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Biomedical subjects

T Ueki

Publications and source records attributed to T Ueki.

At least 91 records · Page 5Linked to original sources
PubMed1996

Positive-negative KG cassettes for construction of multi-gene deletions using a single drug marker.

Positive-negative KG cassettes were developed in order to create a number of independent deletion mutations on the bacterial chromosome using a single drug marker. These cassettes consist of a kanamycin-resistant (KmR) gene for positive screening and a galactokinase gene (galK) for negative screening. Both genes are in an operon driven by the native KmR promoter and are flanked by identical fragments of yeast chromosomal DNA approximately one kb in size. An internal region of a cloned target gene of a bacterium is replaced with a cassette, which is then transformed into the bacterium. The intact gene on the chromosome is replaced with the mutated gene by homologous recombination. From the KmR cells thus obtained, those cells which lose both KmR and galK genes by homologous recombination between the identical yeast DNA fragments are subsequently screened on plates containing 2-deoxygalactose, a non-metabolizable analogue of galactose. This method was applied to isolate a triple-deletion mutant of pkn3, pkn1, and pkn11 from Myxococcus xanthus.

Anti-Bacterial Agents↗
PubMed1996

Modulation of growth and apoptosis response in PC-3 and LNCAP prostate-cancer cell lines by Fas.

Fas/APO-1 is a cell-surface protein, a member of the TNF-receptor family, and it potentially induces apoptosis. In presence of an apoptosis-inducible anti-human Fas MAb, Fas-negative control PC-3 human prostate-cancer cells did not undergo morphological changes, while PC-3 human Fas transfectants showed apoptotic changes in vitro. However, LNCaP human Fas transfectants, as well as Fas-negative control LNCaP human prostate-cancer cells, were Fas-resistant. The growth of Fas-transfected PC-3 tumor was retarded compared with that of control PC-3 tumor in vivo without stimulation of anti-human Fas MAb. Anti-human Fas MAb administration in vivo caused macroscopic Fas-transfected PC-3 tumors formed in BALB/c nude mice to undergo apoptosis.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Cryocrystallography of 3-Isopropylmalate dehydrogenase from Thermus thermophilus and its chimeric enzyme.

The crystal structures of thermostable enzyme, 3-isopropylmalate dehydrogenase of Thermus thermophilus (10T) and a chimeric enzyme between T. thermophilus and Bacillus subtilus with one point mutation (cS82R), were determined at both 100 and 150 K. At the cryogenic condition, the volume of the unit cell decreased by 5% as a result of a contraction in the solvent region. Although the overall structures of both enzymes at low temperature were the same as that of 10T at room temperature, interactions between two domains and between two subunits in a functional dimer of cS82R were significantly altered. The decrease in the average temperature factor of 10T at low temperature and no significant decrease for cS82R suggested that the structure of the engineered enzyme (cS82R) may have many conformational substates even at low temperature, while the native enzyme (10T) at low temperature has a more definite conformation than that at room temperature. The location of water molecules around the enzyme molecule and the calculation of the radii of gyration suggested that cS82R had a weaker hydration than 10T.

Journal Article↗
PubMed1996

Fluorescence polarization study on the dynamics and location of peroxidized fluorescent phospholipids in liposomes.

Motional properties of fluorescent substances produced by lipid peroxidation by a time-resolved fluorescence polarization technique were studied. When liposomes containing phosphatidylethanolamine (PE) and linoleic hydrocarbon chain were incubated at 37 degrees C, fluorophores absorbing maximally at 360 nm and emitting near 430 nm were produced. Their fluorescence anisotropy decay measured at 23 degrees C was fitted well with a sum of a fast relaxation and a time-independent residual term. With the increase of oxidation degree, the time constant of the relaxation term increased. This may be explained by alteration in the membrane structure or by modification of the fluorescent products themselves. Information on the location of the fluorescent products was obtained when their motional property was compared with those of various extrinsic probes that were incorporated at different positions of the lipid bilayer. It was found that the motional property of the fluorescent oxidation products is similar to that of 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene, a rod-shaped hydrophobic probe with a charged terminal. Other probes sensing the polar region or the hydrophobic region of the membrane were characterized by a lower order parameter. It is suggested that the fluorescent oxidation products have a polar moiety located at the membrane surface and attached to the amino group of PE while the tail part being buried in the hydrophobic region of the membrane. This picture is supported by fluorescence quenching experiments with the aqueous quencher Co2+. On the other hand, fluorophores produced by the reaction of malondialdehyde and PE suggested to have a chemical structure in which the angle between the absorption and emission dipole moments is very large. On the basis of these observations, the production pathway of fluorophores in oxidized membranes is discussed.

