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M Shimizu

Publications and source records attributed to M Shimizu.

At least 775 records · Page 43Linked to original sources
PubMed1995

Similarities and differences in tRNA identity between Escherichia coli and Saccharomyces cerevisiae: evolutionary conservation and divergence.

Identity elements, which allow correct recognition of tRNAs by their cognate aminoacyl-tRNA synthetase, have been well elucidated in Escherichia coli to begin to see a pattern for tRNA recognition. We examined the identity elements of several tRNA species from Saccharomyces cerevisiae and Thermus thermophilus using in vitro transcripts. Comparison of identity elements among different organisms indicates not only conservation but also evolutionary divergence of tRNA recognition.

Amino Acyl-tRNA Synthetases↗
PubMed1995

Dynamics in isomerization of intramolecular DNA triplexes in supercoiled plasmids.

Kinetic and thermodynamic nature of two isomers, H-y3 and H-y5, for an intramolecular Pyr.Pur.Pyr triplex in supercoiled plasmid was studied by a specific chemical probe. H-y3 was found to be more stable in terms of enthalpy change by 6 to 10 kcal/mol than H-y5, and the time constant for the conversion from H-y5 to H-y3 was 0.6 hr in the absence of Mg2+, while it was 6 hr in the presence of 50mM Mg2+. The transition probability from the duplex form to either two isomers was also measured: the kinetic barrier was higher for the transition to H-y3 than that to H-y5. These results indicated that Mg2+ acts to increase kinetic barriers of interconversion between H-y3 and H-y5 isomers.

Base Sequence↗
PubMed1994

Rarity of somatic and germline mutations of the cyclin-dependent kinase 4 inhibitor gene, CDK4I, in melanoma.

Evidence from cytogenetics, multipoint linkage analyses of familial melanoma, and loss of heterozygosity studies of familial and sporadic melanomas support localization of a melanoma susceptibility or tumor suppressor gene at chromosomal region 9p21-23. Recently, the inhibitor of cyclin-dependent kinase 4 (CDK4I; also known as p16INK4, multiple tumor suppressor 1, or CDKN2 gene) has been mapped to 9p21 and shown to be mutated or deleted in a large fraction of cell lines derived from many tumor types, including melanoma, suggesting that this gene could be a melanoma suppressor gene. In order to test for somatic mutations in the CDK4I gene in tumors, DNAs from 30 surgically resected melanomas of both cutaneous and uveal origins were sequenced. No mutations were detected in the coding region of the CDK4I gene, while mutations or deletions were detected in 60% (9 of 15) of the cultured melanoma cell line DNAs. Among presumptive familial cases, nine of which were members of families with one or two other documented melanoma cases, no germline mutations were detected by sequence analysis. A deletion in the second exon of the CDK4I gene was found in one germline allele of a familial melanoma patient from a family with eight affected first degree relatives. These results not only support the suggestion that the CDK4I gene is a familial malignant melanoma gene, they also suggest the presence of another suppressor gene locus within 9p21 which is the target of loss of heterozygosity in sporadic melanomas.

Base Sequence↗
PubMed1994

Role of the CCA terminal sequence of tRNA(Val) in aminoacylation with valyl-tRNA synthetase.

All known tRNAs have a universal CCA sequence at the 3'-terminal. To study the role of this terminal sequence in the aminoacylation process, base substitutions were introduced into a transcript of Escherichia coli valine tRNA and the effects on the aminoacylation activity with valyl-tRNA synthetase were evaluated. Substitution of the terminal adenosine residue at position 76 by C or U caused a 5-7-fold decrease of valine charging activity in Vmax/Km, while substitution by G resulted in about a 300-fold decrease. In addition, these mutations gave rise to an appreciable level of misaminoacylation with threonine. ATP hydrolysis activity during threonylation was lower in the terminal adenosine mutants than in the wild-type. Mutations introduced at positions 75 and 74 also caused threonylation instead of reducing valylation, albeit to a much smaller extent. These results indicate that the CCA sequence, especially the base portion of the terminal adenosine residue, plays an important role not only in amino-acylation efficiency with valine but also in preventing misaminoacylation by hydrolyzing misactivated threonyl-tRNA(Val).

