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Biomedical subjects

K Nagata

Publications and source records attributed to K Nagata.

At least 883 records · Page 49Linked to original sources
PubMed1987

Production of recombinant human pancreatic secretory trypsin inhibitor by Escherichia coli.

A synthetic gene for human pancreatic secretory trypsin inhibitor (PSTI) was fused to the coding sequence for the amino-terminal 135 amino acid residues of human interferon-gamma (IFN-gamma) by interposing a methionine codon sequence, and the resulting hybrid gene was efficiently expressed in Escherichia coli cells. Recombinant human PSTI (rHu-PSTI) was separated from the IFN-gamma/PSTI fused protein by cleavage at the methionine residue with cyanogen bromide. Finally, rHu-PSTI was purified by affinity chromatography on a bovine trypsin-CH-Sepharose 4B column. The amino acid composition, partial amino-terminal sequence, disulfide formation, human trypsin inhibitory activity, and immunoreactivity against rabbit anti-human PSTI serum of rHu-PSTI corresponded to those of the natural form.

Amino Acid Sequence↗
PubMed1987

Nucleotide sequence of a human liver cytochrome P-450 related to the rat male specific form.

P-450 human-2 is a human cytochrome P-450 that is immunochemically related to a constitutive male-specific cytochrome P-450 (P-450-male) and the phenobarbital-inducible P-450b/e in rat liver. By screening a human liver cDNA library in bacteriophage lambda gt11, we isolated a clone with an insert length of 1,847 bases (pHY13). The clone was sequenced and shown to code for a protein of 487 amino acids. The N-terminal 11-amino-acid sequence was in agreement with the protein sequence of P-450 human-2. The nucleotide sequence of pHY13 showed less than 50% similarity with those of human cytochrome P-450s, pHP-450(1), HLp, P-450NF, P1-450 4, and P3(450), but the nucleotide sequence of pHY13 is 80% similar to the reported sequence of rat cytochrome P-450, P-450(M-1). In addition, the coding sequence of pHY13 showed close similarity to that of MP-8, which was recently reported as the sequence corresponding to human cytochrome P-450MP, although no apparent similarity was observed in their 3' non-coding sequences except for the first 75 bases and the expected length of the complete sequences. These results, together with the immunochemical data, indicate that P-450 human-2 is closely related, but not identical, to P-450MP, and may belong to the category of developmentally regulated constitutive cytochrome P-450s.

Amino Acid Sequence↗
PubMed1987

Identification of the RNA polymerase-binding site on genome RNA of influenza virus.

The binding sites for influenza viral RNA polymerase on genome RNA segments were investigated. Ribonucleoprotein (RNP) cores containing the RNA polymerase were isolated from detergent-treated virions by glycerol gradient centrifugation. On ApG-primed in vitro transcription by the isolated RNP cores, different levels of RNA transcripts were synthesized for the eight RNP cores, suggesting an uneven distribution of the RNA polymerase. 3'-Terminal labeling of the RNP cores with the use of [32P]pCp and T4-RNA ligase indicated a reciprocal correlation between the levels of the RNA-3' label and RNA synthesis. Centrifugation of detergent-treated virions in a double gradient of cesium trifluoroacetate (or cesium chloride) and glycerol yielded RNA polymerase-RNA complexes devoid of NP, the major RNA-bound protein, but the pattern of RNA-3' labeling remained virtually unaffected. All these observations together indicated that the RNA polymerase is associated near the 3' termini of some viral RNA segments, thereby preventing the in vitro labeling of the RNA-3' ends. The results of foot-printing experiments using RNase V1 and RNase T2 were in agreement with this model.

Adenosine Monophosphate↗
PubMed1987

Induction of murine gamma interferon production by lipopolysaccharide and interleukin-2 in Propionibacterium acnes-induced peritoneal exudate cells.

