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N Doi

Publications and source records attributed to N Doi.

At least 37 records · Page 2Linked to original sources
PubMed1998

Characterization of random-sequence proteins displayed on the surface of Escherichia coli RNase HI.

In a previous study, random-sequence proteins of 120-130 amino acid residues were inserted into the surface loop region of the enzyme, Escherichia coli RNase HI [Doi et al. (1997) FEBS Lett. 402, 177-1801. Here we established that the RNase H activity of the insertion mutants is correlated with their secondary structure contents evaluated by circular dichroism measurement at 222 nm. The random-sequence insert of a mutant enzyme possessing relatively high RNase H activity was detached from the RNase HI scaffold, and its characterization indicated that the random-sequence protein maintains its secondary structure after separation from the scaffold. Thus, the structural features of random-sequence proteins were suggested to be monitored by measuring the activity of the scaffold enzyme into which these proteins have been inserted.

Amino Acid Sequence↗
PubMed1998

Differential effects of various growth factors and cytokines on the syntheses of DNA, type I collagen, laminin, fibronectin, osteonectin/secreted protein, acidic and rich in cysteine (SPARC), and alkaline phosphatase by human pulp cells in culture.

The purpose of this study is to differentiate roles of several growth factors and cytokines in proliferation and differentiation of pulp cells during development and repair. In human pulp cell cultures, laminin and type I collagen levels per cell remained almost constant during the whole culture period (22 days). On the other hand, secreted protein, acidic and rich in cysteine (SPARC/osteonectin) and alkaline phosphatase (ALPase) levels markedly increased after the cultures reached confluence. Laminin and type I collagen, as well as fibronectin, stimulated the spreading of pulp cells within 1 h. Adding transforming growth factor-beta (TGF-beta) decreased laminin and ALPase levels, whereas it increased SPARC and fibronectin levels 3- to 10-fold. Western and Northern blots showed that TGF-beta enhanced SPARC synthesis at the protein and mRNA levels. Basic fibroblast growth factor (bFGF) decreased type I collagen, laminin, SPARC, and ALPase levels without changing the fibronectin level. Platelet-derived growth factor (PDGF) selectively decreased laminin, SPARC, and ALPase levels. Epidermal growth factor (EGF) also decreased SPARC and ALPase levels. Tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) decreased type I collagen and laminin levels, and abolished SPARC and ALPase syntheses. Of these peptides, bFGF and PDGF showed the greatest stimulation of [3H]thymidine incorporation into DNA. TGF-beta, EGF, and TNF-alpha had less effect on DNA synthesis, whereas IL-1beta inhibited DNA synthesis. These findings demonstrated that TGF-beta, bFGF, EGF, PDGF, TNF-alpha, and IL-1beta have characteristically different patterns of actions on DNA, laminin, type I collagen, fibronectin, ALPase, and SPARC syntheses by pulp cells.

Alkaline Phosphatase↗
PubMed1998

Screening of conformationally constrained random polypeptide libraries displayed on a protein scaffold.

The selection of novel proteins or enzymes from random protein libraries has come to be a major objective in current biology, and these enzymes should prove useful in various biological and biomedical fields. New technologies such as in vitro selection of proteins in cell-free systems have high potential to realize evolutionary molecular engineering of proteins. This review highlights an application of insertional mutagenesis of proteins to evolutionary molecular engineering. Random sequence proteins are inserted into the surface of a host enzyme which serves as a scaffold to display random protein libraries. Constraints on random polypeptide conformations owing to the proximity of N- and C-termini on the scaffold would result in greater screening efficiency of libraries. The scaffold enzyme is also used as a probe for monitoring the hill climbing of random sequence proteins on a fitness landscape and navigating rapid protein folding in the sequence space.

Animals↗
PubMed1998

Does increased QT dispersion in the acute phase of anterior myocardial infarction predict recovery of left ventricular wall motion?

QT dispersion has been recognized as an undesirable marker because of its association with arrhythmogenicity in patients with myocardial infarction, but the relation between QT interval dispersion and wall motion abnormalities has not been clarified. After the introduction of reperfusion therapy, it was recognized that T waves were inverted twice in the course of myocardial infarction. An investigation was made of the clinical significance of QT dispersion in relation to the presence of inverted T waves and left ventricular wall motion abnormalities in 34 patients (mean age, 59 years) with acute anterior myocardial infarction who underwent successful reperfusion therapy. The amplitude of the deepest inverted T waves occurring within the first 3 days (T1) and after 3 days (T2) of myocardial infarction were measured in electrocardiographic (ECG) lead V3. On the ECGs on which T1 and T2 were recorded, QT dispersion was calculated (QTd1, QTd2), and T1 and T2 were correlated with QTd1 (r = .65) and QTd2 (r = .47), respectively. The difference between the extent of asynergy in the acute phase and the chronic phase, which was evaluated by the centerline method, was correlated with T1 (r = .63) and QTd1 (r = .67). Patients with a QTd1 of 0.1 second or longer showed a greater change in the extent of asynergy (23.4 +/- 13.1% vs 4.9 +/- 9.8%, P < .01) and less asynergy in the chronic phase (19.9 +/- 15.6% vs 46.5 +/- 14.0%, P < .01) than patients with a QTd1 of less than 0.1 second. Thus, QT dispersion in the acute phase of anterior myocardial infarction indicates recovery of left ventricular wall motion. Prolongation of the local action potential duration of the myocardium that recovers from severe ischemia may be a contributor to the increased QT dispersion that results in inversion of T waves in the acute phase of myocardial infarction.

