Structure and expression of the gene coding for a multicopper enzyme, ascorbate oxidase of cucumber.
Explore the source record for details and available documents.
Ascorbate Oxidase↗
Biomedical subjects
M Takano
Publications and source records attributed to M Takano.
Lysis and aberrant morphology of Bacillus subtilis cells caused by surfactants and their relation to autolysin activity.
The surfactants tested in this study lysed Bacillus subtilis 168 cells at the logarithmic growth phase. Results obtained with inhibitors and a mutant that had defective autolytic enzymes suggested that cell lysis resulted from the deregulation of autolysin activity. The addition of surfactants at sublytic concentrations produced twisted cells, filamented cells, or both. Autolysins extracted with 5 M LiCl from the cell wall fraction and lysozyme added to cells that were treated with surfactants restored the apparently normal cell rod morphology, suggesting that surfactants interfere with the role of autolysins in normal construction of the cell envelope. The rates of cellular autolysis and autolysin activity remaining in growing cells after exposure to a surfactant at a sublytic concentration decreased, although the rate of turnover of cell wall peptidoglycan was the same as that of control cells. Surfactants were suggested to interact with the regulatory system of autolysins and, thus, to affect the activities of autolysins in B. subtilis cells and to cause either morphological changes or cell autolysis, depending on the concentration of surfactants.
Autolysis↗
ATP-dependent decay and recovery of K+ channels in guinea pig cardiac myocytes.
ATP-dependent decay and recovery of the inward rectifier and ATP-sensitive K+ channels were investigated using inside-out patch recording in cardiac myocytes. The solution facing the inner side of the membrane was instantaneously changed with the oil-gate concentration jump method. Both channels were decayed by removing ATP and were recovered by reapplying ATP. The coexistence of Mg2+ was required for the recovery. 5'-Adenylylimidodiphosphate failed to reverse the ATP-dependent decay. The cumulative histograms of survival time and recovery time, obtained from the inward rectifier K+ channel, showed a single exponential distribution, time constants of which were 55 and 43 s, respectively. The time-dependent nature of decay and recovery was also confirmed in the ATP-sensitive K+ channel. The findings indicated that intracellular ATP is one of the factors that determines the activity of the K+ channels. It is most probable that phosphorylation of channel molecules is essential for maintaining the K+ channel in an operative state.
Adenosine Triphosphate↗
[A case of cardiac ischemic lesion detected by a new myocardial perfusion imaging agent SQ30217].
Explore the source record for details and available documents.
Adult↗
Heterogeneity in lectin-binding characteristics of human lymphokine-activated killer cells.
The binding of various lectins to human lymphokine-activated killer (LAK) cells was investigated. Human peripheral blood lymphocytes were cultured for 3 days with recombinant interleukin-2 (rIL-2), and then stained with 20 kinds of lectins. There were four types of lectin-binding patterns: (a) negative staining; (b) weakly positive staining; (c) strongly positive staining; and (d) two populations, one weakly and one strongly positive staining. Among the 20 kinds of lectins, both Lens culinaris agglutinin (LCA) and Pisum sativum agglutinin (PSA) showed two peaks in the staining patterns on LAK cells (type 4). We separated these two populations of LAK cells by using a cell sorter, and assayed them for cytotoxic activity against 51Cr-labeled Hela cells. The LAK cells that were strongly stained by LCA or PSA [LCA (or PSA)-bright LAK cells] showed stronger cytotoxic activity than the LAK cells that were weakly stained by LCA or PSA [LCA (or PSA)-dull LAK cells]. Furthermore, we separated lymphocytes with LCA or PSA before culturing them with rIL-2 (LAK precursor), and we found that both LCA-bright and PSA-bright lymphocytes can develop into LAK cells with higher cytolytic activity. LCA-bright lymphocytes kept their ability to be stained strongly with LCA even after culture with rIL-2 for 3 days. Similarly, LCA-dull lymphocytes were still stained weakly after culture with rIL-2. Two-color assay of LAK cells revealed that 78% of NK (CD16+)-LAK cells and 46% of T (CD3+)-LAK cells were LCA-bright cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Antigens, CD↗
Amino-terminal region of human macrophage colony-stimulating factor (M-CSF) is sufficient for its in vitro biological activity: molecular cloning and expression of carboxyl-terminal deletion mutants of human M-CSF.
