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Biomedical subjects

H Sawai

Publications and source records attributed to H Sawai.

At least 19 recordsLinked to original sources
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

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↗
PubMed2000

Probable involvement of the 5-hydroxytryptamine(4) receptor in methotrexate-induced delayed emesis in dogs.

Delayed emesis in cancer patients undergoing chemotherapy remains a significant problem. The pathogenesis of delayed emesis is still obscure. It was recently demonstrated that methotrexate (MTX), an anticancer drug, evoked delayed emesis in dogs in a manner similar to its actions in humans. We evaluated the antiemetic activity of FK1052, a potent antagonist for both the 5-hydroxytryptamine (HT)(3) and 5-HT(4) receptors, on delayed emesis induced by MTX in beagle dogs. Animal behavior was recorded for 3 days using a video camera. Delayed emesis lasting up to 72 h was observed in dogs treated with MTX (2.5 mg/kg i.v.), but acute emesis did not occur. The following antiemetics, at the dose that prevents cisplatin-induced acute emesis in dogs, were administered i.v. as multiple injections every 12 h during days 2 to 3. FK1052 (1 and 3.2 mg/kg) significantly reduced the emetic episodes caused by MTX, whereas ondansetron (1 mg/kg), a selective 5-HT(3) receptor antagonist, was not effective. The emetic episodes induced by MTX were also inhibited by another 5-HT(3/4) receptor antagonist, tropisetron (1 mg/kg). CP-122,721 (0. 1 mg/kg), a potent selective tachykinin NK(1) receptor antagonist, significantly reduced the emetic responses to MTX. Copper sulfate-induced emesis in dogs was also prevented by FK1052, tropisetron, and CP-122,721 but not by ondansetron. FK1052, tropisetron, and ondansetron had negligible affinity for the NK(1) receptor at 1 microM. These results suggest that the 5-HT(4) receptor may be in part involved in the production of delayed emesis induced by MTX in dogs and that FK1052 may be a useful drug against both acute and delayed emesis induced by cancer chemotherapy.

Animals↗
PubMed1999

Function of the cloned putative neutral sphingomyelinase as lyso-platelet activating factor-phospholipase C.

Sphingolipids such as ceramide and sphingosine have been regarded as novel signal mediators in cells. However, the mechanisms of generation of these lipids upon various stimulation remain to be elucidated. Neutral sphingomyelinase (N-SMase) is one of the key enzymes in the generation of ceramide, and recently the cloning of a putative N-SMase was reported. Because the function of the protein was unclear in the previous report, we investigated the role it plays in cells. N-SMase activity in cells overexpressing the protein with hexa-histidine tag was immunoprecipitated with anti-hexa-histidine antibody. The metabolism of ceramide and SM was not apparently affected in overexpressing cells. Radiolabeling experiments using [(3)H]palmitic acid or [(3)H]hexadecanol demonstrated an accumulation of 1-O-alkyl-sn-glycerol and a corresponding decrease of 1-alkyl-2-acyl-sn-glycero-3-phosphocholine in overexpressing cells. In vitro studies showed that both 1-acyl-2-lyso-sn-glycero-3-phosphocholine (lyso-PC) and 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine (lyso-platelet activating factor (lyso-PAF)) are good substrates of the protein. In further radiolabeling experiments, 1-acyl-lyso-PC was predominantly and equally metabolized into diacyl-PC in both vector and overexpressing cells. On the other hand, 1-O-alkyl-lyso-PC (lyso-PAF) was metabolized into both diradyl-PC and 1-O-alkyl-glycerol in overexpressing cells but only into diradyl-PC in vector cells. These results suggest that the protein acts as lyso-PAF-PLC rather than lyso-PC-PLC or N-SMase in cells.

Animals↗
PubMed1999

Immunohistological studies of metabotropic glutamate receptor subtype 6-deficient mice show no abnormality of retinal cell organization and ganglion cell maturation.

Immature retinal ganglion cells (RGCs) initially show a multistratified dendritic pattern, and, during the postnatal period, these dendrites gradually monostratify into ON and OFF sublaminae. The selective agonist of group III metabotropic glutamate receptors (mGluR), L-2-amino-4-phosphonobutyrate (L-AP-4), hyperpolarizes ON bipolar cells and reduces glutamate release. On the basis of L-AP-4-evoked inhibitory effects on ON-OFF segregation of developing RGCs, it has been hypothesized that glutamate-mediated synaptic activity is crucial for formation of the ON-OFF network. Gene-targeted ablation of mGluR6 specifically expressed in ON bipolar cells blocks normal ON responses but has been predicted to enhance glutamate release from ON bipolar cells. The mGluR6 knock-out mouse therefore provides a unique opportunity to investigate whether glutamate release and ON responses are important factors in the development of ON-OFF segregation. The combination of several different morphological analyses indicates that ON bipolar cells, as well as several distinct amacrine cells, in mGluR6 knock-out mice are normally distributed and correctly extend their terminals to defined retinal laminae. Importantly, both alpha and delta RGCs in adult mGluR6 knock-out mice are found monostratified into cell type-specific layers. Furthermore, no difference between wild-type and mGluR6 knock-out mice is observed in the maturation and dendritic stratification of developing RGCs. Hence, despite a deficit in normal ON responses, mGluR6 deficiency causes no abnormality in the retinal cellular organization nor in the stratifications of both ON bipolar cells and developing and mature RGCs. Based on these findings, we discuss several possible mechanisms that may underlie ON-OFF segregation of RGCs.

Animals↗
PubMed1999

Prenatal diagnosis of thanatophoric dysplasia by mutational analysis of the fibroblast growth factor receptor 3 gene and a proposed correction of previously published PCR results.

Thanatophoric dysplasia (TD) is the most frequent form of neonatal lethal skeletal dysplasia. Recently. mutations in the fibroblast growth factor receptor 3 (FGFR3) gene that cause two subtypes of this disorder, type I (TDI) and type II (TDII), have been identified. This discovery has now made it possible to make a definite diagnosis of TD by molecular methods. To date, prenatal diagnosis of TD has been accomplished by ultrasonography in the second trimester. However, it is not always possible to distinguish TD fetuses it utero from the other osteochondrodysplasias by ultrasonography or radiography. We report on the prenatal diagnosis of a TD fetus, showing severe shortness of limbs and polyhydramnios, by identification of a mutation in the FGFR3 gene. Genomic DNA was isolated from the amniotic fluid and then subjected to PCR amplification. The common TDI mutation, C-->T transition at nucleotide 742 in the FGFR3 gene, was identified using restriction enzyme analysis. This information was critical in obstetric management decisions later in pregnancy. However, although the mutation responsible for TDI was detected previously, we noticed some inconsistencies in the published PCR results and have proposed a correction.

Adult↗