Biomedical subjects

C Hara

Publications and source records attributed to C Hara.

At least 19 recordsLinked to original sources

PubMed2005

Involvement of mitogen-stimulated p70-S6 kinase in the development of sensitization to the methamphetamine-induced rewarding effect in rats.

The neural plasticity associated with behavioral sensitization following repeated administration of a psychostimulant methamphetamine (METH) is thought to require synthesis of new proteins. The aim of the present study was to investigate the role of p70-S6 kinase (p70-S6K) phosphorylation, which contributes to the selective translation of a unique family of mRNA, in mediating both the METH-induced rewarding effect and its sensitization. An intra-nucleus accumbens (N.Acc.) pre-injection with 0.025 pmol/rat of a selective p70-S6K inhibitor rapamycin failed to affect the METH-induced conditioned place preference. However, this treatment clearly abolished the development of sensitization of the METH-induced conditioned place preference. Consistent with the behavioral assay, the level of the immunoreactivity of phosporylated-p70-S6K was not changed in the cytosolic fraction of the N.Acc. obtained from rats that had revealed the METH-induced rewarding effect. In contrast, the immunoreactivities in the cytosolic preparation for Western blotting and immunohistochemical density of phosphorylated-p70-S6K were significantly increased in the N.Acc. obtained from METH-sensitized rats as compared with those with chronic saline treatment. However, the immunoreactivities of phosphorylated-extracellular signal-regulated kinase and phosphorylated-ribosomal S6 protein were not significantly altered in the N.Acc. under the same condition. The present data provide evidence for the change in the translation rate, which can be regulated by S6K phosphorylation, in the N.Acc. during the development of sensitization to METH-induced rewarding effects in rats.

Animals↗

PubMed2004

Implications of protein kinase C in the nucleus accumbens in the development of sensitization to methamphetamine in rats.

Repeated treatment with methamphetamine leads to an enhancement in the methamphetamine-induced dopamine release and its related behaviors. This phenomenon is called sensitization or reverse tolerance. Protein kinase C (PKC) controls numerous signaling cascades by virtue of its ability to phosphorylate target proteins that include other kinases. The purpose of study was then to investigate the implication of PKC in the development of sensitization to the rewarding effect and to the extracellular dopamine release induced by methamphetamine in rats. The conditioned place preference paradigm and in vivo microdialysis assay were performed in the present study. An intra-nucleus accumbens injection of a selective PKC inhibitor chelerythrine chloride abolished the enhancement of the methamphetamine-induced place preference following repeated treatment with methamphetamine. Furthermore, intra-nucleus accumbens injection of chelerythrine chloride blocked the development of sensitization to dopamine release and to the decrease in the major dopamine metabolites, 3'4-dihydroxyphenylacetic acid and homovanillic acid, in the nucleus accumbens induced by repeated methamphetamine treatment. Under these conditions, the immunoreactivity of the cytosolic phosphorylated conventional- or classic-type PKC in the limbic forebrain region including the nucleus accumbens was slightly, but significantly increased in methamphetamine-sensitized rats. The present data provide evidence for the implication of PKC in the nucleus accumbens in the development of sensitization to the methamphetamine-induced rewarding effect, dopamine release and inhibition of dopamine metabolism/re-uptake in rats.

Animals↗

PubMed2001

Transcription analysis of rpoH in Pseudomonas putida.

We previously determined the complete DNA sequence of the rpoH gene encoding the heat-shock sigma factor (sigmaH) of Pseudomonas putida. In the present study, the transcriptional start sites of rpoH were determined to be 41 nucleotides (T1), 153 nucleotides (T2) and 157 nucleotides (T3) upstream from the translational start codon (AUG) of rpoH by rapid amplification of cDNA 5'-ends. Based on the locations of T2 and T3, a sigma70-type promoter (P2) was determined to be located in the open reading frame region of upstream ftsX in addition to the sigmaE-type promoter (P1; DNA Res. 6 (1999) 241). In the in vitro transcription assay with reconstituted RNA polymerases (Esigma70, EsigmaE, EsigmaH and EsigmaS) of Pseudomonas aeruginosa, EsigmaE transcribed rpoH from T1 and Esigma(70) transcribed it from T2 and T3. In both cases, the level of transcription was higher at 42 degrees C than at 30 degrees C. No transcript was detected when EsigmaH or EsigmaS was used. These results indicate that EsigmaE and Esigma70 recognize P1 promoter and P2 promoter, respectively, and also prove that the synthesis of rpoH mRNA is inducible upon heat shock.

