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Biomedical subjects

T Shimada

Publications and source records attributed to T Shimada.

At least 415 records · Page 23Linked to original sources
PubMed1997

[Recent topics in the medical treatment of reflux esophagitis].

Reflux esophagitis is one of the most common disorders of the upper gastrointestinal tract. It can lead to obstruction through stricture formation, in more severe form to bleeding through ulceration, and to cancer development through the association of Barrett's esophagus. The vast majority of esophagitis can be managed medically. Medical management is separated into two categories: (1) life style modification and (2) drug therapy. Drug therapy includes antacids, prokinetics, sucralfate, H2 receptor antagonists (H2-RAs) and proton pump inhibitors (PPIs). Among these, antisecretory therapy is the mainstay for the treatment. PPIs are shown to be superior to H2-RAs in healing of esophagitis and symptom relief. Recurrence, particularly of erosive esophagitis, is common without maintenance therapy. PPIs are also consistently superior to H2-RAs in maintenance of esophagitis healing. Interestingly, a recent report has suggested that curing Helicobacter pylori infection may provoke reflux esophagitis, raising the possibility that the gastroesophageal reflux diseases become more common in the future. Therefore, treatment strategy for reflux esophagitis needs to be re-established in terms of the future cost-effectiveness evaluation and quality-of-life assessments.

Antacids↗
PubMed1996

Gene therapy for adult T cell leukemia using human immunodeficiency virus vector carrying the thymidine kinase gene of herpes simplex virus type 1.

Adult T cell leukemia/lymphoma (ATL) is derived from CD4+ T cells and has a poor prognosis because of its resistance to chemotherapy. To evaluate the effectiveness of gene therapy for ATL, the effect of ganciclovir on ATL cell lines transfected with the thymidine kinase gene of herpes simplex virus type 1 (HSV-TK) was analyzed. To transfer the HSV-TK gene to ATL cells, a human immunodeficiency virus (HIV) vector that has specific infectivity to CD4+ cells was used. HSV-TK was inserted into the long terminal repeats of HIV-1 and driven by the SL3 promoter HXBSL3TK. HXBSL3TK was co-transfected with HXBCAT as a reporter into MT2 or HUT102 cells by DEAE-dextran. The cells were incubated with ganciclovir, and chloramphenicol acetyltransferase (CAT) activity was analyzed. The CAT activity of the MT2 cells and HUT102 cells transfected with HXBSL3TK decreased dose-dependently with ganciclovir. HXBSL3TK was also co-transfected into COS cells with an HIV-1 packaging vector that has gag, pol, and env driven by a cytomegalovirus promoter. The supernatant was transferred to MT2 cells or Raji cells and incubated with ganciclovir. Ninety percent of the MT2 cells transduced by HXBSL3TK and incubated with ganciclovir were killed, but Raji cells were not killed. In addition, HXBTK that expresses the HSV-TK gene and Tat gene driven by the LTR of HIV-1 was constructed. HXBTK had a higher expression of the HSV-TK gene and higher sensitivity to ganciclovir than did HXBSL3TK.

Animals↗
PubMed1996

Two-step gene transfer using an adenoviral vector carrying the CD4 gene and human immunodeficiency viral vectors.

Human immunodeficiency virus-1 (HIV-1) belongs to the lentivirus subfamily of retroviruses and has several interesting features, including T cell tropism and the ability to infect nondividing cells. Replication-incompetent HIV vectors were developed and were shown to be capable of targeted gene transfer into CD4+T cells. This strict T cell tropism may be important for the development of gene therapy of acquired immunodeficiency syndrome (AIDS), but it hampers the use of the HIV vector for other gene transfer applications. To expand the host range of the HIV vector, we established the two-step gene transfer system, which allows us to transduce non-T cells stably. In the first step, the CD4 gene was introduced into target cells using a replication-defective adenoviral vector. Transient but high-level expression of CD4 molecules was detected in both adherent and floating cells. In the subsequent step, the cells were incubated with HIV vectors. Stable integration of the HIV vector was demonstrated in cells transduced with the adenoviral vector. These results indicate that transient expression of CD4 molecules by the adenoviral vector is sufficient to render non-T cells susceptible to HIV-mediated gene transfer. This two-step gene transfer strategy may be used as a general method to transduce various types of human cells stably including nondividing cells.

