Biomedical subjects

K Imai

Publications and source records attributed to K Imai.

At least 307 records · Page 17Linked to original sources

PubMed1999

A Na+-H+ exchange inhibitor (SM-20550) protects from microvascular deterioration and myocardial injury after reperfusion.

Na+-H+ exchange inhibitors may reduce myocardial damage after reperfusion. However, their effects on microvascular deterioration are not known. We examined the potency of a novel Na+-H+ exchange inhibitor, SM-20550 [ N-(Aminoiminomethyl)-1,4-dimethyl-1H-indole-2-carboxamide methanesulfonate], and its effects on microvascular damage after reperfusion. In an in vitro study, the Na+-H+ exchange inhibiting activity of SM-20550 was about 10 times greater than that of ethylisopropyl amiloride. In in vivo experiments, we occluded the left circumflex coronary artery in 29 dogs for 2 h and then reperfused for 5 h. SM-20550 was administered either before ischemia (n = 11) or before reperfusion (n = 7). Another 11 dogs served as controls. We found that SM-20550 not only improved coronary vasodilator responses to acetylcholine and adenosine after reperfusion, but also reduced infarct size (P < 0.01). Intramyocardial bleeding, which should reflect microvascular damage, was not found in dogs with SM-20550 treatment. Infarct size was correlated inversely with collateral blood flow in control (both, P < 0.01) but not in SM-20550-treated animals. Furthermore, SM-20550 significantly suppressed ventricular fibrillation during both ischemia and reperfusion. These results suggest that protective effects of Na+-H+ exchange inhibitors on reperfused myocardium are due at least in part to microvascular protection.

Acetylcholine↗

PubMed1999

Detection of DBD-carbamoyl amino acids in amino acid sequence and D/L configuration determination of peptides with fluorogenic Edman reagent 7-[(N,N-dimethylamino)sulfonyl]-2,1,3-benzoxadiazol-4-yl isothiocyanate.

A method for amino acid sequence and D/L configuration identification of peptides by using fluorogenic Edman reagent 7-[(N, N-dimethylamino)sulfonyl]-2,1,3-benzoxadiazol-4-yl isothiocyanate (DBD-NCS) has been developed. This method was based on the Edman degradation principle with some modifications. A peptide or protein was coupled with DBD-NCS under basic conditions and then cyclized/cleaved to produce DBD-thiazolinone (TZ) derivative by BF3, a Lewis acid, which could significantly suppress the amino acid racemization. The liberated DBD-TZ amino acid was hydrolyzed to DBD-thiocarbamoyl (TC) amino acid under a weakly acidic condition and then oxidized by NaNO2/H+ to DBD-carbamoyl (CA) amino acid which was a stable and had a strong fluorescence intensity. The individual DBD-CA amino acids were separated on a reversed-phase high-performance liquid chromatography (RP-HPLC) for amino acid sequencing and their enantiomers were resolved on a chiral stationary-phase HPLC for identifying their D/L configurations. Combination of the two HPLC systems, the amino acid sequence and D/L configuration of peptides could be determined. This method will be useful for searching D-amino-acid-containing peptides in animals.

Amino Acid Sequence↗

PubMed1999

Notochord-dependent expression of MFH1 and PAX1 cooperates to maintain the proliferation of sclerotome cells during the vertebral column development.

During axial skeleton development, the notochord is essential for the induction of the sclerotome and for the subsequent differentiation of cartilage forming the vertebral bodies and intervertebral discs. These functions are mainly mediated by the diffusible signaling molecule Sonic hedgehog. The products of the paired-box-containing Pax1 and the mesenchyme forkhead-1 (Mfh1) genes are expressed in the developing sclerotome and are essential for the normal development of the vertebral column. Here, we demonstrate that Mfh1 like Pax1 expression is dependent on Sonic hedgehog signals from the notochord, and Mfh1 and Pax1 act synergistically to generate the vertebral column. In Mfh1/Pax1 double mutants, dorsomedial structures of the vertebrae are missing, resulting in extreme spina bifida accompanied by subcutaneous myelomeningocoele, and the vertebral bodies and intervertebral discs are missing. The morphological defects in Mfh1/Pax1 double mutants strongly correlate with the reduction of the mitotic rate of sclerotome cells. Thus, both the Mfh1 and the Pax1 gene products cooperate to mediate Sonic hedgehog-dependent proliferation of sclerotome cells.

Animals↗

PubMed1999

BAG-1 accelerates cell motility of human gastric cancer cells.

