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At least 865 records · Page 48Linked to original sources
PubMed1994

Hepatic and extrahepatic bioactivation and GSH conjugation of aflatoxin B1 in sheep.

Whole-body autoradiography of [3H]aflatoxin B1 ([3H]AFB1) in lamb showed a localization of bound labelling, in addition to the liver, in the nasal olfactory and respiratory mucosa, in the mucosa of the nasopharynx, pharynx, oesophagus, larynx, trachea, bronchi and bronchioles and in the palpebral and bulbar conjunctiva. Microautoradiography revealed that the bound material was confined to specific cell types in extrahepatic tissues. Whole-body autoradiography also showed a labelling of pigmented tissues (such as the eye melanin), which can be ascribed to a melanin affinity of AFB1. In vivo experiments, performed with microsomal preparations of tissues from ewe and lamb showed that several of the extrahepatic tissues were more efficient than the liver in forming DNA-bound AFB1 metabolites. The nasal olfactory mucosa was by far the most effective tissue in this respect. AFB1 induced a high number of gene mutations in Salmonella typhimurium TA100 when incubated with supernatant preparations (9000 g) of the nasal olfactory mucosa, whereas incubations with preparations of the liver resulted in a lower effect. It has been reported that AFB1 can induce nasal tumours in sheep. When microsomal preparations of various tissues were incubated in the presence of reduced glutathione (GSH), but without any addition of cytosolic glutathione-S-transferase (GST), a drastic decrease in the AFB1-DNA binding was seen. Analyses of the water-soluble metabolites formed in the microsomal incubations supplemented with GSH showed fluorescent and ninhydrin-positive metabolites that were not present in the absence of GSH. These results indicate that sheep tissues have intrinsic microsomal GST or cytosolic GSTs associated with the microsomal fraction with a high capacity to catalyse the conjugation of bioactivated AFB1 to GSH. The results of the present study show that several extrahepatic tissues of sheep have a potent capacity to bioactivate AFB1 and also a high capacity to GSH conjugate the bioactivated AFB1.

Aflatoxin B1↗
PubMedDate not supplied

Heterogeneity of lipid A.

In a previous study, lipid A from Shigella flexneri was separated into eight distinct fractions by thin-layer chromatography (TLC). In the present investigation, lipid A from different gram-negative bacteria was analyzed by thin-layer chromatography for its heterogeneity and compared with lipid A of S. flexneri. Purified lipid A preparations from six different enterobacterial species were heterogeneous, but based on Rf, comparable bands were found among all preparations. Changing the culture conditions by using two other culture media resulted in very minor changes in the TLC appearance. Different serotypes of Shigella grown under the same conditions also showed very minor differences. Two bands (5 and 8) in each preparation of lipid A comigrated with lysophosphatidylethanolamine and phosphatidylethanolamine, respectively, and both bands were stained with ninhydrin and molybdenum blue. Therefore, phosphatidylethanolamine, and probably lysophosphatidylethanolamine, are common constitutents of unfractioned lipid A preparations, and both phospholipids can occur even in highly purified unfractionated lipid A.

Chromatography, Thin Layer↗
PubMedDate not supplied

A labetalol metabolite with analytical characteristics resembling amphetamines.

A metabolite of labetalol that is responsible for previous reports of false-positive assays for amphetamines by thin-layer chromatography and immunoassay has been identified. The compound, 3-amino-1-phenylbutane (APB), an oxidative metabolite of labetalol, was initially identified in a patient's urine by gas chromatography-mass spectrometry and confirmed by analysis of the pure material. Gas chromatography showed a single major peak with relative retention time indistinguishable from that of methamphetamine, both underivatized and as the pentafluoropropionyl derivative. By classical thin-layer chromatography, APB migrated identically to methamphetamine but showed ninhydrin color development characteristic of amphetamine. APB was shown to cross-react approximately 2% with the Abbott TDx amphetamine/methamphetamine II kit, 10% with the Syva EMIT d.a.u. polyclonal amphetamine class kit, and 3% with the Syva EMIT d.a.u. monoclonal amphetamine kit. This degree of cross-reactivity is sufficient to cause false-positive immunoassays when cutoffs of 300 ng/mL are used.

