Protein-Ligand Interaction Analyses with Nuclear Magnetic Resonance Spectroscopy Enhanced by Dissolution Triplet Dynamic Nuclear Polarization.

Solution-state nuclear magnetic resonance spectroscopy (NMR) is a powerful method for the analysis of intermolecular interactions within a biomolecular system. However, low sensitivity is one of the major obstacles of NMR. We improved the sensitivity of solution-state 13C NMR for the observation of intermolecular interactions between protein and ligand using hyperpolarized solution samples at room temperature. Eutectic crystals composed of 13C-salicylic acid and benzoic acid doped with pentacene were hyperpolarized by dynamic nuclear polarization using photoexcited triplet electrons, and a 13C nuclear polarization of 0.72 ± 0.07% was achieved after dissolution. The binding of human serum albumin and 13C-salicylate was observed with several hundred times sensitivity enhancement under mild conditions. The established 13C NMR was applied for pharmaceutical NMR experiments by observation of the partial return of the 13C chemical shift of salicylate by competitive binding with other non-isotope-labeled drugs.

Expression of heat shock proteins, Hsp70 and Hsp25, in the rat gingiva after irradiation with a CO2 laser in coagulation mode.

BACKGROUND AND OBJECTIVE: The therapeutic rationale of low-energy pulsed CO(2) laser coagulation mode has not been clarified yet. We conducted this study to characterize the effect of low-energy pulsed CO(2) laser coagulation mode irradiation of the rat gingiva in terms of the expression of heat shock proteins. MATERIAL AND METHODS: Laser irradiation was achieved with the parameters of 5 W, 600 mus pulse duration, and fluence of 326 J/cm(2). The gingiva dissected at different times after irradiation was processed for immunohistochemical examination of the expression of the heat shock proteins, Hsp70 and Hsp25. RESULTS: One hour after irradiation, the epithelial keratinocytes facing the laser wound exhibited an overexpression of Hsp70 in their nucleus. The connective tissue cells facing the laser wound, which included fibroblasts and capillary endothelial cells, showed de novo expression of Hsp70 at 3 h post-irradiation, the level of which peaked at 1 d and thereafter decreased. An enhanced and/or de novo expression of Hsp25 in the connective tissue cells facing the laser wound became evident at 3 h after irradiation, and after 1 d the Hsp25-expressing cells increased in number and spread over the wound as wound repair progressed. There was a temporospatial difference in the expression pattern between Hsp70 and Hsp25, with only a few cells appearing to co-express both heat shock proteins. CONCLUSION: The CO(2) laser treatment in coagulation mode produced the expression of heat shock proteins, and the findings suggest that while Hsp70 mainly conferred cell protection, Hsp25 was involved in the progress of wound repair as well as cell protection.

Exogenous ACC enhances volatiles production mediated by jasmonic acid in lima bean leaves.

We report the synergistic effects of exogenous 1-aminocyclopropane-1-carboxylic acid (ACC) and jasmonic acid (JA) on production of induced volatiles by excised lima bean leaves. Application of ACC alone to leaves induced trace amounts of volatiles. ACC positively affected three JA-induced volatiles, (E)- and (Z)-beta-ocimene, and (Z)-3-hexenyl acetate. The ethylene inhibitor, silver thiosulfate, inhibited the production of these compounds. The results suggest synergistic effects of JA and ACC on inducible volatile production by lima bean leaves. Furthermore, lima bean leaves treated with JA plus ACC became more attractive to predatory mites, Phytoseiulus persimilis, than those treated with JA alone.

Intracellular signal transduction of PBAN action in the silkworm, Bombyx mori: involvement of acyl CoA reductase.

In the silkworm, Bombyx mori, production of the sex pheromone bombykol is regulated by a neurohormone termed PBAN. We have detected the activity of acyl CoA reductase in the pheromone gland of B. mori by using palmitoyl CoA as a substrate. The acyl CoA reductase requires NADPH, but not NADH, as a proton dono. When the pheromone gland was incubated with the PBAN fragment peptide TKYFSPRLamide, palmitoyl CoA was incorporated and converted into the corresponding C16 alcohols. Radio HPLC analysis revealed that these C16 alcohols were hexadecan-1-ol (81.2%), (Z)-11-hexadecen-1-ol (12.3%), and (E, Z)-10, 12-hexadecadien-1-ol (= bombykol, 6.5%). The production of C16 alcohols in the pheromone gland was inhibited by the known bombykol biosynthesis inhibitors EDTA, LaCl3, W-7, trifluoperazine, p-nitrophenyl phosphate, NaF and compactin. By contrast, when the pheromone gland homogenate was incubated in the presence of palmitoyl CoA and NADPH, production of C16 alcohols was affected by compactin, W-7 and trifluoperazine, but not by EDTA, LaCl3, p-nitrophenyl phosphate and NaF. These results indicate that compactin, W-7 and trifluoperazine directly suppress the step catalyzed by acyl CoA reductase, whereas EDTA, LaCl3, pNPP, and NaF inhibit bombykol production by affecting other biochemical steps in the signal transduction of PBAN action. The present results also imply that PBAN regulates the step catalyzed by acyl CoA reductase and that palmitoyl CoA could be used as a substrate of the acyl CoA reductase that regulates bombykol biosynthesis.

