Biomedical subjects

Y Obata

Publications and source records attributed to Y Obata.

At least 19 recordsLinked to original sources

PubMed2006

Paternal dual barrier by Ifg2-H19 and Dlk1-Gtl2 to parthenogenesis in mice.

The functional difference between the maternal and paternal genome, which is characterized by epigenetic modifications during gametogenesis, that is genomic imprinting, prevents mammalian embryos from parthenogenesis. Genomic imprinting leads to nonequivalent expression of imprinted genes from the maternal and paternal alleles. However, our research showed that alteration of maternal imprinting by oocyte reconstruction using nongrowing oocytes together with deletion of the H19 gene, provides appropriate expression of maternally imprinted genes. Here we discuss that further alteration of paternally imprinted gene expressions at chromosomes 7 and 12 allows the ng/fg parthenogenetic embryos to develop to term, suggesting that the paternal contribution is obligatory for the descendant.

Animals↗

PubMed2005

Reactive oxygen species production by mitochondria in endothelial cells exposed to reoxygenation after hypoxia and glucose depletion is mediated by ceramide.

In endothelium, reoxygenation after hypoxia (H/R) has been shown to induce production of reactive oxygen species (ROS) by complex III of the mitochondrial respiratory chain. The purpose of the present study was to test the involvement of ceramide in this phenomenon. Human umbilical vein endothelial cells underwent 2 h of hypoxia (PO2, approximately 20 mmHg) without glucose and 1 h of reoxygenation (PO2, approximately 120 mmHg) with glucose. ROS production was measured by the fluorescent marker 2',7'-dichlorodihydrofluorescein diacetate, and cell death by propidium iodide. We showed that 1) after 1 h of reoxygenation, fluorescence had risen and that ROS production was inhibited by desipramine, an inhibitor of sphingomyelinase, an enzyme responsible for ceramide production (126 +/- 7% vs. 48 +/- 12%, P < 0.05); 2) administration of ceramide (N-acetylsphingosine) per se (i.e., in the absence of H/R) induced ROS production (65 +/- 3%), which was inhibited by complex III inhibitor: antimycin A (24 +/- 3%, P < 0.0001), or stigmatellin (31 +/- 2%, P < 0.0001); 3) hypoxia/reoxygenation-induced ROS production was not affected by either ceramide-activated protein kinase inhibitor dimethyl aminopurine or mitochondrial permeability transition inhibitor cyclosporin A but was significantly inhibited by the antiapoptotic protein Bcl-2 (82 +/- 8%, P < 0.05); 4) ceramide-induced ROS production was also inhibited by Bcl-2 (41 +/- 4%, P < 0.0001). These results demonstrate that in endothelial cells submitted to hypoxia and glucose depletion followed by reoxygenation with glucose, the pathway implicated in mitochondrial complex III ROS production is ceramide dependent and is decreased by the antiapoptotic protein Bcl-2.

Cell Hypoxia↗

PubMed2004

Multivariate spline interpolation as a novel method to optimize pharmaceutical formulations.

One of the difficulties in the quantitative approach for formulation design is the difficulty in understanding the actual relationship between causal factors and individual pharmaceutical responses. In this regard, several techniques were applied to determine the relationship between causal factors and the pharmaceutical responses. The generation of response surfaces using multivariate spline interpolation (MSI) has provided rapid and detailed information. Nevertheless, no application of MSI in the pharmaceutical field has been reported to date, even though it promises potential applications. To overcome the shortcomings of the classical response surface method, we newly developed a multi-objective simultaneous optimization method, in which MSI had been incorporated. The method was applied to the optimization problem of a transdermal hydrogel formulation for ketoprofen containing several chemical enhancers. Results suggested a superior function of the MSI approach.

Administration, Topical↗

PubMed2002

Vasopressin and epinephrine are equally effective for CPR in a rat asphyxia model.

Epinephrine has been administered as a drug essential for cardiopulmonary resuscitation (CPR). Recently, vasopressin has been reported to be more effective than epinephrine for CPR in a ventricular fibrillation (VF) model. As a different myocardial pathology is speculated to exist between the VF model and the asphyxia model, we investigated whether vasopressin is also effective in a rat asphyxia model. Twenty-one Sprague-Dawley male rats were divided into three groups: vasopressin 0.8 U/kg (Vaso-Gr), epinephrine 0.05 mg/kg (Epi-Gr), and saline same volume as the other two drugs (Sal-Gr). Five minutes after suffocation induced by obstruction of the tracheal tube, CPR was performed using each drug. Although only one animal survived (17%) in the Sal-Gr, 6/7 (85%) survived in both Vaso-Gr and Epi-Gr (P<0.01). Vasopressin is as effective as epinephrine for CPR in asphyxia-induced rats.

