[Basis for Insulin Lispro: physicochemical approach to fast-acting insulin analog].
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Animals↗
Biomedical subjects
T Nakazawa
Publications and source records attributed to T Nakazawa.
Glibenclamide-sensitive mechanism is involved in helodermin-produced vasodilatation in rat mesenteric artery.
Helodermin-caused vascular relaxation was simultaneously measured with intracellular Ca2+ concentration ([Ca2+]i) in rat mesenteric artery. Helodermin caused concentration-dependent relaxation in the mesenteric artery preconstricted with norepinephrine (NE). Helodermin-caused relaxation was accompanied by decrease in [Ca2+]i, D-cis-Diltiazem, a Ca2+ channel blocker, also lowered the [Ca2+]i and tension increased by NE. However, helodermin relaxed the artery more efficiently than D-cis-diltiazem, suggesting that the peptide decreased myofilament Ca2+ sensitivity. The vascular relaxation and the corresponding decrease in [Ca2+]i induced by helodermin were partly, but significantly attenuated by glibenclamide. Helodermin-induced vascular responses were mimicked by vasoactive intestinal polypeptide (VIP) or forskolin. Furthermore, helodermin increased cAMP contents in the mesenteric artery. These findings show that vasodilatation induced by helodermin is attributable to lowered [Ca2+]i of arterial smooth muscle partly through the activation of glibenclamide-sensitive K+ channels, and to decrease in the myofilament Ca2+ sensitivity. The increase in the cellular cAMP content probably plays a key role in the peptide-induced vasorelaxation.
Animals↗
[Vitamin D3 induction of differentiation of HL-60 cells to monocyte- like cells and their aggregation--a dynamic culture method and clarithromycin].
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Anti-Bacterial Agents↗
A role of the amino acid residue located on the fifth position before the first aspartate-rich motif of farnesyl diphosphate synthase on determination of the final product.
Farnesyl diphosphate (FPP) synthase catalyzes consecutive condensations of isopentenyl diphosphate with allylic substrates to give FPP, C-15 compound, as a final product and does not catalyze a condensation beyond FPP. Recently, it was observed that, in Bacillus stearothermophilus FPP synthase, a replacement of tyrosine with histidine at position 81, which is located on the fifth amino acid before the first aspartate-rich motif, caused the mutated FPP synthase to catalyze geranylgeranyl diphosphate (C-20) synthesis (Ohnuma, S.-i., Nakazawa, T., Hemmi, H., Hallberg, A.-M., Koyama, T., Ogura, K., and Nishino, T. (1996) J. Biol. Chem. 271, 10087-10095). Thus, we constructed 20 FPP synthases, each of which has a different amino acid at position 81, and analyzed them. All enzymes except for Y81P can catalyze the condensations of isopentenyl diphosphate. The final products and the product distributions are different from each other. Y81A, Y81G, and Y81S can produce hexaprenyl diphosphate (C-30) as their final product. The final product of Y81C, Y81H, Y81I, Y81L, Y81N, Y81T, and Y81V are geranylfarnesyl diphosphate (C-25), and Y81D, Y81E, Y81F, Y81K, Y81M, Y81Q, and Y81R cannot produce polyprenyl diphosphates more than geranylgeranyl diphosphate. Substitution of tryptophan does not affect the product specificity of FPP synthase. The average chain length of products is inversely proportional to the accessible surface area of substituted amino acid. However, no significant relation between the final chain length and the kinetic constants Km and Vmax are observed. These observations strongly indicate that the amino acid does not come into contact with the substrates but directly contacts the omega-terminal of an elongating allylic product. This interaction must prevent further condensation of isopentenyl diphosphate.
Alkyl and Aryl Transferases↗
A genetic analysis system of Burkholderia cepacia: construction of mobilizable transposons and a cloning vector.
