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Biomedical subjects

A Yamaguchi

Publications and source records attributed to A Yamaguchi.

At least 73 records · Page 4Linked to original sources
PubMed2001

ORP150 protects against hypoxia/ischemia-induced neuronal death.

Oxygen-regulated protein 150 kD (ORP150) is a novel endoplasmic-reticulum-associated chaperone induced by hypoxia/ischemia. Although ORP150 was sparingly upregulated in neurons from human brain undergoing ischemic stress, there was robust induction in astrocytes. Cultured neurons overexpressing ORP150 were resistant to hypoxemic stress, whereas astrocytes with inhibited ORP150 expression were more vulnerable. Mice with targeted neuronal overexpression of ORP150 had smaller strokes compared with controls. Neurons with increased ORP150 demonstrated suppressed caspase-3-like activity and enhanced brain-derived neurotrophic factor (BDNF) under hypoxia signaling. These data indicate that ORP150 is an integral participant in ischemic cytoprotective pathways.

Animals↗
PubMed2001

Identification and molecular cloning of germinal vesicle lamin B3 in goldfish (Carassius auratus) oocytes.

A bulk isolation method was developed to collect a large number of germinal vesicles (GV) from postvitellogenic oocytes of goldfish (Carassius auratus). Using this method, we obtained GV lamina which are resistant to high salt and nonionic detergent. 2D PAGE revealed that the goldfish GV lamina contained several spots with similar molecular masses (67 kDa) and slightly different neutral isoelectrofocusing values (pI 5.8-6.2). After trypsin digestion and extraction of a major spot (pI 6.1), the peptide was subjected to RP-HPLC and sequenced. A homology search identified this spot as a nuclear lamin. A cDNA encoding goldfish GV lamin was isolated by RT-PCR using degenerate primers designed from the GV lamin tryptic peptide sequence. The goldfish GV lamin cDNA encodes a predicted molecular mass of 67 455 Da with a pI of 5.84. Phylogenetic analysis indicates that the amino-acid sequence is most similar to Xenopus oocyte-specific GV lamin B3, but differs from somatic lamins (A, B1 or B2). In contrast to somatic lamins, neither goldfish nor Xenopus GV lamin contain conserved phosphorylation sites for nuclear transport, except the nuclear localization sequence. Therefore, we conclude that the goldfish oocyte GV is mainly comprised of GV-type lamin (the homolog of Xenopus lamin B3).

Amino Acid Sequence↗
PubMed2001

beta-Catenin gene mutation in human hair follicle-related tumors.

beta-Catenin, a multifunctional protein related to the adherens junction and to signal transduction, is a key molecule of cell proliferation, and it is central to epithelial architecture, regulating the polarity of cells and tissues. beta-Catenin stabilization may play a key role in epidermal signaling leading to hair development, and its aberrant activation may be implicated in formation of hair tumors. Several investigators have shown that pilomatricomas are frequently associated with beta-catenin mutation. In this study, we confirmed beta-catenin gene (CTNNB1) mutation in human pilomatricomas (100% frequency) from which adequate DNA could be obtained for gene analysis. A novel mutation, D32N, was found in one case of pilomatricoma. A preliminary immunohistological study revealed prominent beta-catenin staining in basophilic cells of pilomatricomas, especially in nuclei. Benign tumors which were considered to be derived from hair matrix or hair follicles, and other benign skin tumors, were also investigated. beta-Catenin mutations were not detected in any of the these tumors. These results seem to indicate that hair matrix cells are key players in hair development. Investigation into gene abnormalities of hair-follicle tumors may elucidate the cause of their neoplastic transformation, and may provide a suggestion for the mechanism of hair development.

Adolescent↗
PubMed2001

Percutaneous extracorporeal life support for treatment of fatal mechanical complications associated with acute myocardial infarction.

