Natural abundance 13C-nuclear magnetic resonance spectroscopic analysis of acyclic polyol and trehalose accumulation by several yeast species in response to salt stress

Abstract: Analysis of seventeen yeast strains by ¹³C-NMR spectroscopy has confirmed the significance of glycerol as the sole osmoregulatory solute under salt-stressed conditions, and has shown arabitol to be present in most of the osmotolerant species. Ribitol was detected in some species, including Debaryomyces hansenii , although ribitol accumulation did not correlate with the osmotic pressure of the medium. Relative amounts of arabitol and ribitol decreased in relation to glycerol when the external osmotic pressure was increased. Trehalose was present during exponential growth of some species.     

13C-nuclear magnetic resonance study of glycophorins AM and AN modified with various pyrylium salts

The environment of the N-terminal amino groups of glycophorins A^M and A^N has been studied using¹³C-NMR spectroscopy and pyrylium salts as amino-blocking agents. The extent of amino blocking was monitored by¹³C-reductive methylation of the residual free amino groups. The pyrylium ions reacted with the N-terminal amino groups of the two glycophorins at almost identical rates, which is thought to indicate that the overriding steric bulk of the pyrylium salt may determine the rate of the reaction. The difference in the rates of modification of lysine residues of glycophorins A^M and A^N by the pyrylium ions did indicate that there may exist an environmental difference around the lysine residues between the two glycophorins. This environmental difference may result from solution aggregation of the glycophorin A molecules or from some differences in the pK_a values of the five lysine residues found in glycophorins A^M and A^N.     

Seed dormancy in red rice. X. A 13C-NMR study of the metabolism of dormancy-breaking chemicals

The metabolism of organic dormancy-breaking chemicals is poorly defined and may provide clues to their mode of action. Therefore, hydrated, dormant seeds of red rice ( Oryza sativa L.) were exposed to dormancy-breaking treatments of propionate-2-¹³C (22 m W ) or propanol-1-¹³C (75 m M ) for 24 h at 30°C. Embryo extracts were analyzed by ¹³C-nuclear magnetic resonance spectroscopy. Metabolism of propionate and propanol to 3-hydroxypropionate, an intermediate of the modified β-oxidation pathway, was detected after 2 and 4 h, respectively. This occurred prior to the onset of dormancy-breaking which required 12 h of chemical exposure. Accumulation of 3-hydroxypropionate was rapid and linear in the embryos of propionate-treated seeds. In the embryos of propanol-treated seeds, the level of 3-hydroxypropionate reached a plateau at 4 h. Following 24 h of contact with propionate, labeled citrate was detected in the embryos. The decrease in tissue pH associated with the dormancy-breaking process was fully accounted for by direct acid uptake and metabolic production of 3-hydroxypropionate.     

In Vitro Culture of Phytomonas Sp. Isolated from Euphorbia characias. Metabolic Studies by 1H NMR

ABSTRACT. We describe the in vitro culture of Phytomonas species isolated from Euphorbia characias . The best choice between tested media was SDM-79, in which promastigotes, after 6 days of culture, reached cell densities as high as 4 × 10⁷ cells/ml. Cells growing in LIT or MTL medium showed longer division times and lower cell densities. We succeeded in obtaining Phytomonas sp. amastigote and spheromastigote forms in modified GRACE's medium, yielding transformation rates of up to 70%. Electron microscopy studies were performed in order to characterize the ultrastructural features of these forms obtained in vitro. On the other hand, metabolic studies based on qualitative (nuclear magnetic resonance spectroscopy) and quantitative metabolic methods (enzymatic assays) showed that promastigote forms secreted mainly ethanol, acetate, glycine, glycerol, piruvate and succinate in SDM-79 medium, whereas the major metabolites found after transformation in modified Grace's medium were ethanol, acetate, glycine, piruvate and smaller amounts of glycerol.     