Acylation↗
PubMed1994

Kinetic measurement of the interaction between an oligosaccharide and lectins by a biosensor based on surface plasmon resonance.

Kinetic measurements of the interaction between an oligosaccharide and various lectins were performed using a biosensor based on surface plasmon resonance (SPR). A glycopeptide, prepared from asialofetuin and having a nearly homogeneous N-linked sugar chain, was immobilized on the surface of a sensor chip via the amino groups of its peptide moiety. The interactions of this bound glycopeptide with six lectins [Sambucus sieboldiana lectin, Maackia amurensis lectin, Aleuria aurantia lectin, Ricinus communis agglutinin-120 (RCA120), Datura stramonium lectin (DSA) and Phaseolus vulgaris leukoagglutinating lectin] were monitored in real-time with the change in the SPR response. Of these lectins, only RCA120 and DSA showed an increase in the SPR response, indicating that these two lectins bound specifically to the immobilized glycopeptide. The other lectins did not show any significant changes in the SPR response. These results are in good agreement with the binding specificity previously demonstrated with affinity chromatography. The association-rate constant (kass) and the dissociation-rate constant (kdiss) for the glycopeptide-RCA120 interaction were 3.4 x 10(5) M-1 s-1 and 2.1 x 10(-3) s-1, respectively. The kass and kdiss determined for DSA were 5.7 x 10(5) M-1 s-1 and 1.3 x 10(-3) s-1, respectively. Furthermore, the relative binding molar ratio to the glycopeptide was three times higher for RCA120 than for DSA, suggesting that this sugar chain possesses three binding sites for RCA120 and one for DSA. These parameters are expected to provide useful information for defining the interaction between oligosaccharides and lectins.

Asialoglycoproteins↗
PubMed1994

Inhibitory effect of imipramine on depolarization-induced increases in intracellular Ca2+ of rat cortical neurons.

We examined the effects of imipramine on the increase in intracellular Ca2+ concentration ([Ca2+]i) induced by elevated K+ in cultured neurons of rat cortex. Imipramine (100 nM-200 microM) produced a concentration-dependent inhibition of [Ca2+]i increases induced by 25 mM K+ with an IC50 value of 32 microM. Imipramine had no effect on resting [Ca2+]i levels. When the cells were incubated with imipramine in the presence of a voltage-sensitive Ca2+ channel (VSCC) blocker, either nicardipine (10 microM), verapamil (10 microM), or omega-conotoxin GVIA (1 microM), the combinations of imipramine and each blocker resulted in an additive inhibition of 25 mM K(+)-induced [Ca2+]i increases. The IC50 values were 44, 29 and 24 microM, respectively, which were similar to those found when incubating the cells with imipramine alone. The presence or the absence of imipramine (30 microM) in an incubation with Bay K 8644 (100 nM), a VSCC agonist, showed similar potentiation of the [Ca2+]i increases induced by 15 mM K+ (66 and 52%, respectively). On the other hand, when the cells were incubated with imipramine in the presence of Ni2+ (300 microM) or La3+ (0.3 microM), inorganic Ca(2+)-channel blockers, the IC50 values of inhibition of 25 mM K(+)-induced [Ca2+]i increases were much lower than with imipramine alone (3.2 and 16 microM, respectively). However, incubations with Ni2+ combined with nicardipine or verapamil resulted in an additive inhibition of 25 mM K(+)-induced [Ca2+]i increases.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1994

Escherichia coli seryl-tRNA synthetase recognizes tRNA(Ser) by its characteristic tertiary structure.

