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Biomedical subjects

T Iida

Publications and source records attributed to T Iida.

At least 163 records · Page 9Linked to original sources
PubMed1998

Method for the separation of the unconjugates and conjugates of chenodeoxycholic acid and deoxycholic acid by two-dimensional reversed-phase thin layer chromatography with methyl beta-cyclodextrin.

A simple and efficient method for the separation of individual unconjugated bile acids and their glycine- and taurine-amidated, 3-sulfated, 3-glucosylated and 3-glucuronidated conjugates is described. The method involves the use of a two-dimensional (2D) reversed-phase (RP) high-performance thin-layer chromatographic (HPTLC) technique with methyl beta-cyclodextrin (Me-beta-CD). Five major unconjugated bile acids, chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), ursodeoxycholic acid and lithocholic acid, and their conjugates were examined as the solutes. A high degree of separation of individual bile acids in each homologous series was achieved on a RP-HPTLC plate by developing with aqueous methanol in the first dimension and the same solvent system containing Me-beta-CD in the second dimension. In particular, all of the six 'difficult-to-separate' pairs, unconjugated CDCA and DCA and their conjugated forms with glycine, taurine, sulfuric acid, D-glucose and D-glucuronic acid, were effectively resolved by adding Me-beta-CD in the aqueous mobile phases with the formers having larger mobilities than the latter. The application of this 2D inclusion RP-HPLC method to the separation of glycine-conjugated bile acids in human bile is also described. The present method would be useful for separating and characterizing these bile acids present in biological materials.

Bile↗
PubMed1998

Establishment of an apoptosis-resistant and growth-controllable cell line by transfecting with inducible antisense c-Jun gene.

F-MEL cells were transfected with the c-jun antisense gene located downstream of a glucocorticoid-inducible MMTV promoter, and the obtained cells were named c-jun AS cells. When the c-jun AS cells were treated with dexamethasone (DEX) in DMEM supplemented with 10% serum, the growth of the cells was completely suppressed for a duration of 16 days with a high cell viability exceeding 86%. The c-jun expression in the c-jun AS cells was suppressed moderately in the absence of DEX and strongly in the presence of DEX. The c-jun AS cells grew well and reached a density of 10(6) cells/mL without supplementation of any serum components. Viability was greater than 80% after the cells had been cultured for 8 days in the absence of DEX. The c-jun AS cells stayed at a constant cell density and high viability above 80% for 8 days when they were cultured in the presence of DEX under serum deprivation. In contrast, the wild type F-MEL cells were unable to grow and died by apoptosis in 3 days under serum deprivation. Internucleosomal cleavage of DNA, a landmark of apoptosis, was clearly detectable. Thus the c-jun AS cell line that is resistant to apoptosis induced by serum deprivation and can reversibly and viably be growth-arrested was established. A dual-signal model was proposed to explain the experimental result, the interlinked regulation of apoptosis, and growth by c-jun.

Animals↗
PubMed1998

Complete nucleotide sequences of 93-kb and 3.3-kb plasmids of an enterohemorrhagic Escherichia coli O157:H7 derived from Sakai outbreak.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7, derived from an outbreak in Sakai city, Japan in 1996, possesses two kinds of plasmids: a 93-kb plasmid termed pO157, found in clinical EHEC isolates world-wide and a 3.3-kb plasmid termed pOSAK1, prevalent in EHEC strains isolated in Japan. Complete nucleotide sequences of both plasmids have been determined, and the putative functions of the encoded proteins and the cis-acting DNA sequences have been analyzed. pO157 shares strikingly similar genes and DNA sequences with F-factor and the transmissible drug-resistant plasmid R100 for DNA replication, copy number control, plasmid segregation, conjugative functions and stable maintenance in the host, although it is defective in DNA transfer by conjugation due to the truncation and deletion of the required genes and DNA sequences. In addition, it encodes several proteins implicated in EHEC pathogenicity such as an EHEC hemolysin (HlyA), a catalase-peroxidase (KatP), a serine protease (EspP) and type II secretion system. pOSAK1 possesses a ColE1-like replication system, and the DNA sequence is extremely similar to that of a drug-resistant plasmid, NTP16, derived from Salmonella typhimurium except that it lacks drug resistance transposons.

DNA, Bacterial↗
PubMed1998

Isozyme function of n-alkane-inducible cytochromes P450 in Candida maltosa revealed by sequential gene disruption.

