Biomedical subjects

Y Abiko

Publications and source records attributed to Y Abiko.

At least 19 recordsLinked to original sources

PubMed1992

Purification of sufficiently gamma-carboxylated recombinant protein C and its derivatives. Calcium-dependent affinity shift in immunoaffinity and ion-exchange chromatography.

Protein C, which is an important anti-thrombotic factor in the blood coagulation cascade, undergoes several post-translational modifications. gamma-Carboxylation on nine glutamic acid residues at the N-terminal region of the light chain [gamma-carboxylated glutamic acid (Gla) domain] is considered to be critical for full anti-clotting activity. It is also known that when recombinant protein C is expressed in animal cells this particular modification is often lost. We were successful in preparing a monoclonal antibody (PC01) which distinguishes the sufficiently gamma-carboxylated protein from the rest by its specific affinity for the Ca(2+)-induced conformational change of the former, and thereby developed a simple process of purifying sufficiently gamma-carboxylated protein C. Culture supernatant of Chinese hamster ovary cell transformants was first applied to Q-Sepharose and recombinant protein C was partially purified. It was then loaded onto a PC01 affinity column in the presence of 5 mM calcium chloride. Sufficiently gamma-carboxylated protein C was retained while insufficient-carboxylated protein C quickly passed through. The former was eluted with 5 mM EDTA efficiently and with high purity, contained eight Gla units per molecule, and had similar anti-clotting activity. The flow-through was relatively impure protein C which contained five Gla units per molecule and showed limited anti-clotting activity. We extended the application of the Ca(2+)-induced conformational change to conventional ion-exchange chromatography. The sufficiently gamma-carboxylated protein C was found to elute earlier in the salt gradient from an anion-exchange column in the presence of 5 mM calcium chloride being fully separated from the insufficiently carboxylated protein C.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗

PubMed1992

Cardioprotective effect of pindolol in ischemic-reperfused isolated rat hearts.

The effects of pindolol and timolol on ischemia reperfusion damage were studied in isolated working rat hearts. Ischemia (15 min) decreased the mechanical function and the energy state, and increased the tissue levels of free fatty acids (FFA). During reperfusion (20 min), the mechanical function did not recover, but the energy state recovered incompletely, whereas FFA increased further. Pindolol (50 microM) accelerated recovery of the mechanical function and the energy state that had been decreased by ischemia during reperfusion, and inhibited the accumulation of FFA during ischemia and reperfusion, especially when it was applied during the whole period of reperfusion. Timolol (50 microM), however, did not accelerate recovery of the mechanical function and the energy state during reperfusion, although it attenuated FFA accumulation during reperfusion. The pindolol-induced recovery of the mechanical function during reperfusion was reduced by timolol. The results suggest that the intrinsic sympathomimetic activity of pindolol may play an important role, at least in part, in producing the cardioprotective effect, especially during reperfusion.

Adenine Nucleotides↗

PubMed1992

Role of Porphyromonas gingivalis 40-kDa outer membrane protein in the aggregation of P. gingivalis vesicles and Actinomyces viscosus.

Porphyromonas gingivalis, an important pathogen in periodontitis, produces extracellular vesicles that aggregate with Actinomyces viscosus cells. A 40-kDa outer membrane protein (OMP)-coding gene from P. gingivalis was cloned and the protein was found to be localized in these vesicles. The recombinant 40-kDa OMP did not show aggregation activity. However, affinity-purified antibody against the recombinant protein significantly inhibited aggregation of P. gingivalis vesicles with A. viscosus cells. The antibody also inhibited cellular coaggregation of several strains of P. gingivalis with A. viscosus cells, but not with other periodontal pathogens. Moreover, aggregation of A. viscosus cells with P. gingivalis vesicles was inhibited in a dose-dependent manner by pre-treatment of the A. viscosus cells with the recombinant protein. These findings suggest that the 40-kDa OMP may be an important aggregation factor of P. gingivalis.

Actinomyces viscosus↗

PubMed1992

Stimulation by interleukin-1 of interleukin-6 production by human periodontal ligament cells.