Amino Acid Sequence↗

PubMed2001

Involvement of corticotropin-releasing factor receptor subtype 1 in morphine withdrawal regulation of the brain noradrenergic system.

Effects of pretreatment with the selective corticotropin-releasing factor (CRF) subtype 1 (CRF(1)) receptor antagonist, 2-(N-(2-methylthio-4-isopropylphenyl)-N-ethyl-amino-4-(4-(3-fluorophenyl)-1,2,3,6-tetrahydropyridin-1-yl)-6-methylpyrimidine (CRA1000) on the behavioral and biochemical changes after naloxone-precipitated morphine withdrawal were examined in ICR mice. Mice were chronically treated with morphine (8-45 mg/kg) for 5 days. Naloxone (3 mg/kg, s.c.) precipitated jumping, diarrhea, and body weight loss in morphine-dependent mice. In addition, 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG) and noradrenaline turnover (MHPG/noradrenaline) levels in the cerebral cortex were increased following naloxone challenge in morphine-dependent mice. However, 5-hydroxytriptamine turnover did not alter the increase following naloxone challenge in morphine-dependent mice. Pretreatment with CRA1000 (20 mg/kg, i.p.) attenuated the incidence of withdrawal signs and naloxone-precipitated increases in noradrenaline turnover. These results suggest that the activation of CRF(1) receptor may play an important role in the elevation of noradrenaline transmission, but not in 5-hydroxytriptamine transmission, in the cerebral cortex, which projects from the locus coeruleus during morphine withdrawal.

Animals↗

PubMed2001

Differential effects of psychological stress on activation of the 5-hydroxytryptamine- and dopamine-containing neurons in the brain of freely moving rats.

We investigated the effects of psychological stress, lacking direct physical stimulus, on the release of 5-hydroxytryptamine (5-HT) and dopamine (DA) in the basolateral nucleus of the amygdala (BLA) and the dorsal raphe nuclei (DRN) in the rat using the in vivo microdialysis technique with dual probes, one in each region of the same animals. Psychological stress was employed using the communication box paradigm. Psychological stress for 1 h significantly increased dialysate 5-HT levels in the DRN and the BLA. Psychological stress-induced 5-HT release in the BLA was significantly greater than those in the DRN, indicating that modifications of the serotonergic neurons in the BLA are more sensitive to psychological stress than are those of the DRN. Psychological stress also increased DA release in the BLA, while the dialysate DA levels in the DRN were unchanged. These results suggest that psychological stress preferentially activates ascending serotonergic neurons from the DRN to the BLA but not those of dopaminergic neurons. Furthermore, our findings indicate that both the serotonergic neurons and the dopaminergic neurons in the BLA may have a distinct role to play in the neuronal responses to psychological stress.

Amygdala↗

PubMed2001

Molecular basis of ocular abnormalities associated with proximal renal tubular acidosis.

