PubMed Health⌕ Search

Biomedical subjects

M Tokumoto

Publications and source records attributed to M Tokumoto.

At least 37 records · Page 2Linked to original sources
PubMed2000

Identification of the goldfish 20S proteasome beta6 subunit bound to nuclear matrix.

Proteasomes are large, multisubunit particles that act as the proteolytic machinery for most of the regulated intracellular protein breakdown in eukaryotic cells. Proteasomes are present in both the nucleus and cytoplasm. When we analyzed the molecular composition of protein constituents of the nuclear matrix preparation of goldfish oocytes by two-dimensional polyacrylamide gel electrophoresis followed by sequence analysis, we found a 26 kDa spot identical in amino acid sequence to the beta6 subunits of the 20S proteasome. No spot of other subunits of 20S proteasome was detected. Here we describe the cloning, sequencing and expression analysis of Carassius auratus, beta6_ca, which encodes one of the proteasome beta subunits from goldfish ovary. From the screening of an ovarian cDNA library, two types of cDNA were obtained, one 941 bp and the other 884 bp long. The deduced amino acid sequences comprise 239 and 238 residues, respectively. These deduced amino acid sequences are highly homologous to those of beta6 subunits of other vertebrates. Immunoblot analysis of nuclear matrix using anti-proteasome antibodies showed only a spot of beta6_ca. These results suggest that the beta6 subunit of the goldfish 20S proteasome, beta6_ca, is responsible for anchoring proteasomes in the nucleus.

Amino Acid Sequence↗
PubMed2000

Two proteins, a goldfish 20S proteasome subunit and the protein interacting with 26S proteasome, change in the meiotic cell cycle.

To investigate the regulatory mechanism for the proteasome in the meiotic cell cycle, we purified the 26S proteasome from immature (in G2-phase) and mature (in M-phase) oocytes, and compared its subunits by immunoblotting. At least two protein bands, at 30 kDa (detected by GC3beta antibody) and 62 kDa (detected by 1-4D5 antibody), differed between 26S proteasomes. A monoclonal antibody, GC3beta cross-reacted with two bands in the 26S proteasome from immature oocytes, however, the upper band was absent in the 26S proteasome from mature oocytes. The 62-kDa protein band detected by 1-4D5 antibody was not detected in the immature oocyte 26S proteasome; however, a band was detected in mature oocyte 26S proteasome. The cDNAs encoding these proteins were isolated by an immunoscreening method using the monoclonal antibodies. The 30-kDa protein was an alpha4 subunit, which is one of the alpha-subunit group of the 20S proteasome, and the 62-kDa protein was a homologue of CCTepsilon, one of the components of eukaryotic molecular chaperones. Phosphatase treatment of the 26S proteasome revealed that a part of the alpha4 subunit of goldfish 20S proteasome, alpha4_ca, is phosphorylated in G2-phase and dephosphorylated in M-phase. A binding assay using a recombinant goldfish CCTepsilon revealed that unmodified CCTepsilon interacts with the 26S proteasome. Fertilization triggers a transition from meiotic metaphase to mitotic interphase. During fertilization, a GC3beta cross-reacting upper band reappeared. The 62-kDa band dissociated from the 26S proteasome. As a result, the 26S proteasome changed to an immature type from a mature type during fertilization. These results suggest that the 26S proteasome is changed reversibly during the meiotic cell cycle by modification of its subunits and interactions between regulators.

Amino Acid Sequence↗
PubMed2000

cDNA cloning and molecular analysis of papaya small GTP-binding protein, pgp1.

In the course of papaya EST collection, one clone (pRA4-3) encoding partial sequence of papaya small GTP-binding protein gene, pgp1, was obtained. Based on the sequence information of pRA4-3, the entire coding region of pgp1 was cloned using the 3'RACE PCR technique. ORF of pgp1 is 636bp long and deduced molecular weight of the protein is 23,311. Phylogenetic analysis showed that PGP1 belongs to YPT/RAB group of the small GTP-binding protein and is a homologue of RAB2. Southern analysis showed that there are several pgp1-related genes in papaya genome. Northern analysis showed that pgp1 was expressed equally in stems of seedlings that were grown under light and dark conditions. This result shows that PGP1 is not involved in the phytochrome-mediated signal transduction as an auxin signal transducer in stems of papaya seedlings.

Amino Acid Sequence↗
PubMed2000

[The microbiology laboratory in the diagnosis of bacteremia associated with catheters].

