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Biomedical subjects

M F Roberts

Publications and source records attributed to M F Roberts.

At least 109 records · Page 6Linked to original sources
PubMed1985

Charged detergents enhance the activity of phospholipase C (Bacillus cereus) towards micellar short-chain phosphatidylcholine.

Phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3) (Bacillus cereus) activity toward diheptanoylphosphatidylcholine is increased 50-100% by low concentrations of both positively and negatively charged detergents. Zwitterionic and nonionic detergents have no such activating effect. This charged detergent activation requires an interface, since comparable detergent concentrations have no effect on the hydrolysis rate of monomeric dihexanoylphosphatidylcholine. From NMR and diacylglycerol solubility studies it is suggested that activation results from detergent interacting with diacylglycerol to accelerate product release from the enzyme.

Bacillus cereus↗
PubMed1985

31P NMR studies of Clostridium thermocellum. Mechanism of end product inhibition by ethanol.

31P NMR studies of intact cells and perchloric acid extracts are used to investigate the effect of ethanol on the bioenergetics and glycolysis of Clostridium thermocellum, an anaerobic bacterium potentially useful for the single step conversion of biomass to ethanol. Whole cells suspended in phosphate buffer and given a carbon source (cellobiose) at 60 degrees C rapidly establish a pH gradient across the membrane that can be monitored by the chemical shifts of inorganic phosphate in the exterior buffer and in the cytoplasm. Peak intensities can be related to phosphate active transport rates. Wild type bacteria and cells grown in inhibiting concentrations of ethanol establish similar pH gradients, but with slower kinetics and slower phosphate transport rates for the cells adapted to growth in ethanol. Direct addition of ethanol does not affect the rate of pH gradient formation or phosphate transport. Thus, while ethanol does not directly affect processes for energy conservation carried out by the membrane, adaptation to ethanol does alter membrane functions such as phosphate transport. 31P NMR spectra of perchloric acid extracts show that when wild type cells are adapted to grow in inhibiting concentrations of ethanol and then energized with cellobiose, sugar phosphate content is increased and the steady state distribution of glycolytic intermediates is altered. Nucleotide triphosphate/nucleotide diphosphate ratios are unaltered in these cells. These results strongly indicate that in C. thermocellum growth inhibition by ethanol is related to a blockage in glycolysis.

Anaerobiosis↗
PubMed1985

31P-NMR studies of the oral pathogen Streptococcus mutans: observation of lipoteichoic acid.

We have used 31P-nuclear magnetic resonance spectroscopy to identify phosphorus-containing compounds in whole cells of two serotype c strains of the oral pathogen Streptococcus mutans. The major resonance, centered at 0 ppm in whole cells, was attributed to lipoteichoic acid on the basis of its chemical shift, insensitivity to pH changes, cellular localization and a comparison with spectra obtained with purified lipoteichoic acid from S. mutans. The linewidths of resonances observed for intact cells and purified lipoteichoic acid were moderately narrowed by increasing the ionic strength, and substantially broadened in the presence of the lectin concanavalin A. Experiments with purified lipoteichoic acid suggest that this compound in whole cells is complexed with divalent cations such as Mg2+. Intracellular pools of other phosphorus-containing metabolites were found to be low when compared to the lipoteichoic acid concentration in both starved and glycolyzing cells.

Cell Membrane↗
PubMed1985

500-MHz 1H NMR studies of ragweed allergen Ra5.

The solution conformation of short ragweed allergen Ra5, a protein of 45 amino acid residues cross-linked with four disulfide bridges, has been investigated by 1H NMR spectroscopy at 500 MHz. The aromatic region, which contains resonances from three tyrosines and two tryptophans, has been partially assigned. Two tyrosines titrate with a pK of 10.2; a third tyrosine is buried under the tryptophan resonances, and its pK could not be determined. The two tryptophans reside in different microenvironments; the resonances of one are very similar to those found in random coil structures while the other has dramatically shifted peaks. Nuclear Overhauser effect (NOE) difference spectroscopy is used to define two distinct spin-diffusion systems for the aromatic residues and to further identify several methyl-containing amino acids involved in these systems. Assignments in the methyl region are based on selective decoupling, chemical shifts, NOE difference spectra, and 2-D J-resolved and 2-D J-correlated spectroscopy (COSY) methodology. A unique ring-current-shifted methyl doublet in the Ra5 spectrum titrates into the bulk methyl region with a pK of 10.2. Examination of the COSY map suggests that this resonance belongs to either leucine-1 or isoleucine-38. Chemical removal of the N-terminal leucine did not affect the ring-current-shifted methyl. Therefore, this unique resonance has been assigned to the methyl of isoleucine-38.(ABSTRACT TRUNCATED AT 250 WORDS)

Allergens↗
PubMed1985

Correlation of 19F-NMR spectra of halothane in rat tumor and non-tumor tissues with membrane alterations.

