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β-D-Fructofuranosyl-(2↔1)-2-acetamido-2-deoxy-α-D-glucopyranoside (N-acetylsucros-amine; SucNAc) is an enzymatic product from sucrose and 2-acetamido-2-deoxy-D-glucose (GlcNAc) and cannot be hydrolyzed by yeast β-fructofuranosidase. Although the distinct binding of SucNAc with the β-fructofuranosidase could not be identified by isothermal titration calorimetry or 1H NMR titration, unambiguous signals indicating their molecular interactions were observed by STD NMR experiments. An inhibitory activity of SucNAc in the hydrolysis of sucrose was also confirmed by 1H NMR spectroscopy. Because hydrolysis of sucrose was unaffected in the presence of GlcNAc, a disaccharide moiety including an acetamido group is considered to be crucial to show an inhibitory activity for the β-fructofuranosidase.

Mycelia of Aspergillus oryzae NBRC100959 contain 2 types of β-fructofuranosidases, transfructosylation-catalyzing enzyme (βFFaseI), and hydrolysis-catalyzing enzyme (βFFaseII). Using βFFaseI extracted from the mycelia of strain NBRC100959, two novel oligosaccharides consisting of GlcNAc and fructose, β- d -fructofuranosyl-(2→1)-β- d -fructofuranosyl-(2↔1)-2-acetamido-2-deoxy-α- d -glucopyranoside (N-acetyl-1-kestosamine, 1-KesNAc) and β- d -fructofuranosyl-(2→1)-β- d -fructofuranosyl-(2→1)-β- d -fructofuranosyl-(2↔1)-2-acetamido-2-deoxy-α- d -glucopyranoside (N-acetylnystosamine, NysNAc), were synthesized from β- d -fructofuranosyl-(2↔1)-2-acetamido-2-deoxy-α- d -glucopyranoside (N-acetylsucrosamine, SucNAc). We next planned to synthesize 1-KesNAc and NysNAc using A. oryzae mycelia. However, it was thought that the presence of βFFaseII is disadvantageous for the production of these oligosaccharides by βFFaseI, because βFFaseII hydrolyzed 1-KesNAc and NysNAc. We succeeded to produce A. oryzae mycelia containing βFFaseI as the major β-fructofuranosidase, by increasing sucrose concentration in the culture medium. Then, using a dried sample of these A. oryzae mycelia, reaction for the oligosaccharide production was performed. As the results, 190 mg of 1-KesNAc and 60 mg of NysNAc were obtained from 0.6 g of SucNAc. This whole-cell catalysis method facilitates the synthesis of 1-KesNAc and NysNAc because extraction and purification of βFFaseI from mycelia are unnecessary.

Two kinds of oligosaccharides, N-acetylraffinosamine (RafNAc) and N-acetylplanteosamine (PlaNAc), were synthesized from N-acetylsucrosamine and melibiose using the transgalactosylation activity of Aspergillus niger α-galactosidase. RafNAc and PlaNAc are novel trisaccharides in which d-glucopyranose residues in raffinose (Raf) and planteose are replaced with N-acetyl-d-glucosamine. These trisaccharides were more stable in acidic solution than Raf. RafNAc was hydrolyzed more rapidly than Raf by α-galactosidase of green coffee bean. In contrast, RafNAc was not hydrolyzed by Saccharomyces cerevisiae invertase, although Raf was hydrolyzed well by this enzyme. These results indicate that the physicochemical properties and steric structure of RafNAc differ considerably from those of Raf. Two kinds of novel trisaccharides were synthesized from N-acetylsucrosamine and melibiose, using transgalactosylation action of Aspergillus niger α-galactosidase.

The WaterLOGSY type experiment with 19F detection was applied to observe the interaction between a fluorinated compound and a macromolecule. The proposed experiment, which was developed based on the 19F{1H} saturation transfer difference experiment, was carried out using the conventional spectrometer equipped with a single high band amplifier and a H/F/C-double tuned probe. The selective 19F detection is advantageous in screening the fluorinated compounds, considering that 19F is a sensitive nucleus in NMR spectroscopy. The effective approach to discriminate binding of the fluorinated compounds to proteins with 19F detection was demonstrated using the complex of diflunisal and human serum albumin.

