Biomedical subjects

T Mukai

Publications and source records attributed to T Mukai.

At least 55 records · Page 3Linked to original sources

PubMed2001

Inhibition of adhesion of food-borne pathogens to Caco-2 cells by Lactobacillus strains.

AIMS: To select adhesive strains among strains of Lactobacillus and to apply them to inhibit adhesion of food-borne pathogens. METHODS AND RESULTS: Twelve Lactobacillus strains (10 from intestine) were examined for adhesion using Caco-2 cell cultures. The two most adhesive strains, Lactobacillus crispatus JCM 8779 and Lact. reuteri JCM 1081, were used to test antiadhesion activity against enterotoxigenic Escherichia coli, Salmonella typhimurium and Enterococcus faecalis strains. Adhesion of the pathogens was inhibited by both Lactobacillus strains. Adhesion of Ent. faecalis was especially strongly inhibited by JCM 8779. Although antimicrobial activity was not detected in the culture supernatant fluid by agar well diffusion assay, the supernatant fluid obtained from the harvested JCM 8779 cell suspension showed bactericidal activity against Ent. faecalis. CONCLUSION: The strong antiadhesion activity of JCM 8779 against Ent. faecalis appears to be due to the combined effect of both bactericidal activity and competition for attachment site. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report that Lact. crispatus produces a bactericidal substance.

Antibiosis↗

PubMed2001

Molecular characterization of Escherichia coli NAD kinase.

NAD kinase was purified to homogeneity from Escherichia coli MG1655. The enzyme was a hexamer consisting of 30 kDa subunits and utilized ATP or other nucleoside triphosphates as phosphoryl donors for the phosphorylation of NAD, most efficiently at pH 7.5 and 60 degrees C. The enzyme could not use inorganic polyphosphates as phosphoryl donors and was designated as ATP-NAD kinase. The N-terminal amino-acid sequence of the purified enzyme was encoded by yfjB, which had been deposited as a gene of unknown function in the E. coli whole genomic DNA sequence database. yfjB was cloned and expressed in E. coli BL21(DE3)pLysS. The purified product (YfjB) showed NAD kinase activity, and was identical to ATP-NAD kinase purified from E. coli MG1655 in molecular structure and other enzymatic properties. The deduced amino-acid sequence of YfjB exhibited homology with that of Mycobacterium tuberculosis inorganic polyphosphate/ATP-NAD kinase [Kawai, S., Mori, S., Mukai, T., Suzuki, S., Hashimoto, W., Takeshi, Y. & Murata, K. (2000) Biochem. Biophys. Res. Commun. 276, 57-63], and those of many hypothetical proteins for which functions have not yet been revealed. The YfjB homologues were considered to be NAD kinases and alignment of their sequences revealed highly conserved regions, XXX-XGGDG-XL and DGXXX-TPTGSTAY, where X represents a hydrophobic amino-acid residue.

Adenosine Triphosphate↗

PubMed2001

Sustained ocular delivery of tilisolol to rabbits after topical administration or intravitreal injection of lipophilic prodrug incorporated in liposomes.

To improve the retention time of tilisolol in the precorneal area or vitreous body, we prepared liposomes incorporating the O-palmitoyl prodrug of tilisolol. O-Palmitoyl tilisolol was completely incorporated in the liposomes. After topical administration of O-palmitoyl tilisolol liposomes to the rabbit eye, O-palmitoyl tilisolol rapidly disappeared from the tear fluid. The inclusion of 2% carmellose sodium slightly prolonged the retention of O-palmitoyl tilisolol in the tear fluid. After intravitreal injection of O-palmitoyl tilisolol liposomes, there was a relatively prolonged retention of O-palmitoyl tilisolol in the vitreous body. At 24 and 48 h after intravitreal injection of O-palmitoyl tilisolol liposomes, the tilisolol concentration in the vitreous body was significantly higher compared with the concentration after intravitreal injection of tilisolol liposomes.

Administration, Topical↗

PubMed2001

Effect of instillation method on the absorption of phenolsulphonphthalein as a model drug from the liver and small intestinal serosal surface in rats.

