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Biomedical subjects

M Sekiguchi

Publications and source records attributed to M Sekiguchi.

At least 253 records · Page 14Linked to original sources
PubMed1995

Mouse methyltransferase for repair of O6-methylguanine and O4-methylthymine in DNA.

cDNA for mouse O6-methylguanine-DNA methyltransferase was expressed in methyltransferase-deficient Escherichia coli mutant cells, and the overproduced mouse enzyme was purified to a homogeneous state. Using this purified product, polyclonal antibodies were prepared and used to estimate amounts of the methyltransferase protein in cells. A single cell of NIH3T3 contained 1.8 x 10(4) molecules of the methyltransferase protein. When mouse fibroblasts were immunostained, it was shown that most of the methyltransferase protein exists in the cytoplasm rather than in the nucleus. Using double-stranded oligomers containing a single O6-methylguanine or O4-methylthymine at predetermined sites, the mouse enzyme repaired O6-methylguanine and O4-methylthymine, at an almost equal efficiency. In the LacZ reversion assay, MNNG-induced A:T to G:C as well as G:C to A:T transition mutations were efficiently suppressed by the function of mouse methyltransferase, in vivo.

3T3 Cells↗
PubMed1995

Sequence-specific DNA recognition of the Escherichia coli Ada protein associated with the methylation-dependent functional switch for transcriptional regulation.

The Escherichia coli Ada protein, a suicidal DNA methyltransferase, is converted into a transcriptional regulator for methylation-resistance genes by the transfer of a methyl group from a DNA methylphosphotriester to its own Cys69 residue. Here, we report the DNA recognition mode and the functional switch mechanism of the N-terminal 16 kDa fragment of the Ada protein. NMR analysis has revealed that the segment from residues 102 to 123 forms a helix-turn-helix structure. A site-directed mutagenesis study has shown that the second helix in the helix-turn-helix structure plays a crucial role in specific recognition of DNA. These results imply that the sequence-specific interaction of the Ada protein with DNA occurs through the helix-turn-helix motif. NMR experiments on the methylated protein-DNA complex showed line broadening for the amide proton signals from the helix-turn-helix motif and for the protons in the vicinity of Cys69. In the case of the nonmethylated protein-DNA complex, signal broadening was observed only for protons from the helix-turn-helix. These findings suggest that the residues in the vicinity of Cys69 come into direct contact with the cognate DNA after methylation. We propose that the direct contact of this region is a major factor for the "switch" that converts the Ada protein from a nonspecific DNA binding form to a transcription factor.

Amino Acid Sequence↗
PubMed1995

Adhesion molecules on eosinophils in acute eosinophilic pneumonia.

We describe the clinical features, eosinophil surface analysis, and concentration of interleukin-5 (IL-5) in bronchoalveolar lavage (BAL) and in peripheral blood (PB) of a young male patient with acute eosinophilic pneumonia. Eosinophilic pneumonia was diagnosed by BAL and by its clinical course. There was no evidence of an infectious etiology, and the patient showed rapid improvement with methylprednisolone pulse therapy (1 gm/d for 3 d). Significant differences in the expression of surface molecules such as CD54, HLA-DR, and CD69 were observed between BAL and PB eosinophils; IL-5 concentrations in BAL and in PB before treatment were 450 pg/ml and 700 pg/ml, respectively. After treatment, IL-5 concentration in PB was reduced to below 100 pg/ml. The patient has shown no recurrence after treatment.

Acute Disease↗
PubMed1995

Periodic angioedema with eosinophilia: increased serum level of interleukin 5.

A 48-year-old man developed episodic non pitting edema and eosinophilia. Symptoms were alleviated promptly when treated with prednisolone. However, major basic protein (MBP), eosinophil cationic protein (ECP), and eosinophil-derived neurotoxin (EDN) levels in the serum as well as the total number of eosinophils remained high. During periods of attack serum levels of interleukin-5 (IL-5) were elevated, but the levels were lowered following treatment, suggesting that IL-5 is involved in periodic angioedema with eosinophilia.

Angioedema↗
PubMed1995

Effect of prone position on apnea severity in obstructive sleep apnea.

We describe a patient with obstructive sleep apnea (OSA) whose apnea-hypopnea index (AHI) improved remarkably in the prone position accompanied by an improved sleep quality, despite a higher AHI in the supine position and even in the lateral position. Magnetic resonance imaging revealed the most dilated upper airway in the prone position, which suggests the role of anatomical narrowing of the upper airway as an important component in the pathophysiology of positional apnea patients. Further studies are needed to determine the therapeutic efficacy of a prone sleeping position in patients with OSA.

Humans↗
PubMed1995

IL-5 predominant in bronchoalveolar lavage fluid and peripheral blood in a patient with acute eosinophilic pneumonia.

