Biomedical subjects

N Okada

Publications and source records attributed to N Okada.

At least 109 records · Page 6Linked to original sources

PubMed1999

Interaction of kyotorphin and brain peptide transporter in synaptosomes prepared from rat cerebellum: implication of high affinity type H+/peptide transporter PEPT2 mediated transport system.

High-affinity type H+/peptide cotransporter PEPT2 is preferentially expressed in the kidney, and is responsible for reabsorption of di- and tripeptides in epithelial tubules. Interestingly, PEPT2 has been recently cloned from rat brain. However, there is very little information available on the peptide transporter activity in the brain. In the present study, we investigated the interaction of kyotorphin (L-tyrosyl-L-arginine) with the peptide transporter using synaptosomes prepared from rat cerebellum. The activity of the peptide transporter was assessed by measuring the uptake of radiolabeled glycyl-sarcosine (Gly-Sar), which is a prototypical substrate for the peptide transporter, in the presence of H+-gradient. Kyotorphin competitively inhibited the uptake of Gly-Sar with an inhibitory constant (Ki) of 30 +/- 4 microM in rat cerebellum synaptosomes. This uptake property is very close to that of PEPT2. Carnosine (beta-alanyl-L-histidine) also inhibited the uptake of Gly-Sar, on the other hand, TRH did not interact with the peptide transporter. RT-PCR using specific primers revealed that PEPT2 mRNA exists in cerebellum in rat. Taken collectively, these results indicate that the functional peptide transport system in rat cerebellum might be the high affinity transporter PEPT2.

Animals↗

PubMed1999

Induction of lacZ mutation by 7,12-dimethylbenz[a]anthracene in various tissues of transgenic mice.

The induction of gene mutations was examined in MutaMouse after an intraperitoneal injection of 7, 8-dimethylbenz[a]anthracene (DMBA) at 20 mg/kg in a collaborative study participated by four laboratories. Although the DMBA dose used was lower than the level that has been reported to induce micronucleated erythrocytes maximally in several mouse strains, a killing effect appeared after day 9 of the post-treatment interval. Mutations in lacZ transgene were detected by the positive selection assay following in vitro packaging of phage lambda from the genomic DNA of the transgenic animals that survived. The mutant induction was evaluated in the bone marrow, liver, skin, colon, kidney, thymus, and testis 7 to 28 days after the treatment. In the bone marrow, the mutant frequency reached a maximum, approximately a 30-fold increase, 14 days after the treatment and the increased frequency persisted at least up to day 28 of the post-treatment. Induction of mutants was detected in the liver, colon, thymus, and skin to lesser extents. Marginal responses were obtained in the kidney and testis. The slight increases in the mutant frequencies in the kidney and testis observed in some laboratories were within laboratory-to-laboratory or animal-to-animal variations. In contrast to the gene mutation induction in the bone marrow, the frequency of micronucleated reticulocytes increased transiently 3 days after the treatment and returned to a control level before day 8 of the post-treatment. It was suggested that DMBA induced gene mutation is fixed in stem cells depending on cell proliferation while DNA damages responsible for chromosome breakage are not transmitted to progeny cells.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1999

TIP120B: a novel TIP120-family protein that is expressed specifically in muscle tissues.

TATA-binding protein (TBP) forms complexes with various nuclear proteins and plays roles in all eukaryotic transcription. We previously identified TBP-interacting protein 120 (TIP120) from rat liver. TIP120 stimulates in vitro transcription generally. Homologs of TIP120 exist in various higher eukaryotes including D. melanogaster, C. elegans, and A. thaliana. Here, we isolated cDNA of a novel rat TIP120-like protein, named TIP120B. Rat TIP120B was composed of 1,235 amino acids and was 60% identical to the original TIP120 (re-named TIP120A). However, TIP120B gene was expressed specifically in the muscle tissues, which was contrary to the ubiquitous expression of TIP120A. Moreover, TIP120B protein was observed exclusively in the muscle tissues. TIP120B is therefore suggested to be a muscle-specific protein. Northern blot analysis of the mouse embryo revealed that the expression of TIP120B was temporarily increased during the embryogenesis, whereas TIP120A maintained a constant expression level. Pull-down assay using GST-fused TBP demonstrated that TBP specifically associated with TIP120B in the nuclear extract. These results indicate that TIP120B is a muscle-specific TIP120 family protein and can also interact with TBP. TIP120B is supposed to have a specific role in muscle tissues, which may be diffrerent from that of TIP120A.