Catheter related sepsis is an outstanding problem in patients in every age group. The microbiological diagnosis should consider the main pathways of infection (catheter-skin interface, endoluminal). With this aim we analysed 1496 central and peripheral short term catheters and 119 episodes of catheter related bacteremia. Catheters were cultured according to the quantitative technique of Brun Buisson (QT), the semiquantitative technique of Maki (SQ) and qualitative broth culture (QL). The following results of sensitivity, specificity, positive predictive value, negative predictive value and Youden index were obtained: SQ = 87%, 88%, 40%, 99%, 0.75; QT (> or = 10(2) CFU/ml) = 88%, 89%, 43%, 99%, 0.77; QT (> or = 10(3) CFU/ml) = 77%, 92%, 48%, 97%, 0.69; QL = 94%, 68%, 20%, 99%, 0.62. Results of SQ and QT > or = 10(2) were comparable, nevertheless, the addition of QT to SQ increased the detection of bacteremia by 12.8%, while in the opposite situation the increase was 10%. According to this, it is advisable to combine routinely SQ and QT. Finally, in 42 episodes of bacteremia related to implanted catheters processed by quantitative differential culture of blood drawn through the catheter and blood drawn through the peripheral vein the relationships were: > 1000 in 79% of cases, between 100 and 1000 in 9% of cases and between 5 and 10 in just 5% of cases.

Bacteremia↗
PubMed1999

Identification of the Xenopus 20S proteasome alpha4 subunit which is modified in the meiotic cell cycle.

The proteasomes are large, multi-subunit particles that act as the proteolytic machinery for most of the regulated intracellular protein degradation in eukaryotic cells. To investigate the regulatory mechanism for the 26S proteasome in cell-cycle events, we purified this proteasome from immature and mature oocytes, and compared its subunits. Immunoblot analysis of 26S proteasomes showed a difference in the subunit of the 20S proteasome. A monoclonal antibody, GC3beta, cross-reacted with two bands in the 26S proteasome from immature oocytes (in G2-phase); however, the upper band was absent in the 26S proteasome from mature oocytes (in M-phase). These results suggest that changes in the subunits of 26S proteasomes are involved in the regulation of the meiotic cell cycle. Here we describe the molecular cloning of one of the alpha subunits of the 20S proteasome from a Xenopus ovarian cDNA library using an anti-GC3beta monoclonal antibody. From the screening, two types of cDNA are obtained, one 856bp, the other 984bp long. The deduced amino-acid sequences comprise 247 and 248 residues, respectively. These deduced amino-acid sequences are highly homologous to those of alpha4 subunits of other vertebrates. Phosphatase treatment of 26S proteasome revealed the upper band to be a phosphorylated form of the lower band. These results suggest that a part of the alpha4 subunit of the Xenopus 20S proteasome, alpha4_xl, is phosphorylated in G2-phase and dephosphorylated in M-phase.

Amino Acid Sequence↗
PubMed1999

Molecular cloning of cDNA encoding a cyclin-selective ubiquitin carrier protein (E2-C) from Carassius auratus (goldfish) and expression analysis of the cloned gene.

Destruction of cyclin B is required for exit from mitosis and meiosis. A cyclin-specific ubiquitinating system, including cyclin-selective ubiquitin carrier protein (E2-C), is thought to be responsible for cyclin B destruction. Here we present the cloning, sequencing and expression analysis of goldfish, Carassius auratus, E2-C which encodes the cyclin-selective ubiquitin carrier protein from goldfish ovary. The cloned cDNA is 677 bp long and encodes 172 amino acids. The deduced amino acid sequence is highly homologous to E2-C from other species. Recombinant goldfish E2-C possesses ubiquitinating activity against cyclin B. The expression of mRNA for E2-C was similar to that of mRNA for cyclin B, occurring at very high level in the ovary. The similarity of the expression pattern of E2-C and cyclin B suggests that E2-C mediates a cyclin-specific ubiquitination.

Amino Acid Sequence↗
PubMed1999

Disappearance of a novel protein component of the 26S proteasome during Xenopus oocyte maturation.

