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T Nishi

Publications and source records attributed to T Nishi.

At least 127 records · Page 7Linked to original sources
PubMed1998

Targeted gene transfer to corneal endothelium in vivo by electric pulse.

A novel method of in vivo targeted gene transfer to intentionally selected areas of the corneal endothelium was developed. Plasmid DNA with the lacZ gene coding for beta-galactosidase was injected into the anterior chamber of adult Wistar rats, and eight pulses of electricity at intensities ranging from 5 to 40 V/cm were delivered for 50 ms to the cornea with a specially designed electric probe in order to determine the effect of gene transfer on the corneal endothelial cells. Gene expression was visualized by enzymatic color reaction using X-gal in enucleated eyes on days 1, 3, 7, 14 and 21 after gene transfer. The treated eyes were then photographed and the X-gal-positive areas were evaluated by an image analyzer. The ratios of the areas (X-gal-positive area/area of entire corneal endothelium x 100%) were then calculated to determine gene transfection efficiency. The expression of beta-galactosidase was clearly detected in the cytoplasm of the corneal endothelial cells as early as day 1 and lasted until day 21. The most intense gene expression was observed on days 1 and 3 (5.21% on day 1 and 6.45% on day 3). The expression of beta-galactosidase on day 3 was most evident following delivery of 20 V electric pulses (0.09% at 5 V, 0.03% at 10 V, 6.45% at 20 V). beta-Galactosidase expression was limited to the corneal endothelial cells in highly selected areas and no beta-galactosidase expression was detected in any other intra- or axtraocular tissues. In addition, no cell damage was apparent in the cornea and no inflammation was detected in any other intraocular tissues. Thus, low-voltage electric pulses successfully transferred the gene of interest to highly selective areas of the corneal endothelium without inducing any pathological changes. This targeted gene transfer method appears to have great potential for use in gene therapy for ocular diseases.

Animals↗
PubMed1998

Guanidinated casein hydrolysate stimulates pancreatic secretagogue release by direct action to the intestine in rats.

Previously, we observed that pancreatic exocrine secretion was strongly enhanced after duodenal administration of guanidinated casein peptic hydrolysate (HGC) in rats with chronic bile-pancreatic juice (BPJ) diversion. Using a perifusion system of isolated dispersed rat intestinal mucosal cells, we investigated whether this phenomenon depends on a direct effect of protein on the intestine to release pancreatic secretagogues, such as cholecystokinin (CCK). Amylase release from isolated pancreatic acini was used as an assay to measure CCK or other pancreatic secretagogues in the effluents. Mucosal effluent with HGC stimulated amylase release from acini with or without soybean trypsin inhibitor (SBTI). Perifused effluent with low-concentrated SBTI did not stimulate amylase release. These results indicate that HGC stimulates release of pancreatic secretagogue from the intestinal mucosal cells independent of remaining trypsin activity in the isolated mucosa. Effluents with intact casein, its peptic digest, and homoarginine, which is a unique amino acid contained in HGC, were unable to stimulate amylase release from acini. Effluent with a high concentration of SBTI, which is rich in arginine residues, stimulated amylase release, but not with the same tryptic inhibitory effect of lima bean trypsin inhibitor, which is poor in arginine residues. These findings suggest that guanidyl residues in protein structure are responsible for release of pancreatic secretagogues from isolated intestinal mucosal cells. Finally, the increment of amylase release from pancreatic acini in response to the perifused effluent with HGC was eliminated in acini treated with a potent CCK antagonist, FK480. We conclude that in rats with BPJ diversion, HGC stimulates CCK release from the intestine by direct action on intestinal mucosa.

Amino Acids↗
PubMed1998

Loss of merlin-p85 protein complex in NF2-related tumors.

