MENS-associated increase of muscular protein content via modulation of caveolin-3 and TRIM72.

Microcurrent electrical neuromuscular stimulation (MENS) is known as an extracellular stimulus for the regeneration of injured skeletal muscle in sports medicine. However, the effects of MENS-associated increase in muscle protein content are not fully clarified. The purpose of this study was to investigate the effects of MENS on the muscular protein content, intracellular signals, and the expression level of caveolin-3 (Cav-3), tripartite motif-containing 72 (TRIM72) and MM isoenzyme of creatine kinase (CK-MM) in skeletal muscle using cell culture system. C2C12 myotubes on the 7th day of differentiation phase were treated with MENS (intensity: 10-20 microA, frequency: 0.3 Hz, pulse width: 250 ms, stimulation time: 15-120 min). MENS-associated increase in the protein content of myotubes was observed, compared to the untreated control level. MENS upregulated the expression of Cav-3, TRIM72, and CK-MM in myotubes. A transient increase in phosphorylation level of Akt was also observed. However, MENS had no effect on the phosphorylation level of p42/44 extracellular signal-regulated kinase-1/2 and 5'AMP-activated protein kinase. MENS may increase muscle protein content accompanied with a transient activation of Akt and the upregulation of Cav-3 and TRIM72.

Lactate increases myotube diameter via activation of MEK/ERK pathway in C2C12 cells.

AIM: Lactate is produced in and released from skeletal muscle cells. Lactate receptor, G-protein-coupled receptor 81 (GPR81), is expressed in skeletal muscle cells. However, a physiological role of extracellular lactate on skeletal muscle is not fully clarified. The purpose of this study was to investigate extracellular lactate-associated morphological changes and intracellular signals in C2C12 skeletal muscle cells. METHODS: Mouse myoblast C2C12 cells were differentiated for 5 days to form myotubes. Sodium lactate (lactate) or GPR81 agonist, 3,5-dihydroxybenzoic acid (3,5-DHBA), was administered to the differentiation medium. RESULTS: Lactate administration increased the diameter of C2C12 myotubes in a dose-dependent manner. Administration of 3,5-DHBA also increased myotube diameter. Not only lactate but also 3,5-DHBA upregulated the phosphorylation level of mitogen-activated protein kinase kinase 1/2 (MEK1/2), p42/44 extracellular signal-regulated kinase-1/2 (ERK1/2) and p90 ribosomal S6 kinase (p90RSK). MEK inhibitor U0126 depressed the phosphorylation of ERK-p90RSK and increase in myotube diameter induced by lactate. On the other hand, both lactate and 3,5-DHBA failed to induce significant responses in the phosphorylation level of Akt, mammalian target of rapamycin, p70 S6 kinase and protein degradation-related signals. CONCLUSION: These observations suggest that lactate-associated increase in the diameter of C2C12 myotubes is induced via activation of GRP81-mediated MEK/ERK pathway. Extracellular lactate might have a positive effect on skeletal muscle size.

Heat shock transcription factor 1-associated expression of slow myosin heavy chain in mouse soleus muscle in response to unloading with or without reloading.

AIM: The effects of heat shock transcription factor 1 (HSF1) deficiency on the fibre type composition and the expression level of nuclear factor of activated T cells (NFAT) family members (NFATc1, NFATc2, NFATc3 and NFATc4), phosphorylated glycogen synthase kinase 3α (p-GSK3α) and p-GSK3ß, microRNA-208b (miR-208b), miR-499 and slow myosin heavy chain (MyHC) mRNAs (Myh7 and Myh7b) of antigravitational soleus muscle in response to unloading with or without reloading were investigated. METHODS: HSF1-null and wild-type mice were subjected to continuous 2-week hindlimb suspension followed by 2- or 4-week ambulation recovery. RESULTS: In wild-type mice, the relative population of slow type I fibres, the expression level of NFATc2, p-GSK3 (α and ß), miR-208b, miR-499 and slow MyHC mRNAs (Myh7 and Myh7b) were all decreased with hindlimb suspension, but recovered after it. Significant interactions between train and time (the relative population of slow type I fibres; P = 0.01, the expression level of NFATc2; P = 0.001, p-GSKß; P = 0.009, miR-208b; P = 0.002, miR-499; P = 0.04) suggested that these responses were suppressed in HSF1-null mice. CONCLUSION: HSF1 may be a molecule in the regulation of the expression of slow MyHC as well as miR-208b, miR-499, NFATc2 and p-GSK3 (α and ß) in mouse soleus muscle.

