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Biomedical subjects

M Itakura

Publications and source records attributed to M Itakura.

At least 91 records · Page 5Linked to original sources
PubMed1996

Detection of different quasispecies of hepatitis C virus core region in cancerous and noncancerous lesions.

The quasispecies of hepatitis C virus (HCV) core region in non-cancerous and cancerous hepatocellular carcinoma (HCC) lesions, respectively, of 7 patients with hepatocellular carcinoma were studied. Multiple fluorescence based-polymerase chain reaction (PCR)-single strand conformation polymorphism exhibited a different set and a larger number of quasispecies in cancerous portions than those in non-cancerous portions. DNA sequencing of PCR-amplified core region substantiated an accumulation of nucleotide substitutions, and a greater number of quasispecies in cancerous portions than those in non-cancerous portions. The deduced amino acid sequences disclosed that at the peptide position 45, Ser is dominant in non-cancerous lesions, and Gly in cancerous lesions, respectively. Thus, HCV in hepatocellular carcinoma includes a large number of specific quasispecies presumably due to their vigorous proliferation. A different set of quasispecies with the amino acid change is presumed to be related to the hepatocarcinogenesis.

Aged↗
PubMed1996

GGAAAT motifs play a major role in transcriptional activity of the human insulin gene in a pancreatic islet beta-cell line MIN6.

The insulin gene is specifically expressed in pancreatic islet beta cells. Various cis-acting DNA elements in the 5'-flanking region of the human insulin gene were examined for their contribution to the transcriptional activity using sensitive human growth hormone (hGH) reporter plasmids. The hGH constructs, having successively deleted human insulin promoter sequences, were transfected to a pancreatic islet beta-cell line MIN6. The deletion of two GGAAAT (GG) motifs, GG2 at -145 to -140 bp and GG1 at -134 to -129 bp, decreased the transcriptional activity to 6.5% of that of the promoter sequence from -156 to +1 bp. The selective mutations in both GG motifs also decreased the transcriptional activity to 5.5%. One-base mutations of GG2 and GG1 decreased the transcriptional activity to 82 and 11%, respectively. The two-base mutations between GG2 and GG1 affected the transcriptional activity more strongly than those just outside the GG motifs. A single set of GG motifs in the upstream of thymidine kinase promoter increased the transcriptional activity to 216% compared to that of thymidine kinase promoter alone in MIN6 cells. With an electrophoretic mobility shift assay (EMSA), a nuclear factor in MIN6 cells was shown to bind the DNA fragments containing two GG motifs. This factor did not bind to another GGAAAT-like sequence at -313 to -305 bp in the human insulin gene. These results suggested that the GG motifs contributed to the cell-specific transcription of the human insulin gene in association with the binding of the sequence-specific nuclear factor.

3T3 Cells↗
PubMed1996

Diabetic damage of selective renal reabsorption assessed by albumin negative charge.

To clarify the pathogenesis of diabetic albuminuria at its onset, the percentage of glycated albumin (%G-albumin) in excreted urinary albumin and its negative charge were assayed. In non-diabetic albuminuria (control-A) group, the high ratio of urinary %G-albumin to serum %G-albumin (urine/serum ratio of %G-albumin) suggested a selective renal excretion of glycated albumin (G-albumin) over albumin as a result of normally-functioning selective reabsorption of albumin over G-albumin in renal tubules. Urinary %G-albumin and albumin negative charge indexing the degree of glycation, which was assayed by the binding capacity of positively-charged Alcian Blue (ABBC), thus negatively correlated with serum %G-albumin. In non-insulin dependent diabetic subjects with albuminuria (DM-A), however, urinary %G-albumin and ABBC positively correlated with serum %G-albumin, and the urine/serum ratio of %G-albumin was low and gradually increased toward 1 as serum %G-albumin increased. Although the strict glycemic control for 3 weeks reduced the increased urinary %G-albumin and ABBC, the decreased urine/serum ratio of %G-albumin remained unaltered. It is concluded that hyperglycemia-induced renal damage starts with the loss of selective tubular reabsorption of albumin over G-albumin, and that this damage cannot be recovered by strict glycemic control for 3 weeks.

Adult↗
PubMed1996

Abnormality in membrane fatty acid compositions of cells measured on erythrocyte in alcoholic liver disease.