Biodegradation of nicotine by newly isolated Pseudomonas sp. CS3 and its metabolites

Aims: Isolation and characterization of nicotine‐degrading bacteria with advantages suitable for the treatment of nicotine‐contaminated water and soil and detection of their metabolites. Methods and Results: A novel nicotine‐degrading bacterial strain was isolated from tobacco field soil. Based on morphological and physiochemical properties and sequence of 16S rDNA, the isolate was identified as Pseudomonas sp., designated as CS3. The optimal culture conditions of strain CS3 for nicotine degradation were 30°C and pH 7·0. However, the strain showed broad pH adaptability with high nicotine‐degrading activity between pH 6·0 and 10·0. Strain CS3 could decompose nicotine nearly completely within 24 h in liquid culture (1000 mg L^?1 nicotine) or within 72 h in soil (1000–2500 mg kg^?1 nicotine) and could endure up to 4000 mg L^?1 nicotine in liquid media and 5000 mg kg^?1 nicotine in soil. Degradation tests in flask revealed that the strain had excellent stability and high degradation activity during the repetitive degradation processes. Additionally, three intermediates, 3‐(3,4‐dihydro‐2H‐pyrrol‐5‐yl) pyridine, 1‐methyl‐5‐(3‐pyridyl) pyrrolidine‐2‐ol and cotinine, were identified by GC/MS and NMR analyses. Conclusions: The isolate CS3 showed outstanding nicotine‐degrading characteristics such as high degradation efficiency, strong substrate endurance, broad pH adaptability, and stability and persistence in repetitive degradation processes and may serve as an excellent candidate for applications in the bioaugmentation process to treat nicotine‐contaminated water and soil. Also, detection of nicotine metabolites suggests that strain CS3 might decompose nicotine via a unique nicotine‐degradation pathway. Significance and Impact of the Study: The advantage of applying the isolated strain lies in broad pH adaptability and stability and persistence in repetitive use, the properties previously less focused in other nicotine‐degrading micro‐organisms. The strain might decompose nicotine via a nicotine‐degradation pathway different from those of other nicotine‐utilizing Pseudomonas bacteria reported earlier, another highlight in this study.     

Biosynthesis of novel polyhydroxyalkanoate containing 3-hydroxy-4-methylvalerate by Chromobacterium sp. USM2

Aims: Polyhydroxyalkanoate (PHA) with enhanced physicochemical properties will be ideal for a wide range of practical applications. The incorporation of 3‐hydroxy‐4‐methylvalerate (3H4MV) into the polymer backbone is known to improve the overall properties of the resulting polymer. However, the most suitable micro‐organism and PHA synthase that can synthesize this monomer efficiently still remain unknown at present. Therefore, we evaluated the abilities of a locally isolated Chromobacterium sp. USM2 to produce PHA containing 3H4MV. Methods and Results: The ability of Chromobacterium sp. USM2 to synthesize poly(3‐hydroxybutyrate‐co‐3‐hydroxy‐4‐methylvalerate) [P(3HB‐co‐3H4MV)] was evaluated under different culture conditions. It was found that Chromobacterium sp. USM2 can synthesize P(3HB‐co‐3H4MV) when glucose and isocaproic acid were fed as carbon source. However, the highest molar fraction of 3H4MV, 22 mol% was detected in Chromobacterium sp. USM2 when isocaproic acid was provided as the sole carbon source. In addition, aeration was identified as a crucial factor in initiating the accumulation of high 3H4MV molar fractions. Conclusions: Chromobacterium sp. USM2 was able to synthesize broad comonomer compositional distribution of P(3HB‐co‐3H4MV). Significance and Impact of the Study: Compared with Cupriavidus necator and Burkholderia sp., Chromobacterium sp. USM2 was found to have better ability to bioconvert isocaproic acid to form 3H4MV unit.     

Identification of organic solutes accumulated by purple and green sulphur bacteria during osmotic stress using natural abundance 13C nuclear magnetic resonance spectroscopy

Abstract Natural abundance ¹³C NMR spectroscopy has identified sucrose and trehalose as the principle compatible solutes accumulated by non-halophilic purple and green sulphur bacteria respectively, in response to osmotic stress. Synthesis of glycine betaine as a compatible solute was rare in non-halophilic phototrophic sulphur bacteria and appears to be limited almost exclusively to halotolerant isolates, although all isolates tested were able to accumulate exogenous glycine betaine from the growth medium in response to osmotic stress. These data support the hypothesis that the degree of halotolerance of a microorganism may be due, at least in part, to the metabolic effects of the compatible solute(s) accumulated.     

