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Biomedical subjects

H Sawai

Publications and source records attributed to H Sawai.

At least 19 recordsLinked to original sources
PubMed2006

Integrin-linked kinase activity is associated with interleukin-1 alpha-induced progressive behavior of pancreatic cancer and poor patient survival.

Cancer cell adhesion and invasion into extracellular matrix are regulated by integrin-linked kinase (ILK) activity in a phosphatidylinositol 3-kinase (PI3-K)-dependent manner. In this study, we demonstrated that ILK and beta(1)-integrin play important roles in interleukin (IL)-1alpha-induced enhancement of adhesion and invasion of pancreatic cancer cells through p38 mitogen-activated protein kinase (MAPK) signaling pathway and activator protein-1 (AP-1) activation. Alteration of ILK kinase activity controlled IL-1alpha-induced p38 MAPK phosphorylation and its downstream AP-1 activation with subsequent regulation of pancreatic cancer cell adhesion and invasion. Overexpressed ILK enhances the IL-1alpha-induced p38 MAPK phosphorylation more strongly through glycogen synthase kinase 3 (GSK-3) activation, and subsequently induces AP-1 activation, which promotes aggressive capabilities of pancreatic cancer cells. In contrast, knockdown of ILK kinase activity inhibits the IL-1alpha-induced activation of MAPK/AP-1 pathway via inhibition of GSK-3 phosphorylation. In immunohistochemical analysis, statistically significant association between strong expression of ILK and poor prognosis of pancreatic cancer patients were observed, and strong expression of ILK in cancerous tissues can be a significant prognostic indicator of pancreatic cancer patients. Our results suggest that ILK is involved with aggressive capability in pancreatic cancer and that these regulations can be helpful to understand biological processes for a better translational treatment for pancreatic cancer patients.

Cell Adhesion↗
PubMed2004

Possible presence of O-linked carbohydrate in the human male reproductive tract CD52.

Male reproductive tract CD52 (mrtCD52) is an antigen recognized by a complement-dependent sperm-immobilizing monoclonal antibody (SI-Abs) derived in an infertile patient. The molecule has been shown to contain a unique N-linked carbohydrate that does not cross-react with other tissues. In this study, we have investigated whether O-linked carbohydrate as well as N-linked carbohydrate is present in mrtCD52 using specific lectins and anti-CD52 core peptide antiserum. The lectin PNA, which recognizes O-linked carbohydrate [Galbeta1-3GalNAc], reacted with mrtCD52 and showed a similar polymorphic reaction pattern to that of the anti-peptide antiserum in western blotting analysis on two-dimensional SDS-PAGE. The PNA-reactive spots disappeared after removal of O-linked carbohydrate, but not after removal of N-linked carbohydrate. These results suggest that O-linked carbohydrate is present in mrtCD52. The moiety may possibly contribute to a specific antigenic epitope of mrtCD52.

Antibodies, Monoclonal↗
PubMed2004

Immunohistochemical analysis of molecular biological factors in intraductal papillary-mucinous tumors and mucinous cystic tumors of the pancreas.

BACKGROUND: To investigate the malignancy and differentiation of intraductal papillary-mucinous tumors (IPMTs) and mucinous cystic tumors (MCTs) of the pancreas, clinicopathologic characteristics and immunohistochemical features were analyzed. METHODS: The clinicopathologic characteristics and immunohistochemical features of 24 patients with IPMT and 8 with MCT who underwent pancreatic resections at our hospital were examined. Immunohistochemical features analyzed included expression of p53 protein, proliferating cell nuclear antigen, integrins, interleukin-1 receptor type I, and hormone-associated receptors, and the factors correlated with malignancy were identified by multiple logistic regression. RESULTS: Among the IPMTs, there were 16 intraductal papillary adenomas, 5 intraductal papillary adenocarcinomas, and 3 moderate dysplasias. Among the MCTs, there were 6 mucinous cyst adenomas and 2 mucinous cyst adenocarcinomas. Multivariate analysis revealed that of the clinicopathologic characteristics, only the presence of mural nodules (odds ratio (OR) 7.12, P = 0.044) was independently correlated with the malignancy of IPMTs, and that of the immunohistochemical features, only alpha integrin subunit expression was independently correlated with malignancy of pancreatic mucinous tumors (OR 15.6, P = 0.036), especially IPMTs (OR 35.7, P = 0.012). CONCLUSION: These results indicate that alpha-containing integrin expression can be a significant marker of malignancy in pancreatic mucinous tumors.

Adenocarcinoma, Mucinous↗
PubMed2003

Epitope analysis for human sperm-immobilizing monoclonal antibodies, MAb H6-3C4, 1G12 and campath-1.