It has been proven that the fatty acids of esterified phospholipids in the cell membrane play an important role in membrane fluidity. Our previous in vitro experiment indicated that the impairment of erythrocyte membrane fluidity might be largely because of the change in fatty acids. The aim of this study is to clarify changes of cell membrane fatty acids in more detail in relation to various stages and pathology of alcoholic liver disease. For the analysis, erythrocyte membranes were exploited on the assumption that their fatty acid compositions may be similar to those of other organs. In alcoholic liver disease, unsaturated fatty acids in the erythrocyte membrane decreased and saturated fatty acids increased. Consequently, the unsaturated fatty acid/saturated fatty acid ratio decreased significantly. When fractions of saturated fatty acids were studied, myristic acid (C14:0) increased markedly in the alcoholic group, and the increase was striking particularly in the cases of alcoholic hepatitis concurrently with hemolysis. Palmitic acid (C16:0) also tended to increase in the alcoholic liver disease group. A longer chain saturated fatty acid, stearic acid (C18:0), showed a moderate but significant increase in the alcoholic fatty liver and hepatic fibrosis group, but it decreased significantly in the alcoholic liver cirrhosis, as with the finding in viral liver cirrhosis. As with unsaturated fatty acids, linoleic acid (C18:2), arachidonic acid (C20:4), and eicosapentanoic acid (C20:5) decreased significantly. The arachidonic acid/linoleic acid ratio, which indicates microsomal elongation activity of liver cells, was found to be broadly distributed. No significant change was found in each group of alcoholic liver disease. However, the cases showing a decrease in this ratio had severe hepatic dysfunction concurrently. Thrombogenic Index, serving as an indicator for fatty acids in food, and that is concerned with formation of thrombus, was studied, using fatty acid fractions of the erythrocyte membrane. The index was significantly increased in alcoholic liver disease. It was suggested that the chronic alcohol intake and the resultant liver diseases might enhance the abnormality of the membrane fatty acid composition. These changes may affect cell membrane fluidity and eventually metabolic functions of the cell.

Alcoholism↗
PubMed1996

Studies on quinolone antibacterials. IV. Structure-activity relationships of antibacterial activity and side effects for 5- or 8-substituted and 5,8-disubstituted-7-(3-amino-1-pyrrolidinyl)-1-cyclopropyl-1, 4-dihydro-4-oxoquinoline-3-carboxylic acids.

A series of 7-(3-amino-1-pyrrolidinyl)-1-cyclopropyl-1,4-dihydro-4- oxoquinoline-3-carboxylic acids bearing various substituents (H, F, C1, Me, OH, OMe, OEt, OCH2F, OCHF2, OCF3, SMe) at the C-8 position was prepared and evaluated for in vitro antibacterial activity against both standard laboratory strains and bacteria resistant to quinolones such as ciprofloxacin (CPFX, 1) and ofloxacin (OFLX, 2) from clinical isolates. The 8-methyl (8a), 8-fluoro (9a), 8-chloro (10a) and 8-methoxy (12a) compounds were 4 times more potent than CPFX (1) against both gram-positive and gram-negative bacteria. But these four compounds caused injury to the chromosomes of mammalian cells at a concentration of 100 micrograms/ml. Next, a series of quinolones having various substituents (H, C1, Me, NH2, NHMe, NMe2) at the C-5 position was prepared and evaluated for antibacterial activity and injurious effect on the chromosome. We found that the 5-amino-8-methyl compound (8d) showed strong antibacterial activity (in vitro antibacterial activity of 8d is 4 times more potent than that of CPFX (1) against both gram-positive and gram-negative bacteria), reduced injury to the chromosome, and reduced quinolone-type toxicity (free from both phototoxicity at a dosage of 30 mg/kg in guinea pigs (i.v.) and convulsion-inducing activity when coadministered with fenbufen at dosage of 100 mg/kg in mice (i.p.)).

4-Quinolones↗
PubMed1996

Studies on quinolone antibacterials. V. Synthesis and antibacterial activity of chiral 5-amino-7-(4-substituted-3-amino-1-pyrrolidinyl)-6- fluoro-1,4-dihydro-8-methyl-4-oxoquinoline-3-carboxylic acids and derivatives.

