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Biomedical subjects

M Yoshida

Publications and source records attributed to M Yoshida.

At least 523 records · Page 29Linked to original sources
PubMed1999

Molecular cloning and expression in Escherichia coli of the extracellular endoprotease of Aeromonas caviae T-64, a pro-aminopeptidase processing enzyme(1).

PA protease (pro-aminopeptidase processing protease) activates the pro-aminopeptidases from Aeromonas caviae T-64 and Vibrio proteolytica by removal of their pro-regions. Cloning and sequencing of the PA protease gene revealed that PA protease was translated as a preproprotein consisting of four domains: a signal peptide; an N-terminal propeptide; a mature region; and a C-terminal propeptide. The deduced amino acid sequence of the PA protease precursor showed significant homology with several bacterial metalloproteases. Expression of the PA protease gene in Escherichia coli indicated that the N-terminal propeptide of the PA protease precursor is essential to obtain the active form of the protease. The N- and C-terminal propeptides of the expressed pro-PA protease were processed autocatalytically.

Aeromonas↗
PubMed1999

Polyamine stimulation of the synthesis of oligopeptide-binding protein (OppA). Involvement of a structural change of the Shine-Dalgarno sequence and the initiation codon aug in oppa mRNA.

We previously suggested that the degree of polyamine stimulation of oligopeptide-binding protein (OppA) synthesis is dependent on the secondary structure and position of the Shine-Dalgarno (SD) sequence of OppA mRNA. To study the structural change of OppA mRNA induced by polyamines and polyamine stimulation of initiation complex formation, four different 130-mer OppA mRNAs containing the initiation region were synthesized in vitro. The structural change of these mRNAs induced by polyamines was examined by measuring their sensitivity to RNase T(1), specific for single-stranded RNA, and RNase V(1), which recognizes double-stranded or stacked RNA. In parallel, the effect of spermidine on mRNA-dependent fMet-tRNA binding to ribosomes was examined. Our results indicate that the secondary structure of the SD sequence and initiation codon AUG is important for the efficiency of initiation complex formation and that spermidine relaxes the structure of the SD sequence and the initiation codon AUG. The existence of a GC-rich double-stranded region close to the SD sequence is important for spermidine stimulation of fMet-tRNA binding to ribosomes. Spermidine apparently binds to this GC-rich stem and causes a structural change of the SD sequence and the initiation codon, facilitating an interaction with 30 S ribosomal subunits.

5' Untranslated Regions↗
PubMed1999

Leptomycin B inactivates CRM1/exportin 1 by covalent modification at a cysteine residue in the central conserved region.

The cellular target of leptomycin B (LMB), a nuclear export inhibitor, has been identified as CRM1 (exportin 1), an evolutionarily conserved receptor for the nuclear export signal of proteins. However, the mechanism by which LMB inhibits CRM1 still remains unclear. CRM1 in a Schizosaccharomyces pombe mutant showing extremely high resistance to LMB had a single amino acid replacement at Cys-529 with Ser. The mutant gene, named crm1-K1, conferred LMB resistance on wild-type S. pombe, and Crm1-K1 no longer bound biotinylated LMB. (1)H NMR analysis showed that LMB bound N-acetyl-L-cysteine methyl ester through a Michael-type addition, consistent with the idea that LMB binds covalently via its alpha, beta-unsaturated delta-lactone to the sulfhydryl group of Cys-529. When HeLa cells were cultured with biotinylated LMB, the only cellular protein bound covalently was CRM1. Inhibition by N-ethylmaleimide (NEM), an alkylating agent, of CRM1-mediated nuclear export probably was caused by covalent binding of the electrophilic structure in NEM to the sulfhydryl group of Cys-529, because the crm1-K1 mutant showed the normal rate for the export of Rev nuclear export signal-bearing proteins in the presence of not only LMB but also NEM. These results show that the single cysteine residue determines LMB sensitivity and is selectively alkylated by LMB, leading to CRM1 inactivation.

Amino Acid Sequence↗
PubMed1999

Targeted disruption of CRE-binding factor TREB5 gene leads to cellular necrosis in cardiac myocytes at the embryonic stage.