Mechanical complications of acute myocardial infarction (AMI), such as free wall rupture, ventricular septal perforation (VSP), and mitral regurgitation due to papillary muscle rupture, are associated with high mortality rates. These complications result in extreme deterioration and increased risk of death in patients who do not receive timely resuscitation and surgical treatment. We studied the effectiveness of percutaneous extracorporeal life support (ECLS) for fatal mechanical AMI complications. Nine patients (7 men and 2 women, mean age 69 +/- 6 years) who suffered circulatory collapse refractory to conventional resuscitation were treated with ECLS. Circulatory collapse was caused by free wall rupture in 4 patients, VSP in 4, and mitral regurgitation due to papillary muscle rupture in 1. All patients were successfully resuscitated by ECLS and underwent surgical repair with conventional cardiopulmonary bypass. Eight patients required ECLS after surgery. Four of the 9 patients (2 with free wall rupture, 1 with VSP, and 1 with papillary muscle rupture) were successfully weaned from ECLS and were discharged. Three of the 4 survivors had no major complications, but the remaining survivor suffered neurological deficit. Four patients died while on devices. The duration of ECLS was from 13 to 167 h (mean 76 +/- 57 h) with a maximum bypass flow of 2.0 to 3.9 L/min (mean 2.9 +/- 0.6 L/min). There were no device-related complications during the support period. Total weaning rate was 56% (5/9), and survival was 44% (4/9). We conclude that ECLS can provide appropriate circulatory support during resuscitation and subsequent postoperative circulatory support for cardiovascular collapse associated with AMI complications.

Aged↗
PubMed2001

L-arginine polymers inhibit the development of vein graft neointimal hyperplasia.

OBJECTIVE: We sought to determine whether L -arginine polymer treatment of vein grafts enhances vascular production of nitric oxide and inhibits the development of neointimal hyperplasia. METHODS: External jugular veins of New Zealand White rabbits (n = 42) were harvested; treated intraluminally for 15 minutes with phosphate-buffered saline solution or L -arginine polymer 5, 7, or 9 at either 10 or 100 micromol/L; and then grafted into the contralateral carotid artery. Rabbits were killed after 28 days, and 5-microm sections of vessels were stained with hematoxylin and scored for intima/media ratio by using computerized morphometric analysis. Separate veins were treated in a similar fashion with biotinylated polymers and phosphate-buffered saline solution to assess for translocation efficiencies. Finally, vein segments pretreated with either phosphate-buffered saline solution or L -arginine polymers were cultured in Dulbecco's modified Eagle's medium containing lipopolysaccharide (100 microg/mL) and interferon gamma (200 U/mL) for 48 hours before measuring nitric oxide levels by means of the Griess reaction. RESULTS: Biotinylated L -arginine polymers demonstrated a dose- and length-dependent uptake into intimal and medial cells of treated vessels. Nitric oxide levels were significantly higher in vein segments treated with 100 micromol/L of L -arginine polymer 9 compared with control segments. Finally, the intima/media ratio also reflected both length- and concentration-dependent inhibition of neointimal hyperplasia.intima/media ratio PBS R5 R7 R9 10 micromol/L 0.909 +/- 0.072 0.920 +/- 0.073 0.861 +/- 0.138 0.710 +/- 0.122 100 micromol/L 0.924 +/- 0.061 0.581 +/- 0.089* 0.529 +/- 0.093* PBS, Phosphate-buffered saline solution; R, L -arginine polymer. *P <.001 versus phosphate-buffered saline solution and L -arginine polymer 5 controls (Bonferroni-corrected value). CONCLUSIONS: Arginine polymers of sufficient length and concentration were effective in increasing nitric oxide levels and reducing neointimal hyperplasia in this vein graft model.

Animals↗
PubMed2001

Monoclonal antibody that binds to the central loop of the Tn10-encoded metal tetracycline/H+ antiporter of Escherichia coli.