Deficiency of heat shock transcription factor 1 suppresses heat stress-associated increase in slow soleus muscle mass of mice.

AIM: Effects of heat shock transcription factor 1 (HSF1) deficiency on heat stress-associated increase in slow soleus muscle mass of mice were investigated. METHODS: Both HSF1-null and wild-type mice were randomly assigned to control and heat-stressed groups. Mice in heat-stressed group were exposed to heat stress (41 °C for 60 min) in an incubator without anaesthesia. RESULTS: Significant increase in wet and dry weights, and protein content of soleus muscle in wild-type mice was observed seven days after the application of the heat stress. However, heat stress had no impact on soleus muscle mass in HSF1-null mice. Neither type of mice exhibited much effect of heat stress on HSF mRNA expression (HSF1, HSF2 and HSF4). On the other hand, heat stress upregulated heat shock proteins (HSPs) at the mRNA (HSP72) and protein (HSP72 and HSP110) levels in wild-type mice, but not in HSF1-null mice. The population of Pax7-positive nuclei relative to total myonuclei of soleus muscle in wild-type mice was significantly increased by heat stress, but not in HSF1-null mice. Furthermore, the absence of HSF1 gene suppressed heat stress-associated phosphorylation of Akt and p70 S6 kinase (p-p70S6K) in soleus muscle. CONCLUSION: Heat stress-associated increase in skeletal muscle mass may be induced by HSF1 and/or HSF1-mediated stress response that activates muscle satellite cells and Akt/p70S6K signalling pathway.

Caffeine activates preferentially α1-isoform of 5'AMP-activated protein kinase in rat skeletal muscle.

AIM: Caffeine activates 5'AMP-activated protein kinase (AMPK), a signalling intermediary implicated in the regulation of glucose, lipid and energy metabolism in skeletal muscle. Skeletal muscle expresses two catalytic α subunits of AMPK, α1 and α2, but the isoform specificity of caffeine-induced AMPK activation is unclear. The aim of this study was to determine which α isoform is preferentially activated by caffeine in vitro and in vivo using rat skeletal muscle. METHODS: Rat epitrochlearis muscle was isolated and incubated in vitro in the absence or presence of caffeine. In another experiment, the muscle was dissected after intravenous injection of caffeine. Isoform-specific AMPK activity, the phosphorylation status of AMPKα Thr(172) and acetyl-CoA carboxylase (ACC) Ser(79) , the concentrations of ATP, phosphocreatine (PCr) and glycogen, and 3-O-methyl-d-glucose (3MG) transport activity were estimated. RESULTS: Incubation of isolated epitrochlearis muscle with 1 mm of caffeine for 15 min increased AMPKα1 activity, but not AMPKα2 activity; concentrations of ATP, PCr and glycogen were not affected. Incubation with 3 mm of caffeine activated AMPKα2 and reduced PCr and glycogen concentrations. Incubation with 1 mm of caffeine increased the phosphorylation of AMPK and ACC and enhanced 3MG transport. Intravenous injection of caffeine (5 mg kg(-1) ) predominantly activated AMPKα1 and increased 3MG transport without affecting energy status. CONCLUSION: Our results suggest that of the two α isoforms of AMPK, AMPKα1 is predominantly activated by caffeine via an energy-independent mechanism and that the activation of AMPKα1 increases glucose transport and ACC phosphorylation in skeletal muscle.

Relationship between oral mucositis and high-dose methotrexate therapy in pediatric acute lymphoblastic leukemia.