We have examined the effect of the instillation method on the absorption of a drug from the liver and the small intestinal serosal surface in rats. We performed continuous microinstillation via an infusion pump and bolus instillation via a syringe, using phenolsulphonphthalein (phenol red) as the model drug. After continuous microinstillation of phenolsulphonphthalein 2.35 mg in 235 microL for 5 min on the liver and small intestinal serosal surface in rats, the AUC (area under the curve) of the plasma concentration profile up to 60 min was significantly higher compared with bolus instillation. A similar trend was observed after continuous microinstillation of phenolsulphonphthalein 2.35 mg in 117.5 microL for 2.5 min. The calculated absorption rate constants (Ka) after continuous microinstillation of phenolsulphonphthalein based on a two-compartment model with first-order absorption were higher than those after bolus instillation on the liver and small intestinal serosal surface at either instillation concentration. Moreover, Ka was increased after continuous microinstillation of 2.35 mg in 117.5 microL at either instillation site. Instillation of phenolsulphonphthalein on the liver surface resulted in a 1.2- to 2.3-fold higher Ka compared with the small intestinal serosal surface. This tendency was marked after continuous microinstillation of 2.35 mg in 117.5 microL. In conclusion, absorption could be enhanced by instilling a small amount of drug solution on the liver surface gradually and continuously, suggesting a promising approach for instillation site-selective drug delivery in the peritoneal cavity.

Algorithms↗

PubMed2001

Establishment of a mass-production system for NADP using bacterial inorganic polyphosphate/ATP-NAD kinase.

The inorganic polyphosphate/ATP-dependent NAD kinase (Ppnk) of Mycobacterium tuberculosis was applied to the mass-production of NADP from NAD and inorganic polyphosphate (metaphosphate). When Ppnk purified from recombinant Escherichia coli cells overexpressing the M. tuberculosis Ppnk was used, 30 mM (27 g/l) NADP was produced from 50 mM NAD and 100 mg/ml metaphosphate at 37 degrees C and pH 7.0. The recombinant E. coli cells were immobilized in polyacrylamide gel, and treated with acetone to render the cells permeable to substrates and products. When acetone-treated immobilized cells were used, 16 mM (14 g/l) NADP was produced from 50 mM NAD and 100 mg/ml metaphosphate at 37 degrees C and pH 7.0. The isolation of NADP formed in the reaction mixture was easy because of the absence of by-products (ATP degradation compounds), and this NADP production system using purified Ppnk or immobilized recombinant E. coli cells expressing Ppnk is thought to be feasible in the production of NADP on an industrial scale.

Journal Article↗

PubMed2001

Methylation-sensitive genome scanning.

Imprinted genes in mammals are expressed exclusively from one of the parental alleles (1-6). This is regulated by parental-allele-specific CpG methylation. For example, H19 is methylated exclusively on the paternal allele, which is repressed, and is expressed exclusively from the maternal allele, which is unmethylated. Therefore, one way to find imprinted genes is searching for parental-allele-specific CpG methylation. Southern analysis using methylation-sensitive restriction enzymes could be used for such a purpose. However, usually only one gene can be analyzed by one Southern analysis. Moreover, Southern analysis requires one DNA probe for each analysis. These facts indicate at least 300 Southern analyses using 300 different probes are required to find only one imprinted gene, because the population of imprinted genes is estimated to be 0.3%. Therefore, this kind of analysis is not appropriate for searching for new imprinted genes, and the development of a new method that can simultaneously analyze thousands of genes was required.

Animals↗

PubMed2001

Diagnosis of deep vein thrombosis using platelet scintigraphy.

PURPOSE: We reviewed the usefulness of platelet scintigraphy, in which autologous platelets labeled with indium-111-oxine reveal thrombotic activity, for evaluating deep vein thrombosis (DVT). MATERIALS: During the past 2 years, 39 cases with DVT were enrolled in this study. DVT was definitely diagnosed by color duplex scanning, platelet scintigraphy or both in all cases. For semiquantitative analysis, we estimated the ratio of accumulation in the abnormal region to that in the normal vein on the other side, and defined an abnormal accumulation ratio as over 1.2. RESULTS: Abnormal accumulation ratio showing active DVT was recognized in 30 cases (77%), and showed a good correlation with clinical symptoms. In addition, in 19 cases with crural DVT, platelet scintigraphy showed abnormal accumulation ratio in 16 cases (84%), while duplex scanning detected thrombi in 13 cases (68%). In cases with abnormal accumulation ratio, thrombolytic and anticoagulant therapy were very effective for improving clinical symptoms as well inducing regression of the accumulation ratio. CONCLUSIONS: Platelet scintigraphy was very useful for the diagnosis and treatment of DVT and for evaluation of the effect of anticoagulant therapy. Limitations in the definite diagnosis of deep vein thrombosis (DVT) have been apparent for more than three decades. During the last decade, duplex scanning has reached a high level of accuracy and has been considered the gold standard in the diagnosis of DVT, instead of venography. However, duplex scanning and venography demonstrates only the anatomic alterations associated with venous lesions. In contrast, in platelet scintigraphy, the labeled platelets are incorporated directly into the thrombus and can reveal thrombus activity. We noticed that autologous platelets labeled with indium-111-oxine accumulated on fresh lesions of DVT. This observation suggested two applications of this technique: 1) evaluation of the role of platelets in the pathophysiologic characteristics of DVT; and 2) monitoring the effects of anticoagulant therapy.

