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Tomohiro Terada

Publications and source records attributed to Tomohiro Terada.

29 records · Page 2Linked to original sources
PubMed2004

Efflux properties of basolateral peptide transporter in human intestinal cell line Caco-2.

Small peptides and some pharmacologically active compounds are absorbed from the small intestine by the apical H(+)-coupled peptide transporter 1 (PEPT1) and the basolateral peptide transporter. Here we investigated the efflux properties of the basolateral peptide transporter in Caco-2 cells using two strategies, efflux measurements and a kinetic analysis of transepithelial transport of glycylsarcosine (Gly-Sar). [(14)C]Gly-Sar efflux through the basolateral membrane was not affected significantly by the external pH. Both approaches revealed that the basolateral peptide transporter was saturable in the efflux direction, and that the affinity was lower than that in the influx direction. For two peptide-like drugs, there was no difference in substrate recognition by the basolateral peptide transporter between the two sides of the membrane. Using the kinetic parameters of PEPT1 and the basolateral peptide transporter, a computational model of Gly-Sar transport in Caco-2 cells was constructed. The simulation fitted the experimental data well. Our findings suggested that substrate affinity of the basolateral peptide transporter is apparently asymmetric, but pH-dependence and substrate specificity are symmetric for the two directions of transport. The behaviour of Gly-Sar in Caco-2 cells could be predicted by a mathematical model describing the peptide transporters.

Anti-Bacterial Agents↗
PubMed2004

Design and synthesis of novel hydrophilic spacers for the reduction of nonspecific binding proteins on affinity resins.

Tubulin and actin often bind nonspecifically to affinity chromatography resins, complicating research toward identifying the cellular targets. Reduction of nonspecific binding proteins is important for success in finding such targets. We herein disclose the design, synthesis, and effectiveness in reduction of nonspecific binding proteins, of novel hydrophilic spacers (2-5), which were introduced between matrices and a ligand. Among them, tartaric acid derivative (5) exhibited the most effective reduction of nonspecific binding proteins, whilst maintaining binding of the target protein. Introduction of 5 on TOYOPEARL reduced tubulin and actin by almost 65% and 90% compared to that without the hydrophilic spacer, respectively, with effective binding to the target protein, FKBP12.

Actins↗
PubMed2004

Genetic variant Arg57His in human H+/peptide cotransporter 2 causes a complete loss of transport function.

We evaluated the functional consequences of genetic variations in human H(+)/peptide cotransporter 2 (hPEPT2, SLC15A2) resulting in the amino acid changes Arg57His (R57H) and Pro409Ser (P409S). The transport activity of variant R57H was completely abolished, whereas that of variant P409S was comparable with that of wild-type hPEPT2 at pH 5.0-8.0. R57H variant protein was detected in the crude membranes of transiently expressed HEK293 cells by Western blot analysis. The expression of the R57H variant at the plasma membrane was confirmed by indirect immunofluorescence in Xenopus oocytes, suggesting that the loss of transport function of hPEPT2 R57H was not due to a change in membrane protein expression. This is the first demonstration of a functional impairment of the SLC15A family induced by a single nucleotide polymorphism.

Amino Acid Substitution↗
PubMed2004

Surgical resection deteriorates gemcitabine-induced leukopenia in pancreatic cancer.

BACKGROUND: Gemcitabine hydrochloride (GEM) is one of the most effective chemotherapeutic agents for pancreatic cancer; however, factors affecting GEM-induced leukopenia have not been clarified yet. In the present study, we analyzed the relationship between patients backgrounds and GEM-induced leukopenia. METHODS: Thirty-eight patients with pancreatic cancer were analyzed for correlation between the dose of GEM and the blood leukocyte number. Moreover, we compared leukopenia in resected and non-resected patients. RESULTS: The incidence of grade 3 or 4 leukopenia was 25% in the non-resected patients, whereas equivalent leukopenia was observed in 57% of the resected patients ( P = 0.048 by the chi(2) test). The relative decrease in blood leukocytes induced by GEM administration was more severe in resected patients (41.3 +/- 9.9%), as compared to non-resected patients (52.6 +/- 16.0%; P = 0.023 by t-test). CONCLUSION: In the present study, we found that the administration of GEM to patients after surgical resection caused more severe leukopenia, as compared to findings in non-resected patients. These data suggested that more frequent monitoring of the leukocyte count and prolonged intervals between GEM administrations are necessary for resected patients with pancreatic cancer.Gemcitabine hydrochloride (GEM) is one of the most effective chemotherapeutic agents for pancreatic cancer; however, factors affecting GEM-induced leukopenia have not been clarified yet. In the present study, we analyzed the relationship between patients' backgrounds and GEM-induced leukopenia.

