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Biomedical subjects

J Schaefer

Publications and source records attributed to J Schaefer.

At least 55 records · Page 3Linked to original sources
PubMed1997

Structure and dynamics of pentaglycyl bridges in the cell walls of Staphylococcus aureus by 13C-15N REDOR NMR.

Whole cells and cell-wall fractions of Staphylococcusaureus have been labeled by various combinations of [1-13C]glycine, [15N]glycine, L--6-13C-lysine, L--6-15N-lysine, D--1-13C-alanine, and D--15N-alanine. The resulting materials have been examined using 13C and 15N solid-state, magic-angle spinning NMR techniques including cross-polarization, double cross-polarization, and rotational-echo double resonance. The results of these measurements indicate that the peptidoglycan glycyl bridges are complete (five units long) and form cross-links between three-quarters of all peptide stems. The pentaglycyl bridges are immobilized in lyophilized cell-wall fractions in a compact conformation with inter-residue spacings comparable to those of an alpha helix. The bridges have a similar compact conformation in intact whole cells, regardless of whether the cells have been lyophilized or were hydrated and frozen at -10 degrees C. The bridges are also in a time-averaged compact conformation in whole cells at 0 degrees C but with sizable structural fluctuations associated with local mobility. A small fraction of bridges are in extended-chain conformations.

Cell Wall↗
PubMed1997

Closed form of liganded glutamine-binding protein by rotational-echo double-resonance NMR.

Rotational-echo double-resonance NMR has been used to determine internuclear distances in the complex of glutamine-binding protein and its ligand, l-glutamine. The distances between the ligand and Tyr185 are consistent with the results of molecular dynamics simulations constrained by three REDOR-determined distances to His156. This model is also consistent with six other REDOR-determined internuclear distances, most of which agree with values from the first report of an X-ray structure of the complex of glutamine-binding protein and l-glutamine.

Carrier Proteins↗
PubMed1997

REDOR dephasing by multiple spins in the presence of molecular motion.

Closed-form, numerical algorithms are presented for calculating REDOR dephasing for three general cases: (i) collections of isolated I-S spin pairs; (ii) many S spins coupled to an I spin; and (iii) an I-S spin pair in relative motion. For the case when more than one S spin is dipolar coupled to an I spin, the calculation assumes that the S-S homonuclear interaction does not affect REDOR dephasing. Full numerical simulations show that this assumption is true if the S-spin lineshapes are inhomogeneously broadened, the S-spin chemical shifts are far from rotational resonance, and a version of REDOR is used which minimizes the number of S-spin pi pulses. For the rapidly rotating -CF3 group of poly(trifluoroethyl methacrylate), the formalisms of (ii) and (iii) are combined to calculate the dephasing. The experimentally measured dephasing matches theory when the wiggling motion of the -OCH2CF3 moiety of the polymer is taken into account.

Algorithms↗
PubMed1997

Differential expression of multiple somatostatin receptors in the rat cerebellum during development.

The present study describes the expression pattern of somatostatin receptor genes during the development of rat cerebellum. Characterization of somatostatin receptors was carried out by binding studies using receptor subtype-selective ligands in the germinative epithelium and granule cell layer of the cerebellum from postnatal day 4 (P4) to P21 and in granule cell cultures. Quantitative reverse transcription-PCR carried out for the five receptor subtype mRNAs in cerebellar extracts showed that sst1 mRNAs are predominant at the end of gestation. A transient high expression of the sst2 gene was observed from P7 to P14. In parallel, high levels of binding sites sensitive to sst2 ligands were detected in the granule cell germinative epithelium and in granule cell cultures. sst3 mRNAs rapidly increased from P14 and became the predominant form at P21, but respective binding sites were not detected. Whereas sst4 mRNA levels were generally low, those of sst5 were nearly undetectable. Reverse transcription-PCR carried out in granule cell cultures revealed the relative abundance of sst mRNAs as follows: sst2 > sst1 > sst3 = sst4. sst5 mRNA was undetectable. The results show the expression of four somatostatin receptor genes, but only three receptors (sst1, sst4, and mainly sst2) were detected as binding sites during cerebellar development.

Animals↗
PubMed1997

Characterisation of carnitine palmitoyltransferases in patients with a carnitine palmitoyltransferase deficiency: implications for diagnosis and therapy.