TREB5 (hXBP-1) is a basic region leucine zipper protein which binds to a CRE-like element in both human T-cell leukemia virus type 1 and MHC class II genes. To study the function(s) of TREB5 in normal development, we have generated TREB5 deficient mice by gene targeting. Heterozygous mutant mice have not exhibited any obvious abnormalities; however, homozygous mutant embryos die between embryonic days 10.5 and 14.5. The major defect responsible for lethality is cellular necrosis of cardiac myocytes located at the atrium and the truncus arteriosus with its following ventricle. Necrotic alteration was not observed in either the endocardial cushion or the conotruncal ridge. These results indicate that TREB5 plays an essential role in maintenance and/or growth of cardiac myocytes during cardiogenesis.

Animals↗
PubMed1999

D allele of the angiotensin-converting enzyme gene is a risk factor for secondary cardiac events after myocardial infarction.

We retrospectively examined the relationship between the genotype of the angiotensin-converting enzyme (ACE) gene or the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene, and the secondary cardiac events after myocardial infarction. The study population consisted of 176 patients (ACE genotype: deletion homozygote (DD)=20, insertion/deletion heterozygote (ID)=91, insertion homozygote (II)=65; MTHFR genotype: valine homozygote (VV)=37, valine/alanine heterozygote (VA)=71, alanine homozygote (AA)=68) with acute or recent myocardial infarction at the start of the follow-up. We defined the occurrence of cardiac death, recurrent myocardial infarction, or admission due to unstable angina as the endpoint. Cardiac events related coronary intervention were excluded from the endpoints. During the follow-up (1903+/-1414 days), four patients had cardiac death, 12 patients had recurrent myocardial infarction and 13 patients had admission due to unstable angina. A Cox analysis revealed that the endpoints were significantly associated with diabetes mellitus (RR=4.423), total cholesterol level (RR=1.025) and the genotype of the ACE gene (RR=4.490). The ID or DD genotype of the ACE gene was associated with higher occurrence of the endopoints. The MTFHR gene was not associated with the endopoint. The present results suggest that the presence of the deletion allele of the ACE gene may be a risk factor for secondary cardiac events after myocardial infarction.

Alleles↗
PubMed1999

On the maximum size of proteins to stay and fold in the cavity of GroEL underneath GroES.

GroEL encapsulates non-native protein in a folding cage underneath GroES (cis-cavity). Here we report the maximum size of the non-native protein to stay and fold in the cis-cavity. Using total soluble proteins of Escherichia coli in denatured state as binding substrates and protease resistance as the measure of polypeptide held in the cis-cavity, it was estimated that the cis-cavity can accommodate up to approximately 57-kDa non-native proteins. To know if a protein with nearly the maximum size can complete folding in the cis-cavity, we made a 54-kDa protein in which green fluorescent protein (GFP) and its blue fluorescent variant were fused tandem. This fusion protein was captured in the cis-cavity, and folding occurred there. Fluorescence resonance energy transfer proved that both GFP and blue fluorescent protein moieties of the same fused protein were able to fold into native structures in the cis-cavity. Consistently, simulated packing of crystal structures shows that two native GFPs just fit in the cis-cavity. A fusion protein of three GFPs (82 kDa) was also attempted, but, as expected, it was not captured in the cis-cavity.

Animals↗
PubMed1999

Tax protein of HTLV-1 inhibits CBP/p300-mediated transcription by interfering with recruitment of CBP/p300 onto DNA element of E-box or p53 binding site.

Tax protein of human T-cell leukemia virus type 1 (HTLV-1) is a potent transcriptional regulator which can activate or repress specific cellular genes and has been proposed to contribute to leukemogenic processes in adult T-cell leukemia. The molecular mechanism of Tax-mediated trans-activation has been well investigated. However, trans-repression by Tax remains to be studied in detail, although it is known to require a specific DNA element such as E-box or p53 binding site. Examining possible mechanisms of trans-repression, we found that co-expression of E47 and p300 activated E-box dependent transcription and this activation was efficiently repressed by Tax. In this system, Tax bound to p300 and decreased the level of p300 complexed on the E-box element. Similarly, Tax inhibited transcription directed by p53 and CBP, reducing the level of CBP on the p53 binding site. These results indicate that Tax interferes with recruitment of CBP/p300 into protein complexes on E-box and p53 binding site through its binding to CBP/p300. In contrast to these findings, we observed that Tax increased the level of CBP on the viral 21-bp enhancer which is trans-activated by Tax. From these observations, we propose a universal mechanism for Tax-mediated trans-repression and trans-activation of transcription in which Tax binds to CBP/p300 and determines the accessibility of CBP/p300 to protein complexes on specific DNA element.

Binding Sites↗
PubMed1999

Rotation of Escherichia coli F(1)-ATPase.