An n-alkane-assimilating yeast Candida maltosa contains multiple n-alkane-inducible forms of cytochromes P450 (P450alk), which can be assumed to catalyze terminal hydroxylation of n-alkanes in the assimilation pathway. Eight structurally related P450alk genes have been identified. In the present study, the function of four major isoforms of P450alk (encoded by ALK1, ALK2, ALK3, and ALK5 genes) was investigated by sequential gene disruption. Auxotrophic markers used for the selection of disrupted strains were regenerated repeatedly through either mitotic recombination between heterozygous alleles of the diploid genome or directed deletion of the marker gene, to allow sequential gene disruptions within a single strain. The strain depleted of all four isoforms could not utilize n-alkanes for growth, providing direct evidence that P450alk is essential for n-alkane assimilation. Growth properties of a series of intermediate disrupted strains, plasmid-based complementation, and enzyme assays after heterologous expression of single isoforms revealed (i) that each of the four individual isoforms is alone sufficient to allow growth on long chain n-alkane; (ii) that the ALK1-encoding isoform is the most versatile and efficient P450alk form, considering both its enzymatic activity and its ability to confer growth on n-alkanes of different chain length; and (iii) that the ALK5-encoding isoform exhibits a rather narrow substrate specificity and thus cannot support the utilization of short chain n-alkanes.

Alkanes↗
PubMed1998

Cloning and characterization of an n-alkane-inducible cytochrome P450 gene essential for n-decane assimilation by Yarrowia lipolytica.

A gene encoding cytochrome P450 involved in n-alkane utilization was cloned from an n-alkane assimilating yeast, Yarrowia lipolytica CX161-1B. The RT-PCR was performed on the mRNA prepared from the cells grown on n-alkane as a template using degenerated PCR primers designed for the conserved amino acid sequences of the CYP52 family. The RT-PCR amplified fragment was then used as a probe to isolate genes coding for P450 of the CYP52 family from the genomic DNA library of the strain CX161-1B. The nucleotide sequence of one of the positive clones was determined. An open reading frame which had the same nucleotide sequence as the RT-PCR-amplified fragment was identified. It was of 523 amino acid residues, 60.2 kDa in molecular mass, and had 30-45% sequence identity with the other members of the CYP52 family of Candida species so far analysed. The expression of the P450 gene that was named as YlALK1 was induced by n-tetradecane and repressed by glycerol. A YlALK1 gene disruptant did not grow well on n-decane, but grew on longer-chain n-alkanes such as hexadecane as a sole carbon source. Introduction of YlALK1 on a plasmid to the disruptant restored the decane assimilation. These results suggest that the YlALK1 gene product is the major P450A1k to metabolize short-chain n-alkanes such as decane and dodecane in Y. lipolytica.

Alkanes↗
PubMed1998

Fas antigen expression and apoptosis of lymphocytes in macaques infected with simian immunodeficiency virus strain mac.

To investigate the role of apoptosis in the pathogenesis of HIV infection we used macaques infected with simian immunodeficiency virus (SIV) as a primate model and examined the characteristics of the apoptosis of lymphocytes in SIV mac-infected macaques. In vitro apoptosis was more strongly induced in peripheral blood mononuclear cells (PBMC) from SIV mac239-infected macaques than those from uninfected controls. We found that the frequency of Fas antigen-positive cells was higher in PBMC from SIV mac-infected macaques than from uninfected controls, and in vitro apoptosis of PBMC was suppressed by an inhibitor of the interleukin-1 beta converting enzyme (ICE) family proteases. In biopsied lymph nodes, the number of apoptotic nuclei in T cell-dependent areas was higher in SIV mac-infected macaques than in uninfected controls. A higher number of apoptotic nuclei in lymph nodes of SIV mac-infected macaques was observed in the stage of persistent general lymphadenopathy than in those with AIDS-related complex, while there was no significant difference in the extent of apoptosis of cultured PBMC among the SIV mac-infected macaques. These results suggest that in vitro apoptosis is mediated by the Fas/Fas ligand and ICE system and that apoptosis in lymph nodes may be more closely related to the stage of SIV mac infection than is that of cultured PBMC.

Animals↗
PubMed1998

Structure-retention correlation of isomeric bile acids in inclusion high-performance liquid chromatography with methyl beta-cyclodextrin.