OBJECTIVE: Oral mucositis is a major toxicity in the high-dose methotrexate (HD-MTX) treatment for children with acute lymphoblastic leukemia (ALL). The first aim of this study was to evaluate the relationship between the MTX serum concentration and occurrence of oral mucositis in pediatric ALL patients. The second aim was to clarify the relationship between MTX exposure and epidermal keratinocyte cell injury using an in vitro study. METHODS: 49 patients were treated according to the Japan Association of Childhood Leukemia Study (JACLS) ALL-HR02 protocol. This protocol involves HD-MTX treatment (3 g/m2 for 24-h i.v. infusion). The MTX serum concentrations were measured by a fluorescence polarization immunoassay. The relationship between oral mucositis and MTX serum concentrations 48 and 72 h after administration was determined. The cell toxicity of MTX for human epidermal keratinocytes was analyzed by using a cell viability assay (WST-1 assay). In addition, pharmacokinetic evaluation for clearance, AUC extrapolated from 48 h to infinity (AUC48h-inf) and elimination half-life (t1/2b) were done using the 1-compartmental models. RESULTS: Oral mucositis occurred in 24 patients (49.0%), in whom 20 patients (83.3% in oral mucositis group) showed WHO severity Grade 1 or 2. Only 4 patients (16.7% in oral mucositis group) showed Grade 3 severity. 22 patients (44.9%) had oral mucositis in the group with a concentration under 10-6 M 48 h after MTX administration. There was no significant deference among the cell viabilities in the concentrations of 10-6 M, 10-5 M and 10-4 M 48 h after the MTX exposure. However, the cell viability obtained 24 h after the MTX exposure was significantly different from the respective cell viability 48, 72 and 96 h after the MTX exposure. In the group with oral mucositis, the clearance decreased significantly (p = 0.042), and the t1/2b (p = 0.025) and AUC48h- yen (p = 0.025) increased significantly compared with the non-symptom group. CONCLUSIONS: It seems that there is no significant relationship between the serum MTX concentration and oral mucositis. This in vitro study has demonstrated that the cell injury was related to the duration of MTX exposure rather than a high MTX concentration.

Pioglitazone opposes neurogenic vascular dysfunction associated with chronic hyperinsulinaemia.

BACKGROUND AND PURPOSE: We previously demonstrated that chronic hyperinsulinaemia induced by drinking high levels of fructose augments adrenergic nerve-mediated vasoconstriction and suppresses vasodilatation mediated by calcitonin gene-related peptide (CGRP)-containing (CGRPergic) vasodilator nerves. In this study, the effects of pioglitazone on vascular responses induced by stimulation of adrenergic nerves, CGRPergic nerves and vasoactive agents were investigated in pithed rats given 15% fructose solution to drink (FDR). EXPERIMENTAL APPROACH: To assess the effect of pioglitazone on the altered vascular responsiveness in the hyperinsulinaemic state in vivo, changes in vascular responses to spinal cord stimulation (SCS) and intravenous bolus injections of noradrenaline, angiotensin II and CGRP were evaluated in pithed control rats and FDR either untreated or treated with pioglitazone. KEY RESULTS: In the pithed FDR, vasoconstrictor responses to SCS and to injections of noradrenaline and angiotensin II were significantly greater than those of pithed control rats. In pithed FDR with artificially increased blood pressure and blockade of the autonomic ganglia, the vasodilator responses to SCS and CGRP injection were significantly smaller than those of pithed control rats. Oral administration of pioglitazone to FDR for two weeks markedly decreased plasma levels of insulin, triglycerides and blood glucose. In FDR pioglitazone diminished the augmented vasoconstrictor responses to SCS, noradrenaline and angiotensin II, and ameliorated the decrease in vasodilator responses to SCS. CONCLUSIONS AND IMPLICATIONS: The present results suggest that pioglitazone improves not only insulin resistance, but also the dysfunctions in vascular control regulated by adrenergic and CGRPergic nerves in the hyperinsulinaemic state.

Delta 9-tetrahydrocannabinol-induced catalepsy-like immobilization is mediated by decreased 5-HT neurotransmission in the nucleus accumbens due to the action of glutamate-containing neurons.

