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Y Sugiyama

Publications and source records attributed to Y Sugiyama.

At least 19 recordsLinked to original sources
PubMed2006

Effect of co-culturing with embryonic fibroblasts on IVM, IVF and IVC of canine oocytes.

We studied the effects of mouse embryonic fibroblasts (MEF) and canine embryonic fibroblasts (CEF) on IVM, IVF and IVC of canine oocytes. Cumulus-oocyte complexes were harvested from ovaries by slicing, and in vitro maturation was evaluated in three different conditions: culture media only (control), co-culture with MEF, or co-culture with CEF. The oocytes were cultured for 48 or 72 h. Only oocytes larger than 100 microm in diameter with a homogeneous dark cytoplasm and two or more layers of cumulus cells were used. The culture medium was TCM 199+10% fetal bovine serum (FBS) with 100 IU/mL penicillin and 100 microg/mL streptomycin. After 48 h of IVM, the oocytes were fertilized in vitro with fresh canine spermatozoa that had been selected by a swim-up method, and the oocytes and spermatozoa were co-cultured in modified Krebs-Ringer bicarbonate solution (TYH) for up to 20 h in 5% CO2 in air at 38.5 degrees C. After insemination, oocytes were transferred to three different conditions (the same as for IVM) and were cultured. After 48 or 72 h of maturation in vitro, the maturation rate of MII oocytes cultured in co-culture of MEF and CEF was higher than for oocytes cultured in control (P<0.05). Although the rate that reached the MII stage was not different in the 48 and 72 h cultures, the percentage of degenerated oocytes was greater at 72 h in all three treatment groups. The proportion of monospermic and polyspermic oocytes was not different among the three treatment groups. Cleavage rates were higher in the MEF and CEF treatment groups than in the control group (P<0.05). Co-culture with CEF developed the embryo up to the 16-cell stage, and with MEF up to morula stage. In conclusion, co-culture of embryonic fibroblast cells enhanced nuclear and cytoplasmic maturation of canine oocytes.

Animals↗
PubMed2006

Determination of bovine serum low-density lipoprotein cholesterol using the N-geneous method.

The N-geneous method is a recently developed method for determination of low-density lipoprotein cholesterol (LDL-C) in human serum. In the present study, we attempted to adapt this method to bovine serum. The values of LDL-C obtained using the N-geneous method were highly correlated with those from the method using ultracentrifugation and heparin sepharose affinity chromatography (r = 0.934, p < 0.001). The reproducibility of this method was acceptable (intra-assay CV 4.2%, inter-assay CV 7.6%) for clinical use. Using the N-geneous method, serum LDL-C was evaluated in cows around parturition, and in cows with fatty liver induced by fasting. The concentration of LDL-C decreased significantly in cows close to parturition. A reduced concentration of LDL-C was also observed in cows with fatty liver. In both cases, the changes of LDL-C were similar to those of apolipoprotein B (apoB)-100, and the values of LDL-C were highly correlated (r = 0.876, p < 0.001) with those of apoB-100. These results suggest that the concentration of LDL-C reflects the level of apoB-100. The N-geneous method is simple and rapid, and might to be a useful tool to elucidate the clinical significance of LDL-C in bovine serum.

Animals↗
PubMed2006

[Total anomalous pulmonary venous connection showing an unusual shape of the pulmonary vein successfully repaired in a neonate; report of a case].

A 10-day-old neonate with Darling type Ia of total anomalous pulmonary venous connection is presented. The left pulmonary vein entered the right thoracic cavity behind the right atrium and drained into the innominate vein together with the right lower, middle and upper pulmonary veins. The patient was treated with right-sided approach. The postoperative course was uneventful. To our knowledge, this case is the first successful correction of this subtype of anomaly in Japan.

Cardiac Surgical Procedures↗
PubMed2005

Bio-sorption of acidic gelatine hydro-gels implanted in the back tissues of Fisher's rats.