We previously demonstrated that 5-amino-7-(3-amino-1-pyrrolidinyl) -1-cyclopropyl-1,4-dihydro-8-methyl-4-oxoquinoline-3-carboxylic acid (7) has strong in vitro antibacterial activity even against quinolone-resistant bacteria. We examined optimization of the 3-aminopyrrolidine moiety of 7 by introduction of C-alkyl (Me, Et, Pr, di-Me, cyclopropyl) and N-alkyl groups (Me, di-Me). C-Alkylation at the 4-position of the 3-aminopyrrolidine moiety enhanced in vitro and in vivo antibacterial activity. (S)-5-Amino-7-(7-amino-5-azaspiro[2.4]hept-5-yl)-1-cyclopropyl-pyr rolidinyl) -1-cyclopropyl-6-fluoro-1,4-dihydro-8-methyl-4-oxoquinoline-3-carboxylic acid (15b) showed strong antibacterial activity (in vitro antibacterial activity including quinolone-resistant bacteria is 4 times more potent than that of ciprofloxacin (CPFX) (1); in vivo antibacterial activity is 1.5 to 20 times more potent than that of CPFX (1)) and reduced quinolone toxicity (free from both phototoxicity at a dosage of 30 mg/kg in guinea pigs (i.v.) and convulsion when coadministered with 4-biphenylacetic acid at a dosage of 20 micrograms in rats (i.c.v.)). Their selectivity between DNA topoisomerase II (derived from eukaryotic cells) and DNA gyrase (derived from bacterial cells) was about 3000-fold.

Animals↗
PubMed1996

Absence of mutations at codon 768 of the RET proto-oncogene in sporadic and hereditary pheochromocytomas.

Sixteen sporadic pheochromocytomas, 3 pheochromocytomas in neurofibromatosis 1, and 4 pheochromocytomas in multiple endocrine neoplasia (MEN) 2A or 2B were screened for mutations at codon 768 of the RET proto-oncogene by AluI digestion of polymerase chain reaction PCR products and mutations in exon 13 by PCR-single strand conformation polymorphism (SSCP) analysis. Although mutations at codon 768 (GAG --> GAC; Glu --> Asp) of the RET proto-oncogene were recently reported to be found in 40% of sporadic medullary thyroid carcinomas (MTCs), the absence of missense mutations at codon 768 was confirmed both with PCR-restriction fragment length polymorphism (RFLP) and PCR-SSCP analysis in all examined cases of pheochromocytomas. These results suggest that mutations at codon 768 of the RET proto-oncogene do not represent a frequent mechanism of tumorigenesis for both sporadic and hereditary pheochromocytomas.

Adrenal Gland Neoplasms↗
PubMed1996

Obvious mRNA and protein expression but absence of mutations of the RET proto-oncogene in parathyroid tumors.

The study on the expression of the RET proto-oncogene in parathyroid tumors disclosed obvious mRNA expression by the reverse transcription (RT)-polymerase chain reaction (PCR) method and protein expression by Western blotting. To find out whether mutations in the cysteine-rich regions or tyrosine kinase domain of the RET proto-oncogene are etiological for parathyroid tumorigenesis, sporadic parathyroid adenomas and carcinomas, parathyroid tumors from multiple endocrine neoplasia 1, familial isolated hyperparathyroidism or hereditary hyperparathyroidism-jaw tumor syndrome were screened by PCR-single strand conformation polymorphism and PCR restriction fragment length polymorphism. Missense mutations in the cysteine-rich region, or codons 768 or 918 in the tyrosine kinase domain of the RET proto-oncogene, were not detected in any of the examined cases of parathyroid tumors. These results suggest that mutations of the RET proto-oncogene do not represent a frequent mechanism of tumorigenesis for parathyroid tumors.

Adult↗
PubMed1996

Multiple fluorescence-based PCR-SSCP analysis using internal fluorescent labeling of PCR products.

The method to internally label PCR products with multiple colored fluorescent dyes was developed and applied to multiple fluorescence-based PCR single-stranded conformational polymorphism (MF-PCR-SSCP) analysis. PCR-amplified fluorescent DNA fragments, which were internally labeled by adding fluorescent dUTPs ([F]dUTPs) to the PCR mixture, were heat-denatured and applied to a nondenaturing polyacrylamide gel (SSCP gel) set on an automated DNA sequencer with a gel temperature-controlling system. The image data were analyzed by GENESCAN 672 software. In spite of differences in species and amount of integrated [F]dUTPs, the SSCP profiles were not significantly affected, even by the different labeling methods used-internal labeling or post-labeling at the 3' ends-in regard to the three different [F]dUTPs examined. However, the salt concentration of the solution containing the DNA samples affected the SSCP profiles. The internally labeled [F]dUTP-containing DNA fragments beyond 1000 bp in length were successfully digested with restriction endonucleases and subjected to SSCP analysis. MF-PCR-SSCP analysis with internal fluorescence labeling affords a simple and sensitive method to detect alterations in DNA sequences.

DNA Mutational Analysis↗
PubMed1996

Lack of CD54 expression and mutation of p53 gene relate to the prognosis of childhood Burkitt's lymphoma.

