Cytoprotection by pyruvate through an anti-oxidative mechanism in cultured rat calvarial osteoblasts.

Although we have previously shown drastic cell death by pyruvate deficiency in osteoblasts at the proliferative stage, the exact mechanism remains unclear so far. Cell survivability was significantly decreased in rat calvarial osteoblasts cultured for 0 to 3 days in vitro (DIV) following replacement of the eutrophic alpha-modified minimum essential medium (alpha-MEM) with Dulbecco's modified eagle medium (DMEM) for cultivation. The addition of pyruvate enriched in alpha-MEM, but not in MEM, entirely prevented cell death induced by the medium replacement throughout a culture period from 0 to 3 DIV. Both cysteine and reduced glutathione protected cell death in cells cultured for 3 DIV without significantly affecting that in cells cultured for 1 DIV, however, while none of lactate, acetate and insulin significantly prevented the cell death irrespective of the culture period up to 3 DIV. A marked increase was detected in intracellular reactive oxygen species (ROS) levels 4 h after the medium replacement. In osteoblasts cultured in alpha-MEM for 3 DIV, but not in those for 7 DIV, hydrogen peroxide (H2O2) markedly decreased cell survivability when exposed for 2 to 24 h. Furthermore, H2O2 was effective in significantly decreasing cell survivability in osteoblasts cultured in DMEM for 7 DIV. Pyruvate at 1 mM not only prevented cell death by H2O2, but also suppressed the generation of intracellular ROS in osteoblasts exposed to H2O2. These results suggest that pyruvate could be cytoprotective through a mechanism associated with the anti-oxidative property rather than an energy fuel in cultured rat calvarial osteoblasts.

Possible expression of functional glutamate transporters in the rat testis.

Neither expression nor functionality is clear in peripheral tissues with the molecular machineries required for excitatory neurotransmitter signaling by L-glutamate (Glu) in the central nervous system, while a recent study has shown that several Glu receptors are functionally expressed in the rat testis. This fact prompted us to explore the possible functional expression in the rat testis of the Glu transporters usually responsible for the regulation of extracellular Glu concentrations in the brain. RT-PCR revealed the expression, in the rat testis, of mRNA for five different subtypes of Glu transporters, in addition to that for particular subtypes of ionotropic and metabotropic Glu receptors. Glutamate transporter-1 (GLT-1) was different in the brain from that in the testis in terms of molecular sizes on Northern and Western blot analyses. In situ hybridization as well as immunohistochemical analysis showed localized expression of glutamate aspartate transporter at interstitial spaces and GLT-1 at elongated spermatids in the rat testis respectively. The expression of mRNA was localized for excitatory amino acid transporter-5 at the basal compartment of the seminiferous tubule in the rat testis. [(3)H]Glu was accumulated in testicular crude mitochondrial fractions in a temperature- and sodium-dependent saturable manner with pharmacological profiles similar to those shown in brain crude mitochondrial fractions. These results suggested that particular subtypes of central Glu transporters for the regulation of extracellular Glu concentrations in the rat testis could be constitutively and functionally expressed.

Effects of volatile and intravenous anesthetics on the uptake of GABA, glutamate and dopamine by their transporters heterologously expressed in COS cells and in rat brain synaptosomes.

Although the neurotransmitter uptake system is considered a possible target for the presynaptic action of anesthetic agents, observations are inconsistent concerning effects on the transporter and their clinical relevance. The present study examined the effects of volatile and intravenous anesthetics on the uptake of GABA, glutamate and dopamine in COS cells heterologously expressing the transporters for these neurotransmitters and in the rat brain synaptosomes. Halothane and isoflurane, but not thiamylal or thiopental, significantly inhibited uptake by COS cell systems of GABA, dopamine and glutamic acid in a concentration-dependent manner within clinically relevant ranges for anesthesia induced by these agents. Similarly, in synaptosomes halothane and isoflurane but not thiopental significantly suppressed the uptake of GABA and glutamic acid, respectively. These results do not support the hypothesis that volatile and intravenous anesthetics exert their action via specific inhibition of GABA uptake to enhance inhibitory GABAergic neuronal activity. Rather, they suggest that presynaptic uptake systems for various neurotransmitters including GABA may be the molecular targets for volatile anesthetic agents.