By applying the same method used for F(1)-ATPase (TF(1)) from thermophilic Bacillus PS3 (Noji, H., Yasuda, R., Yoshida, M., and Kinosita, K., Jr. (1997) Nature 386, 299-302), we observed ATP-driven rotation of a fluorescent actin filament attached to the gamma subunit in Escherichia coli F(1)-ATPase. The torque value and the direction of the rotation were the same as those observed for TF(1). F(1)-ATPases seem to share common properties of rotation irrespective of the sources.

Actin Cytoskeleton↗
PubMed1999

Down-regulation of the INK4 family of cyclin-dependent kinase inhibitors by tax protein of HTLV-1 through two distinct mechanisms.

Tax oncoprotein of human T-cell leukemia virus type 1 (HTLV-1) affects multiple regulatory processes of infected cells through activation and repression of specific transcription and also through modulation of functions of cell cycle regulators. Previously, we found that Tax binds to p16ink4a, a member of the INK4 family of cyclin-dependent kinase inhibitors, and counteracts its inhibitory activity, resulting in cell cycle progression. In this study, we examined the effects of Tax on other members of the INK4 family and found that Tax can bind to p15ink4b similarly to p16ink4a, but not to p18ink4c and p19ink4d. Tax binding to p15ink4b inactivated its function and restored CDK4 kinase activity. Accordingly, Tax-expressing cells became resistant to p15ink4b-mediated growth arrest induced by TGFbeta. On the other hand, expression of p18ink4c was transcriptionally repressed by Tax through the E-box element of the promoter, which may contribute to the marked reduction of p18ink4c mRNA in HTLV-1-infected T-cells. These observations indicate that Tax suppresses the inhibitory activities of INK4 family members through two independent mechanisms: functional inhibition of two INK4 proteins and repression of expression of another INK4 protein. These effects may play roles in HTLV-1-induced deregulation of the cell cycle, possibly promoting cellular transformation.

Carrier Proteins↗
PubMed1999

Inositol 1,3,4-trisphosphate acts in vivo as a specific regulator of cellular signaling by inositol 3,4,5,6-tetrakisphosphate.

Ca2+-activated Cl- channels are inhibited by inositol 3,4,5, 6-tetrakisphosphate (Ins(3,4,5,6)P4) (Xie, W., Kaetzel, M. A., Bruzik, K. S., Dedman, J. R., Shears, S. B., and Nelson, D. J. (1996) J. Biol. Chem. 271, 14092-14097), a novel second messenger that is formed after stimulus-dependent activation of phospholipase C (PLC). In this study, we show that inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) is the specific signal that ties increased cellular levels of Ins(3,4,5,6)P4 to changes in PLC activity. We first demonstrated that Ins(1,3,4)P3 inhibited Ins(3,4,5,6)P4 1-kinase activity that was either (i) in lysates of AR4-2J pancreatoma cells or (ii) purified 22,500-fold (yield = 13%) from bovine aorta. Next, we incubated [3H]inositol-labeled AR4-2J cells with cell permeant and non-radiolabeled 2,5,6-tri-O-butyryl-myo-inositol 1,3, 4-trisphosphate-hexakis(acetoxymethyl) ester. This treatment increased cellular levels of Ins(1,3,4)P3 2.7-fold, while [3H]Ins(3, 4,5,6)P4 levels increased 2-fold; there were no changes to levels of other 3H-labeled inositol phosphates. This experiment provides the first direct evidence that levels of Ins(3,4,5,6)P4 are regulated by Ins(1,3,4)P3 in vivo, independently of Ins(1,3,4)P3 being metabolized to Ins(3,4,5,6)P4. In addition, we found that the Ins(1, 3,4)P3 metabolites, namely Ins(1,3)P2 and Ins(3,4)P2, were >100-fold weaker inhibitors of the 1-kinase compared with Ins(1,3,4)P3 itself (IC50 = 0.17 microM). This result shows that dephosphorylation of Ins(1,3,4)P3 in vivo is an efficient mechanism to "switch-off" the cellular regulation of Ins(3,4,5,6)P4 levels that comes from Ins(1,3, 4)P3-mediated inhibition of the 1-kinase. We also found that Ins(1,3, 6)P3 and Ins(1,4,6)P3 were poor inhibitors of the 1-kinase (IC50 = 17 and >30 microM, respectively). The non-physiological trisphosphates, D/L-Ins(1,2,4)P3, inhibited 1-kinase relatively potently (IC50 = 0.7 microM), thereby suggesting a new strategy for the rational design of therapeutically useful kinase inhibitors. Overall, our data provide new information to support the idea that Ins(1,3,4)P3 acts in an important signaling cascade.