Recent advance in tissue engineering therapy requires new scaffold materials. Acidic gelatine powders (10 wt%) were, thus, dissolved in water, were or were not cross-linked, and freeze-dried. After sterilization, prepared small sponges were implanted in 7-week-old Fisher's rats' subcutaneous tissues for up to 2 weeks. Sponges absorbed body fluid and changed into hydro-gels in vivo. Non-cross-linked hydro-gels were absorbed within 3 days, while cross-linked hydro-gels were eliminated after 7 days' implantation. Histological observations revealed that the common captivation process was mild while granulocytes and macrophages were encountered. Because acidic gelatine sponges can accommodate various basic growth factors, it can be speculated that prepared sponges might be used as short-time hydro-gel scaffolds and growth-factor carriers.

Absorbable Implants↗
PubMed2004

The complete nucleotide sequence and multipartite organization of the tobacco mitochondrial genome: comparative analysis of mitochondrial genomes in higher plants.

Tobacco is a valuable model system for investigating the origin of mitochondrial DNA (mtDNA) in amphidiploid plants and studying the genetic interaction between mitochondria and chloroplasts in the various functions of the plant cell. As a first step, we have determined the complete mtDNA sequence of Nicotiana tabacum. The mtDNA of N. tabacum can be assumed to be a master circle (MC) of 430,597 bp. Sequence comparison of a large number of clones revealed that there are four classes of boundaries derived from homologous recombination, which leads to a multipartite organization with two MCs and six subgenomic circles. The mtDNA of N. tabacum contains 36 protein-coding genes, three ribosomal RNA genes and 21 tRNA genes. Among the first class, we identified the genes rps1 and psirps14, which had previously been thought to be absent in tobacco mtDNA on the basis of Southern analysis. Tobacco mtDNA was compared with those of Arabidopsis thaliana, Beta vulgaris, Oryza sativa and Brassica napus. Since repeated sequences show no homology to each other among the five angiosperms, it can be supposed that these were independently acquired by each species during the evolution of angiosperms. The gene order and the sequences of intergenic spacers in mtDNA also differ widely among the five angiosperms, indicating multiple reorganizations of genome structure during the evolution of higher plants. Among the conserved genes, the same potential conserved nonanucleotide-motif-type promoter could only be postulated for rrn18-rrn5 in four of the dicotyledonous plants, suggesting that a coding sequence does not necessarily move with the promoter upon reorganization of the mitochondrial genome.

Base Sequence↗
PubMed2004

Temporal expression profiles of organic anion transport proteins in placenta and fetal liver of the rat.

Physiological cholestasis linked to immature hepatobiliary transport systems for organic anions occurs in rat and human neonates. In utero, the placenta facilitates vectorial transfer of certain fetal-derived solutes to the maternal circulation for elimination. We compared the ontogenesis of organic anion transporters in the placenta and the fetal liver of the rat to assess their relative abundance throughout gestation and to determine whether the placenta compensates for the late maturation of transporters in the developing liver. The mRNA of members of the organic anion transporting polypeptide (Oatp) superfamily, the multidrug resistance protein (Mrp) family, one organic anion transporter (OAT), and the bile acid carriers Na(+)-taurocholate cotransporting polypeptide (Ntcp) and bile salt export pump (Bsep) was quantified by real-time PCR. The most abundant placental transporters were Oatp4a1, whose mRNA increased 10-fold during gestation, and Mrp1. Mrp1 immunolocalized predominantly to epithelial cells of the endoplacental yolk sac, suggesting an excretory role that sequesters fetal-derived solutes in the yolk sac cavity, and faintly to the basal syncytiotrophoblast surface. The mRNA levels of Oatp2b1, Mrp3, and Bsep in the placenta exceeded those in the fetal liver until day 20 of gestation, suggesting that the fetus relies on placental clearance of substrates when expression in the developing liver is low. Mrp3 immunolocalized to the epithelium of the endoplacental yolk sac and less abundantly in the labyrinth zone and endothelium of the maternal arteries. The placental expression of Oatp1a1, Oatp1a4, Oatp1a5, Oatp1b2, Oat, Ntcp, Mrp2, and Mrp6 was low.