Animals↗

PubMed2002

Vasopressin with delayed combination of nitroglycerin increases survival rate in asphyxia rat model.

Recently, vasopressin has been reported as a more effective drug than epinephrine (adrenaline) for cardiopulmonary resuscitation (CPR). However, vasopressin decreases myocardial blood flow (MBF) because of its strong vasoconstriction, to maintain better coronary perfusion pressure (CPP) compared with epinephrine. Nitroglycerin is well known to be able to maintain MBF and increase survival rate. In a VF model, vasopressin combined with nitroglycerin maintained CPP; however, low survival rates were observed compared with vasopressin alone. We investigated the effectiveness of the delayed use of nitroglycerin combined with vasopressin in a severe asphyxia model. Fourteen Sprague-Dawley male rats were divided into two groups: vasopressin 0.8 U/kg alone (V-Gr.), and nitroglycerin 0.3 microg/kg 45 s after the administration of 0.8 U/kg vasopressin (VN-Gr.). Six min after asphyxia induced by obstructing the tracheal tube, CPR was performed in two ways. Three animals resuscitated in the V-Gr. (42%) and six/seven (84%) in the VN-Gr. (P<0.05). In the 6 min of asphyxia rat model, vasopressin combined with delayed nitroglycerin is more effective than vasopressin alone.

Animals↗

PubMed2002

Efficient cloning and expression of HLA class I cDNA in human B-lymphoblastoid cell lines.

Analysis of HLA restriction specificity is one of the important steps in characterizing T cell clones. This usually requires either a panel of HLA-typed cells or HLA cDNA transfectants. Although preparation of HLA cDNA transfectants is laborious, utilization of transfectants is advantageous when a suitable panel is not available due to linkage disequilibrium or rarity of the HLA allele of interest. In this report, we describe an efficient and rapid HLA cloning and expression system. Three sets of PCR primers specific for HLA-A, B and C loci were designed by extensively sequencing 5'- and 3'- untranslated regions of HLA class I genes. The PCR-amplified products were introduced into modified Phoenix retrovirus vectors containing a puromycin resistant gene under the control of a LTR promotor. Gibbon ape leukemia virus (GALV)-pseudotyped retrovirus was produced and infected into B-lymphoid cell lines. Following expansion in selection media, more than 80% of cells expressed transduced HLA at a comparable level to that normally expressed. These results indicate that locus-specific PCR cloning and utilization of GALV-pseudotyped retroviral vector can be an effective and relatively efficient tool for constructing a panel of different HLA transfectants.

Antibodies, Monoclonal↗

PubMed2001

The binding of thymus leukemia (TL) antigen tetramers to normal intestinal intraepithelial lymphocytes and thymocytes.

Thymus leukemia (TL) Ags belong to the family of nonclassical MHC class I Ags and can be recognized by both TCRalphabeta and TCRgammadelta CTL with TL, but not H-2 restriction. We previously reported that the CTL epitope is TAP independent, but the antigenic molecule(s) presented by TL has yet to be determined. In the present study, TL tetramers were prepared with T3(b)-TL and murine beta(2)-microglobulin, not including antigenic peptides, and binding specificity was studied. CTL clones against TL Ags were stained with the T3(b)-TL tetramer, and the binding shown to be CD3 and CD8 dependent. Normal lymphocytes from various origins were also studied. Surprisingly, most CD8(+) intraepithelial lymphocytes derived from the small intestines (iIEL), as well as CD8(+) and CD4(+)CD8(+) thymocytes, were stained, while only very minor populations of CD8(+) cells derived from other peripheral lymphoid tissues, such as spleen and lymph nodes, were positive. The binding of T3(b)-TL tetramers to CD8(+) iIEL and thymocytes was CD8 dependent, but CD3 independent, in contrast to that to TL-restricted CTL. These results altogether showed that TL-restricted CTL can be monitored by CD3-dependent binding of T3(b)-TL tetramers. In addition, CD3-independent T3(b)-TL tetramer binding to iIEL and thymocytes may imply that TL expressed on intestinal epithelium and cortical thymocytes has a physiological function interacting with these tetramer(+)CD8(+) T lymphocytes.

Animals↗

PubMed2001

Two consecutive zinc fingers in Sp1 and in MAZ are essential for interactions with cis-elements.