Human T lymphoblastoid cell line CEM-ON belongs to a helper/inducer subclass and secretes M-CSF into medium constitutively. We have isolated a full-length cDNA clone for this factor from a cDNA library of this cell line. The cDNA was 2.5 kb and coded for a 554 amino acid polypeptide precursor including signal sequence. The Northern blot analysis showed that the major transcript of M-CSF is about 4.2 kb. We have studied the expression of not only the wild type plasmid, but also the five C-terminal deletion mutants encoding N-terminal 154, 163, 170, 177, and 185 amino acid residues in monkey COS-1 cells. The results showed that at least C-terminal 377 amino acid residues of human M-CSF are not essential to manifest the in vitro biological activity on murine bone marrow cells.
Amino Acid Sequence↗
CCA [N-(2-carboxyphenyl)-4-chloroanthranilic acid disodium salt], a newly developed immunomodulating drug, suppresses T-cell activation by acting on macrophages.
The cellular mechanism of action of a newly developed drug, CCA, N-(2-carboxyphenyl)-4-chloroanthranilic acid disodium salt, on PHA-, autologous mixed lymphocyte reaction (AMLR)-, and phorbol myristate acetate (PMA)-stimulated T-cell proliferation was investigated. Addition of 50 micrograms of CCA per milliliter suppressed PHA- and AMLR-stimulated T-cell proliferation. In contrast, CCA failed to suppress PMA-stimulated macrophage-depleted T-cell proliferation. After treatment of T cells or macrophages with CCA for 12 h, recombined T cells and macrophages were stimulated with phytohemagglutinin. [3H]Thymidine incorporation by T cells was suppressed when macrophages but not T cells were treated with CCA. These results indicate that CCA suppresses T-cell proliferation by acting on macrophages. The mechanism involved in this suppression of CCA was due to the loss of Ia antigen on macrophages and the loss of interleukin-1 (IL-1) secretion from macrophages.
Adjuvants, Immunologic↗
A novel concatenated dimer of recombinant bovine somatotropin.
A novel protein concatenated dimer structure was generated during the folding/oxidation of inclusion bodies of recombinant bovine somatotropin synthesized in Escherichia coli. The structure of this dimeric molecule was determined by peptide mapping with trypsin, and limited proteolysis by thrombin. Peptide mapping demonstrated that the two disulfide pairs in bovine somatotropin dimer were identical to those in monomer. Limited proteolysis with thrombin resulted in the cleavage of only a single peptide bond between arginine-132 and alanine-133 in bovine somatotropin dimer. This single peptide bond cleavage was sufficient to convert this dimer to a monomeric molecular weight species as analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and HPLC. Since the single cleaved peptide bond is present in the large disulfide loop of bovine somatotropin, these data demonstrate that the dimeric molecule exists as a novel concatenated structure through the interlocking of the disulfide loops of this protein.
Animals↗
Preoperative computed tomography staging of rectal carcinoma.
Pelvic computed tomography (CT) was analyzed in 48 patients with rectal carcinoma. Air was insufflated into the rectum before CT scanning. The areas of tumor (T) and rectosigmoid lumen (L) were determined for T/L ratio. Changes of the perirectal fatty tissues on CT were classified into five patterns: shaggy, granular, linear, clubbed, and wavy appearances. The T/L ratio and changes of the perirectal fatty tissue were correlated with transmural tumor extension. When the T/L ratio was above 1, tumors were frequently classified as Dukes' staging B and C, whereas a T/L ratio above 2 suggested Dukes' C classification. Clubbed or wavy appearance in the perirectal fatty tissues suggested that the tumor extended beyond the submucosa involving the muscularis propria. These findings were very useful for surgery.