Aged↗
PubMed1995

Peritrophic envelopes of Lutzomyia spinicrassa (Diptera: Psychodidae).

The secretion, morphology, and chemical composition of the peritrophic envelope were studied in the phlebotomine sand fly, Lutzomyia spinicrassa Morales, OsornoMesa, Osorno & Hoyos, a suspected vector of Leishmania braziliensis in Colombia and Venezuela. Viewed under light microscopy, the envelope matured rapidly and could be dissected from the blood bolus as early as 12 h and until 36 h after feeding; subsequently it began to degrade. The envelope was initially a closed sac around the blood meal, but opened posteriorly in most flies by 6 h. The posterior opening may facilitate the migration and establishment of Le. braziliensis in the hindgut. Secretion of envelope precursors was from the entire midgut epithelium. Electron microscopy revealed that electron-dense precursor material (possibly chitin) was present, bathing the microvilli during the first 12 h after blood feeding. This secretion appeared to originate from the bases of the microvilli. From 1 to 36 h, an electron-lucid precursor material (possibly protein) was secreted from the entire length of microvilli and from their bases. Both precursors appeared to be formed at the epithelial surface, not associated with secretory vesicles. The envelope developed rapidly from precursor material, and by 6 h a defined electron-lucid structure was present above the microvilli. Most mature envelopes (12-36 h) were 0.5-2.1 microns thick, multilayered, wholly electron-lucid, and composed of microfibrils and granules. Electron-dense components were seen in some envelopes at 24-36 h. An anterior hyaline plug was present from 12 to 36 h. Envelopes were composed of chitin, protein, and glycoprotein, based on chemical and histochemical tests. The likely presence of several amino acids (lysine, aspartic acid, and glutamic acid) that may cross-link chitin and protein was demonstrated by a positive ninhydrin-Schiff test. This study constitutes the first ultrastructural investigation of peritrophic envelope development by a New World sand fly.

Animals↗
PubMed2005

Dietary S-methylmethionine, a component of foods, has choline-sparing activity in chickens.

Acid hydrolysis of dehulled soybean meal (SBM) and corn gluten meal (CGM) followed by chromatographic amino acid analysis (ninhydrin detection) revealed substantial quantities of S-methylmethionine (SMM) in both ingredients (1.65 g SMM/kg SBM; 0.5 g SMM/kg CGM). Young chicks were used to quantify the methionine- (Met) and choline-sparing bioactivity of crystalline L-SMM, relative to L-Met and choline chloride standards in 3 assays. A soy isolate basal diet was developed that could be made markedly deficient in Met, choline, or both. When singly deficient in choline or in both choline and Met, dietary SMM addition produced a significant (P < 0.01) growth response. In Assay 2, dietary SMM did not affect (P > 0.10) growth of chicks fed a Met-deficient, choline-adequate diet. A standard-curve growth assay revealed choline bioactivity values (wt:wt) of 14.2 +/- 0.8 and 25.9 +/- 5.1 g/100 g SMM based on weight gain and gain:food responses, respectively. A fourth assay, using standard-curve procedures, showed choline bioactivity values of 20.1 +/- 1.1 and 22.9 +/- 1.7 g/100 g SMM based on weight gain and gain:food responses, respectively. It is apparent that SMM in foods and feeds has methylation bioactivity, and this has implications for proper assessment of dietary Met and choline requirements as well as their bioavailability in foods and feeds.

Animals↗
PubMed1998

Substrate specificity of human monoamine (M)-form phenol sulfotransferase: preparation and analysis of Dopa 3-O-sulfate and Dopa 4-O-sulfate.