The zinc finger proteins Sp1 and Myc-associated zinc finger protein (MAZ) are transcription factors that control the expression of various genes. Regulation of transcription by these factors is based on interactions between GC-rich DNA-binding sites (GGGCGG for Sp1 and GGGAGGG for MAZ) and the carboxyl-terminal zinc finger motifs of the two proteins. Sp1 and MAZ have three and six zinc fingers, respectively, and the details of their interactions with cis-elements remain to be clarified. We demonstrate here that Sp1 and MAZ interact with the same GC-rich DNA-binding sites, apparently sharing DNA-binding sites with each other. We found that the DNA binding activities of Sp1 and MAZ depended mainly on consecutive zinc fingers, namely the second and third zinc fingers in Sp1 and the third and fourth zinc fingers in MAZ. Furthermore, the interactions of the zinc finger proteins with the same cis-elements appear to play a critical role in the regulation of gene expression. It seems plausible that two consecutive zinc finger motifs in a zinc finger protein might be essential for interaction of the protein with DNA.

3T3 Cells↗

PubMed2001

Disruption of imprinted expression of U2afbp-rs/U2af1-rs1 gene in mouse parthenogenetic fetuses.

The present study shows that the U2afbp-rs gene, a paternally expressed imprinted gene, is activated and expressed in a biallelic manner from maternal alleles in parthenogenetic mouse fetuses on day 9.5 of gestation. The mean expression was detected to be 88% (31-134%) of that in control biparental fetuses, using real-time quantitative reverse transcription and polymerase chain reaction. This disrupted expression of the gene was associated with changes in the chromatin structure but not with the methylation pattern in the regulation region. The present results show that parental specific expression of imprinted genes is not always maintained in uniparental embryos. This suggests that both parental genomes are necessary to establish parental specific expression of the U2afbp-rs gene.

Animals↗

PubMed2001

Evaluation and structure-activity relationship of synthesized cyclohexanol derivatives on percutaneous absorption of ketoprofen using artificial neural network.

The effect of 35 newly synthesized O-ethylmenthol (MET) derivatives on percutaneous absorption of ketoprofen was investigated in rats. In order to understand the relationship between the structure of compounds and promoting activity (structure-activity relationship), an artificial neural network (ANN) was employed. In the in vivo percutaneous absorption study, male Wistar rats, weighing 160-180 g, were used. The apparent penetration rate (Rp) was estimated based on a pharmacokinetic model with a constant rate of penetration through the skin after a lag time. As an index of the promoting activity of each compound, an enhancement factor (Ef), defined as follows, was used: Ef=Rp(with enhancer)/Rp(without enhancer). An irritation evoked on rat skin was microscopically judged at the end of the in vivo percutaneous absorption experiment and evaluated as a total irritation score (TIS). Ef and TIS were selected as output variables to determine the ANN structure. Calculated logP, molecular weight, steric energy (SE), van der Waals area, van der Waals volume, dipole moment, highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) were used as factors to determine the structural nature of cyclohexanol derivatives. Among these parameters, logP, SE and LUMO significantly affected the prediction of Ef and TIS. The predicted values of Ef and TIS coincided well with in vivo percutaneous absorption experimental values. However, results observed with a linear regression method were poor compared with the ANN approach. The contribution index of logP was approximately 50% in the prediction of Ef, suggesting that lipophilicity among physicochemical properties contributes most of the promoting activity of these compounds.

Animals↗

PubMed2001

Simultaneous optimization based on artificial neural networks in ketoprofen hydrogel formula containing O-ethyl-3-butylcyclohexanol as percutaneous absorption enhancer.

The influence of the amounts of additives including 1-O-ethyl-3-n-butylcyclohexanol (OEBC), diisopropyl adipate (DIA), and isopropanol (IPA) on the penetration rate (R(p)) of ketoprofen from hydrogels through rat skin in vivo was investigated. Skin irritation evoked by the application of hydrogels was evaluated based on a microscopic observation of skin cross-sections. Both optimization techniques incorporating an artificial neural network (ANN) and a second-order polynomial regression analysis were applied to the optimization of ketoprofen hydrogel formulations. Findings indicated that the R(p) and total irritation score (TIS) of the skin were predicted quantitatively as a function of quantities of OEBC, DIA, and IPA, employing ANN. In contrast, the prediction ability of the polynomial regression equation was somewhat poorer compared with that of ANN. The observed results of R(p) and TIS in the optimal formulation coincided well with the predictions in the simultaneous optimization technique incorporating ANN.

Animals↗

PubMed2001

Localization of gelatinolytic activity can be detected in breast cancer tissues by film in situ zymography.