[3H]N-[1-(2-Thienyl)cyclohexyl]-3,4-Piperidine ([3H]TCP) Binding in Human Frontal Cortex: Decreases in Alzheimer-Type Dementia

We studied [3H]N-[1-(2-thienyl)cyclohexyl]-3,4-piperidine [( 3H]TCP) binding to human frontal cortex obtained at autopsy from 10 histologically normal controls and eight histopathologically verified cases with Alzheimer-type dementia (ATD). Extensively washed membrane preparations were used to minimize the effects of endogenous substances. In ATD frontal cortex, the total concentration (Bmax) of [3H]TCP binding sites was significantly reduced by 40-50%. The apparent dissociation constant (KD) values showed no significant change. The reduction in binding capacity was also apparent in Triton X-100-treated membrane preparations, and there was a linear correlation between the number of [3H]TCP binding sites and that of N-methyl-D-aspartate (NMDA)-sensitive [3H]glutamate binding sites. [3H]TCP binding sites spared in ATD brains retained the affinity for the ligand and the reactivity to NMDA, L-glutamate, and glycine. These results suggest that the primary change in NMDA receptor-ion channel complex in ATD brains is the reduction of its number, possibly reflecting the loss of neurons bearing these receptor complexes, and that the functional linkage within the receptor complexes spared in ATD brains remains normal.     

Natural abundance of 13C and 15N in C3 and C4 vegetation of southern Africa: patterns and implications

The mean annual rainfall in southern Africa is found to explain over half of the observed variance in the stable nitrogen (N) isotopic signatures of C₃ vegetation in southern Africa (r²=0.54, P<0.01). The inverse relationship between the stable N isotopic signatures of foliar samples from C₃ vegetation and long‐term southern African rainfall is found on a scale larger than previously observed. A modest relationship is found between stable carbon (C) isotopic signatures of C₃ vegetation and rainfall across the region (r²=0.20, P<0.01). No such relationship is found between stable C and N isotopic signatures of C₄ vegetation and rainfall. The explanation of the relationship between ¹⁵N in C₃ vegetation and the mean annual rainfall presented here is that nutrient availability varies inversely with water availability. This suggests that water‐limited systems in southern Africa are more open in terms of nutrient cycling and therefore the resulting natural abundance of foliar ¹⁵N in these systems is enriched. The use of this relationship may be of value to those researchers modeling both the dynamics of vegetation and biogeochemistry across this region. The use of the isotopic enrichment in C₃ vegetation as a function of rainfall may provide an insight into nutrient cycling across the semi‐arid and arid regions of southern Africa. This finding has implications for the study of global change, especially as it relates to vegetation responses to changing regional rainfall regimes over time.     

[3H]Neurotensin(8–13) Binds in Human Brain to the Same Sites as Does [3H]Neurotensin but with Higher Affinity

The binding of [3H]neurotensin(8-13) to membranes from human frontal cortex at 0 degree C was time dependent, specific, saturable, and reversible. Saturation isotherms provided an equilibrium dissociation constant (KD) of 0.52 nM, and the maximal number of binding sites (Bmax) was 3.5 pmol/g original wet weight of tissue. Scatchard analysis yielded a straight line, and the Hill coefficient was equal to 1, a result indicating that [3H]neurotensin(8-13) bound to single, noncoopertive sites. The KD values of several analogs of neurotensin determined in competition with [3H]neurotensin(8-13) were similar to those previously determined in competition with [3H]neurotensin. The regional distribution of binding sites for [3H]neurotensin(8-13) was also similar to that for [3H]neurotensin. These results suggest that [3H]neurotensin(8-13) binds to the same sites as [3H]neurotensin and that [3H]neurotensin(8-13) has a higher affinity than [3H]neurotensin for these sites in human brain.     

The vacuolar localization of a basic peroxidase isoenzyme responsible for the synthesis of α-31,41-anhydrovinblastine in Catharanthus roseus (L.) G. Don Leaves

Partially purified protein extracts of Catharanthus roseus leaves were able to couple catharanthine and vindoline to produce α-3′,4′-anhydrovinblastine (AVLB) in a reaction strictly dependent on H₂O₂. This result, and the co-purification of peroxidase with AVLB synthetase activity, strongly suggest a peroxidase-like nature for the coupling enzyme. Only one peroxidase isoenzyme was detected in C. roseus leaves, and it was shown that this isoenzyme consists of a molecularly-heterogeneous basic peroxidase (EC 1-11-1-7) mainly located in the vacuole. These results suggest that a basic peroxidase located in the vacuole may be the main enzyme responsible for AVLB synthesis in C. roseus leaves. This isoenzyme was also found in cell walls where a peroxidase inhibitor was detected.     