Amino Acid Sequence↗

PubMed1999

sigma Receptor ligand-induced up-regulation of the H(+)/peptide transporter PEPT1 in the human intestinal cell line Caco-2.

We determined the effects of (+)pentazocine, a selective sigma(1) ligand, on the uptake of glycylsarcosine (Gly-Sar) in the human intestinal cell line Caco-2 which expresses the low affinity/high capacity peptide transporter PEPT1. Confluent Caco-2 cells were treated with various concentrations of (+)pentazocine for desired time (mostly 24 hr). The activity of PEPT1 was assessed by measuring the uptake of [(14)C]Gly-Sar in the presence of a H(+) gradient. (+)Pentazocine increased the uptake of [(14)C]Gly-Sar mediated by PEPT1 in a concentration- and time-dependent manner. Kinetic analyses have indicated that (+)pentazocine increased the maximal velocity (V(max)) for Gly-Sar uptake in Caco-2 cells without affecting the Michaelis-Menten constant (K(t)). In addition, semi-quantitative RT-PCR revealed that treatment of (+)pentazocine increased PEPT1 mRNA in Caco-2 cells in a concentration-dependent manner. These data suggest that sigma(1) receptor ligand (+)pentazocine up-regulates PEPT1 in Caco-2 cells at the level of increased mRNA, causing an increase in the density of the transporter protein in the cell membrane.

Base Sequence↗

PubMed1999

Structure of rat gamma-tubulin and its binding to HP33.

Gamma-tubulin is localized at the microtubule organizing center and is thought to participate in the organizing of the microtubule network. In this study, we isolated a cDNA of rat gamma-tubulin. The rat gamma-tubulin cDNA encoded 451 amino acids, the same number as that of its counterpart in other vertebrates, and its structure was found to be highly conserved in vertebrates. In a previous work, we identified HP33 (hepatocarcinogenesis- and hepatocellular proliferation-related 33-kDa protein) that was localized at the centrosome of hepatic cells and that exhibited MAP-like activity. In vitro GST pull-down assay using highly purified recombinant HP33 and bacterially expressed gamma-tubulin demonstrated that HP33 bound to gamma-tubulin directly. These results suggest that HP33 is localized at the centrosome via association with both the microtubule and its minus end-specific component, gamma-tubulin.

Amino Acid Sequence↗

PubMed1999

Inhibition of a membrane complement regulatory protein by a monoclonal antibody induces acute lethal shock in rats primed with lipopolysaccharide.

Rats pretreated with traces of LPS developed acute fatal shock syndrome after i.v. administration of a mAb that inhibits the function of a membrane complement regulatory molecule. Such a shock was not observed after the administration of large amounts of LPS instead of the mAb following LPS pretreatment. The lethal response did not occur in rats depleted of either leukocytes or complement, and a C5a receptor antagonist was found to inhibit the reaction. Furthermore, LPS-treated rats did not suffer fatal shock following the injection of cobra venom factor, which activates complement in the fluid phase so extensively as to exhaust complement capacity. Therefore, complement activation on cell membranes is a requirement for this type of acute reaction.

Acute Disease↗

PubMed1999

Complement-mediated anti-HIV-1 effect induced by human IgM monoclonal antibody against ganglioside GM2.

