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Cyclohexadepsipetides enniatin B, B1 and G were isolated from the cultivation broth of Fusarium dimerum Penzig, an endophyte of Magnolia x soulangeana. Their production was about 350 mg l(-1) after 96 h of submerged cultivation in Sabouraud maltose medium. Isolated enniatins inhibited growth of selected microorganisms and activity of 12-lipoxygenase with IC50 = 0.73 mg l(-1).

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Chiral beta-cyclodextrin was used to separate diazepam conformers. Several mobile phases of the composition acetonitrile/acetate buffer 200 mmol/l (pH=3.3, 5.5, 6.5) were employed for this purpose. As follows, the influence of addition of chiral beta-cyclodextrin to the mobile phase on diazepam separation was studied. The interconversion was a concurrence process of separation, resulting from stereolability of the diazepam molecule. The influences of temperature, flow rate, pH, and ionic strength of the mobile phase on interconversion and chromatographic parameters (retention factor and selectivity coefficient) were studied. Complementary off-line NMR measurements were carried out with the goal to confirm the structure of diazepam in the presence of an acid mobile phase.

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In vivo magnetic resonance spectroscopy (MRS) studies of glial brain tumours reported that higher grade of astrocytoma is associated with increased level of choline-containing compounds (Cho) and decreased levels of N-acetylaspartate (NAA) and creatine and phosphocreatine (Cr). In this work, we studied the metabolism of glioma tumours by in vitro proton magnetic resonance spectroscopy (1H-MRS). 1H-MR spectra were recorded in vitro from perchloric acid extracts of astrocytoma (WHO II) and glioblastoma multiforme (WHO IV) samples. We observed differences between astrocytoma and glioblastoma multiforme in the levels of Cho, alanine, lactate, NAA, and glutamate/glutamine. In astrocytoma samples, we found higher MR signal of NAA and lower signal of Cho and alanine. MR spectra of glioblastoma samples reported significantly higher levels of lactate and glutamate/glutamine. In contrast, levels of Cr were the same in both tumour types. We also determined NAA/Cr and Cho/Cr ratios in the tumour samples. The NAA/Cr ratio was higher in astrocytomas than in glioblastomas multiforme. Conversely, the Cho/Cr ratio was higher in glioblastoma multiforme. The results indicate that MRS is a promising method for distinguishing pathologies in human brain and for pre-surgical grading of brain tumours.

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Twenty four crossbred (Large White, White Meaty, Pietrain, Hampshire) pigs were tested by DNA probe for a mutation on the ryanodine receptor RYR1 (malignant hyperthermia-MH). An equal number of pigs heterozygote (monomutant-MON) and normal on MH (nonmutant-NON) were used in the experiment. The pigs were fed finisher feed (control group) or finisher feed supplemented with magnesium (3.6 g MgO per pig per day; MgO group) for 5 days prior to slaughter. Pigs fed the diet supplemented with MgO had higher plasma Mg concentrations. Phosphorus nuclear magnetic resonance ((31)P NMR) measurements on postmortem (15 min) muscle samples (longissimus muscle) showed the highest phosphocreatine levels in normal pigs fed MgO (P<0.05). The MgO supplementation caused increased Ca(2+) uptake and Ca(2+) ATPase activity only in normal (NON) pigs. ATPase activity was lowest (P<0.05) in heterozygote control pigs. Pigs fed MgO supplemented diet had higher pH (45 min postmortem). A significant lower pH (P<0.05) was obtained in heterozygous (MON) control pigs. Also pigs fed with MgO had lower percentage of drip losses and significant differences (P<0.05) were obtained between heterozygous (MON) pigs. The results indicate that dietary MgO supplementation can improve parameters of muscle energetic metabolism, Ca(2+) uptake and meat quality (pH, drip loss).