Upon two-dimensional thin-layer separation, the sulfated L-3, 4-dihydroxyphenylalanine (L-DopaS) generated enzymatically was found to co-migrate with only one of the two ninhydrin-stained spots corresponding to the two sulfated forms (3-O-sulfate and 4-O-sulfate) of synthetic L-DopaS. To clarify precisely the identity of the enzymatically generated L-DopaS, the two sulfated forms of synthetic L-DopaS were separated and purified using high performance liquid chromatography. Purified L-Dopa 3-O-sulfate and L-Dopa 4-O-sulfate were identified by 1H-nuclear magnetic resonance (NMR) spectrometry and used as standards in the analysis of the L-DopaS generated during metabolic labeling of HepG2 human hepatoma cells or enzymatic assay using recombinant human monoamine (M)-form phenol sulfotransferase. The results obtained demonstrated unequivocally the generation of L-Dopa 3-O-sulfate, indicating the specificity of the M-form phenol sulfotransferase being for the meta-hydroxyl group of L-Dopa.

Arylsulfotransferase↗
PubMed1988

Some chemical properties of maitotoxin, a putative calcium channel agonist isolated from a marine dinoflagellate.

Maitotoxion, a putative Ca2+ channel agonist, was isolated from cultures of the marine dinoflagellate Gambierdiscus toxicus as a colorless amorphous solid. The toxin reacted positively to Dragendorff's reagent but not to ninhydrin reagent. The mouse lethality of maitotoxin determined by intraperitoneal injection was 0.13 micrograms/kg. Chemical features of the toxin were elucidated mainly by various spectroscopic measurements. The molecular weight of maitotoxin as a disodium salt was estimated to be 3,424.5 +/- 0.5 from the negative fast atom bombardment (FAB) mass spectrum. The presence of two sulfate ester groups in the molecule was apparent from the IR and mass spectra, and from analyses of solvolysis products. Despite of its large size, maitotoxin seems to have no known repeating units, such as amino acids and sugars, no carbonyl groups, no side chains other than methyls or an exomethylenes, and no carbocycles.

Animals↗
PubMed1976

The structure and function of ribonuclease T1. XXI. Modification of histidine residues in ribonuclease T1 with iodoacetamide.

1. When ribonuclease T1 [EC 3.1.4.8] (0.125% solution) was treated with a 760-fold molar excess of iodoacetamide at pH 8.0 and 37 degrees, about 90% of the original activity was lost in 24 hr. The half-life of the activity was about 8 hr. The binding ability for 3'-GMP was lost simultaneously. Changes were detected only in histidine and the amino-terminal alanine residues upon amino acid analyses of the inactivated protein and its chymotryptic peptides. The inactivation occurred almost in parallel with the loss of two histidine residues in the enzyme. The pH dependences of the rate of inactivation and that of loss of histidine residues were similar and indicated the implication of a histidine residue or residues with pKa 7.5 to 8 in this reaction. 3'-GMP and guanosine showed some protective effect against loss of activity and of histidine residues. The reactivity of histidine residues was also reduced by prior modification of glutamic acid-58 with iodoacetate, of lysine-41 with maleic or cis-aconitic anhydride or 2,4,6-trinitrobenzenesulfonate or of arginine-77 with ninhydrin. 2. Analyses of the chymotryptic peptides from oxidized samples of the iodoacetamide-inactivated enzyme showed that histidine-92 and histidine-40 reacted with iodoacetamide most rapidly and at similar rates, whereas histidine-27 was least reactive. Alkylation of histidine-92 was markedly slowed down when the Glu58-carboxymethylated enzyme was treated with iodoacetamide. On the other hand, alkylation of histidine-40 was slowed down most in the presence of 3'-GMP. These results suggest that histidine-92 and histidine-40 are involved in the catalytic action, probably forming part of the catalytic site and part of the binding site, respectively, and that histidine-27 is partially buried in the enzyme molecule or interacts strongly with some other residue, thus becoming relatively unreactive.