Expression of the intercellular adhesion molecule-1 (CD54) as well as the mutations of p53 gene were studied in childhood Burkitt's lymphoma (BL). Expression of CD54 was identified in 6 of 15 fresh BL cases. Mutations of p53 gene, analyzed by polymerase chain reaction-single stranded chain polymorphism followed by sequencing, were found in 5 of 14 cases examined. Interestingly, all the cases with p53 mutation were CD54 negative. This high frequency of p53 mutation in the CD54 negative group prompted us to analyze the clinical features of these cases. Six of 15 cases died within 21 months after initiation of therapy and five of these were CD54 negative. In addition, four of these had p53 mutation. These results suggest that the lack of CD54 by BL cells may provide the background for the mutation of p53 gene to occur which could result in the transformation to a more aggressive phenotype.

Adolescent↗
PubMed1996

[Evaluation of risk factors of interferon-associated retinopathy in patients with type C chronic active hepatitis].

The risk factors of retinopathy associated with administration of interferon have not been fully clarified. We prospectively examined the retinal condition in 50 patients with type C chronic active hepatitis during alpha-interferon treatment. 43 patients (86%) were shown to have retinopathy during the course of interferon treatment, and were divided into three groups. Grades I, II and III were patients having a single episode of transient retinopathy with soft exudate or hemorrhage (34%), frequent episodes of retinopathy (42%), and exacerbating retinopathy requiring change or cessation of interferon treatment (10%), respectively. The patients with grade II and III were found to have the first retinal changes within 8 weeks after initiation of the interferon therapy. Early onset of retinopathy and presence of systemic disease such as diabetes mellitus or hypertension were risk factors for serious retinopathy with statistical significance. The grades of retinopathy were also well correlated with dosage and duration of interferon treatment. These results suggest that careful fundus examination is required up to 8 weeks after initiation of interferon treatment, especially for the patients with risk factors such as early onset of retinopathy, presence of systemic diseases, and large dosages and long duration of interferon therapy, in order to prevent serious ocular complications.

Adult↗
PubMed1996

Loss of ganciclovir sensitivity by exclusion of thymidine kinase gene from transplanted proinsulin-producing fibroblasts as a gene therapy model for diabetes.

To establish a practical method of somatic gene therapy, we aimed to develop a regulatory system at the cellular level using a suicide vector. We introduced the herpes simplex virus type 1 thymidine kinase (HSV-tk) gene into the human proinsulin-producing Ltk-cells and examined whether ganciclovir (GCV) administration could control proinsulin production in vivo. The cells transfected with the HSV-tk gene showed more than 100-fold increase in sensitivity to GCV compared with the parent cells. Analysis of blood glucose in diabetic nude mice with transplanted cells showed that proinsulin production by these cells was strongly suppressed by GCV treatment in vivo as reflected by the reversal to hyperglycemia. However, in the in vivo experiment, the plasmid containing the HSV-tk gene was spontaneously lost from the transplanted cells in one of six cases resulting in the resistance to GCV as reflected by the persistent hypoglycemia and increased tumor size. This system of HSV-tk and administration of GCV may be applicable to gene therapy as a suicide vector, but the system of stable expression of the HSV-tk gene must be established.

Animals↗
PubMed1996

[Metabolism of purine nucleotides and the production of uric acid].

The molecular and biochemical aspects of purine nucleotide biosynthesis through de novo and salvage pathways, the production of uric acid, and their regulation mechanisms are reviewed for further understanding of hyperuricemia and gout. The metabolic rate of purine nucleotide biosynthesis is chiefly determined by the regulation of the de novo pathway, especially amidophosphoribosyltransferase and PRPP synthetase, and the accumulation of uric acid results from the acceleration of de novo biosynthesis and catabolism of purine nucleotide or the decrease in urinary excretion of uric acid. Moreover, several enzyme mutations of purine nucleotide metabolism are also clinically important including gout with hyperactive HPRT and the deficiency of HPRT (Lesch-Nyhan syndrome), adenylosuccinate lyase, xanthine oxidase, APRT, PNP, or ADA (SCID) with gene therapy.

Amidophosphoribosyltransferase↗
PubMed1996

[Amidophosphoribosyltransferase].

Amidophosphoribosyltransferase (ATase) is the supposed regulatory allosteric enzyme of de novo purine nucleotide biosynthesis. ATase cDNAs and genomic DNAs were cloned from 15 different species. The chicken, rat, and human ATase genes and AIRC (aminoimidazole ribonucleotide carboxylase) genes are closely linked and divergently transcribed from an intergenic regions of 0.2-0.6 kb. The crystal structure of B. subtilis ATase was determined. The ATase tetramer is a doughnut-shaped molecule and each ATase subunit is organized in two domains of approximately equal size. The four [4 Fe-4 S] clusters are located at the corners of the tetramer. The activity of ATase is regulated positively by PRPP and negatively by GMP and AMP.

