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N Okada

Publications and source records attributed to N Okada.

At least 127 records · Page 7Linked to original sources
PubMed1999

TTG as the initiation codon of Salmonella slyA, a gene required for survival within macrophages.

The slyA gene, which has been implicated in the virulence of Salmonella serovar Typhimurium and its survival in macrophages, is widely distributed among different Salmonella serovars. In this study, we cloned and sequenced the translational initiation region of the slyA gene from nine different serovars and found sequence differences in the previously proposed ATG initiation codon but not in a TTG triplet, another putative initiation codon in the slyA gene. Therefore, we determined the actual translational initiation site of the slyA gene by analyzing slyA genes with defined mutation in either the ATG or TTG sequences in an in vitro translation assay and a quantitative hemolytic assay in Escherichia coli. The replacement of TTG by TTC in the slyA gene significantly reduced both the amount of protein synthesized and the hemolytic activity of a transformed strain of E. coli, while replacement of ATG by ATC had no effect in these assays. In addition, the amino acid sequence analysis of the His-tagged SlyA protein showed that it was identical with the amino acid sequence deduced from the 5' end of the slyA gene with a TTG initiation codon. Our results suggest that TTG serves as the translational initiation codon for the slyA gene of Salmonella.

Amino Acid Sequence↗
PubMed1999

mRNA expression of complement components and regulators in rat arterial smooth muscle cells.

The presence of C5b-9 complexes, some complement regulators, and abundant cytokines in atherosclerotic lesions has been reported. However, it is unclear whether these complement-associated proteins are produced by vascular smooth muscle cells (SMCs) and how they are influenced by the cytokines. In the present study, we demonstrated, by the reverse transcription-polymerase chain reaction method, the mRNA expression of complement components (C3, C4, and C5) and membrane regulators (decay-accelerating factor, membrane cofactor protein, Crry, and CD59) in cultured SMCs derived from the rat carotid artery. The expression of C9 mRNA was also induced upon stimulation by interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) and/or lipopolysaccharide (LPS). Northern blot analysis showed that the mRNA expression of C3, C4, DAF and Crry was up-regulated, but that of CD59 was down-regulated by IFN-gamma, TNF-alpha and/or LPS alone or by synergy. The increase of C3 mRNA by TNF-alpha or LPS and that of C4 mRNA by IFN-gamma was induced in a dose-dependent manner. The results indicate that the arterial SMCs of rat have the ability to produce complement components and regulators, which is affected by cytokines and/or LPS. Since atherosclerosis is characterized by the intimal proliferation of SMCs, the complement system including its regulators may be involved in the pathogenesis of the disease.

Animals↗
PubMed1999

Arginine carboxypeptidase (CPR) in human plasma determined with sandwich ELISA.

There are two types of carboxypeptidases present in human blood, carboxypeptidase N (CPN) and arginine carboxypeptidase (CPR). CPR is generated during coagulation from a precursor (proCPR) which can be converted to the active form by trypsin in vitro. Since it is difficult to distinguish the two types of carboxypeptidases in human blood by the measurement of enzyme activity, we established a quantitative sandwich ELISA by which CPR can be quantitated. The amount of CPR in plasma, fresh serum and heated serum were essentially the same. Therefore the ELISA assay does not distinguish proCPR, activated CPR and inactivated CPR. With the ELISA method, CPR was quantitated in plasma from fifty patients with rheumatoid arthritis and eleven patients with severe hepatitis as well as healthy individuals. The amount of CPR in plasma obtained from patients with rheumatoid arthritis was not found to be lower than that of normal subjects. Furthermore, the patients who suffered severe hepatitis and had very low levels of CPR-total were fatal. This suggests that a decrease of CPR level might be a good indication of a patient's prognosis to death by hepatitis.

Antibodies, Monoclonal↗
PubMed1999

Human IgM monoclonal antibody to ganglioside GM2 and complement suppress virus propagation in ex vivo cultures of lymphocytes from HIV-1 infected patients.

HIV-1 infection induces aberrant ganglioside GM2 expression on infected cell lines, and human IgM anti-GM2 monoclonal antibody (L55 Ab) together with normal fresh human serum (FHS) as a source of complement causes complement mediated cytolysis of HIV-1 infected cells as well as HIV-1 particles. We report here that high expression of GM2 was also detected on HIV-1 infected lymphocytes from HIV-1 seropositive patients. L55 Ab effectively suppressed the generation of HIV in the presence of FHS in primarily cultured lymphocytes from HIV-1 infected patients in ex vivo experiments, and the suppression was enhanced additively by AZT. These data suggest that L55 Ab may increase the therapeutic effect of chemotherapy.