The NF2 tumor suppressor gene product, designated merlin, belongs to the family of molecules that links membranous protein with the cytoskeleton. We have previously shown that merlin was co-immunoprecipitated with a cellular protein, p85, in cultured cell. To analyze the alteration of merlin and associated proteins in surgical specimens, we developed a new method for biotin-labeling of whole cellular proteins. Screening of tumor tissues using our method showed that none of malignant gliomas and half of the NF2-related tumors had altered p85 and merlin. Our detection method seems useful for the screening of merlin alterations in NF2-related tumors.

Avian Proteins↗
PubMed1998

[TNM classification--pediatric tumors].

The clinical and postsurgical TNM classifications (cTNM and pTNM) for neuroblastoma (NB), nephroblastoma (WT) and soft tissue sarcomas were presented in 1982 by the TNM Committee in UICC in collaboration with SIOP. The Japanese TNM Committee proposed new pTNM systems (J-pTNM) for NB and WT, and new cTNM and pTNM system for primary liver carcinoma in infants and children (HT). These pTNMs were based on the staging systems developed by the Malignant Tumor Committee of the Japanese Society of Pediatric Surgeons. The proposal of subdivision of M category in NB was presented for testing the new telescopic ramifications of TNM. The TNM for HT was added as a new classification recommended for testing. The effectiveness of these TNM systems was assessed using NB, WT and hepatoblastoma (HB) cases which were registered in collaborating institutes. The analyses suggested that pTNM, especially the J-pTNM system in NB, WT and HT were effective for the assessment of prognoses, although cTNM systems were not enough to assess the extent of the disease.

Child↗
PubMed1998

Pancreatoblastoma: optically clear nuclei in squamoid corpuscles are rich in biotin.

Pancreatoblastoma is a rare pancreatic neoplasm that generally occurs in children and demonstrates unique histopathologic features, including squamoid corpuscles. We encountered five cases of pancreatoblastoma and unexpectedly found the presence of optically clear nuclei (OCN) only in tumor cells of squamoid corpuscles. Recent studies showed that OCN seen in some types of neoplastic and non-neoplastic tissues were rich in biotin and revealed false-positive immunostaining by the avidin-biotin peroxidase complex method. We conducted an investigation to see if the same were true for pancreatoblastoma. As a result, the OCN were positively stained when the tissue section was incubated with peroxidase-labeled avidin or streptavidin and developed in a chromogen solution. The reaction was completely blocked by pretreatment with free avidin. An identical nuclear staining pattern was observed by the indirect immunoperoxidase method with the antibiotin antibody. Ultrastructurally, the OCN were occupied by filamentous substructures of chromatin measuring approximately 10 to 12 nm in diameter. These results suggested to us that the OCN in pancreatoblastomas were part of the unique nuclear change in which abundant biotin accumulated in association with the characteristically arranged chromatin substructure. When immunostaining with biotin as the linkage agent is performed in pancreatoblastomas, the false positivity in the OCN should be kept in mind to avoid any misinterpretation of the results.

Biotin↗
PubMed1997

Expression cloning of cDNA encoding a human beta-1,3-N-acetylglucosaminyltransferase that is essential for poly-N-acetyllactosamine synthesis.

The structure and biosynthesis of poly-N-acetyllactosamine display a dramatic change during development and oncogenesis. Poly-N-acetyllactosamines are also modified by various carbohydrate residues, forming functional oligosaccharides such as sialyl Lex. Herein we describe the isolation and functional expression of a cDNA encoding beta-1,3-N-acetylglucosaminyltransferase (iGnT), an enzyme that is essential for the formation of poly-N-acetyllactosamine. For this expression cloning, Burkitt lymphoma Namalwa KJM-1 cells were transfected with cDNA libraries derived from human melanoma and colon carcinoma cells. Transfected Namalwa cells overexpressing the i antigen were continuously selected by fluorescence-activated cell sorting because introduced plasmids containing Epstein-Barr virus replication origin can be continuously amplified as episomes. Sibling selection of plasmids recovered after the third consecutive sorting resulted in a cDNA clone that directs the increased expression of i antigen on the cell surface. The deduced amino acid sequence indicates that this protein has a type II membrane protein topology found in almost all mammalian glycosyltransferases cloned to date. iGnT, however, differs in having the longest transmembrane domain among glycosyltransferases cloned so far. The iGnT transcript is highly expressed in fetal brain and kidney and adult brain but expressed ubiquitously in various adult tissues. The expression of the presumed catalytic domain as a fusion protein with the IgG binding domain of protein A enabled us to demonstrate that the cDNA encodes iGnT, the enzyme responsible for the formation of GlcNAcbeta1 --> 3Galbeta1 --> 4GlcNAc --> R structure and poly-N-acetyllactosamine extension.