Adenoviridae↗
PubMed1996

Doppler estimation of pulmonary artery end-diastolic pressure using contrast enhancement of pulmonary regurgitant signals.

Pulmonary artery (PA) end-diastolic pressure is used as an estimate of PA wedge pressure. We evaluated contrast enhanced pulmonary regurgitant signals in the assessment of PA end-diastolic pressure in 24 patients in a critical care unit. Right atrial pressure was estimated by the percent decrease of the inferior vena caval diameter with inspiration. Weak or absent pulmonary regurgitant signals were enhanced by sonicated albumin (Albunex) in 23 patients (96%). The Doppler-determined PA end-diastolic pressure (the sum of the pulmonary regurgitant pressure gradient at end-diastole and the right atrial pressure) was significantly correlated with the catheter-determined PA end-diastolic pressure (y = 0.85x + 1.72, r = 0.93). Compared with invasive hemodynamic monitoring, the contrast-enhanced Doppler technique using Albunex is effective for measuring PA end-diastolic pressure, even in critically ill patients.

Aged↗
PubMed1996

Lack of electron transfer from cytochrome b5 in stimulation of catalytic activities of cytochrome P450 3A4. Characterization of a reconstituted cytochrome P450 3A4/NADPH-cytochrome P450 reductase system and studies with apo-cytochrome b5.

Many catalytic activities of cytochrome P450 (P450) 3A4, the major human liver P450 enzyme, require cytochrome b5 (b5) for optimal rates. The stimulatory effect of b5 on P450 reactions has generally been thought to be due to transfer of electrons from ferrous b5 to the ferrous P450-O2-substrate complex. We found that apo-b5, devoid of heme, could substitute for b5 in stimulating two prototypic activities, testosterone 6beta hydroxylation and nifedipine oxidation. The stimulatory effect was not seen with albumin, hemoglobin, catalase, or cytochrome c. Apo-b5 could not substitute for b5 in a testosterone 6beta hydroxylation system composed of NADH-b5 reductase and P450 3A4. Rates of electron transfer from NADPH-P450 reductase to ferric P450 3A4 were too slow (<2 min-1) to support testosterone 6beta hydroxylation ( approximately 14 min-1) unless b5 or apo-b5 was present, when rates of approximately 700 min-1 were measured. The oxidation-reduction potential (Em,7) of the ferric/ferrous couple of P450 3A4 was unchanged ( approximately -310 mV) under different conditions in which the kinetics of reduction were altered by the addition of substrate and/or apo-b5. Rapid reduction of P450 3A4 required substrate and a preformed complex of P450 3A4, NADPH-P450 reductase, and b5; the rates of binding of the proteins to each other were 2-3 orders of magnitude less than reduction rates. We conclude that b5 can facilitate some P450 3A4-catalyzed oxidations by complexing with P450 3A4 and enhancing its reduction by NADPH-P450 reductase, without directly transferring electrons to P450.

Animals↗
PubMed1996

hMutSbeta, a heterodimer of hMSH2 and hMSH3, binds to insertion/deletion loops in DNA.