BAG-1 is a Hsp70/Hsc70-binding protein that interacts with Bcl-2, Raf-1, steroid hormone receptors, Siah-1, and hepatocyte growth factor (HGF) receptors, implying multiple functions for the BAG-1 protein. Here, we provide evidence that gene transfer-mediated overexpression of BAG-1 markedly enhances the motility of human gastric cancer cells. Two independent in vitro migration assays showed that the BAG-1-expressing MKN74 cells exhibited more active migration compared with control transfectants or parent MKN74 cells. In MKN74 cells, the overexpression of BAG-1 affected neither cell adhesion capability nor migration responses to HGF. The promotive effect of BAG-1 on cell migration was similarly observed in transfectants of another human gastric cancer MKN45 cell line. In BAG-1 transfected gastric cancer MKN74 cells, BAG-1 colocalized with cytokeratin as well as actin filaments, and was concentrated at membrane ruffles induced by lysophosphatidic acid (LPA). Taken together, these studies demonstrate that BAG-1 has a novel function as promoter of cell migration in human gastric cancer cells, possibly through cooperation with cytoskeletal proteins.

Actins↗

PubMed1999

Enantiomeric determination of D- and L-lactate in rat serum using high-performance liquid chromatography with a cellulose-type chiral stationary phase and fluorescence detection.

A method is described for the quantitative determination of d- and L-lactate in 10 microl of rat serum, which includes fluorescence derivatization of D- and L-lactate with 4-(N, N-dimethylaminosulfonyl)-7-piperazino-2,1,3- benzoxadiazole (DBD-PZ) followed by O-acetylation. The derivatives are separated by HPLC on an octadecylsilica, and, via column switching, on a cellulose-type chiral column. Levulinic acid was used as the internal standard. The enantiomers of lactate were separated with the separation factor (alpha) of 1.27 and the resolution (Rs) of 2.72, while the linearity for the detection was over the range of 10 nmol/ml to 20 micromol/ml (r = 0.999). Interday precision values for D-lactate in rat serum were 5.8, 5.3, and 4.1% for 10, 100, and 1000 nmol/ml, and accuracy values were 109.6, 98.2, and 103.1%, respectively (n = 5). The reduction of d-lactate concentration in rat serum by fasting was observed with the method.

Acetylation↗

PubMed1999

Simultaneous automatic determination of catecholamines and their 3-O-methyl metabolites in rat plasma by high-performance liquid chromatography using peroxyoxalate chemiluminescence reaction.

A highly specific and sensitive automated high-performance liquid chromatographic method for the simultaneous determination of catecholamines (CAs; norepinephrine, epinephrine, and dopamine) and their 3-O-methyl metabolites (normetanephrine, metanephrine, and 3-methoxytyramine) is described. Automated precolumn ion-exchange extraction of diluted plasma is coupled with HPLC separation of CAs and their 3-O-methyl metabolites on an ODS column, postcolumn coulometric oxidation, fluorescence derivatization with ethylenediamine, and finally peroxyoxalate chemiluminescence reaction detection. The detection limits were about 3 fmol for norepinephrine, epinephrine, and dopamine, 5 fmol for normetanephrine, and 10 fmol for metanephrine and 3-methoxytyramine (signal-to-noise ratio of 3). Fifty microliters of rat plasma was used and 4-methoxytyramine was employed as an internal standard. The relative standard deviations for the method (n = 5) were 2.5-7.6% for the intraday assay and 6.3-9.1% for the interday assay. The method was applicable to the determination of normetanephrine and metanephrine in 50 microl of rat plasma.

Animals↗

PubMed1999

Involvement of wiskott-aldrich syndrome protein in B-cell cytoplasmic tyrosine kinase pathway.

Bruton's tyrosine kinase (Btk) has been shown to play a role in normal B-lymphocyte development. Defective expression of Btk leads to human and murine immunodeficiencies. However, the exact role of Btk in the cytoplasmic signal transduction in B cells is still unclear. This study represents a search for the substrate for Btk in vivo. We identified one of the major phosphoproteins associated with Btk in the preB cell line NALM6 as the Wiskott-Aldrich syndrome protein (WASP), the gene product responsible for Wiskott-Aldrich syndrome, which is another hereditary immunodeficiency with distinct abnormalities in hematopoietic cells. We demonstrated that WASP was transiently tyrosine-phosphorylated after B-cell antigen receptor cross-linking on B cells, suggesting that WASP is located downstream of cytoplasmic tyrosine kinases. An in vivo reconstitution system demonstrated that WASP is physically associated with Btk and can serve as the substrate for Btk. A protein binding assay suggested that the tyrosine-phosphorylation of WASP alters the association between WASP and a cellular protein. Furthermore, identification of the phosphorylation site of WASP in reconstituted cells allowed us to evaluate the catalytic specificity of Btk, the exact nature of which is still unknown.