Single nucleotide polymorphisms (SNPs) assay using reversible association and dispersion of DNA-linked colloidal nanoparticles.

The amphiphilic copolymer consisting of oligodeoxyribonucleotide (ODN; dT12) as the hydrophilic part and thermo-responsive poly(N-isopropylacrylamide) (polyNIPAAm) as the hydrophobic part was prepared. The copolymers formed DNA-linked colloidal nanoparticles above the phase transition temperature of polyNIPAAm part. The nanoparticles aggregated rapidly when the complementary ODN (dA12) was added into the dispersion. In contrast, they kept dispersed in the absence of the complementary ODN and in the presence of the point-mutated ODN ((dA6)dT(dA5)). These distinct phenomena may be applied for an oligonucleotides discrimination system in gene diagnosis.

Regulation of aggregated structure of dye molecules by DNA hybridization.

Dye H-aggregates were successfully created by introducing multiple Methyl Red molecules consecutively in the single stranded DNA. By the presence of its complementary DNA, however, the H-band disappeared and new peak appeared at longer wavelength, indicating that aggregated structure changed by the duplex formation. Thus, supramolecular structure of the dye aggregates was successfully controlled by use of DNA.

Catalytic hydrolysis of peptides by cerium(IV).

Oligopeptides are efficiently hydrolyzed by Ce(IV) to the corresponding amino acids under mild conditions. The pseudo first-order rate constants for the hydrolysis of H-Gly-Phe-OH and H-Gly-Gly-OH at pH 7.0 and 50 degrees C are 3.5 x 10(-1) and 2.8 x 10(-1) h(-1), with [Ce(NH4)2(NO3)6]0=10mM (the half-lives are 2.0 and 2.5 h). The catalytic activity of the Ce(IV) is far greater than those of other lanthanide ions and non-lanthanide ions. No oxidative cleavage was observed under the reaction conditions. Catalytic turnover of the Ce(IV) was also evidenced. The hydrolysis is fast especially when the substrates have no metal-coordinating side chains. Tripeptides and tetrapeptides are hydrolyzed at the similar rates as the dipeptides. In the hydrolysis of tripeptides, the amide linkage near the N-terminus is preferentially hydrolyzed. Neither the N-carbobenzyloxy derivative nor the amide of H-Gly-Phe-OH is hydrolyzed to a measurable extent, showing that both the terminal amino group and the carboxylate are coordinated to the Ce(IV) ion. This complexation is further confirmed by 1H NMR spectroscopy. The Ce(IV) ion is therefore one of the most active catalysts for peptide hydrolysis.

Propofol anaesthesia in mice is potentiated by muscimol and reversed by bicuculline.

We have examined the role of gamma-aminobutyric acid (GABA) neurones in propofol anaesthesia in mice using the righting reflex. Propofol i.p. increased the percentage of loss of the righting reflex in a dose-dependent manner with an ED50 value of 140 (95% confidence limits 123-160) mg kg-1 (n = 40; eight animals per dose, five doses per dose-response curve). The ED50 for propofol decreased significantly to 66 (58-75) mg kg-1 in the presence of the GABAA receptor agonist muscimol 1 mg kg-1 i.p. (n = 40) (P < 0.05). In contrast, the ED50 increased significantly to 240 (211-274) mg kg-1 in the presence of the antagonist bicuculline 5 mg kg-1 i.p. (n = 40) (P < 0.05). Our results suggest that propofol anaesthesia may be mediated, at least in part by GABA neurons.

Mechanistic analogy between metal ion-catalyzed self-splicing of protein and RNA.

Recent studies have shown that RNA hydrolysis is remarkably promoted by metal ions and suitably designed metal complexes. We have found that autocleavage of dipeptides X-Ser and a tripeptide Gly-Ser-Ala is catalyzed by Zn(II) at 70 degrees C and pH 7.0. A mechanistic analogy between metal ion-catalyzed hydrolysis of peptides and RNA is suggested.

Frequency analyses of EMG power spectra of anterior temporal and masseter muscles in children and adults.