A genetic analysis system of Burkholderia cepacia (Bc) was developed which included transposon mutagenesis and complementation of mutation with the cloned genes of interest. To deliver the transposon in this multidrug-resistant microorganism, two plasmids, pKN30 and pKN31, were constructed which contained Tn5 derivatives, Tn5-30Tp and Tn5-31Tp, respectively, carrying KmR and TpR genes. The plasmids have the origin of ColE1 replication and the mobilization gene of RP4. Tn5-31Tp was mobilized to Bc KF1, a strain isolated from a pneumonia patient, by the transfer system of RP4 integrated in the chromosome of Escherichia coli (Ec). Selection with trimethoprim resulted in generation of a number of transposants of Bc KF1. Fourteen protease-deficient mutants were isolated, all of which contained a single transposon marker in the chromosome. Thirteen protease-deficient mutants were also lipase deficient. An Ec-Bc shuttle plasmid, pTS1209, was constructed that consists of oriColE1, oripSa, ApR and CmR genes, and several unique restriction sites for cloning. Plasmid pTS1209 was successfully employed for cloning genes of Bc involved in protease production.
Burkholderia cepacia↗
Conversion from farnesyl diphosphate synthase to geranylgeranyl diphosphate synthase by random chemical mutagenesis.
Prenyltransferases catalyze the consecutive condensation of isopentenyl diphosphate (IPP) with allylic diphosphates to produce prenyl diphosphates whose chain lengths are absolutely determined by each enzyme. In order to investigate the mechanisms of the consecutive reaction and of the determination of ultimate chain length, a random mutational approach was planned. The farnesyl diphosphate (FPP) synthase gene of Bacillus stearothermophilus was subjected to random mutagenesis by NaNO2 treatment to construct libraries of mutated FPP synthase genes on a high-copy plasmid. From the libraries, the mutants that showed the activity of geranylgeranyl diphosphate (GGPP) synthase were selected by the red-white screening method (Ohnuma, S.-i., Suzuki, M., and Nishino, T. (1994) J. Biol. Chem. 268, 14792-14797), which utilized carotenoid synthetic genes, phytoene synthase, and phytoene desaturase, to visualize the formation of GGPP in vivo. Eleven red positive clones were identified from about 24,300 mutants, and four (mutant 1, 2, 3, and 4) of them were analyzed for the enzyme activities. Results of in vitro assays demonstrated that all these mutants produced (all-E)-GGPP although the amounts were different. Each mutant was found to contain a few amino acid substitutions: mutant 1, Y81H and L275S; mutant 2, L34V and R59Q; mutant 3, V157A and H182Y; mutant 4, Y81H, P239R, and A265T. Site-directed mutagenesis showed that Y81H, L34V, or V157A was essential for the expression of the activity of GGPP synthase. Especially, the replacement of tyrosine 81 by histidine is the most effective because the production ratios of GGPP to FPP in mutant 1 and 4 are the largest. Based on prediction of the secondary structure, it is revealed that the tyrosine 81 situates on a point 11 approximately 12 A apart from the first DDXXD motif, whose distance is similar to the length of hydrocarbon moiety of FPP. These data might suggest that the aromatic ring of tyrosine 81 blocks the chain elongation longer than FPP. Comparisons of kinetic parameters of the mutated and wild type enzymes revealed several phenomena that may relate with the change of the ultimate chain length. They are a decrease of the total reaction rate, increase of Kmfor dimethylallyl diphosphate, decrease of Vmax for dimethylallyl diphosphate, and allylic substrate dependence of Km for IPP.
Alkyl and Aryl Transferases↗
Induction of muscarinic receptor subtypes in monocytic/macrophagic cells differentiated from EoL-1 cells.
We demonstrated that eosinophilic leukemia cell line-1 (EoL-1 cells) differentiated into monocytic/macrophagic cells by a treatment with interferon-gamma. Muscarinic receptor mRNA was not detected in untreated EoL-1 cells, and the cytosolic concentration of Ca2+ ([Ca2+]i) did not rise either in these cells. Interestingly, when EoL-1 cells were treated with interferon-gamma, mRNAs for muscarinic M3 and M5 receptors could be detected in these cells, along with an increase in [Ca2+]i and chemotaxis induced by carbachol that could be blocked with 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) and pirenzepine. These data support the functional importance of muscarinic M3 and M5 receptors in monocytic/macrophagic cells differentiated from EoL-1 cells. This model also provides evidence of a significant functional interaction between muscarinic M3 and M5 receptors.