Human monoclonal antibody, MAb H6-3C4, possesses strong sperm immobilizing activity. MAb H6-3C4 has been suggested by several research groups to react with a carbohydrate moiety of male reproductive tract CD52 (mrtCD52). In the present study, we analysed the epitope on mrtCD52 for MAb H6-3C4 and found that it was polymorphic in Western blot analysis and disappeared after enzymatic removal of the N-linked carbohydrate moiety. Two other monoclonal antibodies (1G12, campath-1) with sperm-immobilizing activity recognized mrtCD52 in a polymorphic manner similar to MAb H6-3C4. Further analysis showed that 1G12 recognized a structure formed by the peptide and/or a glycosylphosphatidylinositol (GPI) anchor portion as does campath-1. Results of a lectin binding assay suggested the presence of O-linked carbohydrates on mrtCD52. Our results also indicated that the peptide portion of CD52 could serve as an epitope for sperm-immobilizing antibodies. It was concluded that the epitope of MAb H6-3C4 is similar to, but distinct from, those of 1G12 and campath-1, and that mrtCD52 contains different antigenic epitopes.

Antibodies, Monoclonal↗
PubMed2002

Effect of Bcl-2 overexpression on establishment of ipsilateral retinocollicular projection in mice.

During perinatal development in rodents, ipsilateral retinofugal projection spreading over the superior colliculus is eventually restricted to the rostromedial region. Since this restriction is accompanied by the apoptotic death of more than half of the retinal ganglion cells (RGCs), cell death is believed to play a major role in the restriction of transient ipsilateral projection from the retina to the superior colliculus. To determine the role of RGC death in the establishment of ipsilateral retinofugal projection, we examined the projection pattern in the superior colliculus and the dorsal lateral geniculate nucleus of transgenic mice overexpressing the human bcl-2 gene, which protects against cell death in the CNS. Retrograde labeling of RGCs showed that the number of ipsilaterally projecting RGCs in adult transgenic mice was approximately twice that in adult wild-type mice, indicating that the naturally occurring death of RGCs was prevented in these mutant mice. However, anterograde labeling of ipsilateral retinofugal pathways revealed that the innervation of retinogeniculate and retinocollicular projections was as restricted in transgenic mice as in wild-type mice. From these results we suggest that restriction of ipsilateral retinofugal projection during development is due to retraction or elimination of excessive terminals rather than to naturally occurring RGC death.

Animals↗
PubMed2001

Topological specificity in reinnervation of the superior colliculus by regenerated retinal ganglion cell axons in adult hamsters.

In normal rodents there is a precise topology of the retinocollicular projection, the nasotemporal and ventrodorsal axes of the retina being respectively projected onto the caudorostral and mediolateral axes of the contralateral superior colliculus (SC). We evaluated the distribution of regenerated retinal ganglion cell (RGC) axon terminals in the SC of adult hamsters in which an unbranched peripheral nerve graft was directed from the retina to the contralateral SC. Responses to visual stimulation of individual RGCs were recorded from terminal arbors of their regenerated axons in the reinnervated SC. Retinal positions of these RGCs were inferred from the locations of their visual receptive fields. At some sites in the reinnervated SC, axon terminal arbors converged from widely separated RGCs. Conversely, axon terminal arbors at widely separated sites in the SC could emanate from contiguous RGCs. To assess whether any tendency for order was superimposed on the apparent disorganization of the regenerated projection, we evaluated the relative positions of pairs of RGC terminals in the SC in relation to the relative retinal locations of the corresponding pairs of RGCs. Among the 983 pairs of RGCs able to be evaluated from nine animals studied 30-60 weeks after grafting, there was a statistically significant 3/2 tendency for the more nasally situated of two RGCs to project its terminal more caudally in the SC than that of the more temporally situated RGC. A similar tendency toward appropriate organization was not found with respect to the ventrodorsal axis of the retina and the mediolateral axis of the SC.

Animals↗
PubMed2001

Effect of the terminal amino group of a linker arm and its length at the C5 position of a pyrimidine nucleoside on the thermal stability of DNA duplexes.

2'-Deoxyuridine derivatives bearing a substituent at the C5-position, which has a different chain length and a different functional group (methyl or amino), were synthesized and incorporated into oligodeoxyribonucleotides. The effect of the substituent groups in the major groove on the stability of the duplexes was investigated by UV melting experiments. It was found that the stabilization of these duplexes by a terminal amino group depended on the length of a linker arm.

DNA↗
PubMed2001

Enzymatic synthesis of modified DNA by PCR.

We synthesized various 5'-triphosphates of C5-substituted 2'-deoxyuridine derivatives bearing methylene linker at C5-alpha position. We examined whether the C5-substituted 2'-deoxyuridine 5'-triphosphates (dUTP) can work as a substrate for the modified DNA synthesis by PCR. We found that only KOD dash DNA polymerase, a thermostable DNA polymerase from extremely thermophilic archaeum, accepted the modified substrates in place of TTP for PCR forming the corresponding modified DNAs. On the other hand, no other DNA polymerase could accept these TTP analogues.

