Single-Molecule FRET to Measure Conformational Dynamics of DNA Mismatch Repair Proteins.

Single-molecule FRET measurements have a unique sensitivity to protein conformational dynamics. The FRET signals can either be interpreted quantitatively to provide estimates of absolute distance in a molecule configuration or can be qualitatively interpreted as distinct states, from which quantitative kinetic schemes for conformational transitions can be deduced. Here we describe methods utilizing single-molecule FRET to reveal the conformational dynamics of the proteins responsible for DNA mismatch repair. Experimental details about the proteins, DNA substrates, fluorescent labeling, and data analysis are included. The complementarity of single molecule and ensemble kinetic methods is discussed as well.

Possible role of prostaglandin E1 on adrenergic neurotransmission in the guinea-pig taenia coli.

The effects of prostaglandin (PG) E1 were investigated on the responses to adrenergic and non-adrenergic inhibitory nerve stimulation using the perivascular nerve-taenia preparation of the guinea pig. PGE1 caused a rapid and sustained contraction and markedly inhibited the response to adrenergic but not to non-adrenergic inhibitory nerve stimulation. It was also observed that PGE1 had some desensitizing action to exogenous noradrenaline on the postjunctional site. Although indomethacin decreased the tone of the preparation, it potentiated the response to adrenergic nerve stimulation without any effects on the response to non-adrenergic inhibitory nerve stimulation. From these observations, it was concluded that endogenous PGE1 may also play a regulatory role in adrenergic inhibitory neurotransmission, mainly by inhibitory action on noradrenaline release and partly by a similar action on the postjunctional site.

Effects of prostaglandin E1 and indomethacin on the stimulation-evoked overflow of 3H-noradrenaline from guinea-pig taenia.

Prostaglandin E1 (5.8 X 10(-8) M) markedly and reversibly reduced the stimulation-evoked overflow of total tritium, while it had no effects on basal outflow. Indomethacin (8.4 X 10(-6) M) increased the stimulation-evoked overflow of total tritium at low frequencies (2--5 HZ), while it had no effect at high frequencies of stimulation (more than 10 HZ). It was concluded that endogenous prostaglandin E1 also plays a regulatory role in adrenergic inhibitory neurotransmission by inhibiting the noradrenaline release from adrenergic nerve terminals of the guinea-pig taenia caecum.

Antitumor effects of SEF19, a new nonsteroidal aromatase inhibitor, on 7,12-dimethylbenz[a]anthracene-induced mammary tumors in rats.

The antitumor and endocrine effects of a new nonsteroidal aromatase inhibitor, 2-(imidazol-1-yl)-4,6-dimorphorino-l, 3, 5-triazine (SEF19) were examined in female Sprague-Dawley rats bearing estrogen dependent 7,12-dimethylbenz[a]anthracene(DMBA)-induced mammary tumors, and the effects were compared with those of CGS20267. The rats bearing DMBA-induced mammary tumors within 6-15 weeks after the DMBA administration were divided into the treatment groups once a week every week, and they were treated with SEF19, CGS20267 and vehicle for 4 weeks. One hundred rats were sacrificed 4 hours after the last administration, and the remaining 60 rats were sacrificed after a 4-week recovery period. During the treatment and recovery period, the tumor size was generally smaller in the SEF19 and CGS20267-treated subgroups than in the control subgroup. Tumor sizes in the subgroups treated with high doses of SEF19 (25 mg/kg/day and 50 mg/kg/2 days) were reduced to the size of the CGS20267-treated subgroup. The CGS20267-treated rats showed decrease in the serum estradiol level and an increase in the serum testosterone level. Their uterine weights were reduced. SEF19 treatment failed to show any effect on the serum levels of estrone, estradiol, testosterone and androstenedione, but it suppressed uterine weight in a dose-dependent manner. After the recovery period, no effect was detected in the serum concentrations of steroid hormones and the weight of the organs. At every dose used in the present study the aromatase inhibitory activity of SEF19 was weaker than that of CGS20267, but the inhibitory effect on mammary tumor growth of SEF19 at high doses was comparable to that of CGS20267. We conclude that the antitumor effect of SEF19 is not due to aromatase inhibition but mainly to its direct cytotoxicity.