The structure-retention correlation of various C24 bile acid isomers was studied by the addition of methyl beta-cyclodextrin (Me-beta-CD) to mobile phases in reversed-phase high-performance liquid chromatography (HPLC). The compounds examined include a series of monosubstituted bile acids related to cholanoic acids differing from one another in the position and configuration of an oxygen-containing function (hydroxyl or oxo group) at the position C-3, C-6, C-7, or C-12 and the stereochemistry of the A/B-ring fusion (trans 5 alpha-H and cis 5 beta-H) in the steroid nucleus. The inclusion HPLC with Me-beta-CD was also applied to biologically important 4 beta- and 6-hydroxylated bile acids substituted by three to four hydroxyl groups in the 5 beta-steroid nucleus. These bile acid samples were converted into their fluorescence prelabeled 24-pyrenacyl ester derivatives and chromatographed on a Capcell Pak C18 column eluted with methanol-water mixtures in the presence or absence of 5 mM Me-beta-CD. The effects of Me-beta-CD on the retentions of each compound were correlated quantitatively to the decreasing rate of capacity factors and the relative strength of host-guest interactions. On the basis of the retention data, specific and nonspecific hydrogen-bonding interactions between the bile acids and the Me-beta-CD were discussed.

Bile Acids and Salts↗
PubMed1998

Indocyanine green angiographic features of idiopathic submacular choroidal neovascularization.

PURPOSE: To clarify the indocyanine green angiographic features of idiopathic submacular choroidal neovascularization. METHODS: We performed fluorescein and indocyanine green angiography in 16 eyes of 16 patients (nine men, seven women; mean age +/- SD, 35.6 +/- 6.9 years; range, 24 to 48 years) with idiopathic submacular choroidal neovascularization. During the mean follow-up of 11.4 +/- 7.9 months (range, 1 to 28 months), angiography was repeated (mean, 2.6 +/- 0.5 times; range, 2 to 3 times) in 12 eyes and performed only once in four eyes. RESULTS: In the early phase of indocyanine green angiography, a network of choroidal neovascularization was observed in 11 of the 16 eyes with well-defined choroidal neovascularization seen by fluorescein angiography. Choroidal hyperfluorescent areas were noted in 10 of the 16 eyes in the late-phase angiography. Three of the 10 eyes showed focal dilatation of choroidal veins within the hyperfluorescent areas. Choroidal neovascular membrane was located within the hyperfluorescent areas in eight of the 10 eyes with choroidal hyperfluorescent areas. A dark rim surrounding choroidal neovascularization was observed in 13 of the 16 eyes at the initial examination. Indocyanine green angiography was repeated in nine of the 13 eyes and in the other three eyes without dark rim initially. During the follow-up period, the dark rim became prominent in eight of the nine eyes and developed in one eye without dark rim initially. Choroidal neovascular membrane regressed in these nine eyes. Choroidal neovascularization remained active or enlarged in three eyes in which the dark rim was stationary or invisible. CONCLUSIONS: The dark rim surrounding the choroidal neovascularization in indocyanine green angiograms appeared to reflect regression of idiopathic choroidal neovascularization. Choroidal vascular abnormalities such as hyperfluorescent areas or focal venous dilatation seem to be the background lesion predisposing to the choroidal neovascularization.

Adult↗
PubMed1998

Nicking sites in a subunit of cholera toxin and Escherichia coli heat-labile enterotoxin for Vibrio cholerae hemagglutinin/protease.

We analyzed the nicking site of the A subunit of Escherichia coli heat-labile enterotoxin for hemagglutinin/protease produced by Vibrio cholerae non-O1 (NAG-HA/P). The determined nicking site was the Thr193-Ile194 junction, which was distinct from that for a protease of V. cholerae (Ichinose et al., European Journal of Epidemiology 8, 743-747, 1992). We further analyzed proteolytic cleavage by NAG-HA/P of a synthetic peptide corresponding to the nicking region of cholera toxin A subunit and determined the cleavage site to be preferentially between Ser194 and Met195, and in addition between Ser193 and Ser194.

Bacterial Toxins↗
PubMed1998

Nuclear factor kappa B dependent induction of gamma glutamylcysteine synthetase by ionizing radiation in T98G human glioblastoma cells.

