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Biomedical subjects

J Schaefer

Publications and source records attributed to J Schaefer.

At least 109 records · Page 6Linked to original sources
PubMed1987

Metabolism of glyphosate in an Arthrobacter sp. GLP-1.

The metabolism of glyphosate [N-(phosphonomethyl)glycine] in a bacterium tentatively identified as an Arthrobacter sp., capable of growth on this herbicide as its sole phosphorus source, has been investigated using solid-state NMR techniques as well as radiotracer analysis. The pathway involves the conversion of glyphosate to glycine, a C1 unit and phosphate. The phosphonomethyl carbon is specifically incorporated into the amino acids serine, cysteine, methionine, and histidine, as well as into purine bases and thymine, indicating the involvement of tetrahydrofolate in single-carbon transfer reactions. Glycine derived from glyphosate is utilized in purine and protein biosynthesis. This pathway for glyphosate degradation in a gram-positive bacterium is similar to that previously reported for Pseudomonas sp. PG2982 [Jacob et al. (1985) J. Biol. Chem. 260, 5899-5905] and is distinct from that reported for soil metabolism of glyphosate where aminomethylphosphonic acid has been shown to be a major metabolite. Preliminary evidence is presented which indicates that the conversion of glyphosate to glycine and the C1 unit involves the intermediate formation of sarcosine. Thus, the primary event in glyphosate degradation by Arthrobacter sp. GLP-1 is the cleavage of its C-P bound. This report constitutes the first demonstration of the metabolism of glyphosate in a gram-positive bacterium.

Arthrobacter↗
PubMed1987

Aromatic cross-links in insect cuticle: detection by solid-state 13C and 15N NMR.

Cross-polarization magic-angle-spinning nuclear magnetic resonance spectroscopy has been used to determine insect cuticle composition and cross-link structure during sclerotization or tanning. Unsclerotized cuticle from newly ecdysed pupae of the tobacco hornworm, Manduca sexta L., had a high protein content with lesser amounts of lipid and chitin. Concentrations of chitin, protein, and catechol increased substantially as dehydration and sclerotization progressed. Analysis of intact cuticle specifically labeled with carbon-13 and nitrogen-15 revealed direct covalent linkages between ring nitrogens of protein histidyl residues and ring carbons derived from the catecholamine dopamine. This carbon-nitrogen adduct was present in chitin isolated from cuticle by alkaline extraction and is probably bound covalently to chitin. These data support the hypothesis that the stiffening of insect cuticle during sclerotization results primarily from the deposition of protein and chitin polymers and their crosslinking by quinonoid derivatives of catecholamines.

Animals↗
PubMed1987

Solid-state NMR studies of regulation of N-(phosphonomethyl)glycine and glycine metabolism in Pseudomonas sp. strain PG2982.

Lyophilized samples of Pseudomonas sp. PG2982 grown on 13C- and 15N-labeled glyphosate have been analyzed by single and double cross-polarization 13C NMR. Both the carbon and nitrogen metabolism of glyphosate are significantly influenced by the nitrogen source used for the growth of the organism. When ammonium sulfate is the source of nitrogen, the glycyl moiety of glyphosate is utilized intact for the biosynthesis of purines and proteins. But when the organism is grown on glycine as the source of nitrogen, the carbons and nitrogen of glyphosate are scrambled, consistent with incorporation into serine and pyruvate, and hence participation in general metabolism. When both ammonium and glycine are present in the growth medium, regulation of the metabolic fluxes along each of the two major pathways appears to be determined by the intracellular glycine concentration.

Ammonia↗
PubMed1987

Solid-state NMR studies of Klebsiella pneumoniae grown under nitrogen-fixing conditions.

The carbon and nitrogen metabolism of Klebsiella pneumoniae M5a1 has been characterized using 13C and 15N labeling with detection by cross-polarization magic-angle spinning solid-state NMR. Cells grown on ammonium typically require some 20 h to derepress fully for nitrogenase when transferred to medium devoid of any source of fixed nitrogen. We have established that during this period some cellular proteins are catabolized with the liberated nitrogen being used for the synthesis of purines needed for formation of ribosomal RNA. The 20-h derepression period can be shortened to 6 h by the introduction of fixed nitrogen in certain specific forms. Serine is the most successful agent we have examined for shortening the derepression period and glycine among the least successful. We attribute this difference to the advantage of serine over glycine in providing both specific and nonspecific carbon and nitrogen sources for complete purine synthesis. These determinations were made by tracing the metabolism of 13C- and 15N-labeled chemical bonds from the 2 amino acids during derepression.