Heterologous expression of polyhydroxyalkanoate depolymerase from <Emphasis Type="Italic">Thermobifida</Emphasis> sp. in <Emphasis Type="Italic">Pichia pastoris</Emphasis> and catalytic analysis by surface plasmon resonance

A polyhydroxyalkanote depolymerase gene from Thermobifida sp. isolate BCC23166 was cloned and expressed as a C-terminal His₆-tagged fusion in Pichia pastoris. Primary structure analysis revealed that the enzyme PhaZ-Th is a member of a proposed new subgroup of SCL-PHA depolymerase containing a proline–serine repeat linker. PhaZ-Th was expressed as two glycosylated forms with apparent molecular weights of 61 and 70 kDa, respectively. The enzyme showed esterase activity toward p-nitrophenyl alkanotes with V _max and K _m of 3.63 ± 0.16 μmol min⁻¹ mg⁻¹ and 0.79 ± 0.12 mM, respectively, on p-nitrophenyl butyrate with optimal activity at 50–55°C and pH 7–8. Surface plasmon resonance (SPR) analysis demonstrated that PhaZ-Th catalyzed the degradation of poly-[(R)-3-hydroxybutyrate] (PHB) films, which was accelerated in (R)-3-hydroxyvalerate copolymers with a maximum degradation rate of 882 ng cm⁻² h⁻¹ for poly[(R)-3-hydroxybutyrate-co-3-hydroxyvalerate] (12 mol% V). Surface deterioration, especially on the amorphous regions of PHB films was observed after exposure to PhaZ-Th by atomic force microscopy. The use of P. pastoris as an alternative recombinant system for bioplastic degrading enzymes in secreted form and a sensitive SPR analytical technique will be of utility for further study of bioplastic degradation.     

Characteristics of [14C]Guanidinium Accumulation in NG 108-15 Cell Exposed to Serotonin 5-HT3 Receptor Ligands and Substance P

Abstract: In the presence of substance P (SP; 10 μM), serotonin (5-HT; 1 μM) triggered a cation permeability in cells of the hybridoma (mouse neuroblastoma X rat glioma) clone NG 108-15 that could be assessed by measuring the cell capacity to accumulate [¹⁴C]guanidinium for 10-15 min at 37°C. In addition to 5-HT (EC₅₀, 0.33 μM), the potent 5-HT₃ receptor agonists 2-methyl-serotonin, phenylbiguanide, and m-chlorophenylbiguanide, and quipazine, markedly increased [¹⁴C]guanidinium uptake in NG 108-15 cells exposed to 10 μM SP. In contrast, 5-HT₃ receptor antagonists prevented the effect of 5-HT. The correlation (r= 0.97) between the potencies of 16 different ligands to mimic or prevent the effects of 5-HT on [¹⁴C]guanidinium uptake, on the one hand, and to displace [³H]zacopride specifically bound to 5-HT₃ receptors on NG 108-15 cells, on the other hand, clearly demonstrated that [¹⁴C]guanidinium uptake was directly controlled by 5-HT₃ receptors. Various compounds such as inorganic cations (La³⁺, Mn²⁺, Ba²⁺, Ni²⁺, and Zn²⁺), D-tubocurarine, and memantine inhibited [¹⁴C]guanidinium uptake in NG 108-15 cells exposed to 5-HT and SP, as expected from their noncompetitive antagonistic properties at 5-HT₃ receptors. However, ethanol (100 mM), which has been reported to potentiate the electrophysiological response to 5-HT₃ receptor stimulation, prevented the effects of 5-HT plus SP on [¹⁴C]guanidinium uptake. The cooperative effect of SP on this 5-HT₃-evoked response resulted neither from an interaction of the peptide with the 5-HT₃ receptor binding site nor from a possible direct activation of G proteins in NG 108-15 cells. Among SP derivatives, [D-Pro⁹]SP, a compound inactive at the various neurokinin receptor classes, was the most potent to mimic the stimulatory effect of SP on [¹⁴C]guanidinium uptake in NG 108-15 cells exposed to 5-HT. Although the cellular mechanisms involved deserve further investigations, the 5-HT-evoked [¹⁴C]guanidinium uptake appears to be a rapid and reliable response for assessing the functional state of 5-HT₃ receptors in NG 108-15 cells.     