Mouse monoclonal antibodies were prepared using His-tagged Tn10-encoded metal-tetracycline/H+ antiporter [TetA(B)His] as an antigen. From them, those reacting equally with His-tagged and wild-type TetA(B) were selected and named TCL-1. Cysteine-scanning mutants were used to determine the TCL-1 binding site on the TetA(B) protein. First, 12 Cys mutants of TetA(B) in which one residue in a protruding loop region was replaced by cysteine were constructed. Western blot analysis revealed the binding of TCL-1 to all of these Cys-mutants except for R186C. Then, we constructed 13 cysteine-scanning mutants, F179C to T191C. Among them, eight mutants, F179C to T182C, N184C, and T189C to T191C, exhibited TCL-1 binding, whereas the other five, K183C, T185C, R186C, D187C, and N188C, exhibited no or lower TCL-1 binding. These results clearly indicate that the sequence recognized by TCL-1 is 183Lys-X-Thr-Arg-Asp-Asn188 in the central loop region of TetA(B). TCL-1 is the first reported antibody that binds to a region other than the C-terminus of TetA(B), and the recognized amino acid sequence was identified.

Amino Acid Sequence↗
PubMed2001

Hexosaminidase-altered aberrant crypts, carrying decreased hexosaminidase alpha and beta subunit mRNAs, in colon of 1,2-dimethylhydrazine-treated rats.

Aberrant crypt foci (ACF), consisting of morphologically irregular crypts, are thought to be precancerous lesions for colon cancers. For their molecular analysis, it is necessary to avoid contamination with adjacent normal crypts and stromal cells. Decreased hexosaminidase activity in ACF, which has been histochemically demonstrated, was used in the present study to classify isolated crypts in combination with morphological changes. The length, rim diameter, and width (average SD, microm) of hexosaminidase-positive (Hex + ) crypts were 238.6 +/- 40.4, 89.5 +/- 22.9, and 57.6 +/- 14.0, respectively. For hexosaminidase-negative (Hex - ) crypts, the values were 314.4 +/- 77.8, 140.3 +/- 45.7, and 97.3 +/- 34.7, the width being 1.69 times greater (P < 0.0001). Crypts wider than 115 microm (approximately 2 times the average size of Hex + crypts) were all from ACF, judging from hexosaminidase staining. To analyze transcription levels of Hex alpha and beta subunits (Hexa and Hexb, respectively), real-time relative quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis was performed using the LightCycler system. In aberrant crypts, both Hexa and Hexb were significantly down-regulated to 0.266 (P < 0.002) and 0.131 (P < 0.001) units, respectively, compared with those in morphologically normal crypts, with beta-actin as the internal standard. This decrease could be a molecular marker for precancerous enzyme-altered ACF.

1,2-Dimethylhydrazine↗
PubMed2001

Type II topoisomerase mutations in fluoroquinolone-resistant clinical strains of Pseudomonas aeruginosa isolated in 1998 and 1999: role of target enzyme in mechanism of fluoroquinolone resistance.

The major mechanism of resistance to fluoroquinolones for Pseudomonas aeruginosa is the modification of type II topoisomerases (DNA gyrase and topoisomerase IV). We examined the mutations in quinolone-resistance-determining regions (QRDR) of gyrA, gyrB, parC, and parE genes of recent clinical isolates. There were 150 isolates with reduced susceptibilities to levofloxacin and 127 with reduced susceptibilities to ciprofloxacin among 513 isolates collected during 1998 and 1999 in Japan. Sequencing results predicted replacement of an amino acid in the QRDR of DNA gyrase (GyrA or GyrB) for 124 of the 150 strains (82.7%); among these, 89 isolates possessed mutations in parC or parE which lead to amino acid changes. Substitutions of both Ile for Thr-83 in GyrA and Leu for Ser-87 in ParC were the principal changes, being detected in 48 strains. These replacements were obviously associated with reduced susceptibilities to levofloxacin, ciprofloxacin, and sparfloxacin; however, sitafloxacin showed high activity against isolates with these replacements. We purified GyrA (The-83 to Ile) and ParC (Ser-87 to Leu) by site-directed mutagenesis and compared the inhibitory activities of the fluoroquinolones. Sitafloxacin showed the most potent inhibitory activities against both altered topoisomerases among the fluoroquinolones tested. These results indicated that, compared with other available quinolones, sitafloxacin maintained higher activity against recent clinical isolates with multiple mutations in gyrA and parC, which can be explained by the high inhibitory activities of sitafloxacin against both mutated enzymes.