Interleukin-1(IL-1), a cytokine present in the gingiva and crevicular fluid of patients with periodontitis and in the periodontal ligament (PDL) of experimentally moved teeth, has multiple biological activities, including the ability to elicit bone resorption. Interleukin-6, also found in the gingiva of patients with periodontitis, may induce osteoclastic bone resorption through an effect on osteoclastogenesis. Here IL-6 production and its gene expression in response to recombinant IL-1 beta were examined in primary cultures of PDL cells. IL-1 beta stimulated IL-6 production by these cells in a dose- and time-dependent manner; this increase in IL-6 production was much higher than that in human gingival fibroblasts. In situ hybridization, using a synthetic oligonucleotide DNA probe of the IL-6 gene, revealed that most PDL cells expressed IL-6 mRNA in response to IL-1 beta treatment. The finding that IL-6 is produced by PDL cells and is regulated by IL-1 beta has revealed a potentially important mechanism for controlling alveolar bone resorption.

Cells, Cultured↗

PubMed1992

Cloning of the gene encoding a glycylprolyl aminopeptidase from Porphyromonas gingivalis.

A genomic library of Porphyromonas gingivalis 381 was constructed in the cosmid vector pHC79. A clone, pSN1, was identified by the expression of glycylprolyl-naphthylamide hydrolysing activity. The DNA insert contained within the cosmid pSN1 was subcloned into the plasmid vector pBR328 to create the recombinant plasmid pSN11 containing a 2.9 kb EcoRV insert. An Escherichia coli transformant containing pSN11 produced a protein having a molecular weight of 75 kDa. Southern-blot hybridization revealed that the 2.9 kb EcoRV DNA hybridized with an identical sized Eco RV DNA fragment in the chromosomal DNA of P. gingivalis 381.

Blotting, Southern↗

PubMed1992

Gene cloning of Porphyromonas gingivalis specific antigens recognized by serum of adult periodontitis patient.

1. Porphyromonas gingivalis is believed an important pathogen of adult periodontitis. A gene library of P. gingivalis 381 was constructed in lambda phage vector L47.1. The library was probed with serum obtained from patients of severe adult periodontitis. Two clones, lambda MDBG101 and lambda MDBG103 which were expressed, 200 and 160 kDa respectively, were selected and further studied. 2. The expressed antigens in these two clones were also reacted with rabbit antiserum against whole cells, capsular fraction and cell surface fraction of P. gingivalis. 3. Genes coding protein antigens in lambda MDBG101 and lambda MDBG103 were subcloned into high-copy-number plasmid vector pACYC184 and subclones obtained were designated as MD101 and MD103. Recombinant plasmids, pMD101 and pMD103, differed in their restriction endonuclease digestion. 4. Immunodiffusion analysis showed that cloned proteins from MD101 and MD103 reacted with antiserum against P. gingivalis but did not react with antiserum against Prevotella intermedia, Prevotella loescheii and Prevotella asaccharolyticus. 5. These data suggest that P. gingivalis species-specific antigens has been successfully cloned and expressed in Escherichia coli. Since these cloned specific antigens were recognized by adult periodontitis patient sera, the recombinant antigen will be useful material for the development of serodiagnosis of P. gingivalis infection in adults periodontitis.

Adult↗

PubMed1992

Suppressive effect of soluble factor(s) derived from Prevotella loescheii ATCC 15930 on proliferation of human lymphocytes.

Soluble sonic extracts of Prevotella loescheii caused a dose-dependent inhibition of human peripheral blood lymphocyte proliferation by mitogen and of the proliferation of a leukemic cell line, BALL-1, when assessed by DNA synthesis (3H-thymidine incorporation). RNA (3H-uridine incorporation) and protein (3H-leucine incorporation) synthesis were similarly altered after exposure to the extract. There was no effect on cell viability as measured by either trypan blue exclusion or extracellular release of the cytoplasmic enzyme lactate dehydrogenase. Preliminary characterization indicates the suppressive factor(s) derived from P. loescheii to be a protein since it is heat-labile and trypsin-sensitive. The factor eluted in a peak on a high-pressure liquid chromatography gel filtration corresponding to a molecular weight of approximately 32,000. Since black-pigmented anaerobic rods have been implicated in the pathogenesis of periodontal disease, the data suggest that P. loescheii contributes to the disease process by suppressing lymphocyte function.