Glioblastoma is one of the most malignant of all neoplasms, and often shows resistance to chemotherapy and radiation therapy. Ionizing radiation activates transcriptional factors, such as nuclear factor kappa-B (NF-kappa B). Previously we found that glutathione (GSH) synthesis is induced by cytokines mediated by NF-kappa B (Urata et al. J. Biol. Chem., 1996). Here, we present direct evidence that NF-kappa B activated by ionizing radiation induces the expression of gamma-glutamylcysteine synthetase (gamma-GCS), the rate limiting enzyme of GSH synthesis, using T98G human glioblastoma cells. T98G cells have approximately 14-times the level of intracellular GSH of NB9 cells, radiation-sensitive neuroblastoma cells. In T98G cells, 30-Gy of ionizing radiation was required for the activation of NF-kappa B on an electrophoretic mobility shift assay and the induction of gamma-GCS mRNA on Northern blots and a nuclear run-on assay. However, when T98G cells were treated with buthionine sulfoximine, 3-Gy of ionizing radiation stimulated the DNA-binding activity of NF-kappa B and the expression of gamma-GCS. We constructed chimeric genes containing various regions of gamma-GCS promoter gene and the coding region for Luciferase. T98G cells transiently transfected with a plasmid containing the gamma-GCS promoter-luciferase construct showed increased luciferase activity when treated with ionizing radiation. The luciferase activity stimulated by ionizing radiation was found in the gamma-GCS promoter containing the NF-kappa B binding site, whereas not in that containing its mutated site. These results suggest that GSH synthesis is upregulated by ionizing radiation mediated by NF-kappa B and a high concentration of GSH in T98G cells causes downregulation of the NF-kappa B-DNA binding activity in response to ionizing radiation. The irresponsiveness of the intracellular signal transduction cascade to irradiation may be a factor in the resistance of T98G cells to radiation therapy.

Acetylcysteine↗
PubMed1998

Nicardipine normalizes elevated levels of antioxidant activity in response to xanthine oxidase-induced oxidative stress in hypertensive rat heart.

It has been reported that the production of oxygen radicals mediated by xanthine oxidase (XO) is stimulated in hypertensive cardiovascular endothelium, suggesting involvement of oxidative stress in pathogenesis of hypertension. In this study we estimated the effect of nicardipine, a calcium blocker, on the oxidative stress and antioxidant activities in left ventricles from spontaneously hypertensive rat (SHR) and stroke-prone SHR (SHRSP). The activity of XO increased 3.5-fold in SHR and 6.2-fold in SHRSP compared to that in normal controls (WKY). Interestingly, the levels of glutathione (GSH) and the activity of its synthesizing enzyme (gamma-glutamylcysteine synthetase, gamma-GCS) elevated concomitantly in SHR and SHRSP: the level of GSH increased 1.2-fold in SHR and 1.3-fold in SHRSP. The activity of gamma-GCS was elevated 1.5-fold in SHR and 2.4-fold in SHRSP, accompanying an increase in the expression of its mRNA. Treatment of these rats with nicardipine, for 4 weeks improved blood pressure, from 176 +/- 10 to 140 +/- 8 mmHg in SHR, and from 201 +/- 11 to 167 +/- 5 mmHg in SHRSP, respectively, and decreased wet weight of heart, levels of GSH, and the activities of XO and gamma-GCS. Nicardipine reduced the expression of gamma-GCS mRNA. Collectively, these results suggest that reactive oxygen species produced by XO in hypertensive rat heart cause induction of the expression of gamma-GCS and nicardipine plays a role in reducing the oxidative stress in hypertensive heart.

Animals↗
PubMed1998

The effects of a thromboxane A2 synthesis inhibitor and a prostaglandin I2 analogue on experimental acute necrotizing pancreatitis in rats.

To elucidate the role of thromboxane A2 (TxA2) and prostaglandin I2 (PGI2) in acute necrotizing pancreatitis (ANP) in rats and to determine the effect of the TxA2 synthesis inhibitor OKY-046 and the PGI2 analogue OP-2507, the levels of two prostanoids (TxB2, 6-keto PGF1alpha) and two types of phospholipase A2 (PLA2) activity (cytosolic and secretory) were measured in plasma and three tissues (pancreas, lung, and kidney) after injection of a mixed solution of 5% sodium taurocholate and 0.1% trypsin into the pancreatic duct to induce ANP. The survival rate 24 h after inducing ANP was 33.3% in the nontreated group, versus 83.3 and 58.3% in the groups treated with OKY-046 and OP-2507, respectively. Only the group treated with OKY-046 showed significant improvement compared with the nontreated group. The plasma, pancreatic, and pulmonary TxB2 levels decreased significantly in the group treated with OKY-046, and the histopathological changes were not as severe. The levels of pancreatic and pulmonary cytosolic PLA2 activities decreased, and plasma and pancreatic secretory PLA2 activities also decreased. In conclusion, the levels of both types of PLA2 activity and TxA2 production decreased, and the survival rate improved as a result in the group treated with OKY-046, but OP-2507 had no effect on ANP. TxA2 and two types of PLA2 activity play an important role in the process of aggravation of acute pancreatitis.