Antibodies, Monoclonal↗
PubMed1999

Intracellular interleukin-1 alpha production in human gingival fibroblasts is differentially regulated by various cytokines.

Interleukin-1 (IL-1) may play a critical role in immune and inflammatory responses in inflamed gingiva, and it is synthesized by a wide variety of host cells. In this study, we examined the regulatory effects of various cytokines on bioactive membrane IL-1 and intracellular IL-1 alpha production in cultured human gingival fibroblasts (HGF). Recombinant human (rh) IL-1 beta stimulated membrane IL-1 activity, which was mainly attributed to IL-1 alpha. rhIL-1 beta and rh tumor necrosis factor (TNF)-alpha stimulated HGF to produce intracellular IL-1 alpha, whereas rh interleukin-6 (IL-6), rh interleukin-4 (IL-4), and rh interferon (IFN)-gamma did not do so. Intracellular IL-1 alpha production induced by rhIL-1 beta or rhTNF-alpha may be partially related to protein kinase C (PKC) activation, because rhIL-1 beta or rhTNF-alpha-induced intracellular IL-1 alpha production was stimulated by pre-treatment with 12-o-tetradecanoylphorbol 13-acetate (TPA), a PKC activator, but was suppressed by the pre-treatment with 1-(5-isoquinoline-sulfonyl) -2-methylpiperazine dihydrochloride (H-7), which is a PKC inhibitor. rhIL-4 inhibited rhIL-1 beta- or rhTNF-alpha-induced intracellular IL-1 alpha production, but rhIL-6 had no effect on this production. Pre-treatment with rh IFN-gamma remarkably enhanced intracellular IL-1 alpha production induced by subsequent treatment with rhIL-1 beta or rhTNF-alpha. Simultaneous treatment with rhIFN-gamma and rhIL-1 beta inhibited rhIL-1 beta-induced intracellular IL-1 alpha production, but co-treatment with rhIFN-gamma and rhTNF-alpha enhanced rhTNF-alpha-induced intracellular IL-1 alpha production. These results suggest that in inflamed gingiva, pro-inflammatory cytokines such as IL-1 beta and TNF-alpha may induce bioactive intracellular IL-1 alpha production in human gingival fibroblasts and that this production can be differentially modulated by T-cell-derived cytokines such as IFN-gamma or IL4.

Adult↗
PubMed1999

Enhanced permeability of insulin across the rat intestinal membrane by various absorption enhancers: their intestinal mucosal toxicity and absorption-enhancing mechanism of n-lauryl-beta-D-maltopyranoside.

We have examined the in-vitro permeability characteristics of insulin in the presence of various absorption enhancers across rat intestinal membranes and have assessed the intestinal toxicity of the enhancers using an in-vitro Ussing chamber method. The absorption enhancing mechanism of n-lauryl-beta-D-maltopyranoside was studied also. The permeability of insulin across the intestinal membranes was low in the absence of absorption enhancers. However, the permeability was improved in the presence of enhancers such as sodium glycocholate and sodium deoxycholate in the jejunum, and sodium glycocholate, sodium deoxycholate, n-lauryl-beta-D-maltopyranoside, sodium caprate and ethylenediaminetetraacetic acid (EDTA) in the colon. Overall, the absorption enhancing effects were greater on the colonic membrane than on the jejunal membrane. The intestinal membrane toxicity of these enhancers was characterized using the release of cytosolic lactate dehydrogenase from the colonic membrane. A marked increase in the release of lactate dehydrogenase was observed in the presence of sodium deoxycholate and EDTA. The release of lactate dehydrogenase in the presence of these absorption enhancers was similar to that seen with sodium dodecyl sulphate (SDS), used as a positive control, indicating high toxicity of these enhancers to the intestinal membrane. In contrast, sodium glycocholate and sodium caprate caused minor releases of lactate dehydrogenase, similar to control levels, suggesting low toxicity. In addition, the amount of lactate dehydrogenase in the presence of n-lauryl-beta-D-maltopyranoside was much less than that seen with sodium deoxycholate, EDTA and SDS. Therefore, sodium glycocholate, sodium caprate and n-lauryl-beta-D-maltopyranoside are useful absorption enhancers due to their high absorption enhancing effects and low intestinal toxicity. To investigate the absorption enhancing mechanisms of n-lauryl-beta-D-maltopyranoside, the transepithelial electrical resistance (TEER), voltage clamp experiments and the circular dichroism spectra were studied. n-Lauryl-beta-D-maltopyranoside decreased the TEER values in a dose-dependent manner, suggesting that the enhancer may open the tight junctions of the epithelium, thereby increasing the permeability of insulin via a paracellular pathway. This speculation was supported by the findings that 20 mM n-lauryl-beta-D-maltopyranoside produced a greater increase in the paracellular flux rate than in the transcellular flux rate by the voltage clamp studies. Evaluating the circular dichroism spectra we found that insulin oligomers were not dissociated to monomers by the addition of n-lauryl-beta-D-maltopyranoside, but dissociation did occur with the addition of sodium glycocholate. Thus, the dissociation of insulin was not a major factor in the absorption enhancing effect of n-lauryl-beta-D-maltopyranoside. These findings provide basic information to select the optimal enhancer for the intestinal delivery of peptide and protein drugs including insulin.