HIV-infected cells aberrantly express a high level of antigenic glycosidic structures such as GM2 and Gg4. Some normal sera containing natural IgM Abs to GM2 and/or Gg4 cause C-mediated cytolysis of HIV-infected cells. In the present study we demonstrated that a human IgM anti-GM2 mAb (L55 Ab) can induce cytolysis of HIV-infected cells. Increased GM2 expression by HIV-1 infection of a human T cell line (MOLT4), a human monocyte cell line (U937), and human lymphoblastoid cells was confirmed by immunofluorescence staining with L55 Ab. These infected cells were readily lysed by L55 Ab in the presence of fresh human serum as a C source that alone did not cause cytolysis. L55 Ab also had the ability to destroy HIV-1 particles via C-mediated lysis. By adding L55 Ab together with human C to mixed culture of HIV-infected cells and naive cells, HIV-1 replication was significantly suppressed, and this effect was synergistic when L55 Ab was combined with a reverse transcriptase inhibitor and a proteinase inhibitor. Therefore, a human IgM anti-GM2 mAb may be effective in treating HIV-infected patients, especially when used together with chemotherapeutic agents.

Anti-HIV Agents↗

PubMed1999

Glomerular hypertension as one cause of albuminuria in type II diabetic patients.

AIMS/HYPOTHESIS: Results from animal models of glomerular hypertension have suggested that this disorder is one cause of albuminuria in diabetic nephropathy. We evaluated this hypothesis clinically. METHODS: The subjects were 20 patients with Type II (non-insulin-dependent) diabetes mellitus but without uraemia or hypertension: 8 had normoalbuminuria and 12 had albuminuria (> or = 20 micrograms/min). In the 2-week study, patients were on a diet with ordinary amounts of sodium for 1 week and on a sodium-restricted diet for 1 week. Urinary excretion of sodium and albumin and the systemic blood pressure were measured daily. Intrarenal haemodynamics, in terms of the glomerular pressure and resistance of afferent and efferent arterioles, were calculated from renal clearance, the plasma total protein concentration, and the pressure-natriuresis relation. In 8 of the 12 patients with albuminuria, an angiotensin-converting enzyme inhibitor, cilazapril, was given orally (2 mg/day) and the 2-week study was repeated. RESULTS: In patients with albuminuria, resistance of efferent arterioles and the glomerular pressure were higher than in patients with normoalbuminuria (glomerular pressure, 53 +/- 5 vs 43 +/- 5 mmHg, means +/- SD, p < 0.001). Urinary excretion of albumin correlated (n = 20, r = 0.675, p < 0.001) with the glomerular pressure but not with systemic pressure. The increased glomerular pressure and the albuminuria were decreased by cilazapril but systemic pressure was not. CONCLUSIONS/INTERPRETATION: These findings are consistent with the hypothesis that glomerular hypertension is present in Type II diabetic patients with early nephropathy and can cause albuminuria.

Adult↗

PubMed1999

Functional electrical stimulation for detrusor instability.

The clinical efficacy of functional electrical stimulation (FES) for female detrusor instability (DI) is reviewed. Various types of FES methods (including anogenital long-term stimulation, short-term maximal stimulation, implantable stimulation and transcutaneous stimulation) have been reported. The therapeutic effects of these approaches were similar: the percentage of patients improved has been reported to be in the range 50%-90%. FES for female DI is a non-destructive procedure with a curative effect and very few side effects. This curative effect (re-education) is a major benefit of FES.

Electric Stimulation Therapy↗

PubMed1999

Primary natural killer/T-cell lymphomas of the oral cavity are aggressive neoplasms.