Animals↗
PubMed2002

Expression and function of the ST2 gene in a murine model of allergic airway inflammation.

BACKGROUND: We have recently reported that soluble ST2 protein levels are elevated in the sera of patients with asthma, and correlate well with the severity of asthma exacerbation. However, the role, function, and kinetics of soluble ST2 expression in asthma remain unclear. OBJECTIVE: The objective of the present study was to clarify the function and kinetics of soluble murine (m) ST2 expression in a murine asthma model. METHODS: We analyzed the kinetics of gene and protein expression of mST2 in sera or lung tissue after allergen (ovalbumin; OVA) challenge in a murine model of allergic airway inflammation, the effects of mST2 protein on OVA-induced Th2 cytokine production in vitro from splenocytes of sensitized mice, and the effects of soluble mST2 on Th2-dependent allergic airway inflammation by in vivo gene transfer of mST2. RESULTS: Serum mST2 protein levels increased to the maximal level 3 h after the allergen challenge, before serum IL-5 levels peaked. The mRNA expression of mST2 in lung tissue was induced after the allergen challenge, while that in the spleen was constitutively detected. Furthermore, pre-treatment with mST2 protein significantly inhibited the production of IL-4 and IL-5, but not IFN-gamma, from OVA-stimulated splenocytes in vitro, and intravenous mST2 gene transfer resulted in a drastic reduction in the number of eosinophils and in the levels of IL-4 and IL-5 in bronchoalveolar lavage fluid, compared with those in response to transfer of non-coding plasmid vector or of lipid alone. CONCLUSION: These results suggest that increases in endogenous mST2 protein after allergen exposure may modulate Th2-mediated airway inflammation, and that in vivo gene transfer of mST2 can be applicable to use in a novel immunotherapy for allergic diseases.

Allergens↗
PubMed2001

Osmotic stress up-regulates aquaporin-3 gene expression in cultured human keratinocytes.

Of ten members of the aquaporin family (AQP), the mRNA expression and regulation of AQP1, AQP3, AQP4 and AQP9 in cultured human keratinocytes were examined by an RNase protection assay. AQP3 mRNA was expressed in growing and differentiating cells, while AQP9 mRNA was only detected in differentiating cells. The epidermis in skin-equivalent cultures expressed both AQP3 and AQP9 mRNA. However, neither AQP1 nor AQP4 mRNA was detectable in either monolayer or skin-equivalent cultures. Incubation of keratinocytes in sorbitol-added hypertonic medium increased AQP3 mRNA expression. This was confirmed using other solutes such as NaCl, mannitol, glucose and sucrose. The effect of sorbitol was reversible, dose-dependent and maximal at 24 h after addition. However, AQP1, AQP4 and AQP9 mRNA expression were unchanged under any of the hypertonic conditions examined. These findings indicated that osmotic stress up-regulates AQP3 gene expression in cultured keratinocytes.

Aquaporin 1↗
PubMed2001

Long-circulating poly(ethylene glycol)-poly(D,L-lactide) block copolymer micelles with modulated surface charge.

Reactive polymeric micelles consisting of an alpha-acetal-poly(ethylene glycol)-poly(D,L-lactide) block copolymer (acetal-PEG-PDLLA) with a narrow size distribution were prepared in this study to conjugate small peptidyl ligands, tyrosine (Tyr) and tyrosyl-glutamic acid (Tyr-Glu), through reductive amination after converting the alpha-acetal group to an aldehyde group, allowing modulation of the surface charge of the micelles from neutral (Tyr-) to anionic (Tyr-Glu-). Both of these micelles showed a significantly long circulating time in the blood compartment with 25% of injected dose still circulating even at 24 h. Further, an appreciably lowered uptake into the liver and spleen was demonstrated for the anionic Tyr-Glu-conjugated PEG-PDLLA micelle compared with a neutral Tyr-conjugated micelle, suggesting a substantial role of the slight anionic charge on the micelle surface in avoiding non-specific organ uptake. Stability of the micelle form in the blood compartment was directly observed for the Tyr-PEG-PDLLA micelle by a gel filtration assay of a plasma sample collected from the micelle-injected mice at 24 h. These results demonstrated that a surface-modulated PEG-PDLLA micelle with a suitable size and a narrowly distributed nature has promising potential as a long-circulating carrier system with desirable biocompatibility and biofunctionality.