Algorithms↗
PubMed1999

HP33: hepatocellular carcinoma-enriched 33-kDa protein with similarity to mitochondrial N-acyltransferase but localized in a microtubule-dependent manner at the centrosome.

Using a new subtraction method and chemically induced rat hepatocellular carcinomas, we identified a hepatocellular carcinogenesis and hepatocyte proliferation-related gene designated hp33 that encoded a 33-kDa protein. The predicted protein was similar to the bovine aralkyl N-acyltransferase and arylacetyl N-acyltransferase. HP33 was restrictively expressed in the liver and kidney, and its gene expression was stimulated in the regenerating liver as well as in hepatocellular carcinoma. Interestingly, it was demonstrated in various hepatic cells that HP33 was localized in regions surrounding the centrosome, where mitochondria were not concentrated. Moreover, its centrosomal localization was evident in the interphase but not in the mitotic phase of the cell cycle. The centrosomal localization of HP33 was dependent on microtubules, and ectopically expressed HP33 was seen at centrosomes even in fibroblasts, which do not exhibit a typical staining pattern of HP33. The centrosomal localization of HP33 became invisible by nocodazole treatment, whereas the mitochondrial staining pattern was not affected by it. In vitro cosedimentation experiments using purified microtubules indicated that HP33 bound to MTs directly and that its MT-binding ability was dependent on the C-terminal basic domain of the protein. These results suggest that, different from early predictions based on its primary structure, HP33 has a growth- and carcinogenesis-related function that may be independent of mitochondrial function.

Acyltransferases↗
PubMed1999

Prolongation of the effective duration of cytomedical therapy by re-injecting SK2 hybridoma cells microencapsulated within alginate-poly(L)lysine-alginate membranes into human interleukin-6 transgenic mice.

We previously reported that SK2 hybridoma cells that secreted anti-human interleukin-6 (hIL-6) monoclonal antibodies (SK2 mAb) were microencapsulated within alginate-poly(L)lysine-alginate (APA) membranes (APA-SK2 cells) for immunoisolation, and a single intraperitoneal injection of these APA-SK2 cells remarkably improved IgG1 plasmacytosis in hIL-6 transgenic mice (hIL-6 Tgm). However, the duration of the effectiveness of APA-SK2 cells as a cytomedicine was unfortunately limited. In this study, we attempted to re-inject APA-SK2 cells into hIL-6 Tgm for the purpose of prolonging the cytomedical therapy. In hIL-6 Tgm re-injected with APA-SK2 cells, the plasma IgG1 level did not show any increase in 37 week old mice, and their survival time was at least three times longer than those of untreated hIL-6 Tgm. These results suggest that re-injected APA-SK2 cells survived and secreted SK2 mAb in the allogeneic mice. Thus, the limited duration of the cytomedical effects of APA-SK2 cells was probably caused by the disappearance of the inner space of microcapsules for cell proliferation, not by the rejection of the host's immune system. Therefore, if we can regulate the proliferation of the cells microencapsulated within a semipermeable membrane, we may be able to develop a cytomedicine which will continue its function longer after a single injection.

Alginates↗
PubMed1999

Creatinine clearance as a substitute for the glomerular filtration rate in the assessment of glomerular hemodynamics.

