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Biomedical subjects

M Wada

Publications and source records attributed to M Wada.

At least 379 records · Page 21Linked to original sources
PubMed1995

A supprescin from a phytopathogenic fungus deactivates transcription of a plant defense gene encoding phenylalanine ammonia-lyase.

Both elicitor and supprescin (suppressor) are present in the pycnospore germination fluid of a pea pathogen Mycospharella pinodes. A nuclear run-on assay revealed that supprescin rapidly deactivated elicitor-triggered transcription of the gene encoding phenylalnine ammonia-lyase in pea epicotyl tissues. The mechanism underlying the deactivation of the plant defense gene by signal molecules secreted from the fungal pathogen was investigated. Cis-acting sequences and trans-acting factors responsive to supprescin in a TATA-proximal region of a member of the phenylalanine ammonia-lyase gene family in pea were examined in vitro. Gel mobility-shift assays and DNase I footprinting analysis revealed that the promoter region of PSPAL2 was modified by the binding of nuclear factors at multiple sites that were possibly involved in supprescin-mediated deactivation. The prominent changes by supprescin were observed at boxes 2 and 4 and near exonic sequences.

Ascomycota↗
PubMed1995

KatG sequence deletion is not the major cause of isoniazid resistance in Japanese and Yemeni Mycobacterium tuberculosis isolates.

One of the mechanisms of isoniazid resistance to Mycobacterium tuberculosis has been proved to be the chromosomal deletion of katG. Based on this finding, 22 isoniazid-resistant isolates of M. tuberculosis obtained in Japan and Yemen were analysed for katG by polymerase chain reaction and catalase activity. Only six (27%) of the 22 isolates were compatible with the mechanism (lack of amplification of katG and loss of catalase activity). In contrast, eight isolates (36%) were katG positive but catalase activity-negative and eight (36%) were positive for both factors, indicating that isoniazid resistance is multifactorial and the deletion of katG was not the major cause of resistance in the isolates examined in this study.

Bacterial Proteins↗
PubMed1995

Isomyosin patterns of single type IIB, IID and IIA fibres from rabbit skeletal muscle.

The present study demonstrates for the first time isomyosin patterns of the three fast-twitch fibre types IIB, IID/X, and IIA. Single muscle fibres were dissected from freeze-dried fibre bundles of rabbit adductor magnus, extensor digitorum longus, and psoas muscles. Pure fibre types, expressing only one myosin heavy chain isoform (MHCIIb, MHCIId/x, MHCIIa), were delineated by electrophoresis of fibre fragments under denaturing conditions. Pieces of the same fibres were then subjected to electrophoresis under non-denaturing conditions. A three-band pattern of fast isomyosins, representing the LC3f homodimer (FM1), the LC1f/LC3f heterodimer (FM2), and the LC1f homodimer (FM3), was detected in each of the three pure fibre types. Therefore, three isomyosins, different in their light chain complement, coexist in each pure fibre. The relative mobilities of the three bands, migrating in the order FM1 > FM2 > FM3, were the same in the three fibre types. The absolute electrophoretic mobilities of the MHCIIb-, MHCIId- and MHCIIa-based isomyosin triplets differed in the order MHCIIb triplets > MHCIId triplets > MHCIIa triplets. The proportions of FM1, FM2, and FM3 varied in type IIB, IID, and IIA fibres. FM2 was the dominant isomyosin in all three fibre types, but fibre type-related differences existed in the FM1 to FM3 ratio. This ratio was lowest in IIA fibres and highest in IIB fibres which agrees with our previous observations that the LC3f/(LC1f + LC3f) fraction is lowest in type IIA and highest in type IIB fibres.

Animals↗
PubMed1995

Leukocytapheresis therapy, performed with leukocyte removal filter, for inflammatory bowel disease.