Adult↗
PubMed1997

Enzymatic characterization of human alpha1,3-fucosyltransferase Fuc-TVII synthesized in a B cell lymphoma cell line.

The human alpha1,3-fucosyltransferase, Fuc-TVII, a key enzyme in the biosynthesis of selectin ligands, was expressed as a soluble protein-A chimeric form in a human B cell lymphoma cell line, Namalwa KJM-1, and purified using IgG-Sepharose. The enzymatic properties of recombinant soluble Fuc-TVII were then examined. Its enzyme activity was highest at pH 7.5, and the presence of 25 mM Mn2+ was required for full activity. Fuc-TVII exhibits an acceptor specificity restricted to alpha2,3-sialylated type 2 oligosaccharides, and the apparent Km values for alpha2,3-sialyl lacto-N-neotetraose and GDP-fucose were 3.08 mM and 16.4 microM, respectively. The inhibitory effects of various nucleotides on the activity of Fuc-TVII reflected its donor specificity for the nucleotide portion of GDP. Fuc-TVII was demonstrated to be useful for the synthesis of a sialyl Lewis x hexasaccharide from lacto-N-neotetraose in combination with an alpha2, 3-sialyltransferase, ST3Gal IV. Polyethylene glycols enhanced the thermal stability of Fuc-TVII, leading to increased formation of the reaction product.

Burkitt Lymphoma↗
PubMed1997

The efficiency of antioxidants delivered by liposomal transfer.

Phenolic antioxidants of the hydroxychroman class, alpha-tocopherol (alpha-TOC) and 2,2,5,6,7-pentamethyl-6-hydroxychroman (PMHC), and the hindered phenols 2,3-dihydro-5-hydroxy-2,2,4-trimethylnaphtho[1,2-b]furan (NFUR), 2,6-di-tert-butyl-4-methoxyphenol (DBHA), and 2,6-di-tert-butyl-4-methyl phenol (BHT), were delivered into oxidizable (ACCEPTOR) liposomes of dilinoleoylphosphatidylcholine (DLPC) or 1-palmitoyl-2-linoleoyl-phosphatidylcholine (PLPC) from saturated DONOR liposomes of dimyristoylphosphatidylcholine (DMPC) by liposomal transfer. The antioxidant activities, k(inh), by the inhibited oxygen uptake method were compared with the k(inh)s determined when the antioxidants were introduced into the liposomes by coevaporation from organic solvents. The peroxidations were initiated using either thermal initiators, water-soluble azo-bis-amidinopropane hydrochloride (ABAP), lipid-soluble azo-bis-2,4-dimethylvaleronitrile (ADVN) and di-tert-butylhyponitrite (DBHN), or the photoinitiator benzophenone. The antioxidants PMHC, NFUR, DBHA, and BHT transferred rapidly between liposomes, but several hours of incubation were needed to transfer alpha-TOC. The average k(inh)s in liposomes, in the relative order NFUR approximately DBHA > PMHC > BHT approximately alpha-TOC, were markedly lower than known values in organic solvent. k(inh) values in liposomes appear to be controlled by effects of hydrogen bonding with water and by restricted diffusion of antioxidants, especially in the case of alpha-TOC. Product studies of the hydroperoxides formed during inhibited oxygen consumption were carried out. The cis,trans/trans,trans (c,t/t,t) product ratios of the 9- and 13-hydroperoxides formed from PLPC during inhibited peroxidation by PMHC were similar for both the coevaporated and liposomal transfer procedures. The c,t/t,t ratio for the same concentration of alpha-TOC, 1.52, compares to a value of 1.69 for PMHC at the start of the inhibition period. The higher c,t/t,t ratio observed for NFUR in DLPC, which varied between values of 7.0 at the start of the inhibition to about 1.8 after the break in the induction period, is a reflection of the increased hydrogen atom donating ability of the antioxidant plus the increased concentration of oxidizable lipid provided by DLPC.

