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Biomedical subjects

N Okada

Publications and source records attributed to N Okada.

At least 235 records · Page 13Linked to original sources
PubMed1995

Beneficial effects of a novel anti-hypoxemic agent, TEI-7322, on bleomycin-induced experimental hypoxemia in rats.

Almitrine bismesylate is known to be an anti-hypoxemic agent that acts via the enhancement of hypoxic pulmonary vasoconstriction. However, screening for this class of compounds has been minimal, owing, in part, to a lack of convenient hypoxemic models in small animals. The present study was designed to establish a convenient model of hypoxemia induced by bleomycin and to evaluate anti-hypoxemic agents including a newly synthesized compound. TEI-7322, 2-allylamino-4-tert-butyl-amino-7-methyl-7H-pyrrolo[2,3- d]pyrimidine hydrochloride by using this model. Bleomycin was intratracheally instilled into rats. After 3 weeks, the arterial blood gas pressures were monitored in the animals in the conscious state. Then, prednisolone, doxapram, almitrine or TEI-7322 was administered to the bleomycin-treated rats to monitor changes in arterial blood gas pressures. Bleomycin-treated rats showed a decrease in the arterial blood O2 pressure (PaO2). The blood CO2 pressure (PaCO2) increased, along with an increase in the alveolar-arterial oxygen difference (AaDO2). These blood gas pressures in bleomycin-treated rats were not affected by treatment with prednisolone. Doxapram decreased the PaCO2 but did not change the PaO2. However, administration of almitrine or TEI-7322 significantly improved the PaO2 of bleomycin-treated rats with a decrease in the PaCO2. In conclusion, (1) bleomycin-induced lung injury causes hypoxemia in rats, probably resulting from ventilation-perfusion inequality; thus this model may be useful for evaluating anti-hypoxemic agents; and (2) TEI-7322, as well as almitrine, showed anti-hypoxemic effects in this model with different properties from those of doxapram, possibly due to improvement of ventilation-perfusion inequality, indicating that TEI-7322 may be a potent candidate for the treatment of hypoxemia.

Almitrine↗
PubMed1995

Mollusk genes encoding lysine tRNA (UUU) contain introns.

New intron-containing genes encoding tRNAs were discovered when genomic DNA isolated from various animal species was amplified by the polymerase chain reaction (PCR) with primers based on sequences of rabbit tRNA(Lys). From sequencing analysis of the products of PCR, we found that introns are present in several genes encoding tRNA(Lys) in mollusks, such as Loligo bleekeri (squid) and Octopus vulgaris (octopus). These introns were specific to genes encoding tRNA(Lys)(CUU) and were not present in genes encoding tRNA(Lys)(CUU). In addition, the sequences of the introns were different from one another. To confirm the results of our initial experiments, we isolated and sequenced genes encoding tRNA(Lys)(CUU) and tRNA(Lys)(UUU). The gene for tRNA(Lys)(UUU) from squid contained an intron, whose sequence was the same as that identified by PCR, and the gene formed a cluster with a corresponding pseudogene. Several DNA regions of 2.1 kb containing this cluster appeared to be tandemly arrayed in the squid genome. By contrast, the gene encoding tRNA(Lys)(CUU) did not contain an intron, as shown also by PCR. The tRNA(Lys)(UUU) that corresponded to the analyzed gene was isolated and characterized. The present study provides the first example of an intron-containing gene encoding a tRNA in mollusks and suggests the universality of introns in such genes in higher eukaryotes.

Animals↗
PubMed1995

Multiple isoforms of guinea pig decay-accelerating factor (DAF) generated by alternative splicing.