OBJECTIVES: Carnitine palmitoyltransferase (CPT) deficiency is one of the most common defects of mitochondrial fatty acid oxidation. Two different enzymes (CPT-I and CPT-II) are involved. Due to problems in measuring enzyme activity, relatively little is known about the substrate specificity of each of the human enzymes. This is of considerable importance in the treatment of patients. The objectives were to establish a reliable method for the measurement of CPT activity in whole cells, to use this to characterise the substrate specificity of each enzyme, and finally, to determine if medium chain triglycerides would be of benefit in the treatment of deficient patients. METHODS: A simple permeabilisation technique was used which allows the measurement of CPT activity in a small amount of cultured skin fibroblasts or peripheral blood cells. Using this technique three patients were identified with CPT deficiency. In two of these patients, one with CPT-I deficiency and one with CPT-II deficiency, a complete substrate specificity profile of the mitochondrial carnitine acyltransferases was established for all saturated even chain acyl-CoA esters. RESULTS: For both enzymes the highest CPT activity was with C12-CoA. About 70% of total cellular carnitine octanoyltransferase activity was due to mitochondrial CPT. As CPT is involved in the transport of medium chain fatty acids the metabolic response of a patient with CPT-II deficiency to dietary medium chain triglycerides was assessed. Despite the normal production of ketone bodies there was a significant medium chain dicarboxylic aciduria in the patient, indicating a limited capacity of the CPT independent mitochondrial uptake of medium chain fatty acids. CONCLUSIONS: CPT deficiency can easily be diagnosed in permeabilised cultured skin fibroblasts. Both CPT-I and CPT-II are more active with medium chain length substrates than previously assumed. Care should therefore be taken in the treatment of these patients with medium chain triglycerides.

Adult↗
PubMed1996

Identification of a novel hypothalamic neuropeptide Y receptor associated with feeding behavior.

Neuropeptide Y (NPY) plays important roles in the central control of appetite and energy balance, but the receptor subtype responsible for this function has not been cloned. Here we report the cloning by expression of a novel NPY receptor subtype from a rat hypothalamus cDNA library. The novel receptor, referred to as the NPY Y5 receptor, has a transcript of approximately 2.6 kilobases with an open reading frame of 1335 base pairs that encodes a 445-amino acid protein. The amino acid sequence deduced from the rat Y5 cDNA clone shows only 30-33% identity to other NPY receptors, including Y1, Y2, and Y4/PP1. Using the rat Y5 receptor cDNA probe, the human homologue was obtained by low stringency hybridization. The human Y5 amino acid sequence has 88% identity to the rat Y5 receptor. Importantly, pharmacological analysis shows that the rat and human Y5 receptors have high affinity for the peptides that elicit feeding (e.g. NPY, PYY, (2-36)NPY, and (LP)NPY) and low affinity for nonstimulating peptides (e.g. (13-36)NPY and rat PP), suggesting that it is the NPY feeding receptor subtype.

Amino Acid Sequence↗
PubMed1996

Secondary structure and location of a magainin analogue in synthetic phospholipid bilayers.

Magainins are cationic, membrane-active peptides which show broad-spectrum antimicrobial activity. We have investigated the secondary structure and location of an analogue of magainin 2 in synthetic phospholipid bilayers using a combination of Fourier transform infrared (FTIR) spectroscopy and solid-state nuclear magnetic resonance (NMR) spectroscopy. Ala19-magainin 2 amide exhibits both alpha-helix and beta-sheet secondary structures in lipid bilayers containing either dipalmitoylphosphatidylglycerol (DPPG) or a 1:1 molar mixture of DPPG and dipalmitoylphosphatidylcholine (DPPC). The combination of FTIR and solid-state NMR results suggests that there are two populations of peptide. The secondary structure of one population is alpha-helix while that of the other population is beta-sheet. We demonstrate that the solid-state NMR technique, rotational-echo double resonance (REDOR), can be used to measure both intra- and intermolecular dipole-dipole interactions in membrane-bound peptides. Our REDOR experiments indicate that alpha-helical Ala19-magainin 2 amide is bound near the phospholipid head groups.

Amino Acid Sequence↗
PubMed1996

In situ analysis of peptidyl DOPA in mussel byssus using rotational-echo double-resonance NMR.

Rotational-echo double-resonance (REDOR) 13C NMR spectra with 2H dephasing have been obtained from plaques and threads from the byssus of the marine mussel Mytilus edulis labeled by sea-water exposure to L-[ring-4-(13)C]tyrosine and L-[ring-d4]tyrosine for 2 days. Specific isotopic enrichment of tyrosine in protein reached 25% in both 13C and 2H. Fifteen percent of the total 13C label was incorporated as diphenolic carbon. Based on REDOR dephasing, about one-tenth of tyrosine rings in both intact plaques and threads are within 4 A of each other or rings of 3,4-dihydroxy-phenylalanine (DOPA). However, there is no direct evidence for the formation of covalent linkages between or among tyrosine and DOPA rings in either plaques or threads.