A method for the clinical assessment of glomerular hemodynamics has been published previously. We here examined whether, when using this method, renal creatinine clearance (Ccr) can be substituted for the glomerular filtration rate (GFR). The study subjects comprised 57 inpatients from Osaka City General Hospital: 30 with type 2 diabetes mellitus and 27 with chronic glomerulonephritis. During the 2-wk study, patients received a high-salt diet for 1 wk and a low-salt diet for 1 wk. Urinary sodium excretion and systemic blood pressure were measured daily. The renal plasma flow, Ccr, and plasma total protein concentration were also evaluated simultaneously on the last day of the high-salt diet. The GFR was also calculated from the fractional renal accumulation of 99mTc-diethylenetriaminepentaacetic acid (DTPA). Glomerular hemodynamics, represented by the glomerular capillary hydraulic pressure and the resistance of afferent and efferent arterioles, were calculated using the renal clearance, the plasma total protein concentration, and the pressure-natriuresis relationship. Values for renal hemodynamics with the Ccr-derived GFR were compared with those from the 99mTc-DTPA-derived GFR. Ccr values of 53 to 169 ml/min correlated with the 99mTc-DTPA-derived clearance of 39 to 179 ml/min (n=57, r=.71, p<.001). Values for the glomerular pressure and the resistances of afferent and efferent arterioles calculated using the Ccr-derived GFR correlated significantly with those calculated using the 99mTc-DTPA-derived GFR (r=.99, p<.001 and r=.99, p<.001, respectively). These results indicate that the Ccr is an accurate representation of the GFR for use in glomerular hemodynamic analysis of the pressure-natriuresis relationship.

Chronic Disease↗
PubMed1999

Detection of porcine epidemic diarrhea virus using polymerase chain reaction and comparison of the nucleocapsid protein genes among strains of the virus.

Two pairs of PCR primers were designed to perform nested PCR targetting of a 540 bp fragment of the nucleocapsid (N) protein gene (N gene) of porcine epidemic diarrhea virus (PEDV). The N gene of PEDV was amplified with 4 PEDV strains and 11 small intestines of PEDV-infected piglets collected from 2 farms in Kagoshima prefecture, Japan. Nucleotide sequences of the PCR products from a Korean and two Japanese strains (KKN96-1 and S1) of PEDV isolated in 1993 and 1996, respectively, were almost identical. These results suggest that the PCR is an available tool for detection of PEDV from pigs in the field, and that the two Japanese strains (KKN96-1 and S1) were genetically similar to the Korean strain.

Animals↗
PubMed1999

Cyclosporin A mono-therapy in nephrotic syndrome with contra-indication of steroid therapy.

We describe three cases of nephrotic syndrome with a contra-indication for steroid therapy successfully treated with cyclosporin A (CsA). A 21-year-old man with focal segmental glomerulosclerosis (FSGS) complicated by necrosis of the femoral head, and a 34-year-old woman and a 48-year-old man with minimal change disease (MCD) complicated by psychogenic reaction and diabetes mellitus, respectively, were given CsA at initial dosages of 3.8-5.0 mg/kg/day and immediately remitted completely. However, two of these patients suffered relapses when CsA was tapered. They are currently maintained in complete or partial remission on CsA at dosages of 3.2-4.7 mg/kg/day. These findings suggest that CsA mono-therapy may be useful in nephrotic syndrome patients contra-indicated for steroid therapy.

Adult↗
PubMed1999

[Correlation of risk area and reverse redistribution of 99mTc-sestamibi SPECT in acute myocardial infarction following direct PTCA].

Redistribution of 99mTc-sestamibi is negligible in usual circumstances, but recent reports demonstrated reverse redistribution is detectable in acute myocardial patients. Correlation of risk area, observed in 99mTc-sestamibi "freezed" SPECT image at onset, and delayed images at 5-25 days after onset (post-PTCA image) is evaluated in 19 acute myocardial infarction patients treated with direct PTCA. Reverse redistribution was observed in 85% of reperfused area. Linear relationship of % uptake in each SPECT segment between onset and post-PTCA images (taken at 0.5, 4, and 6 hours after injection) is evaluated and the relationship improves over time course. The correlation coefficient between onset and 6 hours-delayed image is 0.88, and the visual concordance shows 77% of score matching. Delayed 99mTc-sestamibi SPECT image on reperfused AMI seems to represent risk area with some underestimation. It may be useful to estimate both risk and salvaged areas on early and delayed SPECT with a single 99mTc-sestamibi injection.

Adult↗
PubMed1999

Efficient detection of deletions induced by a single treatment of mitomycin C in transgenic mouse gpt delta using the Spi(-) selection.

