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Biomedical subjects

T Imanaka

Publications and source records attributed to T Imanaka.

At least 163 records · Page 9Linked to original sources
PubMed1994

Angiotensin II binding activity in cultured porcine arterial endothelial cells.

Angiotensin II (A II) binding activity was detected in the particulate fraction (100,000 g, 60 min precipitate) of cultured porcine aortic endothelial cells. Scatchard analysis of the binding activity indicated a single class of binding sites with a dissociation constant (Kd) of 1.1 nM and a total binding capacity (Bmax) of 125 fmol/mg protein. The binding of [125I]A II was inhibited by excess unlabelled A II, A II analogues ([Sar1, Ile8]A II and [Sar1, Ala8]A II), A I (angiotensin I) and A III (angiotensin III), but not by bradykinin. Type specific A II receptor antagonists, losartan (type 1 angiotensin II receptor) and PD123319 (type 2 angiotensin II receptor), did not inhibit the binding. These results suggest that the A II specific binding protein(s) or receptor(s) is present in arterial endothelial cells, and that it is different from typical type 1 and type 2 angiotensin II receptors.

Angiotensin II↗
PubMed1994

A monoclonal antibody against oxidized lipoprotein recognizes foam cells in atherosclerotic lesions. Complex formation of oxidized phosphatidylcholines and polypeptides.

In this study we have used homogenates of human atheromatous plaque as immunogen to establish a murine monoclonal antibody which recognizes oxidized low density lipoprotein (LDL). This monoclonal antibody, FOH1a/DLH3, reacted with oxidized LDL, but enzyme-linked immunosorbent assay showed that it had no reaction with native, acetylated, or malonaldehyde-treated LDL. The antibody cross-reacted with oxidized high density lipoprotein, suggesting that specific sequences of the apolipoprotein B are not essential for antigen recognition by the antibody. Immunohistochemical analysis of thin paraffin sections from human coronary arteries showed that foam cells derived from macrophages in atherosclerotic lesions were heavily stained by this antibody. Several other structures in the lesions, including swollen collagen fibers, cellular debris in necrotic cores, and endothelial cells, were moderately stained. The epitope of this antibody was characterized by a model antigen-generating system using ferrous ion-induced peroxidation of lipids. When the total lipid fraction extracted from LDL was treated with the ferrous ion-induced peroxidation system, the reaction mixture was recognized by the antibody. Antigenic product(s) was produced only when phosphatidylcholine (PC) was treated with the ferrous ion-induced peroxidation, other lipids failed to react to the antibody. To investigate the possible formation of a complex of antigenic product with a polypeptide, a synthetic peptide and a rabbit antiserum against the peptide were used. Reaction mixture of ferrous ion-induced peroxidation of PC in the presence of the peptide was added to a microtiter well precoated with the monoclonal antibody FOH1a/DLH3. After washing, the peptide remaining in the well was detected with rabbit antiserum against the peptide, whereas no reactivity was observed when peptide alone was added to the well. Binding of the antigenic complex to the precoated monoclonal antibody was competed by oxidized PC produced in the absence of any polypeptide. We conclude that oxidized phospholipid product(s) is the epitope of the monoclonal antibody and that the oxidized phospholipid forms complexes with polypeptides. The antigenic materials are detected in foam cells in atherosclerotic lesions.

Amino Acid Sequence↗
PubMed1994

Construction of a new host-vector system in Arthrobacter sp. and cloning of the lipase gene.

Arthrobacter sp. strain MIS38 was transformed with a shuttle vector containing the kanamycin resistant gene kan (derived from Tn5) by an electroporation method. This shuttle vector is from Brevibacterium lactofermentum and Escherichia coli, pULRS8. The following optimal condition of electroporation was determined. A square wave pulse of 1 kV/cm electric field strength for 0.5 ms duration yielded 3 x 10(5) transformants/micrograms plasmid DNA. The number of transformants increased with the amount of DNA over the range 0.01-5 micrograms. This host-vector system was then used successfully to clone and express a lipase gene from Arthrobacter sp. strain MIS38 into both Arthrobacter sp. MIS38 and E. coli JM109.

Amino Acid Sequence↗
PubMed1994

Non-Hodgkin's lymphomas with chromosomal translocations involving 3q27 band and immunoglobulin gene loci: report of two cases.