Drug Carriers↗
PubMed2001

Elevated soluble ST2 protein levels in sera of patients with asthma with an acute exacerbation.

Previous studies have reported that ST2 is preferentially expressed on Th2 cells and plays a critical part in controlling airway inflammation in murine models of asthma. However, the clinical role of ST2 in patients with bronchial asthma remains unclear. In our study, we examined 56 patients with atopic asthma in a nonattack phase and 200 nonatopic normal volunteers for healthy control, and analyzed the relationship of their serum ST2 levels to asthma severity, pulmonary function, and laboratory data. Of the 56 patients with atopic asthma, 30 exhibited asthmatic exacerbation, and their serum ST2 levels were also analyzed. The serum ST2 levels were low, but a statistical difference was found between patients with nonattack asthma and the healthy control group (p < 0.05). We also found a differential rise of serum ST2 level that correlates well with the severity of asthma exacerbation. Furthermore, the serum ST2 levels during asthma exacerbation statistically correlated with the percentage of predicted peak expiratory flow (r = -0.634, p = 0.004) and Pa(CO(2)) (r = 0.516, p = 0.003). These results suggest that soluble human ST2 protein in sera may be related to Th2-mediated allergic inflammation inducing acute exacerbation in patients with atopic asthma.

Acute Disease↗
PubMed2001

A novel integrin-linked kinase-binding protein, affixin, is involved in the early stage of cell-substrate interaction.

Focal adhesions (FAs) are essential structures for cell adhesion, migration, and morphogenesis. Integrin-linked kinase (ILK), which is capable of interacting with the cytoplasmic domain of beta1 integrin, seems to be a key component of FAs, but its exact role in cell-substrate interaction remains to be clarified. Here, we identified a novel ILK-binding protein, affixin, that consists of two tandem calponin homology domains. In CHOcells, affixin and ILK colocalize at FAs and at the tip of the leading edge, whereas in skeletal muscle cells they colocalize at the sarcolemma where cells attach to the basal lamina, showing a striped pattern corresponding to cytoplasmic Z-band striation. When CHO cells are replated on fibronectin, affixin and ILK but not FA kinase and vinculin concentrate at the cell surface in blebs during the early stages of cell spreading, which will grow into membrane ruffles on lamellipodia. Overexpression of the COOH-terminal region of affixin, which is phosphorylated by ILK in vitro, blocks cell spreading at the initial stage, presumably by interfering with the formation of FAs and stress fibers. The coexpression of ILK enhances this effect. These results provide evidence suggesting that affixin is involved in integrin-ILK signaling required for the establishment of cell-substrate adhesion.

Actinin↗
PubMed2001

Biosynthetic studies on the cyclopentane ring formation of allosamizoline, an aminocyclitol component of the chitinase inhibitor allosamidin.

Allosamizoline (1) is an aminocyclitol component of allosamidin, a Streptomyces metabolite, and has a cyclopentane ring originated from D-glucosamine. Biosynthesis of the cyclopentane ring was studied by feeding experiments with a variety of deuterium-labeled glucosamine and glucose. In the feeding experiments with [3-(2)H]- and [4-(2)H]-D-glucosamine and [1-(2)H]-D-glucose, deuterium was incorporated into C-3, C-4, and C-1 of 1, respectively. On the other hand, feeding experiments with [5-(2)H]- and [6,6-(2)H(2)]-D-glucosamine showed that deuterium on C-5 and one of the two deuterium atoms on C-6 of glucosamine were lost during the cyclopentane ring formation of 1. In the feeding experiments with (6R)- and (6S)-[6-(2)H(1)]-D-glucose, the (6R)-deuterium of glucose was incorporated into the proS position on C-6 of 1, but the (6S)-deuterium of glucose was not incorporated into 1. These results suggested that an intermediate with a 6-aldehyde group is involved in the biosynthesis of the cyclopentane ring moiety of 1 and overall inversion of stereochemistry of the C-6 methylene group occurred by stereospecific oxidation and reduction on C-6 during the formation of 1. The 6-aldehyde intermediate may play a key role in the biosynthetic step(s) of cyclization to form the cyclopentane ring and/or deoxygenation at C-5.