Animals↗
PubMed1999

Analysis of interaction between chaperonin GroEL and its substrate using fluorescence correlation spectroscopy.

Fluorescence correlation spectroscopy (FCS) provides information about translational diffusion properties of fluorescent molecules in tiny detection volume and allows the analysis of binding processes of biomolecules in homogeneous solution. In this study, FCS was used to measure equilibrium binding constants of disulfide-reduced apo-alpha-lactalbumin (rLA), denatured pepsin, and apo-cytochrome c (apo-cyt c) bound by chaperonin GroEL at different salt concentrations. The results indicate that apo-cyt-c has a much stronger affinity to GroEL than denatured pepsin and rLA have. Titration experiments of GroEL to each substrate with various concentrations of four kinds of salts (K+, Na+, Ca2+, and Mg2+) show that the binding constant of denatured pepsin and rLA to GroEL depends on the salt concentration. The dependence of denatured pepsin binding to GroEL on salt concentration is much stronger than that of rLA. However, the interaction of positively charged apo-cyt c with GroEL is not affected by the salt concentration. Furthermore, the divalent cation promotes the binding of GroEL to denatured pepsin and rLA more strongly than does the monovalent cation.

Animals↗
PubMed1999

Conserved and divergent expression patterns of the proteolipid protein gene family in the amphibian central nervous system.

The recent discovery of a proteolipid protein gene family has revealed that its members are in fact widely distributed and are not exclusively associated with myelination. To date, three different gene products, DMalpha/DM-20/PLP, DMbeta/M6a, and DMgamma/M6b, have been isolated from certain primitive fish species, mouse, and human central nervous system (CNS). We cloned Xenopus laevis orthologues of DMbeta/M6a and DMgamma/M6b and investigated the expression patterns of these gene transcripts as well as that of PLP in developing Xenopus CNS. As is the case in shark and mouse, the mRNA encoding the major myelin integral protein, PLP, is first detected at stage 42/43 in tadpoles and is exclusively found in morphologically recognizable oligodendrocytes throughout the brain, while DMbeta mRNA is solely expressed in young presumptive neurons in the gray matter. There exist two distinct DMgamma mRNAs and, in contrast to these evolutionarily conserved expression patterns, DMgamma mRNAs distribute uniquely within the ventricular zone in young tadpoles (stage 25) through maturity. Furthermore, both DMbeta and DMgamma are expressed in the developing retina, and their distributions are different from one other. In Xenopus CNS, therefore, the expression patterns of three proteolipid proteins, PLP, DMbeta, and DMgamma, are distinct from each other, implying very different roles for their protein products within the cell populations in which they are expressed.

Amino Acid Sequence↗
PubMed1999

Hepatotoxicity and consequently increased cell proliferation are associated with flumequine hepatocarcinogenesis in mice.

It has been reported that flumequine (FLU) induces hepatic tumors in mice when given orally for 18 months. We investigated possible underlying mechanisms using a two-stage mouse hepatocarcinogenesis model. After initiation with a single intraperitoneal injection of 100 mg/kg body weight diethylnitrosamine (DEN) or saline, male CD-1 mice were given 4000 ppm FLU in the diet or 500 ppm phenobarbital (PB) in drinking water for 9, 19, 24 or 30 weeks. Toxicity, evidenced by centrilobular swollen and polar hepatocytes with fatty droplets, infiltration of inflammatory cells and increased numbers of mitosis in hepatocytes, was apparent in the livers of mice treated with FLU at all time points, but its severity declined towards the termination. FLU did not induce cytochrome P-450 enzymes such as 1A1, 2B1 and 3A2 as assessed immunohistochemically, while positive expression of 8-hydroxy-2'-deoxyguanosine (8-OHdG) was increased in hepatocytes of both DEN + FLU and FLU groups compared with the relevant controls. In animals given PB, eosinophilic swelling of hepatocytes was prominent, and the hepatocytes showed strongly positive reactions for CYP 1A1 and 3A2. Altered cell foci were induced in the livers of FLU-treated animals both with and without DEN initiation, especially the former, and their development paralleled the degree of hepatic toxicity. These results suggest that FLU hepatocarcinogenicity in mice is dependent on hepatotoxic damage and consequently increased cell proliferation. Oxidative damage to DNA may also be a crucial factor.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1999

Transplacental administration of diethylstilbestrol (DES) causes lesions in female reproductive organs of Donryu rats, including endometrial neoplasia.