Intracellular signal transduction of PBAN action in the common cutworm, Spodoptera litura: effects of pharmacological agents on sex pheromone production in vitro.

Pheromone biosynthesis activating neuropeptide (PBAN) regulates sex pheromone production in the pheromone glands of many species of female moths. In order to probe the biochemical steps as well as underlying mechanisms regulated by PBAN, we have tested the effects of pharmacological agents on sex pheromone production of the common cutworm, Spodoptera litura, using an in vitro assay. Among the pharmacological agents we tested, ionomycin (calcium ionophore) alone stimulated sex pheromone production, while LaCl3 (calcium channel blocker), W-7, trifluoperazine (calmodulin inhibitor), NaF, and p-nitrophenyl phosphate (phosphatase inhibitor) suppressed the pheromone production by a pheromonotropic peptide, TKYFSPRLamide. By contrast, forskolin (adenylate cyclase activator), phorbol 12-myristate 13-acetate (protein kinase C activator), and cyclic nucleotides alone failed to stimulate sex pheromone production. These results suggest that Ca2+/calmodulin complex and phosphoprotein phosphatase are involved in the signal transduction of PBAN action in S. litura.

Intracellular signal transduction of PBAN action in lepidopteran insects: inhibition of sex pheromone production by compactin, an HMG CoA reductase inhibitor.

Pheromone biosynthesis activating neuropeptide (PBAN) regulates sex pheromone production in the pheromone glands of many species of female moths. In order to probe the biochemical steps as well as underlying mechanisms regulated by PBAN, we have tested the effect of chemicals on sex pheromone production by using an in vitro assay. Among the chemicals we tested here, compactin, a specific 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitor, clearly inhibited the pheromone biosynthesis in the silkworm, Bombyx mori, and the common cutworm, Spodoptera litura. Since the activation of HMG CoA reductase occurs by dephosphorylation mediated by a specific phosphatase and the biochemical step regulated by PBAN in bombykol biosynthesis is similar to the one catalyzed by HMG-CoA reductase in cholesterol biosynthesis, the present results support the idea that phosphoprotein phosphatase has a significant role to regulate bombykol production in the intracellular transduction of PBAN action in B. mori.

Involvement of calcineurin in the signal transduction of PBAN in the silkworm, Bombyx mori (Lepidoptera).

In several moth species sex pheromone production in the pheromone gland is regulated by a neurohormone, pheromone biosynthesis activating neuropeptide (PBAN). In Bombyx mori it is suggested that PBAN, after binding to the cell-surface receptor, primarily activates a plasma membrane receptor-activated Ca2+ channel to increase cytosolic levels of Ca2+, and Ca2+/calmodulin complex directly or indirectly activates a phosphoprotein phosphatase, which in turn elicits activation of acyl CoA reductase (the key enzyme under PBAN control) through dephosphorylation, resulting in pheromone (bombykol) production. The effect of cyclosporin A (CsA) and FK 506, specific inhibitors of calcineurin (phosphoprotein phosphatase 2B) was studied on the sex pheromone production, in B. mori. The in vitro experiments showed that both chemicals exerted a dose-dependent inhibitory action when they were co-incubated with TKYFSPRL amide (Hez-PBAN fragment peptide). Practically, no difference was detected between the two chemicals in the tested doses (0.025-1250 microM). When effects of CsA or FK 506 were studied on cell-free production of bombykol by using microsomal fraction no inhibition was detected. Since microsomal fraction contains the acyl CoA synthetase, the rate-limiting acyl CoA reductase and the precursor, bombykol is produced if supplied with CoA, ATP and NADPH. Thus, the inhibitory action of CsA and FK506 under in vitro conditions should occur before the step of acyl group reduction and the effect is likely to be attributable to the inhibition of calcineurin in the signal transduction cascade mechanism of PBAN, in B. mori. The existence of calcineurin in the pheromone gland by using Western blot analysis is also demonstrated.

Epitope mapping on the ookinete surface antigen Pbs21 of Plasmodium berghei: identification of the site of binding of transmission-blocking monoclonal antibody 13.1.