Proximal renal tubular acidosis associated with ocular abnormalities such as band keratopathy, glaucoma, and cataracts is caused by mutations in the Na(+)-HCO(3)(-) cotransporter (NBC-1). However, the mechanism by which NBC-1 inactivation leads to such ocular abnormalities remains to be elucidated. By immunological analysis of human and rat eyes, we demonstrate that both kidney type (kNBC-1) and pancreatic type (pNBC-1) transporters are present in the corneal endothelium, trabecular meshwork, ciliary epithelium, and lens epithelium. In the human lens epithelial (HLE) cells, RT-PCR detected mRNAs of both kNBC-1 and pNBC-1. Although a Na(+)-HCO(3)-cotransport activity has not been detected in mammalian lens epithelia, cell pH (pH(i)) measurements revealed the presence of Cl(-)-independent, electrogenic Na(+)-HCO(3)-cotransport activity in HLE cells. In addition, up to 80% of amiloride-insensitive pH(i) recovery from acid load in the presence of HCO(3)(-)/CO(2) was inhibited by adenovirus-mediated transfer of a specific hammerhead ribozyme against NBC-1, consistent with a major role of NBC-1 in overall HCO(3)-transport by the lens epithelium. These results indicate that the normal transport activity of NBC-1 is indispensable not only for the maintenance of corneal and lenticular transparency but also for the regulation of aqueous humor outflow.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed2000

Involvement of caspase 3-like protease in methylmercury-induced apoptosis of primary cultured rat cerebral microglia.

Methylmercury (MeHg) has been implicated to induce massive neurodegeneration by disruption of neuron-glia interactions besides a direct potent neurotoxicity. In the present study, we examined potential cytotoxic effects of MeHg on primary cultured rat microglia. Following treatment with a relatively low concentration (0.5 microM) of MeHg, microglia had induced cell death accompanied by DNA fragmentation and an activation of caspase-3-like protease. MeHg-induced microglial death was significantly suppressed by the caspase-3-like protease inhibitor benzyloxycarbonyl-Try-Val-Ala-Asp-fluoromethyl-ketone indicating the occurrence of caspase-3-like protease-executed apoptosis. The aspartic protease inhibitor pepstatin A had a partial but significant inhibitory effect on MeHg-induced microglial apoptosis. These results indicate that a relatively low concentration of MeHg predominantly induces caspase-3-like protease-executed apoptosis of microglia, while the endosomal/lysosomal system is also partially involved in the cell death pathway.

Animals↗

PubMed2000

Intracellular pH regulatory mechanism in a human renal proximal cell line (HKC-8): evidence for Na+/H+ exchanger, CI-/HCO3- exchanger and Na+-HCO3- cotransporter.

In the present study we investigated whether an immortalized human renal proximal cell line, HKC-8, expresses a recently cloned Na+-HCO3- cotransporter (NBC-1) and, if so, which isoform (kNBC-1 from kidney or pNBC-1 from pancreas) is expressed in this cell line. Cell pH (pHi) measurements using a pH-sensitive fluorescence probe in the absence of HCO3-/CO2 revealed the presence of a Na+/H+ exchanger that required high concentrations of amiloride for full inhibition. In the presence of HCO3-/CO2 another pHi recovery process, dependent on Na+ but independent of Cl-, was identified. This process was electrogenic and was inhibited by 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulphonic acid (DIDS), being consistent with the Na+-HCO3- cotransporter. In addition, the pHi responses to Cl- removal were compatible with the presence of a Na+-independent Cl-/HCO3- exchanger that was also inhibited by DIDS. Reverse transcriptase polymerase chain reaction (RT-PCR) using primers designed for specific and common regions detected mRNAs of both kNBC-1 and pNBC-1 and Western blot analysis confirmed the expression of NBC-1 protein. These results indicate that HKC-8 has transport activities similar to intact proximal tubules and also suggest that both kNBC-1 and pNBC-1 may contribute to the Na+-HCO3- cotransport activity in this cell line.

Acids↗

PubMed2000

Incubation in tissue culture media allows isolated rabbit proximal tubules to regain in-vivo-like transport function: response of HCO3-absorption to norepinephrine.