Adipose Tissue↗
Decreased transport of p-aminohippurate in renal basolateral membranes isolated from rats with acute renal failure.
Transport of D-glucose, p-aminohippurate (organic anion), and tetraethylammonium (organic cation) was studied in the renal basolateral membrane vesicles isolated from rats with acute renal failure (ARF). ARF was induced by a single injection of uranyl nitrate. Carrier-mediated transport of p-aminohippurate, estimated under anion-anion exchange condition, was significantly decreased in basolateral membrane vesicles isolated from ARF rats. In contrast, there were no significant differences in D-glucose and tetraethylammonium uptake between normal and ARF rats. When normal basolateral membrane vesicles were incubated in vitro with uranyl nitrate, no significant inhibition in p-aminohippurate uptake was observed. These results suggest that organic anion transport is decreased in renal basolateral membranes from ARF rats, and this transport dysfunction cannot be explained by the direct interaction of uranyl nitrate with the organic anion carrier.
Acute Kidney Injury↗
Primary structure of cucumber (Cucumis sativus) ascorbate oxidase deduced from cDNA sequence: homology with blue copper proteins and tissue-specific expression.
cDNA clones for ascorbate oxidase were isolated from a cDNA library made from cucumber (Cucumis sativus) fruit mRNA. The library was screened with synthetic oligonucleotides that encode the NH2-terminal sequence of this enzyme. Nucleotide sequence analysis of the cloned cDNA inserts revealed a 1761-base-pair open reading frame that encoded an NH2-terminal signal peptide of 33 amino acids and a mature enzyme of 554 amino acids (Mr, 62,258). The amino acid sequence deduced from nucleotide sequence analysis agrees with the NH2-terminal amino acid sequence of the purified ascorbate oxidase, as determined by microsequencing methods. Cucumber ascorbate oxidase contained four histidine-rich regions with striking sequence homology to the corresponding parts of the other multicopper oxidases such as Neurospora crassa laccase and human ceruloplasmin and, to some extent, to a low molecular weight copper protein such as plastocyanin. Moreover, these data further support the hypothesis that the small blue copper proteins and the multicopper oxidases have evolved from the same ancestral gene. By RNA blot hybridization analysis, the mRNA for the ascorbate oxidase was found to be abundant in cucumber fruit tissue while expressed at very low levels in leaf and root tissues.
Amino Acid Sequence↗
Molecular cloning and expression of rat interleukin-1 alpha cDNA.
A cDNA sequence coding for rat interleukin-1 alpha (IL-1 alpha) has been isolated from a cDNA library that was prepared with mRNA derived from LPS-stimulated rat peritoneal macrophages by using human IL-1 alpha cDNA as a probe. The rat cDNA encodes a 270 amino acid residue protein which is homologous (65%) to human IL-1 alpha. The rat cDNA sequence under SV40 early promoter directed the synthesis of biologically active IL-1 in monkey COS-1 cells. Rat IL-1 alpha mRNA is not expressed in spleen, lung, liver or brain, and is also not expressed in these organs of LPS-treated rat except spleen. This suggests that IL-1 alpha is not produced constitutively in various tissues and LPS is not sufficient to induce IL-1 alpha in most tissues. Our data indicate that the IL-1 activities which have been reported to be produced in the brain are not of alpha type. We have constructed a plasmid expressing the carboxy terminal 156 amino acids in Escherichia coli. Recombinant rat IL-1 alpha produced in COS cells or E. coli has cytotoxic activity against the human melanoma cell line A375S1 (GIF activity), which has been reported to be sensitive to human IL-1 alpha and IL-1 beta. This suggests that GIF activity is common to IL-1s derived from various sources.
