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Biomedical subjects

I Kobayashi

Publications and source records attributed to I Kobayashi.

At least 19 recordsLinked to original sources
PubMed1995

Restriction-modification systems as genomic parasites in competition for specific sequences.

Restriction-modification (RM) systems are believed to have evolved to protect cells from foreign DNA. However, this hypothesis may not be sufficient to explain the diversity and specificity in sequence recognition, as well as other properties, of these systems. We report that the EcoRI restriction endonuclease-modification methylase (rm) gene pair stabilizes plasmids that carry it and that this stabilization is blocked by an RM of the same sequence specificity (EcoRI or its isoschizomer, Rsr I) but not by an RM of a different specificity (PaeR7I) on another plasmid. The PaeR7I rm likewise stabilizes plasmids, unless an rm gene pair with identical sequence specificity is present. Our analysis supports the following model for stabilization and incompatibility: the descendants of cells that have lost an rm gene pair expose the recognition sites in their chromosomes to lethal attack by any remaining restriction enzymes unless modification by another RM system of the same specificity protects these sites. Competition for specific sequences among these selfish genes may have generated the great diversity and specificity in sequence recognition among RM systems. Such altruistic suicide strategies, similar to those found in virus-infected cells, may have allowed selfish RM systems to spread by effectively competing with other selfish genes.

Binding, Competitive↗
PubMed1995

Polyarginine and eosinophil-derived major basic protein increase microvascular permeability independently of histamine or nitric oxide release.

We tested the hypothesis that cationic peptides (polyarginine; poly-argn) and eosinophil-derived major basic protein (MaBP) increase permeability by stimulating the release of histamine and/or nitric oxide. We used intravital microscopy, clearance, and integrated optical intensity (IOI), using FITC-dextran 150 (FITC-dx 150) as a tracer, to evaluate changes in microvascular permeability in the hamster cheek pouch. Poly-argn at 1 microM (topical) increased the clearance of FITC-dx 150 from 610 to 3240 nl/60 min/g. In contrast, 1 microM polyglutamic acid (poly-glun; and anionic peptide) did not affect the clearance of FITC-dx 150 (605 nl/60 min/g). At 0.5 and 1.0 nM, poly-argn increased the clearance of FITC-dx 150 from 610 to 1722 and 2396 nl/60 min/g (P < 0.05). Similarly, 0.5 nM MaBP increased clearance from 591 +/- 38 to 1967 +/- 168 nl/60 min/g. L-NAME at 10(-4) M did not prevent the 0.5 nM MaBP-induced elevation in clearance (1784 +/- 350). Poly-argn at 1 nM increased net mean IOI by 21.5 +/- 7.2 units. This elevation was not inhibited by topical 10(-4) M L-NMMA (27.5 +/- 8.4). Using 0.5 nM poly-argn as agonist, we assayed suffusate samples for their histamine concentration using a competitive enzyme immunoassay and found no detectable histamine. Pyrilamine, an H1 antagonist, did not inhibit the 0.5 nM poly-argn-induced elevation in clearance of FITC-dx 150. We conclude that (1) cationic peptides and MaBP increase microvascular permeability and (2) the increase in microvascular permeability produced by low concentrations of poly-argn and by MaBP is independent of the release of histamine and does not require nitric oxide.

Animals↗
PubMed1995

Colonic mucosal hemodynamics and tissue oxygenation in patients with ulcerative colitis: investigation by organ reflectance spectrophotometry.

Colonic mucosal hemodynamics were investigated at the rectosigmoidal region of the colon in 46 patients with ulcerative colitis and in 18 normal subjects by organ reflectance spectrophotometry under colonoscopy. The value for the index of mucosal hemoglobin concentration (IHb) was significantly higher, and value for the index of mucosal hemoglobin oxygen saturation (ISO2) was significantly lower in patients with active ulcerative colitis than values in the normal controls or in patients with inactive ulcerative colitis. The results indicate mucosal congestion and hypoxemia in patients with active ulcerative colitis. The changes in IHb and ISO2 correlated well with the severity of ulcerative colitis scored by endoscopic findings and with the number of infiltrating inflammatory cells in the mucosa analyzed histologically in biopsy samples. In conclusion, the colonic mucosal microcirculation in patients with active ulcerative colitis was disturbed and showed congestion and hypoxemia. The analysis of hemodynamic changes may be helpful for assessing the activity of ulcerative colitis.