Using a new stop-flow perfusion technique with microspectrofluorometric determination of luminal fluid pH, we have studied which substrates or incubation conditions allow isolated rabbit proximal tubules to attain in-vivo-like rates of HCO3- absorption (J(HCO3)) and maximal responses of J(HCO3) to norepinephrine (NE). Essentially three incubation media were tested: plasma-like HCO(3-)-Ringer solution containing 5 mmol/l D-glucose (G-Ringer sol.), the same solution also containing 10 mmol/l lactate and 5 mmol/l L-alanine, (LAG-Ringer sol.), and two tissue culture media (DMEM and RPMI 1640). Compared to G-Ringer sol., application of LAG-Ringer sol. in the bath and/or lumen, or application of DMEM or RPMI 1640 in the bath either slightly increased or decreased J(HCO3) with borderline significance. However, RPMI 1640 plus 1 mmol/l pyruvate stimulated J(HCO3) by 55%. While NE (10(-5) mol/l), if applied in G-Ringer sol., had no effect, in the presence of LAG-Ringer sol. it increased J(HCO3) by approximately =40%, and in the presence of DMEM or RPMI 1640 it increased J(HCO3) by approximately =100%. This stimulation by NE followed Michaelis-Menten kinetics with an EC50 value of 0.25 micromol/l and was probably mediated by alpha1-adrenergic receptors. Additional cell pH measurements suggest that NE stimulates the basolateral Na+-HCO3- cotransporter which then becomes susceptible to inhibition by cAMP. We conclude that incubation in tissue culture media allows isolated proximal tubules to maintain a better functional state than the commonly used solutions with unphysiologically high substrate concentrations.

Absorption↗

PubMed2000

Dopamine inhibits renal Na+:HCO3- cotransporter in rabbits and normotensive rats but not in spontaneously hypertensive rats.

BACKGROUND: Dopamine (DA) is thought to regulate renal proximal transport through the inhibition of the Na+,K+-ATPase and/or Na+/H+ exchanger. Defects in this dopaminergic system are proposed to be a pathogenic factor of genetic hypertension. However, microperfusion studies have not consistently confirmed direct tubular effects of DA. METHODS: Isolated proximal straight tubules were perfused peritubularly with Dulbecco's modified Eagle's tissue culture medium (DMEM) containing norepinephrine (NE) to improve incubation conditions. Intracellular Na+ concentrations ([Na+]i) and cell pH (pHi) were measured with fluorescence probes. RESULTS: When incubated in DMEM plus NE, DA increased [Na+]i in rabbit tubules. Inhibition of Na+,K+-ATPase could not explain this response, as it was not suppressed by ouabain. An analysis of pHi responses to bath HCO3- reduction revealed that DA, SKF 38393 (a DA1 agonist), and adenosine 3',5'-cyclic monophosphate (cAMP) inhibited the basolateral Na+:HCO3- cotransporter in rabbit and Wistar-Kyoto rat (WKY), if its transport stoichiometry was converted to 3 HCO3-:1 Na+ by DMEM plus NE incubation. The inhibitory effect of DA was abolished by SCH 23390, a DA1 antagonist, but not by (-)-sulpiride, a DA2 antagonist. In spontaneously hypertensive rats (SHRs), however, DA and SKF 38393 failed to inhibit the cotransporter, although the inhibitory effects of cAMP and parathyroid hormone were comparable to those in WKY. CONCLUSION: These results indicate that DA inhibits the Na+:HCO3- cotransporter in renal proximal tubules and also suggest that dysregulation of the cotransporter, possibly through the defect in DA1 receptor signaling, could play an important role in development of hypertension in SHRs.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗

PubMed2000

Odor distinctiveness between enantiomers of linalool: difference in perception and responses elicited by sensory test and forehead surface potential wave measurement.