[Physiological role of prostaglandins in adrenergic and non-adrenergic inhibitory neurotransmission in guinea pig taenia coli].

Prostaglandin (PG) E1, PGE2 and PGF2alpha and their biosynthesis inhibitor, indomethacin, were tested for their effects on the inhibitory responses of taenia induced by electrical stimulation of adrenergic and/or non-adrenergic inhibitory nerves in the perivascular nerve-taenia preparation isolated from guinea-pig caecum. The response to adrenergic nerve stimulation was considerably reduced by PGE1 and PGE2, while it was little affected by PGF2alpha. Although PGE1 and PGE2 produced contraction of taenia, it may be possible to dismiss from consideration their action of contraction of taenia in accounting for their inhibitory effect on the response to nerve stimulation since the following observations were made, 1) when the taenia were contracted by histamine, the response to adrenergic nerve stimulation was not reduced and 2) in the presence of polyphloretin phosphate, PGE1 and PGE2 did not contract taenia but reduced the response to adrenergic nerve stimulation. In the presence of indomethacin, the response to adrenergic nerve stimulation was increased greatly. On the contrary, the inhibitory response to non-adrenergic inhibitory nerve stimulation was not affected by application of PGE1, PGE2, PGF2alpha and indomethacin. These results suggest that endogenous PG of E series in guinea-pig taenia may play a role in modulating adrenergic neurotransmission. Attempts to demonstrate that PG could operate on a non-adrenergic inhibitory neurotransmission by a negative feedback mechanism were without success.

[Simulation of K-contracture curve in smooth muscle (author's transl)].

40 mM K-induced isometric contracture of guinea pig taenia coli in the presence of 30 microM dantrolene showed a rapidly rising peak followed by a plateau and then a low sustained tonic contraction. The plateau was delayed by low Ca so that two phasic contractions, fast and slow, were separated from each other. In K-contracture after 15 sec contact with normal Ca following low Ca condition, the fast phasic contraction but not the slow one regained its tension depressed by preceding low Ca, while the slow phasic contraction recovered from its delay. In the presence of 0.2 microM verapamil, K-contracture consisted of the fast phasic contraction without plateau and of the low tonic contraction. The results suggest that K-contracture in the normal state consists of three components, the fast and slow phasic contractions and the tonic contraction, and that dantrolene inhibits a tonic contraction, whereas verapamil inhibits the slow phasic and the tonic contraction. Thus, the contraction curve of each component was tentatively expressed by exponential function, and for simulation of the observed curves a computer was utilized to synthesize the curves using the components of varying parameters. The simulation was successful when based on the above suggestion.

Investigation on SCH00013, a novel cardiotonic agent with Ca++ sensitizing action. 1st communication: phosphodiesterase III inhibitory effect and class III antiarrhythmic effect in guinea-pig heart.

