Search PubMed⌕ Search

Biomedical subjects

T Imanaka

Publications and source records attributed to T Imanaka.

At least 199 records · Page 11Linked to original sources
PubMed1991

Promoter sequence analysis in Bacillus and Escherichia: construction of strong promoters in E. coli.

Many derivatives of the nprM promoter of Bacillus stearothermophilus and the strong early promoter, A3, of coliphage T3 were designed and chemically synthesized. These promoters consisted of some or all of the AT box, consensus sequence, tac promoter sequence, spacer, and lac operator. The promoter activities were assessed by their ability to express the cat gene in Escherichia coli. One of the derivatives of the A3 promoter, which contained the lac operator, was much stronger (about 3.5 times) than the tac promoter. The promoter activities in E. coli were considerably modified by the substitutions in the -43 (AT box) and -35 regions.

Base Sequence↗
PubMed1991

A novel 57 kDa peroxisomal membrane polypeptide detected by monoclonal antibody (PXM1a/207B).

BALB/c mice were immunized with peroxisomal membranes prepared from rat liver. Spleen cells were fused with myeloma cells (P3/U1) and the hybridomas were selected using peroxisomal membranes. A monoclonal antibody (PXM1a/207B) which recognized peroxisomal membranes was selected. Using the antibody, a novel 57 kDa polypeptide was identified in the peroxisomal membrane fraction. Immunoblot analysis of the subcellular fractions demonstrated that the 57 kDa polypeptide was present exclusively in peroxisomal membranes. The 57 kDa polypeptide was partially digested by trypsin and chymotrypsin under conditions where peroxisomal particles remained intact, indicating that the polypeptide is exposed to the cytosolic face of the peroxisomal membrane. The amount of 57 kDa polypeptide increased in parallel with proliferation of peroxisomes by administration of clofibrate.

Animals↗
PubMed1991

Analysis of the active center of Bacillus stearothermophilus neopullulanase.

The active center of the neopullulanase from Bacillus stearothermophilus was analyzed by means of site-directed mutagenesis. The amino acid residues located in the active center of the neopullulanase were tentatively identified according to a molecular model of Taka-amylase A and homology analysis of the amino acid sequences of neopullulanse, Taka-amylase A, and other amylolytic enzymes. When amino acid residues Glu and Asp, corresponding to the putative catalytic sites, were replaced by the oppositely charged (His) or noncharged (Gln or Asn) amino acid residue, neopullulanase activities toward alpha-(1----4)- and alpha-(1----6)-glucosidic linkages disappeared. When the amino acids corresponding to the putative substrate-binding sites were replaced, the specificities of the mutated neopullulanases toward alpha-(1----4)- and alpha-(1----6)-glucosidic linkages were obviously different from that of the wild-type enzyme. This finding proves that one active center of neopullulanase participated in the dual activity toward alpha-(1----4)- and alpha-(1----6)-glucosidic linkages. Pullulan is a linear glucan of maltotriosyl units linked through alpha-(1----6)-glucosidic linkages. The production ratio of panose from pullulan was significantly increased by using the mutated neopullulanase which exhibited higher specificity toward the alpha-(1----4)-glucosidic linkage. In contrast, the production ratio of panose was obviously decreased by using the mutated neopullulanse which exhibited higher specificity toward the alpha-(1----6)-glucosidic linkage.

Amino Acid Sequence↗
PubMed1991

Residual neutron-induced radionuclides in samples exposed to the nuclear explosion over Hiroshima: comparison of the measured values with calculated values.

Residual radionuclides induced by neutrons from the Hiroshima atomic bomb have been measured at Kanazawa University for 14 years. The results of 152Eu, 154Eu and 60Co are reviewed in this paper. Where appropriate, an attempt is made to provide our new data with the aim of reinterpreting our published data. From the comparison of the measured values with the calculated values by DS86 methodology, we may point out here: (1) that close agreement was found between measured and calculated values for the specific radioactivity of 152Eu in the samples exposed at ground ranges between 320 m and 720 m; (2) that the calculated/measured ratios for the specific radioactivity of 152Eu were, however, larger than unity in the vicinity of ground zero and smaller than unity at locations more than 1000 m apart from ground zero; (3) that, in the vicinity of ground zero, epithermal neutron fluence evaluated from a set of measured specific radioactivities of 152Eu, 154Eu and 60Co showed a close agreement with the calculated result, whereas thermal neutron fluence evaluated in the same way was different from the calculated result; and (4) that the depth distribution of the specific radioactivity of 152Eu in a wall sample which was exposed at the location 320m from ground zero approximately agreed with the calculated result.