Leukocytapheresis (LCAP), performed with a leukocyte removal filter, was administered five times, at 1-week intervals, for 5 weeks of intensive therapy and five times, at approximately 1-month intervals, for approximately 5 months of maintenance therapy, to 13 patients with inflammatory bowel disease (IBD) diagnosed as ulcerative colitis (UC) in 8 and Crohn's disease (CD) in 5. Clinical and blood examinations showed no side effects in any of the patients. During the intensive therapy, excellent or moderate clinical response was recognized in 11 of the 13 patients (84.6%), of whom 6 had a dramatic response; the excellent or moderate clinical response continued throughout the maintenance therapy in 8 of the patients (61.5%). Flow cytometry showed that the patients who had improved generally had high values for percentages of HLADR+, HLADR+CD3+, and HLADR+CD8+ cells before the first LCAP, and that these values and the C-reactive protein levels and erythrocyte sedimentation rates had decreased to the normal range by the end of both intensive and maintenance therapy. In the patients who showed poor response, in contrast, all the above values had been at or near normal before the initial LCAP administration. The clinical improvement in the absence of any additional medical treatment suggests that LCAP has the capacity to influence the causal mechanism(s) of IBD and that IBD is strongly associated with the cell-mediated immune response.

Adolescent↗
PubMed1995

A new model of diabetic pregnancy with genetic hypertension: pregnancy in spontaneously hypertensive rats with neonatal streptozotocin-induced diabetes.

OBJECTIVE: We designed this study to develop a new animal model of high-risk pregnancy complicated by hypertension and diabetes mellitus. STUDY DESIGN: Female spontaneously hypertensive rats were injected intraperitoneally with 75 mg/kg streptozotocin or vehicle as control at 2 days of age. They were mated with untreated male spontaneously hypertensive rats at 4 to 5 months of age. RESULTS: Hyperglycemia, defined as > 20 mmol/L plasma glucose level, was maintained during pregnancy in streptozotocin-treated spontaneously hypertensive rats, and systolic blood pressure was significantly higher in the streptozotocin-treated group than in controls before delivery (p < 0.01). Furthermore, urinary albumin excretion was significantly increased in the streptozotocin-treated group during and after pregnancy compared with the prepregnant level (p < 0.05), whereas in controls it remained unchanged. The incidence of low birth weight was significantly higher in male neonates from streptozotocin-treated mothers than those from control mothers (p < 0.05). CONCLUSION: Neonatally streptozotocin-treated spontaneously hypertensive rats may be useful for studying the combined effect of hypertension and diabetes mellitus during pregnancy.

Animals↗
PubMed1995

Efficacy of a skeletal muscle-powered dynamic patch: Part 1. Left ventricular assistance.

In this study, we examined the capability of a skeletal muscle-powered, dynamic patch to provide left ventricular assistance. An actuator was developed that used linear traction power furnished by the latissimus dorsi muscle and liquid as the medium for power transfer. The proximal portion of the muscle was dissected and was reattached to the actuator. The left ventricular apex was excised, and the dynamic patch lined with autologous pericardium was implanted during cardiopulmonary bypass. Hemodynamic studies were performed in 8 dogs after weaning from cardiopulmonary bypass. Muscle stimulation was found to significantly increase the systolic aortic pressure (91.6 versus 112.1 mm Hg; p < 0.01), the mean aortic pressure (65.2 versus 73.0 mm Hg; p < 0.01), and aortic blood flow (0.77 versus 0.92 L/min; p < 0.01). The left atrial pressure decreased from 17.9 to 16.6 mm Hg (p < 0.01). This "hybrid" left ventricular assist device possesses notable clinical advantages because of its remarkable efficacy in assisting circulation. Further experimental studies using preconditioned skeletal muscle are necessary to assess the long-term effects of this technique.

Animals↗
PubMed1995

Efficacy of a skeletal muscle-powered dynamic patch: Part 2. Right ventricular assistance.

The purpose of this study was to assess the feasibility of using a skeletal muscle-powered dynamic patch to assist the failing right ventricle. Seven adult mongrel dogs were used in the study. The proximal portion of the left latissimus dorsi muscle was harvested and reattached to the actuator to serve as a skeletal muscle energy convertor. The right ventricular free wall was fully excised and the dynamic patch was implanted under cardiopulmonary bypass. After weaning from cardiopulmonary bypass, the latissimus dorsi muscle was stimulated using a burst frequency of 33 Hz, a burst duration of 200 ms, and 1:2 synchronous mode stimulation with the native R wave. Latissimus dorsi muscle stimulation increased systolic aortic pressure (78 versus 91 mm Hg; p < 0.01), mean aortic pressure (56 versus 62 mm Hg; p < 0.05), aortic blood flow (0.73 versus 0.97 mL; p < 0.01), and systolic right ventricular pressure (41 versus 56 mm Hg; p < 0.01). The mean right atrial pressure decreased from 14 to 9.6 mm Hg (p < 0.01). Our results demonstrate that the use of a right ventricular dynamic patch powered by a skeletal muscle linear-type actuator can not only function as a right ventricular free wall substitute but also lead to the augmentation of right ventricular and global cardiac function.