The effects of transplacental administration of diethylstilbestrol (DES) on female reproductive organs were investigated using Donryu rats. The animals were given subcutaneous injections of DES dissolved in olive oil at doses of 0.01 or 0.1 mg/kg on days 17 and 19 of gestation. In female offspring, clinical signs, body weights and estrous cycles were continuously assessed until all survivors were killed at month 18. A low mean litter size and shortening of period of pregnancy were recognized in the 0.1 mg/kg group. Disorder and/or suspension of the estrous cycle (so called persistent estrus) also appeared very early in the 0.1 mg/kg group. Macroscopically, the incidences of hypoplasia of the oviduct, cystic dilatation of the uterus and small size of the uterine cervix were higher in the 0.1 mg/kg group than those in the control group. Histologically, in the ovary, the incidence and degree of atrophy were increased in both 0.01 and 0.1 mg/kg groups. In the uterus, total incidences of endometrial hyperplasias were about the same in all groups. However, endometrial adenocarcinomas were dose-dependently increased in the treated groups, the incidence in the 0.1 mg/kg group being significant, compared to that in the control. In the vagina, mucification was more prominent in the treated animals, especially at the higher dose, but no tumors were observed. The present results indicate that prenatal exposure to DES can produce uterine adenocarcinomas in rats, as reported earlier for mice, although its carcinogenic activity is not so strong. Increase of endometrial adenocarcinoma incidence might depend on hormonal imbalance resulting from the ovarian atrophy due to transplacental treatment of DES.

Adenocarcinoma↗
PubMed1999

Intramolecular chaperone and inhibitor activities of a propeptide from a bacterial zinc aminopeptidase.

An aminopeptidase from Aeromonas caviae T-64 was translated as a preproprotein consisting of three domains; a signal peptide (19 amino acid residues), an N-terminal propeptide (101 residues) and a mature region (273 residues). We demonstrated that a proteinase, which was isolated from the culture filtrate of A. caviae T-64, activated the recombinant pro-aminopeptidase by removal of the majority of the propeptide. Using L-Leu-p-nitroanilide as a substrate, the processed aminopeptidase showed a large increase in kcat when compared with the unprocessed enzyme, whereas the Km value remained relatively unchanged. The similar Km values for the pro-aminopeptidase and the mature aminopeptidase indicated that the N-terminal propeptide of the pro-aminopeptidase did not influence the formation of the enzyme-substrate complex, suggesting the absence of marked conformational changes in the active domain. In contrast, the marked difference in kcat suggests a significant decrease in the energy of one or more of the transition states of the enzyme-substrate reaction coordinate. Moreover, we showed that the activity of the urea-denatured pro-aminopeptidase could be recovered by dialysis, whereas the activity of the urea-denatured mature aminopeptidase, which lacked the propeptide, could not. Further to this, the propeptide-deleted aminopeptidase formed an inclusion body in the cytoplasmic space in Escherichia coli and was not secreted at all. These results suggested that the propeptide of the pro-aminopeptidase acted as an intramolecular chaperone that was involved with the correct folding of the enzyme in vitro and was required for extracellular secretion in E. coli.

Aeromonas↗
PubMed1999

Heat-inactivated proteins are rescued by the DnaK.J-GrpE set and ClpB chaperones.

Functional chaperone cooperation between Hsp70 (DnaK) and Hsp104 (ClpB) was demonstrated in vitro. In a eubacterium Thermus thermophilus, DnaK and DnaJ exist as a stable trigonal ring complex (TDnaK.J complex) and the dnaK gene cluster contains a clpB gene. When substrate proteins were heated at high temperature, none of the chaperones protected them from heat inactivation, but the TDnaK.J complex could suppress the aggregation of proteins in an ATP- and TGrpE-dependent manner. Subsequent incubation of these heated preparations at moderate temperature after addition of TClpB resulted in the efficient reactivation of the proteins. Reactivation was also observed, even though the yield was low, if the substrate protein alone was heated and incubated at moderate temperature with the TDnaK.J complex, TGrpE, TClpB, and ATP. Thus, all these components were necessary for the reactivation. Further, we found that TGroEL/ES could not substitute TClpB.

Bacterial Proteins↗