Delta(9)-tetrahydrocannabinol (THC) has been reported to induce catalepsy-like immobilization, but the mechanism underlying this effect remains unclear. In the present study, in order to fully understand the neural circuits involved, we determined the brain sites involved in the immobilization effect in rats. THC dose-dependently induced catalepsy-like immobilization. THC-induced catalepsy-like immobilization is mechanistically different from that induced by haloperidol (HPD), because unlike HPD-induced catalepsy, animals with THC-induced catalepsy became normal again following sound and air-puff stimuli. THC-induced catalepsy was reversed by SR141716, a selective cannabinoid CB(1) receptor antagonist. Moreover, THC-induced catalepsy was abolished by lesions in the nucleus accumbens (NAc) and central amygdala (ACE) regions. On the other hand, HPD-induced catalepsy was suppressed by lesions in the caudate putamen (CP), substantia nigra (SN), globus pallidus (GP), ACE and lateral hypothalamus (LH) regions. Bilateral microinjection of THC into the NAc region induced catalepsy-like immobilization. This THC-induced catalepsy was inhibited by serotonergic drugs such as 5-hydroxy-L-tryptophan (5-HTP), a 5-HT precursor, and 5-methoxy-N,N-dimethyltryptamine (5-MeODMT), a 5-HT receptor agonist, as well as by anti-glutamatergic drugs such as MK-801 and amantadine, an N-methyl-d-aspartate (NMDA) receptor antagonist. THC significantly decreased 5-HT and glutamate release in the NAc, as shown by in vivo microdialysis. SR141716 reversed and MK-801 inhibited this decrease in 5-HT and glutamate release. These findings suggest that the THC-induced catalepsy is mechanistically different from HPD-induced catalepsy and that the catalepsy-like immobilization induced by THC is mediated by decreased 5-HT neurotransmission in the nucleus accumbens due to the action of glutamate-containing neurons.

p51/p63 Inhibits ultraviolet B-induced apoptosis via Akt activation.

The epidermis must be protected against excess apoptotic cell death in response to ultraviolet-B (UV-B) irradiation. p53 is known to be critical for this protection. Although the p53 family member DeltaNp51B/DeltaNp63alpha (an N terminal-deleted form of p51/p63) is abundantly expressed in keratinocytes, its contribution to UV-B-dependent apoptosis is largely unknown. We found that, after a transient increase, DeltaNp51B is downregulated in UV-B-irradiated keratinocytes undergoing apoptosis, whereas p53 is upregulated with delayed kinetics. Furthermore, the reduction of DeltaNp51B by small interfering RNAs augmented UV-B-dependent apoptosis in keratinocytes, indicating that DeltaNp51B blocks keratinocyte apoptosis. Although the exogenous expression of DeltaNp51B in keratinocytes did not further block the UV-B-dependent apoptosis, to our surprise the expression of TAp51B (an isoform with a full NH(2)-terminal transactivation domain that is structurally and functionally similar to p53) decreased apoptosis significantly. The blockade of keratinocyte apoptosis by the p51 was dependent on the phosphorylation of Akt, resulting in the activation of a survival pathway. Thus, in addition to its indispensable roles in epithelial development, p51 acts in adult cells to protect the epidermis against UV-B irradiation by preventing excess depletion of keratinocytes.

Docetaxel enhances the cytotoxicity of tetrahydropyranyladriamycin in a sequence-dependent manner.

BACKGROUND: Taxanes and anthracyclines are active against breast cancer. In this study we investigated the combined antitumor activity of these drugs, with particular regard to sequence-dependency. MATERIALS AND METHODS: The combined antitumor activity of docetaxel and tetrahydropyranyladriamycin (THP) against two human breast cancer xenografts was assessed using an in vitro histoculture drug-response assay. The sequence-dependency of the combined cytotoxicity was evaluated by isobologram. RESULTS: A synergistic antitumor activity of combined docetaxel + THP was exhibited against the R-27 xenograft when docetaxel was given first or simultaneously with THP. However, this synergism was diminished when THP was used before docetaxel. While an additive effect of combined docetaxel + THP was observed against MX-1 xenograft when docetaxel was given first or simultaneously with THP, this effect was not marked using the THP/docetaxel sequence. CONCLUSION: Docetaxel increased the antitumor activity of THP, but only when administered before or simultaneously with THP.

Ameliorative effect of vasopressin-(4-9) through vasopressin V(1A) receptor on scopolamine-induced impairments of rat spatial memory in the eight-arm radial maze.

