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Biomedical subjects

I Fujii

Publications and source records attributed to I Fujii.

At least 55 records · Page 3Linked to original sources
PubMed1996

Effects of antidepressants on intracellular Ca2+ mobilization in CHO cells transfected with the human 5-HT2C receptors.

Serotonin 5-HT2C receptor-mediated intracellular Ca2+ mobilization was investigated in 5-HT2C receptors expressed in Chinese hamster ovary (CHO) cells; and fura-2/AM was used to investigate the regulation of 5-HT2C receptor function. CHO cells, transfected with a cDNA clone for the 5-HT2C receptor, expressed 287 fmol/mg of the receptor protein as determined by mianserin-sensitive [3H]-mesulergine binding (kd = 0.49 nM). The addition of 5-HT mobilized intracellular Ca2+ in a dose-dependent fashion, ranging from basal level of 99 +/- 1.8 nM up to 246 +/- 21.2 nM, with an EC50 value for 5-HT of .015 microM. Exposure to 5-HT, a 5-HT receptor agonist, mCPP [1-(3-chlorophenyl)piperazine dihydrochloride], a 5-HT2C agonist, and DOI [1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane], a 5-HT2C and 5-HT2 agonist, resulted in increased intracellular Ca2+ levels. Mianserin, mesulergine, ritanserin, and ketanserin each blocked 5-HT-mediated intracellular Ca2+ mobilization more effectively than spiperone. Mianserin and amoxapine inhibited 5-HT-mediated intracellular Ca2+ mobilization completely; amitriptyline, nortriptyline, and imipramine reduced it about 50%. These results suggest that antagonism in CHO cells transfected with human 5-HT2C receptors is a component of the serotonergic properties of a number of established antidepressants.

Animals↗
PubMed1996

Cloning of chalcone-flavanone isomerase cDNA from Pueraria lobata and its overexpression in Escherichia coli.

Chalcone-flavanone isomerase (CHI) cDNA was isolated from Pueraria lobata by combination of cDNA library screening using Phaseolus vulgaris CHI cDNA as a probe and polymerase chain reaction techniques. Analysis of nucleotide sequence of the cloned cDNA revealed a 675-bp open reading frame encoding a 225-amino-acid polypeptide with a molecular weight of 23,803 Da. The CHI cDNA coding region was cloned into pET-3d expression vector and successfully overexpressed in Escherichia coli cells as active CHI enzyme. The recombinant CHI was then purified to apparent homogeneity by DEAE-cellulose column chromatography. Replacement of Cys-119 with Ala was carried out by site-directed mutagenesis and the result that the mutant CHI showed CHI enzyme activity confirmed that Cys-119 is not involved in the CHI catalytic active site.

Amino Acid Sequence↗
PubMed1996

Neuronal and vascular pathology produced by verocytotoxin 2 in the rabbit central nervous system.

To study the pathogenesis of the central nervous system (CNS) involvement associated with verocytotoxin-producing Escherichia coli infection, we developed an animal model by administering verocytotoxin 2 to rabbits either intravenously or intrathecally. After an interval of 2-9 days, the rabbits became paralyzed in a dose-dependent manner and in the absence of renal impairment. The minimal intravenous and intrathecal doses that produced these neurological signs were 250 and 0.4 ng/kg, respectively. After intravenous administration, most of the toxin was cleared from the serum within 24 h, with concomitant transition of a small amount into the cerebrospinal fluid. Pathological examination revealed that neurons in various CNS regions showed atrophy, cytoplasmic hyperchromasia and nuclear pyknosis as early as 6 h after administration. The distribution of affected neurons was constant and irrespective of the route of administration. Abnormalities of the blood vessels, such as the thickening of arterioles walls, were noted from 2 days after administration. The vascular lesions became more prominent after the intrathecal injection, which caused thrombosis and multiple infarction. Selective deposition of the toxin on the vessel walls was demonstrated immunohistochemically. Thus, the pathological manifestations of verocytotoxin 2 neurotoxicity consisted essentially of two types of lesions, early neuronal and late vascular, both of which might have developed under the influence of the toxin that had entered the CNS by crossing or circumventing the blood-brain barrier.