Electrocardiography↗
PubMed1998

Inhibition of rat oxytocin and vasopressin supraoptic nucleus neurons by nociceptin in vitro.

The effects of nociceptin (orphanin FQ) on the excitability of electrophysiologically-identified oxytocin and vasopressin neurons were investigated in rat hypothalamic supraoptic nucleus slices in vitro, using whole-cell patch-clamp recording techniques. Nociceptin inhibited the spontaneous discharge of 9/20 (45%) of supraoptic nucleus neurons tested, while in the remaining 11/20 neurons it inhibited firing rate and induced repetitive burst-firing. There were no differences between the effects of nociceptin on oxytocin and vasopressin neurons. When recordings were made using EGTA-containing patch pipettes, nociceptin caused inhibition in all 30 supraoptic nucleus neurons tested, and burst-firing was not seen. The inhibitory effects of nociceptin persisted in low Ca, Co medium, and were not antagonized by naloxone at concentrations sufficient to antagonize the inhibitory actions of morphine and U50488. The actions of nociceptin on supraoptic nucleus neurons are therefore likely to be mediated by postsynaptic opioid receptor-like (ORL1) receptors that are distinct from known opioid receptors. The inhibitory responses to nociceptin were also insensitive to naloxone benzoylhydrazone, which itself had no effect on the spontaneous discharge of the supraoptic nucleus neurons. Our findings demonstrate that endogenous nociceptin may have a functional role in regulating oxytocin and vasopressin secretion through its actions on hypothalamic supraoptic nucleus neurons.

Animals↗
PubMed1998

Induction of granulomas in interferon-gamma gene-disrupted mice by avirulent but not by virulent strains of Mycobacterium tuberculosis.

To gain a better understanding of the pathological role of interferon-gamma (IFN-gamma) in specific granuloma formation, IFN-gamma gene-deficient mice (BALB/c and C57BL/6) were produced. The IFN-gamma gene in embryonic stem (ES) cells was disrupted by inserting the beta-galactosidase gene (lacZ) and the neomycin resistance gene (neo) at the translation initiation site in exon 1 by homologous recombination. Six-week-old IFN-gamma-deficient and wild-type mice were inoculated with 10(3)-10(7) bacilli of various strains of Mycobacterium tuberculosis (Kurono, H37Rv, H37Ra and BCG Pasteur) through their tail veins. The mice were examined 7 weeks later for granuloma formation. The avirulent BCG Pasteur and H37Ra strains (10(3)-10(4) bacilli/ml) induced granulomas in the spleen, liver and lungs of IFN-gamma-deficient mice. The granulomas consisted of epithelioid macrophages and Langhans multinucleate giant cells, but lacked caseous necrosis. The virulent Kurono and H37Rv strains induced disseminated abscesses but not granulomas in various organs of IFN-gamma-deficient mice and Mac-3-positive macrophages were not detected in the abscess lesions. These results suggest that IFN-gamma may be primarily responsible for macrophage activation and that other factor(s) may be involved in the granuloma formation mechanism.

Animals↗
PubMed1998

[Therapeutic efficacy of benzoxazinorifamycin KRM-1648 against experimental murine tuberculosis: (1). A study on the efficacy of short course treatment with the intratracheal and intravenous infection model].