Amino Acid Sequence↗
Interaction of the fluorescent dye 1-N-phenylnaphthylamine with Escherichia coli cells during heat stress and recovery from heat stress.
The fluorescent dye 1-N-phenylnaphthylamine permeated Escherichia coli cells after exposure to a heat stress at 55 degrees C in Tris/Mg2+ buffer, pH 8.0. The rate of dye permeation increased with time during heat treatment and decreased gradually during subsequent incubation at 37 degrees C in a minimal medium. The initial level of rapid adsorption of the dye also increased with heating time, although it remained roughly constant during post-heating incubation. The results obtained suggest that the permeability barrier to the dye in the outer membrane was damaged by heat stress and was repaired after sublethal heating. RNA, protein and lipid syntheses, as well as an energy-yielding process, appeared to be necessary for the repair of impermeability to the dye.
1-Naphthylamine↗
Two populations of mouse lymphokine-activated killer cells separated by use of soybean agglutinin.
We separated lymphokine-activated killer (LAK) cells induced from spleen cells of BALB/c mice by culturing with recombinant interleukin-2 (rIL-2) into soybean agglutinin-positive (SBA+) and soybean agglutinin-negative (SBA-) fractions with a cell sorter at the time when LAK activity reached a maximum (day 3). We found that the cells with LAK activity were enriched in the SBA+ fraction. Analysis of cell surface phenotypes revealed that the SBA+ cells are of non-T cell origin, while the SBA- fraction consists of T cells. We also found that the large granular lymphocyte (LGL) fraction of spleen cells obtained with a Percoll gradient became SBA+ after culture for 3 days with rIL-2, whereas cells of high density did not. The change in SBA binding sites was also examined on C57BL/6 mouse spleen cells and we found that NK1.1+ non-T cells selectively acquire SBA binding sites at an early stage of activation with rIL-2. On the other hand, sorted SBA- cells gradually acquired SBA binding sites on extended culturing with rIL-2. These results suggest that the expression of SBA binding sites is related to the stage of cell activation with rIL-2, though cells of the NK-lineage became SBA+ at an earlier period of a culture with rIL-2 than cells of the T-lineage. Utilizing these findings, we separated NK-derived LAK cells by means of SBA-binding, and used the separated cells for adoptive immunotherapy for experimental pulmonary metastasis. We found that SBA(+)-NK cell-derived LAK cells showed stronger activity for the inhibition of experimental pulmonary metastasis than SBA(-)-T cell-derived LAK cells.
Animals↗
Transport of cephalosporin antibiotics in rat renal basolateral membranes.
Transport mechanisms of cephalosporin antibiotics in rat renal basolateral membranes have been studied in isolated membrane vesicles. Uptake of [14C]p-aminohippurate, a typical organic anion, by basolateral membrane vesicles was enhanced when membrane vesicles were preloaded with unlabelled p-aminohippurate (counter-transport). Cephalosporins such as cephradine, cephalexin, and cefazolin inhibited the counter-transport of [14C]p-aminohippurate, as did unlabelled p-aminohippurate and probenecid. In contrast, cephalexin did not affect the uptake of [3H]tetraethylammonium, an organic cation, by basolateral membrane vesicles. These results suggest that most cephalosporins are transported via organic anion transport systems in rat renal basolateral membranes.
Animals↗
The Mg2+ block and intrinsic gating underlying inward rectification of the K+ current in guinea-pig cardiac myocytes.