The effects on humans of inhalation of optically active linalools were examined in terms of sensory tests and portable forehead surface electroencephalographic (IBVA-EEG) measurements in order to assess their odor distinctiveness by chiral isomers. (R)-(-)-Linalools with specific rotation of [alpha](D) = -15.1 degrees were isolated by repeated flash column chromatography from lavender oil, while (S)-(+)-linalools with [alpha](D) = +17.4 degrees and (RS)-(+/-)-linalools with [alpha](D) = 0 degrees and content of (R)-form 50.9% and (S)-form 49.1% were obtained from coriander oil and commercial linalool, respectively, by using the same method. With the use of an inhalator, each was administered to subjects both before and after 10 min of work. It was found that administration after work evoked different subjective impressions when compared with that before work depending on the configuration of the isomers and the type of work employed. For instance, inhalation of (R)-(-)-linalool after hearing environmental sounds not only produced a much more favorable impression in the sensory test but was also accompanied by a greater decrease in beta waves after work in comparison with that before work. This is in contrast to the case of mental work, which resulted in a tendency for agitation accompanied by an increase in beta waves. These findings led us to conclude that enantiomeric stereospecificity of linalool evoked different odor perception and responses not only with chiral dependence but also with task dependence. In addition, in comparing these sensory profiling features and IBVA-EEG tendencies between hearing environmental sound and mental work, a tendency was observed for (R)-(-)-linalool to coincide with (RS)-(+/-)-linalool but not with (S)-(+)-linalool.

Acyclic Monoterpenes↗

PubMed1999

Alteration of perceived fragrance of essential oils in relation to type of work: a simple screening test for efficacy of aroma.

The perceptional change of fragrance of essential oils is described in relation to type of work, i.e. mental work, physical work and hearing environmental (natural) sounds. The essential oils examined in this study were ylang ylang, orange, geranium, cypress, bergamot, spearmint and juniper. In evaluating change in perception of a given aroma, a sensory test was employed in which the perception of fragrance was assessed by 13 contrasting pairs of adjectives. Scores were recorded after inhaling a fragrance before and after each type of work, and the statistical significance of the change of score for 13 impression descriptors was examined by Student's t-test for each type of work. It was confirmed that inhalation of essential oil caused a different subjective perception of fragrance depending on the type of work. For example, inhalation of cypress after physical work produced a much more favorable impression than before work, in contrast to orange, which produced an unfavorable impression after physical work when compared with that before work. For mental work, inhalation of juniper seemed to create a favorable impression after work, whereas geranium and orange both produced an unfavorable impression then. From these studies, together with those conducted previously with lavender, rosemary, linalool, peppermint, marjoram, cardamom, sandalwood, basil and lime, we thus concluded that the sensory test described here might serve not only as a screening test for efficacy of aroma but also as a categorized table for aroma samples which can act as a reference to each other.

Adult↗

PubMed1998

Interleukin-9 receptor alpha chain mRNA formation in CD8+ T cells producing anti-human immunodeficiency virus type 1 substance(s).

A search for gene(s) associated with anti-human immunodeficiency virus type 1 (HIV-1) activity of CD8+ T cells was attempted using molecular cloning and the relation between the anti-HIV activity of CD8+ T cells and the interleukin-9 receptor alpha chain (IL-9R-alpha) mRNA expression from the cDNA clones obtained was examined. The anti-HIV-1 activity of CD8+ T cell culture supernatants was assessed by measuring the level of HIV-1 replication of a CD4+ T cell line transfected with an infectious HIV-1 DNA clone. IL-9R-alpha mRNA was assayed by reverse transcriptase-polymerase chain reaction (RT-PCR). Of 5 cases showing high level of anti-HIV-1 activity (more than 80% suppression of HIV-1 replication), the mRNA was detected in 4 cases. Of 10 cases showing low level of anti-HIV-1 activity (less than 80% suppression of HIV-1 replication), the mRNA was detected in one case. Soluble recombinant human IL-9 receptor (rhIL-9sR) did not suppress HIV-1 replication at a concentration of 1 microgram/ml. These data suggest that the IL-9R-alpha mRNA formation in CD8+ T cells may correlate with and play some role in the anti-HIV-1 activity of CD8+ T cells from HIV-1-infected individuals.