Thirty-four cases of primary non-Hodgkin's lymphoma of the oral cavity were investigated for their clinical findings, histopathological features, immunophenotypes and association with Epstein-Barr virus (EBV). Four cases (12%) were natural killer/T-cell lymphomas, 3 (9%) were T-cell lymphomas and 27 (79%) were B-cell lymphomas. Compared with T- and B-cell lymphomas, NK/T-cell lymphomas had a male predominance (M:F 4:0), and most presented as ulceration of the palate and/or maxillary gingiva. Histologically, the lesions showed diffuse infiltration of medium-sized or large lymphoid tumour cells. Angiocentricity and/or angioinvasion were found in all 4 cases. The immunophenotypes of the NK/T-cell lymphomas were CD3+, CD43+, CD45RO+, CD56+ and TIA-1+. EBV was detected in 2 NK/T-cell lymphomas by in situ hybridization (ISH) and polymerase chain reaction (PCR) methods, and was not detected in T- and B-cell lymphomas. The survival rate of patients with NK/T-cell lymphoma was zero, but the survival rates for patients with T-cell and B-cell lymphomas were 67% and 38%, respectively. It appears that NK/T-cell lymphomas of the oral cavity have a predilection for originating in the palate and maxillary gingiva and are aggressive neoplasms. EBV positivity might be associated with more aggressive behaviour.

Adolescent↗

PubMed1999

Neuropeptide Y overexpression in the preweanling Zucker (fa/fa) rat.

Hypothalamic preproNPY overexpression in the Zucker fatty (fa/fa) rat was examined. In situ hybridization was used to determine the relative level of preproNPY mRNA in the arcuate nucleus of +/+, +/fa, and fa/fa pups aged postnatal day 2 (P2), 5, 9, 12, or 25. The relative optical density (ROD) of probe hybridization in the arcuate, the area of hybridization (A), and the product of ROD x A (a measure of total arcuate preproNPY mRNA hybridization) were measured. Values were normalized to the mean +/fa value within each litter. Initial analysis showed that preproNPY mRNA hybridization (ROD x A) in fa/fa pups was significantly higher than +/fa and +/+ pups on P9, 12, and 25, and significantly higher than +/fa on P5. No significant difference between lean (+/+ and +/fa) genotypes, however, were observed at any age tested. Values from the lean genotypes were, therefore, pooled, and data were normalized to the mean value of lean animals for analysis. This analysis revealed that preproNPY mRNA hybridization in fa/fa pups was higher than lean littermates as early as P2.

Aging↗

PubMed1999

Role of complement regulatory membrane proteins in ischaemia-reperfusion injury of rat gastric mucosa.

BACKGROUND: The role of complement in ischaemia-reperfusion injury has not been well investigated. 5I2 is a monoclonal antibody (mAb) directed against a rat membrane inhibitor of the C3 convertase step, which is the rat counterpart of mouse Crry/p65. 6D1 is a mAb against rat CD59 which inhibits the formation of membrane attack complexes. METHODS: We visualized the tissue distribution of these membrane inhibitors in rat gastrointestinal tract by immunohistochemical staining with the appropriate mAb. Then, we tested the hypothesis that complement regulatory proteins protect rat gastric mucosa against ischaemia-reperfusion stress by using these mAbs. Gastric mucosal integrity was continuously monitored by measuring the blood-to-lumen clearance of [51Cr]-labelled ethylenediaminetetraacetic acid (EDTA) under control conditions, during ischaemia and after reperfusion. RESULTS: Rat 6D1 and 5I2 antigens were both widely distributed and predominantly expressed on smooth muscle and endothelial cells in gastrointestinal tracts. Blockade of complement regulatory proteins with 5I2 and 6D1 mAbs resulted in a significant increase in [51Cr]-EDTA clearance after reperfusion. CONCLUSIONS: These findings support the hypothesis that endogenous complement regulatory proteins may act as important protective factors against ischaemia-reperfusion stress in rat gastric mucosa.

Animals↗

PubMed1999

Crry, a complement regulatory protein, modulates renal interstitial disease induced by proteinuria.