Fluorescence Polarization↗
PubMed1996

Molecular cloning and characterization of a newly identified member of the cadherin family, PB-cadherin.

We have isolated cDNA clones encoding novel proteins belonging to the cadherin family. These novel proteins are encoded by two distinct mRNA species generated by alternative splicing from a single gene, and based on preferential expression in the pituitary gland and brain, we named it PB-cadherin. One mRNA species encodes long type PB-cadherin composed of 803 amino acid residues with a longer cytoplasmic domain, whereas the other species encodes short-type PB-cadherin composed of 694 amino acid residues with a shorter cytoplasmic domain. Both long and short type PB-cadherin contain five repeats of a cadherin motif in the extracellular domain, the transmembrane domain, and the cytoplasmic domain, and the deduced amino acid sequences have a 30% homology to those of E-, N-, and P-cadherins. Although the primary structure of N-terminal amino acids is identical between long and short type PB-cadherin, the following structures in the cytoplasmic regions are completely different. The long type PB-cadherin but not the short type contains the putative catenin-binding domain. When these two distinct forms of PB-cadherins were stably expressed in L cells, L cells expressing long type PB-cadherin or short type PB-cadherin both acquired a Ca2+-dependent cell adhesion property, thereby indicating that both types of PB-cadherin are responsible for Ca2+-dependent cell adhesion. Persistent expression of PB-cadherin mRNA was found in the brain of rat embryos at least from embryonic day 15 to the postnatal period. In situ localization of PB-cadherin mRNA in the adult rat brain indicated that PB-cadherin mRNA is expressed in the inner granular layer of the olfactory bulb, Purkinje cell layer of the cerebellum, and in the pineal gland. PB-cadherin may play an important role in morphogenesis and tissue formation in neural and non-neural cells for the development and maintenance of the brain and neuroendocrine organs by regulating cell-cell adhesion.

Aging↗
PubMed1996

Distribution of activated complement, C3b, and its degraded fragments, iC3b/C3dg, in the colonic mucosa of ulcerative colitis (UC).

The third component of complement (C3) is central to both the classical and alternative pathways in complement activation. In this study, involvement of C3 activation in the mucosal injury of UC was investigated. We examined the distribution of activated (C3b) and degraded fragments (iC3b/C3dg) of C3, terminal complement complex (TCC), and complement regulatory proteins in normal and diseased colonic mucosa including UC and other types of colitis using immunohistochemical techniques at the level of light and electron microscopy. While C3b and iC3b/C3dg staining was negligible in the normal mucosa, iC3b/C3dg and, to a lesser extent, C3b were deposited in UC mucosa along the epithelial basement membrane. The deposition was enhanced in relation to the severity of mucosal inflammation (C3b, P less than 0.05; iC3b/C3dg, P less than 0.01). Epithelial deposition of TCC was not observed in most UC mucosa. Immunoelectron microscopy showed that C3b and iC3b/C3dg were distributed mainly along the epithelial basement membrane and the underlying connective tissue in a granular, studded manner, and weakly present along the basolateral surface of epithelial cells. These C3 fragments were also deposited in inflammatory control mucosa such as ischaemic and infectious colitis. Our findings suggest that deposition of the C3 fragments occurs in inflamed colonic mucosa of diverse etiologies, including UC, but to define a role of the deposition in the development of mucosal injury in UC awaits direct study.

Adult↗
PubMed1996

Crystal structure of H2-proteinase from the venom of Trimeresurus flavoviridis.

The crystal structure of the zinc-protease, H2-proteinase, isolated from the venom of Trimeresurus flavoviridis has been determined. The crystallographic R factor is 0.176 for 10,635 reflections with Fobs > 2sigma(Fobs) in the 8.0 to 2.2 Angstrom resolution range. The enzyme has two domains with a cleft in which a catalytic zinc atom is located. The N-terminal domain is composed of four helices around a central five-stranded beta-sheet. The irregularly folded C-terminal domain contains one helix and two disulfide bridges. These two domains are linked by a disulfide bridge. In the zinc environment, the catalytic zinc atom forms a distorted tetrahedral coordination with three histidines and one catalytic water molecule, and the methionine-containing turn is structurally conserved. These are distinctive features of the metzincins, one of the zinc metalloprotease superfamilies. The entire structure shows good agreement with that of two Crotalus snake venom proteases, adamalysin II and atrolysin C. The H2-proteinase, however, contains no structural calcium ions, and differences of disulfide configurations and the coordination of the catalytic water molecule exist as compared with the other two proteases.