Base Sequence↗
PubMed2001

A phycocyanin-deficient mutant of synechocystis PCC 6714 with a single-base substitution upstream of the cpc operon.

The structure and expression of the cpc operon encoding phycocyanin subunits and linker polypeptides in a phycocyanin-deficient mutant (PD-1) and the wild-type of Synechocystis PCC 6714 were analyzed. The results of sequence and Northern blot analyses of the wild type indicate that the cpc operon consists of cpcB, cpcA, cpcC1, cpcC2 and cpcD, in that order. The levels of the transcripts in PD-1 were one-tenth to one-sixth as high as those in the wild type. In the PD-1 genome, a single-base substitution of C for T has occurred at base 259 upstream of the translational initiation codon of cpcB (at three bases downstream of the putative -10 region). To evaluate the in vivo transcription activities of these promoters in a cyanobacterium, we constructed vectors for the transformation of Synechococcus PCC7942, pANY1 and pANY2, which contain the upstream region of cpcB of the wild type (pANY1) or PD-1 (pANY2) and the promoter-less luxAB fusion. The bioluminescence of the transformants with pANY2 was one-tenth to one-sixth as high as that with pANY1. The coincidence of the results of Northern analysis and the promoter assay shows that the phycocyanin deficiency of PD-1 is due to the single-base substitution in the upstream region of the cpc operon.

Base Sequence↗
PubMed2001

Alteration of integrins by interleukin-1alpha in human pancreatic cancer cells.

INTRODUCTION: Adhesion of tumor cells to extracellular matrix (ECM) proteins plays an important role in tumor invasion and metastasis. AIMS: To investigate the expression of integrins in human pancreatic cancer cell lines and its alteration by interleukin (IL)-1alpha to examine the mechanism of adhesion of metastatic human pancreatic cancer cells to ECM proteins. METHODOLOGY: The expression of integrin subunits and their alteration by IL-1alpha were examined by flow-cytometric analysis and cellular enzyme-linked immunosorbent assay in three metastatic human pancreatic cancer cell lines (AsPC-1, BxPC-3, and SW1990) and two nonmetastatic cancer cell lines (PaCa-2 and PANC-1). In addition, assays of cancer cell adhesion to ECM proteins were performed to investigate if increased integrin expression actually affected the adhesive interaction between cancer cells and the putative integrin ECM ligands. RESULTS: The alpha(6) subunit expressed in metastatic cancer cells was enhanced by IL-1alpha. Metastatic cancer cells also showed preferential adherence to laminin compared with nonmetastatic cancer cells, and this was enhanced by IL-1alpha. CONCLUSION: In pancreatic cancer, the enhancement of alpha(6)beta(1) integrin by IL-1alpha through IL-1 receptor type I, as well as the expression of alpha(6)beta(1) integrin, plays an important role in metastasis formation.

Antigens, CD↗
PubMed2000

Identification of ISC1 (YER019w) as inositol phosphosphingolipid phospholipase C in Saccharomyces cerevisiae.

Sphingolipids have emerged as novel bioactive mediators in eukaryotic cells including yeast. It has been proposed that sphingomyelin (SM) hydrolysis and the concomitant generation of ceramide are involved in various stress responses in mammalian cells. The yeast Saccharomyces cerevisiae has inositol phosphosphingolipids (IPS) instead of SM and glycolipids, and synthesis of IPS is indispensable to its growth. Although the genes responsible for the synthesis of IPS have been identified, the gene(s) for the degradation of IPS has not been reported. Here we show that ISC1 (YER019w), which has homology to bacterial neutral sphingomyelinase (SMase), encodes IPS phospholipase C (IPS-PLC). First, we observed that overexpression of ISC1 greatly increased neutral SMase activity, and this activity was dependent on the presence of phosphatidylserine. Cells deleted in ISC1 demonstrated negligible neutral SMase activity. Because yeast cells have IPS instead of SM, we investigated whether IPS are the physiologic substrates of this enzyme. Lysates of ISC1-overexpressing cells demonstrated very high PLC activities on IPS. Deletion of ISC1 eliminated endogenous IPS-PLC activities. Labeling yeast cells with [(3)H]dihydrosphingosine showed that IPS were increased in the deletion mutant cells. This study identifies the first enzyme involved in catabolism of complex sphingolipids in S. cerevisiae.

Blotting, Western↗
PubMed2000

Interaction of UvrA and UvrB proteins with a fluorescent single-stranded DNA. Implication for slow conformational change upon interaction of UvrB with DNA.

