Biomedical subjects

Tetsuya Hasegawa

Publications and source records attributed to Tetsuya Hasegawa.

14 recordsLinked to original sources

PubMed2006

A modular strategy for tailoring fluorescent biosensors from ribonucleopeptide complexes.

Fluorescent biosensors that facilitate reagentless sensitive detection of small molecules are crucial tools in the areas of therapeutics and diagnostics. However, construction of fluorescent biosensors with desired characteristics, that is, detection wavelengths and concentration ranges for ligand detection, from macromolecular receptors is not a straightforward task. An ATP-binding ribonucleopeptide (RNP) receptor was converted to a fluorescent ATP sensor without chemically modifying the nucleotide in the ATP-binding RNA. The RNA subunit of the ATP-binding RNP and a peptide modified with a pyrenyl group formed a stable fluorescent RNP complex that showed an increase in the fluorescence intensity upon binding to ATP. The strategy to convert the ATP-binding RNP receptor to a fluorescent ATP sensor was applied to generate fluorescent ATP-binding RNP libraries by using a pool of RNA subunits obtained from the in vitro selection of ATP-binding RNPs and a series of fluorophore-modified peptide subunits. Simple screening of the fluorescent RNP library based on the fluorescence emission intensity changes in the absence and presence of the ligand afforded fluorescent ATP or GTP sensors with emission wavelengths varying from 390 to 670 nm. Screening of the fluorescence emission intensity changes in the presence of increasing concentrations of ATP allowed titration analysis of the fluorescent RNP library, which provided ATP sensors responding at wide concentration ranges of ATP. The combinatorial strategy using the modular RNP receptor reported here enables tailoring of a fluorescent sensor for a specific ligand without knowledge of detailed structural information for the macromolecular receptor.

Adenosine Triphosphate↗

PubMed2006

Conversion of FAD to FMN and riboflavin in plasma: effects of measuring method.

The stability of flavin adenin dinucleotide (FAD) in plasma was studied under a low-intensity light and FAD was found to be converted to flavin mononucleotide (FMN) and riboflavin (RF) in both human and rat plasma. The hydrolysis rates of FAD in plasma at 4 degrees C were lower than those at 37 degrees C. In addition, the hydrolysis rates were markedly inhibited when EDTA, known as an anticoagulant, was added to plasma. These results indicated that plasma samples in pharmacokinetic studies should be pretreated with EDTA, extracted at the earliest convenience and lower temperature like 4 degrees C to keep a high stability. The pharmacokinetic study after intravenous administration of FAD at a dose of 500 nmol/kg as FAD in rats was performed with plasma samples after addition of EDTA under strict light and temperature control. A measurable amount of FAD in plasma together with rapid conversions of FAD to FMN and RF were observed in rat plasma. The AUC values (mean+/-S.D. of 4 rats) for FAD, FMN and RF were 707+/-378, 3643+/-958 and 30095+/-3544 nmol x min/l, respectively. Using excess EDTA under strict temperature and light control may be useful for assessment of vitamin B2 in the in vivo study.

Animals↗

PubMed2005

Cultured skin loaded with tetracycline HCl and chloramphenicol as dermal delivery system: mathematical evaluation of the cultured skin containing antibiotics.

Dermal patches consisting of cultured human skin with antibiotics, which have a protective effect on wound skin as well as a preventative effect on second infection of the skin, were prepared and mathematically analyzed as a new drug delivery system (DDS) that can be applied to serious skin defects such as severe burns. In the present study, a three-dimensional cultured human skin model (living skin equivalent-high, LSE-high) was used as a cultured skin membrane and tetracycline HCl (TC-HCl) and chloramphenicol (CP) were used as antibiotics. At first, antibiotics were entrapped in the LSE-high from the dermal side through culture medium in order to obtain a drug-loaded LSE-high. The antibiotic release from the drug-loaded LSE-high was then examined and the resulting release data were used to calculate the effective diffusion coefficient of the antibiotics (D(LSE)) and initial loading concentration of the antibiotics (C0) in the LSE-high. The release profile of TC-HCl was represented by general diffusion-limited kinetics, whereas an initial burst effect was found in the release profile of CP. Therefore, the burst effect was taken into account for analyzing the release profile of CP. Stripped skin excised from hairless rats was used as a wound model, and the antibiotic permeation through the skin from aqueous solution was examined and evaluated using differential equations for Fick's second law of diffusion to obtain the effective diffusion coefficient of the antibiotics in the wound skin (D(skin)). Furthermore, the antibiotic permeation profile through the excised stripped skin from the drug-loaded LSE-high was measured and theoretically evaluated by Fick's second law of diffusion with previously obtained parameters (C0, D(LSE), D(skin)) using a newly constructed two- or three-layered diffusion model. The calculated concentrations of TC-HCl and CP in the upper epidermis of the model wound skin were over their minimum inhibitory concentration (MIC) for several hours against various bacteria, suggesting that this dosage system is useful for the treatment of severe burns. In addition, the present analytical method and diffusion model, with the drug-loaded LSE-high and stripped rat skin, are useful tools for evaluating this new DDS.