Amino Acid Sequence↗
PubMed1996

[Urothelial carcinoma of the navicular fossa].

We report a case of transitional cell carcinoma arising in the fossa navicularis. The patient was a 74-year-old man. He had no history of sexually transmitted disease or urethral stricture. Clinically, the tumor was suspected to be a condyloma acuminatum; however, the pathological diagnosis yielded an unexpected result: transitional cell carcinoma. Radiological examinations and cystoscopy showed no other tumor besides the primary cancer in the fossa navicularis. Partial resection of the urethra was performed and the patient has been without evidence of disease for 3 years.

Aged↗
PubMed1996

[Three-dimensional-CT pancreatography under balloon-ERP in the pancreatic diseases--its method and usefulness].

Three-dimensional-CT pancreatography (3D-CTP) under balloon-ERP was carried out in 13 patients with the pancreatic diseases. Tapering stenosis of pancreatic duct in 2 patients out of 2 with pancreatic cancer, shape of cyst and relationship between cyst and pancreatic duct in 7 patients out of 7 with pancreatic cysts, and irregularity of wall of pancreatic duct in 2 patients out of 3 with chronic pancreatitis was reconstructed by 3D-CTP, stereographically. Moreover, the confluence of cyst and pancreatic duct in 3 out of 7 pancreatic cysts did not become clear on balloon-ERP, but it was distinct on 3D-CTP. It is suggested that 3D-CTP is useful in understanding pancreatic diseases stereographically, and can be applied to operative simulation, interventional radiology and differential diagnosis on them.

Adult↗
PubMed1995

Dispersion state of phospholipids and fluorescence production with peroxidation in organic solvents: investigated by time-resolved fluorescence technique.

Fluorescent substances were found to be produced efficiently when phospholipids containing phosphatidylethanolamine (PE) and linoleic chains were autoxidized in non-polar solvents. By using these fluorescent substances as intrinsic probes, the dispersion state of phospholipids was investigated in various organic solvents. Fluorescence anisotropy decay measurements indicated that the aggregation size of phospholipids was much larger in hexane than in chloroform, methanol and tert-butyl alcohol. The average diameter of phospholipid aggregates in hexane was calculated to be 4-6 nm, which was dependent on the lipid composition. A consistent result was obtained when N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-gly cer o-3- phosphoethanolamine (NBD-PE) was used as an extrinsic probe. Comparison of the fluorescence data with small-angle X-ray scattering (SAXS) data suggested that a reverse micellar structure of phospholipids formed in hexane. It was shown that phospholipid aggregation enhanced the extent of peroxidation as well as the production yield of fluorescent substances of phospholipid.

Chromatography, Thin Layer↗
PubMed1995

Structural characterization of the molten globule and native states of apomyoglobin by solution X-ray scattering.

Compactness and shape are two of the critical properties that describe the degree of protein folding. Solution X-ray scattering is an effective technique for measuring these properties quantitatively. Structural characteristics of various conformational states of horse myoglobin were studied in terms of size and shape by solution X-ray scattering. The radius of gyration for native holomyoglobin was 17.5 A, while that of the apomyoglobin native state was 19.7 A. Corresponding to the increase in the radius of gyration, the largest dimension of the molecule also increased from 47.5 A to 62.5 A. Both states are globular in shape. The scattering profiles in the high angle region suggest that the apomyoglobin native state has a distinct tertiary structure, and that packing of alpha-helices in the apomyoglobin native state would be looser than that of holomyoglobin. These observations indicate that the native state of apomyoglobin is expanded from that of holomyoglobin, and that the conformations of the two are not identical. The radii of gyration for the acid-unfolded state and the denaturant-unfolded state were 30 A and 35 A, respectively. Both unfolded states have chain-like conformations without any tertiary structures. The radius of gyration and the largest dimension of the molten globule stabilized by trichloroacetate were 23.1 A and 72.5 A, respectively. The molten globule is expanded from the native state although it is globular, and is much more compact than the unfolded state. The bimodal distance distribution function and scattering profile at high-angle region suggest that the structure of the apomyoglobin molten globule contains a core comprising a cluster of multiple alpha-helices and flaring tail(s), which would be a common structural property of the compact denatured state appearing during the folding process. The compactness of each conformational state is highly correlated with the extent of formation of the alpha-helix.