The membrane environments in normal and tumor rat tissue and the effect of hyperthermia thereon are studied with 19F-NMR spectroscopy of the general anesthetic halothane. Normal and tumor cell types are clearly differentiated by the halothane resonance. A hydrophobic environment prominent in tumor tissue is more sensitive to heat treatment than the corresponding environments of normal cells. Studies of extracted lipids suggest that this may be due in part to the considerable difference in lipid temperature response which exists between normal and kidney tumor cells.

Adenocarcinoma↗
PubMed1985

Methyl branching in short-chain lecithins: are both chains important for effective phospholipase A2 activity?

Several seven-carbon fatty acyl lecithins with varied acyl chain branching have been synthesized and characterized as potential phospholipase A2 substrates. Micellar bis(4,4-dimethylpentanoyl) phosphatidylcholine, bis(5-methylhexanoyl)phosphatidylcholine, bis(3-methylhexanoyl)phosphatidylcholine, and bis(2-methylhexanoyl)phosphatidylcholine are poor substrates for phospholipase A2 (Naja naja naja). These branched lecithins also inhibit the hydrolysis of diheptanoylphosphatidylcholine by the enzyme with Ki values comparable to or smaller than the apparent Km of the linear compound. The terminally branched lecithins are excellent substrates for another surface-active hydrolytic enzyme, phospholipase C from Bacillus cereus. When only one acyl chain bears a methyl group, the hybrid lecithins 1-heptanoyl-2-(2-methylhexanoyl)phosphatidylcholine and 1-(3-methylhexanoyl)-2-heptanoylphosphatidylcholine are substrates comparable to diheptanoylphosphatidylcholine. Analysis of micellar structure and dynamics by 1H and 13C NMR spectroscopy, quasi-elastic light scattering, and comparison of critical micellar concentrations indicates little significant difference in the conformation and dynamics of these seven-carbon fatty acyl lecithin micelles, even when the methyl groups are adjacent to the carbonyls. Phospholipase A2 UV difference spectra induced by phospholipid binding imply different enzyme conformations or aggregation states caused by linear-chain and asymmetric-chain lipids compared to bis(methylhexanoyl)phosphatidylcholines. The differences in hydrolytic activity of phospholipase A2 against the branched-chain micellar lecithins can then be attributed to an enzyme-lipid interaction at the active site. The species with both fatty acyl chains branched bind to phospholipase A2 but are not turned over rapidly. Since poor enzymatic activity only occurs for lecithins with both chains methylated, the interaction of both chains with the enzyme must be important for catalytic efficiency.

Kinetics↗
PubMed1985

Amount and chain length of polyphosphates in Escherichia coli depend on cell growth conditions.

Anaerobiosis induced an accumulation of polyphosphates (poly Pi) in a phosphate-rich medium by an alkaline-phosphatase constitutive mutant of Escherichia coli. The total poly Pi content was maximum at around 6 h of anaerobic growth. Both trichloroacetic acid- and NaOH-soluble poly Pi were found to be present. The acid-soluble fraction consisted mainly of a linear polymer of about 20 +/- 5 phosphate units, whereas the alkali-extractable poly Pi fraction contained at least four molecular species of higher chain length as determined by gel filtration. The majority of poly Pi extracted at 6 h had lower chain lengths than those extracted from cells incubated for 24 h. In vivo 31P nuclear magnetic resonance spectra of E. coli cells as a function of growth conditions were consistent with the in vitro extract results.

Chromatography, Gel↗
PubMed1985

Interaction of local and reflex thermal effects in control of forearm blood flow.

We measured forearm blood flow (ABF) bilaterally on six subjects during 15-min periods of leg exercise and the first 10 min of recovery. One forearm (control) was kept at about 33 degrees C skin temperature in all experiments. In experiments at ambient temperature (Ta) of 15 degrees C, the other arm (experimental) was kept at about 26, 33, and 40 degrees C, respectively, during three successive cycles of exercise and recovery. ABF in the 26 degrees C forearm was linearly related to and averaged 42% of control. The relation of ABF in the 40 degrees C forearm to control ABF showed a bend at control ABF of 4-5 ml X 100 ml-1 X min-1. Below the bend, experimental ABF average 213% of control. Above the bend, experimental ABF averaged 5.09 ml X 100 ml-1 X min-1 above control. In four subjects, after heating the experimental forearm to 40 degrees C, we measured ABF for 25-30 min at rest in Ta of both 15 and 25 degrees C. At 25 degrees C Ta, ABF in the heated forearms rose gradually, but control ABF showed little change. At 15 degrees C Ta, the effect on ABF of local heating to 40 degrees C was much reduced, apparently due to reflex vasoconstrictor signals.