Amidines↗
PubMed1997

4-aminopyridine and l-cis-diltiazem block the cGMP-activated K+ channels closed by light in the molluscan extra-ocular photoreceptors.

The cGMP-activated K+ channels closed by light lead to the depolarizing photocurrent of photoreceptors in the Onchidium ganglion. Whole-cell current records showed that external application of 100-200 microM 4-aminopyridine or 200-400 microM l-cis-diltiazem completely blocked the macroscopic photocurrent at any depolarizing and hyperpolarizing potentials. Single-channel current recordings suggested that both 4-aminopyridine and l-cis-diltiazem act to block the cGMP-activated K+ channels in their open state from inside the cell.

4-Aminopyridine↗
PubMed1997

Analysis of initial attachment of B cells to endothelial cells under flow conditions.

This study examined the adhesive interactions of peripheral blood B cells with TNF-alpha-activated endothelial monolayers, and analyzed the roles of E-selectin, P-selectin, or VCAM-1 molecules expressed on activated HUVEC. B cell interaction occurred on activated HUVEC, but not on resting HUVEC under flow conditions. The majority of peripheral blood B cells expressed P-selectin glycoprotein ligand-1 and alpha4 integrin. However, the expression of cutaneous lymphocyte Ag on B cells was low. Under flow conditions, B cells could bind to P-selectin-coated tubes and VCAM-1-transfected Chinese hamster ovary cells. In contrast, B cells could not bind to E-selectin-coated tubes. Adhesion activity of B cells to P-selectin-coated tubes was weaker than that of T cells. Furthermore, adhesion activity of B cells to VCAM-1-transfected Chinese hamster ovary cells was similar to that of T cells. Treatment of activated HUVEC with anti-VCAM-1 mAb reduced interaction of B cells under flow conditions. However, the treatment of activated HUVEC with anti-P-selectin mAb did not reduce interaction. These data indicated that the interaction of VCAM-1 with alpha4 integrin plays a major role in an initial attachment of B cells to endothelial monolayers under flow conditions.

Animals↗
PubMed1997

Postnatal ontogeny of striatal-enriched protein tyrosine phosphatase (STEP) in rat striatum.

The present study was undertaken to examine developmental change in expression of striatal-enriched protein tyrosine phosphatase (STEP) in the postnatal striatum of rats. For this purpose, immunohistochemical staining and transimmunoblotting analyses were carried out using a cDNA-generated polyclonal antibody to the STEP with a molecular weight of 46 kDa. Immunostaining showed that in neonatal striatum STEP-immunoreactivity was found in discrete patches composed of many immature cells, which corresponded to the tyrosine hydroxylase-immunopositive "dopamine islands." With development there was an increase in staining intensity and in the number of positively reacting cells. By 4 weeks postnatally, STEP-immunoreactivity was almost homogeneously distributed throughout the striatum, as was seen at the adult stage. Immunoblotting analysis showed that STEP protein expression abruptly increased from 2 to 4 weeks postnatally when it reached the adult level. These findings suggest that STEP is involved in development and maturation of the striatal neurons.

Animals↗
PubMed1997

High-level expression and purification of a recombinant human alpha-1, 3-fucosyltransferase in baculovirus-infected insect cells.