Calcium↗
Hydrogen peroxide secreted by tumor-derived macrophages down-modulates signal-transducing zeta molecules and inhibits tumor-specific T cell-and natural killer cell-mediated cytotoxicity.
Although alterations in CD3-associated signal-transducing molecules in tumor-infiltrating T cells of patients with advanced cancer have been previously described, the mechanism behind these changes is not known. We demonstrate that macrophages isolated from metastatic lymph nodes of patients with malignant melanoma down-regulate levels of CD3 zeta in autologous peripheral blood T cells. Lipopolysaccharide (LPS)- or phorbol 12-myristate 13-acetate (PMA)-stimulated monocytes derived from peripheral blood of healthy donors also induced decreased expression of CD3 and CD16-associated zeta chains similar to that observed in T cells and natural killer (NK) cells of patients with advanced cancer. Co-culture with activated monocytes impaired Ca2+ mobilization in peripheral blood derived-T cells when stimulated with monoclonal antibodies to CD3 and also strongly inhibited melanoma-specific cytotoxic T lymphocyte (CTL) activity and NK activity. The presence of catalase, a scavenger of H2O2, during co-culture almost totally abrogated the inhibitory effect of activated monocytes on melanoma-specific CTL lines and on NK cells. Pre-treatment of CTL or NK cells with nontoxic concentrations (1 x 10(-5) M) of H2O2 also severely reduced their cytotoxic activity which could be prevented by catalase. The decrease in CD3 zeta and in CD16 zeta expression, induced by macrophages isolated from metastatic lymph nodes or by LPS-stimulated monocytes, was also prevented by catalase when maintained throughout the co-culture period. The possibility that monocyte/macrophage-derived reactive oxygen metabolites contribute directly to alterations in signal transducing molecules of T cells and NK cells and to the mechanism of immunosuppression in individuals with cancer should be considered.
CD3 Complex↗
Effect of leukotriene C4 exposure on ciliated cells of the nasal mucosa.
To clarify the effects of leukotriene C4 (LTC4) on human ciliated epithelium, ciliary activity of the ethmoid sinus mucosa was measured photoelectrically in tissue culture. At concentrations ranging from 10(-6)M to 10(-9)M, LTC4 showed minimal effects on the ciliated epithelium during the initial 30 minutes of exposure; thereafter, ciliary inhibition was observed in a concentration- and time-dependent manner. Irrigation of the mucosa with culture medium 15 minutes after exposure prevented the LTC4-induced ciliary inhibition. However, irrigation 60 minutes after exposure failed to inhibit 10(-8)M LTC4-induced ciliary dysfunction and mucosal damage. The LTC4-induced ciliary inhibition was blocked in the presence of FPL-55712 and/or Ly-171883, both leukotriene receptor antagonists. L-serine and sodium tetraborate complex (SBC), a gamma-glutamyl transpeptidase (gamma-GTP) inhibitor, also inhibited the LTC4-induced ciliary inhibition. These findings indicate that LTC4 is converted to LTD4 by gamma-GTP during 60 minutes of exposure, and LTC4 itself has minimal direct effects on the ciliated cells.
Acetophenones↗
Vasopressin reduces taurochenodeoxycholate-induced hepatotoxicity by lowering the hepatocyte taurochenodeoxycholate content.