BACKGROUND: Extracellular matrix-degrading proteinases secreted by malignant tumor cells have been thought to play an essential role in the processes of invasion and metastasis. However, existence and localization of gelatinase activity in breast cancer tissues have not been clarified. METHODS: We developed a novel film for highly reproducible detection and the localization of gelatinolytic activity. This film has a gelatin layer with a constant thickness 7 microm, and adequate crosslinking to control the speed of degradation by proteases. Cryosections of several breast cancer tissues were put on this gelatin film and incubated for 16 hrs at 37 degrees C. After staining with ponceau 3R dye, the digested area was evaluated under light microscopy. RESULTS: Digestion of gelatin was detected in more than 90%of breast cancer specimens, although it varied in degree and area for each case. In most cases, the gelatinolytic activity was located within cancer nests, and was not detected in stromal cells surrounding cancer cells. The gelatinolytic activity was inhibited by 1,10-phenanthroline, an inhibitor of matrix metalloproteinases (MMPs). CONCLUSIONS: In this study, the localization of net MMP activity was confirmed in breast cancer nest using film in situ zymography. Detailed analysis on the relationship between the strength or distribution of MMP activity and malignancy are anticipated in the future.

Azo Compounds↗

PubMed2001

Limited association between a catechol-O-methyltransferase (COMT) polymorphism and breast cancer risk in Japan.

BACKGROUND: Catechol-O-methyltransferase (COMT) inactivates the estradiol metabolites, 2-hydroxy estradiol and 4-hydroxy estradiol. To date, three studies in Caucasians and one study in Chinese have been conducted to determine the association with breast cancer risk of a functional polymorphism (G-to-A, Val158Met) of this enzyme, but the results were inconsistent. In order to examine the impact of this polymorphism on breast cancer risk in Japan, a case-control study was conducted, at Aichi Cancer Center Hospital. METHODS: The cases were 150 patients with histologically confirmed breast cancer who had been diagnosed within 4 years before enrollment at this hospital. The controls were 165 non-cancer patients, mainly from the gastroenterology and breast surgery clinics at the hospital. COMT-H (Val) is the wild-type allele, with high enzyme activity, while the COMT-L (Met) allele has low activity. Genotyping was conducted by a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method. RESULTS: The allele frequency of COMT-L was 36.0% for cases and 33.0% for controls. Age-adjusted odds ratios relative to the COMT-HH genotype were 1.46 (95% confidence interval [CI], 0.90-2.36) for COMT-HL, and 0.99 (95% CI, 0.49-2.02) for the COMT-LL genotype. Significant odds ratios were not observed for any subgroup stratified by menopausal status, age at menarche, age at birth of first child, body mass index, and breast cancer history of mother and/or sister(s). CONCLUSION: The present study suggested that any association of the COMT polymorphism with breast cancer risk is limited in Japanese.

Adult↗

PubMed2001

Identification of a new imprinted gene, Rian, on mouse chromosome 12 by fluorescent differential display screening.

Systematic screening of differentially expressed genes among androgenetic, parthenogenetic, and normal embryos by means of fluorescent differential display revealed five imprinted genes. One of them, named Rian, was expressed exclusively from the maternal allele and was closely linked to an imprinted gene, Meg3(Gtl2), mapped to the distal end of chromosome 12. The Rian transcript did not have any apparent open reading frame, and its transcript was exclusively localized to the nucleus.

Animals↗

PubMed2001

Promoting mechanism of menthol derivative, 1-O-ethyl-3-buthylcyclohexanol, on the percutaneous absorption of ketoprofen.

Menthol derivatives were synthesized and evaluated for their promoting activity on the percutaneous absorption of ketoprofen and skin irritation in vivo, choosing O-ethylmenthol (MET) as the mother compound. The compound having a C-3 positionned n-butyl group (1-O-ethyl-3-n-buthylcyclohexanol, OEBC) indicated the most promoting activity and caused relatively little skin irritation. In order to understand enhancement mechanism of OEBC an in vitro permeation study of ketoprofen was performed. The time course of the cumulative amounts of drug permeated through the rat skin exhibited a linear relation after an initial lag time. This was analyzed in membrane diffusion model and the diffusion and partition parameters of ketoprofen were estimated. Both parameters were remarkably enhanced when a hydrogel containing a small quantity of OEBC (0.5%) was applied. Furthermore, to clarify the site of action of OEBC, we also investigated in vitro permeation study of ketoprofen employing different skins of state, reversed skin and stratum corneum stripped skin. When OEBC was added to the hydrogels which were applied to the reversed and stripped skins, almost no changes of the flux were observed compared with the control (without OEBC). These results suggested that the site of action of OEBC was stratum corneum. Morphological changes of the stratum corneum surface were microscopically observed with 0-2% OEBC. The spaces between the stratum corneum cells treated with 0.5-2% OEBC became extended and the shape of each cell became clear. This may suggest that the site of action of OEBC was the intercellular of stratum corneum. Furthermore, an electron spin resonance study was performed to investigate the effect of OEBC on the intercellular lipid bilayer fluidity of the stratum corneum and the rotational correlation times were calculated. 2,2,6,6-Tetramethylpiperidine-1-oxyl (TEMPO) and 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL) were used as the spin label. In use of OEBC, the fluidity of TEMPO labeled the stratum corneum lipid increased as the addition of OEBC. The results suggested that OEBC promote the penetration of drugs by enhancing fluidity of the local lipid bilayers around TEMPO.