UvrA and UvrB proteins play key roles in the damage recognition step in the nucleotide excision repair. However, the molecular mechanism of damage recognition by these proteins is still not well understood. In this work we analyzed the interaction between single-stranded DNA (ssDNA) labeled with a fluorophore tetramethylrhodamine (TMR) and Thermus thermophilus HB8 UvrA (ttUvrA) and UvrB (ttUvrB) proteins. TMR-labeled ssDNA (TMR-ssDNA) as well as UV-irradiated ssDNA stimulated ATPase activity of ttUvrB more strongly than did normal ssDNA, indicating that this fluorescent ssDNA was recognized as damaged ssDNA. The addition of ttUvrA or ttUvrB enhanced the fluorescence intensity of TMR-ssDNA, and the intensity was much greater in the presence of ATP. Fluorescence titration indicated that ttUvrA has higher specificity for TMR-ssDNA than for normal ssDNA in the absence of ATP. The ttUvrB showed no specificity for TMR-ssDNA, but it took over 200 min for the fluorescence intensity of the ttUvrB-TMR-ssDNA complex to reach saturation in the presence of ATP. This time-dependent change could be separated into two phases. The first phase was rapid, whereas the second phase was slow and dependent on ATP hydrolysis. Time dependence of ATPase activity and fluorescence polarization suggested that changes other than the binding reaction occurred during the second phase. These results strongly suggest that ttUvrB binds ssDNA quickly and that a conformational change in ttUrvB-ssDNA complex occurs slowly. We also found that DNA containing a fluorophore as a lesion is useful for directly investigating the damage recognition by UvrA and UvrB.

Adenosine Triphosphatases↗
PubMed2000

Molecular analysis of familial androgen insensitivity syndrome due to replacement of glutamic acid 802 by lysine.

We studied a Japanese family presenting at least two cases of complete androgen insensitivity syndrome (CAIS) and negative androgen receptor binding. The index subject showed a 46, XY karyotype and a complete female phenotype. For the purpose of further diagnosis and genetic counseling, molecular analysis of the androgen receptor (AR) gene was performed. Direct sequencing of the AR gene identified a mutation at nucleotide 2935 (A-->G). This replacement was a novel missense mutation, resulting in the substitution of glutamic acid 802 by lysine which deleted a recognition site for EcoRI in exon 6 of the AR gene. We identified another affected individual, using chromosome and molecular analysis of the AR gene at exon 6. Furthermore, although heterozygote carriers could not be identified on clinical grounds, molecular identification of healthy individuals and heterozygote carriers in the family members provided definitive information for genetic counseling. We believe that the molecular analysis of familial CAIS is very informative for both the affected individuals and other family members.

Adolescent↗
PubMed2000

Nonenzymatic template-directed condensation of short-chained oligouridylates on a poly(A) template.

An oligouridylate with chain-length of more than three was condensed on a polyadenylate [poly(A)] template with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) in imidazole buffer. The condensation reaction proceeds via the phosphorimidazolide of the oligouridylate as an intermediate. Pentauridylate [(pU)5] was converted to decauridylate [(pU)10] in 10% yield at 0 degree C for 7 days in the presence of the poly(A) template, while no coupling product was obtained in the absence of the poly(A) template. The resulting linkage of the (pU)10 was mainly 2'-5' linkage.

Base Sequence↗
PubMed2000

Synthesis and properties of a new type DNA dendrimer.

We have designed a new type of a DNA dendrimer which has rigid branched structure. The branching molecule was prepared from 1,3,5-tribromobenzene. The dendrimer unit, in which three oligonucleotide-chains, two molecules of T15 and one molecule of A15, linked to the branching molecule, was synthesized by an automated DNA synthesizer. The properties of the dendrimer unit and dendrimer formation by inter-molecular association of T15 and A15 chains of the dendrimer unit will be presented.

Benzene Derivatives↗
PubMed2000

Stereospecific synthesis of alpha-anomeric pyrimidine nucleoside.

A facile stereospecific synthetic method for alpha-anomeric 2'-deoxypyrimidine nucleoside unit utilizing aminooxazoline derivative of ribofuranose was investigated. Thus, easily accessible riboaminooxazoline derivative prepared by ribose and cyanamid was allowed to react with ethyl alpha-bromoethylacrylate to give corresponding adduct. The adduct was cyclized by strong base such as potassium t-butokiside. The resulted 2,2'-cyclonucleoside was then treated with acetyl bromide followed by n-butyltin hydride to give alpha-anomeric 3',5'-di-O-acetylthymidine. 3',5'-Di-O-acety groups of the nucleoside were easily removed by the action of excess of triethyl amine in methanol. Essentially same procedure afforded corresponding 2'-deoxyuridine, which was further, converted to alpha-anomeric 2'-deoxycytidine.

Deoxycytidine↗