Adult↗
PubMed1984

The fluorinated anesthetic halothane as a potential NMR biologic probe.

Fluorinated anesthetics such as halothane preferentially partition into hydrophobic environments such as cell membranes. The 19F-NMR spectrum of halothane in a rat adenocarcinoma (with known altered lipid metabolism and membrane composition) shows an altered chemical shift pattern compared to the anesthetic in normal tissue. In eight tumor samples examined, the 19F-NMR spectra exhibit two distinct resonances, compared to a single resonance observed in normal tissues. This is explained by an enhanced or altered hydrophobic component in the tumor tissue giving rise to two discrete halothane environments. Another fluorinated anesthetic, isoflurane, shows similar behavior in distinguishing normal from diseased tissue. Given the large chemical shift range of fluorine and the inherent sensitivity of this nucleus, 19F-NMR spectra of fluorinated anesthetics can also be used to follow anesthetic degradation by the liver. The ability of fluorinated anesthetics to discriminate tissues and to monitor metabolic processes is potentially useful for in vivo 19F-NMR surface coil and imaging studies.

Adenocarcinoma↗
PubMed1984

Spontaneous formation of stable unilamellar vesicles.

Stable unilamellar vesicles form spontaneously upon mixing aqueous suspensions of long-chain lecithins (fatty acid chain lengths 14 carbons or longer) with small amounts (20 mol %) of micellar synthetic short-chain lecithins (fatty acid chain lengths 6-8 carbons). These vesicles are potentially ideal for any experiment (i.e., membrane protein reconstitution, drug delivery, etc.) that requires an easily formed, nonleaky unilamellar structure.

Lipid Bilayers↗
PubMed1984

Reflex and local thermal control of rabbit ear blood flow.

We measured thermal conductance (K) of the ears of 12 New Zealand White rabbits during periods of core heating in which mean skin temperature and ear temperatures (Te) were controlled. Right Te was always held at 28 degrees C; left Te was 15, 20, 24, 28, 31, 33 degrees C in separate core heating periods. Core heating was obtained by perfusing a previously implanted intravascular heat exchanger with hot water. Blood temperature (Tbl) was measured with a thermocouple that had been previously placed in contact with the common carotid artery. During heating, K rose with rising Tbl, but the relation between K and Tbl depended on Te. At high Te, K rose rapidly in the initial stages of core heating and soon reached its maximal value. At cool Te, K rose slowly with rising Tbl until high Tbl was reached; then K rose rapidly to maximal values. The maximal value of K reached at high Tbl was independent of Te. The results suggest that local temperature affects ear blood flow by affecting the way that ear blood vessels react to sympathetic reflex signals.

Animals↗
PubMed1983

The interaction of dialkyl ether lecithins with phospholipase A2 (Naja naja naja).

Dialkyl lecithins are nonhydrolyzable substrate analogues for phospholipase A2 (Naja naja naja). Short chain dialkyl lecithins (which form monomers and micelles), and long chain ether-linked lecithins (which form bilayers or can be solubilized in detergent micelles) have been used to study phospholipase A2/phospholipid interactions. The results of gel filtration, 1H and 31P NMR spectroscopy, kinetic assays and UV difference spectroscopy show that binding of the ether-linked lecithins is independent of metal ion in pure lecithin systems (monomer, micelle, or bilayer), but requires Ca2+ for tight binding when Triton X-100 is used to solubilize the lecithin in mixed micelles. The apparent KD for dihexadecyl phosphatidylcholine is 2 mM in sonicated vesicles, but is increased to 6 mM in Triton-mixed micelles in the presence of Ca2+. When phospholipase A2 binds monomeric and micellar dialkyl lecithin, the lecithin phosphate resonance is broadened considerably, indicating that head group motion is restricted. Ca2+ enhances this interaction with the head group in these pure lipid systems. In similar 31P NMR experiments with dihexadecyl phosphatidylcholine solubilized in Triton X-100 mixed micelles, phospholipase A2 does not cause comparable broadening of the phosphate resonance, even in the presence of Ca2+ where gel filtration shows binding occurs. The kinetic effect of the ether-linked lipid inhibitors on phospholipase activity toward ester-linked lecithins is also modulated by the presence of Triton X-100. Ether-linked lecithins are much more potent inhibitors in two-component (dialkyl- diacyl-) lecithin micelles and bilayers than in complex mixed micelles with Triton X-100. Detergent-induced changes in the characteristics of the micellar interface are implicated in this inhibition phenomenon. The interaction of phospholipase A2 with sonicated vesicles of dihexadecyl phosphatidylcholine has also been examined by 1H and 31P NMR spectroscopy. When phospholipase A2 binds to vesicles, it promotes loss of encapsulated lanthanide ions, and appears to accelerate transbilayer phospholipid "flip-flop" rates. These results are discussed in terms of membrane asymmetry studies using phospholipase A2.