Anti-Infective Agents↗
PubMed2001

Novel macrolide-specific ABC-type efflux transporter in Escherichia coli.

In the Escherichia coli genome, five putative open reading frame (ORF) clusters, mdlAB, ybjYZ, yddA, yojHI, and yhiH, have been assumed to be possible genes for ABC drug efflux transporters (I. T. Paulsen, M. K. Sliwinski, and M. H. Saier, Jr., J. Mol. Biol. 277:573-592, 1998). We cloned all of these ORFs in multicopy plasmids and investigated the drug resistance of drug-supersensitive host cells lacking constitutive multidrug efflux transporter genes acrAB. Among them, only ybjYZ gave significant erythromycin resistance and significantly decreased the accumulation of [(14)C]erythromycin. Therefore, ybjYZ was renamed macAB (macrolide-specific ABC-type efflux carrier). Plasmids carrying both the macA and -B genes conferred resistance against macrolides composed of 14- and 15-membered lactones but no or weak resistance against 16-membered ones. Neither of the two genes produced resistance alone. The DNA sequence suggests that MacB is an integral membrane protein with four transmembrane segments and one nucleotide-binding domain, while MacA belongs to a membrane fusion protein (MFP) family with a signal-like sequence at its N terminus. The expression of the histidine-tagged proteins confirmed that MacB is an integral membrane protein and MacA is a peripheral membrane protein. In addition, MacAB required TolC for its function in a way similar to that of most of the MFP-dependent transporters in E. coli. MacB is thus a novel ABC-type macrolide efflux transporter which functions by cooperating with the MFP MacA and the multifunctional outer membrane channel TolC. This is the first case of an experimentally identified ABC antibiotic efflux transporter in gram-negative organisms.

ATP-Binding Cassette Transporters↗
PubMed2001

Analysis of a complete library of putative drug transporter genes in Escherichia coli.

The complete sequencing of bacterial genomes has revealed a large number of drug transporter genes. In Escherichia coli, there are 37 open reading frames (ORFs) assumed to be drug transporter genes on the basis of sequence similarities, although the transport capabilities of most of them have not been established yet. We cloned all 37 putative drug transporter genes in E. coli and investigated their drug resistance phenotypes using an E. coli drug-sensitive mutant as a host. E. coli cells transformed with a plasmid carrying one of 20 ORFs, i.e., fsr, mdfA, yceE, yceL, bcr, emrKY, emrAB, emrD, yidY, yjiO, ydhE, acrAB, cusA (formerly ybdE), yegMNO, acrD, acrEF, yhiUV, emrE, ydgFE, and ybjYZ, exhibited increased resistance to some of the 26 representative antimicrobial agents and chemical compounds tested in this study. Of these 20 ORFs, cusA, yegMNO, ydgFE, yceE, yceL, yidY, and ybjYZ are novel drug resistance genes. The fsr, bcr, yjiO, ydhE, acrD, and yhiUV genes gave broader resistance spectra than previously reported.

ATP-Binding Cassette Transporters↗
PubMed2001

Overexpression of the response regulator evgA of the two-component signal transduction system modulates multidrug resistance conferred by multidrug resistance transporters.

Overexpression of evgA, a response regulator of a two-component system, increased multidrug efflux in Escherichia coli. Since overexpression of the emrKY operon, which is controlled by evgAS, could account only for deoxycholate resistance, the evgAS locus apparently controls expression of at least one other multidrug efflux operon.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2001

Somatic lamins in germinal vesicles of goldfish (Carassius auratus) vitellogenic oocytes.