To investigate the sequence requirements of Escherichia coli tRNA(Ser) for recognition by seryl-tRNA synthetase, various mutants of unmodified tRNA(Ser) were constructed. Substitution of G2.C71 by C2.G71, but not by A2.U71 or U2.A71, impaired the serine-accepting activity, indicating that this position is not involved in recognition by seryl-tRNA synthetase, but contributes to discrimination from other tRNAs processing C2.G71 such as tRNA(Leu). Other nucleotides characteristic of tRNA(Ser), including the discriminator base, were not involved in recognition by seryl-tRNA synthetase. The anticodon was not involved, as suggested by its sequence variety within the isoacceptors. The long variable arm composed of over ten nucleotides, which is a characteristic feature of tRNA(Ser) together with tRNA(Leu) and tRNA(Tyr), was stem-length-specifically, but not sequence-specifically, important for recognition. In order to introduce a sufficient serine-accepting activity to a tRNA(1LEU) transcript in vitro, besides the change from C2.G71 to G2.C71, the following elements had to be changed to those characteristic of tRNA(Ser): the sequence in the D-loop, the stem pairing pattern of the variable arm, the tertiary base-pair 15.48 and the nucleotide at position 59 in the T psi C-loop. None of the nucleotides at these changed positions was involved in base-specific recognition, indicating that seryl-tRNA synthetase selectively recognizes tRNA(Ser) on the basis of its characteristic tertiary structure rather than the nucleotides specific to tRNA(Ser).

Base Sequence↗
PubMed1994

Determination of tomatine in foods by liquid chromatography after derivatization.

A liquid chromatographic method for measuring tomatine levels in tomatoes and tomato products was developed. Tomatine was extracted with 1% acetic acid and purified on a C18 cartridge. Tomatine in the eluate was acetylated with acetic anhydride and isolated on a C18 cartridge. The solvent in the eluate was evaporated and the residue was dissolved in acetonitrile. An aliquot was injected into an Inertsil ODS-2 HPLC column and the acetylated tomatine was measured at 205 nm using a UV detector. The limit of determination was 1 microgram g-1. Tomatine was detected in the green portions of tomatoes and in tomato ketchups and juices at levels below 7 micrograms g-1.

Acetylation↗
PubMed1994

Detailed study of sequence-specific DNA cleavage of triplex-forming oligonucleotides linked to 1,10-phenanthroline.

We introduced eight bases, including four base analogs, into 15-mer triplex-forming oligonucleotides (TFOs) [d-psTTTCTTTNTTTTCTT; ps = thiophosphate; N = A, G, C, T, 2'-deoxyinosine (I), 2'-deoxyxanthosine (X), 5-methyl-2'-deoxycytidine (m5C), or 5-bromo-2'-deoxyuridine(br5U)] to investigate the Hoogsteen-like hydrogen bonding to the base in the target 34-mer strand (d-TGAGTGAGTAAAGAAARAAAAGAATGAGTGCCAA.d-TTGGCACTCATTCTTTTYTTTCT TTACTCACTCA; RY = AT, GC, TA, or CG). We examined the thermal stability of 15-mer triplexes in buffer containing 100 mM sodium acetate and 1 M NaCl at pH 5.0. The triplexes with typical triplets of T.AT (51.3 degrees C), br5U.AT (52.4 degrees C), C+.GC (66.7 degrees C), and m5C+.GC (66.8 degrees C) at the central position showed relatively higher Tm values, as expected. The relatively high stability of the X.AT triplex (39.8 degrees C) was observed. Among the N.TA triplets, G.TA (44.8 degrees C) was thermally the most stable, and moreover, the data showed that the N.TA triplet was also stabilized by I in the N position (40.7 degrees C). Furthermore, the TFOs were converted to DNA-cleaving molecules by introducing a newly synthesized 1,10-phenanthroline (OP) derivative on the thiophosphate group at the 5' end. Cleavage reactions of the 32P-labeled DNA (34-mer) were carried out. The cleavage efficiencies were compared to the Tm values of triplexes with or without an OP derivative. Results showed that the increased cleavage yields reflect the higher thermal stability of the triplex formed in most cases, but a few exceptional cases existed. Especially, the G-containing TFO did not show the above correlation between thermal stability and cleavage yield.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Mercaptopropionic Acid↗
PubMed1994

Multiple hepatic hemangiomas with significant arterioportal venous shunting.

