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M F Roberts

Publications and source records attributed to M F Roberts.

At least 91 records · Page 5Linked to original sources
PubMed1989

Beta-aminoglutaric acid is a major soluble component of Methanococcus thermolithotrophicus.

13C- and 15N-NMR spectroscopy have been used to identify beta-aminoglutaric acid (beta-glutamic) as a major soluble component of the thermophilic, autotrophic marine methanogen Methanococcus thermolithotrophicus. This rare, non-protein amino acid has been recognized as a major dissolved free amino acid in marine sediments, but the microorganism responsible for its production has not previously been identified. The concentration of beta-aminoglutarate (beta-glutamate) is about one half that of free alpha-glutamate and increases (relative to the alpha-isomer) as cells enter the stationary phase. Analysis of the 13C label distribution in a 13CO2-pulse/12CO2-chase experiment shows that label enters the beta-aminoglutarate pool after it has decayed from other small soluble molecules. This implies that beta-aminoglutarate is a catabolic product of the cells. Preliminary biosynthesis studies with labeled precursors indicate that only a single acetate moiety is incorporated in this unusual compound. This information is used to suggest possible biosynthetic pathways.

Anaerobiosis↗
PubMed1989

The effect of beta-sitosterol on spore germination and germ-tube elongation of Aspergillus niger and Botryodiplodia theobromae.

The effect of beta-sitosterol on spore germination and prevention of elongation of germ-tube of Aspergillus niger and Botryodiplodia theobromae was studied. The antifungal activity of the compound showed a percentage inhibition, of the fungal spore germination, of about 40% at a concentration of 50 micrograms/ml while inhibition on the elongation of germ-tubes was as high as 65% at the same concentration. The ED50 for inhibition of germ-tube elongation in A. niger was about 31 micrograms/ml. The role of the compound in disease resistance is discussed.

Aspergillus niger↗
PubMed1989

Effect of temperature on alpha-adrenoceptor affinity and contractility of rabbit ear blood vessels.

We have studied contractile responses to norepinephrine (NE) and K+ of isolated rabbit ear arteries and veins in an effort to determine how alpha-adrenoceptor affinity and smooth muscle contractility affect responsiveness at different temperatures. Arteries have predominantly alpha 1-adrenoceptors and veins have predominantly receptors of the alpha 2-type. We obtained dose-response curves to NE at 9, 16, 23, 30, 37 and 42 degrees C in the absence and presence of the irreversible alpha-adrenoceptor blocker phenoxybenzamine (POB). In both vessels, affinity of the alpha-adrenoceptors was determined by comparing equieffective doses of NE before and after blockade of the receptors. In other experiments, we obtained contractions to 80 mM KCl at the same temperatures. Affinity of the ear arterial alpha 1-receptors was maximal at 23 degrees C, while affinity of ear venous alpha 2-receptors increased linearly with cooling to 9 degrees C. Responses to K+ in both vessels decreased with cooling. Taken together, the changes in receptor affinity and response to K+ may explain the shape of curves relating contractile strength to temperature.

Animals↗
PubMed1987

Enzymatic hydrolysis of short-chain lecithin/long-chain phospholipid unilamellar vesicles: sensitivity of phospholipases to matrix phase state.

Short-chain lecithin/long-chain phospholipid unilamellar vesicles (SLUVs), unlike pure long-chain lecithin vesicles, are excellent substrates for water-soluble phospholipases. Hemolysis assays show that greater than 99.5% of the short-chain lecithin is partitioned in the bilayer. In these binary component vesicles, the short-chain species is the preferred substrate, while the long-chain phospholipid can be treated as an inhibitor (phospholipase C) or poor substrate (phospholipase A2). For phospholipase C Bacillus cereus, apparent Km and Vmax values show that bilayer-solubilized diheptanoylphosphatidylcholine (diheptanoyl-PC) is nearly as good a substrate as pure micellar diheptanoyl-PC, although the extent of short-chain lecithin hydrolysis depends on the phase state of the long-chain lipid. For phospholipase A2 Naja naja naja, both Km and Vmax values show a greater range: in a gel-state matrix, diheptanoyl-PC is hydrolyzed with micellelike kinetic parameters; in a liquid-crystalline matrix, the short-chain lecithin becomes comparable to the long-chain component. Both enzymes also show an anomalous increase in specific activity toward diheptanoyl-PC around the phase transition temperature of the long-chain phospholipid. Since the short-chain lecithin does not exhibit a phase transition, this must reflect fluctuations in head-group area or vertical motions of the short-chain lecithin caused by surrounding long-chain lecithin molecules. These results are discussed in terms of a specific model for SLUV hydrolysis and a general explanation for the "interfacial activation" observed with water-soluble phospholipases.