A human alpha-1,3-fucosyltransferase (Fuc-TVII) was expressed by recombinant baculovirus-infected insect Sf9 cells as a secretory fusion protein. The fusion protein consisted of the human granulocyte colony-stimulating factor signal peptide followed by an IgG-binding domain of protein A, a Fuc-TVI-derived peptide, and the putative catalytic domain of Fuc-TVII. The signal peptide was correctly cleaved and the recombinant Fuc-TVII was secreted into the culture medium at a concentration of 10 micrograms/ml. The recombinant Fuc-TVII could be highly purified in a single-step purification procedure, i.e., IgG-Sepharose column chromatography. The enzymatic properties of the Sf9-produced Fuc-TVII were compared with the properties of that expressed by a human B-cell line, Namalwa KJM-1, transfected with an episomal plasmid carrying the fusion Fuc-TVII cDNA. Both recombinant proteins showed alpha-1,3-fucosyltransferase activity toward a type II oligosaccharide with a terminal alpha-2,3-linked sialic acid among various acceptors. The apparent Km values of Sf9-produced Fuc-TVII for GDP-fucose and its acceptor substrate were slightly lower than those of the Fuc-TVII produced by Namalwa KJM-1 cells. Sf9-produced Fuc-TVII has N-linked carbohydrate chains whose molecular weights are lower than those linked to Namalwa KJM-1-produced Fuc-TVII. This difference in carbohydrate structure hardly affects the thermal stability of Fuc-TVII. The baculovirus expression system is available for high-level expression of stable and enzymatically active secretory Fuc-TVII.

Amidohydrolases↗
PubMed1997

Treatment of esophageal cancer in patients over 80 years old.

A total of 828 patients with esophageal cancer were treated at the Second Department of Surgery of Tokai University in the 20-year period from 1975 to June 1994, including 45 patients over 80 years old. We reviewed these elderly patients to assess the optimum therapeutic approach for such individuals. In recent years, the number of elderly patients with esophageal cancer has steadily been increasing. Advanced cancer is more common among this group, but early cancer has also been detected more frequently in recent years. Of the 45 elderly patients (80%) in our series, 36 were encountered in the last 10 years. As 28.9% of the patients had multiple cancers, a careful workup was necessary preoperatively. Since most patients (88.9%) had complications and were also in a poor general condition, limited surgery was recommended in consideration of the postoperative quality of life. The indications for endoscopic mucosal resection (EMR) may be able to be extended to submucosal1 (sm1) cancer without lymph node swelling. Postoperative complications occurred in 60% of those undergoing surgical resection or esophageal bypass, although death only resulted in 1 case. The 5-year survival rate after surgical resection was 30.8%. These results therefore support the use of surgical treatment for selected elderly patients with esophageal cancer.

Adult↗
PubMed1997

Cervical cord ependymoma with numerous microrosettes.

"Microrosette ependymoma," which is ependymoma with numerous microrosettes throughout the tumor, has rarely been reported. We describe an autopsy case of cervical cord ependymoma with two unusual features: the presence of numerous microrosettes and the formation of trabecular architecture. The tumor originated in the C2 segment of a man aged 23 years and gradually expanded over the following 15 years and 10 months until the entire cervical cord was involved. Beside the low grade of malignancy, the tumor cells exhibited a strong tendency to form microrosettes and trabecular architecture, which formed many perivascular pseudorosettes. The microrosettes mostly consisted of only two or a few more cells, in the absence of large rosettes. Thus the constituent cells were those forming perivascular pseudorosettes. Electron microscopy and immunohistochemistry characterized the ependymal properties of the microrosettes, whose lumina frequently contained fibril bundles similar to those of the Reissner's fiber fibrils, in addition to cilia and microvilli. The pathogenesis of the occurrence of numerous microrosettes is unknown; however, a defect in the mechanism of regulation of rosette formation and enlargement is the most likely explanation.

Adult↗
PubMed1997

The complete nucleotide sequence and characterization of the linear DNA plasmid pRS64-2 from the plant pathogenic fungus Rhizoctonia solani.