Adult↗

PubMed2001

IL-12 plays a pivotal role in LFA-1-mediated T cell adhesiveness by up-regulation of CCR5 expression.

The chemokine receptor CCR5 has been implicated in the recruitment of T cells to inflammatory sites. However, the regulation of CCR5 induction on T cells and its contribution to T cell adhesiveness are poorly understood. Using a Th1 clone, 2D6, that can be maintained with interleukin (IL)-12 or IL-2 alone (designated 2D6(IL-12) or 2D6(IL-2), respectively), we investigated how CCR5 is induced on T cells and whether CCR5 is responsible for up-regulating the function of adhesion molecules. 2D6(IL-12) grew, forming cell aggregates, in culture containing IL-12. This was due to lymphocyte function-associated antigen (LFA)-1-intercellular adhesion molecule (ICAM)-1 interaction, because 2D6(IL-12) expressed both LFA-1 and ICAM-1 and cell aggregation was inhibited by anti-ICAM-1 monoclonal antibody. Despite comparable levels of LFA-1 and ICAM-1 expression, 2D6(IL-2) cells did not aggregate in culture with IL-2. It is important that there was a critical difference in CCR5 expression between 2D6(IL-12) and 2D6(IL-2); the former expressed high levels of CCR5, and the latter expressed only marginal levels. Both types of cells expressed detectable albeit low levels of RANTES (regulated on activation, normal T expressed and secreted) mRNA. Unlike IL-12 or IL-2, IL-18 induced high levels of RANTES mRNA expression without modulating CCR5 expression. Therefore, combined stimulation with IL-12 and IL-18 strikingly up-regulated 2D6 cell aggregation. Notably, LFA-1-mediated aggregation of 2D6(IL-12) cells was suppressed by anti-CCR5 antibody. These results indicate that IL-12 plays a critical role in CCR5 expression on Th1 cells and consequently contributes to CCR5-mediated activation of LFA-1 molecules.

Animals↗

PubMed2000

Identification of novel first exons in Ad4BP/SF-1 (NR5A1) gene and their tissue- and species-specific usage.

It has been demonstrated that the mammalian Ad4BP/SF-1 (NR5A1) gene is regulated precisely in sex, tissue, and developmental stage specific manners. To clarify the complex transcriptional regulation, we investigated in the present study whether the gene transcription is regulated by multiple promoters accompanied by noncoding first exons. Novel first exons (Io and Ig) were identified downstream of the already identified exon Ia. Nucleotide sequences revealed that Ia and Ig exons were well conserved, whereas Io exon was less conserved among the mouse, rat, and human genes. Interestingly, the splice donor of the mouse and human Io and human Ig exons do not satisfy the consensus sequence. Transcripts containing Ia, Io, and Ig were detected in all rat tissues examined, while the transcript containing Io was undetectable in the corresponding tissues of mice. The lack of exon Io usage in the mouse was confirmed by transient transfection assays with cultured cells. Quantitative RT-PCR analysis revealed that the transcript containing Ig exon was the main product in the pituitary but significantly less in the spleen, suggesting that the regulation of Ad4BP/SF-1 gene transcription in the pituitary and spleen is distinct from that of other tissues. The above findings, together with the structural abnormality at the splice donor site, suggest that acquisition of the multiple first exons enables the Ad4BP/SF-1 gene to be regulated differentially in different animal species and in different tissues in the same animal.

Alternative Splicing↗

PubMed2000

C11orf21, a novel gene within the Beckwith-Wiedemann syndrome region in human chromosome 11p15.5.

A novel gene, C11orf2, was identified by BLAST search in the human chromosome 11p15.5 region potentially responsible for Beckwith-Wiedemann Syndrome (BWS) and some cancers. Two cDNA clones with different sizes were obtained, which share a potential ORF of 399bp and are different in their 3' untranslated regions. This gene was revealed to be expressed exclusively in human heart and in almost no other tissues examined by northern blotting. Two transcripts of different sizes, 0.9 and 3.1kb, were identified in heart, consistent with the length of the two cDNA clones. The gene shows biallelic expression (non-imprinted) in fetal liver, although it is located in the imprinted domain of 11p15.5. C11orf21 codes a protein of 132 amino acids as proved by the expression of C11orf21-EGFP fusion protein in cultured cells. The EGFP-fusion protein expressed in cultured cells localized mainly in the cytoplasm.