Administration, Cutaneous↗

PubMed2005

Usefulness of coadministration of bucolome in warfarin therapy: pharmacokinetic and pharmacodynamic analysis using outpatient prescriptions.

Bucolome, a nonsteroidal anti-inflammatory drug, has often been coadministered to patients who take warfarin as an anticoagulant. This combination increases the anticoagulant effect, which is most likely due to the interaction of bucolome with the pharmacokinetics (PK) or pharmacodynamics (PD) of warfarin. More than 30 years ago the mechanism of this interaction was reported to be inhibition of warfarin protein binding by bucolome, and the inhibition of warfarin metabolism by bucolome was also recently reported. Here, we examined daily doses of warfarin and its anticoagulant effect (thrombo-test, TT) in outpatient prescriptions in five hospitals to elucidate the drug interaction and the usefulness of this drug combination. Among the warfarin prescriptions, 78 were for patients also taking bucolome and 99 were for patients not taking bucolome. The daily dose of warfarin in patients taking bucolome was significantly lower than those without bucolome (ca. 40%). TT in patients taking bucolome was significantly lower as compared to those not taking bucolome. Control of the anticoagulant effect was greater with coadministration of bucolome and warfarin than with warfarin alone. PK and PD analysis of our results suggests that the improved therapeutic effect resulting from coadministration of warfarin with bucolome was due to lower and less patient-to-patient variation of intrinsic hepatic clearance (CL(int)) of warfarin, since bucolome decreased the high CL(int) but did not have a great effect on the low CL(int). In conclusion, administration of bucolome in warfarin therapy is useful to control the anticoagulant effect of warfarin. Attention should also be paid to the enzymatic inhibition by bucolome on the PK of coadministered drugs.

Adult↗

PubMed2005

Ribonucleopeptides recognize the phosphotyrosine residue.

Tools for selective recognition and sensing of kinase-catalyzed phosphorylation of Tyr or Ser/Thr residues located on a protein surface are essential for understanding signal transduction cascades of living cell. A stable complex of RNA and peptide (ribonucleopeptide) provides a new approach to tailor a receptor for small molecules. In vitro selection of an RNA-derived pool of ribonucleopeptide afforded a ribonucleopeptide receptor specific for phosphotyrosine (pTyr). Moreover, the ribonucleopeptide receptors are conveniently converted to fluorescent sensors for pTyr.

Binding Sites↗

PubMed2005

Decrease in skin permeation and antibacterial effect of parabens by a polymeric additive, poly(2-methacryloyloxyethyl phosphorylcholine-co-butylmetacrylate).

The inhibitory effect of poly(2-methacryloyloxyethyl phosphorylcholine-co-butylmetacrylate) (PMB) on the in vitro skin permeation of p-hydroxybenzoic acid and its esters (parabens; methylparaben, ethylparaben, n-propylparaben and n-butylparaben) as model compounds was evaluated. Solubility of the parabens in distilled water was increased by addition of PMB, and the increasing ratio was dependent on the concentration of PMB. The increment of the ratio was more marked in lipophilic parabens than hydrophilic ones. Skin permeability of parabens from their aqueous suspensions was decreased by addition of PMB, and the decreasing ratio was dependent on the PMB concentration. Then, antibacterial effect of parabens of their aqueous solutions was determined with and without PMB by measuring minimum inhibitory concentration (MIC) against E. coli and S. aureus. As results, MIC and antibacterial effect were increased and decreased, respectively, by addition of PMB. The contribution of PMB was more marked in butylparaben than methylparaben. These decreases in skin permeation and antibacterial effect of parabens by PMB are probably due to a higher solubility in water and a lower partition to the skin and bacterial membranes of parabens by addition of PMB.

Animals↗

PubMed2004

Anomalous Hall effect governed by electron doping in a room-temperature transparent ferromagnetic semiconductor.