Agammaglobulinaemia Tyrosine Kinase↗

PubMed1999

SEA-scFv as a bifunctional antibody: construction of a bacterial expression system and its functional analysis.

A SEA-antibody single chain Fv (SEA-scFv) fusion protein was produced by bacterial expression system in this study. SEA-scFv has both staphylococcal enterotoxin A (SEA) effects and antibody activity directed at the epithelial mucin core protein MUC1, a cancer associated antigen. It was expressed mostly in the cytoplasm as an insoluble form. The gene product was solubilized by guanidine hydrochloride, refolded by conventional dilution method, and purified using metal-chelating chromatography. The resulting SEA-scFv fusion protein preparation was found to react with MUC1 and MHC class II antigens and had the ability to enhance cytotoxicity of lymphokine activated killer cells with a T cell phenotype against a human bile duct carcinoma cell line, TFK-1, expressing MUC1. This genetically engineered SEA-scFv fusion protein promises to be an important reagent for cancer immunotherapy.

Antibodies, Bispecific↗

PubMed1999

An enzymatic cycling method using pyruvate orthophosphate dikinase and firefly luciferase for the simultaneous determination of ATP and AMP (RNA).

A novel bioluminescent enzymatic cycling assay for ATP and AMP with concomitant use of firefly luciferase and pyruvate orthophosphate dikinase (PPDK) was developed. In this system, AMP and pyrophosphate produced from ATP by firefly luciferase were converted back into ATP by PPDK. This resulted in constant luminescence once the stable phase had been reached. Background luminescence of the reagent was reduced with adenosine phosphate deaminase by degrading ATP and AMP in the reagent. The maximum recycling ratio calculated from the integrated luminescence value was 2.64 cycles/min. The measurable ranges for ATP and AMP were equal and were between 4 x 10(-13) and 4 x 10(-17) mol/assay. The amount of yeast RNA could be estimated in the range of 1 x 10(-8) to 1 x 10(-12) g/assay by estimating the amount of AMP resulting from the degradation of RNA with nuclease P1. Various food samples were subjected to measurement of the amount of ATP + AMP + RNA to provide an index for hygiene monitoring. For beef extract, sensitivity was improved by more than 20 million compared to the previous methods relying only on the amount of ATP as an index.

Adenosine Deaminase↗

PubMed1999

D-Aspartate stimulation of testosterone synthesis in rat Leydig cells.

D-Aspartate increases human chorionic gonadotropin-induced testosterone production in purified rat Leydig cells. L-Aspartate, D-,L-glutamate or D-,L-asparagine could not substitute for D-aspartate and this effect was independent of glutamate receptor activation. Testosterone production was enhanced only in cells cultured with D-aspartate for more than 3 h. The increased production of testosterone was well correlated with the amounts of D-aspartate incorporated into the Leydig cells, and L-cysteine sulfinic acid, an inhibitor of D-aspartate uptake, suppressed both testosterone production and intracellular D-aspartate levels. D-Aspartate therefore is presumably taken up into cells to increase steroidogenesis. Intracellular D-aspartate probably acts on cholesterol translocation into the inner mitochondrial membrane, the rate-limiting process in steroidogenesis.

Animals↗

PubMed1999

Development of the arbitrarily primed-representational difference analysis method and chromosomal mapping of isolated high throughput rat genetic markers.

Linkage mapping of quantitative trait loci requires analysis of a large number of animals. Although genetic markers isolated by representational difference analysis (RDA) and its modifications meet the needs, the number of these markers has been limited. In the present study, we established the arbitrarily primed (AP)-RDA method to isolate virtually an unlimited number of the high throughput genetic markers. A representation of the genome, an AP-amplicon, was prepared by AP-PCR with a single primer or with a combination of primers using genomic DNA of the ACI/N (ACI) or BUF/Nac (BUF) rat as a template. By subtracting the AP-amplicon of ACI from that of BUF, a total of 40 polymorphic and independent markers were isolated in seven series of AP-RDA using a single primer. Two series of AP-RDA with primer combination yielded seven additional independent markers. All of the markers gave clear positive/negative signals by hybridization of a filter where AP-amplicons from F2 rats of ACI and BUF were dot-blotted at a high density without any concentration or purification. All of the 47 independent markers were mapped to unique chromosomal positions by linkage analysis, even though some arbitrary primers had very similar sequences. The markers were also informative between other strains of rats. Simultaneous hybridization of multiple filters made it possible to genotype a large number of rats simultaneously for multiple genetic loci. The AP-RDA method promises isolation of a large number of high throughput genetic markers in any species and is expected to facilitate linkage mapping of subtle quantitative trait loci.

