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Biomedical subjects

M Shimizu

Publications and source records attributed to M Shimizu.

At least 847 records · Page 47Linked to original sources
PubMed1993

Escherichia coli single-stranded DNA-binding protein alters the structure of intramolecular triplexes in plasmids.

The ability of the Escherichia coli single-stranded DNA-binding protein (SSB) to recognize structural features associated with intramolecular triplex formation in oligopurine.oligopyrimidine (pur.pyr) inserts in recombinant plasmids was evaluated. The SSB protein binds to supercoiled plasmids and causes a site-preferential increase in OsO4 reactivity of the pyrimidine strand involved in the formation of the Hy-3 isomer of the triplex structure. The E. coli RecA protein showed no reaction with triplexes in similar studies. This behavior is consistent with SSB-mediated unpairing of the H-DNA-forming region.

Base Sequence↗
PubMed1993

Left ventricular functional reserve in patients with syndrome X: evaluation by continuous ventricular function monitoring.

OBJECTIVES: The objective of this study was to evaluate cardiac functional reserve in patients with syndrome X. BACKGROUND: Syndrome X is characterized by stress-induced anginal pain and ST segment depression, normal findings on coronary angiography and normal left ventricular function at rest. Reduced coronary vasodilative reserve and abnormal myocardial lactate metabolism have been described in such patients. METHODS: To assess left ventricular functional reserve in patients with syndrome X, continuous radionuclide monitoring of left ventricular end-diastolic volume, end-systolic volume and ejection fraction was performed in 12 patients and 13 normal control subjects during supine bicycle ergometer exercise. RESULTS: In control subjects, end-diastolic volume increased at peak exercise from 100% to 106.5% (p < 0.01), end-systolic volume decreased from 39.1% to 22.6% (p < 0.01) and ejection fraction increased from 60.9% to 78.6% (p < 0.01). In patients with syndrome X, end-diastolic volume increased at peak exercise from 100% to 106% (p < 0.01), and end-systolic volume decreased at ST segment depression < or = 0.5 mm (the ST point) from 37% to 28.8% (p < 0.01) but increased at peak exercise to 44.7% (p < 0.01 vs. the ST point). Thus, ejection fraction increased at the ST point from 63% to 72.7% (p < 0.01) but decreased at peak exercise to 57.7% (p < 0.01 vs. the ST point and control subjects) in proportion to the degree of ST segment depression. In nine patients (75%), ejection fraction at peak exercise was lower than baseline values. All patients and control subjects showed a rapid ejection fraction increase just after exercise during the recovery period. The degree of ejection fraction "overshoot" in patients was similar to that in control subjects, but the interval from the end of exercise to the overshoot in patients was significantly longer than that in control subjects (118 vs. 65 s, p < 0.01). CONCLUSIONS: In patients with syndrome X subjected to exercise stress, left ventricular function remained normal before the onset of ST segment depression. Once ST segment depression appeared, left ventricular function deteriorated in proportion to the degree of depression, and reduced left ventricular function persisted into the recovery period. Continuous ventricular function monitoring is thus a useful predictor of reduced left ventricular functional reserve in patients with syndrome X.

Adult↗
PubMed1993

Unfolding/refolding studies on bovine beta-lactoglobulin with monoclonal antibodies as probes. Does a renatured protein completely refold?

We investigated whether any local moieties within a protein molecule could completely refold from the denatured state to regain the native conformation. Bovine beta-lactoglobulin (beta-LG) was denatured in the presence of guanidine hydrochloride (GdnHCl) as the denaturant. Renaturation of the denatured beta-LG was attempted by dialyzing to remove GdnHCl. The renatured molecules regained the same retinol binding activity as that of native beta-LG, and physicochemical studies also indicated that refolding of the denatured beta-LG had been almost completely successful. Local structural differences between the native and renatured beta-LG molecules were evaluated by using our panel of four anti-beta-LG monoclonal antibodies (anti-beta-LG mAbs). The structures of the epitope regions in native beta-LG recognized by two of these mAbs were the same as those in renatured beta-LG. However, it is notable that the binding properties of the other two mAbs to native beta-LG indicated a wide structural difference in the epitope regions between the native and renatured beta-LG. These regions unable to completely refold were the same as those that unfolded preferentially to the alpha-helix region, shown in the previous report (Kaminogawa, S., Shimizu, M., Ametani, A., Hattori, M., Ando, O., Hachimura, S., Nakamura, Y., Totsuka, M., and Yamauchi, K. (1989) Biochim. Biophys. Acta 998, 50-56). Complete refolding was never attained by several renaturation conditions such as quicker or slower removal of the denaturant, nor by additional oxidation treatment after reducing the disulfide bonds. These results suggest that some specific moiety(ies) in a protein molecule cannot return to the native conformation from a denatured state, even if the other moieties refold completely, and that such a conformational difference between renatured and native forms has no affect on the biological function of ligand binding.