Animals↗
PubMed1995

Direct 99mTc labeling of human immunoglobulin with an insoluble macromolecular Sn(II) complex.

An insoluble macromolecular Sn(II)(R-Sn) complex which strongly binds Sn(II) by chelation was applied to the direct 99mTc labeling of human immunoglobulin(IgG) to minimize the influence of Sn(II). 99mTc labeling was achieved at greater than 90% yield simply by the short-term mixing of IgGa containing > 2-SH groups per IgG molecule, 99mTc pertechnetate, and the R-Sn complex in pH 7 solution. The 99mTc-IgGa obtained by this labeling method has high stability based on the thiol-specific binding of 99mTc without transchelation from another weakly bound 99mTc-complex.

Animals↗
PubMed1995

Electroporation-mediated transfection of mammalian cells with crude plasmid DNA preparations.

We designed a simple and reproducible electroporation-mediated transfection procedure with which to screen mammalian expression vector-constructed cDNA libraries. Using a specific chamber composed of five parallel electrodes, the recipient cells can be electroporated separately with 40 plasmid DNA preparations in a single experiment. Over 300 crude plasmids prepared from E. coli (DH-5) carrying a pcD2neo-vector-derived cDNA library were tested. The efficiency of stable transfection by electroporation with crude plasmid DNA preparations was 10-times higher than with the CsCl-purified plasmid DNA. When the crude plasmids were digested with RNase, the efficiency of stable transfection markedly decreased, indicating that the contaminating bacterial RNA in the crude plasmid preparations has a strong carrier effect during the electroporation. Even when salmon sperm DNA or genomic DNA from the recipient cells was used as the carrier of the purified plasmid, the efficiency was not higher than that using the crude preparations. This procedure is useful not only for screening a number of cDNAs but also for routinely introducing biologically active foreign genes into cultured mammalian cells.

3T3 Cells↗
PubMed1995

Chemosensitisation of spontaneous multidrug resistance by a 1,4-dihydropyridine analogue and verapamil in human glioma cell lines overexpressing MRP or MDR1.

Multidrug resistance phenotypes in human tumours are associated with the overexpression of the 170 kDa P-glycoprotein encoded by the multidrug resistance 1 (MDR1) gene, and also with that of the non-P-glycoprotein-mediated multidrug resistance gene, MRP, which encodes a 190 kDa membrane ATP-binding protein. We have previously reported that overexpression of MRP appears to be responsible for spontaneous multidrug resistance in some human glioma cell lines (Abe et al., Int. J. Cancer, 58, 860-864, 1994). In this study, we investigated whether chemosensitising agents of P-glycoprotein-mediated multidrug resistance such as verapamil, a biscoclaurine alkaloid (cepharanthine), and a dihydropyridine analogue (NIK250) could also reverse multidrug resistance in human glioma cells. The glioma cell lines were the two MRP-expressing cell lines, T98G and IN500, an MDR1-expressing cell line, CCF-STTG1, and the MRP1 MDR1-non-expressing cell line, IN157. Verapamil and NIK250 almost completely reversed drug resistance to vincristine, etoposide and doxorubicin in T98G cells, while they also reversed drug resistance to vincristine and etoposide, but only partially to doxorubicin in IN500 cells. Cepharanthine as well as verapamil and NIK250 reversed vincristine resistance in CCF-STTG1 cells, but cepharanthine only partially reversed drug resistance in T98G and IN500 cells. The cellular accumulation of [3H]etoposide increased about 2- and 3-fold compared with control in T98G cells in the presence of verapamil and NIK250 respectively. Furthermore, the release of doxorubicin from the nuclei of T98G cells was blocked by NIK250. However, NIK250 and verapamil caused no apparent increase in vincristine accumulation in T98G cells. NIK250 or verapamil might exert inhibitory effects upon MRP function, resulting in a reversal of MRP-mediated spontaneous multidrug resistance in cultured human glioma cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Effects of maternal diabetes on blood pressure and glucose tolerance in offspring of spontaneously hypertensive rats: relation to birth weight.