We describe an acute eosinophilic pneumonia (AEP) patient with bronchoalveolar lavage fluid (BALF). Eosinophil cell number (47%), content of interleukin (IL)-5 (8.22 x 10(2) pg/ml) and eosinophil cationic protein (9.25 micrograms/ml) were high in BALF. No eosinophilia was seen in peripheral blood on admission; however, content of IL-5 was 9.47 x 10(2) pg/ml. After methylprednisolone pulse therapy, he improved rapidly with a reduction in eosinophil cell number (7%) and the content of IL-5 (< 100 pg/ml) in BALF. However, a high content of IL-5 (6.9 x 10(2) pg/ml) and transient eosinophilia (17.5%) were seen in peripheral blood. It is important to distinguish between AEP and infectious pneumonia, because of the differing treatments. If the diagnosis of AEP is doubtful, BALF should be performed early.

Acute Disease↗
PubMed1995

[A case of chronic eosinophilic pneumonia involving IL-5 and GM-CSF].

A 57-year-old woman was admitted because of dry cough and middle-grade fever. Chest rentogenogram showed a diffuse infiltrative shadow in the right middle and lower lung fields. The symptoms disappeared after treatment with minocycline, but a new dense shadow appeared in the right upper and middle lung fields. Eosinophilic pneumonia was diagnosed from the results of bronchoalveolar lavage and transbronchial lung biopsy. The symptoms were alleviated and the infiltrative shadow was effaced after treatment with predonisolone (40 mg/day). The results of a biopsy with human eosinophils suggested that the serum and bronchoalveolar lavage fluid contained IL-5 and GM-CSF.

Bronchoalveolar Lavage Fluid↗
PubMed1995

[A case of chronic pancreatitis with eosinophilic pleural effusion].

A 48-year-old man was admitted for the evaluation of a massive left pleural effusion. Thoracenthesis yielded a bloody excudate with a high percentage of eosinophils (27%) and high values of pancreatic enzymes (amylase 16,000 Somogyi, Elastase 35,000 ng/dl, Lipase 12,800 U/l, Trypsin 77,000 ng/ml). The amylase isozyme of the exudate was 100% pancreatic-type fraction. The blood showed no eosinophilia (4%). A computed tomographic scan and magnetic resonance image of the abdomen revealed a pancreatic pseudocyst in contact with the diaphragm, and thrombi in the inferior vena cava and the splenic vein. After pancreatic cystectomy and splenectomy, the pleural effusion resolved rapidly. Eosinophilic pleural effusion has been reported as a complication of several disorders: pneumonia, lung carcinoma, pulmonary tuberculosis, and pulmonary infarction. However, we know of no previous report of eosinophilic pleural effusion with pancreatitis. In this case, it is interesting that the massive eosinophilic pleural effusion associated with chronic pancreatitis resolved immediately after the operation, and the patient was discharged.

Chronic Disease↗
PubMed1995

[Septic lung caused by methicillin-resistant Staphylococcus epidermidis].

A 55-year-old man was admitted to our department one month after resection of rectal carcinoma, with complaints of fever and general malaise. Shock developed rapidly after admission. The chest X-ray film and computed tomography showed diffuse small nodular shadows and bilateral pleural effusion. Septic lung caused by Methicillin-resistant Staphylococcus epidermidis (MRSE) was diagnosed from the results of a bacteriological study. This bacteria is a Coagulase Negative Staphylococcus (CNS). Chemotherapy with Minocycline and Cefotiam was effective. Characteristic radiologic features of this case may be related to the early stage of adult respiratory distress syndrome (ARDS) and septic pulmonary microembolism.

Anti-Bacterial Agents↗
PubMed1994

Immunosuppression to cardiac allografts and soluble antigens by anti-vascular cellular adhesion molecule-1 and anti-very late antigen-4 monoclonal antibodies.

Effective activation of T cells in immune responses depends on two signals, one from the CD3/TCR complex and another from accessory cell surface proteins. Very late Ag-4 (VLA-4) can exert a costimulatory function upon binding with vascular cellular adhesion molecule-1 (VCAM-1). We investigated the effects of mAbs against VCAM-1 and VLA-4 on cardiac allograft survival and humoral response to soluble Ags. BALB/c hearts were transplanted into C3H/He recipients. mAbs (100 micrograms/day) were administered i.p. for the first 6 days. Graft survival in mice treated with M/K-2 (anti-VCAM-1; median survival: 20 days, n = 6) and those treated with PS/2 (anti-VLA-4; 30 days, n = 6) was greater than that in control mice (8 days, n = 7). Eight of 18 mice treated with both M/K-2 and PS/2 accepted the grafts over 65 days, and five of them accepted the grafts over 100 days. Allografts treated with the two mAbs showed scattered infiltration of leukocytes without evidence of active rejection at 65 days. Mice with long-surviving cardiac grafts were challenged with skin grafts from donor and third-party (C57BL/6) strains. Survival of the donor-type skin was significantly greater than that of the third-party skin. One mouse specifically accepted the donor-type skin indefinitely (> 150 days). FACS analysis of splenocytes at 55 days after transplantation showed complete recovery of VLA-4 expression from a down-regulation observed at day 7. In addition, mice immunized with heat-aggregated human gamma-globulin did not produce specific Ab, even after boost immunization, if PS/2 was administered at the time of the first immunization. This unresponsiveness to xenogeneic protein lasted for more than 50 days. These results indicate that in vivo administration of anti-VCAM-1 and anti-VLA-4 mAbs induces specific immunosuppression to cardiac allografts and soluble Ags.