Lipopolysaccharide (LPS) induces high levels of gamma interferon (IFN-gamma) in the circulation of mice pretreated with heat-killed Propionibacterium acnes. The following results were obtained in the present study. LPS, as well as interleukin-2 (IL-2), was also able to induce IFN-gamma in vitro in peritoneal exudate cells (PEC) from such mice. Splenocytes and lymph node cells from these mice or resident peritoneal cells from control mice produced trace or undetectable amount of IFN-gamma upon exposure to LPS. A synergistic effect on IFN-gamma induction was observed when LPS was added to a culture of PEC together with IL-2. Indomethacin augmented the induction of IFN-gamma by LPS or IL-2, and prostaglandin E2 reversed its effect. Deprivation of plastic-adherent or nylon wool-adherent cells abolished the induction by LPS or IL-2, whereas it did not affect that by concanavalin A. Culture supernatant of plastic-adherent cells incubated with LPS stimulated the nylon wool-nonadherent cells to produce IFN-gamma in the presence of IL-2, but interleukin-1 or phorbol myristic acetate did not replace the LPS-stimulated supernatant. The ability of PEC to produce IFN-gamma measured as a function of time after P. acnes injection increased in proportion to their natural killer (NK)-like activity against YAC-1 cells. Moreover, treatment of PEC with monoclonal anti-Thy-1 antibody or with anti-asialo GM1 antiserum plus complement eliminated the production of IFN-gamma and the NK-like activity simultaneously, whereas treatment with monoclonal anti-Lyt-2 antibody plus complement did not. These results suggest that IL-2 and some unidentified factor released from plastic-adherent cells by LPS stimulation cooperatively induce IFN-gamma production in activated, Thy-1- and asialo GM1-positive NK-like cells appearing in inflammatory reactions and that prostaglandin E2 regulates IFN-gamma production in these cells.

Animals↗
PubMed1987

Immunoelectron microscopic localization of fibronectin in the smooth muscle layer of mouse small intestine.

We studied the ultrastructural distribution of fibronectin in the smooth muscle layer of mouse small intestine with affinity-purified antibodies using the immunogold technique. Fibronectin was present over the pericellular area extending from the cell membrane to the extracellular matrix beyond the basal lamina. Distribution of the glycoprotein over the pericellular area was heterogeneous, i.e., it was localized more abundantly in the narrow space between smooth muscle cells, the gaps having a width of 60-80 nm where the two dense bands in adjacent cells matched each other. Such localization suggests that fibronectin contributes to cell adhesion. Within the basement membrane, gold label was localized both in lamina lucida and lamina densa, more densely in the latter than in the former. Fibronectin was also co-distributed with collagen fibers in the extracellular matrix. Within smooth muscle cells, gold particles were observed on rough endoplasmic reticulum and secretory vesicle-like structures. These results suggest that smooth muscle cells synthesize fibronectin and secrete it as a component of the basal lamina and extracellular matrix.

Animals↗
PubMed1987

[A case of successful surgical treatment of rectal cancer complicated by acute myelomonocytic leukemia].

A 47-year-old man, who had been diagnosed as having acute myelomonocytic leukemia (AMMOL) and had been treated with combination chemotherapy, was admitted to our hospital because he had developed melena. He had been judged to be in complete remission and had shown no signs of recurrence for years, Daunorubicin, vincristine 6-Mercaptopurine and Cyclophosphamide had been administered for maintenance and intensification therapy. He was well until January 1986, when this melena began. A barium enema was given and he was diagnosed as having rectal cancer. Amputation of the rectum and a permanent abdominal colostomy was made safely, mainly because he had been in complete remission, and he recovered normally after the operation. In recent years, the survival of patients with malignancies has improved due to aggressive treatment even in cases of hematological neoplasms. However, the risk of secondary neoplasms in patients treated for cancer has increased. This case suggests that we have to be careful when prescribing treatment for cancer patients, since anti-tumor drugs may have cartinogenic effects.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1987

[The effectiveness of dynamic CT as a tool for the analysis of cerebral hemodynamics].

It is generally accepted that dynamic CT is one of the best way to evaluate cerebral hemodynamics. Employing our original computer simulation method, the present study was undergone to examine the theoretical background of dynamic CT. When a single bolus injection of non-diffusable indicator is intravenously performed, the time density curve of a cerebral tissue consists of the following two components: the time density curve of a carotid artery (g(x)) and the dispersion curve through the cerebral tissue (f(x)). A mean transit time (MTT) of the latter curve can be mathematically calculated by height-over area method. The concept gave us an interesting idea that a computer simulation could produce the time density curve implying an MTT. Convolution technique was applied to synthesize it from f(x) and g(x). The latter curve (g(x)) is influenced by several conditions such as a dispersion through a lung, a cardiac output and systemic arterial pressure. Therefore, it is not essential to measure the definite parameter from the time density curve obtained by dynamic CT. Dynamic CT should be considered as a qualitative method rather than a quantitative one. Several parameters were given by the analysis of a simulated curve. A time to peak (PT), above all, was most linearly correlated to MTT. A prolongation of PT in the insulted area, which means a prolongation of MTT, leads to a decrease of cerebral blood flow. In addition, we examined if Gamma variate fitting curve could apply a substitute for an original time density curve.(ABSTRACT TRUNCATED AT 250 WORDS)

Cerebrovascular Circulation↗
PubMed1986

Purification and characterization of recombinant murine immune interferon.