In order to clarify the mechanism of action of 4,5-dihydro-6-[1-[2-hydroxy-2-(4-cyanophenyl)ethyl]-1,2,5,6- tetrahydropyrido-4-yl]pyridazin-3(2H)-one (SCH00013), a novel cardiotonic agent with Ca++ sensitizing action, its effects on contractile force, atrial rate and action potential, and on the activity of Na+, K(+)-ATPase and phosphodiesterase (PDE) I-IV were studied in the guinea-pig heart. SCH00013 exerted a positive inotropic effect (PIE) on isolated right ventricular papillary muscles in a concentration-dependent manner (EC50 = 9.2 mumol/l): the relative potency was milrinone > SCH00013 > vesnarinone. The PIE of SCH00013 was not influenced by propranolol, a beta-blocker, and SCH00013 did not affect the activity of cardiac Na+, K(+)-ATPase. The PIE of SCH00013 was partially inhibited by carbachol, a muscarinic receptor agonist, which implies a partial contribution of the cAMP-dependent mechanism to the PIE. SCH00013 inhibited the activity of PDE III selectively, but the potency was weak: the IC50 value was 64.9 mumol/l, which was 46 and 3.9 times less potent than those of milrinone and vesnarinone, respectively. SCH00013 and vesnarinone elicited a moderate decrease in the rate of beating of isolated right atria, while milrinone increased it. SCH00013 markedly prolonged the action potential duration and the effective refractory period with no change in the resting membrane potential and dV/dtmax, an indication that SCH00013 may suppress the activity of delayed rectifying K+ channels. These results indicate that SCH00013, that primarily acts as a Ca++ sensitizer, possesses a weak selective PDE III inhibitory effect. The potential positive chronotropic effect of SCH00013 due to PDE III inhibition may be offset by its effect on K+ channels.

Investigation on SCH00013, a novel cardiotonic agent with Ca++ sensitizing action. 2nd communication: in vivo cardiovascular effects and bioavailability in dogs.

In vivo cardiovascular effects and bioavailability of 4,5-dihydro-6-[1-[2-hydroxy-2-(4-cyanophenyl)ethyl]-1,2,5,6- tetrahydropyrido-4-yl]pyridazin-3(2H)-one (SCH00013), a novel cardiotonic agent, were investigated. In anesthetized dogs, intravenous administration of SCH00013 (0.3-10 mg/kg) increased maximum rate of rise in left ventricular pressure (LVdP/dtmax) in a dose-dependent manner with no change in heart rate (HR) and, at the dose of 3 mg/kg or higher, at which the increase in LVdP/dtmax reached the maximum, it decreased blood pressure. In conscious dogs, oral administration of SCH00013 (1-10 mg/kg) also increased LVdP/dtmax dose-dependently with no change in HR. The increase in the plasma concentration of orally administered SCH00013 (3 mg/kg) was parallel to the increase in LVdP/dtmax. The areas under the plasma concentration versus time curve (AUC0-24 h) after oral and intravenous administration of SCH00013 (3 mg/kg) were essentially identical (15.3 +/- 2.0 micrograms.h/ml and 16.5 +/- 2.1 micrograms.h/ml, respectively). These results suggest that oral bioavailability of SCH00013 is notably high. In conclusion, the positive inotropic effect of SCH00013 with neither elevation of HR nor excessive hypotension, as well as the high oral bioavailability of this compound, may provide a beneficial pharmacological treatment of the patients with congestive heart failure.

Investigation on SCH00013, a novel cardiotonic agent with Ca++ sensitizing action. 3rd communication: stereoselectivity of the enantiomers in cardiovascular effects.

The cardiovascular effects of the enantiomers, (+)-SCH00013 and (-)-SCH00013, of a novel cardiotonic agent 4,5-dihydro-6-[1-[2-hydroxy-2-(4-cyanophenyl)ethyl]- 1,2,5,6-tetrahydropyrido-4-yl]pyridazin-3(2H)-one (SCH00013) were investigated in vitro and in vivo. The enantiomers of SCH00013 elicited an equipotent positive inotropic effect in isolated guinea-pig papillary muscles. Both of the enantiomers had a modest negative chronotropic effect in isolated guinea-pig right atria and the difference in the chronotropic effects of the enantiomers was not significant. In anesthetized dogs, both enantiomers increased LVdP/dtmax without change in heart rate and slightly decreased blood pressure. These hemodynamic effects of the enantiomers were not significantly different from each other. (+)-SCH00013 and (-)-SCH00013 increased the extent of cell shortening in association with only a small increase in the Ca++ transients in indo-1-loaded rabbit cardiomyocytes, and both the increases in cell shortening and Ca++ transients were not significantly different between the enantiomers. Both isomers equally shifted the relationships between the increases in the cell shortening and Ca++ transients to the left and upward as compared with the relationships for the elevation of extracellular Ca++ concentration and isoproterenol, which indicates that the effectiveness of the Ca++ sensitizing effects of the enantiomers are almost equivalent. The enantiomers of SCH00013 showed equipotent inhibitory effect on the phosphodiesterase (PDE) III activity. The maximal extent and the potency of prolonging effect of the two enantiomers on the effective refractory period were also the same. Thus, the efficacy and potency of the effects on the cardiovascular parameters such as myofibrillar Ca++ sensitivity, PDE III activity and the effective refractory period for the both enantiomers of SCH00013 are equivalent, indicating that the cardiovascular effects of SCH00013 may be due to equal contribution of both enantiomers.