Alleles↗

PubMed2000

Inorganic Polyphosphate/ATP-NAD kinase of Micrococcus flavus and Mycobacterium tuberculosis H37Rv.

An enzyme with both inorganic polyphosphate [poly(P)]- and ATP-dependent NAD kinase activities was isolated from Micrococcus flavus. The enzyme was a dimer consisting of 34 kDa subunits, and was named poly(P)/ATP-NAD kinase. Internal amino acid sequences of the enzyme showed homologies with some function-unknown proteins released on the GenBank database. Among such proteins, hypothetical Rv1695 protein (Accession No. Z98268-16), which was encoded by a gene named "Rv1695" on genomic DNA of Mycobacterium tuberculosis H37Rv, was proposed to be poly(P)-dependent NAD kinase. By cloning and expression in Escherichia coli, Rv1695 was shown to encode poly(P)/ATP-NAD kinase and named ppnk. The ppnk product, recombinant-poly(P)/ATP-NAD kinase (Ppnk) was purified and characterized. The enzyme was a tetramaer consisting of 35 kDa subunits when expressed in E. coli. Poly(P)/ATP-NAD kinases of M. flavus and Ppnk of M. tuberculosis H37Rv specifically and completely phosphorylated NAD by utilizing commercially available poly(P)s and nucleoside triphosphates as phosphoryl donors.

Amino Acid Sequence↗

PubMed2000

Dendritic cells transduced by multiply deleted HIV-1 vectors exhibit normal phenotypes and functions and elicit an HIV-specific cytotoxic T-lymphocyte response in vitro.

Dendritic cells (DCs) genetically modified to continually express and present antigens may be potent physiologic adjuvants for induction of prophylactic or therapeutic immunity. We have previously shown that an env and nef deleted HIV-1 vector (HIV-1 delta EN) pseudotyped with VSV-G transduced monocyte-derived macrophages as well as CD34(+) precursors of DCs. Here we extended these findings with HIV-1 delta EN to highly differentiated human DCs derived in culture from circulating monocytes (DCs). In addition, a new vector derived from HIV-1 delta EN but further deleted in its remaining accessory genes vif, vpr, and vpu (HIV-1 delta EN V(3)) was also tested. Both vectors efficiently transduced DCs. Transduction of DCs did not significantly alter their viability or their immunophenotype when compared with untransduced DCs. Furthermore, the phagocytic potential of immature DCs, as well as their ability to differentiate into mature DCs capable of stimulating T-cell proliferation, was not affected. Finally, DCs transduced by the HIV-1 delta EN vector were able to elicit a primary antiviral cytotoxic T-cell response in autologous CD8 T cells. These results suggest that HIV-1-based vectors expressing viral antigens may be useful for in vivo active immunization as well as ex vivo priming of cytotoxic T cells for adoptive T-cell therapy. (Blood. 2000;96:1327-1333)

Acquired Immunodeficiency Syndrome↗

PubMed2000

Sequence-based structural features between Kvlqt1 and Tapa1 on mouse chromosome 7F4/F5 corresponding to the Beckwith-Wiedemann syndrome region on human 11p15.5: long-stretches of unusually well conserved intronic sequences of kvlqt1 between mouse and human.

Mouse chromosome 7F4/F5 is a syntenic locus of human 11p15.5 in which many imprinted genes are clustered. Transmission of aberrant human 11p15.5 or duplicated 11p causes Beckwith-Wiedemann syndrome (BWS) depending on which parent the chromosome is derived from. To analyze a syntenic mouse locus corresponding to human 11p15.5, mouse BAC contigs were constructed between Nap2 and Tapa1, in which 390 kb was sequenced between Kvlqt1 and Tapa1. An unexpected finding was that of highly conserved intronic sequences of Kvlqt1 between mouse and human, and their homologies came up to at least 160 kb because the length of this gene extended to 350 kb, suggesting the possibility of some functional constraint due to transcriptional and/or post-transcriptional regulation of this region. Many expressed sequence tags (ESTs) were mapped on this locus. Three genes, Lit1 (Kvlqt1-AS), Mtr1 and Tssc4, were identified and characterized. Lit1 is an antisense-transcript of Kvlqt1 and paternally expressed and maternally methylated throughout the developmental stage. The position where Lit1 exists corresponded to a highly conserved region between mouse and human. This transcript extends at least 60 kb from downstream to upstream of exon 10 in Kvlqt1. Tssc4 and Mtr1 carried putative open reading frames but neither was imprinted. Further characterization of this locus based on the sequence comparison between mouse and human will contribute valuable information towards resolving the mechanism of the occurrence of BWS and the associated childhood tumor.