Animals↗
PubMed1996

Carbon dioxide induced panic attack in panic disorder in Japan.

1. The authors investigated the psychological and biochemical factors associated with challenge by 5% CO2-95% O2 inhalation for 20 min. While fifteen healthy people were used as control, thirteen cases who were diagnosed by DSM-III-R as suffering from panic disorder were used as subjects. CO2 inhalation induced panic in 38% of the panic disorder patients, but did not cause panic in any of the control cases (0%). 2. Acute panic inventory (API), heart rate and breathing rate of the panic group increased significantly after CO2 inhalation compared with the values in the control and non-panic groups. 3. Heart rates and systolic blood pressure were significantly higher those in the panic disorder and non-panic groups than in the control group prior to CO2 inhalation. The cortisol values in the panic and non-panic groups also were significantly higher than those in the control group before and after CO2 inhalation. 4. These results suggest elevated activity of the sympathetic nervous system during panic. The significantly higher heart rate, systolic blood pressure and cortisol values of the panic disorder subjects relative to the control before CO2 inhalation may have been due to circumstantial factors. The present findings of convincing evidence for behavioral, physiological, and biochemical hypersensitivity to CO2 in patients with panic disorders are consistent with a model of interoceptive conditioning in these patients.

Acute Disease↗
PubMed1996

Ligand recognition in mu opioid receptor: experimentally based modeling of mu opioid receptor binding sites and their testing by ligand docking.

For three-dimensional understanding of the mechanisms that control potency and selectivity of the ligand binding at the atomic level, we have analysed opioid receptor-ligand interaction based on the receptor's 3D model. As a first step, we have constructed molecular models for the multiple opioid receptor subtypes using bacteriorhodopsin as a template. The S-activated dihydromorphine derivatives should serve as powerful tools in mapping the three-dimensional structure of the mu opioid receptor, including the nature of the agonist-mediated conformational change that permits G protein-coupling to "second messenger' effector molecules, and in identifying specific ligand-binding contacts with the mu opioid receptor. The analyses of the interactions of some opioid ligands with the predicted ligand binding sites are consistent with the results of the affinity labeling experiments.

Affinity Labels↗
PubMed1996

CCK-4-induced calcium mobilization in T cells is enhanced in panic disorder.

We investigated the effects of brain cholecystokinin (CCK) receptors on the intracellular calcium concentration and protein kinase C in human T cells. CCK-4 produced a transient increase in calcium in the absence of extracellular calcium. CCK-B agonists stimulated calcium mobilization in a dose-dependent manner in T cells. CCK-B antagonists suppressed CCK-4-induced calcium mobilization more potently than CCK-A antagonist. The recovery of desensitization of the CCK-4-induced response was delayed by phosphoserine/phosphothreonine phosphatase inhibitor, calyculin A. The responsiveness to CCK-4 was also reduced by phorbol 12,13-dibutyrate (PDBu), and this effect of PDBu was abolished completely by preincubation with staurosporine. CCK-4-induced calcium mobilization was too small to attribute the desensitization to the protein kinase C transduction pathway. T cells from patients with untreated panic disorder exhibited significantly higher cholecystokinin-4-induced calcium mobilization than those from healthy controls or patients with treated panic disorder. These results suggest that cholecystokinin-B receptor function in T cells of patients with panic disorder is enhanced. Cholecystokinin-4-induced calcium mobilization in T cells may be state dependent and useful as a biological marker of panic disorder.

Adult↗
PubMed1996

Evaluation of unbound serum carbamazepine and carbamazepine-10,11-epoxide concentration prediction methods in polytherapy adult patients with epilepsy.