Ferromagnetic semiconductors are believed to be suitable for future spintronics, because both charge and spin degrees of freedom can be manipulated by external stimuli. One of the most important characteristics of ferromagnetic semiconductors is the anomalous Hall effect. This is because the ferromagnetically spin-polarized carrier can be probed and controlled electrically, leading to direct application for electronics. Control of the Curie temperature and magnetization direction by electronic field, and photo-induced ferromagnetism have been performed successfully using the anomalous Hall effect for group III-V ferromagnetic semiconductors. In these cases, the operation temperature was much below room temperature because of the limited Curie temperature of less than 160 K (ref. 6). Here, we report on the anomalous Hall effect governed by electron doping in a room-temperature transparent ferromagnetic semiconductor, rutile Ti(1-x)Co(x)O(2-delta) (of oxygen deficiency delta). This result manifests the intrinsic nature of ferromagnetism in this compound, and represents the possible realization of transparent semiconductor spintronics devices operable at room temperature.

Journal Article↗

PubMed2004

Ribonucleopeptides: functional RNA-peptide complexes.

Selection of functional RNAs from randomized pool of RNA molecules successfully affords RNA aptamers that specifically bind to small molecules, and that have catalytic activities. Recent structural analyses of the ribosomal RNA complex suggest that the RNA-protein complex would be a new structural candidate for the design of tailor-made receptors and enzymes. We have designed an ATP binding domain that consists of an RNA subunit and a peptide subunit by means of structure-based design approach and successive in vitro selection method. The RNA subunit is designed to consist of two functional domains; an ATP binding domain with 20 randomized nucleotides and an adjacent stem region that serves as a binding site for the RNA-binding peptide. The randomized nucleotide region was placed next to the HIV-1 Rev response element to enable the formation of "ribonucleopeptide" pools in the presence of the Rev peptide. In vitro selection of RNA oligonucleotides from the randomized pool afforded a ribonucleopeptide receptor specific for ATP. The ATP-binding ribonucleopeptide did not share the known consensus nucleotide sequence for ATP aptamers, and completely lost its ATP-binding ability in the absence of the Rev peptide. The ATP-binding activity of the ribonucleopeptide was increased by a substitution of the N-terminal amino acid of the Rev peptide. These results demonstrate that the peptide stabilizes the functional structure of RNA and suggest that amino acids outside the RNA binding region of the peptide participate in the ATP binding. Our approach would provide a new strategy for the design of tailor-made ribonucleopeptide receptors.

Adenosine Triphosphate↗

PubMed2004

Design of sensing ribonucleopeptides for small ligands.

Here we report a simple method to convert synthetic receptors to fluorescent sensors. An RNA-peptide complex (ribonucleopeptide) with a known three-dimensional structure is used as a framework of the receptor. Artificial ribonucleopeptide sensors were created with a combination of in vitro selection method and successive modification of the peptide with a fluorophore. A ribonucleopeptide complex of the fluorophore labeled peptide showed a remarkable fluorescence emission change upon binding cognate ligands.

Amines↗

PubMed2004

Utility of a three-dimensional cultured human skin model as a tool to evaluate the simultaneous diffusion and metabolism of ethyl nicotinate in skin.

The simultaneous diffusion and metabolism of ethyl nicotinate (EN) in a cultured human skin model, Living Skin Equivalent-high, was evaluated by the in vitro skin permeation and metabolism experiments, and esterase distribution was also determined. Theoretical calculations using Fick's 2nd Law of Diffusion with Michaelis-Menten kinetics were performed to obtain the permeation and metabolic parameters together with information on enzyme distribution. The obtained data was compared with the corresponding results in excised hairless rat skin. The partition coefficient of EN from the vehicle to the skin was of the same order of magnitude for the cultured human skin and hairless rat skin, but the diffusion and metabolic parameters were different. Esterase concentration in the epidermal membrane was greater than in the dermis of cultured skin, which was similar to hairless rat skin. Taking into account the similarities and differences between the membranes, the cultured human skin model can be utilized as a model membrane to rapidly predict simultaneous diffusion and metabolism of the prodrug through human skin.

Algorithms↗

PubMed2003

Effect of electric field on the enhanced skin permeation of drugs by electroporation.