Alanine↗
PubMed1977

C-Terminal peptidyl-L-proline hydrolase activity of Aspergillus acid carboxypeptidase.

Aspergillus saitoi acid carboxypeptidase hydrolyzed C-terminal peptidyl-L-proline bonds and released the C-terminal proline from Z-Gly-Pro-Leu-Gly-Pro and Z-Gly-Pro at pH 3.3. Proline liberated by the enzymic reaction was measured by a sensitive colorimetric ninhydrin method in glacial acetic acid at 513 nm. A Km value of 1.0 mM and a kcat value of 0.09 s-1 for Z-Gly-Pro-Leu-Gly-Pro hydrolysis, and a Km value of 5.0 mM and a kcat value of 0.0045 s-1 for Z-Gly-Pro hydrolysis were calculated from Lineweaver-Burk plots.

Amino Acid Sequence↗
PubMed1979

Nature of adenosine triphosphatase accelerating peptide from hydrolysate of fur seal muscle.

Ultrafiltered fur seal muscle hydrolysate was divided into eleven fractions by gel filtration on Sephadex G-15. One of the fractions (Fraction G9) accelerated the ATPase activity of carp myosin B to a rate about two-fold faster than that of the control. Fraction G9 showed a single ninhydrin spot in its silica gel thin layer chromatograph, and gave a positive test for tryptophan by the p-dimethylaminobenzaldehyde method, while tests for tyrosine, and for arginine were negative. The ion exchange amino acid analysis of its acid hydrolysate showed a predominant content of lysine, nearly equivalent to the amount of tryptophan determined from its UV absorbancy and the p-dimethylaminobenzaldehyde method. The N-terminal amino acid analysis gave di-DNP-Lys as the sole DNP-amino acid. The structure of the ATPase accelerating peptide fraction, Fraction G9, was deduced to be Lys-Trp.

Adenosine Triphosphatases↗
PubMed1981

Purification and properties of urate oxidase from Streptomyces cyanogenus.

Urate oxidase [EC 1.7.3.3] was purified to homogeneity from cell-free extracts of a strain of Streptomyces cyanogenus. The enzyme had a molecular weight of 100,000 and consisted of three subunits each with a molecular weight of 32,000. The isoelectric point was at pH 4.0. No evidence was found for the involvement of copper, iron or coenzymes in the urate oxidase reaction. The enzyme was most active at pH 8 and at 35 degrees C, and was stable between pH 6 and 11 (35 degrees C, 1 h) and below 50 degrees C (pH 7.8, 10 min). The enzyme was inhibited by cyanide and sulfhydryl reagents, but only slightly by heavy metal ions and chelating agents. The activity was inhibited by xanthine and 2-hydroxypurine. The enzyme was found not to be inhibited by high concentrations of uric acid when the activity was assayed in terms of hydrogen peroxide formation. Urea and racemic allantoin were formed from uric acid by the enzyme reaction in phosphate buffer, and urea and other ninhydrin-positive materials in borate buffer.

Amino Acids↗
PubMed1985

Histamine and 5-hydroxytryptamine in human scar tissue.

Patients with unstable scars in some areas of the body complain of local itchiness and pain, which may be caused by histamine and serotonin. Therefore, I measured these substances in scar tissue in various conditions taken at the time of operation. The histamine value was determined by the amount of carbon dioxide produced in histidine decarboxylase activity (HDCA); the measurement of 5-hydroxytryptamine (5-HT) was done by the Ninhydrin method. High values of HDCA were usually found in skin flaps and scar tissue around pressure sores. 5-HT activity reached a higher level in unstable scars than in stable scars.

Adolescent↗
PubMed1994

Heat treatment of infant formula: effect on postprandial serum alpha-amino-nitrogen concentrations in very-low-birth-weight infants.