The ookinete surface protein of Plasmodium berghei Pbs21 belongs to a class of sexual stage antigens able to induce in the vertebrate host a transmission-blocking immune response. The effectors of this transmission-blocking immunity are antibody molecules directed against particular protein epitopes. The anti-Pbs21 monoclonal antibody 13.1 is known to bind a linear stretch of amino acids within the primary sequence of Pbs21 and to efficiently block the development of P. berghei in the mosquito gut. To map the 13.1 epitope along the amino acid sequence of Pbs21 we assayed the ability of 13.1 antibody to recognize, in Western blot, a series of Pbs21 deletion mutants as well as the ability of synthetic peptides to inhibit 13.1 binding to full length Pbs21. The epitope was identified within the second EGF-like domain of the Pbs21 molecule.

[Effect of dibucaine and lidocaine on histamine release from mouse bone marrow-derived cultured mast cells].

We examined the effects of dibucaine and lidocaine on histamine release from mouse bone marrow-derived cultured mast cells. The effects of these drugs on intracellular calcium were also monitored by assessing Fura-2 signals. Additionally, the inhibitory effects of lidocaine on IgE dependent and independent stimuli were examined. Though dibucaine induced histamine release and increases in intracellular calcium from mast cells dose-dependently, lidocaine did not. Lidocaine inhibited both the IgE-dependent and independent histamine release from mast cells in a dose dependent manner. However, the ability of lidocaine to inhibit the IgE-dependent response was greater. Lidocaine also inhibited increases in intracellular calcium to a greater extent after IgE-dependent stimulation as compared with IgE-independent stimulation. The degree of the inhibition of histamine release by lidocaine appeared to parallel decreases in calcium mobilization.

Bioactivity of natural O-prenylated phenylpropenes from Illicium anisatum leaves and their derivatives against spider mites and fungal pathogens.

A variety of volatile phenylpropenes, C6-C3 compounds are widely distributed in the plant kingdom, whereas prenylated phenylpropenes are limited to a few plant species. In this study, we analysed the volatile profiles from Illicium anisatum leaves and identified two O-prenylated phenylpropenes, 4-allyl-2-methoxy-1-[(3-methylbut-2-en-1-yl)oxy]benzene [O-dimethylallyleugenol (9)] and 5-allyl-1,3-dimethoxy-2-(3-methylbut-2-en-1-yl)oxy]benzene [O-dimethylallyl-6-methoxyeugenol (11)] as major constituents. The structure-activity relationship of a series of eugenol derivatives showed that specific phenylpropenes, including eugenol (1), isoeugenol (2) and 6-methoxyeugenol (6), with a phenolic hydroxy group had antifungal activity for a fungal pathogen, whereas guaiacol, a simple phenolic compound, and allylbenzene had no such activity. The eugenol derivatives that exhibited antifungal activity, in turn, had no significant toxicant property for mite oviposition. Interestingly, O-dimethylallyleugenol (9) in which the phenolic oxygen was masked with a dimethylallyl group exhibited a specific, potent oviposition deterrent activity for mites. The sharp contrast in structural requirements of phenylpropenes suggested distinct mechanisms underlying the two biological activities and the importance of a phenolic hydroxy group and its dimethylallylation for the structure-based design of new functional properties of phenylpropenes.

[Inhibition of cholinesterase activity induced by pyridaphenthion].

Inhibitory effects on cholinesterase (ChE) activity in blood of the rabbit induced by pyridaphenthion(PD), an organophosphorus compound, and effects of 2-pyridine aldoxime methiodide(PAM) on this inhibition were examined. 1) Experimental results within 24 hr: An oral administration of each dose of PD(100 approximately 750 mg/kg) gradually decreased ChE activity and ChE activity value decreased to 20.5% in the erythrocytes and 21.5% in the plasma 24 hr after administration of 500 mg/kg of PD. However, this value recovered remarkably with an intravenous injection of PAM. The ChE activity value was 55.5% in the erythrocytes and 41.4% in the plasma with a single injection, and respectively 67.8% and 59.1% when PAM was injected three times. 2) Experimental results for 14 days: Following an increase of the administered dose (100 approximately 400 mg/kg) of PD, a decrease in ChE activity was apparent and a recovery to normal values was delayed. This inhibition could be ameliorated quickly with an injection of PAM. Considering that PAM does not have remarkable detoxicative effects on organophosphorus compounds which have a low toxicity, PAM appears to be a promising antidote against PD.

Cell-free production of the silkworm sex pheromone bombykol.