1H nuclear magnetic resonance and magnetic circular dichroism studies of ferric low-spin cytochrome P-450scc

400 MHz ¹H NMR of ferric low-spin cytochrome P-450_scc purified from bovine adrenal cortex was measured for the first time. As compared with ¹H NMR spectra of low-spin P-450_cam and metMb- mercaptan complexes, paramagnetic shifts of low-spin P-450_scc complexes were more divergent, suggesting that there is a subtle difference in the heme environment between P-450_scc and P-450_cam [1]. The paramagnetic shifts of low-spin complexes of P-450_scc caused by adding nitrogenous inhibitors, aminoglutethimide and metyrapone, were different from those caused by adding an intermediate, 20α-hydroxycholesterol, and a detergent, Tween 20 [2]. The paramagnetic shifts of the metMb-mercaptan complexes were convergent compared with those of ferric low-spin P-450_scc and P-450_cam, suggesting that the electronic character and/or the conformation of the internal thiolate ligand in P-450_scc and P-450_cam are different from those of the external thiolate ligand in metMb-thiolate complexes [3]. The paramagetic shifts of the metMb-mercaptan complexes were dependent on the electron donating factor of the alkyl group of the bound mercaptans [4].Magnetic CD(MCD) spectra of ferric low-spin P-450_scc, rabbit liver P-450 complexes and metMb- mercaptan complexes were also observed at various temperatures. The temperature dependences of the Soret MCD bands for the low-spin P-450 and metMb- mercaptan complexes were decidedly less pronounced than those for the low-spin metMb-CN? or imidazole complexes, suggesting that thiolate ligands markedly influence the Soret MCD band of the ferric low-spin complexes [1]. The suggestion described in [2] implied by the ¹H NMR study was reconfirmed from the temperature dependence study of the Soret MCD [2]. The temperature dependences of the Soret MCD bands for low-spin P-450 complexes having a non-nitrogenous ligand were more pronounced than for those having a nitrogenous ligand.     

Epoxypropane biosynthesis by Methylomonas sp. GYJ3: batch and continuous studies

Methylomonas sp. GYJ3 is a methanotrophic bacterium containing methane monooxygenase (MMO), which catalyses the epoxidation of propene to epoxypropane. In this study, the cell suspension of Methylomonas sp. GYJ3 has been used for epoxypropane biosynthesis from propene. When propene is epoxidized, the product epoxypropane is not further metabolized and accumulates extracellularly. Unfortunately, continuous production of epoxypropane is usually difficult due to exhaustion of reductant and the accumulation of toxic products. Hence, in order to address these problems, batch experiments were performed to explore the possibility of producing epoxypropane by a co-oxidation process. Methane was chosen as the most suitable electron-donating co-substrate since it did not result in molecular toxicity and provided abundant reductant for epoxidation. It was found that the maximum production of epoxypropane occurred in an atmosphere of 30% methane. Batch experiments also indicated that continuous removal of product was necessary to overcome the inhibition of epoxypropane. In continuous experiments, optimum mixed gaseous substrates were continuously circulated through the stirred tank bioreactor to remove product from the cell suspension. Initial epoxypropane productivity was 268 mol/day. The bioreactor has been allowed to operate continuously for 12 days without obvious loss of epoxypropane productivity, and more than 96% of initial MMO activity was retained.     