Bacterial Proteins↗
PubMed1987

Influence of ventricular contractility on non-work-related myocardial oxygen consumption.

The relationship between myocardial oxygen consumption (MVO2) and the total pressure-volume area (PVA), which represents the total mechanical work performed during a cardiac cycle, has been shown to be linear and independent of loading conditions: MVO2 = aPVA + b. When inotropic state is enhanced, the MVO2-PVA relation shifts upward (increase in b), and when inotropic state is depressed the relation shifts downward (decrease in b). However, the quantitative relationship between contractility and b (the non-work-related myocardial oxygen consumption) determined over a wide range of contractilities is not known. In seven isolated blood perfused canine hearts, left ventricular (LV) contractility was increased by dobutamine and decreased with nifedipine or reduction of coronary blood flow. At each level of contractility, the end-systolic pressure-volume relationship (ESPVR) and the MVO2-PVA relation were determined. For each heart, the resulting values of b (ml O2/beat) were plotted as a function of Emax (mmHg/ml), an index of contractility defined as the slope of the ESPVR. There was a linear relation between Emax and b over a wide range of contractilities; on average, b (ml O2/beat) = 0.0036 Emax (mmHg/ml) + 0.0101 [r = 0.929-0.978 (95% confidence interval)], when Emax was varied over an average range of 2.8-9.6 mmHg/ml. These results suggest a common underlying determinant of contractility and non-work-related oxygen consumption.

Animals↗
PubMed1987

N and C NMR determination of methionine metabolism in developing soybean cotyledons.

The metabolism of d- and l-methionine by immature cotyledons of soybean (Glycine max, L. cv Elf) grown in culture has been investigated using solid-state (13)C and (15)N nuclear magnetic resonance. d-Methionine is taken up by the cotyledons and converted to an amide, most likely by N-malonylation. About 16% of the l-methionine taken up is incorporated intact into protein, and 25% remains as soluble methionine. Almost two-thirds of the l-methionine that enters the cotyledons is degraded. The largest percentage of this is used in transmethylation of the carboxyl groups of pectin. Methionine is not extensively converted to polyamines. We attribute the stimulation of growth of the cotyledons by exogenous methionine to the bypassing of a rate-limiting methyl-transfer step in the synthesis of methionine itself, and subsequently of pectins and proteins.

Journal Article↗
PubMed1986

Direct measurement of poly(beta-hydroxybutyrate) in a pseudomonad by solid-state 13C NMR.

Four narrow lines are observed in the high-resolution cross-polarization magic-angle spinning 13C NMR spectra of intact, lyophilized samples of Pseudomonas sp. LBr, in addition to the broader lines normally associated with bacterial cellular material. These narrow lines arise from poly(beta-hydroxybutyrate). The cellular carbon contained in this storage material can be measured quantitatively and nondestructively from the solid-state NMR spectra. We find that cells starved for phosphorus store up to 50% of their total carbon as poly(beta-hydroxybutyrate). When such cells are used to inoculate medium containing a source of phosphorus, all of the poly(beta-hydroxybutyrate) is metabolized by the time the culture has reached midlogarithmic growth phase.

Carbon Isotopes↗
PubMed1986

Analysis of chitin structure by nuclear magnetic resonance spectroscopy and chitinolytic enzyme digestion.

Solid-state 13C-NMR analysis of chitin prepared from cuticle of the tobacco hornworm, Manduca sexta (L.), and of crab yielded spectra that demonstrate a high degree of chemical homogeneity (greater than 95%) for the preparations. The chemical shifts of the well-resolved carbon signals from both samples matched closely those of the monomeric unit 2-acetamido-2-deoxy-D-glucopyranoside (GlcNAc). Chromatographic analysis of products from the digestion of chitin by the binary chitinase system (endo splitting chitinase and exo splitting beta-N-acetylglucosaminidase) isolated from M. sexta molting fluid showed that the major product from both chitin preparations is GlcNAc. Also detected was a minor product (product U) that had a chromatographic retention time on the carbohydrate analysis column intermediate between those of chitin penta- and hexasaccharides. Gel filtration chromatography of U indicated that U had an apparent molecular weight intermediate between that of GlcNAc and of N,N'-diacetylchitobiose. Cation-exchange chromatography of U after acid hydrolysis revealed the presence of glucosamine only. Derivatization with trinitrobenzenesulfonate showed the presence of a free amino group in U. Solution proton and carbon NMR spectroscopy were used to identify U as a N-monoacetylchitobiose [O-beta-D-2-amino-2-deoxyglucopyranosyl- (1----4)-2-acetamido-2-deoxy-beta-D-glucopyranose] with the residue at the nonreducing end deacetylated. These studies showed that chitin prepared from alkali- and heat-treated insect or crab cuticle contains trace levels of deacetylated residues that are released as a dead-end product, N-monoacetylchitobiose, after digestion by the binary enzyme system.

