Nuclear Magnetic Resonance Spectroscopic Studies on the Microbial Degradation of Mononitrophenol Isomers

The biochemical pathways followed by a mixed bacterial culture and one of its constituent strains, Sarcina maxima, MTCC 5216 (hitherto unreported) during the degradation of mononitrophenol isomers was studied using extensive nuclear magnetic resonance (NMR) spectroscopy (One- and Two-Dimensional Heteronuclear Multiple Quantum Coherence Transfer-2D HMQCT NMR). NMR investigations revealed that o-nitrophenol (ONP) could be degraded by the consortium to metabolites such as catechol, cis, cis-muconic acid, γ-hydroxymuconic semialdehyde, maleylacetate and β-ketoadipate. The spectra of ONP reaction mixture degraded by S.␣maxima showed that formation of maleylacetate from γ-hydroxymuconic semialdehyde should go through a new metabolite γ-hydroxymaleylacetate, hitherto unreported. The consortium could breakdown m-nitrophenol (MNP) to 4-aminocatechol indicating that it came from 3-hydroxyaminophenol. However, S. maxima MTCC 5216, could convert MNP to hitherto unreported 2-nitrohydroquinone and the subsequent 2-hydroxylaminohydroquinone to 1,2,4-benzenetriol along with γ-hydroxymuconic semialdehyde, muconolactone and maleylacetate. The pathway followed by the consortium during p-nitrophenol (PNP) degradation was by the formation of 4-nitrocatechol, maleylacetate and β-ketoadipate. PNP reaction mixture of S.␣maxima, MTCC 5216 on the other hand, showed that the pathway could proceed through the formation of p-hydroquinone as the initial metabolite. The present study conclusively established the nitrophenol-degrading ability of both the consortium and S. maxima MTCC 5216, including exhibiting slight deviations from the pathways followed by the other reported microorganisms.     

结构基因组学研究与核磁共振

各种生物的基因组DNA测序计划的完成,将结构生物学带入了结构基因组学时代.结构基因组学是对所有基因组产物结构的系统性测定,它运用高通量的选择、表达、纯化以及结构测定和计算分析手段,为基因组的每个蛋白质产物提供实验测定的结构或较好的理论模型,这将加速生命科学各个领域的研究.生物信息学、基因工程、结构测定技术等的发展为结构基因组学研究提供了保证.近年来核磁共振在技术方法上的进展,使其成为结构基因组学高通量结构分析中的一个关键方法.     

17O Nuclear Magnetic Resonance Spectrum of H217O in Frog Striated Muscle

Whole striated muscles from the frog Rana esculenta were bathed in Ringer's solution enriched with H₂¹⁷O; the muscle water was subsequently collected by vacuum distillation. The integrated intensity of the nuclear magnetic resonance (NMR) signal of ¹⁷O in the muscle was measured to be approximately ¾ of the signal observed in the distilled water. The phenomenon may arise either from immobilization of a population of the water molecules which may be a very small fraction or as much as ¼ of the total, or may reflect tumbling of ⅓ of the water molecules in a compartment containing an anisotropic medium. Such an effect was demonstrated for H₂¹⁷O using the model system of sodium linoleate in water.     

Characterization of Metabolites in IntactStreptomyces citricolorCulture Supernatants Using High-Resolution Nuclear Magnetic Resonance and Directly Coupled High-Pressure Liquid Chromatography–Nuclear Magnetic Resonance Spectroscopy

A novel NMR spectroscopic approach to the direct biochemical characterization of bacterial culture broths is presented. A variety of one- and two-dimensional 1H NMR spectroscopic methods were used to characterize low-molecular-weight organic components of broth supernatants from cultures of Streptomyces citricolor. By applying 1H NMR spectroscopy to analyze whole, untreated culture supernatants, it was possible to identify and monitor simultaneously a range of media substrates and excreted metabolites. Identified metabolites include 2-phenylethylamine, trehalose, succinate, acetate, uridine, and aristeromycin, a secondary metabolite with antibiotic properties. Directly coupled HPLC-NMR spectroscopy was also applied to the analysis of broth supernatants for the first time, to aid spectral assignments, especially where signals were extensively overlapped in the 1H NMR spectra of the whole broth mixtures. Two-dimensional NMR methods such as 1H-1H correlation spectroscopy, 1H-13C heteronuclear single quantum correlation, and 1H-13C heteronuclear multiple bond correlation aided the structure elucidation and peak assignments of individual components in the mixtures by providing information on 1H-1H coupling networks and 13C chemical shifts. This work shows that high-resolution NMR spectroscopic methods provide a rapid and efficient means of investigating microbial metabolism directly without invasive or destructive sample pretreatment.     