1. We studied the effects of maternal diabetes on blood pressure and glucose tolerance in the adult female offspring of spontaneously hypertensive rats. 2. Female spontaneously hypertensive rats were rendered diabetic by neonatal streptozotocin treatment, and then were mated with untreated male spontaneously hypertensive rats. Moderately severe hyperglycaemia was maintained during the gestation. 3. The birth weight was significantly lower in the female offspring of the diabetic dams than in the female offspring of the non-diabetic dams. The systolic blood pressure was significantly higher in the offspring from the diabetic dams than that from the control dams at 6 months of age (192 +/- 4 mmHg versus 213 +/- 4 mmHg, P < 0.01). The heart weight was also significantly increased in the offspring of the diabetic dams. Both the blood pressure and heart weight were inversely related to the birth weight. On the other hand, glucose tolerance was unaffected by maternal diabetes. 4. Maternal diabetes aggravated the severity of hypertension in the adult female offspring of spontaneously hypertensive rats. This suggests the importance of the metabolic environment during fetal growth for the development of hypertension.

Animals↗
PubMed1995

Antigenic variation by positional control of major surface glycoprotein gene expression in Pneumocystis carinii.

Major cell surface glycoproteins (MSGs) of Pneumocystis carinii play a crucial role in host-parasite interactions during P. carinii pneumocystosis in AIDS patients. Genes encoding MSGs are repeated and dispersed throughout the genome and are highly polymorphic. MSG gene expression was found to be mediated by a DNA element that was termed the upstream conserved sequence (UCS). The UCS element maps to a single chromosome, is attached to expressed MSG genes, and encodes the sequence found at the 5' ends of most MSG mRNAs. The UCS is not highly repeated, but P. carinii populations contain many different MSG genes attached to the UCS, suggesting that different organisms in the population have different MSG genes attached to the UCS. Such genetic heterogeneity may be generated by recombination between MSG genes at the UCS locus and one or more of the dozens of MSG genes located elsewhere in the genome.

Animals↗
PubMed1995

Effect of a new bombesin receptor antagonist, (E)-alkene bombesin isostere, on amylase release from rat pancreatic acini.

The short-chain pseudopeptide, [D-Phe6, Leu13 psi (CH2NH)Leu14]bombesin(6-14) (RDI), is reported to be a potent antagonist of bombesin, and development of this type of compound has greatly contributed to the investigation of biological actions of bombesin and its related peptides. We recently synthesized (E)-alkene bombesin isostere by replacing the peptide bond with an (E)-double bond: [D-Phe6, Leu13 psi [(E)CH = CH]Leu14] bombesin(6-14) (EABI). The present study examined the effect of EABI on amylase release from rat pancreatic acini. EABI showed no agonistic activity at concentrations up to 1 microM, and RBI showed slight agonistic activity at concentrations > 10 nM. EABI caused a dose-dependent inhibition of amylase release stimulated by 0.1 nM bombesin, with an IC50 of 6.7 +/- 1.7 nM, and induced almost-complete inhibition at 0.3 microM. RDI caused a dose-dependent inhibition of amylase release, with an IC50 of 68.7 +/- 16 nM. EABI caused a parallel and rightward shift of the entire dose-response curve of bombesin-stimulated amylase release, and the degree of the shift was dependent on the concentrations of EABI. EABI (100 nM) and RDI (100 nM) inhibited amylase releases stimulated by gastrin-releasing peptide (1 nM) and neuromedin-C (1 nM). In contrast, amylase release stimulated by cholecystokinin octapeptide (0.1 nM), carbachol (10 microM), vasoactive intestinal peptide (1 nM), and gastrin-17 (10 nM) was not inhibited by EABI and RDI. The results indicate that EABI is a potent and specific bombesin receptor antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

A YAC-based contig of 1.5 Mb spanning the human multidrug resistance gene region and delineating the amplification unit in three human multidrug-resistant cell lines.