We retrospectively evaluated the ability of equations with in vivo population binding parameters of our previous study (Method 1) or an average unbound fraction of 0.25 of Pynnönen (Method 2) to predict the unbound serum carbamazepine (CBZ) concentration in 35 serum samples from 18 adult patients with epilepsy receiving polytherapy. In 9 serum samples from 6 patients, the ability of equations for unbound serum carbamazepine-10,11-epoxide (CBZ-E) concentration prediction was also determined in predictive performance with in vivo population binding parameters of our previous study (Method A) or an average unbound fraction of 0.5 of Pynnönen (Method B). Mean prediction error, mean absolute prediction error (MAE), and root mean squared error (RMSE) were calculated for each method, and these values served as a measure of prediction bias and precision. Method 1 shows a bias to underpredict unbound serum CBZ. The MAE and RMSE were smaller in Method 2 (MAE = 0.454 &mgr;m/L, RMSE = 0.597 &mgr;m/L) than Method 1 (MAE = 0.597 &mgr;m/L, RMSE = 0.721 &mgr;m/L). Method 2 is superior to Method 1 in accuracy and precision. The effects of antiepileptic comedications on predictive performance of Methods 1 and 2 were determined in each group of serum samples with (n = 18, Group 1) or without (n = 17, Group 2) valproic acid (VPA) comedication. The results obtained by Method 1 show a bias to underprediction in Group 1 and no bias to over- or underprediction in Group 2. Results obtained by Method 2 show no bias to over- or underprediction in Groups 1 and 2. The effects of VPA comedication on predictive performance of unbound serum CBZ are relatively larger in Method 1 than Method 2. There was a weak but significant positive relationship between age and unbound serum CBZ fraction by simple regression analysis (n = 35, r = 0.368, p = 0.0297). The determined coefficient indicated that only about 14% of variations in unbound serum CBZ fraction can be explained by age, however. In each of Groups 1 and 2, no significant relationship was observed between age and unbound serum CBZ fraction. The effects of age on predictive performance of unbound serum CBZ are relatively small in patients receiving polytherapy. For unbound CBZ-E prediction, each of Methods A and B has no bias to over- or underprediction. The MAE was larger in Method B (MAE = 0.311 &mgr;m/L) than Method A (MAE = 0.233 &mgr;m/L). The differences in RMSE were small between Methods A (RMSE = 0.349 &mgr;m/L) and B (RMSE = 0.333 &mgr;m/L), however. Each of Methods A and B may have similar accuracy and precision.

Journal Article↗
PubMed1996

Comparison of two binding equations for prediction of the concentration of unbound valproic acid in the serum of adult epileptic polytherapy patients.

Because binding of valproic acid to plasma proteins affects the efficacy of the drug in the treatment of epilepsy (only the unbound fraction of the drug is effective) we have compared two methods which use different binding parameters to predict the in-vivo concentration of unbound valproic acid in serum. The study was performed on 46 serum samples from 29 polytherapy adult patients with epilepsy. Mean prediction error, mean absolute prediction error and root mean squared error were calculated for each method; these values served as a measure of prediction bias and precision. The mean absolute prediction errors and root mean squared errors for the two methods were similar in magnitude (Method 1, mean absolute prediction error = 10.0 microM, root mean squared error = 15.0 microM; Method 2, mean absolute prediction error = 10.3 microM, root mean squared error = 13.5 microM). Method 2 had a general tendency to over-predict unbound valproic acid; both methods had a tendency to over-prediction for total concentrations above 500 microM. Method 1 had a tendency to under-prediction at total concentrations below 250 microM. Within the total concentration range of valproic acid investigated, Method 1 was superior to Method 2 for prediction of unbound serum valproic acid. Our approach using Method 1 may be useful for prediction of unbound serum valproic acid concentration in patients with total valproic acid concentrations ranging from 250 to 500 microM; Method 2 may be useful for patients with total valproic acid below 500 microM. Our results suggest that there is wide and unpredictable variability in valproic acid binding to serum proteins among study populations.

Adolescent↗
PubMed1996

Purification and characterization of sulochrin oxidase from Penicillium frequentans.