In order to clarify the mechanism by which pGlu-Asn-Cys(Cys)-Pro-Arg-Gly-NH(2) (vasopressin-(4-9)), a major metabolite C-terminal fragment of [Arg(8)]-vasopressin (vasopressin-(1-9)), improves learning and memory, we used several different drugs such as an acetylcholine receptor antagonist, a Ca(2+)/calmodulin-dependent protein kinase II inhibitor, vasopressin receptor antagonists and L-type Ca(2+) channel blocker to disrupt spatial memory in rats. Moreover, we examined the effect of vasopressin-(4-9) on acetylcholine release in the ventral hippocampus using microdialysis. Vasopressin-(4-9) (10 fg/brain, i.c.v.) improved the impairment of spatial memory in the eight-arm radial maze induced by scopolamine, pirenzepine and Ca(2+)/calmodulin -dependent protein kinase II inhibitor. Pirenzepine, a vasopressin V(1A) receptor antagonist, and L-type Ca(2+) channel blocker, but not a vasopressin V(2) receptor antagonist, suppressed the effects of vasopressin-(4-9) on scopolamine-induced impairment of spatial memory. Moreover, vasopressin-(4-9) did not affect acetylcholine release in the ventral hippocampus of intact rats or of scopolamine-treated rats as assessed by microdialysis. These results suggest that vasopressin-(4-9) activates vasopressin V(1A) receptors on the postsynaptic membrane of cholinergic neurons, and induces a transient influx of intracellular Ca(2+) through L-type Ca(2+) channels to interact with muscarinic M(1) receptors. The activation of these processes by vasopressin-(4-9) is critically involved in the positive effect of vasopressin-(4-9) on scopolamine-induced impairment of spatial memory.

The earliest stages of B cell development require a chemokine stromal cell-derived factor/pre-B cell growth-stimulating factor.

Environmental factors essential for the first stages of B lymphopoiesis remain elusive. Here, we report that immediately after commitment to B lineage, precursors become dependent on a chemokine SDF-1 and its receptor CXCR4 using mutant and radiation chimeric mice. In bone marrow, generation of the earliest identifiable B cell precursor populations requires CXCR4. In fetal liver, we identified Lin(-)CD19(-)c-kit(+)IL-7Ralpha(+)AA4.1(+), the earliest unipotent B cell precursor population, and found that its development was severely affected in SDF-1(-/-) embryos but not in IL-7(-/-) embryos. Lin(-) T cell progenitors appeared normal in SDF-1(-/-) embryos. Moreover, SDF-1 exhibited specific biologic activities on the earliest B cell precursors. SDF-1 provides the first example of a cytokine responsible for the earliest B lineage stages.

Elucidation of the differences between the 430- and 455-nm absorbing forms of P450-isocyanide adducts by resonance Raman spectroscopy.

Alkylisocyanide adducts of microsomal P450 exist in two interconvertible forms, each giving the Soret maximum around 430 or 455 nm. This is demonstrated with a rabbit liver P450 2B4. Resonance Raman spectra of the 430- and 455-nm forms were examined for typical P450s of the two types as well as for P450 2B4 because the 430-nm form of P450 2B4 is liable to change into P420. P450cam and P450nor were selected as a model of the 430- and 455-nm forms, respectively. For the n-butyl isocyanide (CNBu) adduct, the Fe(II)-CNBu stretching band was observed for the first time at 480/467 cm(-1) for P450cam and at 471/459 cm(-1) for P450nor with their (12)CNBu/(13)CNBu derivatives. For P450cam, but not P450nor, other (13)C isotope-sensitive bands were observed at 412/402, 844/835, and 940/926 cm(-1). The C-N stretching mode was identified by Fourier transform IR spectroscopy at 2116/2080 cm(-1) for P450cam and at 2148/2108 cm(-1) for P450nor for the (12)C/(13)C derivatives. These findings suggest that the binding geometry of isocyanide differs between the two forms-bent and linear structures for P450cam-CNBu and P450nor-CNBu, respectively. In contrast, in the ferric state, the Raman (13)C isotopic frequency shifts, and the IR C-N stretching frequencies (2213/2170 and 2215/2172 cm(-1)) were similar between P450cam and P450nor, suggesting similar bent structures for both.

Effects of molecular structure on the stability of a thermotropic liquid crystal. Gas electron diffraction study of the molecular structure of phenyl benzoate.