Animals↗

PubMed1999

Selective transendothelial migration of hematopoietic progenitor cells: a role in homing of progenitor cells.

To elucidate the mechanisms by which hematopoietic progenitor cells transmigrate via the bone marrow (BM) endothelial cells, we first established endothelial cell lines from BM and lung, and BM fibroblast cell lines; then we established an in vitro model of transendothelial migration of hematopoietic progenitor cells in the presence of chemoattractants secreted by BM fibroblast cells. The BM endothelial cells expressed vascular cell adhesion molecule-1 (VCAM-1), but the lung endothelial cells did not. The BM fibroblast cells secreted chemoattractants including stroma cell-derived factor (SDF)-1, which could attract hematopoietic progenitor cells to BM and activate the adhesion molecules expressed on hematopoietic progenitor cells after rolling along the endothelial cells. Anti-SDF-1 antibody inhibited the transendothelial migration of a hematopoietic progenitor cell line, FDCP-2. FDCP-2 that expressed very late activation antigen-4 (VLA-4) and normal progenitor cells transmigrated through BM endothelial cells but not lung endothelial cells, even if in the presence of chemoattractants produced by BM fibroblasts. Both anti-VLA-4 and anti-VCAM-1 antibodies inhibited the transendothelial migration of FDCP-2 cells and normal hematopoietic progenitor cells. These findings suggest that the transendothelial migration of hematopoietic progenitor cells is characteristic of BM endothelial cells, and that VLA-4/VCAM-1 and SDF-1 play important roles in the transendothelial migration and, consequently, homing of hematopoietic progenitor cells to BM.

Animals↗

PubMed1999

Expression of MUC2 gene in gastric regenerative, metaplastic, and neoplastic epithelia.

It has been reported that MUC2 mucin is expressed in goblet cells of gastric intestinal metaplasia, but not in its normal epithelium. To confirm this finding, we have examined the expression of the MUC2 gene by reverse transcriptase-polymerase chain reaction and immunohistochemical methods in gastric tissues obtained by routine upper gastrointestinal tract endoscopy and compared the results with pathological findings based on hematoxylin and eosin (H&E) staining. In 16.7% of the tissue specimens tested, MUC2 mRNA was detected in spite of the absence of intestinal metaplasia in HE specimens. A possible explanation for this was the identification by immunohistochemistry of MUC2 protein in regenerative gastric mucosal cells in biopsies that did not contain intestinal metaplasia. Sialyl-Le(x) epitope, which is suggested to be located on MUC2 mucin core protein (MUC2 protein), was also immunohistochemically detected in both goblet cells of intestinal metaplasia and regenerative epithelium. With regard to carcinoma, MUC2 protein was predominantly expressed in intestinal-type adenocarcinoma. These data indicate that MUC2 mucin is expressed in gastric regenerative epithelium in addition to intestinal metaplasia and intestinal type adenocarcinoma.

Adult↗

PubMed1999

Development and clinical application of high performance liquid chromatography for the simultaneous determination of plasma levels of theophylline and its metabolites without interference from caffeine.

A high performance liquid chromatography (HPLC) method has been developed for the simultaneous determination of plasma levels of theophylline and its metabolites without interference from caffeine or caffeine metabolites. The method is simple and of practical use because it is applicable even to plasma samples from patients who take caffeine-containing beverages. The method was also reproducible with a coefficient of variation of less than 5% for each analyte. The levels of theophylline, determined by HPLC, were validated by their high correlation to the levels obtained by fluorescence polarization immunoassay. HPLC was used to determine theophylline levels in patients with bronchial asthma. The data revealed that the ratio of 1,3-dimethyluric acid, the major metabolite of theophylline, to theophylline concentration in the plasma was within a narrow range in most patients (0.055 +/- 0.01, n = 66), regardless of the method of theophylline administration or the time of blood sampling. Conversely, this ratio was as low as 0.027 +/- 0.005 in the patient with a long plasma half-life of theophylline. These results suggest that it may be possible to predict the plasma half-life of theophylline for each patient from a single blood sample. This may be useful when planning theophylline administration, especially in patients with abnormal theophylline metabolism.