Two cases of non-Hodgkin's lymphoma (NHL) of B-cell origin with reciprocal chromosome translocations involving the 3q27 band and loci for the immunoglobulin genes are described. In case 1, [t(3;14)(q27;q32)], the 14q32 band for the immunoglobulin heavy chain gene was involved, and in case 2, [t(3;22)(q27;q11)], the reciprocal breakpoint was the 22q11 band where the immunoglobulin lambda light chain gene is located. Although the two patients shared some clinical features, the lymphomas varied in histologic phenotypes; in case 1, the lymphoma being a diffuse large cell subtype, whereas in case 2, the lymphoma being the high grade small noncleaved cell lymphoma. Thus, the consistent association of the 3q27 translocation with a specific subtype of NHL remains to be determined. Southern blot analysis of the lymphoma cells from the two patients showed rearrangements and deletions in immunoglobulin genes with features which cannot be explained by normal immunoglobulin-generating mechanisms and which suggest that the translocations involved these loci at the molecular level. Cloning of the breakpoint using DNA probes from the immunoglobulin genes would prove a novel oncogene at the 3q27 band.

Adult↗
PubMed1994

Stabilization and rational design of serine protease AprM under highly alkaline and high-temperature conditions.

Rational shift of the optimum pH toward alkalinity and enhancement of thermostability were investigated by using a thermostable extremely alkaline protease (optimum pH, 12 to 13) from the alkaliphilic and thermophilic Bacillus sp. strain B18'. The protease gene (aprM) was cloned, and the sequence analysis revealed an open reading frame of 361 amino acids that was composed of a putative signal sequence (24 amino acids), a prosequence (69 amino acids), and a mature enzyme (268 amino acids) (molecular weight, 27,664). The amino acid sequence of this protease was compared with those of other serine proteases. A direct correlation of higher optimum pH with an increase in the number of arginine residues was observed. An even more thermostable mutant enzyme was created by introducing a point mutation. When the position of the beta-turn, Thr-203, was replaced by Pro, the residual activity of this mutant enzyme at 80 degrees C for 30 min was higher than that of the wild-type enzyme (50% versus 10%). The specific activity of this mutant enzyme at 70 degrees C was 105% of that of the wild-type enzyme under nondenaturation condition. These data suggest that the higher content of Arg residues favors the alkalinity of the serine protease and that introduction of a Pro residue into the beta-turn structure stabilizes the enzyme.

Amino Acid Sequence↗
PubMed1994

Cloning of the aapT gene and characterization of its product, alpha-amylase-pullulanase (AapT), from thermophilic and alkaliphilic Bacillus sp. strain XAL601.

A thermophilic and alkaliphilic Bacillus sp. strain, XAL601, was isolated from soil. It produces a thermostable and alkaline-stable enzyme with both alpha-amylase and pullulanase activities. The alpha-amylase-pullulanase gene (aapT) from this Bacillus strain was cloned, and its nucleotide sequence was determined (GenBank accession number D28467). A very large open reading frame composed of 6,096 bases, which encodes 2,032 amino acid residues with an M(r) of 224,992, was found. The deduced amino acid sequence revealed that the four highly conserved regions that are common among amylolytic enzymes were well conserved. These include an active center and common substrate-binding sites of various amylases. In the C-terminal region, a six-amino-acid sequence (Gly-Ser-Gly-Thr-Thr-Pro) is repeated 12 times. The aapT gene was then subcloned in Escherichia coli and overexpressed under the control of the lac promoter. Purification of AapT from this recombinant E. coli was performed, and it was shown that the aapT gene product exhibits both alpha-amylase and pullulanase activities with one active site. The optimum temperature and pH for enzyme activity were found to be 70 degrees C and pH 9, respectively. Furthermore, AapT was found to strongly adsorb to crystalline cellulose (Avicel) and raw corn starch. Final hydrolyzed products from soluble starch range from maltose (G2) to maltotetraose (G4). Only maltotriose (G3) was produced from pullulan. The enzyme also hydrolyzes raw starch under a broad range of conditions (60 to 70 degrees C and pH 8 to 9).

Amino Acid Sequence↗
PubMed1994

Purification and characterization of a thermostable thiol protease from a newly isolated hyperthermophilic Pyrococcus sp.