Cell-free production of bombykol was done by incubating a pheromone gland homogenate in the presence of NADPH, ATP, and CoA. Addition of n-hexane to the reaction mixture stimulated bombykol production, resulting in production of 238 ng of bombykol from the homogenate equivalent to 2 pheromone glands after 23 h. Removal of either NADPH, ATP, or CoA resulted in no stimulation of bombykol production, suggesting that the final step of the bombykol biosynthetic pathway is done by acyl CoA synthetase and reductase, sequentially. Incubation first with ATP or high concentrations of ATP suppressed the production of bombykol. Since incubation with ATP also inhibited conversion of [1-14C]palmitoyl CoA into 1-hexadecanol, the inhibitory action of ATP seemed attributable to inactivation of the acyl CoA reductase by phosphorylation, as mediated by a protein kinase in the homogenate. Our results suggest that the activity of acyl CoA reductase in bombykol biosynthesis is regulated by phosphorylation/dephosphorylation, and that the activation occurs by dephosphorylation as mediated by phosphoprotein phosphatase.

A dose monitoring system in high dose rate intracavitary remote afterloading therapy of carcinoma of the uterine cervix using semi-conductor dosimeter.

Our intention was to develop a high accuracy procedure in high dose rate intracavitary remote afterloading therapy of carcinoma of the uterine cervix similar to the accurate dosimetry used in external irradiation. Semi-conductor in-vivo-dosimetry was carried out in order to determine the extent of errors in the calculated dose at reference points and in the definition of point A. The use of a rapid processing computerized system before the treatment was developed in order to decrease the difference between the dose delivered at point A dose and the planned dose. Errors in calculation of within +/- 10% seemed to be unavoidable in the hgh dose rate intracavitary afterloading therapy of carcinoma of the uterine cervix. Two definitions should be used in patients to compute the point A dose according to the ovoid position. Using the rapid processing system, wo could deliver a point A dose within +/- 5% level of the planned dose. The remotely controlled afterloader was safely operated by means of in-vivo-dosimetry which was backed up in routine clinical practice by a pretreatment calculated dose.

Involvement of jasmonate- and salicylate-related signaling pathways for the production of specific herbivore-induced volatiles in plants.

We compared volatiles from lima bean leaves (Phaseolus lunatus) infested by either beet armyworm (Spodoptera exigua), common armyworm [Mythimna (Pseudaletia) separata], or two-spotted spider mite (Tetranychus urticae). We also analyzed volatiles from the leaves treated with jasmonic acid (JA) and/or methyl salicylate (MeSA). The volatiles induced by aqueous JA treatment were qualitatively and quantitatively similar to those induced by S. exigua or M. separata damage. Furthermore, both S. exigua and aqueous JA treatment induced the expression of the same basic PR genes. In contrast, gaseous MeSA treatment, and aqueous JA treatment followed by gaseous MeSA treatment, induced volatiles that was qualitatively and quantitatively more similar to the T. urticae-induced volatiles than those induced by aqueous JA treatment. In addition, T. urticae damage resulted in the expression of the acidic and basic PR genes that were induced by gaseous MeSA treatment and by aqueous JA treatment, respectively. Based on these data, we suggest that in lima bean leaves, the JA-related signaling pathway is involved in the production of caterpillar-induced volatiles, while both the SA-related signaling pathway and the JA-related signaling pathway are involved in the production of T. urticae-induced volatiles.

A modified dose monitoring system in remote afterloading therapy of carcinoma of the uterine cervix using semi-conductor dosimeter.

A dose monitoring system in high dose rate intracavitary remote afterloading therapy of carcinoma of the uterine cervix using semi-conductor dosimeter was developed in July 1979 at our department. During early experience with the above system, extremely high values of mgh as well as high doses to rectum and bladder were encountered in the cases with poor local anatomy or unsuitable application. Solving this problem, such limitations as to the mgh as well as point-A dose were added to the computerized rapid processing system for the pretreatment dose calculation and correction of the treatment parameters to adjust the point-A dose to within +/- 5% of the planned dose. In the majority of the cases treated with Ralstron, this modified dose monitoring system brought considerable success in the optimization of point-A, mgh, rectal and bladder doses, in a simple manner.

Circadian oscillation of a mammalian homologue of the Drosophila period gene.

Many biochemical, physiological and behavioural processes in organisms ranging from microorganisms to vertebrates exhibit circadian rhythms. In Drosophila, the gene period (per) is required for the circadian rhythms of locomotor activity and eclosion behaviour. Oscillation in the levels of per mRNA and Period (dPer) protein in the fly brain is thought to be responsible for the rhythmicity. However, no per homologues in animals other than insects have been identified. Here we identify the human and mouse genes (hPER and mPer, respectively) encoding PAS-domain (PAS, a dimerization domain present in Per, Amt and Sim)-containing polypeptides that are highly homologous to dPer. Besides this structural resemblance, mPer shows autonomous circadian oscillation in its expression in the suprachiasmaticnucleus, which is the primary circadian pacemaker in the mammalian brain. Clock, a mammalian clock gene encoding a PAS-containing polypeptide, has now been cloned: it is likely that the Per homologues dimerize with other molecule(s) such as Clock through PAS-PAS interaction in the circadian clock system.