Bacillus cereus↗
PubMed1987

31P and 13C NMR analyses of the energy metabolism of the thermophilic anaerobe Clostridium thermocellum.

The energy metabolism of an anaerobic obligate thermophile, Clostridium thermocellum, has been examined as a function of incubation temperature using 31P NMR spectroscopy. Specifically investigated were the generation and availability of ATP as a function of temperature, activation energies for key processes in energy metabolism including formation of a pH gradient across the cell membrane, transport of key nutrients, and initial steps in glycolysis, and the existence of a membrane phase transition in the intact organism. Cells generate ATP via glycolysis at all temperatures examined; hence, limitation of the energy supply is not directly responsible for the lack of growth of this organism at low temperatures. Estimations of activation energies show a distinct hierarchy in the ATP-utilizing reactions examined. Conservation of ATP hydrolysis energy as delta pH has the lowest activation energy (less than or equal to 4 kcal/mol), two transport processes exhibit 10 kcal/mol activation energies, and early phosphorylation steps in glycolysis have significantly higher activation energies (approximately 25 kcal/mol). Neither the membrane-bound ATPase responsible for formation of the pH gradient nor the permease involved in phosphate transport shows evidence of a change in behavior around the phase transition temperature determined for extracted lipids of C. thermocellum. Line widths of inorganic phosphate do show a break in behavior around 35-40 degrees C. Possible explanations for this behavior are discussed.

Adenosine Diphosphate↗
PubMed1987

15N-NMR studies of Methanobacterium thermoautotrophicum: comparison of assimilation of different nitrogen sources.

Methanobacterium thermoautotrophicum can utilize glutamine and urea as well as ammonia as the sole nitrogen source during growth on H2 and CO2. High-field 15N-NMR has been used to compare the assimilation of these different nitrogen sources by this organism. The 15N-NMR spectra of extracts of cells grown in media containing [delta-15N]glutamine as the nitrogen source show that the glutamine amide nitrogen is rapidly converted to glutamate. The 15N-NMR spectra of cell extracts from cells grown on [15N]urea show a marked increase in the labeling of the alpha-NH2 of glutamate concurrent with a decrease in the urea resonance. These two nitrogen sources do not show the metabolic shift to alanine as the major resonance in stationary phase as is seen with 15NH4Cl. This behavior is discussed in terms of the enzymes of nitrogen metabolism.

Amino Acids↗
PubMed1987

Short-chain lecithin/long-chain phospholipid unilamellar vesicles: asymmetry, dynamics, and enzymatic hydrolysis of the short-chain component.

Asymmetric unilamellar vesicles are produced when short-chain phospholipids (fatty acyl chain lengths of 6-8 carbons) are mixed with long-chain phospholipids (fatty acyl chain lengths of 14 carbons or longer) in ratios of 1:4 short-chain/long-chain component. Short-chain lecithins are preferentially distributed on the outer monolayer, while a short-chain phosphatidylethanolamine derivative appears to localize on the inner monolayer of these spontaneously forming vesicles. Lanthanide NMR shift experiments clearly show a difference in head-group/ion interactions between the short-chain and long-chain species. Two-dimensional 1H NMR studies reveal efficient spin diffusion networks for the short-chain species embedded in the long-chain bilayer matrix. The short-chain lecithin is considerably more mobile than the long-chain component but has hindered motion compared to short-chain lecithin micelles. This differentiation in physical characteristics of the two phospholipid components is critical to understanding the activity of phospholipases toward these binary systems.

