Biomedical subjects
Publications and source records attributed to H P Broquist.
The development of buthionine sulfoximine, a selective inhibitor of glutathione biosynthesis, is an important new tool to elucidate the in vivo role of glutathione. Recent investigations have shown that ascorbic acid can serve as an essential antioxidant in the presence of severe glutathione deficiency.
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Swainsonine, an indolizidine alkaloid in the locoweeds (certain species of the Astragalus and Oxytropis genera), was fed to young Holstein bull calves in their milk at high elevation (3090 m), and the incidence of high mountain disease (HMD) was compared with locoweed-fed and control calves. Five of 5 calves fed swainsonine and 5 of 5 calves fed fresh Oxytropis sericea showed outward signs of HMD, which included edema under the jaws, throat area and brisket and gross and microscopic lesions of HMD and locoweed poisoning. Grossly there were HMD lesions, including congestion of the liver, right ventricular hypertrophy, and dilatation and excessive fluid in the thoracic and abdominal cavities. Microscopically, the severe centrilobular lesions in the liver, edema of the pulmonary artery, severe edema and/or fibrosis of the roof of the right atrium were suggestive of HMD. The mild to moderate neurovisceral cytoplasmic foamy vacuolation of selected tissues and cerebellar neuroaxonal dystrophy in all calves fed swainsonine and locoweed were indicative of locoweed poisoning. In control calves, 1 of 6 showed equally severe outward, gross, and microscopic lesions of HMD, but none had any lesions indicative of locoweed poisoning. The ratio of right ventricle to left ventricle wall weights were significantly higher (P = 0.033) for the swainsonine-fed calves (1.4) and the locoweed-fed calves (1.3) compared to the controls (0.9). Scores indicating the severity of HMD from observations prior to necropsy were significantly higher for the swainsonine and locoweed-fed calves compared to controls (P = 0.032).(ABSTRACT TRUNCATED AT 250 WORDS)
The biosynthesis of pipecolic acid from L-lysine in the fungal parasite, Rhizoctonia leguminicola has been reinvestigated. Pipecolate is then utilized to form the toxic octahydroindolizine alkaloids, slaframine and swainsonine. Incorporation studies of L-versus D-[U-14C]lysine into R. leguminicola metabolites confirmed earlier findings that L-lysine is the predominant substrate for pipecolate formation and D-lysine for alpha-N-acetyllysine (concerned in lysine catabolism). However [alpha-15N]lysine, not [epsilon-15N]lysine as previously reported, labeled pipecolate. Such findings implied that delta 1-piperideine-6-carboxylate, not delta 1-piperideine-2-carboxylate, was formed from lysine and was the immediate precursor of pipecolate. Evidence from cell-free enzyme systems established the following biosynthetic events: L-lysine A----saccharopine B----delta 1-piperideine-6-carboxylate C----pipecolate. Products of reactions A and C were identified from biological and chemical considerations. Reaction B was carried out by a previously undescribed flavin enzyme termed saccharopine oxidase. The product of reaction B, which reacted with p-dimethylaminobenzaldehyde, was reduced with Na-CNB2H3. Its NMR spectrum was identical with that of deuteriated pipecolate prepared from authentic delta 1-piperideine-6-carboxylate, but not from authentic delta 1-piperideine-2-carboxylate. Reaction B represents a branching of primary lysine metabolism from saccharopine to a secondary pathway leading to pipecolate and to octahydroindolizine alkaloids in R. leguminicola.
The fungal parasite Rhizoctonia leguminicola produces two indolizidine alkaloids, slaframine and swainsonine, of physiological interest. These alkaloids are biosynthesized from pipecolic acid which in turn is derived from L-lysine in this fungus as shown in the accompanying paper (Wickwire, B.M., Harris, C.M., Harris, T.M., and Broquist, H.P. (1989) J. Biol. Chem. 265, 14742-14747): L-lysine----saccharopine----delta 1----piperideine-6- carboxylate----pipecolate. This paper concerns the discovery, purification, and properties of a flavoenzyme, termed saccharopine oxidase, which carries out the oxidative cleavage of saccharopine as follows: Saccharopine + O2----delta 1-piperidine-6-carboxylate + glutamate + H2O2 The enzyme was purified 2,000-fold to homogeneity (polyacrylamide gel electrophoresis) in 14% yield from R. leguminicola mycelia, and had a native molecular mass of about 45,000 daltons by gel filtration (fast protein liquid chromatography Superose). Evidence for the presence of a flavin in the enzyme was drawn from these considerations: (a) the enzyme, while oxidatively cleaving saccharopine, concomitantly reduces 2,6-dichlorophenolindophenol; (b) the purified enzyme has a fluorescence spectrum typical of flavins; and (c) the enzyme requires oxygen and produces hydrogen peroxide. Good correlation was shown with purified saccharopine oxidase between disappearance of saccharopine with the concomitant appearance of delta 1-piperideine-6-carboxylate plus glutamate. The enzyme has a pH optimum about 6 and a Km for saccharopine of 0.128 mM. The enzyme apparently exists in R. leguminicola to shunt saccharopine, a major lysine metabolite, into a secondary pathway of lysine metabolism leading to pipecolate and subsequently to slaframine and swainsonine.