We have prepared polyclonal antibodies against Xenopus 20S proteasomes. The antibodies cross-react with several proteins that are common to 20S and 26S proteasomes and with at least two proteins that are unique to 26S proteasomes. The antibodies were used to analyze changes in the components of proteasomes during oocyte maturation and early development of Xenopus laevis. A novel protein with a molecular weight of 48 kDa, p48, was clearly detected in immature oocytes, but was found at very low levels in mature oocytes and ovulated eggs. p48 was reduced to low levels during oocyte maturation, after maturation-promoting factor was activated. The amount of p48 in eggs remained low during early embryonic development, but increased again after the midblastula transition. These results show that at least one component of 26S proteasomes changes during oocyte maturation and early development and suggest that alterations in proteasome function may be important for the regulation of developmental events, such as the rapid cell cycles, of the early embryo.

Animals↗
PubMed1999

Differential gene expressions of rice superoxide dismutase isoforms to oxidative and environmental stresses.

Active oxygen species (AOSs) are produced under stress conditions of plant cells. Superoxide dismutase (SOD) catalyzes the first step in the AOS scavenging system. The responses of SOD genes to environmental stresses were analyzed in rice seedlings by the treatments of drought, salinity and chilling. The expressions of abscisic acid (ABA)-inducible genes, Mn-SOD gene (sodA1) and one of the cytosolic Cu/Zn-SOD genes (sodCc2), were strongly induced by the treatment of drought and salinity. While Fe-SOD gene (sodB) and the other cytosolic Cu/Zn-SOD gene (sodCc1) were also induced by ABA. However the mRNA level of sodB was decreased by drought treatment, and sodCc1 gene was not induced by drought and salinity treatments. Plastidic Cu/Zn-SOD gene (sodCp) quickly responded to salinity treatment in the light but not in the dark. In the treatment with hydrogen peroxide, sodCp gene was strongly induced shortly after the treatment. These results suggested that phytohormone and AOSs are associated with the regulation of SOD genes under environmental stresses.

Abscisic Acid↗
PubMed1999

Molecular cloning and characterization of a cDNA for an iron-superoxide dismutase in rice (Oryza sativa L.).

We have isolated a cDNA encoding Fe-SOD from rice (Oryza sativa L.). The deduced amino acid sequence consists of a polypeptide with 255 amino acids, including a putative transit peptide (40 a.a.) in amino-terminal residues. This sequence is similar to the known plant Fe-SODs but not classified in the group of known Fe-SODs. The metal analysis and SOD assays of the partial purified recombinant protein expressed in E. coli showed that this cDNA encodes an iron-containing SOD. However this SOD activity was not inhibited by the treatment with hydrogen peroxide, which was expected to inhibit known Fe-SOD activity. mRNA of rice Fe-SOD was detected in all vegetative tissues examined, being especially abundant in calli, and strongly increased by light induction. These results suggested that this cDNA encodes rice Fe-SOD, which is apparently distinct from known plant Fe-SODs.

Amino Acid Sequence↗
PubMed1999

[Monitoring antibiotic resistance in Argentina. The WHONET program, 1995-1996].

The World Health Organization has implemented a surveillance program for antimicrobial resistance that is known as WHONET. In Argentina the program was developed through a network of 23 public and private hospitals that participate in national and international quality-control programs. Between January 1995 and December 1996, the antimicrobial susceptibility of 16,073 consecutive clinical isolates was determined, using the recommended standards of the National Committee for Clinical Laboratory Standards of the United States of America. More than half of the Escherichia coli urinary isolates were resistant to ampicillin and more than 30% to trimethoprim/sulfamethoxazole (SXT). When the percentage of resistant isolates from outpatients (OPs) was compared to that observed in hospitalized patients (HPs), a marked difference in antimicrobial activity was noted in the case of gentamicin (2% from OPs resistant vs. 8% from HPs resistant), norfloxacin (2% vs. 6%), and third-generation cephalosporins (7% vs. 15%). Of the Klebsiella pneumoniae isolates recovered from blood cultures, 71% and 60% showed resistance to third-generation cephalosporins and to gentamicin, respectively. The overall rate of oxacillin resistance in Staphylococcus aureus was 39%. Around half of the Enterococcus spp. isolates showed high resistance to aminoglycosides, but resistance to glycopeptides was not found. In Argentina, ampicillin and SXT were not suitable for treating diarrhea. Shigella flexneri had a higher number of isolates resistant to both of those drugs (87% and 74%, respectively) than Sh. sonnei did (47% and 71%, respectively). About 40% of the Salmonella spp. isolated in pediatric hospitals were resistant to third-generation cephalosporins. When microorganisms causing bacterial meningitis were examined, Streptococcus pneumoniae showed a resistance rate of 18% to penicillin and Haemophilus influenzae a resistance rate of 19% to ampicillin. These rates are within the intermediate range reported for other countries of the Americas and for Europe.