Hydrolysis↗
PubMed1987

Potentiation of the antimalarial activity of qinghaosu by methoxylated flavones.

Interaction between the flavones casticin and artemetin and the antimalarial activity of chloroquine and qinghaosu (QHS) was examined using an in vitro growth assay based on [3H]hypoxanthine incorporation in synchronized cultures of a cloned line of Plasmodium falciparum. Casticin, and to a lesser extent artemetin, selectively enhanced the inhibition of growth by QHS, but had little effect on the activity of chloroquine. The findings suggest that flavones indigenous to Artemisia annua, from which QHS is isolated, might significantly alter the clinical potential of this novel antimalarial drug in the treatment of chloroquine-resistant malaria.

Animals↗
PubMed1987

Mathematical circulation model for the blood-flow-heat-loss relationship in the rat tail.

A mathematical model for the heat-loss-blood-flow relationship is developed for the rat tail. When supplied with experimental values of heat loss and blood flow, the model allows one to compute the distribution of flow in deep and cutaneous vessels as a function of body core and tail temperature and to determine the savings in heat loss that result from alterations in the pattern of circulation and from counter-current heat transfer. Blood flow in the cutaneous and deep lying veins of the tail is controlled by both central and local temperatures and increases fairly linearly with deep body temperature. However, the distribution of blood flow in the tail is controlled only by local tail temperature and is independent of deep body temperature. The change in venous distribution of flow has a great impact on the conservation of heat and can reduce the heat loss from the circulating blood by more than 50% when venous return is directed to deep lying veins. On the other hand, counter-current heat transfer is of only minor importance in the control of heat loss from the tail, resulting at most in a 10% saving of heat loss, and that only at the smallest rate of blood flow.

Animals↗
PubMed1986

13C NMR spectroscopy of Methanobacterium thermoautotrophicum. Carbon fluxes and primary metabolic pathways.

The flux of 13C-labeled carbons from the soluble metabolite 2,3-cyclopyrophosphoglycerate (CPP), a novel compound found in high concentrations exclusively in methanobacteria and methanobrevibacter, into carbohydrate-containing material has been deduced by solid-state 13C NMR spectroscopy which strongly argues for a role in gluconeogenesis for this unique metabolite. The turnover rates, but not the steady-state levels, of CPP labeled by 13CO2 or [13C]acetate depend dramatically on cell growth conditions. When the demand for carbohydrate synthesis is reduced (i.e. in stationary phase), the rates of CPP biosynthesis and degradation decrease 10-fold, and the disaccharide alpha, alpha-trehalose accumulates. Valinomycin, a metabolic inhibitor of Methanobacterium thermoautotrophicum growth, does not affect steady-state levels of CPP, but does decrease 13C uptake into the CPP pool. The effects of these different conditions on CPP labeling suggest stringent regulation of CPP linked to cellular metabolism. Labeling of CPP by [6-(13)C]glucose, which does not serve as an energy or carbon source for this organism, provides strong evidence that glucose is cleaved by the reverse of the gluconeogenesis pathway. This metabolic pathway linking glucose with triose phosphate type precursors and an analysis of the 13C NMR spectrum of CPP labeled by incubating cells with [U-13C]glucose have established that in vivo phosphoenolpyruvate synthetase must be reversible.

Carbon↗
PubMed1986

31P-NMR spectra of methanogens: 2,3-cyclopyrophosphoglycerate is detectable only in methanobacteria strains.