Human decay-accelerating factor (DAF, CD55), which is one of the regulators of complement activation (RCA), prevents complement activation on homologous cell membranes, resulting in a functional discrimination between self cells and invading microorganisms. DAF is a glycosylphosphatidylinositol (GPI)-anchored glycoprotein and has been identified at the molecular level only in primates. We have isolated guinea pig DAF cDNA clones from a spleen library and identified six different classes. All encode the same four short consensus repeat domains with 58% amino acid sequence identity to human DAF, but show variability in the C-terminal region. Alternative splicing of two optional exons generates transmembrane, GPI-anchored, and secreted forms of guinea pig DAF, and differential usage of splice sites in the single exon composed of internally quintuplicated sequences generates variable Ser/Thr-rich regions. Multiple isoforms were expressed ubiquitously in all tissues and cells tested. Similar variability in the Ser/Thr-rich region has been reported in human membrane cofactor protein (MCP), another membrane inhibitor of the RCA family. There seems to be a common ancestral sequence in DAF and MCP consisting of a Ser/Thr-rich region, but obviously multiplication occurred independently, indicating the importance of variability of the Ser/Thr-rich region for the effective regulation of complement activation.

Alternative Splicing↗
PubMed1995

A neuroprotective compound, aurin tricarboxylic acid, stimulates the tyrosine phosphorylation cascade in PC12 cells.

Aurin tricarboxylic acid (ATA), a general nuclease inhibitor, was reported to prevent PC12 cells from cell death caused by serum starvation (1). In our study, ATA also protected PC12 cells, but not NIH3T3 cells, from serum-starved cell death. When we investigated the mechanism of action of ATA on these cells, ATA was found to increase tyrosine phosphorylation in PC12 cells, but not in NIH3T3 cells. Further investigation on tyrosine-phosphorylated proteins revealed that ATA, similar to nerve growth factor and epidermal growth factor, induced tyrosine phosphorylation of mitogen-activated protein kinases. Since the tyrosine phosphorylation of mitogen-activated protein kinases is thought to play an important role inn growth factor-dependent signal pathways, this finding suggests that the action of ATA on PC12 cells is mediated by tyrosine phosphorylation cascade, similar to growth factor signaling. In addition, we found that Shc proteins, phosphatidylinositol 3-kinase, and phospholipase C-gamma were also phosphorylated in ATA-treated PC12 cells. These key proteins in signal transduction pathways are known to associate with ligand-activated growth factor receptors and are phosphorylated on tyrosine. Thus, the phosphorylation of these three proteins by ATA stimulation supports the speculation that ATA activates a certain receptor tyrosine kinase.

3T3 Cells↗
PubMed1995

A monoclonal antibody that blocks the complement regulatory activity of guinea pig erythrocytes and characterization of the antigen involved as guinea pig decay-accelerating factor.

MCA44 is a mAb with the capacity to sensitize neuraminidase-treated guinea pig E for hemolysis by homologous guinea pig C, and the Fab fragments of this mAb could also sensitize guinea pig E interfering with the function of a membrane inhibitor of C on guinea pig E. Using an immunosorbent column to which MCA44 was coupled, the antigenic molecule termed 44Ag was purified from the glycoprotein fraction extracted from E membranes. C intermediate sheep E treated with guinea pig C1 and C4 after sensitization with Ab (EAC14b cells) lost the ability to generate C3 convertase with C2 after incubation with 44Ag. Treatment of guinea pig E and PBL with phosphatidyl-inositol specific phospholipase C (PIPLC) partially removed 44Ag, as determined by flow cytometric analysis after immunofluorescence staining with MCA44. However, 125I-labeled 44Ag adsorbed to human E was efficiently removed by PIPLC treatment with a slight reduction in M(r). The 44Ag purified on an immunosorbent column showed three bands on SDS-PAGE. However, partial N-terminal amino acid sequences of the 55-kDa, 70-kDa, and 88-kDa bands under nonreducing conditions were identical and the sequence was 55% homologous to the N-terminal sequence of human decay accelerating factor (CD55). Intracutaneous administration of MCA44 or its F(ab')2 fragment resulted in increased capillary permeability, even after 3 days, as determined by the appearance of Evans blue spots after i.v. administration of the dye. Because control Abs including anti-class I-MHC did not cause such increased capillary permeability, the increase in permeability caused by MCA44 was likely induced by blocking the function of 44Ag in vivo, indicating a crucial role for these molecules in preventing over-activation of C at the site.

Amino Acid Sequence↗
PubMed1995

Highly specific and efficient cleavage of squid tRNA(Lys) catalyzed by magnesium ions.