Acetylglucosamine↗
PubMed1986

Characterization of peptidoglycan stem lengths by solid-state 13C and 15N NMR.

Lyophilized whole cells of Aerococcus viridans (Gaffkya homari) grown on a synthetic medium containing D-[2-13C, 15N]Ala, or containing both L-[1-13C]Lys and D-[15N]Ala, have been examined by double cross-polarization magic-angle spinning 13C and 15N nuclear magnetic resonance. Results from the double-labeled alanine experiment confirm the absence of metabolic scrambling of alanine by A. viridans. Results from the combined single-label experiment can be used to count directly the number of adjacent L-Lys and D-Ala units in peptide chains of cell-wall peptidoglycan. This count leads to the conclusion that there are no terminal D-Ala or D-Ala-D-Ala units in uncross-linked chains of the peptidoglycan of A. viridans.

Alanine↗
PubMed1985

Solid-state NMR determination of glyphosate metabolism in a Pseudomonas sp.

The metabolism of the broad-spectrum herbicide, glyphosate (N-phosphonomethylglycine) in a soil Pseudomonas sp. PG2982 has been determined by cross-polarization magic-angle spinning 15N and 13C NMR of intact lyophilized cells. Using samples grown on 13C- and 15N-labeled glyphosate, we find that PG2982 does not metabolize glyphosate to aminomethylphosphonate as has been reported for mixed cultures of soil microbes. Rather, the phosphonomethyl carbon-nitrogen bond in glyphosate is cleaved, releasing glycine. Solid-state NMR analysis reveals that 20% of this glycine is used in the synthesis of purines, 35% is incorporated into protein as glycyl residues, with an additional 35% incorporated as seryl residues. The phosphonomethyl carbon of glyphosate is ultimately incorporated into a number of sites, including the C-2 and C-8 positions of the purine rings of nucleic acids, methyl groups of methionine and thymidine, and the methylene group of serine. The pattern of phosphonomethyl carbon incorporation indicates the involvement of tetrahydrofolate, a coenzyme which facilitates single-carbon transfers. This is the first complete determination of the metabolism of glyphosate in a pure culture, and the first bacterial metabolic study using both single and double cross-polarization solid-state NMR.

Biodegradation, Environmental↗
PubMed1985

Solid-state 13C and 15N nuclear magnetic resonance studies of alanine metabolism in Aerococcus viridans (Gaffkya homari).

Transport and metabolism of D- and L-alanine by Aerococcus viridans (Gaffkya homari) have been studied using cross-polarization magic-angle spinning 13C and 15N NMR spectroscopy of lyophilized whole cells, isolated cell walls, and crude extracts. For equimolar concentrations in the growth medium, about 10 times more D-alanine than L-alanine is transported into the cells. Examination of cells labeled with D-[13C]alanine and L-[epsilon-15N]lysine by double cross-polarization magic-angle spinning 15N NMR indicates that only about 20% of the D-alanine present in cell-wall peptidoglycan comes directly from the growth medium. The rest is produced by de novo synthesis. Most of the labeled D-alanine is found within peptidoglycan precursors or inverted to L-alanyl residues of soluble proteins.

Alanine↗
PubMed1985

Solid-state 15N NMR studies of the effects of penicillin on cell-wall metabolism of Aerococcus viridans (Gaffkya homari).

Lyophilized whole cells and isolated cell walls from Aerococcus viridans (Gaffkya homari) grown on a synthetic medium containing benzylpenicillin, and either L-[epsilon-15N]lysine or 15N-ammonium ion as the only source of label, have been studied using cross-polarization magic-angle spinning 15N nuclear magnetic resonance. The lysine is incorporated directly into protein and cell-wall peptidoglycan and was used to measure cell-wall cross-links. The ammonium ion acts as a non-specific label monitoring general metabolism. Inhibition of cell-wall cross linking by penicillin occurs, but may not be the exclusive cause of cell death and lysis in this microorganism. Instead, the disruption of the mechanism for control of peptidoglycan synthesis probably is a contributing factor.

Cell Wall↗
PubMed1985

N and C NMR determination of allantoin metabolism in developing soybean cotyledons.

