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Biomedical subjects

J Schaefer

Publications and source records attributed to J Schaefer.

At least 37 records · Page 2Linked to original sources
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↗
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↗
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↗
PubMed1984

Relationships between beat-to-beat interval and the strength of contraction in the healthy and diseased human heart.

Twenty-six adult patients, classified by clinical and catheter criteria into groups of those with normal and abnormal left ventricular function, were studied during cardiac catheterization. Right heart pacing was established, and left ventricular dP/dt was measured with end-catheter manometers. By varying the interval preceding a test beat after periods of steady pacing it was confirmed that recovery of left ventricular mechanical function (maximum dP/dt) occurs approximately 800 msec (optimum interval) after a beat. The augmentation of maximum dP/dt of the first 2 beats after an extrasystole, each spaced at the optimum interval, was also studied; the amount of potentiation was varied by alterations in extrasystolic interval. Potentiation decayed from the first to the second postextrasystolic beat with a ratio that was fixed in each individual patient. The ratio (recirculation fraction) was higher in patients with normal than in those with abnormal left ventricular function (mean +/- SD 0.52 +/- 0.10 vs 0.37 +/- 0.11, p less than .005). There was an inverse relationship between this ratio and the degree of potentiation of the first postextrasystolic beat (r = .80, p less than .001). We postulate a disturbance of excitation-contraction coupling mechanisms to explain these effects.

Adult↗
PubMed1983

Direct measurement of peptidoglycan cross-linking in bacteria by 15N nuclear magnetic resonance.

Cross-polarization magic angle spinning 9.12-MHz 15N nuclear magnetic resonance was used to measure the extent of peptidoglycan cross-linking within intact bacterial cells and isolated cell walls of Aerococcus viridans ATCC 10400 grown in the presence of L-[epsilon-15N]lysine. A value of 49% for the cross-linking index was found for normal cells, while for those grown in the presence of low levels of penicillin G (0.1 micrograms/ml), the cross-linking index was reduced to 35%.

Magnetic Resonance Spectroscopy↗
PubMed1983

Lipid bilayer dynamics and rhodopsin-lipid interactions: new approach using high-resolution solid-state 13C NMR.

High-resolution, solid-state 13C NMR spectra have been obtained for unsonicated multilamellar dispersions of 1,2-dilauryl-sn-glycero-3-phosphocholine (DLPC), recombinant membranes containing DLPC and rhodopsin, and native retinal rod disk membranes. The roles of 1H dipolar decoupling, 1H-13C cross-polarization, and magic-angle sample spinning have been investigated. Rotating-frame 13C relaxation times have been measured and are discussed in terms of lipid bilayer dynamics and rhodopsin-lipid interactions.

Fatty Acids, Unsaturated↗
PubMed1983

Electrical and mechanical restitution of the human heart at different rates of stimulation.

Action potential duration and contractility are reduced following premature excitations, and gradually increase as the stimulus interval is lengthened. To examine these phenomena of electrical and mechanical restitution in the human heart, we simultaneously measured action potential duration and the maximum rate of left ventricular pressure in five patients undergoing electrophysiological study. Test beats were introduced at varying intervals after the last of a series of steady state intervals. By plotting action potential duration and maximum rate of left ventricular pressure as a function of the test interval, we formed electrical and mechanical restitution curves. When the rate of steady state pacing was increased, there was a decrease in action potential duration and an increase in the maximum rate of left ventricular pressure for all test intervals; i.e., a change in pacing rate affected action potential duration and maximum rate of left ventricular pressure of test responses in a reciprocal fashion. In addition, a higher steady state pacing rate allowed action potentials and contractile responses to be elicited at shorter test intervals, thereby displacing the electrical and mechanical restitution curves to the left. The magnitude of the leftward shift of both curves corresponded closely to the shortening of the steady state action potential duration induced by the increase in pacing rate. These findings confirm for the human heart that both electrical and mechanical restitution occur after membrane repolarization, i.e., as a function of the electrical diastolic interval preceding a beat, and not the stimulus interval.

Action Potentials↗