Apoproteins↗
PubMed1995

Expression of basic fibroblast growth factor and fibroblast growth factor receptor in advanced gastric carcinoma.

The expression of basic fibroblast growth factor (bFGF) and fibroblast growth factor receptor (FGFR) mRNA was examined in gastric carcinomas by immunohistochemistry and in situ hybridization, respectively. In the 20 advanced carcinomas examined, bFGF was found in 14 (70.0 per cent) and was confined to the tumour cells, whereas FGFR mRNA was demonstrated in 12 (60.0 per cent) and seen in both tumour cells and endothelial cells. The bFGF and FGFR mRNA-positive carcinomas were larger, were more frequently classified as undifferentiated adenocarcinoma, more frequently invaded the serosal layer, and had a higher rate of lymph node metastases than the bFGF and FGFR mRNA-negative carcinomas. Patients with bFGF and FGFR mRNA-positive carcinomas appear to die earlier than those with bFGF and FGFR mRNA-negative tumours. The values for the carcinomas that were positive for either bFGF or FGFR mRNA fell between these two groups. The findings suggest that the autocrine/paracrine bFGF/FGFR channel is associated with undifferentiated gastric carcinomas and may lead to a poorer prognosis.

Adenocarcinoma↗
PubMed1995

Detection of decay-accelerating factor in stool specimens of patients with colorectal cancer.

BACKGROUND & AIMS: Colorectal cancers have an increased expression of decay-accelerating factor (DAF). The aim of this study was to determine whether stool specimens of patients with colorectal cancer contain increased amounts of DAF. METHODS: DAF was measured using an immunoassay in the stool specimens of 40 persons with colorectal cancer, 18 with colorectal adenomatous polyps, 13 with upper gastrointestinal cancer, and 41 without gastrointestinal disease. RESULTS: Stool DAF concentrations in patients with colorectal cancer (0-9.8 ng/g stool; median, 1.6 ng/g) were significantly higher than those in patients with adenoma (0-6.4 ng/g; median, 0 ng/g) (P < 0.05), patients with upper gastrointestinal cancer (0-3.1 ng/g; median, 0 ng/g) (P < 0.05), and subjects without gastrointestinal disease (0-3.4 ng/g; median, 0 ng/g) (P < 0.01). Resection of colorectal cancers caused a marked decrease in stool DAF concentrations. The stool DAF test was positive in a substantial portion of patients with colorectal cancer whose tumors were small ( < 2 cm), at an early TNM stage, or unassociated with fecal occult blood positivity. The sensitivity of the test for colorectal cancer was 55%, and the specificity was 85%. CONCLUSIONS: The measurement of stood DAF deserves evaluation as a test for detection of colorectal cancer.

Adenomatous Polyposis Coli↗
PubMed1995

Intraductal papillary mucinous neoplasms of the pancreas associated with so-called "mucinous ductal ectasia". Histochemical and immunohistochemical analysis of 29 cases.

Twenty-nine patients (20 men, nine women; mean age, 65.9 years; range, 49-77 years) with intraductal papillary mucinous neoplasms associated with so-called "mucinous ductal ectasia" of the pancreas were studied both histochemically and immunohistochemically. These cases included six cases of hyperplasia, 13 adenomas, and 10 adenocarcinomas. The mean sizes of the hyperplasia, adenomas, and adenocarcinomas were 2.0 cm, 3.0 cm, and 4.8 cm in diameter, respectively. Tumor size correlated with the degree of cellular atypia. The proliferative rates were significantly higher in the carcinomatous epithelium with those in the hyperplastic and adenomatous epithelia. The polarity of distribution of carcinoembryonic antigen and carbohydrate antigen 19-9 were better preserved in the hyperplastic epithelia than in the carcinomatous epithelia. On the other hand, the papillary and nonpapillary hyperplastic epithelium contained mainly a neutral periodate-reactive glycoprotein with only trace amounts of sialomucins and sulphomucins. In addition, the adenomatous epithelium contained mostly sialomucins with a small amount of sulphomucins. The carcinomatous epithelium contained predominantly sulphomucin. The results of both the histological and immunohistochemical studies suggested the possibility of a sequential change from nonpapillary and papillary hyperplasia, via adenoma, to carcinoma in intraductal papillary-mucinous neoplasms associated with mucinous ductal ectasia. Moreover, these results, in combination with the histochemical findings, are considered helpful in making an appropriate preoperative diagnosis with endoscopic pancreatic ductal biopsy specimens, thus enabling the surgeon to select the most appropriate surgical procedure.

Adenocarcinoma, Mucinous↗