CD4-Positive T-Lymphocytes↗

PubMed1997

[Coumarin-containing chiral discriminating agents. VII. New crystalline 1H-NMR enantiomeric excess determination reagent for alcohols and amines, (R)-(-)- and (S)-(+)-O-coumarinylmandelic acids].

The Mitsunobu reaction of commercial 4-hydroxycoumarin with both (R)-(-)- and (S)-(+)-tert-butyl mandelates derived from commercial enantiopure mandelic acids furnished (S)-(+)- and (R)-(-)-tert-butyl O-coumarinylmandelates which were, then, treated with trifluoroacetic acid to give novel crystalline optically pure (S)-(+)- and (R)-(-)-O-coumarinylmandelic acids [SCMOH and RCMOH], respectively in good overall yields. Diastereotopic nonequivalence 1H-NMR examination of the resultant esters and amides without any racemization or kinetic resolution by way of a Steglich's procedure (a DCC-DMAP method) has proved each acid to be a useful, efficient and reliable chiral derivatizing agent for the enantiomeric excess determination of chiral alcohols and amines.

Alcohols↗

PubMed1997

[The effect of coronary intervention on renal function in patients with chronic renal failure].

To investigate the effect of coronary intervention on renal function in patients with chronic renal failure (CRF) we investigated 19 patients with CRF [serum creatinine (Scr) > 1.5 mg/dl] who underwent coronary intervention for the treatment of acute myocardial infarction or unstable angina recruited from 516 consecutive patients admitted to the coronary care unit of Nara Medical University Hospital from January, 1992 to July, 1995. Serum creatinine levels were measured at 3 points: on admission, at peak level, and at discharge. Nineteen patients were divided into two groups on the basis of increases in Scr (delta Scr): a worsened group (group A) (delta Scr > or = 1mg/dl after coronary intervention; 6 patients) and an unchanged group (group B) (delta Scr < 1 mg/dl ; 13 patients). In group A, except for one patient, the renal function recovered to the level before coronary intervention after adequate hydration or hemodialysis. The volume of contrast medium in group A (420 +/- 134 ml) was significantly higher than group B (253 +/- 97 ml) (p < 0.01). There was significant positive correlation (r = 0.42, p < 0.05) between delta Scr and the volume of contrast medium. In conclusion, coronary intervention for patients with CRF can be performed safely under treatment with adequate hydration and hemodialysis, even at higher serum creatinine levels of up to 4.0 mg/ml.

Aged↗

PubMed1996

Isolation and characterization of two cDNA clones for mRNAs that are abundantly expressed in immature anthers of rice (Oryza sativa L.).

The relationship between the length of anthers and the stage of development of microspores was examined in rice (Oryza sativa L. cv. Hayayuki). Anthers of < or = 2 mm and 2.1-2.2 mm in length and those ready to dehiscence were determined to be at the uninucleate, binucleate and trinucleate microspore stage, respectively. Two cDNAs (YY1 and YY2), representing genes that are specifically expressed in anthers at the uninucleate microspore stage, were isolated and characterized. YY1 cDNA encoded an open reading frame of 95 amino acids. Eight cysteine residues with the potential to form disulfide bridges were present in the amino acid sequence. There was a hydrophobic region at the N-terminus of the putative protein, suggesting that the YY1 protein might be secreted. This cysteine motif and the hydrophobic N-terminus are conserved among products of several anther-specific genes or cDNAs isolated from various plant species. These proteins are thought to form a superfamily of proteins that are confined to anthers. The YY1 transcript was localized in the tapetal cells and the peripheral cells of the vascular bundle. YY2 cDNA encoded an open reading frame of 389 amino acids and the deduced amino acid sequence exhibited substantial homology to that of chalcone synthase. Expression of YY2 mRNA was confined to the tapetal cells. The genes correspond to YY1 and YY2 cDNAs were shown to exist as single copies in the rice genome.

Amino Acid Sequence↗

PubMed1995

Methylcobalamin attenuates the hypoxia/hypoglycemia- or glutamate-induced reduction in hippocampal fiber spikes in vitro.