To study the functional change of masticatory muscles during growth and development, frequency analyses of surface electromyogram (EMG) power spectra were carried out. The subjects were six children (five males and one female), aged 4.5 +/- 0.2 years, having full deciduous dentition (Hellman's dental age IIA) and six adults (four males and two females), aged 27.7 +/- 3.8 years, having full permanent dentition. EMG signals were recorded bilaterally by using bipolar silver-surface electrodes from the anterior temporal and masseter muscles while the subjects were chewing gum and while performing maximum clenching in the intercuspal position. A fast Fourier transform algorithm was used to obtain the power-spectral density function and the power spectra of the EMG signals. Since the total power value from 62.5 to 1000 Hz was 100 percent, the frequencies at 25, 50, 75, and 90 percent of the cumulative power were calculated. The results showed that the frequencies at every percent of the cumulative power were age-dependent and that the EMG power spectra patterns in adult muscles were shifted to significantly lower frequencies than those in child muscles. The shift was probably caused by differences in the proportion of fiber type and fiber size between muscles of children and adults.

[Dentofacial manifestations in children with vitamin D-dependent Rickets type II].

Calcification of the teeth, size of the teeth and dental arches, facial growth and calcification of the carpal bones were studied in three children with clinically different severities of vitamin D-dependent rickets (DDR), type II, with alopecia, which is 1,25-dihydroxyvitamin D-receptor-defect rickets and is particularly resistant to treatment with calciferol analogues. They were treated in a pediatric clinic with large doses of 1 alpha-hydroxyvitamin D3(1 alpha-(OH)D3) and 2 g/day of calcium lactate. The results were as follows: 1. Hypoplasia of enamel of the deciduous teeth was not found. 2. In the deciduous teeth, large pulp chambers and thin dentin were seen in radiographs before treatment. In patients 1 and 2, these abnormalities were reversed by treatment. In patient 3, who had the severest manifestations, large pulp chambers and thin dentin decreased but still remained. 3. Growth of permanent teeth was retarded before treatment and during resistance to treatment. After effective medication, it caught up and was corrected. 4. Problems concerning maxillary and mandibular growth were not found. However Nasions of three patients were more front and lower and Orbitals were lower than standard. 5. In patients 1 and 2, the calcification of carpal bones was accelerated and in patient 3 retarded. 6. Mesiodistal dimensions of erupted deciduous and permanent teeth were within the standard range, except for patient 3, who had smaller lower deciduous teeth. 7. A ground section of the extracted upper right first deciduous molar from patient 3 showed abundant inter-globular dentin and lack of pre-dentinal layer. From the above findings, it was felt that in all probability dentinogenesis was disturbed by the DDR type II. Abnormally large pulp chambers and thin dentin could be corrected by effective medication. In patients with vitamin D-dependent rickets type II, oral hygiene for caries prevention is the most important procedure, because the pulp will be infected immediately after initiation of dental caries. After effective medication, permanent teeth and jaw bones will probably grow normally.

[Hypoplasia of tooth in children with inborn errors of metabolism].

For the investigation of the clinical dental manifestations of inborn errors of metabolism, the oral examination of twenty one children aged from 5 months to 15 years 5 months was performed at the pediatric clinic of inborn errors of metabolism. The results were as follows: 1. In six of seven histidinemia patients, white spots, pit or hypoplasia of enamel, fused teeth, peg teeth or abnormal tubercles were observed. 2. In a patient with GM1-gangliosidosis, the upper and lower central incisors had severe white spots. 3. In one patient with osteopetrosis, one of two glycogenosis type IX and one of two homocystinuria patients, white spots or hypoplasia of enamel were observed. 4. One patient with hypophosphatemic vitamin D-resistant ricket, one with vitamin D-dependent ricket type II and one with galactosemia patient had no enamel hypoplasia. It was strongly suggested that there is a specific relationship of histidinemia and GM1-gangliosidosis to dental manifestations.

[Frequency analysis of the EMG power spectrum of the anterior temporal and masseter muscles in children and adults].