In human cells, mismatch recognition is mediated by a heterodimeric complex, hMutSalpha, comprised of two members of the MutS homolog (MSH) family of proteins, hMSH2 and GTBP [1,2]. Correspondingly, tumour-derived cell lines defective in hMSH2 and GTBP have a mutator phenotype [3,4], and extracts prepared from these cells lack mismatch-binding activity [1]. However, although hMSH2 mutant cell lines showed considerable microsatellite instability in tracts of mononucleotide and dinucleotide repeats [4,5], only mononucleotide repeats were somewhat unstable in GTBP mutants [4,6]. These findings, together with data showing that extracts of cells lacking GTBP are partially proficient in the repair of two-nucleotide loops [2], suggested that loop repair can be GTBP-independent. We show here that hMSH2 can also heterodimerize with a third human MSH family member, hMSH3, and that this complex, hMutSbeta, binds loops of one to four extrahelical bases. Our data further suggest that hMSH3 and GTBP are redundant in loop repair, and help explain why only mutations in hMSH2, and not in GTBP or hMSH3, segregate with hereditary non-polyposis colorectal cancer (HNPCC) [7].

Base Sequence↗
PubMed1996

Requirements for cytochrome b5 in the oxidation of 7-ethoxycoumarin, chlorzoxazone, aniline, and N-nitrosodimethylamine by recombinant cytochrome P450 2E1 and by human liver microsomes.

NADH-dependent 7-ethoxycoumarin O-deethylation activities could be reconstituted in systems containing cytochrome b5 (b5), NADH-b5 reductase, and bacterial recombinant P450 2E1 in 100 mM potassium phosphate buffer (pH 7.4) containing a synthetic phospholipid mixture and cholate. Replacement of NADH-b5 reductase with NADPH-P450 reductase yielded a 4-fold increase in 7-ethoxycoumarin O-deethylation activity, and further stimulation (approximately 1.5-fold) could be obtained when NADPH was used as an electron donor. Removal of b5 from the NADH- and NADPH-supported systems caused a 90% loss of 7-ethoxycoumarin O-deethylation activities in the presence of NADPH-P450 reductase, but resulted in complete loss of the activities in the absence of NADPH-P450 reductase. Km values were increased and Vmax values were decreased for 7-ethoxycoumarin O-deethylation when b5 was omitted from the NADPH-supported P450 2E1-reconstituted systems. Requirements for b5 in P450 2E1 systems were also observed in chlorzoxazone 6-hydroxylation, aniline p-hydroxylation, and N-nitrosodimethylamine N-demethylation. In human liver microsomes, NADH-dependent 7-ethoxycoumarin O-deethylation, chlorzoxazone 6-hydroxylation, aniline p-hydroxylation, and N-nitrosodimethylamine N-demethylation activities were found to be about 55, 41, 33, and 50%, respectively, of those catalyzed by NADPH-supported systems. Anti-rat NADPH-P450 reductase immunoglobulin G inhibited 7-ethoxycoumarin O-deethylation activity catalyzed by human liver microsomes more strongly in NADPH- than NADH-supported reactions, while anti-human b5 immunoglobulin G inhibited microsomal activities in both NADH- and NADPH-supported systems to similar extents. These results suggest that b5 is an essential component in P450 2E1-catalyzed oxidations of several substrates used, that about 10% of the activities occur via P450 2E1 reduction by NADPH-P450 reductase in the absence of b5, and that the NADH-supported system contributes, in part, to some reactions catalyzed by P450 2E1 in human liver microsomes.

Aniline Compounds↗
PubMed1996

Activation of chemically diverse procarcinogens by human cytochrome P-450 1B1.