Amidophosphoribosyltransferase↗
PubMed1996

[Inherited disorders of uric acid metabolism--classification, enzymatic- and DNA-diagnosis].

Uric acid is the end product of purine metabolism in human. Then, the enzymatic abnormalities, concerning purine metabolism, cause disorders of uric acid metabolism including hyperuricemia and hypouricemia. The superactivity of 5-phosphoribosyl-pyrophosphate (PRPP) synthetase and deficiency of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) caused hyperuricemia. In glycogen storage diseases of type I, III, V, and VII, decreased energy supply induces hyperuricemia by accelerating ATP degradation. Deficiencies of xanthine oxidase (XO), purine nucleoside phosphorylase (PNP), and PRPP were reported causing hypouricemia. Many methods for DNA-diagnosis were developed including Southern blot, Northern blot, PCR-SSCP (polymerase chain reaction-single strand conformation polymorphism), PCR-RFLP (restriction fragment length polymorphism), and allele specific oligonucleotide hybridization etc.

Adenosine Triphosphate↗
PubMed1996

Detection of herpes simplex virus DNA in tear fluid of stromal herpetic keratitis patients by nested polymerase chain reaction.

The value of the polymerase chain reaction (PCR) in the diagnosis of stromal herpetic keratitis was examined using tear fluid specimens; the sensitivity of the nested PCR method in detecting the herpes simplex virus (HSV) genome was 1 plaque-forming unit/mL. PCR assay of 72 tear samples from the eyes of 15 patients with stromal herpetic keratitis detected the HSV genome in 5 (33.3%). No positive band was detected in 20 tear samples from both eyes of 10 healthy volunteers. Seven of 38 tear samples (18.4%) and 1 of 34 samples (2.9%) collected from the diseased eye and the contralateral normal eye, respectively, produced positive results; the difference in rates was statistically significant (P < 0.05). The positive rate of samples collected from the diseased eye during an active phase was 16.0% (4 of 25 samples); in the quiescent phase, 23.1% (3 of 13 samples); the difference was not significant (P = 0.45). In HSV genome-positive cases, the average number of tear collections needed to detect the HSV genome was 3.3. Results indicate that PCR assay of tear fluid provides valuable information for the diagnosis of stromal herpetic keratitis, and that repeated tear samples should be collected regardless of the phase of the phase of disease activity.

Administration, Topical↗
PubMed1995

Cloning, characterization, and tissue distribution of porcine SPAI, a protein with a transglutaminase substrate domain and the WAP motif.

The primary and gene structures and tissue distribution of porcine SPAI-2, a protein that belongs to the WAP protein superfamily and has a sodium-potassium ATPase inhibitory activity, were determined by molecular cloning and Northern analysis. A full-length cDNA clone was isolated from a porcine duodenum cDNA library. The cDNA insert encoded a polypeptide of 187 amino acids, which is composed of three domains: a hydrophobic presequence of 21 amino acids, a prosegment of 105 amino acids ending with Asp126, and the mature SPAI-2 sequence of 61 amino acids beginning with Pro127. The prosegment contained 16 repeats of a hexapeptide that is highly homologous to the repetitive sequence found in the transglutaminase domain of the human elafin, an elastase-specific inhibitor that also belongs to the WAP superfamily. The repetitive sequence was demonstrated to be a good substrate of transglutaminase using a recombinant preparation produced in Escherichia coli. A porcine genomic library was then screened for the SPAI gene. Characterization and sequencing of positive clones indicated that the gene is similar to the elafin gene, having 3 exons encoding the 5'-untranslated region and signal sequence, proSPAI, and 3'-untranslated region, respectively. Northern blot analysis revealed intestine-specific expression of SPAI mRNA; the message was especially abundant in the small intestine. ProSPAI was also found in the circulation. The similarity of proSPAI to elafin in the domain structure, the acid-labile nature of the cleavage site (Asp126-Pro127), and the fact that the major form of SPAI in the plasma is proSPAI strongly suggest that proSPAI is not the precursor but rather it is the native form of SPAI. Like elafin, therefore, SPAI appears to be a new type of biologically active substance with a transglutaminase substrate domain that acts as an anchoring sequence.

Amino Acid Sequence↗