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Multidrug resistance of murine leukaemic cell line L1210/VCR (obtained by adaptation of parental drug-sensitive L1210 cells to vincristine) is associated with overexpression of mdr1 gene product P-glycoprotein (Pgp)-the ATP-dependent drug efflux pump. 31P-NMR spectra of L1210 and L1210/VCR cells (the latter in the presence of vincristine) revealed, besides the decrease of ATP level, a considerable lower level of UDP-saccharides in L1210/VCR cells. Histochemical staining of negatively charged cell surface binding sites (mostly sialic acid) by ruthenium red (RR) revealed a compact layer of RR bound to the external coat of sensitive cells. In resistant cells cultivated in the absence or presence of vincristine, the RR layer is either reduced or absent. Consistently, resistant cells were found to be less sensitive to Concanavalin A (ConA). Moreover, differences in the amount and spectrum of glycoproteins interacting with ConA-Sepharose were demonstrated between sensitive and resistant cells. Finally, the content of glycogen in resistant cells is lower than in sensitive cells. All the above facts indicate that multidrug resistance of L1210/VCR cells mediated predominantly by drug efflux activity of Pgp is accompanied by a considerable depression of oligo- and/or polysaccharides biosynthesis.

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Phosphorus nuclear magnetic resonance ((31)P NMR) measurements were made to determine muscle energetic metabolism on muscle biopsy samples of heterozygote malignant hyperthermia (Nn) and normal (NN) pigs DNA tested on occurrence of mutation in RYR 1 gene. Biopsy samples (approx. 1 g) were obtained by spring-loaded biopsy instrument (Biotech, Slovakia) from Longissimus dorsi (LD) muscle at 80 kg live weight. The spectra were recorded at 121 MHz on a VXR 300 (Varian) spectrometer in 10 mm diameter tube (maintained at 39 °C) for 50 min. pH of bioptates after NMR measurements were also measured at 60 min. The changes in inorganic phosphate (Pi), phosophocreatine (PCr) and adenosine triphosphate (ATP) were faster in heterozygote malignant hyperthermia (MH; 29 crossbred White Meaty×Pietrain) than in normal (13 Duroc, Yorkshire and White Meaty). The values of PCr at 20 min and pH at 60 min after taking biopsy allowed discrimination between NN and Nn pigs and significant (P<0.05) differences were also found between two subgroups in heterozygote MH pigs with different rate of post mortem muscle metabolism. The values of PCr and pH as measured at definite time on the biopsies, were significantly (P<0.05) correlated with the rate of post mortem metabolism (pH) and with meat quality traits (r approx. 0.4-0.6). The (31)P NMR measurements pointed to impaired muscle energetic metabolism connected with the occurrence of mutation on the RYR 1 gene in heterozygote MH pigs.

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The relative configuration of the two xanthene units of neosartorin, a new ergochrome biosynthesised by the soil mould Neosartorya fischeri, was determined using a 1D double-pulsed field gradient spin-echo NOESY experiment. It was found that both units have the same relative stereochemistry. Long-range nonbonding interactions between the substituents of different xanthene units stabilise the nonplanar configuration of the two aromatic rings A and A' connecting both monomer units of neosartorin.

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A mixture of related metabolites (denoted OR-1) was isolated from the fermentation broth of Penicillium funiculosum together with mitorubrinic acid. Structurally OR-1 is glyceric acid esterified with C14-C18 fatty acids. Steady-state studies revealed that OR-1 behaved like an uncompetitive trypsin inhibitor with Ki 17.6 mumol/L.