Investigation on SCH00013, a novel cardiotonic agent with Ca++ sensitizing action. 4th communication: influence on experimentally induced ventricular arrhythmia in dogs.

Influence of 4,5-dihydro-6-[1-[2-hydroxy-2-(4-cyanophenyl)ethyl]- 1,2,5,6,-tetrahydropyrido-4-yl]pyridazin-3(2H)-one (SCH00013) and vesnarinone (CAS 81840-15-5) on the arrhythmia experimentally induced by three different methods was investigated in dogs. In digitalis-induced arrhythmia, SCH00013 (3 mg/kg i.v.) showed a tendency to improve the arrhythmia with a decrease in the arrhythmic ratio and an increase in the conducted beats (CB), though these changes did not reach a significant level; it decreased significantly the blood pressure (BP) with no change in the total heart rate (THR) and atrial rate (AR). Vesnarinone (3 mg/kg i.v.) did not affect these parameters except for BP that was decreased significantly. In two-stage coronary ligation-induced arrhythmia, SCH00013 (1 and 3 mg/kg i.v.) did not change the arrhythmic ratio, CB, AR and BP, while the THR being slightly decreased; the arrhythmic ratio showed a tendency to decrease with SCH00013 when examined at 24 h after coronary ligation. Vesnarinone (3 mg/kg i.v.) did not affect these parameters at 24 and 48 h after ligation. In epinephrine (adrenaline)-induced arrhythmia, both SCH00013 and vesnarinone showed exacerbation of arrhythmia. SCH00013 at 1 mg/kg i.v. did not elicit ventricular fibrillation (VF) in five dogs examined, but at 3 mg/kg i.v. it elicited VF in two of three dogs. Vesnarinone at 1 mg/kg i.v. induced VF in all of three dogs examined. Incidence of VF induced by optical isomers of SCH00013 was not significantly different from each other: both isomers elicited VF in two of six dogs at 1 mg/kg i.v. and at 3 mg/kg i.v. each of them induced VF in two dogs examined. The present results indicate that SCH00013 is a cardiotonic agent that is equivalent to or less arrhythmogenic than vesnarinone in animal models of arrhythmia, such as adrenaline- and digitalis-induced arrhythmia and the two-stage coronary ligation-induced arrhythmia. Optical isomers of SCH00013 were essentially equieffective in eliciting exacerbation of adrenaline-induced arrhythmia in the dog.

Potent and selective aromatase inhibitor: in vitro and in vivo studies with s-triazine derivative SEF19.