The unique compound 2,3-cyclopyrophosphoglycerate occurs at a detectable concentration in the genera Methanobacterium and Methanobrevibacter but not in Methanococcus, Methanospirillum and Methanosarcina, as shown by a 31P-NMR survey of several different methanogens. Metabolic poisons (carbonyl cyanide m-chlorophenylhydrazone and valinomycin) do not decrease the level of the cyclic pyrophosphate in Methanobacterium thermoautotrophicum; therefore, it cannot be a phosphagen, i.e., an energy storage material. 13CO2 is rapidly incorporated into this cyclic compound which represents the major soluble carbon as well as the phosphorus component of this methanobacteria. 13C-NMR analysis demonstrates that the pKa of the 2,3-cyclopyrophosphoglycerate carboxyl group is 2.55. The unusual pseudomurein cell wall structure of methano- and methanobrevibacteria necessitates a high demand on carbohydrate metabolism. For this reason, and the fact that when its concentration is decreased no new phosphorus resonances appear in the high resolution spectra, it is suggested that 2,3-cyclopyrophosphoglycerate has a function in carbohydrate metabolism.

2,3-Diphosphoglycerate↗
PubMed1986

Interaction of short-chain lecithin with long-chain phospholipids: characterization of vesicles that form spontaneously.

Stable unilamellar vesicles formed spontaneously upon mixing aqueous suspensions of long-chain phospholipid (synthetic, saturated, and naturally occurring phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin) with small amounts of short-chain lecithin (fatty acid chain lengths of 6-8 carbons) have been characterized by using NMR spectroscopy, negative staining electron microscopy, differential scanning calorimetry, and Fourier transform infrared (FTIR) spectroscopy. This method of vesicle preparation can produce bilayer vesicles spanning the size range 100 to greater than 1000 A. The combination of short-chain lecithin and long-chain lecithin in its gel state at room temperature produces relatively small unilamellar vesicles, while using long-chain lecithin in its liquid-crystalline state produces large unilamellar vesicles. The length of the short-chain lecithin does not affect the size distribution of the vesicles as much as the ratio of short-chain to long-chain components. In general, additional short-chain decreases the average vesicle size. Incorporation of cholesterol can affect vesicle size, with the solubility limit of cholesterol in short-chain lecithin micelles governing any size change. If the amount of cholesterol is below the solubility limit of micellar short-chain lecithin, then the addition of cholesterol to the vesicle bilayer has no effect on the vesicle size; if more cholesterol is added, particle growth is observed. Vesicles formed with a saturated long-chain lecithin and short-chain species exhibit similar phase transition behavior and enthalpy values to small unilamellar vesicles of the pure long-chain lecithin prepared by sonication. As the size of the short-chain/long-chain vesicles decreases, the phase transition temperature decreases to temperatures observed for sonicated unilamellar vesicles. FTIR spectroscopy confirms that the incorporation of the short-chain lipid in the vesicle bilayer does not drastically alter the gauche bond conformation of the long-chain lipids (i.e., their transness in the gel state and the presence of multiple gauche bonds in the liquid-crystalline state).

Animals↗
PubMed1986

Indirect observation by 13C NMR spectroscopy of a novel CO2 fixation pathway in methanogens.

High-field carbon-13 nuclear magnetic resonance (NMR) spectroscopy has been used to monitor the isotopic dilution of doubly carbon-13-labeled precursors for 2,3-cyclopyrophosphoglycerate, a novel primary metabolite that occurs in certain methanogens. A unique carbon dioxide fixation pathway that gives rise to asymmetric labeling of acetyl coenzyme A has been demonstrated in Methanobacterium thermoautotrophicum. The effect of selected metabolic inhibitors on the labeled species in the pathway has been examined by NMR. These techniques establish a general, sensitive method for the delineation of convergent biosynthetic pathways.

Acetates↗
PubMed1985

Sensitivity of phospholipase C (Bacillus cereus) activity to phosphatidylcholine structural modifications.