Alleles↗

PubMed2000

Extended magnetic reconnection at the Earth's magnetopause from detection of bi-directional jets

Magnetic reconnection is a process that converts magnetic energy into bi-directional plasma jets; it is believed to be the dominant process by which solar-wind energy enters the Earth's magnetosphere. This energy is subsequently dissipated by magnetic storms and aurorae. Previous single-spacecraft observations revealed only single jets at the magnetopause--while the existence of a counter-streaming jet was implicitly assumed, no experimental confirmation was available. Here we report in situ two-spacecraft observations of bi-directional jets at the magnetopause, finding evidence for a stable and extended reconnection line; the latter implies substantial entry of the solar wind into the magnetosphere. We conclude that reconnection is determined by large-scale interactions between the solar wind and the magnetosphere, rather than by local conditions at the magnetopause.

Journal Article↗

PubMed2000

Suppression of paraquat-induced wet dog shakes by nitric oxide synthase inhibitors in rats.

We have found that paraquat (PQ), a widely used herbicide, causes wet dog shakes (WDS), which involve the central opioid system, in rats. A non-selective nitric oxide (NO) synthase (NOS) inhibitor, N(omega)-nitro-L-arginine (L-NA), but not its less active enantiomer, N(omega)-nitro-D-arginine, decreased the PQ-induced WDS in a dose-related manner. A selective neuronal NOS inhibitor in vivo, 7-nitroindazole, also decreased the PQ-induced WDS. Although an opioid receptor antagonist, naloxone, reversed the suppressive effect of these NOS inhibitors on the PQ-induced WDS, L-arginine, an NO precursor, had no effect on it. These findings suggest that the suppression of the PQ-induced WDS by NOS inhibition is associated with the central opioid system and is insusceptible to exogenous L-arginine.

Animals↗

PubMed2000

Highly variable sequences in the env V3 region of HIV type 1 distributing among Thai carriers from 1995 to 1997.

The amino acid sequences of the Env V3 region of HIV-1 subtype E in Thailand were highly variable in the samples obtained from 1995 to 1997, compared with the previously reported sequences in samples obtained from 1990 to 1993. The sequences of the V3 region in the samples from five provinces in Thailand revealed that the variability was much higher in the samples from Bangkok and Ubonrachathani than in those from Chiangmai, Prathumthani, and Trang. There was no apparently different level of diversity at the V3 region in the samples from symptomatic and asymptomatic individuals. The V3 loop motif in most (66.7%) of the samples was GPGQ, although this motif was more heterogeneous in the samples from Bangkok and Ubonrachathani than in those from the other three provinces. The N-linked glycosylation sites in the V3 region among these samples were relatively conserved. There was no apparent difference in the presence of positively charged amino acids at positions 306 and 320 between the samples from symptomatic and asymptomatic individuals.

Amino Acid Sequence↗

PubMed2000

Dual quantum nondemolition measurements via successive soliton collisions

Quantum mechanics allows quantum nondemolition (QND) variables to be measured without being changed. This requires QND variables to be initially in an eigenstate and measurement backaction noise to be directed into conjugate variables. Experimental demonstrations thus require two measurements: the first to collapse variables toward an eigenstate and the second to show results identical to the first. Here, we report results from two successive soliton-collision QND measurements that optical correlation measurements show to be nearly identical.

Journal Article↗

PubMed2000

Spontaneous esophageal rupture treated by conservative therapy.

The prognosis of spontaneous esophageal rupture of the esophagus worsens over time from disease onset to treatment and, in severe cases, may require surgery to save the patient's life. Patients appearing at the hospital considerably after esophageal perforation have no appropriate surgical alternatives and face poor prospects. We conservatively treated a severe case following 2-day lapse of after disease onset, managing a favorable outcome. A 58-year-old man who developed upper abdominal and back pain after vomiting from drinking was transferred to our institute in an emergency due to pain intensifying 2 days after the symptom onset. Chest X-ray revealed a large quantity of bilateral pleural effusion similar to gastrointestinal content, which we withdrew through intrathoracic drainage. Esophagography showed perforation of the esophagus. The patient's poor general condition, including septic shock and adult respiratory distress syndrome, contraindicated radical surgery, so we instituted conservative therapy such as continuous thoracic drainage hyperalimentation. Oral intake was started in month 4 after admission. The patient was discharged in good general condition 7 months after onset.

Drainage↗