Animals↗
PubMed1994

Requirement of the Pro-Cys-His-Arg sequence for O6-methylguanine-DNA methyltransferase activity revealed by saturation mutagenesis with negative and positive screening.

O6-Methylguanine-DNA methyltransferase catalyzes transfer of a methyl group from O6-methylguanine and O4-methylthymine of DNA to a cysteine residue of the enzyme protein, thereby repairing the mutagenic and carcinogenic lesions in a single-step reaction. There are highly conserved amino acid sequences around the methyl-accepting cysteine site in eleven molecular species of methyltransferases. To elucidate the significance of the conserved sequence, amino acid substitutions were introduced by site-directed mutagenesis of the cloned DNA for Escherichia coli Ogt methyltransferase, and the activity and stability of mutant forms of the enzyme were examined. When cysteine-139, to which methyl transfer occurs, was replaced by other amino acids, all of the mutants showed the methyltransferase-negative phenotype. Methyltransferase-positive revertants, isolated from one of the negative mutants, had restored codons for cysteine. Thus the cysteine residue is essential for acceptance of the methyl group and is not replaceable by other amino acids. Using this negative and positive selection procedure, the analysis was extended to other residues near the acceptor site. At the histidine-140 and arginine-141 sites, all the positive revertants isolated carried codons for amino acids identical to those of the wild-type protein. At proline-138, five substitutions (serine, glutamine, threonine, histidine, and alanine) exhibited the positive phenotype but levels of methyltransferase activity in extracts of cells harboring these mutant forms were very low. This suggests that the proline residue at this site is important for maintaining the proper conformation of the protein. With valine-142 substitutions there were seven types of positive revertants, among which mutants carrying isoleucine, cysteine, leucine, and alanine showed relatively high levels of methyltransferase activity. These results indicate that the sequence Pro-Cys-His-Arg is a sine qua non for methyltransferase to exert its function.

Alkylating Agents↗
PubMed1994

A deletion in the second cytoplasmic loop of GluR3 produces a dominant negative mutant of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor.

We have characterized a GluR3 mutant (sGluR3) which has a 33-amino acid deletion in its second cytoplasmic loop (deficit from Tyr-715 to Gly-747 of GluR3-flop). Xenopus oocytes injected with cRNA transcribed from cDNA for this mutant did not respond to kainate and glutamate (each 100 microM) at a holding potential of -70 mV. In oocytes coinjected with cRNAs for this mutant and for normal alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor subunits, the current response to both kainate and glutamate was much weaker than that observed in oocytes injected with cRNA for the normal subunits alone. This inhibitory action was not accompanied by a significant change of ED50 value and shape of the I-V curve and was AMPA receptor-specific. A comparable deletion in GluR1 produced a mutant with properties similar to those of sGluR3, while a 33-amino acid deletion elsewhere in the second cytoplasmic loop of GluR3 (Met-674 to Phe-706) gave a mutant with a weaker effect. These results suggest that sGluR3 acts as a dominant negative mutant on the functional expression of normal AMPA receptors in oocytes by assembling with the normal subunits to produce an essentially nonfunctional receptor complex.

Amino Acid Sequence↗
PubMed1994

Metastatic phenotype in hybrid cells derived from B16 melanoma.

To determine whether or not the metastatic phenotype of mouse melanoma B16 is dominant, two derivative cell lines of B16, high metastatic F10 and low metastatic F1 were hybridized by somatic cell fusion. Prior to fusion, F10 and F1 cells were transfected with plasmid pKOneo or pSV2hph, which confers resistance to G-418 or hygromycin B, respectively. Hybrid clones were isolated by dual resistance to G-418 and hygromycin B. The seven isolated near-tetraploid hybrid clones were examined for metastatic phenotypes. Hybrid cells were injected into the tail vein of syngeneic C57BL/6N mice and the number of metastatic nodules in the lung was counted 21 days later. Four hybrid clones had high metastatic phenotypes while one clone showed an intermediate metastatic phenotype and two low metastatic phenotypes. Most of the hybrid clones retained modally stable chromosome compositions, whereas one of the two low metastatic clones lost chromosomes during in vitro and in vivo proliferations. These results suggest that the high metastatic ability is genetically dominant in B16 melanoma cells and low metastatic hybrid cells may be the result of a loss of a gene(s) responsible for acquisition of the metastatic phenotype in the process of chromosomal rearrangement.

Aneuploidy↗