BACKGROUND/AIMS: Vasopressin has been reported to reduce bile flow, but its effects on bile acid secretion and bile acid-related hepatotoxicity are still unclear. We therefore investigated the influence of vasopressin on the hepatotoxicity and biliary excretion of taurochenodeoxycholic acid in primary cultured rat hepatocytes and isolated perfused rat liver models. METHODS/RESULTS: 1) Addition of vasopressin to hepatocyte cultures significantly decreased lactate dehydrogenase release as compared to cultures exposed to 1 mM taurochenodeoxycholic acid alone, and also reduced intracellular taurochenodeoxycholic acid content from 19.3 +/- 2.2 to 13.0 +/- 1.6 nmol/mg protein. After 30 min of preincubation with 1 mM taurochenodeoxycholic acid, rinsing and reculture of hepatocytes in bile acid-free medium resulted in gradual decrease in the intracellular level of the bile acid, and addition of vasopressin (10(-9) M) to the reculture medium accelerated this process. 2) Superimposition of vasopressin (330 pmol/l) for 10 min on taurochenodeoxycholic acid infusion (1.0 mumol/min: 25 mumol/l) caused a rapid increase in bile flow and biliary excretion of taurochenodeoxycholic acid (697 +/- 42 vs 584 +/- 27 nmol/10 min per g liver) from perfused rat livers, and significantly reduced lactate dehydrogenase release. 3) Superimposition of the PKC blocker H-7 (5 mumol/l) on taurochenodeoxycholic acid infusion (1.0 mumol/min: 25 mumol/l) caused a gradual increase in bile flow and biliary excretion of taurochenodeoxycholic acid. Furthermore, an additional infusion of vasopressin (100 pmol/l) for 10 min in the presence of H-7 produced a greater increase in bile flow and biliary excretion of taurochenodeoxycholic acid as compared with H-7 alone (754 +/- 71 vs. 657 +/- 26 nmol/g liver). 4) Continuous infusion of vasopressin (330 pmol/l) significantly increased the late peak (10-50 min) of horseradish peroxidase excretion from perfused livers (from 8.48 +/- 1.02 to 21.7 +/- 6.02 ng/g liver). CONCLUSIONS: These findings suggest that vasopressin exerts a protective effect against taurochenodeoxycholic acid-induced hepatotoxicity by stimulating the secretion of this bile acid via intracellular vesicular transport systems.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
Effect of cetirizine on antigen-induced tracheal contraction of passively sensitized guinea pigs.
BACKGROUND: Cetirizine dihydrochloride (cetirizine), a potent histamine H1-receptor antagonist, has been developed as an anti-allergy drug. OBJECT: The anti-allergic effects and mechanism of cetirizine were studied using in vitro assay systems. METHODS: We investigated the effect of cetirizine on antigen-induced contractions of isolated tracheal strips and on chemical mediator release from antigen-stimulated lung chips taken from passively sensitized guinea pigs. We examined the antigen-induced mobilization of Ca2+ in MC/9 mast cells sensitized with IgE. RESULTS: Cetirizine inhibited the antigen-induced contraction of isolated guinea-pig trachea concentration dependently. Pyrilamine, another histamine H1-receptor antagonist, delayed the response but did not change the maximum amplitude. Cetirizine at the concentration of 3 microM also inhibited the antigen-induced release of histamine, leukotriene D4, and leukotriene E4 from guinea pig lung chips. Furthermore, it inhibited the antigen-induced Ca2+ increase in MC/9 mast cells, whereas pyrilamine did not. CONCLUSION: These findings suggest that one anti-allergic mechanism of cetirizine may inhibit mediator release which is, at least partially, mediated by a decrease in the transient Ca2+ influx in mast cells.
Animals↗
Uncoupling of biliary lipid from bile acid secretion by formyl-methionyl-leucyl-phenylalanine in the rat.
A neutrophil chemotactic factor N-formyl-methionyl-leucyl-phenylalanine (fMLP), produced by Escherichia coli under conditions of intestinal inflammation, is reported to circulate enterohepatically in the presence of experimental colitis, but its effect on bile secretion is unclear. Therefore, we investigated the effect of fMLP on bile secretion in a single-pass isolated perfused rat liver system. Infusion of fMLP at different concentrations (2 micromol/L, 10 micromol/L, and 20 micromol/L) into the portal vein resulted in excretion into bile in the native form, independent of sodium taurocholate (1 micromol/min) infusion. Excretion of fMLP increased dose dependently, and approximately 12% of the infused dose was detected at each concentration. With constant infusion of sodium taurocholate (1 micromol/min), fMLP (20 micromol/L) increased bile flow but decreased phospholipid and cholesterol secretion. Bile acid secretion was not affected. Phospholipid/bile acid molar ratios decreased from 0.069 +/- 0.002 to 0.038 +/- 0.002, and cholesterol/bile acid molar ratios decreased from 0.0074 +/- 0.0009 to 0.0029 +/- 0.0008. Thus, administration of fMLP resulted in the uncoupling of biliary excretion of phospholipid and cholesterol from that of bile acids; this effect proved reversible. The increase in bile flow caused by fMLP infusion appeared to result from osmotic choleresis. When 25 mg of horseradish peroxidase, a conventional marker of transcytotic vesicle transport pathway, was infused for 1 minute as a pulse load into the portal vein after continuous infusion of taurocholate, its late peak excretion was reduced by fMLP (10 micromol/L) from 9.59 +/- 1.09 to 6.05 +/- 0.66 (ng/g liver). Gel-permeation chromatography of bile showed a specific association of fMLP with bile acids. These results suggest an uncoupling of biliary lipids from bile acids by fMLP because of inhibition of transcellular vesicle transport and interaction between fMLP and bile acid micelles in the bile canaliculus.
