Ultrastructural pulmonary changes induced by intravenously administered 3-methylindole in goats.
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Transmission electron microscopy was used to characterize early pulmonary lesions in goats after a 2-hour intravenous infusion of 0.04 g 3-methylindole (3MI) per kilogram body weight. Groups of 2 or 3 goats were euthanized at 0.5, 2, 4, 8, and 24 hours after the beginning of the infusion. Changes in lung ultrastructure were compared to noninfused and carrier-infused (propylene glycol) controls. By 0.5 hour, mitochondria and intracellular vesicles were swollen in capillary endothelial, alveolar, and nonciliated bronchiolar epithelial cells. Morphologic changes were most severe in the alveolar Type 1 and nonciliated bronchiolar epithelial cells. Interalveolar septums were swollen at 0.5 hour, and interstitial edema was severe at 2 hours. Denuded alveolar epithelial basement membranes were also observed at 2 hours, and some endothelial cells appeared dark and necrotic. Endothelial cells appeared normal after 2 hours. By 4 hours, the remaining intact alveolar Type 1 cells contained larger and more prominent clusters of smooth endoplasmic reticulum, compared with controls. Morphologic changes in alveolar Type 1 and nonciliated bronchiolar epithelial cells became progressively more severe during the 24-hour experiment. These findings demonstrate that 3MI induces a rapid cytotoxic effect primarily on alveolar Type 1 and nonciliated bronchiolar epithelial cells. Proliferation of smooth endoplasmic reticulum in these cells suggests involvement of the mixed function oxidase system in 3MI-induced pneumotoxicity. (+info)
Peptide fragmentation suitable for solid-phase microsequencing. Use of N-bromosuccinimide and BNPS-skatole (3-bromo-3-methyl-2-[(2-nitrophenyl)thio]-3H-indole).
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BNPS-skatole (3-bromo-3-methyl-2-(2-nitrophenyl)thiol-3H-indole) and N-bromosuccinimide have been used to specifically cleave at peptide bonds after amino acids with available C-gamma=C-delta double bonds, i.e. tryptophan, tyrosine and histidine. The resulting C-terminal lactones conveniently attach to amino-glass supports for sequencing with DABITC (4-NN-dimethylaminoazobenzene 4'-isothiocyanate). Also, peptides having such C-termini, i.e. from a chymotryptic digest, can be readily made to react with these reagents and thus be easily attached and sequenced by solid-phase methods. (+info)
D-glyceraldehyde-3-phosphate dehydrogenase. Complete amino-acid sequence of the enzyme from Bacillus stearothermophilus.
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1. The complete amino acid sequence of D-glyceraldehyde-3-phosphate dehydrogenase from the moderate thermophile Bacillus stearothermophilus has been determined. 2. This has been achieved largely by the automated sequence analysis of large fragements derived by chemical cleavage with cyanogen bromide, BNPS-skatole [the product of reaction between N-bromosuccinimide and 2-(nitrophenyl-sulphenyl)-3-methylindole] and hydroxylamine and enzymic hydrolysis with trypsin at arginine residues. 3. The sequence is as follows: (See Text). It has been numbered to maximise homology with the four complete sequences of this enzyme from other sources. Hence the N-terminal residue is numberd 0 and two deletions and two insertions have been introduced. 4. The inability of the B. stearothermophilus apo-enzyme to transfer an acyl moiety from Cys-149 to Lys-183 oberved with muscle enzymes is explained by the replacement of lysine by arginine in the enzyme from the thermophilic organism. 5. The sequences of the S-loop regions, which form the core of the tetrameric enzyme, are similar to each other in B. stearothermophilus and Thermus aquaticus and differ from the highly conserved S-loops of three enzymes from mesophiles. (+info)
Inhibition of ruminal degradation of L-tryptophan to 3-methylindole, in vitro.
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Available evidence supports the view that acute bovine pulmonary edema and emphysema (ABPE) is related to ruminal production of 3-methylindole (3MI) from L-tryptophan (TRP). Ruminal production of 3MI is a two-step process involving the conversion of TRP to indoleacetic acid (IAA) followed by decarboxylation of IAA and 3MI. Reduction in ruminal 2MI production by the inhibition of either of these processes may prevent the onset of ABPE. A closed-system, in vitro ruminal fermentation technique was used to screen 27 compounds for their ability to reduce the conversion of TRP to 3MI. Several compounds tended to reduce 3MI production at both 25 and 5 micrograms/ml. Desoxysalinomycin, X-206, chloral hydrate, nigericin, lasalocid, monensin, narasin and salinomycin all reduced 3MI production by more than 80% at 5 micrograms/ml without reducing total VFA production. All of these compounds, except chloral hydrate, are polyether antibiotics. At least part of the inhibition due to monensin and narasin occurs at the level of TRP conversion to IAA. (+info)
3-Methylindole (skatole) and indole production by mixed populations of pig fecal bacteria.