A hyperthermophilic archaeon strain, KOD1, was isolated from a solfatara at a wharf on Kodakara Island, Kagoshima, Japan. The growth temperature of the strain ranged from 65 to 100 degrees C, and the optimal temperature was 95 degrees C. The anaerobic strain was an S0-dependent heterotroph. Cells were irregular cocci and were highly motile with several polar flagella. The membrane lipid was of the ether type, and the GC content of the DNA was estimated to be 38 mol%. The 16S rRNA sequence was 95% homologous to that of Pyrococcus abyssi. The optimum growth pH and NaCl concentration of the strain KOD1 were 7.0 and 3%, respectively. Therefore, strain KOD1 was identified as a Pyrococcus sp. Strain KOD1 produced at least three extracellular proteases. One of the most thermostable proteases was purified 21-fold, and the molecular size was determined to be 44 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 45 kDa by gel filtration chromatography. The specific activity of the purified protease was 2,160 U/mg of protein. The enzyme exhibited its maximum activity at approximately pH 7.0 and at a temperature of 110 degrees with azocasein as a substrate. The enzyme activity was completely retained after heat treatment at 90 degrees C for 2 h, and the half-life of enzymatic activity at 100 degrees C was 60 min. The proteolytic activity was significantly inhibited by p-chloromercuribenzoic acid or E-64 but not by EDTA or phenylmethylsulfonyl fluoride. Proteolytic activity was enhanced threefold in the presence of 8 mM cysteine. These experimental results indicated that the enzyme was a thermostable thiol protease.

Amino Acid Sequence↗
PubMed1994

Properties and active center of the thermostable branching enzyme from Bacillus stearothermophilus.

Although the branching enzyme (EC 2.4.1.18) is a member of the alpha-amylase family, the characteristics are not understood. The thermostable branching enzyme gene from Bacillus stearothermophilus TRBE14 was cloned and expressed in Escherichia coli. The branching enzyme was purified to homogeneity, and various enzymatic properties were analyzed by our improved assay method. About 80% of activity was retained when the enzyme was heated at 60 degrees C for 30 min, and the optimum temperature for activity was around 50 degrees C. The enzyme was stable in the range of pH 7.5 to 9.5, and the optimum pH was 7.5. The nucleotide sequence of the gene was determined, and the active center of the enzyme was analyzed by means of site-directed mutagenesis. The catalytic residues were tentatively identified as two Asp residues and a Glu residue by comparison of the amino acid sequences of various branching enzymes from different sources and enzymes of the alpha-amylase family. When the Asp residues and Glu were replaced by Asn and Gln, respectively, the branching enzyme activities disappeared. The results suggested that these three residues are the catalytic residues and that the catalytic mechanism of the branching enzyme is basically identical to that of alpha-amylase. On the basis of these results, four conserved regions including catalytic residues and most of the substrate-binding residues of various branching enzymes are proposed.

1,4-alpha-Glucan Branching Enzyme↗
PubMed1993

Immunological detection of F1-ATPase gamma-subunit in rat liver peroxisomes.

The gamma-subunit of F1-ATPase was detected in highly purified rat liver peroxisomes by immunoblot analysis using an antiserum against purified mitochondrial F1-ATPase. Other subunits of F1-ATPase such as alpha, beta and delta were not detected in the peroxisomes. The polypeptide corresponding to the gamma-subunit in peroxisomes was also recognized by affinity purified antibody against F1-ATPase gamma-subunit. The amount of gamma-subunit in liver homogenate increased in parallel with proliferation of peroxisomes after administration of clofibrate in rats, whereas the amounts of alpha, beta and delta-subunits were not increased. These results suggest that the gamma-subunit of F1-ATPase is a constituent of peroxisomes in rat liver.

Animals↗
PubMed1993

Characterization of the membrane sensor PenJ for beta-lactam antibiotics from Bacillus licheniformis by amino acid substitution.

The PenJ protein of the penicillinase gene (penP) expression system from Bacillus licheniformis is an antirepressor and membrane receptor for beta-lactam antibiotics. A putative beta-lactam antibiotic binding site including Ser402 and Lys405, which are homologous to the conserved sequence for the beta-lactam binding site (Ser-X-X-Lys) is present. An amino acid substitution was introduced at Ser402 to Ala, removing the hydroxyl group of the serine. The mutant PenJ, S402A, was still functional. However, two other mutants, S402T (Ser402-->Thr) and K405A (Lys405-->Ala), were not functional. Thus, the hydroxyl group of Ser402 does not appear to be important for penicillin binding. Amino acid substitutions (K539R, D591N and K539R.G541V) were introduced in PenJ in the region of the putative phosphoryl binding domain. None of these mutant PenJ proteins was a functional antirepressor. These results suggested that the putative phosphoryl binding domain might be an important region for signal transduction.

Amino Acid Sequence↗
PubMed1993

Analysis of regions essential for the function of chromosomal replicator sequences from Yarrowia lipolytica.