Amino Acid Sequence↗
PubMed1993

DNA-protein interactions at the S.cerevisiae alpha 2 operator in vivo.

Two homodimeric proteins, alpha 2 and MCM1, are required to repress transcription of a-cell type specific genes in haploid yeast alpha-cells. In vitro studies by others of the interactions of these proteins with operator DNA have suggested that MCM1 binds to the middle and alpha 2 to the ends of the 31 bp operator. We have previously shown that alpha 2 organizes chromatin structure adjacent to the operator; in the presence of alpha 2 repressor, a precisely positioned nucleosome abuts the operator in both minichromosomes and the genome. We present in vivo footprinting evidence consistent with occupancy of the operator by MCM1 in both a- and alpha-cells and by alpha 2 repressor in alpha-cells. Interestingly, our in vivo results differ from previous in vitro work in detail. In contrast to the broad block of reagent accessibility to DNA by the factors seen in vitro, we find a pattern of strand-specific protection or augmented reactivity in vivo. The in vivo results are consistent with genetic data concerning transcriptional regulation of a-cell specific genes and corroborate the crystallographic data of others.

Base Sequence↗
PubMed1993

Recognition nucleotides of Escherichia coli tRNA(Leu) and its elements facilitating discrimination from tRNASer and tRNA(Tyr).

In order to study how Escherichia coli leucyl-tRNA synthetase recognizes tRNA(Leu) and discriminates it from the other two class II tRNAs, tRNA(Ser) and tRNA(Tyr), various mutations were introduced into class II tRNA transcripts. The discriminator base A73, but not the anticodon sequence, was found to serve as a critical recognition element of tRNA(Leu). A base substitution at the invariant nucleotide A14, but not at any of the other nucleotides characteristic of the E. coli tRNA(Leu) isoacceptors among the three class II tRNAs, caused significantly damaged aminoacylation with leucine. A two base-pair deletion in the long variable arm also resulted in no significant decrease of activity. Transplanting the three tertiary elements characteristic of E. coli tRNA(Leu) (i.e. the location of the G18G19 sequence in the D-loop, the A15 U48 base-pair and the stem pairing pattern of the long variable arm) besides the discriminator base change introduced the leucine charging activity in terms of Vmax/Km, up to 0.1 of that for the normal sequence of tRNA(Leu) into both tRNA(Ser) and tRNA(Tyr). These results indicate that A73 and A14 (or its vicinity) are involved in recognition by leucyl-tRNA synthetase, and that several tertiary elements play a significant role in the discrimination of tRNA(Leu) from the other two class II tRNAs.

Base Sequence↗
PubMed1993

A 2-thiouridine derivative in tRNAGlu is a positive determinant for aminoacylation by Escherichia coli glutamyl-tRNA synthetase.

Early investigations into the interaction between Escherichia coli glutamyl-tRNA synthetase (GluRS) and tRNAGlu have implicated the modified nucleoside 5-[(methylamino)methyl]-2-thiouridine in the first position of the anticodon as an important contact for efficient aminoacylation. However, the experimental methods employed were not sufficient to determine whether the interaction was dependent on the presence of the modification or simply involved other anticodon loop-nucleotides, now occluded from interaction with the synthetase. Unmodified E. coli tRNA(Glu), derived by in vitro transcription of the corresponding gene, is a poor substrate for GluRS, exhibiting a 100-fold reduction in its specificity constant (kcat/KM) compared to that of tRNA(Glu) prepared from an overproducing strain. Through the use of recombinant RNA technology, we created several hybrid tRNAs which combined sequences from the in vitro transcript with that of the native tRNA, resulting in tRNA molecules differing in modified base content. By in vitro aminoacylation of these hybrid tRNA molecules and of tRNAs with base substitutions at positions of nucleotide modification, we show conclusively that the modified uridine at position 34 in tRNA(Glu) is required for efficient aminoacylation by E. coli GluRS. This is only the second example of a tRNA modification acting as a positive determinant for interaction with its cognate aminoacyl-tRNA synthetase.