Aged↗
PubMed2004

The diurnal rhythm of the intestinal transporters SGLT1 and PEPT1 is regulated by the feeding conditions in rats.

The intestinal Na(+)/glucose cotransporter 1 (SGLT1) and H(+)/peptide cotransporter 1 (PEPT1) play important roles in the absorption of carbohydrate and protein. Although they exhibit a diurnal rhythm in their expression and function, the factors responsible for this are unclear. In the present study, we examined the effects of various feeding conditions on the diurnal rhythm of intestinal SGLT1 and PEPT1. Rats were divided into 1 of 4 groups: group 1 was fed, group 2 was food deprived for 1-4 d, group 3 was food deprived for 4 d and then refed for 1 or 2 d, and group 4 was fed during the daytime (0900-1500 h) for 10 d. In fed rats, the SGLT1 protein level was significantly higher at 2000 h than at 0800 h. However, in rats deprived of food for 2-4 d, protein levels did not differ between 0800 and 2000 h. In contrast, the SGLT1 messenger RNA (mRNA) level was significantly higher at 2000 h than at 0800 h in rats deprived of food for 4 d. Refeeding for 2 d after 4 d of food deprivation returned the diurnal variation in SGLT1 and PEPT1 protein expressions to normal. Consuming food during the daytime only shifted the peaks of SGLT1 and PEPT1 mRNAs and protein expressions from the dark phase to the light phase. These findings suggest that food intake, rather than the light cycle, greatly affects the diurnal rhythm of SGLT1 and PEPT1 expressions.

Animals↗
PubMed2004

Peptide transporters: structure, function, regulation and application for drug delivery.

Proton-coupled peptide transporters, localized at brush-border membranes of intestinal and renal epithelial cells, play important roles in protein absorption and the conservation of peptide-bound amino nitrogen. These transporters also have significant pharmacological and pharmacokinetic relevance to the transport of various peptide-like drugs such as beta-lactam antibiotics. The identification and molecular characterization of H(+)/peptide cotransporters (PEPT1 and PEPT2) have facilitated the clarification of many aspects of these transporters such as the structure/function relationship and regulation. Recent findings that intestinal PEPT1 can transport l-valine ester prodrugs such as valacyclovir provided a major step forward toward the development of novel drug delivery systems. It has been demonstrated that peptide transporters, which have a similar substrate specificity to PEPT1 and PEPT2, but possess other distinct functional properties, are localized at basolateral membranes of intestinal and renal epithelial cells. This review highlights the recent advances in our knowledge of the cellular and molecular nature of PEPT1, PEPT2 and the basolateral peptide transporters.

Animals↗
PubMed2003

Altered diurnal rhythm of intestinal peptide transporter by fasting and its effects on the pharmacokinetics of ceftibuten.

We previously demonstrated that H+/peptide cotransporter PEPT1 shows a diurnal rhythm in the rat small intestine. In the present study, we examined the effect of food intake on the diurnal rhythm of intestinal PEPT1 using fed and fasted rats and also determined whether such variation affected the pharmacokinetics of peptide-like drugs. In fed rats, PEPT1 protein level was significantly higher at 8:00 PM than at 8:00 AM. However, during fasting for 2 to 4 days, the differences of PEPT1 protein levels between 8:00 AM and 8:00 PM gradually disappeared. Intestinal absorption of an oral antibiotic ceftibuten (CETB), a pharmacological substrate for PEPT1, was also greater at 8:00 PM than at 8:00 AM in fed rats, but not different in 4-day fasted rats. In contrast to PEPT1 protein levels, PEPT1 mRNA levels retained a diurnal rhythm after 4 days of fasting. Pharmacokinetic analyses of CETB after intraintestinal administration demonstrated that both Cmax and area under the plasma concentration-time curve from 0 to 3 h were greater at 8:00 PM than at 8:00 AM in fed rats. In contrast, pharmacokinetic parameters showed no significant difference between 8:00 AM and 8:00 PM for intraintestinal administration in 4-day fasted rats and for intravenous administration in fed and 4-day fasted rats. These findings suggested that the diurnal rhythm of intestinal PEPT1 transport activity was disrupted by fasting and that diurnal variation of intestinal PEPT1 functionality could influence the pharmacokinetics of peptide-like drugs such as CETB.