Seven new glycosides of the campesterol derivative (24R,25S)-ergost-5-ene-3beta,26-diol (1-7) were isolated from the rhizomes of Tacca chantrieri (Taccaceae). Their structures were determined by extensive spectroscopic analysis, including 2D NMR data, and a few chemical transformations.

Diphenylarsinic acid (DPAA) and phenylarsonic acid (PAA), which were degradation products of organoarsenic chemical warfare agents used as sternutatory gas, were detected in the well water at Kamisu, Ibaraki Prefecture, Japan. The standard material of DPAA was synthesized with aqueous arsenic acid and phenylhydrazine in order to determine organic arsenic compounds in well water. The DPAA showed a protonated ion at m/z 263 [M + H]+ and a loss of H2O ion at m/z 245 [M + H − H2O]+ from protonated ion by the electrospray ionization time-of-flight mass spectrometry. The quantitative analysis of DPAA and PAA was performed by high-performance liquid chromatography inductively coupled plasma mass spectrometry and the system worked well for limpid liquid samples such as well water. Copyright © 2005 John Wiley & Sons, Ltd.

Seven new bisdesmosidic triterpene saponins, with up to eight monosaccharides, which were given the trivial names contortisiliosides A–G (1–7), were isolated from Enterolobium contortisiliquum. The structures of the new saponins were determined on the basis of extensive spectroscopic and chromatographic analyses of both intact and acid-hydrolyzed compounds. The isolated saponins were evaluated for their cytotoxic activities against BAC1.2F5 mouse macrophages, EL-4 mouse lymphoma cells, and L-929 mouse fibroblasts. Whereas contortisiliosides A (1) and C (3) were moderately cytotoxic to both BAC1.2F5 macrophages and EL-4 cells, and contortisiliosides D–G (4–7) did not show any apparent cytotoxic activities against the three cell lines, contortisilioside B (2) exhibited selective cytotoxic activity against BAC1.2F5 mouse macrophages, with an IC50 value of 3.4 μM. The macrophage death caused by 2 was shown to be neither necrotic nor apoptosis-inducing based on the unique morphological change of the killed cells, whose cytosols were transformed into large vacuoles, and according to the TUNEL assay.

The two new polyoxygenated spirostanol bisdesmosides 1 and 2 and the new trisdesmoside 3, named hellebosaponin A (1), B (2), and C (3), respectively, were isolated from the MeOH extract of the rhizomes of Helleborus orientalis. The structures of the new compounds were elucidated as (1β,3β,23S,24S)-21-(acetyloxy)-24-[(β-D-fucopyranosyl)oxy]-3,23-dihydroxyspirosta-5,25(27)-dien-1-yl O-β-D-apiofuranosyl-(13)-O-(4-O-acetyl-α-L-rhamnopyranosyl)-(12)-O-[β-D-xylopyranosyl-(13)]-α-L-arabinopyranoside (1), (1β,3β,23S,24S)- 21-(acetyloxy)-24-{[O-β-D-glucopyranosyl-(14)-β-D-fucopyranosyl]oxy}-3,23-dihydroxyspirosta-5,25(27)-dien-1-yl O-β-D-apiofuranosyl-(13)-O-(4-O-acetyl-α-L-rhamnopyranosyl)-(12)-O-[β-D-xylopyranosyl-(13)]- α-L-arabinopyranoside (2), and (1β,3β,23S,24S)-24-[(β-D-fucopyranosyl)oxy]-21-{[O-β-D-glucopyranosyl-(12)-β-D-galactopyranosyl]oxy}-3,23-dihydroxyspirosta-5,25(27)-dien-1-yl O-β-D-apiofuranosyl-(13)-O-(4-O-acetyl-α-L-rhamnopyranosyl)-(12)-O-[β-D-xylopyranosyl-(13)]-α-L-arabinopyranoside (3), respectively, on the basis of detailed spectroscopic studies and chemical evidence.

Reaction of diethoxyphsphoryldifluoromethylzinc bromide (BrZnCF 2 PO 3 Et 2 ) with highly functionalized cyclohex-2-enyl-1-phosphates in the presence of CuBr in THF was examined. The reaction provides a facile method for introducing a difluoro-methylenephosphonate unit to the allylic position within a cyclic array in a stereo- and regioselective manner.