Adult↗
PubMed1995

An immunohistochemical and ultrastructural study of vasomotor nerves in the microvasculature of human dental pulp.

Immunohistochemical methods for S-100 protein and neurone-specific enolase showed two types of nerve endings around the pulp microvasculature: the free endings, which comprise the major neural component and are distributed in all types of microvessel such as arterioles, venules and capillaries; and the varicose endings. The varicose ending was a less frequent, minor component observed only in the arterioles. Both immunohistochemical and ultrastructural observations confirmed that the varicose endings were the terminal axons of efferent vasomotor nerves. Further extensive ultrastructural examinations on the vasomotor nerves added the following new findings to our previous reports. Vasomotor nerves sometimes ramified into more than two terminal axons around arterioles, and most of these ramified axons ended in the adventitia-media junction of the arteriolar wall; however, nerve endings occasionally penetrated into the media. These findings suggest an intimate structural association between vasomotor nerves and arterioles in regulating the arteriolar microcirculation in human dental pulp by stimulating smooth-muscle cells not only of the outermost but also of the inner layers. Furthermore, the deep penetration of terminal axons into the arteriolar wall seems to provide effective regulation of pulpal blood flow under physiological and pathological conditions.

Adult↗
PubMed1995

Immunohistochemical study on the immunocompetent cells of the pulp in human non-carious and carious teeth.

The condition of the pulp tissue was classified into seven groups according to the depth of carious lesions from stage (S) 0 (non-carious teeth) to S6 (exposed pulp). A substantial change in the infiltration of immunocompetent cells occurred between S3 and S4; all types were markedly increased in S4 as compared to S3, with a remarkable increase in the number of helper T lymphocytes, B-lineage cells, neutrophils and macrophages. Therefore, the pulpal immune reaction to carious stimuli could be classified into early (S1-S3) and advanced phases (S4-S6). In the early phase a cellular immunoresponse would be induced by T-lineage cells, and in the advanced phase the humoral immunoresponse is furthered by B-lineage cells concomitant with the destruction of pulp tissue by proteolytic enzymes released from infiltrating neutrophils and macrophages. Human dental pulp is thus equipped with a functional immune response that is sufficient as a biodefensive mechanism. Dental caries should be treated before S4.

Adult↗
PubMed1995

The proliferative activity in oral epithelial dysplasia analyzed by proliferating cell nuclear antigen immunostaining and argyrophilic nucleolar organizer region staining.

The proliferative activity of leukoplakia without dysplastic change (LP), epithelial dysplasia (ED), and squamous cell carcinoma (SCC) in the oral mucosa was examined by means of proliferating cell nuclear antigen (PCNA) immunostaining, silver-binding argyrophilic nucleolar organizer region (AgNOR) staining, and the frequency of mitotic figures. Significant differences in the labeling index of PCNA immunostaining (PI) and mitotic index (MI) were noted between LP and ED and between ED and SCC. The mean numbers of AgNORs (AI) significantly differed between ED and SCC. There was a significant positive correlation between PI and MI in samples of ED. However, there was no significant correlation between AI and other indexes. The number and the distribution of PCNA-positive cells in ED varied among samples. Five samples with higher PI and MI indexes than the mean values were selected from those of dysplasia based on the correlation between PI and MI. Their histological features symptomatic of oral ED as defined by the World Health Organization (WHO) Collaborating Centre in 1978, were investigated and compared with 10 samples with lower indexes. Histological findings, such as "loss of polarity of the basal cells," "an increased nuclear-cytoplasmic ratio," "cellular pleomorphism," and "enlarged nucleoli," were significant histological features of these five samples. This study showed that the four histological components described previously and the increased number of mitotic figures used as the index of proliferating activity were the main histological components related to severe ED of oral mucosa. They will provide a useful means of deciding the histopathological grade of oral ED.

Adult↗
PubMed1995

Cyclosporin A inhibits leukotriene production in intact RBL-1 cells without inhibiting leukotriene biosynthetic enzymes.