Acetylglucosamine↗
PubMed2001

CD40-CD40 ligand (CD154) engagement is required but not sufficient for modulating MHC class I, ICAM-1 and Fas expression and proliferation of human non-small cell lung tumors.

To determine the possible functional significance of CD40 expression on human non-small cell lung carcinomas and to assess the potential of CD40 as a therapeutic target, 18 lung tumor cell lines were established from biopsy tissues and were monitored for phenotypic changes on the cell surface and alterations in tumor cell proliferation after the ligation of CD40 with a trimeric fusion protein complex of CD40 ligand (CD40Lt). CD40 cross-linking resulted in up to a 6-fold increase in the surface expression of major histocompatibility complex (MHC) class I, Fas and intracellular adhesion molecule (ICAM)-1 in a subset of tumors expressing the highest levels of CD40. Suppression of tumor proliferation was seen after the ligation of CD40 on CD40Lt-responsive cell lines. The suppression was dose dependent, reversible and resulted from a delay of the tumor cells entering S-phase. No change in the cell phenotype or in proliferation were observed in CD40-negative tumors or in tumors expressing moderate-to-low levels of CD40 after incubation with CD40Lt. CD40-negative tumors transfected with the CD40 gene expressed high levels of CD40 on their surface, but were also unresponsive to CD40Lt cross-linking of CD40. Our data establish that CD40 is required (but not sufficient) for transducing a signal that results in phenotypic changes in human lung tumors and suppression in their proliferation. We conclude that CD40 on non-small cell lung tumors may represent a potential therapeutic target, but only on a subset of the CD40+ tumors.

Apoptosis↗
PubMed2001

Characterization of bile acid transport mediated by multidrug resistance associated protein 2 and bile salt export pump.

Biliary excretion of certain bile acids is mediated by multidrug resistance associated protein 2 (Mrp2) and the bile salt export pump (Bsep). In the present study, the transport properties of several bile acids were characterized in canalicular membrane vesicles (CMVs) isolated from Sprague--Dawley (SD) rats and Eisai hyperbilirubinemic rats (EHBR) whose Mrp2 function is hereditarily defective and in membrane vesicles isolated from Sf9 cells infected with recombinant baculovirus containing cDNAs encoding Mrp2 and Bsep. ATP-dependent uptake of [(3)H]taurochenodeoxycholate sulfate (TCDC-S) (K(m)=8.8 microM) and [(3)H]taurolithocholate sulfate (TLC-S) (K(m)=1.5 microM) was observed in CMVs from SD rats, but not from EHBR. In addition, ATP-dependent uptake of [(3)H]TLC-S (K(m)=3.9 microM) and [(3)H]taurocholate (TC) (K(m)=7.5 microM) was also observed in Mrp2- and Bsep-expressing Sf9 membrane vesicles, respectively. TCDC-S and TLC-S inhibited the ATP-dependent TC uptake into CMVs from SD rats with IC(50) values of 4.6 microM and 1.2 microM, respectively. In contrast, the corresponding values for Sf9 cells expressing Bsep were 59 and 62 microM, respectively, which were similar to those determined in CMVs from EHBR (68 and 33 microM, respectively). By co-expressing Mrp2 with Bsep in Sf9 cells, IC(50) values for membrane vesicles from these cells shifted to values comparable with those in CMVs from SD rats (4.6 and 1.2 microM). Moreover, in membrane vesicles where both Mrp2 and Bsep are co-expressed, preincubation with the sulfated bile acids potentiated their inhibitory effect on Bsep-mediated TC transport. These results can be accounted for by assuming that the sulfated bile acids trans-inhibit the Bsep-mediated transport of TC.

ATP Binding Cassette Transporter, Subfamily B, Mem↗