As a model of the core of molecules forming liquid crystals, the molecular structure of phenyl benzoate (Ph-C(=O)-O-Ph) at 409 K was determined by gas electron diffraction, and the relationship between the gas-phase structures of model compounds and the nematic-to-liquid transition temperatures was studied. Structural constraints were obtained from RHF/6-31G ab initio calculations. Vibrational mean amplitudes and shrinkage corrections were calculated from the harmonic force constants given by normal coordinate analysis. Thermal vibrations were treated as small-amplitude motions, except for the phenyl torsion, which was treated as a large-amplitude motion. The potential function for torsion was assumed to be V(phi(1),phi(2)) = V(12)(1 - cos 2phi(1))/2 + V(14)(1 - cos 4phi(1))/2 + V(22)(1 - cos 2phi(2))/2, where phi(1) and phi(2) denote the torsional angles around the C-Ph and O-Ph bonds, respectively. The potential constants (V(ij)()/kcal mol(-)(1)) and the principal structure parameters (r(g)/A, angle(alpha)/deg) with the estimated limits of error (3sigma) are as follows: V(12) = -1.3 (assumed); V(14) = -0.5(9); V(22) = 3.5(15); r(C=O) = 1.208(4); r(C(=O)-O) = 1.362(6); r(C(=O)-O) - r(O-C) = -0.044 (assumed); r(C(=O)-C) = 1.478(10); = 1.396(1); angleOCO = 124.2(13); angleO=CC = 127.3(12); angleCOC = 121.4(22); ( angleOCC(cis) - angleOCC(trans))/2 = 3.0(15); ( angleC(=O)CC(cis) - angleC(=O)CC(trans))/2 = 4.8(17), where < > means an average value and C-C(cis) and C-C(trans) bonds are cis and trans to the C(=O)-O bond, respectively. The torsional angle around the O-Ph bond was determined to be 64(+26,-12) degrees. An apparent correlation was found between the contributions of the cores to the clearing point of liquid crystals and the gas-phase structures of model compounds of the cores of mesogens, i.e., phenyl benzoate, trans-azobenzene (t-AB), N-benzylideneaniline, N-benzylideneaniline N-oxide (NBANO), trans-azoxybenzene (t-AXB), and trans-stilbene. The structures of t-AB, NBANO, and t-AXB have been obtained by our research group.

Effects of a thiolate axial ligand on the pi-->pi* electronic states of oxoferryl porphyrins: a study of the optical and resonance Raman spectra of compounds I and II of chloroperoxidase.

Optical absorption and resonance Raman spectra have been investigated for enzymatic intermediates, compounds I and II, of chloroperoxidase (CPO) which contains a thiolate-ligated iron porphyrin. Compound I of CPO (CPO-I), an oxoferryl porphyrin pi cation radical, gave an apparently asymmetric single-peaked Soret band at 367 nm, for which band fitting analyses revealed the presence of two transition bands around 365 and 415 nm. Compound II of CPO (CPO-II), an oxoferryl neutral porphyrin, gave a split Soret spectrum with two bands (blue and red Soret bands) at 373 and 436 nm. Thus both CPO-I and CPO-II can be categorized as hyperporphyrins. The maximum extinction coefficients (epsilon(b) and epsilon(r)) and energies (Eb and Er) of the blue and red Soret bands of CPO-II were found to fall on an epsilon(b)/epsilon(r) versus Eb-Er correlation line derived from data reported for six-coordinate ferrous derivatives of cytochrome P450 and CPO. Corresponding data for CPO-I did not fall on the correlation line. Resonance enhancement of the FeIV=O stretching (vFeO) Raman band was found for CPO-I when Raman scattering was excited at wavelengths within both transition bands around 365 and 415 nm, while the vFeO Raman band was not identified for CPO-II at any of the excitation wavelengths examined here. These findings suggest that the thiolate axial ligand causes Soret band splitting of CPO-II through configuration interaction between the sulfur-->porphyrin e(g)* charge transfer and porphyrin a1u,a2u-->e(g)* transitions, while the FeO portion is important in determining the shape of the Soret band of CPO-I.

The scopolamine-induced impairment of spatial cognition parallels the acetylcholine release in the ventral hippocampus in rats.

We investigated the relationship between the induction of spatial cognition impairment in the 8-arm radial maze task and regional changes (ventral hippocampus (VH), dorsal hippocampus, frontal cortex, and basolateral amygdala nucleus) in brain acetylcholine (ACh) release using microdialysis in rats treated with muscarinic (M) receptor antagonists. In a behavioral study, two M1 antagonists, scopolamine (0.5 mg/kg, i.p. and 20 microg, i.c.v.) and pirenzepine (80 microg, i.c.v.), but not an M2 antagonist, AF-DX116 (40-80 microg, i.c.v.), disrupted spatial cognition in the 8-arm radial maze task. In brain microdialysis with Ringer's solution containing 0.1 mM eserine sulfate, scopolamine and AF-DX116, but not pirenzepine, increased ACh release in the VH. Moreover, in the bilateral injection of scopolamine (2 microg/side), the VH and dorsomedial thalamus nucleus were important regions for scopolamine-induced impairment of spatial cognition. A simultaneous determination of the behavioral changes revealed that scopolamine (0.5 mg/kg, i.p.) markedly decreased the ACh contents and also increased the ACh release in all regions tested. Especially, the changes in the ACh release of the VH closely paralleled the induction of the scopolamine-induced impairment of spatial cognition. These results suggest that the blocking balance between M1 and M2 muscarinic receptor in the VH therefore plays a major role in the spatial cognition impairment induced by scopolamine in the 8-arm radial maze task.