Bacterial Proteins↗

PubMed1992

Positive inotropic and negative chronotropic effects of (-)-cis-diltiazem in rat isolated atria.

1. The cardiovascular effects of (-)-cis-diltiazem, an optical isomer of diltiazem, were studied in the isolated atrium and aortic strip. (-)-cis-Diltiazem (30 microM or more) increased the developed tension of the rat left atrium, while (+)-cis-diltiazem (1 microM or more) decreased it. 2. (-)-cis-Diltiazem (1 to 100 microM) decreased the rate of spontaneous beating in the right atrium as did (+)-cis-diltiazem. 3. The potency of the positive inotropic action of (-)-cis-diltiazem was almost the same as that of ouabain in the rat left atrium, but in the guinea-pig left atrium it was considerably weaker than that of ouabain. 4. In both endothelium-intact and endothelium-denuded aortic strips, (-)-cis-diltiazem relaxed the Ca(2+)-induced contraction. In the endothelium-intact rat aortic strip depolarized by 15 mM KCl, Bay K 8644, a calcium channel agonist, increased the contractile force, whereas (-)-cis-diltiazem did not. 5. These results indicate that (-)-cis-diltiazem has a positive inotropic action in isolated atria in rats and guinea-pigs, but the mode of positive inotropic action of (-)-cis-diltiazem is different from that of ouabain or Bay K 8644.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1992

Role of oxygen radicals in canine myocardial metabolic derangement during regional ischemia.

To clarify the role of oxygen radicals in the development of myocardial injury during ischemia, production of lipid peroxides mediated by oxygen radicals was determined in in vivo dogs subjected to regional ischemia and reperfusion. Myocardial injury was assessed by derangement in energy and carbohydrate metabolism caused by ischemia and reperfusion. The production of lipid peroxides mediated by oxygen radicals considerably increased not only during reperfusion after ischemia but also during ischemia. Removal of oxygen radicals by administration of radical scavengers [recombinant human superoxide dismutase + catalase or N-(2-mercaptopropionyl)glycine] completely prevented the increase in production of lipid peroxides during ischemia. However, the radical scavengers did not attenuate the myocardial energy and carbohydrate metabolic derangements caused by ischemia and reperfusion after ischemia. These results suggest that significant amounts of oxygen radicals are generated during ischemia as well as during reperfusion and that the oxygen radicals and subsequent lipid peroxidation are not major factors in development of myocardial injury during either ischemia or reperfusion after ischemia.

Animals↗

PubMed1992

Heparin stimulates the collagen synthesis in mineralized cultures of the osteoblast-like cell line, MC3T3-E1.

We found that heparin induced an increase in collagen synthesis in MC3T3-E1 cells cultured for 15 and 30 days. Northern blots showed that the effect of heparin on the collagen synthesis was mediated through the mRNA expression of type I collagen (day 15). Although heparin stimulated collagen synthesis over and above that stimulated by the transforming growth factor (TGF)-beta, heparin did not stimulate TGF-beta binding. This study indicates that heparin has special tasks both in bone formation and resorption, since it has the ability to form and degrade collagen. We suggest that heparin assists the regulation of collagen metabolism at bone resorption sites.

Animals↗

PubMed1991

Attenuation of ischemia-induced regional myocardial acidosis by LP-805, a newly developed vasodilator, in dogs.

The effect of 8-tert-butyl-6,7-dihydropyrrolo[3,2-e]-5-methyl- pyrazolo[1,5-a]pyrimidine-3-carbonitrile (LP-805), a newly developed vasodilator, on myocardial acidosis induced by ischemia was studied in anesthetized open-chest dogs. Ischemia was induced by partially occluding the left anterior descending coronary artery. The coronary flow was artificially reduced to about 1/3 of the original flow. Myocardial pH was measured with a glass micro pH electrode inserted into the left ventricular wall perfused by the occluded artery. Myocardial pH decreased from about 7.5 to about 6.9 after the onset of ischemia and remained at this low level until the occluded coronary artery was released. After 30 min of ischemia, either saline containing 0.1 N HCl or 10, 30 or 100 micrograms/kg of LP-805 was injected intravenously. LP-805 attenuated the decrease in myocardial pH induced by ischemia in a dose-dependent manner. In conclusion, LP-805 may reduce the influence of ischemia on the myocardium.