Transport and Metabolism of D-[3H]Adenosine and L-[3H]Adenosine in Rat Cerebral Cortical Synaptoneurosomes

The relationship between transport and metabolism in synaptoneurosomes was examined to determine the metabolic stability of rapidly accumulated D-[3H]adenosine and L-[3H]adenosine and the degree to which metabolism of the accumulated purines affected measurements of apparent KT and Vmax values for adenosine transport. For D-[3H]adenosine, high- and low-affinity accumulation processes were present. For the high-affinity system an inverse relationship was found between transport reaction times and KT and Vmax values. For incubations of 5, 15, and 600 s, which corresponded to 24, 32, and 76% phosphorylation of accumulated D-[3H]adenosine to nucleotides, apparent KT values were 9.4, 8.4, and 4.5 microM, respectively, and Vmax values were 850, 70, and 12 pmol/min/mg of protein, respectively. Pretreatment with 10 microM erythro-9-(2-hydroxy-3-nonyl)adenine, an adenosine deaminase inhibitor, and 5'-iodotubercidin, an adenosine kinase inhibitor, decreased the phosphorylation of accumulated D-[3H]adenosine to 6% with 5-s and 9% with 15-s incubations. This resulted in significantly higher KT values: 36 microM at 5 s and 44 microM at 15 s. At 10-min incubations in the presence of these inhibitors, metabolism of accumulated D-[3H]adenosine was 32%, and apparent KT and Vmax values at this time were not significantly different from those obtained without inhibitors. For L-[3H]adenosine, apparent KT and Vmax values for 20-s incubations were 38.7 microM and 330 pmol/min/mg of protein, respectively. Metabolism (mainly phosphorylation) of accumulated L-[3H]adenosine was observed only at incubations of greater than 30 s.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of 1-Methyl-4-Phenyl-1,2,5,6-Tetrahydropyridine and Related Compounds on the Uptake of [3H]3,4-Dihydroxyphenylethylamine and [3H]5-Hydroxytryptamine in Neostriatal Synaptosomal Preparations

1-Methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) is known to cause a destruction of the dopaminergic nigrostriatal pathway in certain animal species including mice. MPTP and some structurally related analogs were tested in vitro for their capacity to inhibit the uptake of [3H]3,4-dihydroxyphenylethylamine-([3H]DA), [3H]5-hydroxytryptamine ([3H]5-HT), and [3H]gamma-aminobutyric acid [( 3H]GABA) in mouse neostriatal synaptosomal preparations. MPTP was a very potent inhibitor of [3H]5-HT uptake (IC50 value 0.14 microM), a moderate inhibitor of [3H]DA uptake (IC50 value 2.6 microM), and a very weak inhibitor of [3H]GABA uptake (no significant inhibition observed at 10 microM MPTP). In other experiments, MPTP caused some release of previously accumulated [3H]DA and [3H]5-HT, but in each case MPTP was considerably better as an uptake inhibitor than as a releasing agent. The 4-electron oxidation product of MPTP, i.e., 1-methyl-4-phenyl-pyridinium iodide (MPP+), was a very potent inhibitor of [3H]DA uptake (IC50 value 0.45 microM) and of [3H]5-HT uptake (IC50 value 0.78 microM) but MPP+ was a very weak inhibitor of [3H]GABA uptake. These data may have relevance to the neurotoxic actions of MPTP.     

Noninvasive Demonstration of In Vivo 3-Fluoro-3-Deoxy-D-Glucose Metabolism in Rat Brain by 19F Nuclear Magnetic Resonance Spectroscopy: Suitable Probe for Monitoring Cerebral Aldose Reductase Activities

The metabolism of 3-fluoro-3-deoxy-D-glucose (3-FDG) in rat brain in vivo was investigated noninvasively using ¹⁹F nuclear magnetic resonance (NMR) Spectroscopy. Following an intravenous infusion of 3-FDG, 400 mg/kg, four resonances assigned to the α and β anomers of 3-FDG, 3-fluoro-3-deoxy-D-sorbitol, and 3-fluoro-3-deoxy-D-fructose were clearly resolved in brain, a result indicating that 3-FDG is metabolized primarily into the aldose reductase sorbitol (ARS) pathway. An orally administered aldose reductase inhibitor, sorbinil, caused reduction of the flux of 3-FDG into the ARS, an observation suggesting that the method can be applied in quantitative studies of ARS path way activities. Studies of 24-h urine specimens showed that in addition to the two metabolites observed in brain, F^-was excreted into the urine. 3-FDG appears to be a suitable metabolic probe for assessing glucose metabolism in the ARS pathway by in vivo ¹⁹F NMR Spectroscopy.