Calcium↗
PubMed1983

Localization of enzymes and alkaloidal metabolites in Papaver latex.

In continuing studies on the metabolic activity of Papaver somniferum, latex has been examined for its enzyme and alkaloidal metabolite content. After an initial centrifugation of latex at 1000g, the pellet which contained a heterogeneous population of dense organelles was further resolved on sucrose gradients. Of the enzymes monitored, acid phosphatase and L-3,4-dihydroxyphenylalanine decarboxylase were found to be in the latex 1000g supernatant, whereas catecholase (polyphenolase) was localized in two distinct organelles within the 1000g sediment. The lighter organelles, sedimenting at 30% sucrose, contained a soluble enzyme which was readily released on organelle plasmolysis, whereas the catecholase found within the heavier organelles, sedimenting at 55-60% sucrose, was membrane bound and showed significant activity only in the presence of Triton X-100. These latter organelles also contained the alkaloids, including morphine and thebaine, and were observed to readily accumulate [14CH3]morphine. The alkaloid precursor, dopamine, was localized in the same dense vesicle fraction as the alkaloids. The rate of uptake of [7-14C]dopamine into these fractions at room temperature, however, was markedly lower than that of morphine. Electron microscopic examination of the organelles of various densities revealed that they possessed different morphology. The results are consistent with the concept that both the 1000g and supernatant fractions of the latex are required for alkaloid biosynthesis and that a subpopulation of dense organelles found in the 1000g sediment have at least a function as a storage compartment for both alkaloids and their catecholamine precursor.

Alkaloids↗
PubMed1983

Methanophosphagen: Unique cyclic pyrophosphate isolated from Methanobacterium thermoautotrophicum.

A unique cyclic pyrophosphate compound has been detected at 10-12 mM intracellular concentration in Methanobacterium thermoautotrophicum by in vivo(31)P NMR. This compound has been extracted from cells and purified by anion-exchange chromatography. Studies with (1)H, (13)C, and (31)P NMR and fast-atom-bombardment mass spectrometry have identified it as 2,3-cyclopyrophosphoglycerate, an intramolecularly cyclized pyrophosphate of 2,3-diphosphoglycerate. Chemical degradation to 2,3-diphosphoglycerate and synthesis by dicyclohexylcarbodiimide coupling of 2,3-diphosphoglycerate are consistent with this identification. It is suggested that this compound serves as a primary phosphagen in methanogens.

Journal Article↗
PubMed1983

Body temperature control of rat tail blood flow.

Tail blood flow (BF) and heat flow (HF) were measured in five albino rats during transients in rectal temperature (Tre) caused by body heating at rest. During heating, tail temperature (Tt) was kept at 15, 20, 25, 30, 35, or 42 degrees C by enclosing the tail in a water-perfused tube. Thermal conductance (K) was computed as HF/(Tre-Tt). BF was measured by venous occlusion plethysmography. Heating caused a rise in Tre that was accompanied by proportional increases in both K and BF. The ratio R = K/BF represents conductance per unit BF and reflects the amount of heat exchange for a given BF. R can thus be used to estimate the distribution of BF within the tail. R was independent of Tre at all Tt, indicating that BF distribution is controlled by the tail. R was low at low Tt and rose at higher Tt. This suggests that at low Tt, blood flows primarily in central veins of the tail and at higher Tt blood flows in peripheral tail veins.

Animals↗
PubMed1982

Ethanol-induced changes in the membrane lipid composition of Clostridium thermocellum.

When ethanol is added to the growth medium of Clostridium thermocellum ATCC 27405 and C9, a different membrane composition is observed after the period of growth arrest. Changes in fatty acid composition and some unsaturated, branched hydrocarbons have been monitored by GLC-MS. There is a marked increase in normal and anteiso-branched fatty acids at the expense of isobranched fatty acids and an increase in short and unsaturated fatty acids. Thus, an adaptive response to growth in the presence of ethanol induces a membrane containing fatty acids with lower melting points and produces a more 'fluid' membrane. The suggestion is made that these membrane changes may be maladaptive to the performance of C. thermocellum.

Cell Membrane↗
PubMed1982

Vanadium-containing tunicate blood cells are not highly acidic.

The intracellular pH of intact blood cells of the tunicate Ascidia nigra was measured by transmembrane equilibration of [14C] methylamine. The pH of unfractionated blood cells is 7.39 +/- 1.10. The pH of vanadocytes, determined in a fractionation study, is 7.2. Previously used methods, in which pH values less than 3.0 are inferred from cell lysis or vital staining experiments, are shown to be unsuitable for intracellular pH determination due to the chemical composition of these vanadium-containing cells.

Animals↗