The effects of methylcobalamin, a vitamin B12 analogue, on the hypoxia/hypoglycemia- or glutamate-induced reduction in hippocampal CA1 presynaptic fiber spikes elicited by Schaffer collateral stimulation in rat brain slices were evaluated. Hippocampal slices were exposed to 15 min of hypoxia/hypoglycemia, and then these slices were returned to oxygenated and glucose-containing buffer for 3 h. Hypoxia/hypoglycemia reduced CA1 presynaptic potentials in vitro. Treatment with 10 microM methylcobalamin attenuated the impairment of CA1 presynaptic potentials induced by hypoxia/hypoglycemia or glutamate application (10 mM). Daily injection of methylcobalamin (0.5 mg/kg i.p./day) for 3 days in vivo also attenuated the hypoxia/hypoglycemia- or glutamate-induced reduction in presynaptic potentials in hippocampal slices. Pretreatment with cyanocobalamin at 10 microM failed to attenuate the impairment of CA1 presynaptic potentials. However, daily injection of cyanocobalamin (0.5 mg/kg i.p./day) for 3 days caused a protective action against the hypoxia/hypoglycemia- or glutamate-induced functional deficit. Furthermore, co-treatment of L-arginine (100 microM), a substrate for nitric oxide synthase, with methylcobalamin in vitro reversed the methylcobalamin-induced functional recovery. The present results demonstrate that methylcobalamin application in vivo or in vitro leads to functional recovery from hypoxia/hypoglycemia- or glutamate-induced impairment of CA1 presynaptic potentials. Neuroprotection was obtained by in vivo application of cyanocobalamin, but not by its in vitro application. It is reported that in vivo injected cyanocobalamin converted to methylcobalamin in the hepatic cells. Therefore, the results suggest that a transmethylation reaction in the hippocampal regions may be involved in the methylcobalamin-induced functional recovery from ischemic impairment.

Animals↗

PubMed1995

Molecular states of fungal nuclease composed of heterogeneous subunits as estimated from the effects of urea and chelating agents.

Fungal nuclease composed of subunits with molecular weights of 8.0 x 10(4) (80K subunit), 5.0 x 10(4) (50K subunit), and 2.5 x 10(4) (25K subunit) (K. Ito, Y. Matsuura, and N. Minamiura (1994) Arch. Biochem. Biophys. 309, 160-167) was inactivated by urea and dissociated into its subunits. The urea inactivation depended on the concentration of urea, the incubation period and the temperature. The urea-inactivated enzyme had about 25% activity restored by removal of urea, and the native form of the enzyme was also reconstituted. The urea inactivation and the dissociation of subunits were almost completely prevented by Ca2+ but not by glycerol. The enzyme was also inactivated by ethylenediaminetetraacetic acid (EDTA). From this method of inactivation, the 50K and 25K subunits were still associated, but the complex showed no nuclease activity. About 80% of the activity of the EDTA-inactivated enzyme was restored by the addition of Ca2+ or Sr2+ and 20-40% by Mn2+, Ba2+, Mg2+, or Co2+. The reactivation of the enzyme by these metal ions was accompanied by the reconstitution of the native form of the enzyme. The enzyme was inactivated by ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) at much higher concentrations compared with the inactivation by EDTA. On the other hand, dissociation of the subunits by EGTA proceeded in a manner similar to that of the inactivation of the enzyme by EDTA. The 50K and 25K subunits were still associated, and the complex showed nuclease activity. These results indicated that the enzyme contains two kinds of metal ions. One metal ion, represented by Ca2+, is thought to stabilize the quanternary structure of the enzyme, especially to connect the 80K subunit and the complex of the 50K and 25K subunits. Another metal ion, represented by Mg2+, is thought to be bound to the complex of the 50K and 25K subunits and to be required for activity appearance of the complex. Along with these results, possible molecular states of the enzyme under various conditions are proposed.

Aspergillus↗