Humans↗
PubMed1990

Monoclonal antibody EMR1a/212D recognizing site of deposition of extracellular lipid in atherosclerosis. Isolation and characterization of a cDNA clone for the antigen.

We have isolated from a rabbit liver cDNA library, clones coding for the 66-kDa glycoprotein (GP66) characterized previously by EMR1a/212D monoclonal antibody (Nakagami, K., Shimasaki, O., Sato, R., Komine, Y., Ohkuma, S. and Takano, T. (1989) Am. J. Pathol. 135, 93-100), which recognizes the extracellular regions of atherosclerotic arterial wall. The clone spans the sequence coding for the entire GP66 (456 amino acids) and 19 amino acids of signal peptide. GP66 deduced from nucleotide sequence contained an Arg-Gly-Asp cell attachment sequence and had a 76% amino acid sequence homology with human vitronectin. Furthermore, EMR1a/212D recognized rabbit vitronectin purified by heparin-affinity chromatography. RNA blot hybridization detected one transcript of the same size in normal and Watanabe-heritable hyperlipidemic rabbit liver. The levels of plasma GP66 and liver GP66 mRNA were not altered, whereas 9-fold greater accumulation of GP66 was observed in thoracic aorta of Watanabe-heritable hyperlipidemic rabbit. These results suggest that GP66 is rabbit vitronectin and that it is vitronectin which selectively accumulates in thoracic aorta with the development of atherosclerosis.

Amino Acid Sequence↗
PubMed1990

Degradation of newly synthesized apolipoprotein B-100 in a pre-Golgi compartment.

The synthesis and secretion of apolipoprotein (apo) B-100 have been studied in a human hepatoblastoma cell line, the Hep G2 cells. Pulse-chase analysis showed that apoB-100 was not quantitatively recovered in the culture medium. To reveal the intracellular degradation of apoB-100 prior to secretion, cells were incubated with 1 microgram/ml Brefeldin A (BFA) which impeded protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus and the fate of apoB-100 retained in the cells was traced at 37 degrees C. A significant amount of intracellular apoB-100 (40-60%/h) was degraded during the chase period, whereas apoA-1 remained intact. ApoB-100 degradation was temperature dependent, no degradation was observed below 20 degrees C. This degradation process was not inhibited by chloroquine, leupeptin, pepstatin, and chymostatin, suggesting that lysosomal proteases were not involved and that apoB-100 was degraded in a pre-Golgi compartment which is either part of, or closely related to, the ER. Preincubation of cells with low density lipoproteins (LDL) induced a 22-32% increase in the degradation of apoB-100. This result raised the possibility that secretion of apoB-100 might be regulated through the intracellular degradation of apoB-100. These results suggest the existence of the degradation pathway for apoB-100 in a pre-Golgi compartment and an unique regulatory mechanism for apoB-100 secretion.

Anti-Bacterial Agents↗
PubMed1990

The effect of HMG-CoA reductase inhibitor (CS-514) on the synthesis and secretion of apolipoproteins B and A-1 in the human hepatoblastoma Hep G2.

Human hepaotoblastoma cells (Hep G2) were cultured with a competitive inhibitor of HMG-CoA reductase, CS-514. The synthesis of cholesterol was markedly inhibited after 1 h preincubation with CS-514. The synthesis and secretion of apolipoprotein (apo) B and A-1, however, were not affected. A long-term incubation (21-24 h) of cells with CS-514 did not change apo B synthesis and secretion, although a slight depression of apo A-1 synthesis was observed. Hep G2 cells were found to secrete LDL- and HDL-like lipoproteins which were poor in cholesterol when cells were incubated with the drug. These results suggest that the modulation of cholesterol synthesis affects neither the synthesis and secretion of apo B and A-1 nor the formation of lipoproteins.

Apolipoprotein A-I↗
PubMed1990

Transcellular transport of angiotensin II through a cultured arterial endothelial monolayer.

We have studied the mechanisms of angiotensin II (A-II) transport through a cultured arterial endothelial cell monolayer. The transport of 125I-labeled A-II was inhibited by excess unlabeled A-II (50 microM) and [Sar1, Ile8]-A-II (50 microM), but was not inhibited by bradykinin (50 microM). The transport process was shown to be temperature dependent and was inhibited by 10 mM NaN3 plus 50 mM 2-deoxyglucose. Monensin (50 microM), an inhibitor of endocytotic trafficking, reduced the rate of transport of 125I-A-II. It is also shown that the specific pathway for A-II transport was unidirectional from the apical to the basolateral surface of the endothelial cell monolayer.