Electroporation (voltage; 200 V) was applied using an exponentially decaying electric pulse generator on hairless rat skin using different shapes of electrodes, and the in vitro skin permeation of benzoate was measured as an index of the effectiveness of electroporation. Despite the same voltage of application, the skin permeation of benzoate was markedly different by the electrode shapes. Several experiments hereafter suggest that this reason was probably due to different 2-dimensional electric fields in the skin barrier, stratum corneum, and the voltage decreasing pattern applied. To confirm whether this phenomenon was specific for benzoate, diclofenac having a similar pKa was selected to carry out the same kind of examination. As a result, the skin permeability of diclofenac was influenced by 2-dimensional electric fields in the stratum corneum and the voltage decreasing pattern, similar to the benzoate permeation. Next, a rectangular pulse generator, where no decaying pattern was obtained for the application voltage, was used to measure the effect of electroporation on the skin permeation of diclofenac. As expected, the enhanced skin permeation of diclofenac was dependent only on the electric field when using the rectangular pulse generator. These results suggest that the electric field and time profile of the voltage (or AUC of voltage against time) at electroporation are very important factors to increase the efficacy of electroporation, and that the efficacy can be optimized by the shape of electrodes in addition to the application conditions of electroporation.

Animals↗

PubMed2003

Optimization of an ATP-binding ribonucleopeptide receptor.

We describe here a new strategy for the selection and evolution of functional RNA-peptide complexes. An ATP-binding ribonucleopeptide was obtained by means of a structure-based design of ribonucleopeptide domain and by in vitro selection of the RNA subunit of the ribonucleopeptide. Approaches to optimize the ligand-binding selectivity of ribonucleopeptide will be discussed.

Adenosine Triphosphate↗

PubMed2002

Targeting of salicylate to skin and muscle following topical injections in rats.

The process of systemic absorption and tissue targeting efficacy of salicylate (SA) following intracutaneous (i.c.), subcutaneous (s.c.) and intramuscular (i.m.) injections of its sodium salt in rats were evaluated by determining the drug concentration at the injection site and surrounding tissues. After i.c. and s.c. injections, SA was absorbed into the systemic circulation from the muscular vessels as well as the cutaneous or subcutaneous vessels beneath the injection site, and the AUC of the drug in the muscle was extremely high. Following i.m. injection, SA was rapidly absorbed into the systemic circulation mostly from the muscular vein. These results suggested that i.c. and s.c. injections have high degrees of targeting efficacy to the muscle, whereas i.m. injection is not appropriate for drug retention in muscle. In contrast, most of the topically applied drug was absorbed from the cutaneous vessels, and little drug migration to the muscle was observed. Thus, the skin pharmacokinetics of SA after i.c. injection was also markedly different from those after topical application on the skin. These results suggested that the i.c. and s.c. injections may be a good means to improve the targeting ability of drugs to the muscle as well as the skin.

Absorption↗

PubMed2002

Utility of MTT assay in three-dimensional cultured human skin model as an alternative for draize skin irritation test: approach using diffusion law of irritant in skin and toxicokinetics-toxicodynamics correlation.

PURPOSE: A cytotoxicity assay using a three-dimensional cultured human skin model, Living Skin Equivalent-high (LSE-high) was evaluated as an alternative to the Draize skin irritation tests using animals. A relation between the cytotoxicity and calculated concentration of an irritant in skin was also evaluated. METHODS: Colorimetric thiazoyl blue (MTT) conversion assay and a surfactant, cetylpyridinium chloride (CPC), were selected as a cytotoxicity assay and a model irritant. The fraction of dead cell number in the MTT assay or the Draize irritation score (in vitro and in vivo irritation data, respectively) was treated as a function of CPC concentration in the viable skin of LSE-high and guinea pig. Separately, in vitro permeations of CPC through the LSE-high or excised guinea pig skin were determined to calculate the average concentration of CPC in the viable skin using the Fickian diffusion theory. The obtained relations between the irritation scores and CPC concentration were evaluated by the Emax model (Hill equation). RESULTS: CPC concentration showing 50% irritation (IC50) was similar for the MTT assay (18.9%) and Draize test (12.3%), and a good relationship (r = 0.981) was observed between the fraction of dead cell number and the Draize score. In contrast, IC50, 1.32%, for the MTT assay in LSE-high was much lower than that using guinea pig skin. We then corrected the results for the MTT assay using a ratio of IC50 in guinea pig skin against LSE-high, resulting in a good relation between both MTT results in guinea pig skin and LSE-high. CONCLUSION: The present results suggest that the MTT assay using LSE-high may be utilized as an alternative for the Draize test in animals for evaluating skin irritation.

Animals↗