The effects of cyclosporin A (CSA) on arachidonic acid (AA) metabolism were investigated in intact rat basophilic leukemia-1 (RBL-1) cells and cell lysates. Calcium ionophore (A23187)-stimulated synthesis of cysteinyl leukotrienes (LTC4, LTD4, and LTE4), LTB4, and 5-hydroxyeicosatetraenoic acid (5-HETE) in intact cells in the absence or presence of CSA was measured by reversed-phase high-performance liquid chromatography (HPLC). CSA inhibited the production of cysteinyl LTs, LTB4, and 5-HETE in intact cells in a dose-dependent manner. The synthesis of cysteinyl LTs, LTB4, and 5-HETE was also measured after the incubation of cell lysates with free AA in the absence or presence of CSA. CSA did not inhibit synthesis of cysteinyl LTs, but rather stimulated production of LTB4 and 5-HETE in cell lysate. A23187-stimulated release of incorporated [3H]AA from intact cells was not inhibited by CSA. CSA did not inhibit the synthesis of cysteinyl LTs and LTB4 when cells incubated with LTA4 as the substrate. These results indicate that the inhibitory effects of CSA on the synthesis of LTs and 5-HETE in intact cells are attributable to a modulatory action on a step in the series of intracellular events that includes the activation of 5-lipoxygenase, which are initiated by Ca2+ influx and end in the release of metabolites from the cell membrane, rather than to a direct inhibitory action on enzymes in the LT biosynthetic pathway.

Animals↗
PubMed1995

ATP sparing effect of isoflurane during ischaemia and reperfusion of the canine heart.

Sustained dysfunction of myocardial contractility after short periods of coronary artery occlusion and reperfusion has been termed "stunned myocardium". Isoflurane may improve the recovery of regional myocardial contractility in stunned myocardium. The purpose of the present study was to determine if isoflurane prevents depletion of high energy phosphates after myocardial ischaemia-reperfusion and if the reduction in cardiac work during isoflurane anaesthesia contributes to the preservation of high energy phosphate metabolism in an acute canine model. Mongrel dogs were allocated to one of three groups: controls, anaesthetized with urethane and chloralose; ISO group, isoflurane administered before ischaemia; and ISOc group, heart rate and mean arterial pressure controlled to approximately match baseline values. The left anterior descending (LAD) coronary artery was occluded for 15 min and then reperfused for 60 min during 1.5% end-tidal isoflurane anaesthesia. Full thickness samples of myocardium were obtained from the reperfused area (supplied by the LAD) and the non-ischaemic area (supplied by the left circumflex coronary artery). The concentrations of adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), creatine phosphate (CP) and lactate in the endocardial portion of the myocardium were measured. Arterial pressure, aortic flow in the ascending aorta and rate-pressure product decreased significantly after isoflurane. Although the concentration of ATP of the reperfused area in the control group showed a significant reduction 60 min after reperfusion, the ISO and ISOc groups had significantly greater concentrations. Isoflurane anaesthesia maintained myocardial high energy phosphate metabolism in reperfused myocardium. We conclude that the reduction in cardiac work played only a minor role in the ATP-sparing effect of isoflurane.

Adenosine Triphosphate↗
PubMed1995

Evidence for conservative (two-progeny) DNA double-strand break repair.

The double-strand break repair models for homologous recombination propose that a double-strand break in a duplex DNA segment is repaired by gene conversion copying a homologous DNA segment. This is a type of conservative recombination, or two-progeny recombination, which generates two duplex DNA segments from two duplex DNA segments. Transformation with a plasmid carrying a double-strand gap and an intact homologous DNA segment resulted in products expected from such conservative (two-progeny) repair in Escherichia coli cells with active E. coli RecE pathway (recBC sbcA) or with active bacteriophage lambda Red pathway. Apparently conservative double-strand break repair, however, might result from successive events of nonconservative recombination, or one-progeny recombination, which generates only one recombinant duplex DNA segment from two segments, involving multiple plasmid molecules. Contribution of such intermolecular recombination was evaluated by transformation with a mixture of two isogenic parental plasmids marked with a restriction site polymorphism. Most of the gap repair products were from intramolecular and, therefore, conservative (two-progeny) reaction under the conditions chosen. Most were conservative even in the absence of RecA protein. The double-strand gap repair reaction was not affected by inversion of the unidirectional replication origin on the plasmid. These results demonstrate the presence of the conservative (two-progeny) double-strand break repair mechanism. These experiments do not rule out the occurrence of nonconservative (one-progeny) recombination since we set up experimental conditions that should favor detection of conservative (two-progeny) recombination.