Animals↗
Use of intrinsic and extrinsic helper epitopes for in vivo induction of anti-hepatitis C virus cytotoxic T lymphocytes (CTL) with CTL epitope peptide vaccines.
The induction of virus-specific cytotoxic T lymphocytes (CTL) is an important part of vaccine strategy. CTL induction in vivo by two hepatitis C virus (HCV) peptides containing CTL epitopes, one from the NS5 region (P17) and one from the core (C7), was compared. P17 required covalent attachment of a helper peptide (PCLUS3 containing a cluster of epitopes from the human immunodeficiency virus envelope protein), whereas C7 did not. However, the minimal decapeptide of C7, C7A10, alone did not induce CTL. The helper cells induced by PCLUS3-17 or by C7 were shown to be CD4+ and to produce interleukin-2 (IL-2). Thus, help can be supplied by a natural helper epitope intrinsic to the CTL peptide, as in C7, or by attaching a helper epitope from another protein, as in the case of P17. The cluster peptides may be useful promiscuous helper peptides for a variety of CTL epitopes from diverse pathogens.
Amino Acid Sequence↗
The thiJ locus and its relation to phosphorylation of hydroxymethylpyrimidine in Escherichia coli.
Extracts from Escherichia coli K-12 contained two distinct enzymes capable of catalysing the phosphorylation of hydroxymethylpyrimidine (HMP) to HMP monophosphate: pyridoxine kinase (EC 2.7.1.35) and an enzyme that has not previously been genetically analysed, HMP kinase (EC 2.7.1.49). Two distinct genes, pdxL and thiJ, specify the activities of the former and latter enzymes, respectively. The inactivation of both genes by independent mutations in the same cell resulted in the complete loss of HMP kinase activity. Experiments with a series of strains that carry mutations in thiC, thiC pdxB, thiC pdxB pdxL and thiC pdxB pdxL thiJ revealed that the ability of the double mutant (pdxL thiJ) to utilize HMP in thiamin pyrophosphate biosynthesis was restored by introducing the wild-type allele corresponding to the thiJ mutation. The thiJ locus was mapped on the chromosome near the thiD and thiM loci, which govern the activities of phosphomethylpyrimidine kinase (EC 2.7.4.7) and hydroxyethylthiazole kinase (EC 2.7.1.50), respectively.
Chromosome Mapping↗
CD27/CD70 interaction directly induces natural killer cell killing activity.
The CD27 molecule is expressed on a portion of natural killer (NK) cells as well as T and B cells. To provide the functional capacity of CD27 molecule on NK cells, we here highly purified CD3- CD56+ NK cells by flow cytometry (purity > 98%), and investigated their NK cell activity via CD27/CD70 interaction using a CD70-transfectant by a 4 h 51Cr-release assay. The enhancement of NK activity by purified NK cells in the presence of interleukin-2 (IL-2) or interleukin-12 (IL-12) against CD70/Nalm-6 was not recognized as compared to against mock/Nalm-6. However, after a coculture with the fixed CD70/300-19, the purified NK cells increased the NK cell activity against K562, the value being 10 to 20% higher than coculture with the mock/300-19 in the presence of IL-2 or IL-12. The enhancement of NK activity was blocked by the addition of anti-CD70 monoclonal antibody (mAb). In addition, conjugation of NK cells to the target was increased by coculture with the CD70/300-19 without increased expression of adhesion molecules. In the parallel experiment, there was no increase in the killing capacity of NK cells. These results strongly show that CD27/CD70 interaction directly enhances NK activity in the presence of IL-2 or IL-12 by increasing the effector and target conjugate formation, indicating that CD27/CD70 interaction plays an important role in the cytotoxic function of NK cells.