Cephalosporins↗
PubMed1999

[In vitro activity of trovafloxacin, of other fluoroquinolones and of related antimicrobials against clinical isolates. Grupo colaborativo WHONET-Argentina].

The in vitro activity of trovafloxacin (TRV) has been evaluated in comparison with that of other antimicrobial agents against 5671 clinical isolates recovered by representative institutions of different provinces in our country. The resistance percentage to gentamicin and third generation cephalosporins among enterobacteriaceae was high: 17% and 16% respectively, with a considerable variation according to the analyzed species. The resistance to ciprofloxacin (CIP) and TRV affected approximately 9% of the isolates, without significant differences between both drugs. Fluoroquinolones (FQ) presented excellent activity on 166 isolates of Salmonella spp., 208 of Shigella flexneri and 76 of Shigella sonnei, where only one S.sonnei isolate was resistant to CIP, but susceptible to TRV. About half the isolates of Salmonella spp. and S.sonnei and almost all S.flexneri isolates were resistant to ampicillin, and more than 60% of Shigella spp. isolates were resistant to trimethoprim-sulfamethoxazole. A 41% of Staphylococcus aureus and 55% of coagulase-negative staphylococci isolates were resistant to oxacillin, presenting a highly associated multi-resistance. The resistance to FQ was also strongly related to oxacillin resistance, but the resistance to TRV was significantly lower than the CIP resistance: 9% vs 57% for S.aureus and 4% vs 41% for coagulase-negative staphylococci. A similar behavior was observed with Enterococcus spp., where 54% of the isolates were resistant to norfloxacin and only 13% were resistant to TRV. Neither Streptococcus pneumoniae (n = 193) nor Haemophilus influenzae (n = 139) isolates presented resistant to TRV.

Anti-Bacterial Agents↗
PubMed1997

Initiation of cyclin B degradation by the 26S proteasome upon egg activation.

Immediately before the transition from metaphase to anaphase, the protein kinase activity of maturation or M-phase promoting factor (MPF) is inactivated by a mechanism that involves the degradation of its regulatory subunit, cyclin B. The availability of biologically active goldfish cyclin B produced in Escherichia coli and purified goldfish proteasomes (a nonlysosomal large protease) has allowed the role of proteasomes in the regulation of cyclin degradation to be examined for the first time. The 26S, but not the 20S proteasome, digested recombinant 49-kD cyclin B at lysine 57 (K57), producing a 42-kD truncated form. The 42-kD cyclin was also produced by the digestion of native cyclin B forming a complex with cdc2, a catalytic subunit of MPF, and a fragment transiently appeared during cyclin degradation when eggs were released from metaphase II arrest by egg activation. Mutant cyclin at K57 was resistant to both digestion by the 26S proteasome and degradation at metaphase/anaphase transition in Xenopus egg extracts. The results of this study indicate that the destruction of cyclin B is initiated by the ATP-dependent and ubiquitin-independent proteolytic activity of 26S proteasome through the first cutting in the NH2 terminus of cyclin (at K57 in the case of goldfish cyclin B). We also surmise that this cut allows the cyclin to be ubiquitinated for further destruction by ubiquitin-dependent activity of the 26S proteasome that leads to MPF inactivation.

Amino Acid Sequence↗
PubMed1997

Ocular and cerebellar defects in zebrafish induced by overexpression of the LIM domains of the islet-3 LIM/homeodomain protein.

Islet-3 is an LIM/homeodomain protein that is expressed specifically in the eyes and the presumptive tectum in the central nervous system of zebrafish (Danio rerio) embryos. Overexpression of the protein (LIM(Isl-3)) consisting only of the Islet-3 LIM domains in embryos specifically prevented formation of the optic vesicles; caused abnormal termination of the expression of wnt1, engrailed2, and pax2 in the mesencephalic and metencephalic region between 14 hr and 20 hr postfertilization; and severely impaired morphogenetic movement in this region between 20 hr and 26 hr, which should normally lead to formation of the cerebellar primordium. Such defects were all rescued by simultaneous overexpression of Islet-3, suggesting that LIM(Isl-3) acted as a specific dominant-negative variant of Islet-3. These data, combined with the results of mosaic analyses, suggest that Islet-3 is activated by putative LIM-binding cofactors and functions to promote evagination of the optic vesicles and to maintain reciprocal interaction between the mesencephalon and the mesencephalic-metencephalic boundary essential for normal development of this region.

Animals↗