A patient with multiple hepatic hemangiomas and significant arterioportal shunting who was successfully treated by a left-hepatic lobectomy is presented herein. In hemangiomas with significant arterioportal venous shunting, it is essential to differentiate between hepatocellular carcinoma and hypervascular metastatic tumors because few typical findings of hemangioma can be recognized. Moreover, an increased hepatic arterial flow can increase the risk of spontaneous rupture as well as portal hypertension due to the increased inflow to the portal vein. Therefore, a definitive diagnosis should be made by liver biopsy when imaging alone is not sufficient to rule out malignancy. Furthermore, hepatic resection for hemangiomas with significant arterioportal venous shunting may be indicated due to the higher risk of rupture and the possibility of progressive portal hypertension.

Angiography↗
PubMed1994

The loop sequence plays crucial roles for isomerization of intramolecular DNA triplexes in supercoiled plasmids.

The effect of base composition in the central region of polypurine.polypyrimidine (Pur.Pyr) tracts on the formation of intramolecular DNA triplexes in plasmids was examined using chemical probes (diethyl pyrocarbonate and OsO4), and two-dimensional (2-D) agarose gel electrophoresis. Two isomers exist for an intramolecular triplex: one with the 3'-half of the Pyr strand as the third strand (H-y3) and the other with the 5'-half of the Pyr strand as the third strand (H-y5). It was shown that the content and position of G + C residues in the triplex loop region (the center of Pur.Pyr tracts) are primary determinants for the isomerization between the H-y3 and H-y5 triplexes. Divalent metal ions such as Mg2+ and negative supercoiling also modulate the isomerization: the H-y5 conformation is stabilized by the divalent metal ions and/or under relatively lower negative supercoiling. 2-D gel analyses revealed that two isomers, H-y3 and H-y5, are topologically non-equivalent: the H-y3 formation relaxes one more supercoil turn than H-y5. As the G + C content in the center of Pur.Pyr tracts increases, the triplex requires more supercoil energy for formation. Therefore, the base-pair opening in the center of Pur.Pyr tracts is the initial and critical step in the pathway for the formation of triplex as well as the isomerization. The role of the triplex loop sequence is explained by a model in which the nucleation process of H-y3 formation requires a wide range of base-pair opening compared to that of H-y5: such unwinding would not be favored for the central region of the duplex with high G + C content and so it would be in the presence of Mg2+, and thereby the H-y5 formation is promoted.

Base Composition↗
PubMed1994

In vitro growth suppression and morphological change in a human renal cell carcinoma cell line by the introduction of normal chromosome 3 via microcell fusion.

Cytogenetic and molecular studies of human renal cell carcinoma (RCC) have suggested that the genetic and functional losses of one or more putative tumor suppressor genes on the short arm of chromosome 3 play a crucial role in the development of this disease. To examine whether the introduction of chromosome 3 has any effects on the biology of RCC cells, we introduced either chromosome 3, 7, or 11 from normal human fibroblasts into a newly established human RCC cell line with loss of heterozygosity for 3p, via microcell-mediated chromosome transfer. Microcell hybrids containing an introduced, intact chromosome 3 showed a significant reduction in in vitro growth rate and saturation density together with morphological alteration; these properties were not altered in microcell hybrids containing an introduced chromosome 7 or 11. During long-term cultivation, one of the clones that had lost the introduced chromosome 3 showed growth properties and morphology similar to those of the parental cell lines. Thus, our findings provide additional evidence for the presence of a putative tumor suppressor gene or genes on normal chromosome 3p and indicate that the gene is a dominant, negative growth regulator whose loss promotes progressive features of the neoplastic phenotype.

