Investigation of skeletal muscle denervation and reinnervation using magnetic resonance spectroscopy.

OBJECTIVE: To determine changes in skeletal muscle metabolism after nerve transection and repair and to correlate metabolic changes with functional recovery. STUDY DESIGN AND SETTING: Male Wistar rats were divided into 6 experimental groups plus a control group. The posterior tibial nerve was transected and reapproximated. At varying times after surgery (1, 2, 4, 6, or 8 weeks) animals were sacrificed, the gastrocnemius muscle was harvested, and proton nuclear magnetic resonance (NMR) spectroscopy was performed. Functional recovery was measured using the sciatic function index. RESULTS: Animals undergoing nerve repair all showed functional recovery whereas the nonrepaired nerve group did not. Concentration of glucose and lactate increased after denervation and then returned toward normal. Choline concentration decreased and then returned toward normal. In animals not undergoing nerve repair, the metabolic abnormalities persisted and showed no sign of recovery over the 8-week observation period. CONCLUSIONS: 1H NMR spectroscopy is a potentially useful tool to study changes in skeletal muscle metabolism after motor nerve injury. SIGNIFICANCE: NMR spectroscopy is rapidly developing into a clinically useful tool. High-field magnets have improved resolution and data acquisition. Basic experiments, such as those described here, will help guide the use of NMR spectroscopy in clinical medicine and will also lead to a better understanding of basic mechanisms of nerve injury and repair.

Physical and structural characterization of yersiniophore, a siderophore produced by clinical isolates of Yersinia enterocolitica.

Clinical isolates of Yersinia enterocolitca, which belong to mouse-lethal serotypes, produce the siderophore yersiniophore. Siderophore production was shown to be iron regulated and to reach maximum production in late log phase. Yersiniophore is a fluorescent siderophore with maximum excitation at 270 nm and a major emission peak at 428 nm. Absorption maxima were seen at 210 and 250 nm with a low broad peak from 280 to 320 nm. Purification of unchelated yersiniophore for structural analysis was made difficult by low yields (1-2 mg mg-1), and susceptibility to acid hydrolysis, oxidation and possibly polymerization. Yersinophore was therefore purified as an Al3+ chelate, which was found to be stable in solution for several weeks. To purify Al(3+)-yersinophore, unchelated yersiniophore was first extracted from culture supernatants with dichloromethane, concentrated by rotary evaporation and adsorbed to a DEAE-sephacel column. Al(3+)-yersiniophore was eluted with 0.01 M AlCl3 and further purified by HPLC. The structure was established by a combination of elemental analysis, high resolution mass spectrometry and two-dimensional NMR experiments. Yersiniophore is a phenolate-thiazole siderophore with the formula C21H24N3O4S3Al and a molecular weight of 505.07404 when chelated to Al3+. The structure of yersiniophore was determined to be closely related to the structures of pyochelin, produced by Pseudomonas aeruginosa, and anguibactin, produced by Vibrio anguillarum.

Phosphorus-31 and carbon-13 nuclear magnetic resonance studies of glucose and xylose metabolism in cell suspensions and agarose-immobilized cultures of Pichia stipitis and Saccharomyces cerevisiae.

The metabolism of glucose and xylose as a function of oxygenation in Pichia stipitis and Saccharomyces cerevisiae cell suspensions was studied by 31P and 13C nuclear magnetic resonance spectroscopy. The rate of both glucose and xylose metabolism was slightly higher and the production of ethanol was slightly lower in aerobic than in anoxic cell suspensions of P. stipitis. As well, the cytoplasmic pH of oxygenated cells was more alkaline than that of nonoxygenated cells. In contrast, in S. cerevisiae, the intracellular pH and the rate of glucose metabolism and ethanol production were the same under aerobic and anoxic conditions. Agarose-immobilized Pichia stipitis was able to metabolize xylose or glucose for 24 to 60 h at rates and with theoretical yields of ethanol similar to those obtained with anoxic cell suspensions. Cell growth within the beads, however, was severely compromised. The intracellular pH [pH(int)] of the entrapped cells fell to more acidic pH values in the course of the perfusions relative to corresponding cell suspensions. Of importance was the observation that no enhancement in the rate of carbohydrate metabolism occurred in response to changes in the pH(int) value. In contrast to P. stipitis, agarose-immobilized Saccharomyces cerevisiae showed a dramatic twofold increase in its ability to metabolize glucose in the immobilized state relative to cell suspensions. This strain was also able to grow within the beads, although the doubling time for the entrapped cells was longer, by a factor of 2, than the value obtained for log-phase batch cultures. Initially, the pH(int) of the immobilized cells was more alkaline than was observed with the corresponding S. cerevisiae cell suspensions; however, over time, the intracellular pH became increasingly acidic. As with immobilized P. stipitis, however, the pH(int) did not play a key role in controlling the rate of glucose metabolism.

A N and N Nuclear Magnetic Resonance Study of Nitrogen Metabolism in Shoot-Forming Cultures of White Spruce (Picea glauca) Buds.

Nitrogen-14 and nitrogen-15 nuclear magnetic resonance (NMR) spectra were recorded for freshly dissected buds of Picea glauca and for buds grown for 3, 6 and 9 weeks on shoot-forming medium. Resonances for Glu (and other alphaNH(2) groups), Pro, Ala, and the side chain groups in Gln, Arg, Orn, and gamma-aminobutyric acid could be detected in in vivo(15)N NMR spectra. Peaks for alpha-amino groups, Pro, NO(3) (-) and NH(4) (+) could also be identified in (14)N NMR spectra. Perfusion experiments performed for up to 20 hours in the NMR spectrometer showed that (15)N-labeled NH(4) (+) and NO(3) (-) are first incorporated into the amide group of Gln and then in the alphaNH(2) pool. Subsequently, it also emerges in Ala and Arg. These data suggest that the glutamine synthetase/ glutamate synthase pathway functions under these conditions. The assimilation of NH(4) (+) is much faster than that of NO(3) (-). Consequently after 10 days of growth more than 70% of the newly synthesized internal free amino acid pool derives its nitrogen from NH(4) (+) rather than NO(3) (-). If NH(4) (+) is omitted from the medium, no NO(3) (-) is taken up during 9 weeks and the buds support limited growth by utilizing their endogenous amino acid pools. It is concluded that NH(4) (+) and NO(3) (-) are both required for the induction of nitrate- and nitrite reductase.