Phytochemical analysis of the underground parts of Polianthes tuberosa has resulted in the isolation of four new spirostanol saponins with five monosaccharides (1-4). Their structures were determined by spectroscopic analysis, including extensive 1D and 2D NMR data, and the results of hydrolytic cleavage. The cytotoxic activities of 1-4 against HL-60 human promyelocytic leukemia cells and HSC-2 human oral squamous cell carcinoma cells are reported.

The (1)H-(19) F heteronuclear NMR experiments were achieved using the conventional spectrometer equipped with a single high band amplifier and a (1)H/(19)F/(13) C double-tuned probe. Although double high band amplifiers are generally required to perform such experiments, a simple modification of pathway in the conventional spectrometer was capable of acquiring various (1)H-(19)F heteronuclear spectra. The efficiency of the present technique was demonstrated in an application for (19)F{(1)H} and (1)H{(19)F} saturation transfer difference experiments.

Further phytochemical analysis aimed at the triterpene saponin constituents of the roots of Pulsatilla chinensis has resulted in the isolation of four new bisdesmosidic triterpene saponins whose aglycons are based on the lupane skeleton (1-4), together with three known saponins (5-7). The structures of the new compounds were determined by spectroscopic analysis and acid-catalyzed hydrolysis.

Phytochemical analysis of the leaves of Cestrum nocturnum (Solanaceae) resulted in the isolation of two new flavonol glycosides (1, 2) and seven steroidal saponins (3-9), including four new ones (4, 6, 7, and 9). The structures of the new compounds were determined by spectroscopic analysis, including 2D NMR data, and the results of hydrolytic cleavage. Cytotoxic activities of the isolated compounds against human oral squamous cell carcinoma-(HSC-2) cells and normal human gingival fibroblasts are reported.

4-15N-2,3,5-Trimethylpyrazine (1) was synthesized by dechlorination of 4-15N-6-chloro-2, 3, 5-trimethylpyrazine (5) the key intermediate, derived from 15N-DL-alanine (3). 1-15N-2,3,5-Trimethylpyrazine (2) was prepared by decarboxylation of 1-15N-2, 3, 5-trimethyl-pyrazine-6-carboxylic acid (10) obtained by the Pummerer type rearrangement of 1-15N-tetramethylpyrazine 1-oxide (1) followed by oxidation. 1- And 4-oxides and 1,4-dioxides of the above 15Ntrimethylpyrazines were also obtained by treatment with sodium perborate in acetic acid. 15N NMR spectra of the compounds thus prepared were measured and the assignment of two 15N signals were certified.

The taste of black tea differs according to the different areas in which the tea is grown, even for the same species of tea. A combination of (1)H NMR spectroscopy and partial least-squares discriminate analysis (PLS-DA) was used to assess the quality differences of tea leaves from four cultivation areas with different elevations, RAN > 1800 m, UDA = 1200 m, MEDA = 600 m, and YATA < 300 m, in Sri Lanka. As a result of a statistical analysis, PLS-DA showed a separation between high- and low-quality black teas derived from the four different tea cultivation areas. RAN from the highest elevation showed characteristic trends in the levels of theaflavin and theaflavin 3,3'-digallate that were found only in RAN, and the levels of theanine and caffeine were higher, and the levels of thearubigins, especially thearubigin 3,3'-digallate, were lower in RAN than in UDA, MEDA, and YATA. The structures of these components were determined by 1D and 2D NMR analyses. These results demonstrate that this method can be used to evaluate black tea quality according to the chemical composition or metabolites, which are characteristic of the tea leaves cultivated in four regions with different elevations in Sri Lanka.

Tautomeric structures of title compound(I), 5-chloro-2-hydroxy-3,6-diisobutylpyrazine(II) and 2-hydroxy-3,6-diisobutyl-5-methoxypyrazine (III) are discussed. The oxidation of I, II, and III with permaleic acid gave the corresponding monoxides 2-hydroxy-3,6-diisobutylpyrazine, 5-chloro-2-hydroxy-3,6-diisobutylpyrazine 1-oxide , and 2-hydroxy-3,6-diisobutyl-5-methoxypyrazine 1-oxide, respectively

A novel polyoxygenated spirostanol glycoside (1) was isolated from the rhizomes of Helleborus orientalis and its structure was determined by spectroscopic analysis, including extensive 1D and 2D NMR data, and the results of acid hydrolysis. The new glycoside (1) is unique in structure having oxygen atoms at C-1, C-3, C-21, C-23, and C-24 of the spirostanol skeleton and bearing a new acetylated tetraglycoside, a diglycoside, and a monosaccharide at C-1, C-21, and C-24 of the aglycon, respectively.