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Pig fecal slurries converted added L-tryptophan either to indole without detectable intermediates or to 3-methylindole (skatole) via indole-3-acetate. The initial rate of production of 3-methylindole was greatest at pH 6.5 and less at pH 5.0 and 8.0; the initial rates of indole production were similar at pH 6.5 and 8.0. More than 80% of the tryptophan added was converted to 3-methylindole at pH 5.0; at pH 8.0 85% was converted to indole. Both pathways had similar Km values for tryptophan and similar maximum rates. Indole-3-carbinol and indole-3-acetonitrile completely inhibited the production of 3-methylindole from indole-3-acetate but had no effect on the reactions involving L-tryptophan. (+info)
Localization of sites for ionic interaction with lipid in the C-terminal third of the bovine myelin basic protein.
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The myelin basic protein from bovine brain tissue was purified and the two peptides obtained by cleavage of the polypeptide chain at the single tryptophan residue were isolated. The interaction of these peptides and the intact basic protein with complex lipids was investigated by following the solubilization of lipid-protein complexes into chloroform in a biphasic solvent system. The C-terminal peptide fragment (residues 117-170) and the intact basic protein both formed chloroform-soluble complexes with acidic lipids, but not with neutral complex lipids. The N-terminal fragment (residues 1-115) did not form chloroform-soluble complexes with either acidic or neutral complex lipids. The molar ratio of lipid to protein that caused a 50% loss of protein from the upper phase to the lower chloroform phase was the same for the intact basic protein as for the smaller C-terminal peptide fragment. Phosphatidylserine and phosphatidylinositol were approximately twice as efficient as sulphatide at causing protein redistribution to the chloroform phase. The results are interpreted as indicating that the sites for ionic interactions between lipid and charged groups on the basic protein of myelin are located in the C-terminal region of the protein molecule. (+info)
MEK-1 phosphorylation by MEK kinase, Raf, and mitogen-activated protein kinase: analysis of phosphopeptides and regulation of activity.
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MEK-1 is a dual threonine and tyrosine recognition kinase that phosphorylates and activates mitogen-activated protein kinase (MAPK). MEK-1 is in turn activated by phosphorylation. Raf and MAPK/extracellular signal-regulated kinase kinase (MEKK) independently phosphorylate and activate MEK-1. Recombinant MEK-1 is also capable of autoactivation. Purified recombinant wild type MEK-1 and a mutant kinase inactive MEK-1 were used as substrates for MEKK, Raf, and autophosphorylation. MEK-1 phosphorylation catalyzed by Raf, MEKK, or autophosphorylation resulted in activation of MEK-1 kinase activity measured by phosphorylation of a mutant kinase inactive MAPK. Phosphoamino acid analysis and peptide mapping identified similar MEK-1 tryptic phosphopeptides after phosphorylation by MEK kinase, Raf, or MEK-1 autophosphorylation. MEK-1 is phosphorylated by MAPK at sites different from that for Raf and MEKK. Phosphorylation of MEK-1 by MAPK does not affect MEK-1 kinase activity. Several phosphorylation sites present in MEK-1 immunoprecipitated from 32P-labeled cells after stimulation with epidermal growth factor were common to the in vitro phosphorylated enzyme. The major site of MAPK phosphorylation in MEK-1 is threonine 292. Mutation of threonine 292 to alanine eliminates 90% of MAPK catalyzed phosphorylation of MEK-1 but does not influence MEK-1 activity. The results demonstrate that MEKK and Raf regulate MEK-1 activity by phosphorylation of common residues and thus, two independent protein kinases converge at MEK-1 to regulate the activity of MAPK. (+info)
Influence of a gonadotropin-releasing hormone agonist on circulating concentrations of luteinizing hormone and testosterone and tissue concentrations of compounds associated with boar taint.
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Twenty-four Yorkshire boars and six barrows approximately 8 mo of age were used to evaluate the effects of a GnRH agonist (GnRH-A) on circulating concentrations of LH and testosterone (T), concentrations of skatole in fat, and concentrations of 16-androstene steroids in fat and salivary glands. Pigs were assigned to five groups of six pigs each: L200, boars injected with 200 micrograms of GnRH-A/kg BW; L50, boars injected with 50 micrograms of GnRH-A/kg BW; Boar, boars treated with vehicle; Castrate, males castrated on the same day that Groups 1 to 3 received injections; and Barrow, males castrated as neonates. Pigs in L200, L50, and Boar were only given one injection. Blood samples were taken twice daily from d -2 to 28 for determinations of T and LH concentrations and at 15-min intervals for 6 h on d -1, 1, 7, 14, 21, and 28 for characterization of pulsatile LH release. Pigs were slaughtered on d 30, and samples of backfat and salivary glands were collected for quantification of skatole and 16-androstene steroid concentrations. The LH concentrations increased immediately following GnRH-A injection in L200 and L50 (P < .001) and then declined to pretreatment values by approximately 29 h after treatment. Serum T concentrations in L200 and L50 increased after GnRH-A treatment (P < .0001) and then decreased to less than pretreatment concentrations by d 5 in L200 (P < .001) and by d 8 in L50 (P < .01). Concentrations of 16-androstene steroids in fat were less (P < .05) in L200 and Barrow than in Boar.(ABSTRACT TRUNCATED AT 250 WORDS) (+info)