Base Sequence↗
PubMed1993

Crystallographic characterization of a PHO4-DNA complex.

Crystals have been obtained of the DNA-binding domain of the yeast transcription factor PHO4 in complexes with several synthetic fragments of DNA with appropriate cognate sequences. Crystals suitable for X-ray diffraction studies were produced in the case of a complex of the protein with a 17 base-pair fragment of DNA from a solution in polyethylene glycol and calcium chloride. The crystals have the space group of P4(1)2(1)2 or P4(3)2(1)2 with unit cell dimensions a = b = 56.7 A, c = 447.8 A. The diffraction data at 3 A resolution were collected using synchrotron radiation with a Weissenberg camera for macromolecular crystallography.

Base Sequence↗
PubMed1993

Relative bioavailability of two disopyramide capsules in humans based on total, unbound, and unbound enantiomer concentrations.

The relative bioavailability of two 100-mg disopyramide formulations which showed almost an 8- to 10-fold difference in their dissolution rates at pH 1.2 and 6.8 was determined in eight healthy subjects using a randomized block design. Although no significant differences in relative bioavailability were observed between the two formulations when based on the total disopyramide concentration, an almost 30 per cent difference in the extent of bioavailability was observed when assessed in terms of the unbound (+/-)- and (-)-disopyramide concentration, due probably to stereoselective nonlinear plasma protein binding. This suggests that unbound enantiomer parameters would be more sensitive to differences in bioavailability between two disopyramide formulations. Therefore, the type of concentration used would be an important factor for precise evaluation of the relative bioavailability of racemic drugs.

Adult↗
PubMed1993

Asymmetrical septal hypertrophy in patients with hypertension: a type of hypertensive left ventricular hypertrophy or hypertrophic cardiomyopathy combined with hypertension?

To determine whether asymmetrical septal hypertrophy (ASH) in patients with essential hypertension (HT) is a type of hypertensive left ventricular (LV) hypertrophy or hypertrophic cardiomyopathy (HCM) combined with HT, we investigated a group of 7 hypertensive patients with ASH compared with 12 HCM patients and 10 healthy controls using radionuclide angiography and right ventricular endomyocardial biopsy. The LV time-volume curve and its first and second derivative curves were constructed from cardiac output and time-activity curves constructed by combined forward and reverse-gating from the R wave. The LV wall thickness and ejection fraction were significantly greater in both the HT and HCM groups than in the control group, whereas there were no differences in these indices between the HT and HCM groups. Rapid filling volume index and rapid filling fraction showed significantly lower values in the HCM group than in the control group (p < 0.005). In contrast to the HCM group, these indices in the HT group did not differ from those in the control group. The time to peak filling rate was prolonged in the control, hypertension, and HCM groups in increasing order. Histopathological study revealed a higher incidence of myocardial cell disarray in the HCM than in the HT group. The above results suggest that ASH in hypertensive patients is a type of hypertensive LV hypertrophy.

Analysis of Variance↗
PubMed1993

Horizontal component of electro-oculogram as a parameter of arousal in dementia: relationship between intellectual improvement and increasing arousal under pharmacotherapy.

Although nootropics and metabolically active compounds sometimes attenuate intellectual deficits of dementia patients, this mechanism has not been discussed or investigated. The authors hypothesized that these compounds act to enhance attention and/or concentration by increasing the arousal level, and then improve intellectual functions. Based on this hypothesis, the authors investigated the arousal level and the improvement of intellectual functions in ten patients with Alzheimer type dementia and ten patients with multi-infarct dementia before and after 3 months pharmacotherapy using bifemelane hydrochloride. They then analyzed whether the increases in arousal level involves or relates to the improvement of intellectual functions. On the 20 patients, psychological tests were carried out, and the arousal level was evaluated using the frequency of rapid eye movements in the electro-oculogram (EOG) before and after 3 months administration of bifemelane hydrochloride. As a result, a significant positive correlation was observed between the improvement on the Hasegawa's dementia scale and the rate of increase in rapid eye movements in the EOG. The EOG monitoring can be a great help in establishing an objective evaluation method on drug efficacy.

Aged↗
PubMed1993

Detection of activated platelets in urinary sediments by immunofluorescence using monoclonal antibody to human platelet GMP-140 in patients with IgA nephropathy.