The metabolism of allantoin by immature cotyledons of soybean (Glycine max L. cv Elf) grown in culture was investigated using solid state (13)C and (15)N nuclear magnetic resonance. All of the nitrogens of allantoin were incorporated into protein in a manner similar to that of each other and to the amide nitrogen of glutamine. The C-2 of allantoin was not incorporated into cellular material; presumably it was lost as CO(2). About 50% of the C-5 of allantoin was incorporated into cellular material as a methylene carbon; the other 50% was presumably also lost as CO(2). The (13)C-(15)N bonds of [5-(13)C;1-(15)N] and [2-(13)C;1,3-(15)N]allantoin were broken prior to the incorporation of the nitrogens into protein. These data are consistent with allantoin's degradation to two molecules of urea and one two-carbon fragment. Cotyledons grown on allantoin as a source of nitrogen accumulated 21% of the nitrogen of cotyledons grown on glutamine. Only 50% of the nitrogen of the degraded allantoin was incorporated into the cotyledon as organic nitrogen; the other 50% was recovered as NH(4) (+) in the media in which the cotyledons had been grown. The latter results suggests that the lower accumulation of nitrogen by cotyledons grown on allantoin was in part due to failure to assimilate NH(4) (+) produced from allantoin. The seed coats had a higher activity of glutamine synthetase and a higher rate of allantoin degradation than cotyledons indicating that seed coats play an important role in the assimilation and degradation of allantoin.

Journal Article↗
PubMed1985

Regeneration in alfalfa tissue culture: stimulation of somatic embryo production by amino acids and N-15 NMR determination of nitrogen utilization.

The production of somatic embryos in alfalfa (Medicago sativa L., cv Regen S) is increased 5- to 10-fold by alanine and proline. However, utilization of nitrogen for synthesis of protein from alanine, proline, glutamate, and glycine is not qualitatively different, even though the latter two amino acids do not increase somatic embryo formation. These determinations were made by (15)N labeling with detection by nuclear magnetic resonance. Overall metabolism of the nitrogen of proline, alanine, glutamate, and glycine is also similar in two regenerating and nonregenerating genotypes with similar germplasm, except that the levels of free amino acids are consistently higher in the nonregenerating line. In addition, when regeneration is suppressed in either of the two regenerating lines, the level of intracellular free amino acids increases. This increased level of metabolites is the only direct evidence provided by analysis of nitrogen metabolism of differences between the regenerating and nonregenerating states in alfalfa.

Journal Article↗
PubMed1985

Regeneration in Alfalfa Tissue Culture: Characterization of Intracellular pH During Somatic Embryo Production by Solid-State P-31 NMR.

The composition of phosphorus-containing compounds of intact lyophilized alfalfa tissue has been determined, in part, by solid-state (31)P nuclear magnetic resonance. The tissue (Medicago sativa L., cv Regen S; and some of its crosses) was grown in culture under both nonregenerating and regenerating conditions, the latter enhanced by the addition of specific amino acids. Analysis of the (31)P nuclear magnetic resonance spectra shows that regeneration is favored when metabolism occurs without the production of a low average intracellular pH.

Journal Article↗
PubMed1984

Application of statistical methods for the analysis or interval related cardiac performance variations during cardiac arrhythmia in man.

To study the influence of the sequence of stimulation intervals on cardiac performance indices the relationship between properties of succeeding arrhythmic aortic pressure pulses of patients with atrial fibrillation has been analysed by methods of correlation and regression analysis. There are high correlations between properties of succeeding pulses and it can be shown that the pressure amplitude of one pulse correlates to the properties (diastole, diastolic pressure, pressure amplitude) of up to five preceding pulses. Furthermore, the diastolic pressure is also highly correlated with properties of more than one preceding pulse whereas the duration of the diastole and the RR-interval is independent of preceding pulse properties. We, therefore, conclude that there is no beat to beat regulation of intervals during atrial fibrillation. The relations between properties of succeeding pressure pulses have been summarised in a transfer function model, which allows the description of pulse properties from preceding pulse properties without lack of it. The parameters of the transfer function models are estimated from a sequence of arrhythmic pressure pulses and a parameter selection procedure was developed which identifies the optimal number of input variables (= properties of preceding pulses) required to describe the time course of one pulse property. In order to obtain informations about the presence of possible interval dependent potentiation effects, the transfer function model was used to stimulate the behaviour of the aortic pressure for different stimulation sequences, which have been used in the literature to study potentiation effect experimentally. From typical sequences of potentiation phenomena in the stimulated pulse sequence we conclude that frequency potentiation effects exist in the human myocardium.

Aorta, Thoracic↗