. Some derivatives were more effective than salicylamide and ulcerogenic activity was variably lowered.

Analgesics, Non-Narcotic↗

PubMed1986

Immunocytochemical methods for demonstrating canine distemper virus antigen in aldehyde-fixed paraffin-embedded tissue.

The effects of enzymatic digestion, sodium borohydride reduction, acids used in decalcification procedures and techniques for inactivation of endogenous peroxidase were sequentially evaluated for their effect on the immunoreactivity of canine distemper virus in aldehyde-fixed paraffin-embedded tissue. Enzyme digestion improved immunoreactivity while sodium borohydride reduced background staining. Paraformaldehyde-glutaraldehyde-fixed tissues required thioglycolic acid treatment prior to enzyme digestion and sodium borohydride reduction to obtain results comparable to results obtained in formalin-fixed tissues. Detailed protocols for indirect immunofluorescence and the avidin-biotin-peroxidase complex procedure are provided.

Acids↗

PubMed2006

Wheat cells accumulate a syringyl-rich lignin during the hypersensitive resistance response.

The stem rust fungus Puccinia graminis f.sp. tritici is an obligately biotrophic pathogen attacking wheat (Triticum aestivum). In compatible host/pathogen-interactions, the fungus participates in the host's metabolism by establishing functional haustoria in the susceptible plant cells. In highly resistant wheat cultivars, fungal attack is stopped by a hypersensitive response of penetrated host cells. This mechanism of programmed cell death of single plant cells is accompanied by the intracellular accumulation of material with UV-fluorescence typical of phenolic compounds. A similar reaction can be induced in healthy wheat leaves by the application of a rust-derived elicitor. We analysed the biochemical composition of this defense-induced phenolic material. Contents of total soluble and cell wall esterified and etherified phenolic acids were determined in rust-inoculated and elicitor-treated leaves of the fully susceptible wheat cultivar Prelude and its highly resistant, near-isogenic line Prelude-Sr5. While no resistance-related changes occured in any of these fractions, the lignin content as determined by the thioglycolic acid and the acetyl bromide methods increased after elicitor treatment. Nitrobenzene oxidation revealed that the entire increase can be explained by an increase in syringyl units only. These biochemical data were confirmed by fluorescence emission spectra analyses which indicated a defense-induced enrichment of syringyl lignin for cell wall samples both from elicitor-treated wheat leaves and single host cells undergoing a hypersensitive response upon fungal penetration.

Basidiomycota↗

PubMed1993

Conditioning of Parsley (Petroselinum crispum L.) Suspension Cells Increases Elicitor-Induced Incorporation of Cell Wall Phenolics.

The elicitor-induced incorporation of phenylpropanoid derivatives into the cell wall and the secretion of soluble coumarin derivatives (phytoalexins) by parsley (Petroselinum crispum L.) suspension cultures can be potentiated by pretreatment of the cultures with 2,6-dichloroisonicotinic acid or derivatives of salicylic acid. To investigate this phenomenon further, the cell walls and an extracellular soluble polymer were isolated from control cells or cells treated with an elicitor from Phytophthora megasperma f. sp. glycinea. After alkaline hydrolysis, both fractions from elicited cells showed a greatly increased content of 4-coumaric, ferulic, and 4-hydroxybenzoic acid, as well as 4-hydroxybenzaldehyde and vanillin. Two minor peaks were identified as tyrosol and methoxytyrosol. The pretreatment effect is most pronounced at a low elicitor concentration. Its specificity was elaborated for coumarin secretion. When the parsley suspension cultures were preincubated for 1 d with 2,6-dichloroisonicotinic, 4- or 5-chlorosalicylic, or 3,5- dichlorosalicylic acid, the cells exhibited a greatly increased elicitor response. Pretreatment with isonicotinic, salicylic, acetylsalicylic, or 2,6-dihydroxybenzoic acid was less efficient in enhancing the response, and some other isomers were inactive. This increase in elicitor response was also observed for the above-mentioned monomeric phenolics, which were liberated from cell walls upon alkaline hydrolysis and for "lignin-like" cell wall polymers determined by the thioglycolic acid method. It was shown for 5-chlorosalicylic acid that conditioning most likely improves the signal transduction leading to the activation of genes encoding phenylalanine ammonia lyase and 4-coumarate: coenzyme A ligase. The conditioning thus sensitizes the parsley suspension cells to respond to lower elicitor concentrations. If a similar mechanism were to apply to whole plants treated with 2,6-dichloroisonicotinic acid, a known inducer of systemic acquired resistance, one can hypothesize that fungal pathogens might be recognized more readily and effectively.

Journal Article↗

PubMed1989

Direct amino acid analysis of proteins electroblotted onto polyvinylidene difluoride membrane from sodium dodecyl sulfate-polyacrylamide gel.

The band of appropriate proteins (basic pancreatic trypsin inhibitor, soybean trypsin inhibitor, interleukin 2, and human leukocyte interferon alpha A) on a polyvinylidene difluoride (PVDF) membrane, which was electroblotted from sodium dodecyl sulfate (SDS)-polyacrylamide gel and then stained with Coomassie blue R-250, was cut out and directly hydrolyzed in HCl in the presence of thioglycolic acid for amino acid analysis. The analytical values agreed with those expected with recoveries of 29-47%, except that the value for tryptophan was very low or scarcely detected. This method was applied to the identification of human growth hormone (hGH) in a partially purified preparation. The amino acid composition of the band corresponding to about 2 micrograms of hGH agreed with the theoretical values. These results indicate that the band on the PVDF membrane can be directly hydrolyzed for amino acid analysis and that the method can be used for partially purified proteins separated using SDS-polyacrylamide gel electrophoresis.

Amino Acids↗

PubMed2000

Roles of phosphate and an enoyl radical in ferritin iron mobilization by 5-aminolevulinic acid.

5-Aminolevulinic acid (ALA), a heme precursor that accumulates in acute intermittent porphyria (AIP) and lead poisoning, undergoes enolization and subsequent iron-catalyzed oxidation at neutral pH. Iron is released from horse spleen ferritin (HoSF) by both ALA-generated O(2)(.-) and enoyl radical (ALA(z.rad)), which amplifies the chain of ALA oxidation (autocatalysis). Iron chelators such as EDTA, ATP, but not citrate, and phosphate accelerate this process and ALA-promoted iron release from HoSF is faster in horse spleen isoferritins containing larger amounts of phosphate in the core. ALA (+0.377 V versus standard hydrogen electrode) is less effective in releasing iron from ferritin than are thioglycollic acid, 6-hydroxydopamine, and N,N,N', N'-tetramethyl-p-phenylenediamine. During electrochemical one electron oxidation of ALA in a nitrogen atmosphere, spin trapping experiments with 3,5-dibromo-4-nitrosobenzenesulfonic acid demonstrated the formation of a spin adduct characterized by a six line signal, indicating a secondary carbon-centered radical and attributed to a resonant ALA&z.rad; radical. Iron is also released in such anaerobic electrochemical oxidations of ALA in the presence of ferritin, suggesting that, in addition to O(2)(*-), ALA&z.rad; can promote iron mobilization from ferritin. Hence, ALA&z.rad; may amplify the metal-catalyzed oxidation of ALA, damaging ALA-accumulating cells and possibly contributing to the symptoms of porphyria.

Adenosine Triphosphate↗