A human cytochrome P-450 (P450) 1B1 cDNA was expressed in Saccharomyces cerevisiae and the microsomes containing P450 1B1 were used to examine the selectivity of this enzyme in the activation of a variety of environmental carcinogens and mutagens in Salmonella typhimurium TA1535/pSK1002 or NM2009 tester strains, using the SOS response as an end point of DNA damage. We also determined and compared these activities of P450 1B1 with those catalyzed by recombinant human P450s 1A1 and 1A2, which were purified from membranes of Escherichia coli. The carcinogenic chemicals tested included 27 polycyclic aromatic hydrocarbons and their dihydrodiol derivatives, 17 heterocyclic and aryl amines and aminoazo dyes, three mycotoxins, two nitroaromatic hydrocarbons, N-nitrosodimethylamine, vinyl carbamate, and acrylonitrile. Among the three P450 enzymes examined here, P450 lB1 was found to have the highest catalytic activities for the activation of 11,12-dihydroxy-11,12-dihydrodibenzo[a,l]pyrene, 1,2-dihydroxy-1,2-dihydro-5-methylchrysene, (+)-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene, 11,12-dihydroxy-11,12-dihydrobenzo[g]chrysene, 3,4-dihydroxy-3,4-dihydrobenzo[c]phenanthrene, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole, 2-aminoanthracene, 3-methoxy-4-aminoazobenzene, and 2-nitropyrene. P450 1B1 also catalyzed the activation of 2-amino-3,5-dimethylimidazo[4,5-f]quinoline, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, 2-amino-3-methylimidazo[4,5-f]quinoline, 2-aminofluorene, 6-aminochrysene and its 1,2-dihydrodiol, (-)-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene, 1,2-dihydroxy-1,2-dihydrochrysene, 1,2-dihydroxy-1,2-dihydro-5,6-dimethylchrysene, 2,3-dihydroxy-2,3-dihydrofluoranthene, 3,4-dihydroxy-3,4-dihydro-7,12-dimethylbenz[a]anthracene, and 6-nitrochrysene to appreciable extents. However, P450 1B1 did not produce genotoxic products from benzo[a]pyrene, trans- 3,4-dihydroxy-3,4-dihydrobenzo[a]anthracene, trans-8,9-dihydroxy-8,9-dihydrobenzo[a]anthracene, 7,12-dimethylbenz[a]anthracene and its cis-5,6-dihydrodiol, 5-methylchrysene, 11,12-dihydroxy-11,12-dihydro-3-methylcholanthrene, 1,2-dihydroxy-1,2-dihydro-6-methylchrysene, benzo[c]phenanthrene, 2-amino-6-methyldipyridol[1,2-a:3',2'-d]imidazole, 2-acetylaminofluorene, benzidine, 2-naphthylamine, aflatoxin B1, aflatoxin G1, sterigmatocystin, N-nitrosodimethylamine, vinyl carbamate, or acrylonitrile in this assay system. P450 1B1 is expressed constitutively in extrahepatic organs, including fetal tissue samples, and is highly inducible in various organs by 2,3,7,8-tetrachlorodibenzo-p-dioxin and related compounds in experimental animal models. Thus, activation of procarcinogens by P450 lB1 may contribute to human tumors of extrahepatic origin.

Adult↗
PubMed1996

Construction of a human cytochrome P450 1A1: rat NADPH-cytochrome P450 reductase fusion protein cDNA and expression in Escherichia coli, purification, and catalytic properties of the enzyme in bacterial cells and after purification.

A plasmid (pCW) was modified to code for a fusion protein consisting of the complete sequence of human cytochrome P450 (P450) 1A1 (with only the second amino acid changed) in the N-terminal portion connected by a Ser-Thr linker to the portion of rat NADPH-P450 reductase beginning at amino acid 57. This plasmid was used to express the fusion protein in Escherichia coli DH5alpha cells and the protein was purified from detergent-solubilized bacterial membranes using DEAE and 2',5'-ADP agarose chromatography. The purified fusion protein catalyzed benzo[a]pyrene 3-hydroxylation, 7-ethoxyresorufin O-deethylation, and zoxazolamine 6-hydroxylation. Catalytic activity was not increased in the presence of added NADPH-P450 reductase, cytochrome b5, or phospholipid. The fusion protein could also transfer electrons to cytochromes c and b5 but not P450 lA2. The same oxidation products of benzo[a]pyrene were formed with the purified fusion protein and the fusion protein functioning in bacterial cells. The catalytic activity of the human P450 1A1 fusion protein toward several substrates is markedly less than that of a similar fusion protein constructed with rat P450 1A1, in line with the reported differences in catalytic activities of the rat and human P450 1A1 enzymes. The purified fusion protein also oxidized (+)- and (-)-benzo[a]pyrene 7,8-dihydrodiols and eight aryl and heterocyclic amines to genotoxic products, in the absence of added NADPH-P450 reductase. The demonstration of catalytic activities of the human fusion protein within bacterial cells suggests the prospect of utilizing such cellular systems for production of human P450 metabolites.