The presence of activated platelets in the urinary sediments was studied by indirect immunofluorescence using monoclonal antigranular membrane protein (GMP)-140 antibody. GMP-140 is generally expressed on the activated-platelets and -vascular endothelial cells. The purpose of the present study was to determine if the presence of activated platelets in the urinary sediments is correlated with glomerular injuries in patients with IgA nephropathy. Fourteen patients with IgA nephropathy and 11 patients with diffuse mesangial proliferative glomerulonephritis without glomerular IgA deposition (PGN) were examined. The number of activated platelets in the urinary sediments was markedly increased in patients in the advanced stage of IgA nephropathy. The ratio of activated platelets to total platelets in the urinary sediments was also increased in such patients. It appears that the detection of activated platelets in the urinary sediments is useful in determining the degree of histological changes in IgA nephropathy.

Adult↗
PubMed1993

Cryopreservation of porcine blastocysts by vitrification.

The objective of this study was to assess the possibility of cryopreservation of porcine expanded and hatched blastocysts by vitrification. The following four types of vitrification solutions were applied in this study: (i) EFT, a mixture of 7.2 M ethylene glycol, 0.003 M ficoll, and 0.3 M trehalose; (ii) DAP213, 2 M dimethyl sulfoxide, 1 M acetamide, and 3 M propylene glycol; (iii) DAP213-T, DAP213 supplemented with 0.3 M trehalose; and (iv) EPT, 4 M ethylene glycol, 3.1 M propylene glycol and 0.3 M trehalose. The embryos collected on Days 5 to 7 (Day 0 = onset of estrus) were allocated to eight experimental groups according to the types of vitrification solution and the developmental stage. In the toxicity test, the embryos were equilibrated in the respective vitrification solutions either in a single step or in four steps and then transferred to 1 M sucrose in a single step at 22 degrees C without cooling. As a whole, the viabilities of embryos equilibrated in the solutions were lower than those of control embryos. The stepwise equilibration was superior to a single-step equilibration in the in vitro survival of, especially, expanded blastocysts after dilution. No significant difference was observed between the vitrification solutions for the four-step method. In the vitrification test, the embryos were equilibrated in the solutions by the four-step method, loaded into a 0.25-ml plastic straw, and plunged into liquid nitrogen. Although viable embryos were obtained after warming from all of the combinations except the hatched blastocyst-EPT group, viabilities were further reduced by cooling (range of reduction rates: 60 to 100%). The possible cause of low survival after warming is also discussed concerning the cryophysical properties of vitrification solutions.

Acetamides↗
PubMed1993

Differential activation of cyclin and cyclin-dependent kinase genes by adenovirus E1A12S cDNA product.

The differential activation of cyclin and cyclin-dependent kinase genes by the adenovirus E1A gene product (E1A) or serum factors was studied with a rat 3Y1 derivative cell line, g12-21, in which the E1A12S cDNA can be expressed in response to dexamethasone (dex). The induction of DNA synthesis in quiescent g12-21 cells occurred within 12 h after serum stimulation, while it occurred within 8 h after treatment with dex. The expression of cyclin D1 and E genes in the serum-stimulated cells was induced in mid G1 and mid to late G1, respectively, while that of the cyclin D1 gene was not induced and the induction of the cyclin E gene was shifted to the G1/S boundary in the dex-treated cells. The cdk2 gene was induced in late G1 and cdc2 and cyclin A genes at the G1/S boundary in both serum-stimulated and dex-treated cells. These results suggest that E1A skips cell cycle events which normally occur in early to mid G1 and may directly activate late-response genes. Analysis of the transcription factor E2F complexes formed in the promoter regions of cdc2 and dihydrofolate reductase genes showed that the amount of complexes formed is maximal at the G1/S boundary, but decreases in S phase when these genes are transcribed extensively.

Adenovirus E1A Proteins↗
PubMed1993

Some properties and occurrence of cytochrome c-552 in the aerobic photosynthetic bacterium Roseobacter denitrificans.

Characteristics and occurrence of cytochrome c-552 from an aerobic photosynthetic bacterium, Roseobacter denitrificans, were described. Relative molecular mass of the cytrochrome was 13.5 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 15,000 by gel filtration. This cytochrome was a acidic protein having a pI of 5.6 and Em was +215 mV at pH 7.0. Absorption peaks were at 278, 408 and 524 nm in the oxidized form and 416, 523 and 552 nm in the reduced form. Amino acid composition and N-terminal amino acid sequence of cytochrome c-552 determined for 24 residues had low similarities to those of cytochrome c-551 of this bacterium, which is homologous to cytochrome c2, although the physico-chemical properties of these two cytochromes were similar to each other. Cytochrome c-552 was maximally synthesized in the light under aerobic conditions but not in the dark. The synthesis also occurred in the presence of alternative acceptors such as trimethylamine N-oxide (TMAO) and nitrate under anaerobic conditions. Our results suggest that cytochrome c-552 is involved in TMAO respiration and denitrification in R. denitrificans, although the effect of light remains to be solved.