N-methyl-pyridinium formate, a novel component of coffee extracts, inhibited the multiplication of both DNA and RNA viruses. In the presence of the compound, the progeny viral yields of both herpes simplex virus type 1 (HSV-1) and poliovirus in HEp-2 cells and those of influenza virus type A in MDCK cells decreased with increasing concentrations of the compound, although the degree of viral sensitivity to this compound differed. In addition, none of these viruses were directly inactivated by the compound at the concentrations tested. Characterization of the mode of action of this compound against HSV-1 multiplication revealed that it inhibits the viral growth primarily at the initial step of virus multiplication, i.e., within 2 h after the onset of multiplication, although the virus multiplication was affected by the compound throughout the multiplication cycle. In addition, this compound showed a significant cytotoxic effect, although the observed antiviral effect was unlikely to be attributed to the cytotoxic effect.

Urinary excretion of hydroxylysine diglycoside (GGH) or monoglycoside (GH) is known to be a reliable indicator of metabolic status of bone collagen. Although some preparation methods of the standard GGH or GH from different species have been reported, this study demonstrated that the preparation of the GGH or GH from the human urine was more suitable for the analyses of these glycosides. GGH and GH were prepared in two ways : one from the normal human urine and the other from the alkaline hydrolyzate of marine sponge. The adequacy for their use as the standard substances was evaluated by proton NMR and HPLC. The glycosides obtained from the human urine were confirmed to be normal forms. As for the glycosides obtained from marine sponge, it was demonstrated that the glycosides were diastereoisomers. The urinary levels of GGH and GH of the inhabitants in Cd-polluted area were compared with the values obtained from non-polluted subjects. The measurements of GGH and GH were performed by using the glycosides prepared from the normal human urine as the standard substanses. As the results, the urinary GGH or GH levels of the subjects in the Cd-polluted area had wider distribution ranges than those of controls, although there was no significant difference between Cd-polluted subjects and controls.

A new tracer technique, in which a 13C-labeled compound as a biological tracer and 13C nuclear magnetic resonance (NMR) as an analytical tool are used, is proposed. In order to verify the applicability of the method to clinical chemistry. [1-13C]benzoic acid was administered and [1'-13C]hippuric acid excreted in the urine was quantitated by NMR, by using [1-13C]hippuric acid as an internal standard.

The structure of a new monomeric peptidoglycan-related compound with hypotensive and diuretic activities, cymbidine A (1) isolated from the orchid Cymbidium goeringii, was elucidated mainly by spectroscopic analysis. The structure of 1 was shown to involve four amino acids (D-alanin, meso-diaminopimelic acid, D-gultamic acid, and L-valine) and two amino sugars (N-acetylglucosamine and 1,6-anhydro-N-acetylmuramic acid). The sequence of the amino acids and amino sugars was determined by the analysis of 2D NMR data. The absolute stereochemistries of the three amino acids (D-Ala, D-Glu and L-Val) were determined by the modified Marfey's method, and the (6S,10R) configurations of meso-diaminopimelic acid in 1 were indicated on the basis of the CD analysis. The absolute stereochemistry of 1,6-anhydro-N-acetylmuramic acid was also determined by CD data.

Indium-mediated coupling reactions of δ-oxygenated allyl halide derivatives 1 with aldehydes in aqueous media proceeded at the y-position to give diol derivatives 2. Compound 1 is equivalent to an allylic carbanion 4 with a neighboring oxygen functional group generated in aqueous media.

Pi in the medium relieved the toxicity of arsenate against cellular growth of Chlamydomonas reinhardtii. To investigate the relationship between intracellular P contents and arsenate resistance, we determined the intracellular P contents of arsenate-sensitive and arsenate-resistant mutants, which had been generated by random insertional mutagenesis. All 13 arsenate-resistant mutants showed higher P contents than the parent strain, while arsenate-sensitive mutants with high P contents were not found. In one of the arsenate-resistant mutants, AR3, the intracellular P content was about twice that in the wild type during growth in the absence of arsenate. Arsenate incorporation in AR3 was suppressed within 10 min after the addition of 1 mM arsenate, while Pi incorporation continued even after arsenate uptake ceased. Whereas the P content of the wild type decreased to half in the presence of 0.5 mM arsenate, almost the same degree (about 50%) of decrease was observed in AR3 cells grown in the presence of as much as 3 mM arsenate. AR3, in which PTB1, a homolog of a Pi transporter gene, had been disrupted, exhibited a higher activity of a high-affinity Pi transporter, suggesting that it may be due to a compensatory transport activity. These data suggest that the intracellular level of P is one of the important factors of arsenate resistance.