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Glutamic acid decarboxylase (GAD) activity was measured in homogenates of conidia and both submerged and aerial mycelia of Trichoderma viride. The GAD activity in conidia had a temperature optimum at 30 degrees C and a pH optimum at pH 4. GAD was stimulated by EDTA (2 mM) and was insensitive to treatment with calmodulin antagonists calmidazolium (10 microM) or phenothiazine neuroleptics (60 microM). Cyclosporin A (up to 300 microM) partially inhibited GAD in the homogenate, but not in the supernatant obtained after centrifuging the homogenate. Attempts to release GAD activity from the homogenate using high ionic strength, detergents, or urea failed. Freezing-thawing led to the partial increase of activity in the conidial homogenate. These results indicate that GAD is a membrane-bound enzyme. The highest specific activity of GAD was present in the mitochondrial/vacuolar organellar fraction. Germination of conidia in the submerged culture led to a temporary decrease in GAD activity. After prolonged cultivation, the activity displayed quasi-oscillatory changes. The stationary state was characterized by a high GAD activity. The presence of gamma-aminobutyric acid in the submerged mycelia was demonstrated. In surface culture in the dark, GAD activity increased in a monophasic manner until conidia formation. The illumination of dark-cultivated mycelia by a white-light pulse caused a dramatic increase in GAD activity. Light-induced changes were not observed in mutants with delayed onset of conidiation. In the dark or upon illumination by light pulse, the increase of GAD activity preceded the appearance of conidia. Thus, GAD activity in T. viride is closely associated with its developmental status and may represent a link between differentiation events and energy metabolism.

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1. In vivo 1H and 31P magnetic resonance spectroscopy techniques were applied to reveal biochemical changes in the rat brain caused by prolonged ethanol consumption. 2. Three models of ethanol intoxication were used. 3. 1H MRS showed a significant decrease in the concentration of myo-inositol in the brain of rats fed with 20% ethanol for 8 weeks. This change is consistent with perturbances in astrocytes. On the other hand, N-acetyl aspartate and choline content did not differ from controls. 4. 31P MRS did not reveal any significant changes in the high-energy phosphates or intracellular free Mg2+ content in the brain of rats after 14 weeks of 20% ethanol drinking. The intracellular pH was diminished. 5. By means of a 31P saturation transfer technique, a significant decrease was observed for the pseudo first-order rate constant k(for) of the creatine kinase reaction in the brain of rats administered 30% ethanol for 3 weeks using a gastric tube. 6. The 1H MRS results may indicate that myo-inositol loss, reflecting a disorder in astrocytes, might be one of the first changes associated with alcoholism, which could be detected in the brain by means of in vivo 1H MRS. 7. The results from 31p MRS experiments suggest that alcoholism is associated with decreased brain energy metabolism. 8. 31P saturation transfer, which provides insight into the turnover of high-energy phosphates, could be a more suitable technique for studying the brain energetics in chronic pathological states than conventional 31P MRS.

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Creatine kinase reaction rates were measured by the magnetisation transfer technique in brains of healthy adult and aged rats and in rats with chronic cerebral ischemia and chronic ethanol intoxication. These measurements indicated that the rate constant of the creatine kinase reaction is significantly reduced in the case of severe chronic cerebral ischemia in aged rats. In the adult rats, during chronic ethanol intoxication after 3 weeks of administration of 3 ml of 30% ethanol once a day via a gastric tube, a significant decrease in the pseudo first-order rate constant k(for) of the creatine kinase reaction was also found. In contrast, mild chronic cerebral ischemia in adult rats produced an increase in the reaction rate 4 weeks after occlusion. At the same time, corresponding conventional phosphorus magnetic resonance spectra showed negligible changes in signal intensities.

