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Biomedical subjects

A Tsuji

Publications and source records attributed to A Tsuji.

At least 55 records · Page 3Linked to original sources
PubMed2001

Racemization kinetics of meluadrine tartrate in aqueous solution.

The kinetics of racemization of meluadrine tartrate in aqueous solution was investigated by HPLC, over the pH range of 1.2 to 12 at 40, 60 and 80 degrees C. The racemization was followed by measuring both the residual (R)-enantiomer and formed (S)-enantiomer of meluadrine. The pH-racemization rate profile of meluadrine showed a minimum racemization rate constant between pH 4 and 6, and the rate constant increased rapidly with decreasing pH below pH 3. In the basic region, the racemization rate constant showed a maximum at around pH 9. Four pathways of racemization of meluadrine are proposed, depending on the dissociation state of the phenolic and amino groups. The calculated racemization rate-pH profile agreed well with the observations. The values of activation energy (95-115 kJ/mol) suggest that a similar quinoid intermediate is involved throughout the pH range examined. The objective of this study is to clarify the racemization mechanism of benzylalcohol derivative with phenoric group. This study offers fundamental knowledge for the other benzylalcohol derivatives, which have similar moiety structure.

Adrenergic beta-2 Receptor Agonists↗
PubMed2001

Cloning, sequencing, and expression of the gene encoding an intracellular beta-D-xylosidase from Streptomyces thermoviolaceus OPC-520.

The intracellular beta-xylosidase was induced when Streptomyces thermoviolaceus OPC-520 was grown at 50 degrees C in a minimal medium containing xylan or xylooligosaccharides. The 82-kDa protein with beta-xylosidase activity was partially purified and its N-terminal amino acid sequence was analyzed. The gene encoding the enzyme was cloned, sequenced, and expressed in Escherichia coli. The bxlA gene consists of a 2,100-bp open reading frame encoding 770 amino acids. The deduced amino acid sequence of the bxlA gene product had significant similarity with beta-xylosidases classified into family 3 of glycosyl hydrolases. The bxlA gene was expressed in E. coli, and the recombinant protein was purified to homogeneity. The enzyme was a monomer with a molecular mass of 82 kDa. The purified enzyme showed hydrolytic activity towards only p-nitrophenyl-beta-D-xylopyranoside among the synthetic glycosides tested. Thin-layer chromatography analysis showed that the enzyme is an exo-type enzyme that hydrolyze xylooligosaccharides, but had no activity toward xylan. High activity against pNPX occurred in the pH range 6.0-7.0 and temperature range 40-50 degrees C.

Amino Acid Sequence↗
PubMed2001

[Immunohistochemical study of p53 and Ki-67 overexpression in grade 3 superficial bladder tumor in relationship to tumor recurrence and prognosis].