Bacteriophage lambda↗
PubMed1995

Dependence of frequency of homologous recombination on the homology length.

The frequency of homologous recombination is believed to be a linear function of the length (N bp) of homology between DNAs. Here, the N intercept is believed to be determined by a threshold length below which some physical constraint is effective. In the mammalian gene targeting systems, however, the frequency depends more steeply than linearly on the homology length. To explain both the linear dependence and the steeper dependence, we propose a model where the branch point of a reaction intermediate is assumed to "walk randomly" along the homologous region until it is processed. The intermediate is assumed to be destroyed if the branch point ever reaches either end of the homology. In this model, the length dependence is governed by a parameter, h, which is defined as efficiency of processing of the intermediate and reflects unlikelihood of the destruction at either end of the homology. We find that the frequency is proportional to N3 for smaller N and is a linear function of N for larger N. Where the shift from the N3 dependence to the linear dependence takes place is determined by the parameter h. The range of N showing the N3 dependence becomes narrower as h becomes larger. The dependence steeper than linear dependence, which is observed not only in the mammalian gene targeting system but also in bacteriophage T4, Escherichia coli and yeast systems, agrees well with the predicted N3 dependence. The N intercept is determined not by physical (or structural) constraints but only by the parameter h in this model.

Animals↗
PubMed1995

Sleep disturbance in elderly patients with cognitive impairment, decreased daily activity and periventricular white matter lesions.

We investigated how sleep disturbance is correlated with brain lesions, cognitive impairment and decreased daily activity (ADL). Two hundred and four chronically ill patients including patients with vascular dementia were classified into eight groups based on cognitive function, ADL and grades of PVL (periventricular lucency) as shown by computerized tomography. Visual monitoring of the sleep/wake state was performed hourly for 14 consecutive days and the daytime sleep hours and the nighttime sleep hours were determined based on detected disturbances in the sleep/wake pattern. Whether any specific infarcted regions were correlated with sleep disturbance was also evaluated. We found that daytime sleep hours were increased and nighttime sleep hours were decreased by three single factors: cognitive impairment, decreased ADL and the severe grade of PVL. As for an interactive effect of the two factors, daytime sleep hours were increased by dementia with decreased ADL (independent of PVL) and increased by decreased ADL with the severe grade of PVL (independent of dementia). Although three factors affected sleep independently, ADL has an interactive effect on sleep with dementia and with PVL. No specific cortical regions related to sleep disturbance were found.

Activities of Daily Living↗
PubMed1995

Comparative effects of losartan, captopril, and enalapril on murine acute myocarditis due to encephalomyocarditis virus.

Losartan, a recently developed nonpeptide angiotensin II (AII) receptor antagonist, was orally administered for 14 days to mice with viral myocarditis, beginning 7 days after encephalomyocarditis virus inoculation. The angiotensin-converting enzyme inhibitors (ACEI) captopril and enalapril were also administered in the same manner to compare the therapeutic effects of these three drugs on the degree of myocarditis, acute heart failure, and left ventricular (LV) hypertrophy. Heart weight and the heart weight/body weight ratio were reduced by losartan (60 mg/kg/day) and captopril (7.5 mg/kg/day), but not by enalapril (1 mg/kg/day). LV wall thickness and cavity dimension were decreased in the losartan and captopril groups. Captopril reduced both myocardial necrosis and inflammation, whereas enalapril reduced myocardial necrosis but not inflammation. However, none of the studied losartan doses (1.2, 12, 60 mg/kg/day) influenced myocardial necrosis and inflammation resulting from viral infection. Thus, specific blockade of AII is beneficial in congestive heart failure (CHF) and LV hypertrophy but is not effective in viral-evoked inflammation and injury.