Two lysine isoacceptor tRNAs corresponding to the codons AAA and AAG, respectively, were isolated from squid (Loligo bleekeri), and their nucleotide sequences were determined. During this analysis, we discovered that the tRNA with the anticodon CUU was efficiently cleaved at a specific site in the presence of magnesium ions, whereas the tRNA with the anticodon UUU was not. Cleavage occurred almost exclusively at the phosphodiester linkage between G15 and D16 (p16). The most remarkable feature of this cleavage reaction is that the end product was not a 2',3'-cyclic phosphate but was mainly a 3'-phosphate. Thus, this reaction was distinct from the well characterized cleavage of yeast tRNA(Phe) by lead and from reactions catalyzed by various other metalloribozymes. The presence of a cytidine residue at position 60 was required for efficient cleavage but was not crucial for the reaction, and the entire tRNA molecule had to be intact for this specific and efficient cleavage reaction. The present study provides evidence that there exists a new catalytic mechanism for cleavage of tRNA that exploits biologically ubiquitous ions rather than toxic, nonessential ions such as lead.

Animals↗
PubMed1995

Membrane cofactor protein (CD46) is a keratinocyte receptor for the M protein of the group A streptococcus.

The pathogenic Gram-positive bacterium Streptococcus pyogenes (group A streptococcus) is the causative agent of numerous suppurative diseases of human skin. The M protein of S. pyogenes mediates the adherence of the bacterium to keratinocytes, the most numerous cell type in the epidermis. In this study, we have constructed and analyzed a series of mutant M proteins and have shown that the C repeat domain of the M molecule is responsible for cell recognition. The binding of factor H, a serum regulator of complement activation, to the C repeat region of M protein blocked bacterial adherence. Factor H is a member of a large family of complement regulatory proteins that share a homologous structural motif termed the short consensus repeat. Membrane cofactor protein (MCP), or CD46, is a short consensus repeat-containing protein found on the surface of keratinocytes, and purified MCP could competitively inhibit the adherence of S. pyogenes to these cells. Furthermore, the M protein was found to bind directly to MCP, whereas mutant M proteins that lacked the C repeat domain did not bind MCP, suggesting that recognition of MCP plays an important role in the ability of the streptococcus to adhere to keratinocytes.

Antigens, Bacterial↗
PubMed1995

Identity elements of Saccharomyces cerevisiae tRNA(His).

Recognition of tRNA(His) by Saccharomyces cerevisiae histidyl-tRNA synthetase was studied using in vitro transcripts. Histidine tRNA is unique in possessing an extra nucleotide, G-1, at the 5' end. Mutation studies indicate that this irregular secondary structure at the end of the acceptor stem is important for aminoacylation with histidine, while the requirement of either base of this extra base pair is smaller than that in Escherichia coli. The anticodon was also found to be required for histidylation. The regions involved in histidylation are essentially the same as those in E.coli, whereas the proportion of the contributions of the two portions distant from each other, the anticodon and the end of the acceptor stem, makes a substantial difference between the two systems.

Acylation↗
PubMed1995

Evolution of the active sequences of the HpaI short interspersed elements.

Ninety-nine members of the salmonid HpaI and AvaIII families of short interspersed repetitive elements (SINEs) were aligned and a general consensus sequence was deduced. The presence of 26 correlated changes in nucleotides (diagnostic nucleotides) from those in the consensus sequence allowed us to divide the members of the HpaI family into 12 subfamilies and those of the AvaIII family into two subfamilies. On the basis of the average sequence divergences and the phylogenetic distributions of the subfamilies, the relative antiquity of the subfamilies and the process of sequential changes in the respective source sequences were inferred. Despite the higher mutation rates of CG dinucleotides in individual dispersed members, no hypermutability of CG positions was observed in changes in the source sequences. This result suggests that sequences of SINEs located in a nonmethylated or hypomethylated genomic region could have been selected as source sequences for retroposition and/or that some CG sites are the parts of recognition sequences of retropositional machineries.

Animals↗
PubMed1995

Effects of 17 beta-oestradiol and 5 alpha-dihydrotestosterone on the expression of the muscle and heart types of lactate dehydrogenase isozymes in the masseter muscle of developing mice.