PURPOSE: The major drawback of the current treatment for superficial bladder tumor is the high rate of recurrence. Especially, the tumor with grade 3 component has a tendency to recur and progress in stage. However, we have difficulty in predicting tumor recurrence and stage progression accurately by conventional clinicopathological factors. We evaluated the efficacy of p53 and Ki-67 overexpression as a predictor of recurrence or prognosis in patients with superficial bladder tumor of grade 3. MATERIALS AND METHODS: Samples were obtained from 41 patients with superficial transitional cell carcinoma of the bladder of grade 3 who were treated by transurethral resection (TUR). The immunohistochemical study was performed using the antibodies against the p53 protein and Ki-67 antigen on formalin-fixed, paraffinembedded tissue specimens from initial tumors. We evaluated the correlation between these results and several clinicopathological factors. RESULTS: The p53 index and the Ki-67 index in pTa, pT1a and pT1b tumors were 26.4 +/- 30.1%, 28.6 +/- 30.0%, and 34.6 +/- 32.6% (p53) and 20.5 +/- 22.5%, 20.0 +/- 29.3%, and 29.2 +/- 28.4% (Ki-67). There was no significant difference between the each index and tumor stage. Eighteen cases (43.9%) had intravesical recurrence. The p53 index of the initial tumor from the tumor free cases (n = 23), recurrent cases without stage progression (n = 12), and stage progression cases (n = 6) were 19.7 +/- 28.2%, 42.0 +/- 28.7%, and 42.5 +/- 32.0%. Between the recurrence-free cases and the recurrent cases without progression, the p53 index of the initial tumor had statistical significance (p < 0.05). The Ki-67 index was shown to be the same pattern as the p53 index, but there was not statistical significance. Four of patients with stage progression had tumor progression within six months. Three of the patients with tumors with stage progression died of the cancer. In multivariate analysis, tumor multiplicity (p = 0.01), BCG intravesical instillation (p = 0.04), p53 index (p = 0.01) and Ki-67 index (p = 0.02) were the positive risk factors for tumor recurrence, but only the p53 index was the positive risk factor for prognosis fo the patients (p = 0.03). CONCLUSION: These results suggest that the immunohistochemical study of p53 overexpression is a useful predictor for tumor recurrence and prognosis in patients with superficial bladder tumor with grade 3.

Adult↗
PubMed2001

Rapid N transport to pods and seeds in N-deficient soybean plants.

Non-nodulated soybean (Glycine max (L.) Merr.) plants were cultivated hydroponically under N-sufficient (5 mM NaNO(3)) or N-deficient (0.5 mM NaNO(3)) conditions. (13)N- or (15)N- labelled nitrate was fed to the cut end of the stems, and the accumulation of nitrate-derived N in the pods, nodes and stems was compared. Real-time images of (13)N distribution in stems, petioles and pods were obtained using a Positron Emitting Tracer Imaging System for a period of 40 min. The results indicated that the radioactivity in the pods of N-deficient plants was about 10 times higher than that of N-sufficient plants, although radioactivity in the stems and nodes of N-deficient versus N-sufficient plants was not different. A similar result was obtained by supplying (15)NO(3) to cut soybean shoots for 1 h. The fact that the N translocation into the pods from NO(3) fed to the stem base was much faster in N-deficient plants may be due to the strong sink activity of the pods in N-deficient plants. Alternatively, the redistribution of N from the leaves to the pods via the phloem may be accelerated in N-deficient plants. The temporal accumulation of (13)NO(3) in nodes was suggested in both N-sufficient and N-deficient plants. In one (13)NO(3) pulse-chase experiment, radioactivity in the stem declined rapidly after transferring the shoot from the (13)NO(3) solution to non-labelled NO(3); in contrast, the radioactivity in the node declined minimally during the same time period.

Amino Acids↗
PubMed2000

Molecular and functional characterization of organic cation/carnitine transporter family in mice.

Carnitine is essential for beta-oxidation of fatty acids, and a defect of cell membrane transport of carnitine leads to fatal systemic carnitine deficiency. We have already shown that a defect of the organic cation/carnitine transporter OCTN2 is a primary cause of systemic carnitine deficiency. In the present study, we further isolated and characterized new members of the OCTN family, OCTN1 and -3, in mice. All three members were expressed commonly in kidney, and OCTN1 and -2 were also expressed in various tissues, whereas OCTN3 was characterized by predominant expression in testis. When their cDNAs were transfected into HEK293 cells, the cells exhibited transport activity for carnitine and/or the organic cation tetraethylammonium (TEA). Carnitine transport by OCTN1 and OCTN2 was Na(+)-dependent, whereas that by OCTN3 was Na(+)-independent. TEA was transported by OCTN1 and OCTN2 but not by OCTN3. The relative uptake activity ratios of carnitine to TEA were 1.78, 11.3, and 746 for OCTN1, -2, and -3, respectively, suggesting high specificity of OCTN3 for carnitine and significantly lower carnitine transport activity of OCTN1. Thus, OCTN3 is unique in its limited tissue distribution and Na(+)-independent carnitine transport, whereas OCTN1 efficiently transported TEA with minimal expression of carnitine transport activity and may have a different role from other members of the OCTN family.