In fish and amphibians, B-type lamins are divided into somatic (B1, B2) and oocyte-type (B3) lamins. In this study, we purified nuclear lamins from rainbow trout erythrocytes, raised an anti-lamin monoclonal antibody (L-200) that recognizes goldfish somatic-lamins, and isolated cDNAs encoding goldfish B-type lamins (B1 and B2) from a goldfish cell culture cDNA library. Goldfish B-type lamins are structurally similar to lamins found in other vertebrates with minor amino acid substitutions in the conserved region. Western blot analysis showed that goldfish oocytes contained mainly GV-lamin B3 as well as some somatic lamins. Laser-confocal microscope observations revealed that lamin B3 was present only in GV nuclear lamina, whereas somatic lamins were present in dense fibrillar structures throughout nuclear gels of isolated GVs. Similar nuclear filamentous structures were also observed in GVs of paraffin embedded oocytes. Epitope mapping indicated that L-200 recognized a conserved region containing a short stretch of the alpha-helix coiled-coil rod domain (Y(E/Q)(Q/E)LL). A similar motif is also present in other cytoplasmic intermediate filaments (i.e., vimentin, desmin, peripherin and GFAP). Taken together, these findings suggest that lamins or lamin-related intermediate filaments are an important component of the interior architecture of goldfish vitellogenic oocyte nuclei (GVs).

Amino Acid Sequence↗
PubMed2001

Emulsifying potency of new amino acid-type surfactant (II): stable water-in-oil (W/O) emulsions containing 85 wt% inner water phase.

The ternary phase diagram for N-[3-lauryloxy-2-hydroxypropyl]-L-arginine L-glutamate (C12HEA-Glu), a new amino acid-type surfactant, /oleic acid (OA)/water system was established. The liquid crystal and gel complex formations between C12HEA-Glu and OA were applied to a preparation of water-in-oil (W/O) emulsions. Stable W/O emulsions containing liquid paraffin (LP) as the oil and a mixture of C12HEA-Glu and OA as the emulsifier were formed. The preparation of stable W/O emulsions containing 85 wt% water phase was also possible, in which water droplets would be polygonally transformed and closely packed, since the maximum percentage of inner phase is 74% assuming uniformly spherical droplets. Water droplets would be taken into the liquid crystalline phase (or the gel complex) and the immovable water droplets would stabilize the W/O emulsion system. The viscosity of emulsions abruptly increased above the 75 wt% water phase (dispersed phase). The stability of W/O emulsions with a lower weight ratio of OA to C12HEA-Glu and a higher ratio of water phase was greater. This unusual phenomenon may be related to the formation of a liquid crystalline phase between C12HEA-Glu and OA, and the stability of the liquid crystal at a lower ratio of oil (continuous phase). W/O and oil-in-water (O/W) emulsions containing LP were selectively prepared using a mixture of C12HEA-Glu and OA since the desirable hydrophile-lipophile balance (HLB) number for the emulsification was obtainable by mixing the two emulsifiers.

Algorithms↗
PubMed2001

Glucose sensing based on interdigitated array microelectrode.

A micro glucose sensor consisting of an interdigitated array gold microelectrode was developed. The interdigitated array structure, which has 10 microns band width and 10 microns band gap, was fabricated in a small region (2.5 x 5 mm2) on a quartz substrate. Glucose oxidase was chemically fixed onto the electrode surface through self-assembled monolayer of 11-mercaptoundecanoic acid; ferroceneacetic acid was used as electron mediator. Electrochemical properties of the glucose oxidase-immobilized microelectrode were investigated by cyclic voltammogram measurements. Results confirmed that the reductive ferroceneacetic acid generated at counter electrode diffuses through a narrow band gap (10 microns) and can reach the working electrode surface.

Acetic Acid↗
PubMed2001

Antitumor activity of ER-51785, a new peptidomimetic inhibitor of farnesyl transferase: synergistic effect in combination with paclitaxel.