X-ray crystal structure and catalytic properties of Thr252Ile mutant of cytochrome P450cam: roles of Thr252 and water in the active center.

The structure-function relationship in cytochrome P450cam monooxygenase was studied by employing its active site mutant Thr252Ile. X-ray crystallographic analyses of the ferric d-camphor-bound form of the mutant revealed that the mutation caused a structural change in the active site giving an enlarged oxygen-binding pocket that did not contain any hydrophilic group such as the OH group of Thr and H(2)O. The enzyme showed a low monooxygenase activity of ca. 1/10 of the activity of the wild-type enzyme. Kinetic analyses of each catalytic step revealed that the rate of proton-coupled reduction of the oxygenated intermediate of the enzyme, a ternary complex of dioxygen and d-camphor with the ferrous enzyme, decreased to about 1/30 of that of the wild-type enzyme, while the rates of other catalytic steps including the reduction of the ferric d-camphor-bound form by reduced putidaredoxin did not change significantly. These results indicated that a hydrophilic group(s) such as water and/or hydroxyl group in the active site is prerequisite to a proton supply for the reduction of the oxygenated intermediate, thereby giving support for the operation of a proton transfer network composed of Thr252, Asp251, and two other amino acids and water proposed by previous investigators.

Formation of compound I in the reaction of native myoglobins with hydrogen peroxide.

Reaction of ferric native myoglobin (Mb) with hydrogen peroxide (H(2)O(2)) was studied by the aid of stopped-flow rapid-scan spectrophotometry. In contrast to the results in previous studies where compound I was reported to be undetectable, both sperm whale and horse heart metmyoglobins (metMbs) formed a significant quantity of compound I, an oxoferryl porphyrin pi-cation radical (Por(+)-Fe(IV)(O)), during their reactions with H(2)O(2). With both kinds of Mbs, formation of compound I was more clearly observed in D(2)O than in H(2)O. The compound thus formed was capable of performing monooxygenation of thioanisole to methyl phenyl sulfoxide and a 2-electron oxidation of H(2)O(2) giving O(2) and H(2)O as products. It was also converted into ferryl myoglobin (Por-Fe(IV)(O)-globin(+)) spontaneously. Rate constants for these reactions and that for a direct conversion of metMb to ferryl Mb through the homolysis of H(2)O(2) were determined. These results established unambiguously that native metMb can form both compound I and ferryl Mb upon reaction with H(2)O(2) and that these high valent iron compounds serve as essential intermediates in Mb-assisted peroxidative reactions. The observed deuterium effect on the apparent stability of compound I was attributable to that effect on the hydrogen abstraction step in the 2-electron oxidation of H(2)O(2) by compound I.

Changes in diffusion-weighted MRI after status epilepticus.

A 3-year-old male developed hemiplegia and aphasia after convulsive status epilepticus. Diffusion-weighted magnetic resonance images demonstrated cytotoxic edema in the white matter 6 days after the seizure episode and subsequently in the gray matter after an additional 7 days. Diffusion-weighted magnetic resonance images demonstrated a subacute evolution of the pathologic process after the status epilepticus.

Gastrointestinal stromal tumors treated by laparoscopic surgery: report of three cases.

Three cases of gastrointestinal stromal tumors (GIST) were treated by a laparoscopic wedge resection of the stomach. The tumor characteristics were confirmed to be nonepithelial, nonlymphomatous, nonmyogenic, and nonneurogenic gastrointestinal neoplasms with an uncertain origin which were CD34-positive and actin- and S-100-negative. The malignant potential was estimated based on the mitotic figures and growth rates. The results suggest that laparoscopic surgery is an adequate strategy for gastric submucosal tumors including GIST, and also indicates this technique to be a curative, safe, and minimally invasive procedure for both diagnosis and treatment.