Animals↗
PubMed1996

Assessment of a possible genotoxic environmental risk in sheep bred on grounds with strongly elevated contents of mercury, arsenic and antimony.

A part of Northern Palatinate country (Germany) was formerly influenced by mercury mining. Today, in many cases agricultural and housing areas are placed onto or near to former dump grounds of rubble. In the soil of these areas the concentration of mercury, arsenic and antimony was found ranging from basic natural contents up to strongly elevated levels. In a biomonitoring project, sheep bred on grounds contaminated with mercury (range 1-435 mg Hg/kg dry matter), arsenic (range 17-147 mg As/kg dry matter) and antimony (range 2-15 mg Sb/kg dry matter) were taken as example on the uptake of these elements from the environment and for possible effects of this exposure. Significantly elevated mercury levels were found in wool of one collective of exposed sheep (0.107 mg/kg mean vs. 0.048 mg/kg mean, p < 0.001, U-test). Surprisingly, the arsenic content of wool taken from sheep bred in the urban referential area was approx. 10 times higher than that of the sheep bred on the grounds contaminated with arsenic (0.57 mg/kg mean vs. 0.051 mg/kg mean, p < 0.001, U-test). In general, element concentrations in the examined blood samples were low and the differences between the collectives were small: mercury was found in concentrations ranging from 0.9 microgram/l up to 2.0 micrograms/l (means), arsenic and antimony were generally found in concentrations below 1 microgram/l. Neither in the alkaline elution technique nor in the sister chromatid exchange (SCE) analysis significant increases in the rate of DNA-damaging effects between the different sheep collectives were detected. This indicates that the transfer rate of genotoxic compounds of mercury, arsenic or antimony from the environment is too low to register effects with AFE and SCE although the soil was highly contaminated.

Animals↗
PubMed1996

Ligand geometry of the ternary complex of 5-enolpyruvylshikimate-3-phosphate synthase from rotational-echo double-resonance NMR.

The 46-kDa enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the condensation of shikimate 3-phosphate (S3P) and phosphoenolpyruvate (PEP) to form EPSP. The reaction is inhibited by N-(phosphonomethyl)glycine (Glp), which, in the presence of S3P, binds to EPSP synthase to form a stable ternary complex. As part of a solid-state NMR characterization of this structure, we have used dipolar recovery at the magic angle (DRAMA) and rotational-echo double resonance (REDOR) to determine intra- and interligand internuclear distances. DRAMA was used to determine the single 31P-31P distance, while REDOR was used to determine one 31P-15N distance and five 31P-13C distances. These experimental distances were used as restraints in molecular dynamics simulations of an S3P-Glp complex to examine the geometry of the two ligands relative to one another in the ternary complex. The simulations were compared to unrestrained simulations of the EPSP synthase tetrahedral intermediate and its phosphonate analog. The results suggest that Glp is unlikely to bind in the same fashion as PEP, a conclusion that is consistent with recent studies that have questioned the role of Glp as a transition-state or intermediate analog.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed1996

Intersubunit communication in tryptophan synthase by carbon-13 and fluorine-19 REDOR NMR.

The beta subunits of the 143-kDa alpha2beta2 tetrameric enzyme tryptophan synthase have been labeled by L-[ring-4-19F]phenylalanine and L-[phenol-4-13C]tyrosine in an effort to monitor the positions of these residues on ligand binding. Of the 13 phenylalanine and 11 tyrosine residues in the beta subunit, only three pairs have labels with 13C-19F separations of less than 6 angstrom. The beta subunit residues Tyr279 and Phe280 (each members of one of the three Tyr-Phe proximate pairs) have been suggested as possible conformational gates on ligand binding. The 188-MHz 19F NMR spectrum of the microcrystalline, double-labeled enzyme complex has five resolved lines under 5-kHz magic-angle spinning and 80-kHz proton dipolar decoupling. The distribution of beta-subunit 19F isotropic shifts is altered by addition of L-[3-13C]-serine to the mother liquor in contact with the microcrystals, consistent with a conformational rearrangement. The 13C label from serine is detected at 28 ppm as a methyl tautomer of bound aminoacrylate. The change in aromatic 19F chemical shifts on binding of serine indicates an alteration in local electric field gradients within the beta subunit. However, rotational-echo double-resonance 13C NMR (with 19F dephasing) shows that the average 13C-19F distance for the three phenylalanine-tyrosine proximate pairs in the beta subunit is changed by less than 1 angstrom.