Animals↗
PubMed1996

A new strategy for large-scale preparation of high-titer recombinant adeno-associated virus vectors by using packaging cell lines and sulfonated cellulose column chromatography.

The extensive testing of adeno-associated virus (AAV) as a vector for human gene therapy has been hampered by low efficiency of the current packaging system, which is based on transient transfection with plasmid DNAs and infection with adenovirus in permissive cells. In an effort to resolve this problem, HeLa cell-based packaging cell lines were established. These packaging cells carry multiple copies of the AAV genome lacking the inverted terminal repeat (ITR) sequences. The AAV genes were silent in these cells but inducibly expressed by adenovirus infection. When the AAV vector plasmid containing the neoR gene flanked by the ITRs was also integrated into these cells, efficient production of the recombinant AAV particles occurred after adenovirus infection. AAV vector particles in cell lysates could be concentrated by sulfonated cellulose column chromatography. Using the packaging cells and the column chromatography technique, it is possible to prepare AAV vectors with the titer of higher than 10(8) cfu/ml or 5 x 10(10) particles/ml. This new strategy should be useful for testing AAV vectors in vivo.

Cellulose↗
PubMed1996

7-Ethoxycoumarin O-deethylation catalyzed by cytochromes P450 1A2 and 2E1 in human liver microsomes.

7-Ethoxycoumarin O-deethylation has been used widely as a marker activity for assessing substrate specificities of cytochromes P450 (P450) in liver microsomes of mammals, and extensive studies have shown that in rats and mice the major catalysts are P450 1A1, 1A2, and 2B enzymes. In contrast to findings in experimental animal models, P450 2E1 has been reported to be a principal enzyme involved in 7-ethoxy-coumarin O-deethylation in human livers. In this study, we further examined the roles of individual forms of human P450 involved in 7-ethoxycoumarin O-deethylation using microsomes from different human liver samples and from human lymphoblastoid cells expressing human P450 enzymes and purified P450 enzymes isolated from the membrane of Escherichia coli expressing modified P450 proteins. Kinetic analysis showed that there were at least two different enzymes involved in 7-ethoxycoumarin O-deethylation in different human samples. Samples that contained high amounts of P450 2E1 in liver microsomes showed biphasic curves for O-deethylation with relatively high turnover numbers, whereas P450 1A2-rich samples tended to have low Km values with low Vmax values. Anti-human P450 2E1 antibodies inhibited markedly (P < 0.05) the 7-ethoxycoumarin O-deethylation activities catalyzed by human liver microsomes particularly when examined at a high substrate concentration (200 microM). However, we also found that anti-P450 1A2 antibodies suppressed O-deethylation activities only at a low substrate concentration (10 microM). Recombinant human P450 1A2 was found to have a low Km value for 7-ethoxycoumarin O-deethylation, whereas P450 2E1 showed a high Km value. Of the P450 enzymes examined, P450 1A1 gave the highest O-deethylation activities with a low Km value, although this enzyme is reported to be expressed extrahepatically in humans. Other human P450 enzymes, including P450 2A6, 2C10, 2D6, 3A4, and 3A5, did not show significant O-deethylation activities except that P450 2B6, a minor P450 component in human livers, was found to have a Vmax value similar to that of P450 1A2 and a Km value similar to that of P450 2E1. These results suggest that P450 1A2 is a low Km enzyme for 7-ethoxycoumarin O-deethylation in human liver microsome, although it has a low Vmax value than P450 2E1.