Adolescent↗

PubMed1999

Changes of plasma L-arginine levels in spontaneously hypertensive rats under induced hypotension.

Nicardipine, a dihydropyridine type calcium channel blocker, was infused at two flow-rates into spontaneously hypertensive (SH) and control normotensive Wistar-Kyoto (WKY) rats (young, 6-week-old and adult, 23-week-old, n = 5) under pentobarbital anesthesia, to cause hypotension. Mean arterial blood pressure and the concentrations of plasma amino acids and norepinephrine (NE) were measured before infusion and at each step of the infusion. The reduction in blood pressure caused by nicardipine induced a decrease in plasma L-arginine concentration in both young and adult SH rats, this effect being larger in adult rats. There was no significant change in plasma levels of L-arginine in age-matched WKY rats. The concentration of other amino acids did not change in both rat strains. On the contrary, there was an increase in plasma NE concentration in both SH and WKY rats after infusion with nicardipine. Plasma L-arginine concentration showed a good inverse correlation with the logarithm of plasma NE concentration in SH and WKY rats and the correlation was expressed as Y = -alpha log(X) + m (Y, plasma L-arginine concentration (nmol/mL); X, plasma NE concentration (pmol/mL); alpha, a slope; and m, an intercept). alpha, 43.0 and 4.35 for 23-week-old SH and WKY rats, respectively, and 17.0 and 4.0 for 6-week-old SH and WKY rats, respectively. The present data together with previous data suggest a direct noradrenergic stimulation of the synthesis of nitric oxide (NO) from L-arginine. The findings also indicate an impairment of the L-arginine metabolism or pools in SH rats compared with WKY rats. The deficiency of L-arginine increases with the age of SH rats and could be related to the development and maintenance of hypertension due to inefficient production of NO.

Animals↗

PubMed1999

Determination of aspartic acid enantiomers in bio-samples by capillary electrophoresis.

Enantiomeric separation and detection of D,L-aspartic acid (Asp) derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) by capillary electrophoresis (CE) using modified cyclodextrins as chiral selectors was studied. Heptakis(2,3, 6-tri-O-methyl)-beta-cyclodextrin(TM-beta-CD) was most effective for enantiomeric separation of NBD-D,L-Asp with optimum conditions of 30 mM TM-beta-CD in 50 mM phosphate buffer (pH 4.0) and the limit of detection (LOD) attained was 100 nM for each enantiomer. The method proposed in the present study was convenient for both D- and L-Asp determination since the other amino compounds migrated differently and D-Asp in bio-samples such as rat pineal gland and foods was determined with a simple sample pretreatment and a short analysis run time.

Animal Feed↗

PubMed1999

Fluorimetric determination of D- and L-lactate derivatized with 4-(N, N-dimethylaminosulfonyl)-7-piperazino-2,1,3-benzoxadiazole (DBD-PZ) by high-performance liquid chromatography.

A highly sensitive method, based on fluorescence derivatization with 4-N,N-dimethylaminosulfonyl-7-piperazino-2,1,3-benzoxadiazol e (DBD-PZ), was developed for the determination of D- and L-lactic acid. High-performance liquid chromatographic separation of the lactic acid derivative was achieved using an octadecylsilica column followed by enantiomeric separation on a phenylcarbamoylated beta-cyclodextrin chiral column. The separation factor for D, L-lactic acid derivatives was 1.30 using MeOH/H(2)O (80/20) as a mobile phase, and the detection limits were approximately 360 and 300 fmol on column for D- and L-lactic acid derivative, respectively. The proposed method was applied to determine D- and L-lactate in a wine and a lactic drink. Both D- and L-lactate could be determined simultaneously, with the precisions ranging from 3.50 to 5.78% for intra-day and 4.28-9.92% for inter-day determinations. The relative recovery was in the range of 91.6-100%.

Calibration↗

PubMed1999

Enhanced theophylline metabolism in patients with bronchial asthma at age 4 and under.

The plasma levels of theophylline (TP) and its metabolites were measured in patients with bronchial asthma who were treated with a slow-release preparation of TP. The ratios of the plasma levels of these metabolites to TP levels in the group aged 1-4 years were larger than those in the group aged 5 years and older, suggesting enhanced activity of drug-metabolizing enzymes during infancy.

Adolescent↗