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Swainsonine and swainsonine-containing plants produce biochemical and neurological changes in several mammalian species. The toxin is a potent inhibitor of liver lysosomal alpha-D-mannosidase and Golgi mannosidase II. The inhibition of the latter enzyme causes the production of abnormal glycoproteins containing hybrid oligosaccharides instead of complex types in a variety of cultured cells. In view of the widespread occurrence and biological importance of N-linked glycoproteins in the central nervous system, we initiated studies to determine the structure of oligosaccharides in glycoproteins prepared from the brain of control, swainsonine-fed, and locoweed-fed animals. The results presented here indicate that the feeding led to alteration in the structure of brain glycoproteins. Over 25% of the glycoproteins which presumably contained complex-type oligosaccharides were modified and now contained hybrid oligosaccharides. The structure of the N-linked oligosaccharide (glycopeptide) was established by (a) studying the binding properties of the glycopeptide to immobilized lectins of known sugar specificity, and (b) comparing the size of the glycopeptide before and after treatment with exo- and endoglycosidases. The production of hybrid oligosaccharides occurred despite the apparent absence of mannosidase II in brain. The relationships of the altered structure of brain glycoproteins, accumulation of mannose-rich oligosaccharides in the brain, and abnormal behavior of the animals administered swainsonine or locoweed are discussed.
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Two trials were initiated to determine if slaframine (SF) can be used to alter fluid digesta flow and fermentation patterns in the rumen. In trial 1, a preliminary experiment, four Dorset X Barbados Black-belly ruminal-cannulated wethers (avg weight 41.6 8.7 kg) given ad libitum access to a pelleted concentrate/hay diet were injected intramuscularly with 0, 12, 24 or 48 micrograms SF/kg body weight (BW) in a 4 X 4 Latin-square design. Ruminal fluid dilution rate was determined using a single intraruminal infusion of polyethylene glycol (7 g), followed by seven hourly ruminal fluid samples. The administration of 48 micrograms SF/kg BW increased (P less than .10) ruminal volume and outflow by 27 and 25%, respectively, compared with controls. In trial 2, two Hereford and two Angus ruminal cannulated steers (avg weight 568 +/- 93 kg) were injected with 0, 6, 12 or 24 micrograms SF/kg BW at 8-h intervals over a 24-h period in a 4 X 4 Latin-square design. Steers were fed a concentrate diet at twice maintenance in 24 equal portions daily. Ruminal fluid dilution was measured using a single intraruminal infusion of cobalt-ethylenediamine tetraacetic acid (20 g) administered 9 h after the initial SF injection. Ruminal fluid was collected each hour during 8 to 24 h after the initial SF injection and analyzed for pH, osmolality and volatile fatty acids (VFA).(ABSTRACT TRUNCATED AT 250 WORDS)
Slaframine (SF), 1-acetoxy-6-aminooctahydroindolizine a parasympathomimetic with a high affinity for the gastrointestinal tract, was administered by oral intubation daily to 240 broiler chicks at either 0, 8.9, or 17.8 micrograms/kg body weight.75 (BW.75) in saline for 21 days. Throughout the experimental period weight, feed intake, and fecal output were measured. On Day 21 birds were killed, eviscerated, and wet organ weights were obtained. Pancreas and small intestine digesta were homogenized with saline and frozen for analyses of trypsin, chymotrypsin, amylase, and lipase activity as well as total protein. Weight, feed intake and utilization, pancreatic weight, liver weight, and small intestine digesta weight were not affected by SF treatment. Protein content of the digesta decreased 16.6% with the 17.8 micrograms SF/kg BW.75 treatment. Digesta lipase activity was 13.3% (P greater than .05) and specific activity 24% less (P less than or equal to .02) in 17.8 micrograms/kg BW.75 treated birds in comparison with those of controls, and activities decreased in a linear fashion across treatment levels (P less than or equal to .04). Digesta trypsin-specific activity decreased linearly with SF treatment (P less than or equal to .05), averaging 5.5 to 16.9% lower than control treated birds. Pancreatic chymotrypsin-specific activity was not significantly different among treatments. These results suggest that relatively small dosages of SF may affect digestive function of broiler chicks.
The ability of slaframine (SF), a parasympathomimetic, to alter blood growth hormone (GH) and glucose concentrations in broiler chicks was investigated. Eighty male broiler chicks (average weight 225 g) were divided into 10 groups and dosed with either saline (control) or 1 mg SF/kg of body weight by oral intubation. Plasma samples were obtained from separate groups of chicks at 1, 2, 4, 8, and 12 hr after SF administration and analyzed for growth hormone and glucose. One hour after SF administration, glucose increased (P less than .05) 21.4% compared with controls. Growth hormone increased (P less than .05) 449 to 948% from 8 to 12 hr after SF administration. Administration of SF at 1 mg/kg of body weight was associated with increased plasma GH.