7-Alkoxycoumarin O-Dealkylase↗
PubMed1996

Genomic organization and expression of the human MSH3 gene.

We have studied the expression and genomic organization of the human MSH3 gene, which encodes a human homologue of the bacterial DNA mismatch repair protein MutS. This gene is located upstream of the dihydrofolate reductase (DHFR) gene. Northern analysis has demonstrated that the hMSH3 gene is expressed in a variety of human tissues at low levels, like the DHFR gene. Characterization of cosmid clones has shown that the hMSH3 gene consists of 24 exons spanning at least 160 kb. All exon-intron junction sequences match the classical GT/AG rule, except that intron 6 has AT and AA at the ends. Two major transcripts of 5.0 and 3.8 kb have been shown to be derived from the differential use of two polyadenylation sites. Elucidation of the complete genomic organization and the nucleotide sequences of the introns of the hMSH3 gene should be useful for studying the function of this gene and the possible involvement of specific mutations of the hMSH3 gene in some diseases.

Animals↗
PubMed1996

Roles of cytochrome b5 in the oxidation of testosterone and nifedipine by recombinant cytochrome P450 3A4 and by human liver microsomes.

NADH-dependent testosterone 6 beta-hydroxylation and nifedipine oxidation activities could be reconstituted in systems containing cytochrome b5 (b5), NADH-b5 reductase, and bacterial recombinant cytochrome P450 (P450) 3A4 with a synthetic phospholipid mixture, cholate, MgCl2, and reduced glutathione. Replacement of NADH-b5 reductase with NADPH-P450 reductase produced an eightfold increase in testosterone 6 beta-hydroxylation activity. Further stimulation could be obtained when NADPH was used as an electron donor instead of NADH. Removal of b5 from the NADH- and NADPH-supported systems caused a 90% loss of testosterone 6 beta-hydroxylation activities in the presence of NADPH-P450 reductase but resulted in complete loss of the activities in the absence of NADPH-P450 reductase. These results suggested that about 10% of the activities was due to electron flow from NADPH-P450 reductase to P450 3A4 in the absence of b5. In the presence of testosterone and MgCl2, P450 3A4 was reduced by b5 and NADH-b5 reductase, although the rate of P450 3A4 reduction was much slower than that by NADPH-P450 reductase. Anti-human b5 immunoglobulin G (IgG) (purified using rabbit b5 affinity chromatography) inhibited testosterone 6 beta-hydroxylation activity catalyzed by human liver microsomes more strongly in NADH- than in NADPH-supported reactions. However, anti-rat NADPH-P450 reductase IgG inhibited microsomal activities in both NADH- and NADPH-supported systems to similar extents. Addition of NADH enhanced NADPH-supported testosterone and nifedipine oxidations in human liver microsomes. MgCl2 stimulated rates of reduction of b5 by NADPH-P450 reductase, but not by NADH-b5 reductase, in reconstituted systems. These results suggest that b5 is an essential component in P450 3A4-catalyzed testosterone hydroxylation and nifedipine oxidation in human liver microsomes. Our previous observation that rates of reduction of ferric P450 3A4 by NADPH-P450 reductase are accelerated by complexation with substrates and b5 is supported in this study.

Animals↗
PubMed1996

First-trimester prenatal molecular diagnosis of infantile hypophosphatasia in a Japanese family.

We obtained a prenatal molecular diagnosis during the first trimester in a Japanese woman whose first child (the proband) had been a compound heterozygote for infantile hypophosphatasia. We examined chorionic villus DNA samples obtained at 10 weeks of gestation for the base substitutions detected in the proband DNA using polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) and PCR-allele-specific oligonucleotide (ASO) analysis. The genotype of the fetus was the same as that of the proband. The same mobility shift patterns of single strand conformation polymorphism (SSCP) bands were observed in the fetus and the proband. This molecular approach to prenatal diagnosis appears to be more accurate than the enzymatic method and also more accurate and more rapid than the conventional RFLP method.

Alleles↗