6-Ketoprostaglandin F1 alpha↗
PubMed1998

Two cases of unilateral axillary Paget's disease.

We herein report two cases of unilateral axillary Paget's disease. Extramammary Paget's disease commonly occurs in the anogenital area. Unilateral axillary occurrence is relatively rare. One defect was reconstructed with a pedicled scapular flap, and the other was reconstructed with split-skin graft after excision of the tumor. No recurrence was seen in either patient three years postoperatively. However, although the patient who had been reconstructed with a pedicled flap recovered uneventfully, the other patient, who had been reconstructed with a split-skin graft, suffered from axillary contracture due to the shrinkage of the grafted skin after surgery. A skin graft requires fixation for a long period to prevent shrinkage, which causes axillary contracture. Therefore, from the viewpoint of quality of life, reconstruction using flaps is recommended for axillary lesions.

Aged↗
PubMed1998

Bullous pemphigoid associated with Shy-Drager syndrome.

We report a patient with Shy-Drager syndrome who developed multiple tense blisters mainly on the extremities. Circulating anti-basement membrane zone autoantibodies were detected by the indirect immunofluorescence method. Immunoblot analysis using normal human epidermal extracts demonstrated that this patient's serum reacted only with 230 kD bullous pemphigoid antigen (BPAG1). Concerning the pathoetiology of the association of bullous pemphigoid and Shy-Drager syndrome, we discuss a sequence similarity between BPAG1 and dystonin, a candidate gene for dystonia musculorum.

Autoantibodies↗
PubMed1998

A case of nonscarring inflammatory epidermolysis bullosa acquisita: characterization of IgG autoantibodies by immunofluorescence, immunoblotting and immunogold electron microscopy.

We report a case of nonscarring inflammatory epidermolysis bullosa acquisita in a 59-year-old Japanese woman. She developed blisters and erosions on her lip, trunk and extremities. Sodium aurothiomalate was effective for the skin lesions. The patient had been free from bullous skin lesions for the last 13 years and had shown no scarring. Indirect immunofluorescence (IF) study on 1 M NaCl-split skin revealed IgG autoantibodies against the dermal side of the split skin. Immunoblotting using normal human dermal extracts disclosed IgG autoantibodies reactive with the 290 and 145 kD antigens. Circulating IgG autoantibodies were deposited on the lamina densa by immunoelectron microscopy. IF mapping using several antibodies for the components of the basement membrane zone revealed blister formation at the lamina densa. These results suggest that the cleavage at the lamina lucida does not necessarily exclude the diagnosis of EBA and that the definite diagnosis of EBA should be confirmed by immunoblotting or immunoelectron microscopic study.

Antirheumatic Agents↗
PubMed1998

Polymorphisms of HLA class II genes in Japanese patients with pemphigus vulgaris.

Pemphigus vulgaris (PV) is an autoimmune disease mediated by autoantibodies that bind to a keratinocyte adhesion molecule, desmoglein-3. The purpose of this study was to identify critical amino acid residues of PV-associated HLA class II genes. Haplotype and allele distributions, along with molecular polymorphisms, of HLA class II genes were analyzed based on the polymerase chain reaction-restriction fragment length polymorphism in 17 Japanese PV patients. Each patient had one or two alleles of DRB1*04 (*0403, *0404, *0406) or DRB1*14 (*1401, *1405, *1406) subtypes. All DRB1*04 and DRB1*14 alleles carried by PV patients with different ethnic backgrounds reported to date, including DRB1*0402, which confers strong susceptibility to PV among Jewish populations, have amino acid residues Phe26, Leu67 or Ile67, and Val86, as well as hydrophilic amino acid residues at positions 70 and 71 of the DRB1 beta chain.

Adult↗