17 beta-oestradiol (E2) and/or 5 alpha-dihydrotestosterone (5 alpha-DHT) had no effect on the expression of isozymes of lactate dehydrogenase (LDH) in the masseter muscle of intact male mice. However, treatment with E2 restored the level of the muscle (M) type of LDH isozyme, which had been reduced by testectomy, to that found in intact male mice treated with vehicle only. Moreover, 5 alpha-DHT alone was more effective than E2 in increasing the relative level of this isozyme in testectomized mice. 5 alpha-DHT had a more significant effect on the increase in the relative level of the M-type LDH isozyme when combined with E2. These results suggest that androgens promote, in the presence of oestrogens, the postnatal changes in the characteristics of the masseter muscle of developing male animals.

Animals↗
PubMed1995

Construction and evaluation of a virG thyA double mutant of Shigella flexneri 2a as a candidate live-attenuated oral vaccine.

A virG thyA double mutant of Shigella flexneri 2a was constructed as a candidate live-attenuated oral vaccine. In the keratoconjunctivitis model it did not provoke any adverse reaction by itself on guinea pigs' eyes and completely protected them from provoking keratoconjunctivitis. When (2.7-4.8) x 10(10) of the vaccine was inoculated intragastrically after 1 day fasting in cynomolgus monkeys three times at weekly intervals, a watery stool was observed at 40% as a side-effect. Upon intragastric challenge after 1 day fasting with 7.5 x 10(9) of the virulent strain four weeks after the last vaccination, a statistically significant difference was obtained in the mortality rate but not in the morbidity rate between the vaccine and the control group, although the clinical findings were less severe in the vaccine group than in the control group. These results together with the histopathological and immunological findings indicate that the vaccine deserve further detailed studies.

Animals↗
PubMed1995

Role of a rat membrane inhibitor of complement in anti-basement membrane antibody-induced renal injury.

In the kidneys of anti-glomerular basement membrane (anti-GBM) antibody disease, binding of antibodies to tubular basement membrane (TBM) is often observed. The present work was performed to explore the mechanisms of binding of anti-GBM antibodies to TBM in vivo with special reference to 5I2Ag, a rat membrane inhibitor of complement which regulates complement activation at C3 convertase level. To suppress functions of renal 5I2Ag, F(ab')2 fragment of 5I2 (a neutralizing mAb against 5I2Ag) was perfused in the left kidney and then blood circulation was restored. Mild proteinuria ( < 10 mg/16 hr) was observed during first several days. Five days later, there were tubulointerstitial injuries defined by tubular vimentin staining and leukocyte infiltration. Significant deposition of C3 was observed in the capillaries and in TBM. In rats intravenously injected with rabbit anti-rat GBM antibodies five minutes after kidney perfusion with 5I2, strong binding of rabbit IgG to TBM was observed at one and five days after injection. Although these rats showed mild proteinuria comparable to those perfused with 5I2 and those injected with normal rabbit serum, tubulointerstitial injury was significantly enhanced at Day 5. In contrast, rats perfused with irrelevant mAb and injected with anti-GBM antibodies did not show any significant binding of antibodies to TBM nor tubulointerstitial injury. Furthermore, rats which were made proteinuric by puromycin aminonucleoside and injected with anti-GBM antibodies did not show any significant binding of rabbit IgG to TBM. These results indicate that 5I2Ag, a rat membrane inhibitor of complement at the C3 convertase level, regulates vascular permeability in the living kidney, and that dysfunction or decreased expression of this molecule leads to increased accessibility of anti-GBM antibodies to TBM.

Albuminuria↗
PubMed1995

A quantative in vivo method of analyzing human tumor-induced angiogenesis in mice using agarose microencapsulation and hemoglobin enzyme-linked immunosorbent assay.