A saponin-enriched fraction prepared from the MeOH extract of the roots of Clematis chinensis showed cytotoxic activity against HL-60 promyelocytic leukemia cells, from which five new triterpene saponins (1−5) based on oleanolic acid, along with three known saponins (6−8), were isolated. The structures of the new saponins were determined on the basis of spectroscopic analysis, including extensive 1D and 2D NMR data and hydrolysis followed by chromatographic and spectroscopic analysis. Among the isolated saponins, monodesmosidic saponins exhibited cytotoxic activities against cultured tumor cells.

Seven new bisdesmosidic triterpene saponins, with up to eight monosaccharides, which were given the trivial names contortisiliosides A–G (1–7), were isolated from Enterolobium contortisiliquum. The structures of the new saponins were determined on the basis of extensive spectroscopic and chromatographic analyses of both intact and acid-hydrolyzed compounds. The isolated saponins were evaluated for their cytotoxic activities against BAC1.2F5 mouse macrophages, EL-4 mouse lymphoma cells, and L-929 mouse fibroblasts. Whereas contortisiliosides A (1) and C (3) were moderately cytotoxic to both BAC1.2F5 macrophages and EL-4 cells, and contortisiliosides D–G (4–7) did not show any apparent cytotoxic activities against the three cell lines, contortisilioside B (2) exhibited selective cytotoxic activity against BAC1.2F5 mouse macrophages, with an IC50 value of 3.4 μM. The macrophage death caused by 2 was shown to be neither necrotic nor apoptosis-inducing based on the unique morphological change of the killed cells, whose cytosols were transformed into large vacuoles, and according to the TUNEL assay.

Treatment of 1-diethylphosphonyl- or 1-phenylsulfonyl-2-(iodomethyl)cyclopropane-1-carboxylate with Et(3)B leads to an unsymmetrical allylated active methine radical species that gives functionalized cyclopentane derivatives with high stereoselectivity through iodine atom transfer [3 + 2] cycloaddition reaction with alkenes.

Thermal unfolding of ribonculease (RNase) T1 was studied by 1H nuclear Overhauser enhancement spectroscopy (NOESY) and 1H- 15N heteronuclear single-quantum coherence (HSQC) NMR spectroscopy at various temperatures. Native RNase T1 is a single-chain molecule of 104 amino acid residues, and has a single alpha-helix and two beta-sheets, A and B, which consist of two and five strands, respectively. Singular value decomposition analysis based on temperature-dependent HSQC spectra revealed that the thermal unfolding of RNase T1 can be described by a two-state transition model. The midpoint temperature and the change in enthalpy were determined as 54.0 degrees C and 696 kJ/mol, respectively, which are consistent with results obtained by other methods. To analyze the transition profile in more detail, we investigated local structural changes using temperature-dependent NOE intensities. The results indicate that the helical region starts to unfold at lower temperature than some beta-strands (B3, B4, and B5 in beta-sheet B). These beta-strands correspond to the hydrophobic cluster region, which had been expected to be a folding core. This was confirmed by structure calculations using the residual NOEs observed at 56 degrees C. Thus, the two-state transition of RNase T1 appears to involve locally different conformational changes.

Reaction of diethoxyphsphoryldifluoromethylzinc bromide (BrZnCF 2 PO 3 Et 2 ) with highly functionalized cyclohex-2-enyl-1-phosphates in the presence of CuBr in THF was examined. The reaction provides a facile method for introducing a difluoro-methylenephosphonate unit to the allylic position within a cyclic array in a stereo- and regioselective manner.

The diastereoselective synthesis of β-substituted α-hydroxyphosphinates was achieved by hydrophosphinylation of α-oxy aldehydes and α-amino aldehydes with ethyl allylphosphinate catalyzed by lithium phenoxide.