Acute Disease↗
PubMed1995

[Heterogeneity of lipopolysaccharide chain size of Pseudomonas aeruginosa isolated from different clinical sources--with reference to gentamicin-susceptibility and serotype].

Lipopolysaccharide (LPS) compositions of P. aeruginosa isolated from from clinical sources such as blood, urine, pus, sputum, and feces were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and aminoglycoside-susceptibility and serotype of these isolates were investigated in this study. Fifty-nine isolates tested were divided into three groups according to the difference in their LPS compositions; 35 strains with the long-LPS chain (B-band LPS), 14 strains with the short chain (A-band LPS) and 10 LPS-deficient strains. The relationship between the LPS compositions and their sources of 59 strains were investigated. The majority of clinical isolates (12 of the 13 strains) from the blood samples possessed the long-LPS chain (B-band LPS) and the remaining possessed the short-LPS chain (A-band LPS). About 67% each of the isolates from urine and feces possessed the long-LPS chain, and the minor part of both groups possessed the short-LPS chain. In isolates from both sputum and pus samples, the long- and short-LPS chains were found at almost the same rate, and the LPS-deficient isolates were found in the sputum samples at a considerably high rate of 42%. The 19 of the 35 isolates with the long-LPS chain were susceptible to gentamicin (54%) and 12 isolates were resistant (34%). On the other hand, the 14 isolates with the short chain were divided roughly into three groups, gentamicin-resistant, and -susceptible groups and intermediate groups. It was also notable that 7 of the 10 LPS-deficient isolates were resistant to gentamicin.(ABSTRACT TRUNCATED AT 250 WORDS)

Drug Resistance, Microbial↗
PubMed1995

Gene targeting with a replication-defective adenovirus vector.

Wide application of the gene-targeting technique has been hampered by its low level of efficiency. A replication-defective adenovirus vector was used for efficient delivery of donor DNA in order to bypass this problem. Homologous recombination was selected between a donor neo gene inserted in the adenovirus vector and a target mutant neo gene on a nuclear papillomavirus plasmid. These recombinant adenoviruses allowed gene transfer to 100% of the treated cells without impairing their viability. Homologous recombinants were obtained at a level of frequency much higher than that obtained by electroporation or a calcium phosphate procedure. The structure of the recombinants was analyzed in detail after recovery in an Escherichia coli strain. All of the recombinants examined had experienced a precise correction of the mutant neo gene. Some of them had a nonhomologous rearrangement of their sequences as well. One type of nonhomologous recombination took place at the end of the donor-target homology. The vector adenovirus DNA was inserted into some of the products obtained at a high multiplicity of infection. The insertion was at the end of the donor-target homology with a concomitant insertion of a 10-bp-long filler sequence in one of the recombinants. The possible relationship between these rearrangements and the homologous recombination is discussed. These results demonstrate the applicability of adenovirus-mediated gene delivery in gene targeting and gene therapy.

Adenoviridae↗
PubMed1995

Central and reflex neural control of genioglossus in subjects who underwent laryngectomy.

Inspiratory activation of the genioglossus (GG) may occur by central drive or as a reflex to negative airway pressure. To distinguish between these, we studied seven laryngectomy patients who breathe via tracheal stomas. Negative pressure stimuli (-15 and -25 cmH2O for 500 ms) were applied 1) at functional residual capacity and 2) during early inspiration via (i) the upper airway (UA) and (ii) the tracheal stoma. Intraoral surface GG electromyogram was quantified, as described previously (R. L. Horner, J. A. Innes, K. Murphy, and A. Guz, J. Physiol. Lond. 436: 15-29, 1991). Phasic GG activity was also measured from an integrated electromyogram during spontaneous and inspiratory loaded breathing. Reflex GG activation occurred with negative UA pressure both at functional residual capacity and during inspiration (P < 0.001), but pressure stimuli at the stoma caused no significant activation (P = 0.07). Phasic inspiratory activation occurred in four patients at rest and in all seven patients during inspiratory loading (P < 0.02). These patients demonstrate 1) reflex activation of the GG by negative UA pressure without airflow or respiratory effort and 2) central inspiratory GG activation that is not mediated by negative airway pressure.

Aged↗