Amino Acid Sequence↗
PubMed1993

Epithelial properties of human intestinal Caco-2 cells cultured in a serum-free medium.

Human intestinal Caco-2 cells were cultured under serum-free conditions on an insoluble collagen and FCS matrix (Caco-2-SF), and a comparison was made between several characteristics of Caco-2 and Caco-2-SF cells. Their morphological appearance was identical. Slight differences were found in cell growth and expression of brush border enzymes between Caco-2 and Caco-2-SF cells. Similar levels of activity of Gly-Gly transport were expressed in both types of cell. Caco-2 cells cultured on permeable filters showed high transepithelial electrical resistance (TEER), indicating the high monolayer integrity. The transepithelial transport activity for glucose, alanine and Gly-Gly was detected by measuring the change in short-circuit current (delta Isc) after adding each of these nutrients to the apical chamber. In Caco-2-SF cells, such parameters as TEER and delta Isc were reduced drastically, suggesting that the monolayer integrity and cell polarity that are important for transepithelial transport were not attained. These parameters, however, could be restored by adding FCS or by milk whey. The result suggested that FCS and milk whey contain factors which regulate the formation of the tight junctions and, consequently, the development of cell polarity. Thus the Caco-2-SF cell-culture system will provide a useful model for studying factors which regulate the intestinal transepithelial transport functions.

Adenocarcinoma↗
PubMed1993

Effects of diethylene glycol monomethyl ether on pregnancy and postnatal development in rats.

The effects of oral treatment of Wistar rats with diethylene glycol monomethyl ether (diEGME) were examined. In a preliminary dose-finding study with non-pregnant rats, diEGME treatment at doses up to 4,000 mg/kg/day on 11 consecutive days decreased relative weights of thymus and pituitary gland, white and red blood cell counts, hemoglobin concentrations and hematocrit levels. In pregnant rats, treatment at doses of > 3,000 mg/kg/day (over gestation days 7-17) caused total resorption of all litters. In teratology and postnatal studies, pregnant rats were treated with diEGME at doses of 0, 200, 600, and 1,800 mg/kg/day from day 7 through 17 of gestation. At 200 mg/kg, there were no adverse effects on either dams, fetuses, or neonates. At 600 mg/kg, dams were not affected, but fetal body weights were decreased, and fetal thymus and ossification were adversely affected. At 1,800 mg/kg, maternal thymus weights and food consumption were decreased, and visceral malformations of the cardiovascular system were seen in 28.0% of the fetuses. Only 6.3% of the pups delivered by dams treated with 1,800 mg/kg of diEGME survived for 4 days after birth. Thus, diEGME was teratogenic in Wistar rats, but the spectrum differed from that in Sprague-Dawley rats. In addition to teratogenicity, diEGME had significant adverse effects on postnatal development. The most sensitive organ to diEGME was the thymus in both dams and fetuses.

Abnormalities, Drug-Induced↗
PubMed1993

Enamelins in the newly formed bovine enamel.

The possibility of using the antisera raised in rabbits against the porcine 25 kDa amelogenin, 32 and 89 kDa enamelins, and the 13-17 kDa nonamelogenin for the differentiation and identification of the protein components in bovine immature enamel was examined. Although the immunoreactivities of these antisera against bovine enamel proteins were weaker than those against the porcine proteins, it was found that these antisera could differentiate and demonstrate immunohistochemically a characteristic distribution of three different kinds of enamel protein components in the bovine secretory stage enamel similar to those observed in the porcine immature enamel. Of the several high molecular weight proteins being reactive to the anti-porcine 32 and 89 kDa enamelin sera, the 130 kDa protein, having the highest molecular weight, was extracted and purified from the bovine enamel sample which was obtained by peeling approximately 30-microns thickness of the outermost layer of the secretory stage enamel. The amino acid composition of the 130 kDa protein was similar to the known bovine enamelins, and was rich in aspartic acid, glutamic acid, proline, and glycine. The results could suggest that the enamelins of lower molecular weight than this protein, which are found in the bovine secretory stage enamel, are derived from this precursor protein.

Amelogenin↗