UNLABELLED: Crry, a complement regulatory protein, modulates renal interstitial disease induced by proteinuria. BACKGROUND: Recent studies have suggested a role for urinary complement components in mediating tubulointerstitial damage, which is known to have a good correlation with progression of chronic renal diseases. Although accumulating evidence suggests that complement regulatory proteins play an important protective role in glomeruli, their role in renal tubules remains unclear. In order to establish the role of a complement regulatory protein, Crry, in renal tubular injury, we employed a molecular biological approach to block the expression of Crry in tubules of animals with proteinuria induced with puromycin aminonucleoside nephritis (PAN). Methods and Results. Two different antisense oligodeoxynucleotides (ODNs) against Crry were designed and applied to cultured rat mesangial cells in vitro in order to establish their efficacy. Antisense ODN treatment resulted in decreased expression of Crry protein associated with increased sensitivity to complement attack in cell lysis assays compared with control ODN treatment or no treatment (44.7, 1.50, and 1.34%, respectively). Antisense ODNs did not affect the expression of Thy1 as a control, confirming the specificity of our ODNs. In vivo, we performed selective right renal artery perfusion to administer antisense ODNs to the kidney and showed prominent uptake of ODNs by proximal tubular cells. Reduced expression of Crry protein was demonstrated in proximal tubular cells in antisense ODNs-treated kidneys. Normal rats treated with the antisense ODNs did not show any pathological changes. However, in PAN, rats with massive proteinuria showed increased deposition of C3 and C5b-9 in tubules in antisense-treated kidneys, and histological assessment revealed more severe tubulointerstitial injury in antisense-treated animals compared with controls. CONCLUSION: These results establish a pathogenic role for complement in leading to tubulointerstitial injury during proteinuria and, to our knowledge for the first time, show a protective role of a complement regulatory protein, Crry, in renal interstitial disease.

Animals↗

PubMed1999

Comparative study of two ribozymes and DNA-enzyme against the same RNA target.

Two ribozymes, hammerhead ribozyme and hairpin ribozyme, and a DNA-enzyme were designed to cleave a same RNA target, the same site of the rat complement regulatory factor 512 antigen mRNA. The kinetic properties of these RNA-cleaving enzymes were measured and compared under the same conditions, using multiple turnover kinetics and competition kinetics. The catalytic efficiencies of these enzymes, and also the order of these enzymes will be discussed.

Animals↗

PubMed1999

Genealogy of families of SINEs in cetaceans and artiodactyls: the presence of a huge superfamily of tRNA(Glu)-derived families of SINEs.

Several novel (sub)families of SINEs were isolated from the genomes of cetaceans and artiodactyls, and their sequences were determined. From comparisons of diagnostic nucleotides among the short interspersed repetitive elements (SINEs) in these (sub)families, we were able to draw the following conclusions. (1) After the divergence of the suborder Tylopoda (camels), the CHRS family of SINEs was newly created from tRNA(Glu) in a common ancestor of the lineages of the Suina (pigs and peccaries), Ruminantia (cows and deer), and Cetacea (whales and dolphins). (2) After divergence of the Suina lineage, the CHR-1 SINE and the CHR-2 SINE were generated successively in a common ancestor of ruminants, hippopotamuses, and cetaceans. (3) In the Ruminantia lineage, the Bov-tA SINE was generated by recombination between the CHR-2 SINE and Bov-A. (4) In the Suina lineage, the CHRS-S SINE was generated from the CHRS SINE. (5) In this latter lineage, the PRE-1 family of SINEs was created by insertion of part of the gene for tRNA(Arg) into the 5' region of the CHRS-S family. The distribution of a particular family of SINEs among species of artiodactyls and cetaceans confirmed the most recent conclusion for paraphyly of the order Artiodactyla. The present study also revealed that a newly created tRNA(Glu)-derived family of SINEs was subjected both to recombination with different units and to duplication of an internal sequence within a SINE unit to generate, during evolution, a huge superfamily of tRNA(Glu)-related families of SINEs that are now found in the genomes of artiodactyls and cetaceans.

Animals↗

PubMed1999

Retropositional parasitism of SINEs on LINEs: identification of SINEs and LINEs in elasmobranchs.