Transgenic rodent mutation assays permit the detection and molecular analysis of various types of gene mutations, such as base changes and frameshifts, in a number of tissues. It is reported, however, that deletion mutations are not efficiently detected by the assays, in particular those using lambda phage shuttle vectors. Recently, a new transgenic mouse model, i.e., gpt delta, has been developed to selectively detect some types of deletions by Spi(-) selection. Spi(-) selection has an advantage over the other selections to preferentially identify deletions because only lambda phages deficient in both the red and gam gene functions are allowed to form phage plaques. In this study, we examined whether in vivo deletions induced by the treatment of mitomycin C (MMC) are detectable by the Spi(-) assay in the mouse model. The mice were treated with MMC (0.5, 1.0, 2.0, and 4.0 mg/kg, single intraperitoneal injection) and sacrificed 14 days after the dosing. The treatment at 4.0 mg/kg approximately doubled the mutant frequency of Spi(-) in the bone marrow, i.e., 2.52 x 10(-6) vs. 1.31 x 10(-6). The molecular analyses using polymerase chain reaction (PCR) and DNA sequencing indicated that seven Spi(-) mutants at 4.0 mg/kg group had deletions with molecular sizes from 0.8 kilo basepairs (kb) to 8.5 kb, whereas no such deletions were observed in the Spi(-) mutants in the control group. The results suggest that deletions induced by MMC in the bone marrow are efficiently detectable by Spi(-) selection and also that the molecular analyses are useful to evaluate the significance of a marginal increase in mutant frequency in the transgenic rodent mutation assays.

Animals↗
PubMed1999

Quantitative evaluation of salivary gland scintigraphy in Sjörgen's syndrome.

UNLABELLED: This study compared the quantitative characteristics of salivary gland scintigraphy in patients with Sjogren's syndrome with the histopathologic grading of labial biopsy. METHODS: Thirty-nine patients with Sjögren's syndrome were studied by salivary gland scintigraphy and labial biopsy. Twelve normal volunteers were also studied as a control group for scintigraphic parameters. After injection of 370 MBq 99mTc sodium pertechnetate, dynamic salivary scintigraphy with lemon juice stimulation was performed for 50 min. Functional parameters for the parotid and submandibular glands were calculated, and scintigraphic and histopathologic results were compared. RESULTS: With the progression of histopathologic grades 1-4, the velocity of tracer secretion decreased in the parotid gland (P < 0.05), and the quantity of tracer accumulation decreased in the submandibular gland (P < 0.05). The histopathologic grade in patients with Sjögren's syndrome was correlated with these scintigraphic parameters (P < 0.05): Histopathologic grade = 2.304 - 0.014 x (submandibular gland maximum accumulation) + 0.196 x (parotid gland time interval of tracer secretion). CONCLUSION: Decreased secretion velocity in the parotid gland and decreased accumulation in the submandibular gland were sensitive indicators of salivary gland disease in Sjögren's syndrome. The histopathologic grade was correlated with these scintigraphic parameters.

Adult↗
PubMed1999

Cell surface regulators of complement, 5I2 antigen, and CD59, in the rat eye and adnexal tissues.

PURPOSE: Cell surface complement regulatory proteins have been identified in high levels in ocular tissues, but no experimental model is available for examining their physiological roles. To develop such a model, the distribution of 5I2 antigen, a protein possessing the functions of the human decay-accelerating factor (DAF [CD55]) and membrane cofactor protein (MCP [CD46]), and rat inhibitory protein (CD59), the homologue of the human membrane inhibitor of reactive lysis (MIRL[CD59]) were characterized in the rat eye and ocular adnexal structures. METHODS: After euthanasia of female Wistar rats, followed by orbital exenteration, eyelids and orbital tissue including the lacrimal gland were separated from the globes and immediately snap-frozen in liquid nitrogen at -70 degrees C. Tissues then were sectioned at -20 degrees C and examined immunohistochemically for 5I2 antigen and rat CD59. RESULTS: Both molecules were found to be present in high levels in multiple sites. Corneal and conjunctival epithelia showed moderate to intense labeling for both regulators. Fibroblasts in the corneal stroma, conjunctiva, and sclera labeled similarly. Corneal endothelial cells showed intense labeling for rat CD59 but not for 5I2 antigen. The iris and ciliary body showed intense labeling for both proteins. The retina showed labeling at multiple levels, with that of rat CD59 being more intense than that of 5I2 antigen. The lacrimal gland labeled for both regulators. Vessels, muscle, and nerves in the orbit labeled intensely for both antigens. In the eyelid, conjunctiva, sebaceous glands, and muscle and nerve tissues labeled moderately to intensely for both molecules, whereas skin epithelium labeled less intensely. CONCLUSIONS: 5I2 antigen and rat CD59 are expressed in high levels and distributed similarly in the rat eye and lacrimal gland to DAF, MCP, and MIRL in the human eye and lacrimal gland. These findings establish the rat ocular surface as a model for studying the role of cell surface complement regulators in this site. This first identification of copious expression of these proteins in eyelid structures, which also participate in protection of the ocular surface, further suggests an important role for surface complement regulatory proteins in this location.

Animals↗