Adenosine Triphosphate↗
PubMed1990

Angiotropic lymphoma with histologic features of neoplastic angioendotheliomatosis presenting with predominant respiratory and hematologic manifestations. Report of a case and review of the literature [corrected].

Neoplastic angioendotheliomatosis (NAE) is a rare fatal disease characterized by widespread intravascular proliferations of neoplastic mononuclear cells. Clinically, dermatologic and bizarre neurologic manifestations usually predominate. The origin of the neoplastic cells remains still undetermined. The authors report a patient with NAE peculiar with respect to the following points: (1) the patient predominantly manifested respiratory symptoms and hematologic findings and lacked cutaneous or neurologic manifestations; and (2) immunohistochemical and molecular genetic studies showed the B-cell nature of the neoplastic cells, although previous cases with predominant respiratory or hematologic manifestations were reported to be of endothelial origin. Despite the rarity, this type of NAE or angiotropic [corrected] lymphoma should be recognized because it is easily confused with other disorders, particularly vasculitis or thrombotic thrombocytopenic purpura.

Aged↗
PubMed1990

Does single-amino-acid replacement work in favor of or against improvement of the thermostability of immobilized enzyme?

Thermostabilities of kanamycin nucleotidyltransferase and of its mutants that became thermostable, in the free state, because of single-amino-acid replacements were studied after immobilization of the enzymes on cyanogen bromide-activated Sephadex G-200 particles. Lys in place of Gln at position 102 decreased the thermostability of the immobilized enzyme, whereas replacement with other amino acids enhanced it.

Amino Acid Sequence↗
PubMed1990

Cloning and expression of the Clostridium thermosulfurogenes glucose isomerase gene in Escherichia coli and Bacillus subtilis.

The gene that encodes thermostable glucose isomerase in Clostridium thermosulfurogenes was cloned by complementation of glucose isomerase activity in a xylA mutant of Escherichia coli. A new assay method for thermostable glucose isomerase activity on agar plates, using a top agar mixture containing fructose, glucose oxidase, peroxidase, and benzidine, was developed. One positive clone, carrying plasmid pCGI38, was isolated from a cosmid library of C. thermosulfurogenes DNA. The plasmid was further subcloned into a Bacillus cloning vector, pTB523, to generate shuttle plasmid pMLG1, which is able to replicate in both E. coli and Bacillus subtilis. Expression of the thermostable glucose isomerase gene in both species was constitutive, whereas synthesis of the enzyme in C. thermosulfurogenes was inducible by D-xylose. B. subtilis and E. coli produced higher levels of thermostable glucose isomerase (1.54 and 0.46 U/mg of protein, respectively) than did C. thermosulfurogenes (0.29 U/mg of protein). The glucose isomerases synthesized in E. coli and B. subtilis were purified to homogeneity and displayed properties (subunit Mr, 50,000; tetrameric molecular structure; thermostability; metal ion requirement; and apparent temperature and pH optima) identical to those of the native enzyme purified from C. thermosulfurogenes. Simple heat treatment of crude extracts from E. coli and B. subtilis cells carrying the recombinant plasmid at 85 degrees C for 15 min generated 80% pure glucose isomerase. The maximum conversion yield of glucose (35%, wt/wt) to fructose with the thermostable glucose isomerase (10.8 U/g of dry substrate) was 52% at pH 7.0 and 70 degrees C.

Aldose-Ketose Isomerases↗
PubMed1990

Nucleotide sequence and cloning in Bacillus subtilis of the Bacillus stearothermophilus pleiotropic regulatory gene degT.

The regulatory gene (degT) from Bacillus stearothermophilus NCA1503 which enhanced production of extracellular alkaline protease (Apr) was cloned in Bacillus subtilis with pTB53 as a vector. When B. subtilis MT-2 (Npr- [deficiency of neutral protease] Apr+) was transformed with the recombinant plasmid, pDT145, the plasmid carrier produced about three times more alkaline protease than did the wild-type strain. In contrast, when B. subtilis DB104 (Npr- Apr-) was used as a host, the transformant with pDT145 could not exhibit any protease activity. After construction of the deletion plasmids, DNA sequencing was done. A large open reading frame was found, and nucleotide sequence analysis showed that the degT gene was composed of 1,116 bases (372 amino acid residues, molecular weight of 41,244). A Shine-Dalgarno sequence was found nine bases upstream from the open reading frame. A B. subtilis strain carrying degT showed the following pleiotropic phenomena: (i) enhancement of production of extracellular enzymes such as alkaline protease and levansucrase, (ii) repression of autolysin activity, (iii) decrease of transformation efficiency for B. subtilis (competent cell procedure), (iv) altered control of sporulation, (v) loss of flagella, and (vi) abnormal cell division. When B. stearothermophilus SIC1 was transformed with the recombinant plasmid carrying degT, the transformants exhibited abnormal cell division. These phenomena are similar to those of the phenotypes of degSU(Hy) (hyperproduction), degQ(Hy), and degR mutants of B. subtilis. However, the amino acid sequence of the degT product (DegT) is different from those of the reported gene products. Furthermore, DegT includes a hydrophobic core region in the N-terminal portion (amino acid numbers 50 to 160), a consensus sequence for a DNA binding region (amino acid numbers 160 to 179), and a region homologous to transcription activator proteins (amino acid numbers 351 to 366). We discuss the possibility that the membrane protein DegT functions as a sensor protein and transfers the signal of environmental stimuli to the regulatory region of target genes to activate or repress transcription of the genes.