We found a potent aromatase inhibitor through the screening of agents for estrogen-dependent breast cancer. SEF19 (2-(imidazol-1-yl)-4,6-dimorphorino-1,3,5-triazine) decreased 50% of human placental aromatase activity in vitro at the concentration of 5.3 nM. In order to clarify the selectivity of SEF19 for enzyme inhibition, we determined the effect of SEF19 on the activities of four steroidogenic cytochrome P450 enzymes in porcine adrenal gland, P450SCC(side-chain cleavage of cholesterol), P450(11 beta) (11 beta-hydroxylase), P450(17 alpha)(17 alpha-hydroxylase/C17,20 lyase) and P450C21 (21-hydroxylase). SEF19 failed to inhibit the activities of porcine adrenal P450SCC, P450(17 alpha) and P450C21 up to the concentration of 100 microM and showed some inhibition on P450(11 beta) activity at 100 microM, while SEF19 completely nullified the aromatase activity at 1 microM. We also determined the potency of SEF19 for the suppression of aromatase activity in vivo. SEF19 suppressed dose-dependently the uterine hypertrophy of immature rats caused by administration of androstenedione (30 mg/kg, s.c.). The ED50 of SEF19 for the suppression of uterine hypertrophy was 0.8 mumol/kg. These results suggest that SEF19 may serve as a potent and selective agent for the treatment of estrogen-dependent breast cancer.

In vitro cytotoxicity of imidazolyl-1,3,5-triazine derivatives.

We examined in vitro cytotoxic activity of imidazolyl-1,3,5-triazine derivatives using human breast cancer cell lines (MCF-7, R-27, T-47D and ZR-75-1) and murine leukemia cell line (P388). The percentage of viable cells was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazorium bromide (MTT) assay. Hexamethylmelamine (HMM), a 1,3,5-triazine derivative has previously been recognized as an antitumor agent effective against lung, ovarian and breast cancer, but failed to show a significant cytotoxic activity in the present study. In contrast, four imidazolyl-1,3,5-triazine derivatives, 2-(1-imidazolyl)-4,6-bis(morpholino)-1,3,5-triazine, 2-(1-imidazolyl)-4-morpholino-6-(3-thiazolidinyl)-1,3,5-triazine, 2-(4-cyano-4-phenylpiperidino)-4-(1-imidazolyl)-6-morpholino-1,3,5-triaz ine and 2-(1-imidazolyl)-4-(N-methyl-N-phenylamino)-6-morpholino-1,3,5-triazine showed cytotoxic activity for most cell lines, which was significantly greater than the activity of hydroxymethylpentamethylmelamine (HMPMM), a major metabolite of HMM.

Synthesis and antitumor activity of benzimidazolyl-1,3,5-triazine and benzimidazolylpyrimidine derivatives.

Triamino-substituted 1,3,5-triazine and pyrimidine derivatives were synthesized and tested for antitumor activities using some human cancer cell lines and murine leukemia cell lines. All the compounds having benzimidazolyl and morpholino groups as substituents on the 1,3,5-triazine ring showed antitumor activity. Pyrimidine derivatives having the same groups as substituents also showed antitumor activity. Among them, the compounds having 1-benzimidazolyl, morpholino and cis-2,3-dimethylmorpholino groups as substituents on the 1,3,5-triazine ring or pyrimidine ring exhibited the most potent antitumor activity, and these compounds exhibited no or very weak aromatase inhibitory activity. In contrast, the compounds having imidazolyl group instead of benzimidazolyl group as a substituent on the 1,3,5-triazine ring showed a potent aromatase inhibitory activity.

Pneumatosis cystoides intestinalis and trichloroethylene exposure.

A case-control study was carried out to assess the association between pneumatosis cystoides intestinalis (PCI) and working conditions, including occupational exposure to organic solvents. Thirteen patients with primary PCI were individually matched with controls by sex, age, and admission year. It was found that there was a close association between the development of primary PCI and occupational exposure to trichloroethylene (TCE). Twelve of 13 patients with PCI (92.3%) were found to have been exposed occupationally to TCE, and the healing and recurrence of PCI in these patients substantially paralleled the profile of their occupational exposure to TCE. Two pairs of patients with PCI had been working in the same factories, where they had degreased camera lenses with TCE. These results suggest that chronic exposure to TCE could be one of the etiological factors in PCI.