Animals↗
PubMed2003

Inhibitory effect of zinc on PEPT1-mediated transport of glycylsarcosine and beta-lactam antibiotics in human intestinal cell line Caco-2.

PURPOSE: The aim of this study was to examine the effects of zinc on the intestinal peptide transporters (PEPT1 and basolateral peptide transporter) and to elucidate the mechanism of the interactions. METHODS: Caco-2 cells were pretreated with zinc, and the uptake studies were carried out. RESULTS: Zinc treatment resulted in the inhibition of [14C]glycylsarcosine (Gly-Sar) uptake via PEPT1 in a concentration-dependent manner, whereas it showed moderate inhibitory effect on the basolateral peptide transporter. Zinc also inhibited the uptake of oral beta-lactam antibiotics such as ceftibuten and cephradine by PEPT1. Kinetic analysis showed that zinc treatment increased Km values without affecting Vmax values of the [14C]Gly-Sar uptake. The inhibition of [14C]Gly-Sar uptake induced by zinc was observed in the presence of an H+ gradient but not in the absence of an H+ gradient. CONCLUSIONS: These results indicate that zinc is a competitive inhibitor of PEPT1. Zinc inhibited the PEPT1 function, possibly by interacting with histidine residues of PEPT1 that are part of an H+-binding site. These findings would provide important information for clinical, physiologic, and biochemical aspects of peptide transporters.

Animals↗
PubMed2002

Diurnal rhythm of H+-peptide cotransporter in rat small intestine.

In mammals, most physiological, biochemical, and behavioral processes show a circadian rhythm. In the present study, we examined the diurnal rhythm of the H+-peptide cotransporter (PEPT1), which transports small peptides and peptide-like drugs in the small intestine and kidney, using rats maintained in a 12-h photoperiod with free access to chow. The transport of [14C]glycylsarcosine (Gly-Sar), a typical substrate for PEPT1 by in situ intestinal loop and everted intestine, was greater in the dark phase than the light phase. PEPT1 protein and mRNA levels varied significantly, with a maximum at 2000 and minimum at 800. Similar functional and expressional diurnal variations were observed in the intestinal Na+-glucose cotransporter (SGLT1). In contrast, renal PEPT1 and SGLT1 showed little diurnal rhythmicity in protein and mRNA expression. These findings indicate that the intestinal PEPT1 undergoes diurnal regulation in its activity and expression, and this could affect the intestinal absorption of dietary protein.

Absorption↗
PubMedDate not supplied

A quantitative analysis and chemical approach for the reduction of nonspecific binding proteins on affinity resins.

Tubulin and actin often bind nonspecifically to affinity chromatography resins, complicating research toward identifying the cellular targets of small molecules. Reduction of nonspecific binding proteins is important for the success of such biochemical approaches. To develop strategies to circumvent this problem, we quantitatively investigated the binding of tubulin and actin to a series of affinity resins bearing 15 variant ligands on 3 commercially available polymer supports. Nonspecific protein binding was proportional to the hydrophobicity of the affinity resins and could be quantitatively correlated to the CLOGP values of the ligands, which are a measure of compound hydrophobicity. When compounds had CLOGP values greater than 1.5, (amount of tubulin) = 0.73 x CLOGP - 1.1 (n = 7, r = 0.97), and (amount of actin) = 0.42 x CLOGP - 0.79 (n = 7, r = 0.99). On the basis of these studies, we designed a novel hydrophilic poly(ethylene glycol) (PEG) spacer (26) for the conjugation of ligands to chromatography resins. As predicted by our binding algorithm, introduction of this spacer reduced the amount of nonspecific protein binding in proportion to the number of ethylene glycol units.

Actins↗