Amino Acid Sequence↗
PubMed2000

Cancer cell-targeted drug delivery utilizing oligopeptide transport activity.

To study the drug delivery to tumor by utilization of an oligopeptide transport activity, we examined the accumulation of dipeptides and the peptide-mimetic anti-cancer drug, bestatin, a substrate of oligopeptide transporter PepT1. Firstly, we established HeLa cells stably expressing human peptide transporter (hPepT1) (HeLa-hPepT1). Secondly, we constructed an experimental model by inoculation of HeLa-hPepT1 cells subcutaneously into Balb/c nu/nu mice to demonstrate the contribution of PepT1 to the tissue-selective drug delivery. The accumulations of a hydrolysis-resistant dipeptide [(3)H]carnosine and bestatin in solid tumors formed by HeLa-hPepT1 or HeLa-pcDNA3, which are transfected with vector DNA (pcDNA3) were measured. After I.V. administration, tissue-to-plasma concentration ratios (K(p)) of both compounds, in HeLa-hPepT1 tumor was significantly greater than that of [(14)C]inulin, a marker for extracellular fluid space, those of dipeptides in muscle, or those in HeLa-pcDNA3 tumor. Furthermore, bestatin exhibited growth inhibition of HeLa-hPepT1 in vitro. In vivo, repeated oral administration of bestatin for 28 days suppressed the growth of HeLa-hPepT1 tumor specifically. When HT-1080 cells, which may naturally express oligopeptide transport activity, were transplanted, K(p) of [(3)H]carnosine was significantly increased in comparison with that in muscle. In addition, oligopeptide transport activities among various human cell lines were examined. These results provide the first demonstration for the selective delivery of oligopeptides to tumors by specific oligopeptide transport activity.

Animals↗
PubMed2000

Anion antiport mechanism is involved in transport of lactic acid across intestinal epithelial brush-border membrane.

Intestinal epithelial membrane transport of L-lactic acid was characterized using rabbit jejunal brush-border membrane vesicles (BBMVs). The uptake of L-[(14)C]lactic acid by BBMVs showed an overshoot phenomenon in the presence of outward-directed bicarbonate and/or inward-directed proton gradients. Kinetic analysis of L-[(14)C]lactic acid uptake revealed the involvement of two saturable processes in the presence of both proton and bicarbonate gradients. An arginyl residue-modifying agent, phenylglyoxal, inhibited L-[(14)C]lactic acid transport by the proton cotransporter, but not by the anion antiporter. The initial uptakes of L-[(14)C]lactic acid which are driven by bicarbonate ion and proton gradients were inhibited commonly by monocarboxylic acids and selectively by anion exchange inhibitor 4, 4'-diisothiocyanostilbene-2,2'-disulfonic acid and protonophore carbonylcyanide p-trifluoromethoxyphenylhydrazone, respectively. These observations demonstrate that L-lactic acid is transported across the intestinal brush-border membrane by multiple mechanisms, including an anion antiporter and a previously known proton cotransporter.

Animals↗
PubMed2000

Molecular identification and characterization of novel members of the human organic anion transporter (OATP) family.

We identified three novel transporters structurally belonging to the organic anion transporting polypeptide (OATP) family in humans. Since previously known rat oatp1 to 3 do not necessarily correspond to the human OATPs in terms of either tissue distribution or function, here we designate the newly identified human OATPs as OATP-B, -D and -E, and we rename the previously known human OATP as OATP-A. OATP-C proved to be identical with the recently reported LST1/OATP-2. Expression profiles of the five OATPs and the prostaglandin transporter PGT (a member of OATP family) in human tissues showed that OATP-C is exclusively localized in liver, OATP-A and PGT are expressed in restricted ranges of tissues, and OATP-B, -D and -E show broad expression profiles. OATP-B, -C, -D and -E exhibited transport activity for [(3)H]estrone-3-sulfate as a common substrate. OATP-C has a high transport activity with broad substrate specificity.