A contig of 21 nonchimeric yeast artificial chromosomes (YACs) has been assembled across 1.5 Mb of the multidrug resistance (MDR) gene region located at 7q21, and formatted with four previously reported probes, six newly isolated probes, and three sequence-tagged sites (STSs) from internal and end fragments of YACs. A physical map of rare cutter restriction enzyme sites across the region was also constructed by pulsed-field gel electrophoretic (PFGE) analysis of four overlapping YAC clones. The amplification unit of this region in different cell lines was then determined by Southern blot analysis on the basis of the physical map and probes. Amplified DNA was located in extrachromosomal elements in human MDR cell lines studied here, and the size of the amplification unit was determined to be discrete in one MDR amplification but variable in others.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed1995

Functional expression of yeast artificial chromosome-human multidrug resistance genes in mouse cells.

Multidrug resistance (MDR) genes, which are ATP-binding cassette family genes, encode the cell surface glycoprotein, P-glycoprotein, which functions as an energy-dependent drug efflux pump. Two relevant human genes, PGY1 and PGY3, are located on human chromosome 7, and three relevant mouse genes, mdr1a, mdr1b, and mdr2, are located on mouse chromosome 5. An LMD1 cell line was established after the transfer of a 580-kb yeast artificial chromosome (YAC) clone carrying the human MDR locus into mouse L cells; the cell line was shown to have stably integrated YAC DNA in an apparent intact form. Using LMD1 cells as the parental cell line, five vincristine-resistant sublines, designated LMD1-V50, LMD1-V100, LMD1-V200, LMD1-V500, and LMD1-V1000, were isolated by exposure to increasing concentrations of the drug. LMD1-V50, LMD1-V100, LMD1-V200, LMD1-V500, and LMD1-V1000 showed 3-, 7-, 13-, 45-, and 110-fold higher resistance to the cytotoxic effects of vincristine, respectively, than their parental counterpart, LMD1. Immunofluorescence, Western blot, and Northern blot analyses revealed that the human PGY1 gene or its product was overexpressed, accompanied by gene amplification. The human PGY3 gene was also overexpressed in the LMD1-V20, LMD1-V100, and LMD1-V1000 cell lines. Southern blot and fluorescence in situ hybridization (FISH) analyses demonstrated that although essentially the entire YAC DNA was integrated in mouse genome and amplified, the endogenous mouse mdr genes were not amplified in these drug-resistant cell lines. Similar results were obtained by the analyses of vincristine-resistant cell lines isolated from four independent subclones of LMD1 cells. Thus, in contrast to their mouse counterparts, the integrated human MDR genes retained susceptibility to both gene activation and amplification, during the selection of drug-resistant mouse cell lines. The possibility that transferred YACs may retain regulatory properties observed in the cells of origin, and may have a chromatin structure that favors augmented expression, is discussed.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed1995

Characterization of nuclear factors for elicitor-mediated activation of the promoter of the pea phenylalanine ammonia-lyase gene 1.

The nuclear factors presumably associated with the activation of the gene encoding phenylalanine ammonia-lyase by a fungal elicitor were characterized in pea (Pisum sativum L.) epicotyls. The TATA-proximal region was dissected and putative cis-regulatory elements in the promoter of pea phenylalanine ammonia-lyase gene 1 were examined by gel-mobility shift and in vitro footprinting analyses. Specific binding of the nuclear factors to the promoter-proximal regions of pea phenylalanine ammonia-lyase gene 1 associated with elicitor-mediated activation was detected at a region containing consensus sequence motifs of boxes 2 and 4 and other AT-rich sequences. The analyses of DNA fragments containing the deleted promoter regions suggested that a residue from -183 to -173 (ATTAGTAAGTGAT) was essential for a maximal activity of forming low-mobility complex (LMC) in the gel-mobility shift assay, and synthetic oligonucleotides confirmed the presence of at least one nuclear component associated with the formation of an active LMC. Competition experiments and treatment with Hoechst 33258 provided direct evidence that the formation of LMC with the promoter fragments from genes encoding phenylalanine ammonia-lyase and chalcone synthase in pea contained one or more of the same proteins that recognize AT-rich sequence motifs for binding. It also suggests that common high-mobility group-like proteins might be involved in the regulation of elicitor-inducible genes in pea.

Base Sequence↗