Acidosis↗

PubMed1991

Effects of MCI-176, a new quinazolinone calcium antagonist, on myocardial energy and carbohydrate metabolism in ischemic dog hearts.

The effect of 2-(2,5-dimethoxyphenylmethyl)-3-(2-dimethylaminoethyl)- 6-isopropoxy-4(3H)-quinazolinone hydrochloride (MCI-176), a calcium antagonist, on ischemic myocardial metabolism was studied in dog hearts subjected to an occlusion of the left anterior descending coronary artery (LAD) for 3 or 30 min. MCI-176 (0.03 or 0.1 mg/kg), when injected i.v. 5 min before occlusion, increased coronary blood flow and decreased systemic aortic pressure. When the LAD was ligated, the levels of creatine phosphate, ATP, total adenine nucleotides and energy change potential decreased in the ischemic myocardium. Three minutes after ischemia, MCI-176 (0.1 mg/kg) significantly (P less than 0.05) diminished these impairments of energy metabolism. Even 30 min after ischemia, pretreatment with MCI-176 tended to lessen the depletion of ATP and total adenine nucleotides, although these effects were not statistically significant. Myocardial ischemia produced a breakdown of glycogen, an accumulation of lactate, and an inhibition of glycolytic flux through phosphofructokinase reaction. MCI-176 (0.1 mg/kg) significantly (P less than 0.05) reduced these alterations of carbohydrate metabolism after 3 min of ischemia. These results suggest that pretreatment with MCI-176 reduces the impairments of myocardial energy and carbohydrate metabolism in ischemic dog hearts, suggesting that the drug is capable of improving the imbalance between oxygen supply and oxygen demand in the ischemic myocardium.

Adenine Nucleotides↗

PubMed1991

Purification and immunochemical characterization of a recombinant outer membrane protein from Bacteroides gingivalis.

1. Bacteroides gingivalis is thought to be one of the most virulent microorganisms in relation to adult periodontitis. A gene clone, MD125, is an Escherichia coli host which produces an outer membrane protein of B. gingivalis. 2. The recombinant outer membrane protein (rOMP) was purified to homogeneity from cell sonicate of MD125 by four chromatographic steps. The molecular weight of the purified rOMP was estimated to be approximately 40 kDa. 3. Immunodiffusion analysis showed that antiserum against whole cells of B. gingivalis reacted not only with B. gingivalis cells but also with other Bacteroides cells. Antiserum against the purified recombinant protein reacted with cells of B. gingivalis, whereas this antiserum did not react with all of the other Bacteroides species tested. 4. These data suggest that the rOMP may be a B. gingivalis-specific antigen and that the purified rOMP will be useful material for serodiagnosis and for the development of a vaccine against B. gingivalis infection.

Bacterial Outer Membrane Proteins↗

PubMed1991

Purification and characterization of recombinant cell surface protein antigen A of Streptococcus sobrinus B13N.

1. It has been reported that immunization of rhesus monkeys with the surface protein antigen I/II from Streptococcus mutans significantly reduced dental caries. 2. The surface protein antigen A (SpaA) from Streptococcus sobrinus is known to correspond antigenically to I/II. MD51 is an Escherichia coli host containing pMD51, a plasmid encoding the SpaA gene from Streptococcus sobrinus B13N. 3. The recombinant SpaA (rSpaA) was purified from cell extracts of Escherichia coli clone MD51. 4. The purified recombinant SpaA was homogeneous with a molecular weight of 210 kDa according to SDS-PAGE and had an isoelectric point of 4.2 based on isoelectric focusing. 5. Amino acid composition of rSpaA showed a relatively high amount of hydrophobic amino acids (39.7%).

Amino Acids↗

PubMed1991

Platelet-activating factor stimulates production of prostaglandin E2 in murine osteoblast-like cell line MC3T3-E1.