Amino Acid Sequence↗
PubMed2000

Real-time monitoring of in vitro transcriptional RNA synthesis using fluorescence resonance energy transfer.

We have developed a novel method for real-time monitoring of RNA synthesis in in vitro transcription reactions using fluorescence resonance energy transfer (FRET). Two 15mer DNAs, either of which was labeled with Bodipy493/503 as a donor or Cy5 as an acceptor, were prepared. When the two fluorescent DNAs hybridized to adjacent locations on Xenopus: elongation factor 1-alpha (xelf1-alpha) RNA, the distance between the two fluorophores became very close, causing FRET to occur and resulting in changes in fluorescence spectra. A high accessibility 30mer site of xelf1-alpha RNA was found and excess amounts of a pair of donor and acceptor DNA probes that were complementary to the site were added to the in vitro transcription reaction solution. Changes in fluorescence spectra were observed in response to progression of xelf1-alpha RNA synthesis that showed that the fluorescent probes hybridized to the synthesized RNA. Furthermore, when probes hybridizing to the synthesized xelf1-alpha RNA with less efficiency were used to monitor the reaction, spectral changes in response to RNA synthesis were also observed. This result suggests that the probes hybridized to synthesizing RNA molecules before they folded to form secondary structure and that there is no need to select sites on the RNA for the probes, which is required for probes hybridizing to folded RNA molecules.

Animals↗
PubMed2000

Meltrin beta (ADAM19) gene: cloning, mapping, and analysis of the regulatory region.

Meltrin beta (ADAM19) is a member of the metalloprotease-disintegrin family. We report here chromosomal mapping of the mouse and rat meltrin beta genes and cloning and analysis of the mouse upstream regulatory regions. The meltrin beta transcript shows a spatially and temporally restricted expression pattern during morphogenesis, indicating that the actions of this membrane-bound protease are regulated, at least in part, at the transcriptional level. Analysis of the promoter revealed positive and negative regulatory regions upstream of the gene. The former includes a GC-box that appears to be a critical cis-element for activation of the promoter in muscle cells.

ADAM Proteins↗
PubMed2000

p-aminohippuric acid transport at renal apical membrane mediated by human inorganic phosphate transporter NPT1.

Organic anions are secreted into urine via organic anion transporters across the renal basolateral and apical membranes. However, no apical membrane transporter for organic anions such as p-aminohippuric acid (PAH) has yet been identified. In the present study, we showed that human NPT1, which is present in renal apical membrane, mediates the transport of PAH. The K(m) value for PAH uptake was 2.66 mM and the uptake was chloride ion sensitive. These results are compatible with those reported for the classical organic anion transport system at the renal apical membrane. PAH transport was inhibited by various anionic compounds. Human NPT1 also accepted uric acid, benzylpenicillin, faropenem, and estradiol-17beta-glucuronide as substrates. Considering its chloride ion sensitivity, Npt1 is expected to function for secretion of PAH from renal proximal tubular cells. This is the first molecular demonstration of an organic anion transport function for PAH at the renal apical membrane.

Anions↗
PubMed2000

Two novel missense mutations of the OCTN2 gene (W283R and V446F) in a patient with primary systemic carnitine deficiency.

Primary systemic carnitine deficiency (SCD) is an autosomal recessive disorder of fatty acid oxidation caused by defective cellular carnitine transport. The disease is characterized by metabolic derangement simulating Reye's syndrome, hypoglcaemia, progressive cardiomyopathy and skeletal myopathy. Recently, it was shown that SCD is caused by mutations in the organic cation/carnitine transporter OCTN2 (SLC22A5). We report two novel mutations, W283R and V446F, which are both missense mutations in an affected infant. In vitro expression studies demonstrated that both are actually function-loss mutations with virtually no uptake activity. This is the first report of compound heterozygosity for two missense mutations in a patient with SCD. Hum Mutat 15:118, 2000.