The complete nucleotide sequence of one of three linear DNA plasmids (pRS64-2) from the plant pathogenic fungus Rhizoctonia solani was determined. The pRS64-2 DNA consisted of 2877 nucleotides. The nucleotide sequences of the middle 2.2-kb regions of the other two plasmids (pRS64-1 and pRS64-3) were also determined. Comparison of the nucleotide sequences among the three plasmid DNAs indicated the presence of four regions with more than 86% sequence homology, suggesting the development of three plasmid DNAs from a common ancestor. A computer-based study of the pRS64-2 DNA-folding at both termini predicted hairpin loop structures. The hairpin loops consisted of the left- and right-hand terminal 113 and 105 nucleotides, respectively, and had no sequence homology. They had not undergone flip-flop inversion. The hairpins form cruciform base-paired structures.

Base Sequence↗
PubMed1997

Differential binding of peptides substituted at putative C-terminal anchor residue to HLA-DQ8 and DQ9 differing only at beta 57.

HLA-DQ8 (DQA1*0302-DQB1*0302: DQ beta 57 Ala) and (DQA1*0302-DQB1*0303: DQ beta 57 Asp) differ only at beta 57, at which polymorphism reportedly confers distinct susceptibility to insulin-dependent diabetes mellitus (IDDM). To identify the differential peptide binding affected by beta 57, we determined DQ9-binding peptides by affinity-based selection of a phage random peptide library using the biotinylated DQ9 complex. Nonconservative single-residue substitution of high-affinity DQ8- and DQ9-binding peptide (1KLPDYVLWSSSTVVGLGAAGA21) at the underlined residues significantly decreased the peptide binding to DQ8 and DQ9. Affinities of the wild-type 21-mer K4DYVLWSSSTV13 and K4AYAAWAAATA13 to DQ8 and DQ9 were practically the same. The K4DYVLWSSSTV13-based analogue peptides with substitutions at 12T showed that residues R, K, H, E, D, Q, N, T, S, V, L, I, F, M, W, and Y permitted binding to DQ8, whereas only R, T, V, L, I, F, M, W, and Y did so to DQ9. Thus, significant differences exist between DQ9 and DQ8, in that the majority of polar residues, regardless of their static charges at the residue 12, permitted binding to IDDM-susceptible DQ8, which is not the case for DQ9. The affinities of K4DYVLWSSSXV13 AND K4AYAAWAAAAX13 (where X is T, A, K, D, or I) were almost equal to DQ8 and DQ9, suggesting the DQ8- and DQ9-binding peptide motifs could accept both the 8-mer and 9-mer frames depending on intervening sequences between N- and C-terminal anchor residues. The biochemical basis of peptide-HLA interactions determined by DQ beta 57 is discussed.

Amino Acid Sequence↗
PubMed1997

Cloning, characterization and developmental expression of alpha2,8 sialyltransferase (GD3 synthase, ST8Sia I) gene in chick brain and retina.

GD3 and GM2 synthases act on ganglioside GM3 at the branching point of the pathway of synthesis of gangliosides in which the "a", "b" and "c" families are produced. The relative activities of these enzymes are important for regulating the ganglioside composition of a given tissue. In the present work, we report the cloning and characterization of a chick GD3 synthase cDNA. The cloned cDNA directed the synthesis of a functionally active enzyme in transiently transfected CHO-K1 cells and was highly homologous to mammalian GD3 synthases. In Northern blot experiments the cDNA detected a single specific GD3 synthase mRNA of about 9.0 kb both in the chicken brain and retina. The abundance of the specific mRNA transcript declined steadily from E7-E9 to very low values around PN2. The levels of enzyme activities measured at the same developmental stages roughly followed the changes of specific mRNA levels in both tissues. In situ hybridization of embryonic neural retina cells in culture showed that both glial- and neuron-like cells expressed the specific GD3 synthase mRNA, although with different intensities. Results indicate that transcription and/or stability of the specific GD3 synthase mRNA constitute a level of control of the expression of GD3 synthase and indirectly of the ganglioside composition in the developing chicken central nervous system (CNS).

Amino Acid Sequence↗