We found that platelet-activating factor (PAF) stimulated the production of prostaglandin (PG) E2 in MC3T3-E1 cells in a time- and dose-dependent manner. 1.0 microM PAF gave a maximal stimulation of PGE2 production by MC3T3-E1 cells after a 4 hr PAF-treatment. Furthermore, the PAF-induced PGE2 production was abolished by the pre-treatment of the cells with a PAF receptor antagonist, 1-O-hexadecyl-2-acetyl-sn-glycero-3-phospho(N,N,N-trimethyl)hexanolamine, which occupied the same receptor site as PAF. These results suggest that PAF stimulates the PGE2 synthesis through a PAF receptor mediated pathway. Possibly PAF modulates bone metabolism by stimulating PGE2 synthesis.

Animals↗

PubMed1991

Effects of thoracic epidural anesthesia on myocardial pH and metabolism during ischemia.

The effect of thoracic epidural anesthesia (TEA) on the ischemic myocardium was examined in open-chest dogs anesthetized intravenously. Ischemia induced by brief coronary artery occlusion caused an elevation of the ST segment in epicardial ECG and a reduction in myocardial pH and contractile force. TEA with 0.15 ml/kg of 0.4% bupivacaine solution attenuated an ischemia-induced decrease in myocardial pH and an increase of the ST segment in epicardial ECG. This attenuation was maintained even after the restoration of blood pressure and heart rate, which had been decreased significantly after TEA, to pre-TEA levels, suggesting that a beneficial effect of TEA should not be confined to its hemodynamic changes such as decreased blood pressure and heart rate. In contrast, the subendocardial contents of ATP, creatine phosphate (CP) and lactate were not affected by TEA, either in the presence or the absence of 5 min LAD occlusion. These results suggest that neither hemodynamic nor metabolic changes are responsible for the reduced myocardial ischemic acidosis induced by TEA after brief coronary artery occlusion. The acidosis-saving property of TEA is favorable for the ischemic heart.

Adenosine Triphosphate↗

PubMed1991

The inhibitory effect of bevantolol on the accumulation of non-esterified fatty acids during ischaemia in the dog heart in situ.

1. The left anterior descending coronary artery (LAD) was completely ligated for 90 min (i.e. myocardial ischaemia was produced) in the dog anaesthetized with pentobarbital. 2. Bevantolol, a beta 1-adrenoceptor antagonist, was injected (1 mg/kg, intravenously) 5 min before LAD occlusion. The bevantolol injection decreased heart rate without affecting blood pressure. 3. The myocardial samples were taken from the LAD area immediately after the end of LAD occlusion, and were subjected to analysis of the myocardial levels of non-esterified fatty acids (NEFA). 4. In dogs in which saline was injected, ischaemia produced accumulation of NEFA, especially arachidonic and palmitoleic acids, in the myocardium. 5. In dogs in which bevantolol was injected, the accumulation of NEFA induced by ischaemia was almost completely inhibited. 6. It is concluded that bevantolol inhibits ischaemia-induced accumulation of NEFA in the myocardium, and that stimulation of the beta 1-adrenoceptors is probably responsible for NEFA accumulation induced by ischaemia.

Adrenergic beta-Antagonists↗

PubMed1991

Vacuoles and vesicles in the rat junctional epithelium: a study with serial ultrathin sections.

To investigate whether the electron-lucent structures resembling vesicles and vacuoles in the rat molar junctional epithelium (JE) are in fact intracellular or extracellular, a study using serial ultrathin sections was carried out. In one series of experiments, the animals were not treated before the tissues were conventionally fixed; in another, anesthetized animals were administered horseradish peroxidase 20 min before the tissues were fixed. A large number of electron-lucent structures resembling vesicles and vacuoles were detected in both the peripheral and central cytoplasm of the JE localized at enamel and connective tissue sites. These were 70 to 800 nm in diameter and had a lucency similar to that of the extracellular space in untreated specimens fixed with conventional fixative. Serial ultrathin sectioning revealed that the electron-lucent structures gradually became part of the extracellular space in the following sections. There were also found in the middle portion of the cytoplasm in specimens pretreated with horseradish peroxidase. Numerous vacuole-like structures containing peroxidase-positive materials were found to be contiguous with the extracellular space. A small number of vesicles, also containing peroxidase-positive materials, did not appear in the previous or following sections. These results indicate that almost all electron-lucent structures resembling vesicles and vacuoles in the JE are located at the end of a long infolding, and are still in contact with the extracellular space.

Animals↗