Carnitine↗
PubMed2000

Transporter-mediated permeation of drugs across the blood-brain barrier.

Drug distribution into the brain is strictly regulated by the presence of the blood-brain barrier (BBB) that is formed by brain capillary endothelial cells. Since the endothelial cells are connected to each other by tight junctions and lack pores and/or fenestrations, compounds must cross the membranes of the cells to enter the brain from the bloodstream. Therefore, hydrophilic compounds cannot cross the barrier in the absence of specific mechanisms such as membrane transporters or endocytosis. So, for efficient supply of hydrophilic nutrients, the BBB is equipped with membrane transport systems and some of those transporter proteins have been shown to accept drug molecules and transport them into brain. In the present review, we describe mainly the transporters that are involved in drug transfer across the BBB and have been molecularly identified. The transport systems described include transporters for amino acids, monocarboxylic acids, organic cations, hexoses, nucleosides, and peptides. Most of these transporters function in the direction of influx from blood to brain; the presence of efflux transporters from brain to blood has also been demonstrated, including P-glycoprotein, MRPs, and other unknown transporters. These efflux transporters seem to be functional for detoxication and/or prevention of nonessential compounds from entering the brain. Various drugs are transported out of the brain via such efflux transporters, resulting in the decrease of CNS side effects for drugs that have pharmacological targets in peripheral tissues or in the reduction of efficacy in CNS because of the lower delivery by efflux transport. To identify the transporters functional at the BBB and to examine the possible involvement of them in drug transports by molecular and physiological approaches will provide a rational basis for controlling drug distribution to the brain.

Animals↗
PubMed2000

Direct observation of specific messenger RNA in a single living cell under a fluorescence microscope.

We observed the expression of human c-fos mRNA in a living transfected Cos7 cell under a fluorescence microscope by detecting hybrid formed with two fluorescently labeled oligodeoxynucleotides (oligoDNAs) and c-fos mRNA in the cytoplasm. Two fluorescent oligoDNAs were prepared, each labeled with a fluorescence molecule different from the other. When two oligoDNAs hybridized to an adjacent sequence on the target mRNA, the distance between the two fluorophores became very close and fluorescence resonance energy transfer (FRET) occurred, resulting in changes in fluorescence spectra. To find sequences of high accessibility of c-fos RNA to oligoDNAs, several sites that included loop structures on the simulated secondary structure were selected. Each site was divided into two halves, and the pair of fluorescent oligoDNAs complementary to the sequence was synthesized. Each site was examined for the efficiency of hybridization to c-fos RNA by measuring changes in fluorescence spectra when c-fos RNA was added to the pair of oligoDNAs in solution. A 40 mer specific site was found, and the pair of oligoDNAs for the site were microinjected into Cos7 cells that expressed c-fos mRNA. To block oligoDNAs from accumulating in the nucleus, oligoDNA was bound to a macromolecule (streptavidin) to prevent passage of nuclear pores. Hybridization of the pair of oligoDNAs to c-fos mRNA in the cytoplasm was detected in fluorescence images indicating FRET.

Animals↗
PubMed2000

Sudden unexpected death with dysplastic change in the atrioventricular node artery.

We report an autopsy case of a 19-year-old male who died suddenly. Death happened while he was sleeping after drinking alcoholic beverages. His heart revealed concentric hypertrophy of the left ventricle without asymmetric septal hypertrophy or mitral regurgitation. Upon microscopic examination, the epicardial atrioventricular (AV) node artery revealed stenosis with intimal thickening before it entered the ventricular septum and acute ischemic change was observed beneath the conduction system in the upper ventricular septum. This finding suggests that death occurred after some preceding localized ischemic event within the ventricular septum. We therefore consider that the cause of death was fatal arrhythmia due to dysplastic change to the AV node artery.