Several different types of heat treatment are used in the production of commercial infant formulas. The extent of heat treatment can have different physicochemical effects on the milk proteins and will affect their solubility and digestibility. In the present study, 40 very-low-birth-weight infants were randomized at the age of 2 weeks to be given one test feed of either fresh, human milk protein-fortified human milk, a conventional canned sterilized liquid formula, a spray-dried powder formula, or an ultra-high-temperature (UHT)-treated liquid formula. The mean volume of the nasogastric test feed was adjusted so that protein intake was the same in each study group (0.45 g/kg). Venous blood samples were taken preprandially and at 30, 60, and 120 min after the test meal. alpha-amino-nitrogen in serum was assayed by the ninhydrin reaction. The concentration of alpha-amino-nitrogen rose rapidly after the fortified human milk feed, reaching a peak at 30 min. After the peak was reached, the serum values progressively declined, reaching baseline values at 120 min after the start of the test meal. The areas under the curve for all formulas were significantly different from that found for fortified human milk. At 30 min, all formulas had significantly lower alpha-amino-nitrogen values than fortified human milk; at 60 min, all formulas had significantly higher mean concentrations than fortified human milk. At 120 min, none of the formula-fed infants had reached baseline values; in particular, canned sterilized formula was still significantly above baseline values.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1998

Changes in collagen cross-linking in degenerative disc disease and scoliosis.

STUDY DESIGN: Biochemical study of the changes in the collagen cross-link profile of human intervertebral discs collected at surgery from patients with either low back pain associated with disc degeneration or scoliosis. OBJECTIVE: To determine whether changes occur in the collagen cross-link profile in the disc of patients with either low back pain associated with disc degeneration or scoliosis, which may well influence matrix integrity. Such changes in the cross-link profile of a tissue indicates increased matrix turnover and tissue remodeling and may have implications for the progression of these disorders. SUMMARY OF BACKGROUND DATA: The diseases of the intervertebral disc, degenerative disc disease and scoliosis, are both characterized by changes in the extracellular matrix components that will affect the mechanical function of the tissue. The stability of the collagenous components and hence the mechanical integrity of connective tissues such as the disc is dependent on the degree and type of cross-links between the collagen molecules. This article reports results on the distribution of the different cross-links in the disc and the changes that occur with age, degenerative disc disease, and scoliosis. METHODS: Thirty-three discs were obtained from patients with degenerative disc disease and 29 discs from patients with scoliosis. Samples were acid hydrolyzed and the collagen cross-links analyzed by either fractionation on an amino acid analyzer configured for cross-link analysis using ninhydrin postcolumn detection or fractionation by high-pressure liquid chromatography with fluorescence detection. RESULTS: The reducible cross-links and the mature cross-link all increased from the outer anulus fibrosus through into the nucleus pulposus. The highest levels of the mature cross-link were found in the cartilage end-plate. The nonenzymic derived cross-link, pentosidine, in contrast, showed little difference across the disc, but did show the expected age-related increase. In degenerative disc disease, no change in the levels of the reducible or mature cross-links was found, but a decrease was observed in the levels of the age-related cross-link pentosidine in the more severe disease samples. In scoliosis, significantly higher levels of the reducible cross-links were found on the convex than on the concave side of the scoliotic disc. CONCLUSIONS: These changes in the cross-link profile of the intervertebral disc in degenerative disc disease and scoliosis are indicative of increased matrix turnover and tissue remodeling and likely to have implications for the progression of these disorders.

Adolescent↗
PubMed1981

Production, purification and chemical properties of an antistaphylococcal agent produced by Staphylococcus epidermidis.