Antigens, CD↗
A spectrum for obsession and personality disorders.
Obsession was introduced by Kraepelin in 1915 and has been studied extensively since. When a person with obsession becomes physically exhausted with chronic rumination accompanied by suspicion, he or she is driven to impulsive acts, and develops a personality disorder that displays persistent abnormal activities. Obsession is related closely to depression and schizophrenia. Obsession is induced when uncertainty and instability dominates intelligence and creativity. The current social hierarchy of a strongly controlled society rejects diversity of humanity and often triggers personality disorders. This article reviews obsession and a myth as primitive mentality, normal and abnormal obsession, obsession vs possession, society and obsession/ impulsion/degeneration, obsession and slowness/autism, a recent biological approach to obsession and a spectrum for obsession.
Adult↗
Effect of surface roughness on hemolysis in a pivot bearing supported Gyro centrifugal pump (C1E3).
The blood contacting surface quality is an important pump parameter for blood compatibility and cell damage. This study investigates the surface roughness and the effect it has on hemolysis in a centrifugal blood pump. In vitro hemolysis tests were performed with a pivot bearing supported Gyro centrifugal pump (C1E3) simulating cardiopulmonary bypass (CPB; 5 L/min, 350 mm Hg) and left ventricular assist device (LVAD; 5 L/min, 100 mm Hg) conditions. To produce 4 different grades of surface roughness, the impellers and housings were subjected to vapor polishing, sand papering, fine sand blasting, or coarse sand blasting. Seven pumps were assembled with different impeller and housing surfaces. These surfaces were then examined by a surface profile instrument and a scanning electron microscope. The results of this study are as follows. First, the effect of surface roughness on hemolysis was significantly greater in the CPB condition than in the LVAD condition. Second, surface roughness, regardless of whether it is the impeller or pump housing, had little effect on hemolysis in the LVAD condition. Third, in the CPB condition, the surface roughness of the pump housing has a greater effect on hemolysis than does that of the impeller. From a hemolytic point of view, an extremely smooth pump housing is required for use of an impeller type centrifugal pump as a CPB device. In contrast, it is conceivable that a smooth surface is not always essential for an impeller type centrifugal pump that is used as an LVAD.
Animals↗
The effect of the impeller-driver magnetic coupling distance on hemolysis in a compact centrifugal pump.
Blood trauma is one of the important performance parameters of centrifugal pumps. To investigate the blood trauma induced by these pumps, in vitro hemolysis tests have become an important procedure and are increasingly used for pump development and comparisons. The Baylor compact eccentric inlet port (CIE) centrifugal blood pump was developed as a long-term centrifugal ventricular assist device (VAD) as well as a cardiopulmonary bypass pump (CPB). The Baylor CIE pump incorporates a seal-less design with a blood stagnation-free structure. This pump can provide flows of 5 L/min against 350 mm Hg of total pressure head at 2,600 revolutions per minute. The pump impeller is magnetically coupled to the driver magnet in a seal-less manner. The latest hemolysis study revealed that hemolysis may be affected by the gap distance between the driver and the impeller magnet. The purpose of this study was to verify the effect of the magnetic coupling distance on the normalized index of hemolysis (NIH) with the CIE model and to obtain an optimal gap distance. The NIH value was clearly decreased by alteration of the magnetic coupling distance from 7.7 to 9.7 mm in CPB and left ventricular assist device (LVAD) conditions. The NIH, when using the pump as an LVAD condition, was reduced to a level of 0.0056 from 0.095 when the magnetic coupling distance was extended. The same results were also obtained when the pumps were used in a CPB condition. The magnetic coupling distance is an important factor for the CIE model in terms of hemolysis. Different coupling forces effect the bearings and impeller stability. These results suggest that an optimal driving condition with a proper magnetic coupling and an optimal force between the impeller and driver is necessary to develop an atraumatic centrifugal pump.
Blood Flow Velocity↗