Sulochrin oxidase, an enzyme catalyzing regio- and stereospecific phenol oxidative coupling reaction to form (+)-bisdechlorogeodin from sulochrin, was isolated from Penicillium frequentans CMI 96659. By chromatographies on DEAE-cellulose, hydroxyapatite, Phenyl-Sepharose, Mono P, Mono Q, and HPLC gel filtration columns, sulochrin oxidase was purified to apparent homogeneity. The purified enzyme had a molecular weight of 230 K as estimated by gel filtration and 110 K as estimated by sodium dodecylsulfate-polyacrylamide gel electrophoresis under denaturing conditions, suggesting that the active enzyme was a homodimer. The enzyme showed pI 4.0 and an optimum pH of 6. The enzyme activity was strongly inhibited by the copper-chelating reagent, diethyldithiocarbamate, and did not recover its full activity even after removing the inhibitor by dialysis. However, enzyme activity was fully restored by the addition of Cu2+. Thus, sulochrin oxidase is considered to be a copper protein. The enzyme showed high substrate specificity for benzophenone compounds such as sulochrin and dihydrogeodin.

Alcohol Oxidoreductases↗
PubMed1996

[Catalytic antibodies: generation of a new class of protein catalysts exploiting the immune system].

Since the diversity of the immune response permits antibodies to be generated against virtually any substance, catalytic antibodies have the potential to provide catalysts for many chemical transformations. Such catalysts could be tailored by immunization with rationally designed transition state analogs, which is the main attraction of the catalytic antibody research field. However, when mice are immunized with a hapten conjugated to a carrier protein, a few, and occasionally several, of the dozens of antibodies that bind the hapten are characterized as catalytic. The diversity of the immune response can provide a panel of catalytic antibodies that possess varying degrees of catalytic activity and substrate specificity. These results have raised the following fundamental questions: how different are catalytic antibodies which were generated by immunization with a single hapten on a structural or mechanistic basis? We want to study the correlation between the antigen combining site structures and the chemical properties within a panel of catalytic antibodies elicited against a single hapten, which can potentially provide a more global understanding of the molecular mechanisms by which catalytic antibodies are generated in immune responses. We have demonstrated prodrug activation via catalytic antibodies that catalyze the hydrolysis of the nonbioactive chloramphenicol monoester derivative (1) to generate chloramphenicol (2). Immunization with a KLH conjugate of the transition state analog (3), designed on the basis of the transition state stabilization concept, yielded twelve immunoglobulin G (Ig G) proteins binding to 3, six of which were found to catalyze the hydrolysis with varying degrees of activity. This antibody system seems to be a suitable model for studies of the diversity of catalytic antibodies. Therefore, we have examined the biochemical and structural properties of these catalytic and non-catalytic antibodies, to enhance our understanding of the active site structure and function relationship assignments.

Amino Acid Sequence↗
PubMed1995

Molecular cloning and heterologous expression of the gene encoding dihydrogeodin oxidase, a multicopper blue enzyme from Aspergillus terreus.

Aspergillus terreus dihydrogeodin oxidase (DHGO) is an enzyme catalyzing the stereospecific phenol oxidative coupling reaction converting dihydrogeodin to (+)- geodin. We previously reported the purification of DHGO from A. terreus and raised polyclonal antibody against DHGO. From the first cDNA library constructed in lambda gt11 using mRNA from 3-day-old mycelium of A. terreus, four clones were identified using anti-DHGO antibody, but all contained partial cDNA inserts around 280 base pairs. This cDNA fragment was used as a probe to clone the genomic DNA and cDNA for dihydrogeodin oxidase from A. terreus. The sequence of the cloned DHGO genomic DNA and cDNA predicted that the DHGO polypeptide consists of 605 amino acids showing significant homology with multicopper blue proteins such as laccase and ascorbate oxidase. Four potential copper binding domains exist in DHGO polypeptide. The DHGO gene consists of seven exons separated by six short introns. Expression of the DHGO gene in Aspergillus nidulans under the starch or maltose-inducible Taka-amylase A promoter as an active enzyme established the functional identity of the gene. Also, introduction of the genomic DNA for DHGO into Penicillium frequentans led to the production of DHGO polypeptide as judged by Western blot analysis.