Carbon Isotopes↗
PubMed1996

Structural constraints on the ternary complex of 5-enolpyruvylshikimate-3-phosphate synthase from rotational-echo double-resonance NMR.

The 46 kDa enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the condensation of shikimate-3-phosphate (S3P) and phosphoenolpyruvate to form EPSP. The reaction is inhibited by N-(phosphonomethyl)-glycine (Glp), which in the presence of S3P, binds to EPSP synthase to form a stable ternary complex. As part of a solid-state NMR characterization of this structure, 15N labels were introduced selectively into the lysine, arginine and histidine residues of EPSP synthase and distances to a 13C label in Glp and to the 31P in S3P and Glp were measured by rotational-echo double-resonance NMR. Three lysine and four arginine residues are in the proximity of the phosphate group of S3P and the carboxyl and phosphonate groups of Glp. A single histidine residue is in the vicinity of the binding site (closer to Glp than to S3P) but is more distant than the lysine and arginine residues.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed1996

Deficiency of complex II of the mitochondrial respiratory chain in late-onset optic atrophy and ataxia.

Defects of the mitochondrial respiratory chain are increasingly being recognized as an important cause of neurological disease in humans. In many of these patients, the biochemical defect results from an abnormality of the mitochondrial genome. Respiratory chain defects involving complex II, which is entirely encoded by the nuclear genome, are comparatively rare. We report the clinical and biochemical findings in 2 elderly sisters who presented with late-onset neurodegenerative disease. In both patients, a partial deficiency of complex II (approximately 50% of control values) was shown to be present in mitochondria from muscle and platelets. The enzyme defect was not expressed in cultured skin fibroblasts or immortalized lymphocytes. There was an overexpression of the 70-kd flavoprotein subunit in muscle mitochondria from both patients, although we showed that this subunit is present in normal amounts in mitochondrial membranes. Our studies highlight the diversity of the clinical presentation of respiratory chain disease and that complex II deficiency should enter the differential diagnosis of certain patients with late-onset neurodegenerative disease.

Ataxia↗
PubMed1996

Trifunctional enzyme deficiency: adult presentation of a usually fatal beta-oxidation defect.

Disorders of mitochondrial fatty acid oxidation are a common cause of exercise-induced rhabdomyolysis and myoglobinuria. We report three adult patients from a family with symptoms of recurrent exercise-induced rhabdomyolysis. This presentation closely resembles adult-type carnitine palmitoyltransferase II deficiency except that these patients had an associated peripheral neuropathy. Investigation of fatty acid oxidation in the patients revealed a deficiency of the mitochondrial trifunctional enzyme of beta-oxidation, a newly described fatty acid oxidation disorder with multiorgan involvement and a usually fatal outcome in early childhood. Our cases therefore represent a new phenotype of the disease, which is characterized by recurrent rhabdomyolysis and peripheral neuropathy, but without involvement of other organs, and which is associated with prolonged survival beyond the fourth decade. A low-fat/high-carbohydrate diet proved beneficial in one of the patients, drastically reducing the frequency of rhabdomyolytic episodes. Our findings suggest that mitochondrial trifunctional enzyme deficiency should be considered in patients with recurrent episodes of myoglobinuria and peripheral neuropathy presenting in later life.

Adult↗
PubMed1996

Characterization of two forms of cadmium phosphide by magic-angle spinning 31P NMR.

Annealing of commercial grade cadmium phosphide (CD3P2) at 600 K produces a material which, in magic-angle spinning spin-lattice 31P NMR relaxation experiments, has broad lines and multiple T1 values. By contrast, sublimation at 900 K results in a crystalline material with narrow lines and a single T1. However, both materials have the same Cd-P lattice spacings as determined by rotational-echo, double-resonance 31P NMR with 113Cd dephasing. Both materials also have closely similar X-ray diffraction powder patterns. These results are interpreted in terms of a distribution of lattice vacancies in the annealed material, creating structural heterogeneity but with no substantial change in lattice parameters from those of the sublimed cadmium phosphide.

Cadmium↗
PubMed1996

Distance between phosphine-sulfide sidechains of a disubstituted peptide by DRAMA 31P NMR.

Dipolar restoration at the magic angle (DRAMA) has been used to measure the 31P-31P internuclear distance between phosphine-sulfide substituted sidechanins on the fourth and eighth residues of a 12-residue helical peptide. The 7.4 A distance is the same for the peptide lyophilized in bulk or isolated in a cryo- and lyoprotected matrix of poly(ethylene glycol) and sucrose. However, the 31P linewidth for the undiluted peptide is an order of magnitude greater than for the matrix-isolated peptide indicating charge and hydration heterogeneity in the bulk state.

Chemical Phenomena↗