Carcinoma, Renal Cell↗
PubMed1994

Increased actin cable organization after single chromosome introduction: association with suppression of in vitro cell growth rather than tumorigenic suppression.

We previously showed that introduction of a single human chromosome 1, 6, or 9 derived from normal fibroblasts into HHUA endometrial carcinoma cells resulted in suppression of tumorigenicity. The tumorigenic suppression was accompanied by remarkable morphological changes in the microcell hybrids containing an extra copy of chromosome 1. The study presented here was undertaken to search for target cytoskeletal components affected by chromosome 1 transfer into endometrial carcinoma cells. We found that the microcell hybrids containing an extra copy of chromosome 1 were characterized by intracellular actin bundle formation and an excessive accumulation of actin and vinculin. The latter was a result of increased stabilization of the proteins. Additionally, chromosome 3 introduction into RCC23 human renal carcinoma cells resulted in prolongation of cell division and in senescence of a significant proportion of the microcell hybrids. In these microcell hybrids, the intracellular actin network was also reorganized, but the amounts of actin and vinculin protein were not increased. These findings suggest that the increased actin organization, which appeared not to cause tumorigenic suppression in the microcell hybrids, is associated with complementation of tumor suppressor genes and senescence by multiple mechanisms.

Actin Cytoskeleton↗
PubMed1994

Isolation and characterization of mutants of pseudorabies virus with deletion in the immediate-early regulatory gene.

Pseudorabies virus (PrV) immediate-early regulatory protein (IE180) is a potent and promiscuous activator of viral and cellular gene transcription. A mutant with deletion in the IE180 gene is expected to be useful for elucidating the functions of IE180. In this study, we constructed such mutants. Initially, transformants that express IE180 were established from PK-15 and Vero cells by transfection with the IE180 gene of the wild-type PrV. Two types of the deletion mutant could be obtained by using those transformants. One type was the deletion mutant, represented by AY64, in which the IE180 gene was replaced by insertion of the modified Escherichia coli lacZ gene, which was inserted under the control of the IE180 promoter. The other type, represented by AY1030, was a mutant produced from the former type by subsequent deletion of the lacZ gene. The deletion mutants of both types could replicate in the transformants with the IE180 gene, but not in the parent cells. On the other hand, the lacZ gene in AY64 was functional under the control of the IE180 promoter and could express beta-galactosidase (beta-gal) in not only the transformants but also the parent cells, indicating that the IE180 promoter of the mutants can fulfill the intended function in inoculated cells. The parent cells infected with the AY64 expressed only beta-gal, but neither infectious virus nor viral products relating to PrV. The deletion mutants constructed in this study appeared to provide a helpful model for investigating the functions of the IE180-homologs of other herpesviruses as well as IE180 itself, since PrV has only one IE gene. The applicability of the deletion mutants as a useful and safe vector for the introduction of foreign genes into various cells and animals was discussed.

Animals↗
PubMed1994

Pseudorabies virus immediate-early regulatory protein IE180 expressed by recombinant baculovirus is functional.

IE180, an immediate-early regulatory protein of pseudorabies virus, is required to activate viral early and late genes and is essential for viral growth. We constructed a recombinant baculovirus expressing IE180 in SF21AE insect cells. IE180 expressed by the recombinant migrated on gels as a single band with a molecular weight of 180 kDa and reacted with anti-IE180 peptide serum in Western blotting assay. Immunofluorescence using anti-IE180 peptide antibody demonstrated the accumulation of IE180 in cytoplasm of SF21AE cells infected with the recombinant virus. The IE180 produced in the insect cells was introduced into mammalian Vero cells infected with a PrV mutant with deletion of the IE180 gene by a cell-fusion technique. The growth of the deletion mutant in the fused cells was confirmed by infective assay and immunodetection of the progeny virus. This indicates that the IE180 expressed by the recombinant baculovirus is functional as a transactivator.

Animals↗