Amino Acid Sequence↗
PubMed1995

Comparison of equations for predicting bound serum concentrations of carbamazepine and carbamazepine-10, 11-epoxide after polytherapy in patients with epilepsy.

In a previous study, an equation with in vivo population binding parameters of carbamazepine and carbamazepine-10, 11-epoxide (CBZ-E) to serum proteins for the relation between unbound and bound serum concentrations was defined. A review by Pynnönen indicates that the average bound/unbound plasma fraction ratio is 3.0 for carbamazepine and 1.0 for CBZ-E. In this study, the ability of equations with in vivo population binding parameters of the previous study (method 1) or with the average bound/unbound plasma fraction ratio of 3.0 of Pynnönen (method 2) to predict the bound serum carbamazepine concentration was retrospectively evaluated using 85 serum samples from 46 patients with epilepsy taking carbamazepine polytherapy. In 21 serum samples from 16 patients, the ability of these equations to predict bound serum CBZ-E concentration was also determined with in vivo population binding parameters from the previous study (method A) or with the average bound/unbound plasma fraction ratio of 1.0 of Pynnönen (method B). Mean prediction error, mean absolute prediction error (MAE), and root mean squared error (RMSE) were calculated for each method, and these values served as a measure of prediction bias and precision. Method 1 showed a bias to overpredict bound serum carbamazepine. The MAE and RMSE were significantly smaller with method 2 (MAE = 2.4 mumol/L; RMSE = 3.2 mumol/L) than with method 1 (MAE = 4.1 mumol/L; RMSE = 4.8 mumol/L). Method 2 was superior to method 1 in terms of accuracy and precision. For bound CBZ-E prediction, method B had a bias to underprediction. The MAE and RMSE were smaller with method A (MAE = 0.581 mumol/L; RMSE = 0.796 mumol/L) than with method B (MAE = 0.724 mumol/L; RMSE = 0.905 mumol/L). Method A was superior to method B in terms of accuracy and precision.

Adolescent↗
PubMed1995

Evaluation of equations for unbound serum concentration prediction of carbamazepine and carbamazepine-10,11-epoxide in polytherapy pediatric patients with epilepsy.

We retrospectively evaluated the ability of equations with in vivo population binding parameters of our previous study (Method 1) or an average unbound fraction of 0.25 of Pynnönen (Method 2) to predict the unbound serum carbamazepine (CBZ) concentration in 50 serum samples from 28 polytherapy pediatric patients with epilepsy. In 12 serum samples from 10 patients, the ability of equations for unbound serum carbamazepine-10,11-epoxide (CBZ-E) concentration prediction was also determined in predictive performance with in vivo population binding parameters of our previous study (Method A) or an average unbound fraction of 0.5 of Pynnönen (Method B). Mean prediction error, mean absolute prediction error (MAE), and root mean squared error (RMSE) were calculated for each method, and these values served as a measure of prediction bias and precision. Method 1 shows a bias to underpredict unbound serum CBZ. The MAE and RMSE were lower in Method 2 (MAE = 0.696 microM, RMSE = 0.912 microM) than in Method 1 (MAE = 0.946 microM, RMSE = 1.138 microM). Method 2 is superior to Method 1 in accuracy and precision. The effects of antiepileptic co-medications on predictive performance of Method 1 are relatively larger in a co-medicated group of serum samples with valproic acid (n = 33, MAE = 0.994 microM, RMSE = 1.211 microM) than in a group of serum samples without valproic acid co-medication (n = 17, MAE = 0.853 microM, RMSE = 0.979 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1995

Transformations of Penicillium islandicum and Penicillium frequentans that produce anthraquinone-related compounds.