The recombinant murine immune interferon (rMu-IFN-gamma) was purified to homogeneity from Escherichia coli harboring the expression vector of murine IFN-gamma. The purified rMu-IFN-gamma showed an Mr of 15 000 in SDS-polyacrylamide gel electrophoresis. Results of amino acid analysis, amino- and carboxyl-terminal analyses and peptide mapping of rMu-IFN-gamma suggest that it has the complete protein sequence predicted on the basis of cDNA except for lack of four amino acid residues from the mature carboxyl-terminus.

Amino Acid Sequence↗
PubMed1986

Undermethylation of interferon-gamma gene in human T cell lines and normal T lymphocytes.

The relative levels of DNA methylation at CCGG sequences within and around the interferon-gamma (IFN-gamma) gene in normal human tissues and cell lines were examined by Southern blot analysis using isoschizomeric restriction enzymes, HpaII and MspI. On the test of normal tissues, the IFN-gamma gene was undermethylated only in a small population of T lymphocyte, whereas the gene was fully methylated in T cell-depleted lymphocytes and uterus cells. In TCL-Fuj cell line which is a T cell line producing a high level of IFN-gamma spontaneously, the IFN-gamma gene was undermethylated. Moreover, the extent of DNA methylation was inversely correlated to the level of expression of the IFN-gamma gene in several T cell lines including sublines derived from TCL-Fuj cells. However, partial or complete unmethylation at the CCGG sites of IFN-gamma gene was observed in a promyelocytic leukemia cell line and two epithelial cell lines that fail to produce IFN-gamma irrespective of induction. These results suggest that undermethylation of IFN-gamma gene is necessary but not sufficient for its efficient expression.

Cell Line↗
PubMed1986

Phosphorylation and transformation sensitivity of a major collagen-binding protein of fibroblasts.

Using affinity chromatography with immobilized gelatin and native type I collagen, we have identified the major collagen-binding proteins in Nonidet P-40 extracts of chick embryo fibroblasts labeled with [35S] methionine. After washing the gelatin- or collagen-Sepharose beads with high ionic strength buffer, a 47,000-dalton protein was the only major protein besides fibronectin found to bind to these affinity beads. The isoelectric point of this protein was approximately 9.0, with a closely spaced minor spot. The total amount and the synthesis of this collagen-binding protein were both decreased in Rous sarcoma virus-transformed cells. This collagen-binding protein was found to be phosphorylated by incubating intact cells with [32P]orthophosphate. Phosphoamino acid analysis revealed that serine and threonine residues were phosphorylated, but tyrosine was not. Although quantities of the 47,000-dalton protein labeled with [35S]methionine were decreased by a factor 2.5 after transformation, the incorporation of [32P]orthophosphate/unit of protein was 5-7-fold higher in transformed cells. In temperature-sensitive mutant virus-infected cells, the amount of the 47,000-dalton protein was also decreased at the temperature permissive for transformation, and the incorporation of [32P]orthophosphate/protein was also increased. These studies establish that a major membrane-associated collagen-binding protein of fibroblasts is phosphorylated and that it is altered in both total quantity and degree of phosphorylation after malignant transformation.

Animals↗
PubMed1986

Detection of pyrimidine 5'-nucleotidase deficiency using 1H- or 31P-nuclear magnetic resonance.

We describe here a further Japanese family with pyrimidine 5'-nucleotidase (P5'N) deficiency diagnosed using a nuclear magnetic resonance (NMR) spectrum, in Kumamoto prefecture where two families having the disease have been reported before. The specific spectra in 1H-NMR of P5'N deficient erythrocytes were due to three methyl protons of CDP-choline at 3.22 ppm and to H-2, H-8 and ribose-1' of pyrimidine nucleotide phosphate(s) in the lower fields (at 5.82 and 8.00 ppm). The other specificities in 31P-NMR spectra were due to CDP-choline, CDP-ethanolamine and UDP-glucose. Those spectra were not detected in other types of hemolytic anemia.

5'-Nucleotidase↗