Journal Article↗
PubMed2000

Conceptual changes in cancer chemotherapy: from an oral fluoropyrimidine prodrug, UFT, to a novel oral fluoropyrimidine prodrug, S-1, and low-dose FP therapy in Japan.

The conventional concept in cancer chemotherapy considers that no efficacy can be attained without provoking adverse reactions. We presented concrete descriptions based on a novel concept allowing us to emerge from the old one. Relief of adverse reactions, e.g., diarrhea, stomatitis, anorexia, and H&F syndrome, not only improves QOL of the patient but also allows prolongation of the treatment period without lowering patient compliance. We describe in this paper a therapeutic modality which is based on SRC (self-rescuing concept) featuring dual activity, i.e., effect-enhancing activity and adverse reaction-reducing activity. We present the theory and practice of S-1, a novel oral fluoropyrimidine anticancer agent designed to enhance anticancer activity and reduce gastrointestinal toxicity through the deliberate combination of the following components: an oral fluoropyrimidine agent tegafur; a DPD inhibitor (CDHP) which is more potent than uracil used in UFT; and an ORTC inhibitor (Oxo) which localizes in the gastrointestinal tract. Furthermore, we refer to combination therapy with 5-FU (CIV) and low-dose consecutive CDDP in which CDDP was used as a modulator of 5-FU and to the theory and practice of combination therapy with 5-FU (CVI) intermittent (Monday, Wednesday, and Friday) administration and low-dose CDDP consecutive administration in which a difference in cell cycle between gastrointestinal mucosal cell and tumor cell or between bone marrow cell and tumor cell was utilized. We intend in future to combine the abovementioned therapeutic modalities provoking less adverse reactions and being gentle to patients with cancer in an effort to further increase their life expectancy.

Administration, Oral↗
PubMed2000

Functional clarification of MCT1-mediated transport of monocarboxylic acids at the blood-brain barrier using in vitro cultured cells and in vivo BUI studies.

PURPOSE: To prove the functional significance of monocarboxylic acid transporter, MCT1 at the blood-brain barrier (BBB) for the passage of both endogenous and exogenous monocarboxylic acids into the central nervous system. METHODS: Monocarboxylic acid transport at the BBB was studied in rats by using a newly established immortalized brain capillary endothelial cell (BCEC) line, RBEC1, and the results were compared with those obtained by using primary cultured BCECs, cells stably expressed with rat MCT1, and the in vivo brain uptake index (BUI) method. RESULTS: The cell line, RBEC1 meets various morphological and enzymatic criteria of BCECs and appears to be suitable for the study of BBB transport of monocarboxylic acids. The presence of MCT1-transcript in RBEC1 was confirmnned by the RT-PCR method, as previously observed in isolated brain capillaries. A typical substrate of MCT1, lactic acid, was taken up by RBEC1 in a stereospecific and saturable manner. The value of the kinetic parameter Km showed good agreement with values previously obtained in studies using an in vivo BUI and in vitro MCT1-transfected cells. An organic weak acid, benzoic acid, which has been considered to cross biological membranes by passive diffusion, exhibited carrier-mediated transport properties, such as saturation, pH dependence, and stereospecific inhibition in RBEC1, similar to those we observed in primary cultured rat BCECs. The Km values in RBEC1, in primary cultured BCECs and in the in vivo BUI method were comparable and well agreed with that obtained in MCT1-transfected cells, suggesting that the transport features of benzoic acid observed by in vitro methods well reflect the in vivo transport activity. Furthermore, hybrid depletion of MCT1 in RBEC1 using an antisense oligonucleotide against rat MCT1 abolished the saturable transport of benzoic acid. CONCLUSIONS: These observations show that MCT1 plays a significant role in the transport of monocarboxylic acids, including the exogenous organic weak acid benzoic acid, as well as native lactic acid.

Animals↗