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The effect of the presence of ammonia on [1-13C]glucose metabolism in the rumen fibrolytic bacterium Fibrobacter succinogenes S85 was studied by 13C and 1H nuclear magnetic resonance (NMR). Ammonia halved the level of glycogen storage and increased the rate of glucose conversion into acetate and succinate 2.2-fold and 1.4-fold, respectively, reducing the succinate-to-acetate ratio. The 13C enrichment of succinate and acetate was precisely quantified by 13C-filtered spin-echo difference 1H-NMR spectroscopy. The presence of ammonia did not modify the 13C enrichment of succinate C-2 (without ammonia, 20.8%, and with ammonia, 21.6%), indicating that the isotopic dilution of metabolites due to utilization of endogenous glycogen was not affected. In contrast, the presence of ammonia markedly decreased the 13C enrichment of acetate C-2 (from 40 to 31%), reflecting enhanced reversal of the succinate synthesis pathway. The reversal of glycolysis was unaffected by the presence of ammonia as shown by 13C-NMR analysis. Study of cell extracts showed that the main pathways of ammonia assimilation in F. succinogenes were glutamate dehydrogenase and alanine dehydrogenase. Glutamine synthetase activity was not detected. Glutamate dehydrogenase was active with both NAD and NADP as cofactors and was not repressed under ammonia limitation in the culture. Glutamate-pyruvate and glutamate-oxaloacetate transaminase activities were evidenced by spectrophotometry and 1H NMR. When cells were incubated in vivo with [1-13C]glucose, only 13C-labeled aspartate, glutamate, alanine, and valine were detected. Their labelings were consistent with the proposed amino acid synthesis pathway and with the reversal of the succinate synthesis pathway.

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Short echo time in vivo STEAM 1H MR spectra (4.7 T, TE = 16 ms) of normal rat brain were fitted in the time domain using a VARPRO-like algorithm called AMARES which allows an inclusion of a large amount of prior knowledge. The prior knowledge was derived from phantom spectra of pure metabolite solutions measured under the same experimental conditions as the in vivo spectra. The prior knowledge for the in vivo spectra was constructed as follows: for each VARPRO-fitted phantom spectrum one peak (the most prominent one in the in vivo spectrum) was chosen and left unconstrained in the AMARES fitting while all the other peaks in the metabolite spectrum (i.e. their corresponding parameters--amplitudes, damping factors, frequencies and phases) were fixed to the parameter values of the unconstrained peak via amplitude and damping ratios and frequency and phase shifts. Including N-acetyl-aspartate, glutamate, total creatine, cholines, glucose and myo-inositol into the fits provided results which were in agreement with published data. An inclusion of glutamine into the set of fitted metabolites was also investigated.

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Creatine kinase reaction rates were measured by magnetisation transfer technique in the brain of healthy adult and aged rats and in the rats with mild or severe chronic cerebral ischemia. These measurements indicated that the rate constant of the creatine kinase reaction is significantly reduced in the case of chronic brain ischemia in aged rats. In contrast, occlusion of both carotid arteries in adult rats produced a slight increase in the reaction rate 4 weeks after occlusion. At the same time, corresponding conventional phosphorus magnetic resonance spectra showed negligible changes in signal intensities.

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We investigated the carbon metabolism of three strains of Fibrobacter succinogenes and one strain of Fibrobacter intestinalis. The four strains produced the same amounts of the metabolites succinate, acetate, and formate in approximately the same ratio (3.7/1/0.3). The four strains similarly stored glycogen during all growth phases, and the glycogen-to-protein ratio was close to 0.6 during the exponential growth phase. 13C nuclear magnetic resonance (NMR) analysis of [1-13C]glucose utilization by resting cells of the four strains revealed a reversal of glycolysis at the triose phosphate level and the same metabolic pathways. Glycogen futile cycling was demonstrated by 13C NMR by following the simultaneous metabolism of labeled [13C]glycogen and exogenous unlabeled glucose. The isotopic dilutions of the CH2 of succinate and the CH3 of acetate when the resting cells were metabolizing [1-13C]glucose and unlabeled glycogen were precisely quantified by using 13C-filtered spin-echo difference 1H NMR spectroscopy. The measured isotopic dilutions were not the same for succinate and acetate; in the case of succinate, the dilutions reflected only the contribution of glycogen futile cycling, while in the case of acetate, another mechanism was also involved. Results obtained in complementary experiments are consistent with reversal of the succinate synthesis pathway. Our results indicated that for all of the strains, from 12 to 16% of the glucose entering the metabolic pathway originated from prestored glycogen. Although genetically diverse, the four Fibrobacter strains studied had very similar carbon metabolism characteristics.