OBJECTIVES: This study aims to compare in vivo activity of benzoxazinorifamycin KRM-1648 (KRM) with those rifampicin (RFP) and rifabutin (RBT) against experimental murine tuberculosis. STUDY DESIGN: Mice were infected with Mycobacterium tuberculosis or M. bovis by the intratracheal (i.t.) or intravenous (i.v.) routes, and treated for 10 days with various doses of each drug starting from the 9th or 11th day after the TB-infection. RESULTS: (A) A rapid test for in vivo evaluation of three rifamycins was conducted by examining the survival days of treated mice infected with 106 cfu of M. bovis Ravenel. Mice treated with KRM exhibited 2.13.7 times longer survival times, in comparison with those treated with RFP or RBT. (B) In the i.t.-model of M. bovis Ravenel infection, three rifamycin derivatives gave "distinctive dose-response curves" in the correlation of dose sizes with the mean survival times or "log10CFU/lungs reductions". (C) In M. tuberculosis Kurono infection models, the ranking of the anti-TB activity of the three rifamycins in each organ was as follows: i.t.-and i.v.-lungs: KRM >> RFP not equal to RBT, i.v.-spleen: KRM not equal to RBT > RFP, i.v.-liver: KRM not equal to RBT > RFP. (D) Based on the results of "log10CFU reduction" in different organs in M. tuberculosis Kurono infection models, "characteristic in vivo activity patterns of each rifamycin" were obtained. (E) The therapeutic efficacy of KRM in lungs was greater than in spleen and liver with any dose. In contrast, RBT exhibited more remarkable in vivo activity in the spleen and liver than in lungs. CONCLUSION: The prominent in vivo activity of KRM may allow small dose for effective therapy; 1/3 dose or less in comparison with those of RFP or RBT, or intermittent therapy of tuberculosis.

Animals↗
PubMed1998

[Development of the intratracheal infection model of experimental murine mycobacteriosis: comparison with the intravenous infection model].

An intratracheal infection method of experimental murine mycobacteriosis was developed for an in vivo study of antimycobacterial agents. Two models of intratracheal (IT) and intravenous (i.v.) routes of infection with mycobacteria of the same inoculum dose were compared in terms of the mean survival days of mice or bacterial loads in organs during the course of infection. IT model with either of M. bovis Ravenel, M. tuberculosis Kurono, M. tuberculosis H37Rv or M. intracellular N-256 exhibited a much more distinct lung-specific infection than i.v. model with the same dose of respective mycobacterial strains. The intratracheal infection method presented in this report does not require any special equipment and is a much safer method for the researcher than airborne infection. In this model, following slight anesthetizing of mice, bacillary suspension was injected quantitatively into lungs through the mouth and trachea by using a specially modified needle set with a short fine polyethylene tube. This IT model may be useful not only for the in vivo assessment of anti-mycobacterial agents but also for the comparison of virulence among various mycobacterial strains.

Animals↗
PubMed1997

Insertion of foreign random sequences of 120 amino acid residues into an active enzyme.

Random sequences of 120-130 amino acid residues were inserted into a surface loop region of Escherichia coli RNase HI. This library was screened and about 10% of the clones were found to retain RNase H activity. Subsequent random mutagenesis led to an increase in RNase H activity and solubility of the protein. The inserted regions were found not to contribute to the secondary structure of the mutant protein. The high frequency of insertion of flexible sequences and the increase in the protein's function by further mutagenesis simulate one of the events in protein evolution.

Amino Acid Sequence↗
PubMed1997

Involvement of the anterior urethra in male patients with transitional cell-carcinoma of the bladder undergoing radical cystectomy with simultaneous urethrectomy.

Histological tumor extension into the prostate, urethra and ureters was examined in 52 specimens obtained by cystourethrectomy for transitional cell carcinoma of the bladder with one or more risk factors for urethral involvement. In four (21.1%) of 19 patients with diffuse carcinoma in situ in the bladder extending to the internal urethral orifice and prostatic urethra, the anterior urethra was affected by transitional cell carcinoma, including one specimen with invasion into the corpus spongiosum. However, none of 33 patients without these findings had synchronous anterior urethral transitional cell carcinoma involvement. It is recommended that patients with diffuse carcinoma in situ extending into the prostatic urethra undergo careful preoperative assessment of the anterior urethra before cystectomy. In other cases, the anterior urethra can be preserved and used for orthotopic neobladder replacement.

Adult↗
PubMed1997

Increased plasma concentrations of adrenomedullin correlate with relaxation of vascular tone in patients with septic shock.

OBJECTIVE: To investigate plasma concentrations of adrenomedullin in patients with septic shock and the potential association of these concentrations with relaxation of vascular tone. DESIGN: Prospective, case series. SETTING: Department of Emergency and Critical Care Medicine, Nara Medical University. PATIENTS: Twelve patients who fulfilled the clinical criteria for severe sepsis or septic shock (as defined by the Members of the American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference Committee) and 13 healthy volunteers. INTERVENTIONS: Arterial blood samples were obtained via a 20-gauge cannula inserted into each patient's radial artery. MEASUREMENTS AND MAIN RESULTS: After extraction and purification, plasma adrenomedullin was measured by radioimmunoassay. Systemic vascular resistance index, pulmonary vascular resistance, cardiac index, and stroke volume index were determined with a thermodilution catheter. The mean plasma concentration of adrenomedullin was markedly higher in patients than in controls (226.1 +/- 66.4 [SEM] vs. 5.05 +/- 0.21 fmol/mL, p < .01). Moreover, these concentrations correlated significantly with cardiac index, stroke volume index, and heart rate values, and correlated significantly with decreases in diastolic blood pressure, systemic vascular resistance index, and pulmonary vascular resistance index values. CONCLUSIONS: Enhanced production of adrenomedullin in patients with septic shock may contribute to reduced vascular tone, hypotension, or both. More data are needed to clarify the role of adrenomedullin in the regulation of vascular tone in this patient population.