Two DNA segments exhibiting ARS (autonomously replicating sequence) activity in the dimorphic yeast Yarrowia lipolytica were cloned from its chromosome on an integrative LEU2 plasmid. These ARS segments, designated Y1ARS1 and Y1ARS2, conferred on the hybrid plasmids high transformation efficiency and enabled extrachromosomal transmission of the plasmids in 1 or 2 copies per yeast cell under selective conditions. Deletion analysis showed that at least 728-1003 bp for Y1ARS1 and 1377-1629 bp for Y1ARS2 were required for full function. Both of these regions contained two 10/11 matches to an ARS core consensus in Saccharomyces cerevisiae, whereas neither was similar to the S. cerevisiae centromere sequence. Significantly, both Y1ARS elements contained at, or close to, their boundaries a 13 bp sequence, 5'-TATATTCAAGCAA-3', which resembles the cleavage site for topoisomerase II in Drosophila. A central 524 bp ClaI fragment of Y1ARS2 contained four stretches of a 17 bp direct repeat sequence, 5'-GAAAAACAAAAACAGGC-3', and exhibited the electrophoretic behavior typical of bent DNA.

Base Sequence↗
PubMed1993

Purification and properties of the highly thermostable alkaline protease from an alkaliphilic and thermophilic Bacillus sp.

Thermostable alkaline protease from an alkaliphilic thermophile Bacillus sp. B18' was purified by using DEAE- and CM-Toyopearl 650M column chromatographies. Molecular weights of the enzyme determined by SDS-PAGE and gel filtration were 30,000 and 28,000, respectively. The optimum pH and temperature toward the hydrolysis of casein were pH 12-13 and 85 degrees C, both of which are higher than those of a mesophilic alkaline protease from an alkaliphile, Bacillus sp. B21-2. The enzyme was stable at pH 5.0-12.0 and about 60% of the initial enzymatic activity was retained after a 60 min incubation period at pH 10.0 and 70 degrees C. Thermostability of the enzyme was enhanced by Ca2+. The enzyme activity was inhibited by DFP, suggesting that the enzyme is a serine protease. The NH2-terminal amino acid is Gln, which is that of many subtilisin-type proteases. The 20 residues of the NH2-terminal amino acid sequence have a comparative high homology with those of other alkaline proteases from alkaliphiles (40-50%), especially thermostable alkaline protease from Bacillus sp. No. AH-101 (95%) and Thermoactinomyces sp. HS682 (95%).

Amino Acid Sequence↗
PubMed1993

A new way of producing isomalto-oligosaccharide syrup by using the transglycosylation reaction of neopullulanase.

A new way of producing isomalto-oligosaccharide syrup from starch was developed. Isomalto-oligosaccharides contain one or more alpha-(1-->6)-glucosidic linkages with or without alpha-(1-->4)-glucosidic linkages. The isomalto-oligosaccharide syrups are receiving increased attention as food additives because it is thought that they help prevent dental caries and improve human intestinal microflora, acting as a growth factor for bifidobacteria. The new system for production of isomalto-oligosaccharide syrup is based on the strong alpha-(1-->6)-transglycosylation reaction of neopullulanase. Bacillus subtilis saccharifying alpha-amylase was simultaneously used with neopullulanase to improve the yield of isomalto-oligosaccharides. The yield of isomalto-oligosaccharides was increased to more than 60%, compared with a yield of 45.0% obtained by the conventional system. To reduce the costs, the use of immobilized neopullulanase was investigated. Almost the same yield of isomalto-oligosaccharides was obtained by using immobilized neopullulanase.

Journal Article↗
PubMed1993

A new lipopeptide biosurfactant produced by Arthrobacter sp. strain MIS38.

A biosurfactant termed arthrofactin produced by Arthrobacter species strain MIS38 was purified and chemically characterized as 3-hydroxydecanoyl-D-leucyl-D-asparagyl-D-threonyl-D- leucyl-D-leucyl-D-seryl-L-leucyl-D-seryl-L-isoleucyl-L-isoleucyl-L-as paragyl lactone. Surface activity of arthrofactin was examined, with surfactin as a control. Critical micelle concentration values of arthrofactin and surfactin were around 1.0 x 10(-5) M and 7.0 x 10(-5) M at 25 degrees C, respectively. Arthrofactin was found to be five to seven times more effective than surfactin. The minimum surface tension value of arthrofactin was 24 mN/m at a concentration higher than the critical micelle concentration. According to the oil displacement assay, arthrofactin was a better oil remover than synthetic surfactants, such as Triton X-100 and sodium dodecyl sulfate. Arthrofactin is one of the most effective lipopeptide biosurfactants.

Amino Acid Sequence↗