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1. The high-resolution 1H NMR (MRS) spectra of human brain tumor homogenates revealed a broad resonance at 5.3-5.4 ppm in glioblastoma multiforme (N = 16) and brain metastases (N = 3). The broad resonance was identified as ceramide, a sphingosine-fatty acid combination portion of ganglioside, indicating an elevated abundance of monounsaturated fatty acids. GLC analysis of gangliosides in the highly malignant glioblastoma multiforme revealed that the elevated monounsaturated fatty acid is oleic acid (C18:1). The resonance at 5.3-5.4 ppm region was not detectable in normal human brain (N = 2), in meningiomas (N = 2), or in low-grade astrocytomas (N = 12). In normal human brain the abundance of monounsaturated fatty acid is minimal. 2. This investigation was made possible because the method of producing homogenate resulted in (i) no loss of lipids during the process and (ii) a well-homogenised sample, with (iii) no loss in chemical integrity. 3. The properties of tumor gangliosides include antigenic specificity and immunosuppressive activity and the ceramide, a sphingosine-fatty acid combination, noticeably influences the ganglioside immunosuppressive activity. 4. The observation of 1H NMR ceramide resonance in high-malignant brain tumors emphasizes the dramatic role of aberant gangliosides and ceramide precursors on the grade of malignancy and invasiveness. 5. Further insight into the specific nature of the ceramide portion of gangliosides in grading the malignancy of brain tumors should be investigated further.

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Introduction of nuclear magnetic resonance has enabled direct measurements of muscle metabolism in vitro and in vivo. In the presented paper some theoretical and practical applications mainly 31P NMR spectroscopy in research and diagnostics of muscle skeletal diseases are given. In spite of some limitations, the most important advantage of NMR technique is its non-destructive character, the possibility of simultaneous observation of several phosphorylated metabolites in a muscle tissue and the simple method of sample preparation. 31P NMR is a useful tool for studying and detection of muscle diseases and at the same time for its safety for patients.

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1H-nuclear magnetic resonance spectroscopy was used to study malignant melanoma extract. The aim of this work was to study low molecular weight metabolites soluble in water, which can be helpful for a more detailed understanding of tumor metabolism with a view to using this knowledge for diagnosis. The authors found in a well distinguished spectrum the presence of numerous low molecular weight metabolites such as glutamate, glutamine, choline, inositol, creatine, phosphocreatine, phosphocholine, glucose, acetate, alanine, lactate. It is necessary to correlate these in-vitro findings with a study of the above mentioned metabolites in-vivo. Malignant melanoma is appropriate for this investigative and diagnostic technique because of its superficial localization. (Fig. 1, Ref. 18.)

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1. Fibre type composition and phosphate metabolites were studied in m. longissimus thoracis (MLT), m. rectus femoris (MRF) and m. triceps brachii (MTB) in trained (N = 6) and sedentary (N = 6) pigs. 2. Samples were analyzed histochemically and by means of in vitro 31P NMR spectroscopy. 3. Training (duration 11 weeks) consisted of treadmill running at a speed of 1.1 m/sec. The daily exercise time of trained animals gradually increased from 10 min during the very first days to 60 min at the end of the 4th week. 4. During the final 7 weeks exercise time remained unchanged. Sedentary animals were not subjected to training. 5. A higher proportion of type beta R fibres in MRF, MTB and MLT and a lower proportion of type alpha W fibres were found in the trained group of animals compared to the control group. 6. In MLT no significant differences in the proportion of type alpha W were observed between both groups. 7. No significant differences in average fibre diameter of muscle fibres were found between groups. 8. No differences in concentration of phosphate compounds were observed between trained and sedentary groups. 9. Muscles with a higher proportion of type IIb fibres in both groups of pigs contained higher amounts of phosphocreatine (PCr) and were also characterized by a higher ratio of PCr to inorganic phosphate (Pi).

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