Inhibitors of ras farnesylation have been extensively studied in the preclinical stage, and some of them are being developed in the clinic. Herein, we describe the antitumor activity of a new farnesyl transferase inhibitor, ER-51785. In vitro, ER-51785 selectively inhibited farnesyl transferase activity (IC50 = 77 nM) compared with geranylgeranyl transferase I activity (IC50 = 4200 nM). In cells, ER-51785 inhibited posttranslational processing of H-ras with IC50 = 28 nM, but not that of rap 1A at concentrations up to 50 microM. This compound also strongly inhibited colony formation of H-ras-transformed NIH 3T3 fibroblasts and EJ-1 bladder carcinoma cells. In vivo, ER-51785 showed potent tumor regression activity against EJ-1 xenografts but only modest activity against MIA PaCa-2 xenografts. Treatment of ER-51785 in combination with paclitaxel exhibited synergistic effects against colony formation and tumor growth of MIA PaCa-2 cells. The results presented herein support the idea that farnesyl transferase inhibitors alone and in combination with other chemotherapeutic agents have the potential to be developed as therapies for tumors expressing H-ras or K-ras oncogenes.

3T3 Cells↗
PubMed2001

Somatic alterations of the DPC4 and Madr2 genes in colorectal cancers and relationship to metastasis.

The DPC4 and Madr2 genes are located at 18q21, and the LOH on chromosome 18q21 has been shown to occur frequently in colorectal cancers. To investigate the role of these genes in advanced colorectal cancers, we analyzed 29 colorectal specimens for alterations in the DPC4 and Madr2 genes. Twelve (63.2%) of 19 informative primary colorectal cancers showed allelic loss of chromosome 18q21.3 marker. An alteration of the DPC4 gene sequence was identified in 6 (20.7%) of 29 colorectal carcinomas, and the distinct Madr2 gene mobility shifts were present in 3 (10.3%) cancers. Somatic mutations were identified in these tumors by sequencing analysis. DPC4 gene alterations of 4 cases were detected in Mad homology 2 domains. There was no significant correlation between the somatic alteration of Madr2 and clinicopathological findings. However, the frequency of DPC4 mutation was significantly higher in tumors associated with liver metastasis than in those without such metastasis. Our findings suggest that somatic alteration of the DPC4 gene may play a role in tumorigenesis and liver metastasis of human colorectal cancers.

Chi-Square Distribution↗
PubMed2001

Prognostic factors affecting disease-free survival rate following surgical resection of primary breast cancer.

In order to identify the prognostic factors that significantly influence the disease-free survival rate after surgical resection of primary breast cancers, we determined tumour and lymph node grades, and immunohistochemical staining for estrogen and progesterone receptors (ER and PR), c-erbB-2, p53, bcl-2, bax and PCNA in 76 patients. Univariate analysis showed that increased grade of tumour and lymph nodes, negative immunostaining for ER, positive immunostaining for c-erbB-2, and a high PCNA index (> or = 30%) negatively influenced the disease-free survival rate, but PR, p53, bcl-2 and bax had no predictive value. Although p53 was not an independent prognostic factor by itself, the combination of p53, bcl-2, and bax proved to correlate with the disease-free survival, with the best prognosis noted in tumours negative for p53 and positive for both bcl-2 and bax, intermediate prognosis in tumours negative for p53 and positive for either bcl-2 or bax and worst prognosis in tumors negative for p53 as well as bcl-2 and bax. Tumour grade correlated positively with PCNA index, while positive staining for ER correlated negatively with tumour grade as well as with PCNA index, although this was statistically insignificant. Immunostaining of breast cancers for bcl-2 correlated negatively with tumour grade and PCNA index. Immunostaining for c-erbB-2 correlated positively with PCNA but not with tumour grade. Immunostaining for p53 tended to correlate positively with PCNA, but not with tumour grade. Immunostaining for PR and bax did not correlate with tumour grade and PCNA index. These results suggest that in addition to tumour size and lymph node involvement, immunostaining for ER, c-erbB-2, and a high PCNA index are important prognostic factors in human breast cancer. Wild-type p53 with preserved bcl-2 and bax gene products is also a favorable prognostic factor indicating breast cancer at an early stage of cancer progression.

Adult↗