Citrate and Sugar Cofermentation in Leuconostoc oenos, a (sup13)C Nuclear Magnetic Resonance Study

(sup13)C nuclear magnetic resonance spectroscopy was used to investigate citrate-glucose cometabolism in nongrowing cell suspensions of the wine lactic acid bacterium Leuconostoc oenos. The use of isotopically enriched substrates allowed us to identify and quantify in the end products the carbon atoms derived from each of the substrates supplied; furthermore, it was possible to differentiate between products derived from the metabolism of endogenous carbon reserves and those derived from external substrates. Citrate-sugar cometabolism was also monitored in dilute cell suspensions for comparison with the nuclear magnetic resonance results. A clear metabolic shift of the end products from glucose metabolism was observed when citrate was provided along with glucose: ethanol was replaced by acetate, and 2,3-butanediol was produced. Reciprocally, the production of lactate and 2,3-butanediol from citrate was increased in the presence of glucose. When citrate was cometabolized with glucose, a 10-fold reduction in the intracellular concentration of glucose-6-phosphate was observed, a result in line with the observed citrate-induced stimulation of glucose consumption. The presence of citrate provided additional pathways for NADP(sup+) regeneration and allowed the diversion of sugar carbon to reactions in which ATP was synthesized. The increased growth rates and maximal biomass yields of L. oenos growing on citrate-glucose mixtures resulted from increased ATP synthesis both by substrate-level phosphorylation and by a chemiosmotic mechanism.     

Multiparametric Characterization of Grade 2 Glioma Subtypes Using Magnetic Resonance Spectroscopic,Perfusion, and Diffusion Imaging

BACKGROUND AND PURPOSE: The purpose of this study was to derive quantitative parameters from magnetic resonance (MR) spectroscopic, perfusion, and diffusion imaging of grade 2 gliomas according to the World Health Organization and to investigate how these multiple imaging modalities can contribute to evaluating their histologic subtypes and spatial characteristics. MATERIALS AND METHODS: MR spectroscopic, perfusion, and diffusion images from 56 patients with newly diagnosed grade 2 glioma (24 oligodendrogliomas, 18 astrocytomas, and 14 oligoastrocytomas) were retrospectively studied. Metabolite intensities, relative cerebral blood volume (rCBV), and apparent diffusion coefficient (ADC) were statistically evaluated. RESULTS: The 75th percentile rCBV and median ADC were significantly different between oligodendrogliomas and astrocytomas (P < .0001) and between oligodendrogliomas and oligoastrocytomas (P < .001). Logistic regression analysis identified both 75th percentile rCBV and median ADC as significant variables in the differentiation of oligodendrogliomas from astrocytomas and oligoastrocytomas. Group differences in metabolite intensities were not significant, but there was a much larger variation in the volumes and maximum values of metabolic abnormalities for patients with oligodendroglioma compared with the other tumor subtypes. CONCLUSIONS: Perfusion and diffusion imaging provide quantitative MR parameters that can help to differentiate grade 2 oligodendrogliomas from grade 2 astrocytomas and oligoastrocytomas. The large variations in the magnitude and spatial extent of the metabolic lesions between patients and the fact that their values are not correlated with the other imaging parameters indicate that MR spectroscopic imaging may provide complementary information that is helpful in targeting therapy, evaluating residual disease, and assessing response to therapy.     

Nuclear Magnetic Resonance Evidence using D2O for Structured Water in Muscle and Brain

The electric quadrupole moment of the deuterium nucleus provides a nuclear magnetic resonance (NMR) probe of electric field gradients, and thereby of organization of tissue water. 8-17% of H₂O in rat muscle and brain was replaced by D₂O from 50% deuterated drinking water. The peak height of the steady-state NMR spectrum of D in muscle water was 74% lower than that of an equal concentration of D₂O in liquid water. Longitudinal NMR relaxation times (T₁) of D in water of muscle and brain averaged 0.092 and 0.131 sec, respectively, compared with 0.47 sec in D₂O in liquid water. Transverse NMR relaxation times (T₂) averaged 0.009 and 0.022 sec in D₂O of muscle and brain, respectively, compared with 0.45 sec in D₂O in liquid water. These differences cannot be explained by paramagnetic ions or by magnetic inhomogeneities, which leaves increased organization of tissue water as the only tenable hypothesis. Evidence was also obtained that 27% of muscle water and 13% of brain water exist as a separate fraction with T₂ of D₂O less than 2 × 10^-3 sec, which implies an even higher degree of structure. Each of the two fractions may consist of multiple subfractions of differing structure.     

Synthesis and Nuclear Magnetic Resonance Spectroscopic, Mass Spectroscopic, and Plasma Amine Oxidase Inhibitory Properties of Analogues of 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine

Abstract: Seventeen analogues of l-methyl-4-phenyl-1,2,3,6-tetrahydropyridine were synthesized using three reaction pathways: condensation of phenols with 1-methyl-4-piperidone, reaction of Grignard reagents with 1-methyl-4-piperidone followed by dehydration of the product, and aminomethylation of olefins. The identity of the products of synthesis was established by nuclear magnetic resonance spectroscopy, mass spectroscopy. and elemental analysis. Thirteen analogues were shown to inhibit the oxidation of benzylamine by bovine plasma amine oxidase. Increasing the length of the aliphatic chain of N -substituted analogues resulted in increased inhibition. In 4-phenyl-substituted analogues, both the position and electronic character of the substituent group affected the degree of inhibition.     

Nuclear Magnetic Resonance Studies of Haemoglobin M Milwaukee

Analysis of the NMR spectra of haemoglobin M Milwaukee confirms that ligand binding to the α haems changes the quaternary structure.