Bacillus subtilis↗
PubMed1990

Cloning and nucleotide sequences of the Bacillus stearothermophilus neutral protease gene and its transcriptional activator gene.

Both the neutral protease gene (nprS) and its transcriptional activator gene (nprA) from Bacillus stearothermophilus TELNE were cloned in Bacillus subtilis by using pTB53 as a vector plasmid. The presence of the nprA gene enhanced protease synthesis by about fivefold. The nucleotide sequences of nprS and its flanking regions were determined. nprS was composed of 1,653 base pairs and 551 amino acid residues. A Shine-Dalgarno (SD) sequence was found 9 bases upstream from the translation start site (ATG). The deduced amino acid sequence was very similar to that of another thermostable neutral protease gene, nprM (M. Kubo and T. Imanaka, J. Gen. Microbiol. 134:1883-1892, 1988). the amino acid sequence of the extracellular neutral protease NprS was completely identical to that of NprM. By deletion analysis and substitution of the original promoter with a foreign promoter, it was found that the nprA gene existed upstream of nprS. It was also found that a possible target region (palindromic sequence) of the gene product of nprA existed near the promoter sequence of nprS. The nucleotide sequences of nprA and its flanking regions were determined. The DNA sequence revealed only one large open reading frame, composed of 1,218 base pairs (406 amino acids; molecular weight, 49,097). The SD sequence was found 4 bases upstream from the translation start site (GTG). A possible promoter sequence (TTGAAG for the -35 region and AATTTT for the -10 region) was also found about 20 bases upstream of the SD sequence. The nprA gene was separated from nprS by a typical terminator sequence. By constructing an in-frame fusion between the lacZ gene and the 5' region of the nprA gene, it was demonstrated that the coding region of nprA was indeed translated in vivo. Three palindromic sequences, which were highly homologous with a possible target region by NprA, were also found in the 5' region of the nprA gene. This suggests that eh expression of nprA is autoregulated. From the time course of the production of NprA-LacZ fusion protein, it was indicated that nprA was expressed in late log phase, whereas nprS was expressed in the stationary phase. The NprA protein had consensus regions homologous to the DNA recognition domains of DNA-binding proteins but showed no sequence homology with any other regulatory proteins for protease production. It is inferred that NprA protein binds to the upstream region of nprS promoter and activates transcription of nprS. A new regulatory mechanism by the nprA-nprS genes is discussed.

Amino Acid Sequence↗
PubMed1990

[Three cases of malignant lymphoma developing from chronic empyema wall].

Three patients with non-Hodgkin's lymphoma (NHL) involving the lung or chest wall are reported. All patients had tuberculous pleuritis or received artificial pneumothorax for pulmonary tuberculosis 30 years or more previously. The NHL of these patients developed in or close to the thickened pleura. Histologic examination showed diffuse large cell type (LSG classification) in all cases. Lymphomas of all cases were considered to have originated from B-cell lineage because their neoplastic cells expressed B1 (CD20) antigens. In no case the tumors were resectable, and chemotherapy or radiotherapy were performed. Two died of local NHL at 5 and 6 months respectively after diagnosis. One patient is still alive at 8 months after the first treatment for NHL. Early diagnosis is essential to improve the prognosis of these NHLs although it is difficult because of the co-existent pyothorax. Following features seemed to be useful for diagnosis: 1) chest pain or shoulder pain, 2) elevation of LDH level, 3) mass shadow in computed tomography, and 4) abnormal uptake of gallium 67. As precise assessment of tumor size is difficult because of the co-existent pyothorax and necrosis, follow-up studies by gallium scan or magnetic resonance imaging might be useful for proper treatment.

Chronic Disease↗