Some previously unidentified short interspersed repetitive elements (SINEs) and long interspersed repetitive element (LINEs) were isolated from various higher elasmobranchs (sharks, skates, and rays) and characterized. These SINEs, members of the HE1 SINE family, were tRNA-derived and were widespread in higher elasmobranches. The 3'-tail region of this SINE family was strongly conserved among elasmobranchs. The LINEs, members of the HER1 LINE family, encoded an amino acid sequence similar to that encoded by the chicken CR1 LINE family, and they contained a strongly conserved 3'-tail region in the 3' untranslated region. This tail region of the HER1 LINE family was almost identical to that of the HE1 SINE family. Thus, the HE1 SINE family and the HER1 LINE family provide a clear example of a pair of SINEs and LINEs that share the same tail region. Conservation of the secondary structures of the tail regions, as well as of the nucleotide sequences, between the HE1 SINE family and HER1 LINE family during evolution suggests that SINEs utilize the enzymatic machinery for retroposition of LINEs through the recognition of higher-order structures of the conserved 3'-tail region. A discussion is presented of the parasitism of SINEs on LINEs during the evolution of these retroposons.

3' Untranslated Regions↗

PubMed1999

67Ga whole-body scintigraphy in the evaluation of head and neck squamous cell carcinoma.

The diagnostic accuracy of 67Ga-citrate whole-body scintigraphy for the evaluation of primary tumours, local recurrences, cervical node metastases and distant metastases was investigated in patients with head and neck squamous cell carcinoma (SCC). Altogether, 102 67Ga-citrate whole-body scans were performed on 83 patients with head and neck SCC using a dual-headed gamma camera. The results were compared with those of computed tomography (CT) and final diagnosis. 67Ga scintigraphy correctly diagnosed 28 of 29 primary lesions, a sensitivity of 97% (CT = 97%). For tumour recurrence, 67Ga scintigraphy correctly diagnosed 13 of 15 recurrences and 53 of 58 instances of no recurrence, giving a sensitivity of 87% and a specificity of 91% (CT = 80 and 62%, respectively). Scintigraphy correctly diagnosed 23 of 31 metastatic sides of the neck and all 173 negative sides of the neck, giving a sensitivity of 74% and a specificity of 100% (CT = 90 and 84%, respectively). Finally, scintigraphy correctly diagnosed all 12 distant metastases as well as 86 of 90 cases of no metastasis, resulting in a sensitivity of 100% and a specificity of 96%. Furthermore, in five patients, distant metastases were initially detected on 67Ga scintigraphy. In conclusion, 67Ga whole-body scintigraphy with a dual-headed camera resulted in a high sensitivity and specificity for the detection of primary lesions, recurrences and distant metastases of head and neck SCC. Although the sensitivity for detecting neck node metastases was relatively low, the specificity was high. Thus, 67Ga scintigraphy is an effective technique for the evaluation of head and neck SCC, especially tumour recurrence and distant metastases.

Adolescent↗

PubMed1999

Characterization of mouse DAF on transfectant cells using monoclonal antibodies which recognize different epitopes.

Several membrane proteins prevent host cells from homologous complement attack. In humans, one such protein, decay-accelerating factor (DAF), exists as two isoforms, a GPI anchored form and a secreted form, which are generated by alternative splicing. DAF in mouse is also expressed as two isoforms, a GPI anchored form (GPI-DAF) and a transmembrane form (TM-DAF), which are produced from two separate genes. In this study, we transfected cDNA of mouse GPI-DAF or TM-DAF into Chinese hamster ovary (CHO) cells. Both isoforms of DAF on CHO cells were shown to regulate mouse complement C3 deposition mediated by the classical and alternative pathways and the inhibitory activity of both isoforms was species restricted. The two mouse DAF isoforms were effective against rat complement but not against human and guinea pig complement. Furthermore, we produced hamster mAbs to mouse DAF using GPI-DAF transfectant cells and established seven unique mAbs (RIKO-1-7). Western blotting analysis using RIKO-3, which reacts with both GPI-DAF and TM-DAF, and RIKO-4, which is an anti-GPI-DAF specific mAb, indicated that GPI-DAF was expressed on erythrocytes, spleen and testis, and that TM-DAF was expressed only in testis.

Animals↗