The structural features of a phosphatidylcholine molecule important for binding to phospholipase C (Bacillus cereus) have been examined using kinetic analyses of a series of short-chain phosphatidylcholines and analogues. Lipids examined had varying chain lengths, methyl branched chains, phenyl alkanoate chains, and a single fatty acyl chain (lysophosphatidylcholines). A comparison of Vmax and Km for monomolecularly dispersed dibutyroyl-, dihexanoyl- and diheptanoylphosphatidylcholine indicates that the length of the fatty acyl chains must be at least six carbons for efficient binding of the phosphatidylcholine to the enzyme. Enzymatic rates of hydrolysis for pure short-chain phosphatidylcholine micelles of different chain lengths or detergent mixed micelles with comparable concentrations of short- and long-chain phosphatidylcholines show no dependence on substrate chain length greater than six carbons. Methyl branching of short-chain phosphatidylcholines only inhibits phospholipase C activity when the methyl group is adjacent to the carbonyl (e.g., di(2-methyl)hexanoylphosphatidylcholine). In a similar fashion, phosphatidylcholines with phenylalkanoate chains become poor substrates when the phenyl group is near the acyl linkage. As the phenyl group is moved from C-4 to C-2 a large increase in the micellar apparent Km is observed. Chain specificity (sn-1 and/or sn-2 ester linkages) for binding is not absolute, since phospholipase C will hydrolyze micellar short-chain lysophosphatidylcholines at rates one tenth of phosphatidylcholines. In contrast, substitution of ester linkages with ether moieties yields phosphatidylcholine analogues which are even poorer substrates and not good inhibitors of phospholipase C. These results suggest that the carbonyl group and its immediate environment are important for phospholipid interacting with this water-soluble lipolytic enzyme.

Bacillus cereus↗
PubMed1985

2,3-Cyclopyrophosphoglycerate in methanogens: evidence by 13C NMR spectroscopy for a role in carbohydrate metabolism.

The novel compound 2,3-cyclopyrophosphoglycerate (CPP) is the major small molecule carbon pool in Methanobacterium thermoautotrophicum. High-field 13C NMR 13CO2 pulse/unenriched CO2 chase experiments have shown that the labeled CPP rapidly loses its 13C to an insoluble pool, while the CPP steady-state concentration is maintained (as monitored by 31P NMR spectroscopy). The biosynthesis of CPP from CO2, acetyl coenzyme A, and pyruvate as precursors has been established by a 13C NMR study of ethanol extracts of Mb. thermoautotrophicum fed with 13CO2, [1-13C]- and [2-13C]acetate, and [1-13C]pyruvate. That CPP is a post-phosphoenolpyruvate metabolite has been confirmed by in vitro experiments with cell extracts. A role for CPP in carbohydrate metabolism was established when [1-13C]glucose fed to cells resulted in the formation of [3-13C]CPP exclusively. Possible functions of CPP within the cell are discussed.

2,3-Diphosphoglycerate↗
PubMed1985

Observation of tyrosine-O-phosphate in Drosophila melanogaster larvae by 31P-NMR spectroscopy.

31P-NMR spectra of intact larvae and pupae of Drosophila melanogaster have been obtained at 109.3 MHz. A major resonance in these samples has been identified as tyrosine-O-phosphate. Its chemical shift reflects the hemolymph plasma pH. Upon disruption of the organisms (necessary for chemical analyses of tyrosine-O-phosphate), phosphatases rapidly hydrolyze this phosphate ester, generating inorganic phosphate and free tyrosine.

Animals↗
PubMed1985

Influence of dilution on the physical state of model bile systems: NMR and quasi-elastic light-scattering investigations.

Multinuclear (1H and 31P) nuclear magnetic resonance (NMR) spectroscopy and quasi-elastic light scattering have been used to characterize molecular aggregates formed in dilute sodium taurocholate--egg lecithin solutions. When mixed micelles (1.25 g/dL) are diluted with 150 mM aqueous sodium chloride, light-scattering measurements suggest a transformation from mixed micelles to unilamellar vesicle species. Decreased 1H NMR line widths for bile salt resonances are consistent with predominance of a monomer form. The concurrent appearance of a second phospholipid choline methyl resonance indicates two types of phospholipid environment in slow chemical exchange: this behavior is consistent with small unilamellar vesicles. The appearance of bilayer vesicles in dilute model bile solutions is confirmed by addition of a lanthanide shift reagent (Pr3+), which splits the 1H or 31P head-group peak into two components with distinct chemical shift sensitivities. These mixed micelle and vesicle aggregates are also distinguished by their susceptibility to the lipolytic enzyme phospholipase A2 from cobra venom.

Bile↗