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Four esophageal- and ruminal-cannulated Angus steers (avg weight, 308 kg) were used to investigate how salivation is affected by the administration of purified slaframine (SF; 1-acetoxy-6-aminooctahydroindolizine), a cholinergic secretagogue isolated from Rhizoctonia leguminicola. Steers were fed a concentrate diet at twice the net energy requirement for maintenance in hourly increments. In trial 1, a single injection of SF was administered to four steers intramuscularly at 0, 6, 12 and 24-micrograms/kg body weight (BW) in a 4 X 4 Latin-square design. Saliva was collected via esophageal cannula at 15-min intervals 30 min after each feeding, weighted, sampled and reinfused via ruminal cannula over a 10-h period. At 12- and 24-micrograms SF/kg BW, salivary flow was 31 to 43% greater (P less than .01) than at 0- or 6-micrograms SF/kg. Response peaked within the first 3 h and returned to baseline levels at 8 h. Buffering capacity and pH of saliva were not different (P greater than .10); however, osmolality and Na concentration increased and K concentration decreased (P less than .10) as salivation rates increased. Feed intake of steers did not appear affected at any level of SF administration. In trial 2, 0-, 12- and 24-micrograms SF/kg BW were repeatedly administered intramuscularly to three steers in a 3 X 3 Latin-square design at 8-h intervals for 24 h. Salivary flow was measured and sampled over the entire 24-h period, as in trial 1. Flow rates were increased 50 to 70% (P less than .01) by SF treatment.(ABSTRACT TRUNCATED AT 250 WORDS)
Effect of purified slaframine (SF; 1-acetoxy-6-aminooctahydroindolizine), a parasympathomimetic secretagogue isolated from Rhizoctonia leguminicola, on ruminal motility was investigated in cattle and sheep. In trial 1, four ruminal cannulated wethers, fed a pelleted concentrate and hay diet, were injected intramuscularly with 0, 12, 24 and 48 micrograms SF/kg body weight (BW) in a 4 X 4 Latin-square design. Ruminal motility was recorded 1 h before and 1 to 2 h and 3 to 4 h after SF administration by measuring pressure changes exerted upon a fluid-filled, open-tipped catheter inserted into the dorsal sac of the rumen. The frequencies of both primary and secondary ruminal contractions were decreased as much as 20 to 78% with SF (P less than .05) depending upon the dosage level and time after administration. In trial 2, three ruminal-cannulated steers fed a concentrated diet were injected intramuscularly with 0, 12 and 24 micrograms SF/kg BW in a 3 X 3 Latin-square design. A water-filled balloon inserted into the cranial sac of the rumen was used to measure ruminal pressure changes 1 h before and 1 to 2 h, 3 to 4 h and 7 to 8 after SE administration. Frequency of primary and secondary ruminal contractions decreased with SF as much as 27 to 64% depending on the dosage level and time after administration. The frequency of secondary contractions increased 28% (P less than .05) as compared with control during the 7 to 8 h after administration.(ABSTRACT TRUNCATED AT 250 WORDS)
Swainsonine, a plant toxin, strongly inhibits certain alpha-D-mannosidases but has no effect on others [D. R. P. Tulsiani, T. M. Harris, and O. Touster (1982) J. Biol. Chem. 257, 7936-7939]. The reversible inhibition of jack bean and lysosomal alpha-D-mannosidases has previously been suggested to be similar in nature but quite complex. Specific differences in the action of swainsonine on these two enzymes and on Golgi mannosidase II are reported. (a) The inhibition of the jack bean mannosidase, but not rat liver lysosomal alpha-D-mannosidase or Golgi mannosidase II, is increased by preincubation with the alkaloid. (b) The inhibition of the jack bean and lysosomal enzymes, but not mannosidase II, is competitive at inhibitor concentrations of less than or equal to 0.5 microM. (c) The inhibition of jack bean alpha-mannosidase is largely irreversible, its very limited reversibility being partially dependent upon the swainsonine concentration used and on the time of preincubation with the inhibitor. On the other hand, the inhibition of lysosomal alpha-mannosidase is largely reversible, as shown by dilution experiments and by the use of [3H]swainsonine. Golgi mannosidase II shows intermediate reversibility, the results indicating two modes of binding; one rapid and irreversible, the other much slower and reversible.
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A neurological condition resembling that observed in hereditary mannosidosis occurs in animals ingesting spotted locoweed and plants of the genus Swainsona. Swainsonine has been isolated from these plants and has been suggested to be the primary causative agent in inducing the pathological condition. This alkaloid has also been found to increase tissue acid alpha-D-mannosidase levels in rats while lowering liver Golgi mannosidase II levels. In the present study, the effects of locoweed and swainsonine were directly compared for the first time, with the pig as experimental animal. Both increased most lysosomal acid glycosidase activities in most tissues, decreased liver Golgi mannosidase II levels, increased plasma hydrolase levels, and greatly increased tissue oligosaccharide, especially Man5GlcNAc2 and Man4GlcNAc2. These results indicate that swainsonine is the agent in locoweed responsible for the enzymatic and oligosaccharide changes. The behavior of the animals was also similarly affected by swainsonine and locoweed.