1. The blockade by Mg2+ and intrinsic gating of the channel, which underlie the rectification of the inward rectifier K+ current, was investigated using the oil-gap voltage clamp method in isolated guinea-pig ventricular cells. 2. The inward rectifier K+ current was isolated by subtracting trans-gap currents recorded at an extracellular K+ concentration ([K+]o) of 0 mM from those obtained at 14 mM [K+]o in the presence of a given concentration of intracellular Mg2+ ([Mg2+]i). The reversal potential (V0) of the difference current was near the equilibrium potential for K+ (EK). 3. On repolarization across EK, the inward rectifier K+ current showed a rapid exponential increase. The time constant decreased with increasing hyperpolarization, but it was independent of both [Mg2+]i and the preceding depolarization. 4. When the pre-pulse potential was made progressively positive between V0-20 and V0 + 30 mV, the amplitude of the time-dependent component became larger and the preceding current jump decreased at any [Mg2+]i. With pre-pulses more positive than V0 + 40 mV, the time-dependent component started from almost the zero current level at 2 microM [Mg2+]i. At higher [Mg2+]i (350, 500 and 3000 microM), however, the time-dependent component became smaller as the pre-pulse potential was made more positive than V0 + 40 mV. 5. When the membrane was depolarized from a potential of full activation at 2 microM [Mg2+]i, the initial jump in the outward current was ohmic and was followed by an exponential decay. The time-dependent component of the inward current, recorded on repolarization after increasing durations of the preceding depolarization, developed as the outward current decayed. The time constants of both processes were in good agreement. 6. At 500 microM [Mg2+]i, the outward current on depolarization was instantaneously rectified. The time-dependent component recorded on repolarization developed with prolongation of the pre-pulse with a time course slower than at 2 microM [Mg2+]i. The envelope time course became slower as the potential of the depolarization became more positive. 7. Lowering the temperature from 23 to 15 degrees C slowed the time-dependent current with an apparent Q10 of about 3.5 at V0. 8. Based on the experimental data, kinetic parameters were estimated for a model of Mg2+ block, which well simulated the inward-going rectification of the K+ current. 9. It is concluded that the instantaneous inward rectification on depolarization is due to the Mg2+ block at physiological [Mg2+]i.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Kinetics of ATP-sensitive K+ channel revealed with oil-gate concentration jump method.
Kinetics for gating the ATP-sensitive K+ channel was studied by exposing the inside-out patch to instantaneous changes in the intracellular concentration of ATP ( [ATP]i) using the oil-gate concentration jump technique in guinea pig ventricular cells. The closing time course of the channel after increasing [ATP]i was exponential with a time constant (tau), which decreased with increasing [ATP]i. The linear 1/tau - [ATP]i relation revealed two different binding (closing) rate constants (mu) of 51.7 and 5.6 mM-1.s-1 and predicted a common unbinding (opening) rate constant (lambda) of 3.2 s-1. A variable latent period was observed before channel opening when [ATP]i was decreased. The mechanism of latency is not clear. Once the channel started to open at the change lowering [ATP]i, the opening time course was exponential. Measurements of the exponential tau obtained at 0 mM [ATP]i were divided into two groups with corresponding lambda of 2.8 and 20.1 s-1, respectively. The former agrees with the predicted value of 3.2 s-1, but in the latter case, tau for opening increased as [ATP]i was increased. This increase in tau was attributed to a decrease of lambda, which approached an asymptotic value of 3.2 s-1. We conclude that binding and unbinding of one molecule of ATP determine the gating of ATP-sensitive K+ channel. Different pairs of mu and lambda result in four types of gating patterns and practically two states of sensitivities to ATP.
Adenosine Triphosphate↗
Computed cephalography using laser scan system.
A new imaging system with applications in dentistry has been developed which uses recent advances in both electronic and computer technology. The cephalometric radiogram is based on a newly developed laser scan system offering these characteristic features: 1. A clearer image with a wide latitude can now be obtained compared with the cephalometric radiograms taken by the conventional method. 2. Various imaging processes are available through computer enhancement which enable visualization of the sites of dense and thin bones and even the anatomical morphology of the soft tissues. Such images could be easily observed regardless of the purpose of the radiogram. 3. Data are reduced by conversion into digital signals which can be stored in the computer for long periods. 4. Exposure dose is reduced.
Cephalometry↗