Wild-type strains of Penicillium islandicum and Penicillium frequentans, which produce anthraquinone and related compounds, were transformed to benomyl and hygromycin B resistance. Plasmids pSV50 and pBT6, with benomyl-resistant beta-tublin genes, and plasmids pAN7-1 and pDH25, with a bacterial hygromycin phosphotransferase gene under the control of Aspergillus nidulans sequences, were used respectively. Transformation frequencies with these plasmids were 10-20 transformants per micrograms of DNA per 4-8 x 10(7) viable protoplasts. Integration of plasmid DNAs into chromosomal DNAs was confirmed by Southern-blot analysis. Copy numbers and sites of integration varied among transformants. The integrated plasmid DNAs conferring a drug-resistant phenotype were mitotically stable with or without selection. The demonstration of such transformation systems in the essential first step in the application of recombinant DNA technology to study the biosynthetic genes of anthraquinone and related compounds in P. islandicum and P. frequentans.

Anthraquinones↗
PubMed1995

Rapid desensitization of serotonin 5-HT2C receptor-stimulated intracellular calcium mobilization in CHO cells transfected with cloned human 5-HT2C receptors.

Serotonin 5-HT2C receptor-mediated intracellular Ca2+ mobilization was investigated in Chinese hamster ovary (CHO) cells transfected with 5-HT2C receptors. Fura-2 acetoxymethyl ester was used to investigate the regulation of 5-HT2C receptor function. CHO cells, transfected with a cDNA clone for the 5-HT2C receptor, expressed 287 fmol/mg of the receptor protein as determined by mianserin-sensitive [3H]mesulergine binding (KD = 0.49 nM). The addition of 5-HT mobilized intracellular Ca2+ in a dose-dependent fashion, ranging from a basal level of 99 +/- 1.8 up to 379 +/- 18 nM, with an EC50 value for 5-HT of 0.029 microM. Exposure to 5-HT, 1-(3-chlorophenyl)piperazine dihydrochloride (a 5-HT2C agonist), and 1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane (a 5-HT2C and 5-HT2A agonist) resulted in increased intracellular Ca2+ levels. Mianserin, mesulergine, ritanserin, and ketanserin each blocked 5-HT-mediated intracellular Ca2+ mobilization more effectively than spiperone. The receptor was rapidly desensitized by preexposure to 5-HT in a time- and concentration-dependent manner. Mezerein and phorbol 12-myristate 13-acetate, protein kinase C activators, weakly inhibited the intracellular Ca2+ mobilization induced by 10 microM 5-HT. Furthermore, the protein kinase C inhibitor H-7 partially prevented the protein kinase C activator-induced inhibition of the 5-HT-mediated increase in intracellular Ca2+ concentration. The desensitization induced by pretreatment with 5-HT was blocked by W-7, added in conjunction with 5-HT, and partially inhibited by W-5, a nonselective inhibitor of protein kinases and weak analogue of W-7.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Polyketide synthase gene manipulation: a structure-function approach in engineering novel antibiotics.

Polyketides are produced primarily in microorganisms through a specialized metabolism that is a variation of fatty acid biosynthesis. A strong sequence and mechanistic similarity among many of the fatty acid and polyketide synthase enzymes has led to two paradigms for explaining polyketide biochemistry. In one, polyketides are formed by enzyme complexes consisting of four to seven monofunctional proteins in which the beta-carbonyl groups of the intermediates resulting from the condensation of acetate residues are largely not reduced and cyclization of the intermediates typically produces aromatic compounds. The intermediates in the other model are formed by multifunctional enzymes in which each of the initial condensation products is processed through reduction; reduction and dehydration; or reduction, dehydration, and further reduction cycles to produce highly reduced compounds from acetate, propionate, and butyrate residues. Expression of the genes encoding each type of polyketide synthase, or their mutant forms, has provided much information about the underlying biochemistry and, in some cases, resulted in the formation of novel natural products.

Anti-Bacterial Agents↗