Adrenomedullin↗
PubMed1997

[The effect of coronary intervention on renal function in patients with chronic renal failure].

To investigate the effect of coronary intervention on renal function in patients with chronic renal failure (CRF) we investigated 19 patients with CRF [serum creatinine (Scr) > 1.5 mg/dl] who underwent coronary intervention for the treatment of acute myocardial infarction or unstable angina recruited from 516 consecutive patients admitted to the coronary care unit of Nara Medical University Hospital from January, 1992 to July, 1995. Serum creatinine levels were measured at 3 points: on admission, at peak level, and at discharge. Nineteen patients were divided into two groups on the basis of increases in Scr (delta Scr): a worsened group (group A) (delta Scr > or = 1mg/dl after coronary intervention; 6 patients) and an unchanged group (group B) (delta Scr < 1 mg/dl ; 13 patients). In group A, except for one patient, the renal function recovered to the level before coronary intervention after adequate hydration or hemodialysis. The volume of contrast medium in group A (420 +/- 134 ml) was significantly higher than group B (253 +/- 97 ml) (p < 0.01). There was significant positive correlation (r = 0.42, p < 0.05) between delta Scr and the volume of contrast medium. In conclusion, coronary intervention for patients with CRF can be performed safely under treatment with adequate hydration and hemodialysis, even at higher serum creatinine levels of up to 4.0 mg/ml.

Aged↗
PubMed1995

Novel mutation at the initiation codon in the Norrie disease gene in two Japanese families.

We have identified a new mutation of Norrie disease (ND) gene in two Japanese males from unrelated families; they showed typical ocular features of ND but no mental retardation or hearing impairment. A mutation was found in both patients at the initiation codon of exon 2 of the ND gene (ATG to GTG), with otherwise normal nucleotide sequences. Their mothers had the normal and mutant types of the gene, which was expected for heterozygotes of the disease. The mutation of the initiation codon would cause the failure of ND gene expression or a defect in translation thereby truncating the amino terminus of ND protein. In view of the rarity and marked heterogeneity of mutations in the ND gene, the present apparently unrelated Japanese families who have lived in the same area for over two centuries presumably share the origin of the mutation.

Base Sequence↗
PubMed1995

Acute left ventricular failure with pulmonary edema following pericardiocentesis for cardiac tamponade--a case report.

We describe the complications of pericardiocentesis and their management in an 18 year-old man. This patient was admitted because of dyspnea and was found on echocardiogram to have cardiac tamponade with coexisting left ventricular dysfunction. He developed acute left ventricular failure with severe pulmonary edema immediately after pericardiocentesis. This complication may have been caused by an abrupt increase in venous return to the failing left ventricle following the release of the pericardial compression. Therefore, pericardial fluid must be drained with caution in pericardiocentesis, especially in cardiac tamponade patients with left ventricular dysfunction, and hemodynamics should be monitored both during and after this procedure.

Acute Disease↗
PubMed1993

Embryonic development and postnatal changes in free D-aspartate and D-serine in the human prefrontal cortex.

We have analyzed free chiral amino acids (aspartate and serine) in the human frontal cortex at different ontogenic stages (from 14 weeks of gestation to 101 years of age) by HPLC with fluorometric detection after derivatization with N-tert-butyl-oxycarbonyl-L-cysteine and o-phthaldialdehyde. Exceptionally high levels of free D-aspartate and D-serine were demonstrated in the fetal cortex at gestational week 14. The ratios of D-aspartate and of D-serine to the total corresponding amino acids were also high, at 0.63 and 0.27, respectively. The concentration of D-aspartate dramatically decreased to a trace level by gestational week 41 and then remained very low during all postnatal stages. In contrast, the frontal tip contained persistently high levels of D-serine throughout embryonic and postnatal life, whereas the D-amino acid content in adolescents and aged individuals was about half of that in the fetuses. Because D-aspartate and D-serine are known to have selective actions at the NMDA-type excitatory amino acid receptor, the present data suggest that these D-amino acids might play a pivotal role in cerebral development and functions that are related to the NMDA receptor.

Adolescent↗