For the investigation of the functional change of the masticatory muscles along with growth and development, the frequency analysis of the EMG power spectrum was carried out. The subjects were 6 children (5 males and 1 female) with full deciduous dentition (Hellman's dental age IIA) aged 4.5 +/- 0.2 years and 6 adults (4 males and 2 females) with full permanent dentition aged 27.7 +/- 3.8 years. EMG signals were recorded bilaterally by means of bipolar silver surface electrodes from the anterior temporal and masseter muscles when the subjects were chewing chewing gum or performing maximum clenches in the intercuspal position. A fast Fourier transform (FFT) algorithm was used to obtain the power spectrum of the EMG signal. As the total power value from 62.5 to 1000 Hz was 100 per cent, the mean frequencies at 25, 50, 75 and 90 per cent of the cumulative power were calculated. The results were as follows: 1. The mean frequencies at each ratio of the cumulative power were age-dependent and EMG power spectrum patterns significantly shifted to lower frequencies in the muscles of the adults. 2. No statistically significant differences between the chewing and clenching, the anterior temporal and masseter muscle and the left and right side were observed in each group.

[Muscle action potential and masticatory rhythm of anterior temporal and masseter muscles in children and adults].

For the investigation of the functional change of the masticatory muscles along with growth and development, electromyographic evaluation was carried out. The subjects were 6 children (5 males and 1 female) with full deciduous dentition (Hellman's dental age IIA) aged 4.5 +/- 0.2 years and 6 adults (4 males and 2 females) with full permanent dentition aged 27.7 +/- 3.8 years. EMG signals were recorded bilaterally by means of bipolar silver surface electrodes from the anterior temporal and masseter muscles when the subjects were chewing chewing gum or performing maximum clenches in intercuspal position. The cumulative power values from 62.5 to 1000 Hz in the EMG power spectrum during chewing or clenching were calculated as the muscle action potential. The ratio of the action potential of each muscle to the total action potential of four muscles were analyzed. Masticatory rhythm during chewing was analyzed by means of the time parameter (duration, interval and cycle) and their coefficients of variation. The results were as follows: 1. In children the temporal muscles predominated in chewing and clenching, whereas in adults there were three types with Temporal muscles predominating, Masseter muscles predominating and both muscles sharing equally. 2. No statistically significant differences between children and adults were observed in the duration, interval and cycle. 3. In adults the coefficients of variation of the duration, interval and cycle were smaller and the masticatory rhythm was more stable than in children.

[Electromyographic (EMG) electrode impedance and EMG activity from anterior temporal muscle and masseter muscle].

The value and change with time of the impedance of surface EMG electrodes and the effects of their difference between the bipolar electrodes on the electromyographic activity from the anterior temporal muscle and the masseter muscle in six adult male subjects with normal occlusion were studied. The results were as follows: 1. In the anterior temporal muscle, if the impedance of the electrode was under 20 k omega it was stable from just after the electrode disc was applied to the skin. In the masseter muscle, if the impedance was under 30 k omega it became stable within two minutes after the electrode was applied. 2. The difference of impedance between the bipolar EMG electrodes did not correlate with EMG activity.

[Pharmacological studies of MO-8282, a new antidepressant].

We investigated the pharmacological characteristics of MO-8282 as a novel antidepressant. MO-8282 inhibited the specific binding of 3H-clonidine to cerebro-cortical membrane fractions from rats about five times more potently than mianserin, and it competed with clonidine in the twitch response of the isolated guinea-pig ileum under field stimulation. The results indicated that MO-8282 possessed alpha 2-adrenergic receptor blocking activity. MO-8282 in a dose of 30 mg/kg (p.o.) showed no inhibition against the uptake of noradrenaline, dopamine and serotonin in the rat brain, whereas mianserin inhibited the uptake of serotonin specifically. MO-8282, similar to mianserin, had no effect on spontaneous release of 3H-noradrenaline and slightly stimulated the release of 3H-serotonin from the rat cerebrocortical synaptosome. The turnover rate of noradrenaline in rat brain was accelerated by administration of MO-8282 (30 mg/kg) for 15 days; however, that of dopamine and serotonin was not affected. The above findings indicate that MO-8282, unlike tricyclic antidepressants, mainly exerts alpha 2-adrenoceptor blocking action on the central noradrenergic system, similar to mianserin. In addition, the fact that MO-8282 unlike mianserin showed no inhibition against uptake of serotonin in brain suggests that the alpha 2-adrenoceptor blocking of MO-8282 is more specific and potent than that of mianserin.