Two novel triterpene bisdesmosides, designated as enterolosaponin A (1) and B (2), were isolated from Enterolobium contortisiliquum. The chemical structures of 1 and 2 were determined by analysis of their extensive spectroscopic data, as well as hydrolysis followed by chromatographic study. Enterolosaponins have a 2-amino-2-deoxy-D-glucosyl unit (D-glucosamine) as one of the monosaccharides constituting their oligosaccharide moieties, which have been rarely found in natural product research. Enterolosaponin A (1) exhibited a highly selective cytotoxicity against BAC1.2F5 mouse macrophages, and it should be notable that the macrophage death caused by 1 was shown to be neither necrotic nor due to induction of apoptosis from morphology of the died cells, whose cytosol occurred in vacuolation.

A novel polyoxygenated spirostanol glycoside (1) was isolated from the rhizomes of Helleborus orientalis and its structure was determined by spectroscopic analysis, including extensive 1D and 2D NMR data, and the results of acid hydrolysis. The new glycoside (1) is unique in structure having oxygen atoms at C-1, C-3, C-21, C-23, and C-24 of the spirostanol skeleton and bearing a new acetylated tetraglycoside, a diglycoside, and a monosaccharide at C-1, C-21, and C-24 of the aglycon, respectively.

It was confirmed that the flexible arm region of HU alpha forms an antiparallel beta-sheet and that all of the residues of phenylalanines, together with some of leucines and/or valines, form a hydrophobic core within the dimer of HU alpha. HU alpha protein alone is thermally labile and melts at 38 degrees C, but it becomes remarkably stabilized and melts at 59 degrees C in the presence of DNA. Several resonances from both HU alpha and DNA perturbed by their complex formation, notably those of His C-2 and C-4 protons, downfield shifted C alpha protons in the antiparallel beta-sheet, as well as Arg C delta and Lys C epsilon protons. The results indicated that a beta-sheet region of HU alpha binds to DNA, and also showed that rapid equilibrium occurs on the NMR time scale between bound and unbound states of HU alpha. A few intermolecular nuclear Overhauser effects (NOEs) were also observed between the protein and H1' protons of DNA in the complex, suggesting that HU alpha binds primarily to the minor groove of DNA.

^1H NMR spectroscopy has been used to demonstrate specific binding of rat 80S ribosomes to the major conformer of an antitumor bicyclic hexapeptidic glucoside, RA-XII, isolated from Rubia cordifolia, in a fast exchange process.

(+)-Septorine, a metabolite of Septoria nodorum, was synthesized via alanyl-isoleucyl anhydride (2) from L-isoleucine at the first time. Compound (2) was led to a pyrazine-carboxaldehyde (13), which was treated with p-methoxymethoxy-phenylmagnesium bromide to afford an alcohol (14) in a quantative yield. The alcohol (14) was oxidized to a ketone (16), which was subjected to the Pummerer reaction and the following hydrolysis to give (+)-septorine

Tautomeric structures of title compound(I), 5-chloro-2-hydroxy-3,6-diisobutylpyrazine(II) and 2-hydroxy-3,6-diisobutyl-5-methoxypyrazine (III) are discussed. The oxidation of I, II, and III with permaleic acid gave the corresponding monoxides 2-hydroxy-3,6-diisobutylpyrazine, 5-chloro-2-hydroxy-3,6-diisobutylpyrazine 1-oxide , and 2-hydroxy-3,6-diisobutyl-5-methoxypyrazine 1-oxide, respectively

The changes in energy metabolites and corresponding force curves were studied during muscle fatigue with respect to excitation frequency (20 and 50Hz) for contracting muscle in situ and by means of 31P nuclear magnetic resonance (NMR) technique in vitro. The rat hind limb muscle group was stimulated via the tibial nerve by 0.5ms duration square pulses at different frequencies for a period of three minutes. The force signals recorded during repeated experiments were fed through an A/D converter and averaged by the use of a desk-top computer. 31P-NMR spectra were recorded from gastrochemius and soleus muscle of resting and after stimulation. Results indicated that 1) Iarge and significant decreases in high energy phosphate compounds (e.g., creatine phosphate and adenosine triphosphate (ATP)) were observed during prolonged electric stimulation at 50Hz in both the gastrocnemius and soleus muscles, 2) during 20Hz stimulation, muscle fiber type dependent changes in energy metabolites were observed: i. e., the gastrocnemius manifested a significant decline in creatine phosphate and ATP contents (p