An antibacterial agent produced by Staphylococcus epidermidis strain 5 was isolated from culture supernatants; its spectrum of activity is restricted to staphylococci and micrococci. The bacteriocin-like substance was purified to homogeneity by column chromatography on Servachrome XAD-2, CM-Sephadex C-25 and Sephadex G-50. Chemical analysis showed it to be a peptide with a molecular weight of about 6000 and an isoelectric point of about 10.5. Purified material was rapidly inactivated by trypsin, chymotrypsin and pronase and it was relatively heat-stable. After acidic hydrolysis 10 amino acid residues and two unidentified ninhydrin-positive substances were detected. Production of the bacteriocin-like substance was not inducible with mitomycin C.

Amino Acids↗
PubMed1985

Accumulation of peptides by mycobacillin-negative mutants of Bacillus subtilis B3.

Thirteen mycobacillin-negative (My-) mutants of Bacillus subtilis B3 were isolated from an auxotrophically tagged mycobacillin producer organism. The wild-type producer, three feeble producers and three strictly My- mutants did not accumulate any ninhydrin-positive peptide in the culture medium while the remaining seven My- mutants did accumulate ten such peptides whose amino acid composition indicated that there might be only three different peptides. The N-terminal and C-terminal amino acid residues implicated one of these peptides as a pentapeptide intermediate in mycobacillin synthesis; this was further confirmed by its molecular weight and sequence. Studies on cell-free synthesis showed that only the enzyme system from the wild-type strain synthesized mycobacillin while the defective ones from all the My- mutants synthesized one and the same pentapeptide as found in the culture broth of some of the mutants. Further studies in which the enzymes responsible for mycobacillin synthesis by cell-free extracts were separated into three fractions, A, B and C, showed that seven of the mutants were defective in fraction B whereas the three other mutants had defects in both fractions B and C. Thus the pentapeptide Pro----Asp----Glu----Tyr----Asp appears to be implicated in mycobacillin biosynthesis.

Amino Acid Sequence↗
PubMed1990

Protective activity of glycolipid antigen against infection by Leptospira interrogans serovar canicola.

A protective glycolipid antigen (PAg) was extracted from Leptospira interrogans serovar canicola with chloroform/methanol/water (1:2:0.8, by vol.) and partially purified by silica gel column chromatography. The PAg elicited a protective response in hamsters and in cyclophosphamide-treated mice subsequently challenged with homologous Leptospira. The PAg band was detected as a single smear-like band, corresponding to a protein of 23-30 kDa, by silver-staining in SDS-PAGE. In immunoblots, this band reacted with a monoclonal antibody, A5, which agglutinated serovar canicola and recognized a serovar-specific antigen. Furthermore, the PAg did not migrate on silica gel TLC, but was detected at the origin as a ninhydrin- and naphthol-positive spot. This suggests that PAg is a hydrophilic molecule with a carbohydrate chain that contains amino groups, possibly as amino sugars.

Animals↗
PubMed1976

Intracellular peptide hydrolysis by Pseudomonas putida and Pseudomonas maltophilia.

Amino acids liberated by peptidase hydrolysis of di- and oligopeptides by Pseudomonas putida were measured by trinitrobenzenesulphonate assay and high voltage electrophoresis or paper chromatography followed by ninhydrin spray. Intact bacteria or periplasmic contents released by lysozyme treatment did not hydrolyse peptides. Subcellular fractionation showed that glycylmethionine peptidase activity was cytoplasmic. This enzyme had a Km of 2 mM, and was stimulated fivefold by I mM-Co2+. Crude peptidase extract did not cleave peptides with D-residues, acylated N-terminal amino groups or N-methylated peptide bonds but otherwise showed a wide specificity. Di- or tripeptides with blocked C-terminus were hydrolysed. Leucylleucine (12 mM) and leucylglycylglycine (10 mM) did not compete with glycylmethionine (1-2 mM) and glycylmethionylglycine (1-0 mM), respectively, for hydrolysis. Pseudomonas maltophilia also contained peptidase activity (0-84 mumol amino acid released from glycylmethionylglycine/min/mg protein). Peptidases of both P. putida and P. maltophilia were constitutive.

Cell-Free System↗