This study was conducted to develop a quantitative assay system for use in the in vivo evaluation in mice of angiogenesis induced by human tumor cells. The human epidermoid carcinoma cells, A431 cells, were cultured on microcarriers. Microcarrier-attached A431 cells (A431-MC) were microencapsulated with agarose hydrogel to isolate them from the immune system of the C57BL/6 mice after subcutaneous dorsal midline implantation. The agarose hydrogel-microencapsulated A431 cells (Aga-A431 cells; diameter=300 micron) survived for at least 10 days in vitro, and the proliferation profile of the Aga-A431 cells was indistinguishable from that of non-microencapsulated A431 cells. The Aga-A431 cells were subcutaneously injected into mice with an 18-gauge needle. Ten days later, few vessels had formed at the site implanted with cell-free agarose beads, whereas notable angiogenesis was observed at the site implanted with Aga-A431 cells. The degree of angiogenesis was evaluated by measurement of the hemoglobin content in the implanted site using a mouse hemoglobin (mHb) enzyme-linked immunosorbent assay (ELISA) system. This mHb-ELISA system has the advantages of great simplicity and reproducibility. The measured mHb-content of new blood vessels at the site implanted with agarose beads was in good agreement with the amount of angiogenesis observed under a stereoscopic microscope. This assay system enabled us to evaluate the angiogenesis induced by xenogeneic cells, such as human tumor cells. Thus, our novel method may be useful for the study of the angiogenic potential of various human tumor cells and in research on the anti-angiogenic properties of various agents.

Animals↗
PubMed1995

Eicosapentaenoic acid-induced changes in membrane fluidity and cell adhesion molecules in cultured human keratinocytes.

The effects of eicosapentaenoic acid (EPA, 20:5n-3) on the lipid dynamics of cultured epidermal keratinocytes and their expression of cell adhesion molecules were investigated, and were compared with those of arachidonic acid (AA, 20:4n-6). When keratinocytes were treated with 3 micrograms/ml of EPA or AA for 72 h, these compounds were found to be incorporated into the cells. EPA-induced lipid changes were distinguished by a significant increase in the cellular content of n-3 polyunsaturated fatty acids, whereas AA treatment resulted in an increase in the cellular content of n-6 arachidonic acid. These changes in fatty acid composition were accompanied by an increase in cellular membrane fluidity, which was evaluated by the diffusion coefficient, using the method of fluorescence recovery after photobleaching (FRAP) [from 1.77 +/- 0.34 x 10(-8) cm2/s untreated to 2.23 +/- 0.35 x 10(-8) cm2/s EPA-treated (P < 0.001), and 2.16 +/- 0.35 x 10(-8) cm2/s AA-treated (P < 0.001)]. Intercellular adhesion molecule-1 (ICAM-1) was induced on the keratinocyte membrane in the presence of tumour necrosis factor-alpha and interferon-gamma, and pretreatment with EPA or AA further enhanced the expression, almost to an equal degree, as estimated by flow cytometry (P < 0.05). These results indicate that the modulation of ICAM-1 expression does not seem to be EPA-specific, but is presumably a consequence of increased membrane fluidity due to the increased levels of unsaturated fatty acids of both the n-3 and n-6 series in the membrane.

Arachidonic Acid↗
PubMed1995

Role of the conserved C-repeat region of the M protein of Streptococcus pyogenes.

The surface-located M protein functions to protect Streptococcus pyogenes (the group A streptococcus) from phagocytosis by polymorphonuclear leukocytes. It has been suggested that this protection results from the ability of M protein to bind factor H, a serum protein that can inhibit the activation of complement. Among different serological variants of M protein, the C-repeat domain is highly conserved and is exposed on the bacterial surface. This domain has been implicated in binding to complement factor H and in M-protein-mediated adherence of streptococci to human keratinocytes in the cutaneous epithelium. In this study, we constructed an S. pyogenes mutant strain which expresses an M6 protein from which the entire C-repeat domain was deleted. As predicted, this mutant did not adhere well to human keratinocytes and was unable to bind to factor H. Unexpectedly, the mutant was able to survive and multiply in human blood. Therefore, while the binding of factor H and the facilitation of adherence to keratinocytes appear to involve recognition of the C-repeat domain, a region of the M-protein molecule distinct from the C-repeat domain confers upon S. pyogenes its ability to resist phagocytosis.

Alleles↗