Algorithms↗

PubMed2000

Formula optimization of theophylline controlled-release tablet based on artificial neural networks.

Formulation of the controlled-release tablet containing theophylline was optimized based on the simultaneous optimization technique in which an artificial neural network (ANN) was incorporated. As model formulations, 16 kinds of theophylline tablets were prepared. The amounts of Controse, the mixture of hydroxypropylmethyl cellulose with lactose, cornstarch and compression pressure were selected as causal factors. The release profiles of theophylline were characterized as the sum of the fast and slow release fractions. The initial weight, the rate constant in the fast release fraction and the rate constant in the slow release fraction were estimated as release parameters. A set of release parameters and causal factors were used as tutorial data for ANN and analyzed using a computer. Based on the plasma concentration profiles of theophylline predicted by the pharmacokinetic analysis in humans, a desirable set of release parameters was provided. The simultaneous optimization was performed by minimizing the generalized distance between the predicted values of each response and the desirable one that was optimized individually. The optimization technique incorporating ANN showed a fairly good agreement between the observed values of release parameters and the predicted results.

Chemistry, Pharmaceutical↗

PubMed2000

p300/CBP-dependent and -independent transcriptional interference between NF-kappaB RelA and p53.

p53 and NF-kappaB RelA are activated by various genotoxic agents and mutually suppress each other's ability to activate transcription, most likely through competition for transcriptional coactivators such as CBP or p300. However, we found that the inhibition by RelA of p53 transcriptional activity is not completely restored by CBP/p300 overexpression and that a p53 mutant can not suppress RelA activity despite of its ability to bind CBP/p300. In the present study, we further present evidence that these two transcriptional factors directly interact both in vivo and in vitro. These results therefore indicate that the cross transcriptional interference between p53 and RelA is partly caused by the direct interaction between these two transcription factors which is mediated by their dimerization/tetramerization domains and results in inhibition of each other's transcriptional activity. Finally, cells derived from RelA knockout mice showed enhanced p53 transcriptional activity, suggesting that this cross transcriptional interference is physiologically important in cellular response to genotoxic stress.

Animals↗

PubMed2000

Effect of synthesized cyclohexanol derivatives using L-menthol as a lead compound on the percutaneous absorption of ketoprofen.

L-Menthol was selected as a lead compound to synthesize new candidates for percutaneous absorption enhancers. In a previous study, O-ethylmenthol (MET) was the most effective compound and caused relatively little skin irritation. To develop more effective compounds, mono- or disubstitute groups of cyclohexane with an O-ethyl group were synthesized. Some 35 compounds were synthesized and evaluated for their promoting activity and effect on skin. An in vivo percutaneous absorption study was performed using rats with hydrogel containing ketoprofen and each of the synthesized compounds. The plasma concentration of ketoprofen was determined after the application of hydrogel to the abdominal area of rats. The apparent penetration rate (R(p)) was estimated based on the pharmacokinetic model with a constant rate of penetration through the skin after the lag time. The 2-compartment model was applied to the data obtained from the iv administration. As an index to evaluate the promoting activity of each enhancer, an enhancement factor (E(f)) was defined as follows: E(f) = R(p) (with enhancer)/R(p) (without enhancer). Irritation to skin was pathologically evaluated. The treated area of rat abdominal skin was excised after the in vivo experiment using total irritation score (TIS). The compound having a C-3 positioned iso-butyl group on the chemical structure was the most effective and caused relatively little irritation among mono-substituted compounds. In the case of di-substituted compounds, all had the same effect as or a stronger effect than MET. Furthermore, the promoting activity almost corresponded to irritation. To estimate log P, one of the physicochemical properties of molecules, a computer program 'CAChe' was employed. The log P was calculated using the atom typing scheme. Multiple regression analysis revealed that the relations between E(f) or TIS and log P were parabolic. It was suggested that the optimum logP value reflects the promoting activity to enhance percutaneous absorption of ketoprofen.

Animals↗