1,4-dioxane-fused 4-anilinoquinazoline as inhibitors of epidermal growth factor receptor kinase.

The 4-anilinoquinazoline PD 153035 (1) is a potential antitumor agent which acts by inhibiting tyrosine kinase activity of epidermal growth factor receptor (EFGR) via competitive binding at the ATP site of enzyme. A series of cyclic analogues of PD 153035 bearing the 1,4-dioxane ring was prepared by reaction of 6-chloro derivative 5 with several aniline nucleophiles. These were evaluated for their ability to inhibit the EGFR kinase and the growth of primary human tumor cell cultures. All of the new 4-anilinoquinazolines exhibited less potency than PD 153035 against EGFR kinase. However, compounds 2b, 2c, 2e, 2g, and 2h showed higher inhibitory activities than PD 153035 against the growth of A431 tumor cell line. The compound 2b containing 3-chloroaniline ring was as potent as PD 153035 against EGFR kinase and showed about 5.4-fold better potency than PD153035 in the inhibition of growth of A431 cell line with good selectivity.

Induction of early flowering in Cymbidium niveo-marginatum Mak in vitro.

Many orchids take several years to flower. We have been able to induce early flowering in the temperate orchid Cymbidium niveo-marginatum Mak in vitro. The combined treatment of cytokinin (6-benzylaminopurine), restricted nitrogen supply with phosphorus enrichment, and root excision (pruning) induced transition of the Cymbidium shoot from a vegetative to a reproductive stage. Nearly 100% of the plants flowered within 90 days only when the combined treatment was applied. When root excision and/or 6-benzylaminopurine were omitted from the combined treatments, flower induction was significantly reduced. The auxin transport inhibitor, 2,3,5-triiodobenzoic acid prevented flowering of Cymbidium in vitro, although auxin (α-naphthaleneacetic acid) itself did not induce flowering. Gibberellic acid markedly delayed flowering in C. niveo-marginatum even when the flower-promoting treatment was applied. Paclobutrazol, an anti-gibberellin agent, totally blocked the inductive effects of either cytokinin or pruning. These observations suggest that concerted actions of auxin, cytokinin, and gibberellin, as well as nutrient concentration and putative promoting/suppressing agents, determine the timing of Cymbidium orchid transition from the vegetative to reproductive stage.

Molecular cloning and characterization of the Saccharomyces cerevisiae SAB1 gene that suppresses a temperature-sensitive phenotype of the ARS-binding factor 1 mutant.

A high-copy number suppressor gene of the yeast temperature-sensitive lethal abf1 mutant was isolated and named SAB1 (suppressor of ABF1). Chromoblot hybridization and grid-filter hybridization analyses showed that the SAB1 gene was located on chromosome IV. Deletion analyses of the SAB1 plasmid revealed that the suppressor activity was contained in a 1.1 kb DNA region. The nucleotide sequence of the 1.1 kb DNA fragment was determined and turned out to be identical to that of the yeast phosphoribosylanthranilate isomerase gene (TRP1). A binding site for ARS-Binding Factor 1 was located in the coding sequence of the TRP1 gene, which has been known to be a part of the B domain of yeast autonomously replicating sequence 1 (ARS1). Our results suggest that ABF1 might be important for the transcription of the yeast TRP1 gene in addition to having important roles in the stimulation of replication at the ARS1 locus.

Isolation and characterization of Saccharomyces cerevisiae SAB2, a suppressor gene for temperature-sensitive phenotype of ARS-binding factor 1 mutant.

A high-copy number suppressor of yeast abf1-5 mutant, a temperature-sensitive lethal mutant, was isolated and named SAB2 (suppressor of ABF1). Hybridization to a yeast chromoblot and to prime clone grid filters revealed that the SAB2 gene was located near the yeast SUP3 on chromosome XV. The suppressor activity was contained in a 2.5 Kbp DNA region of the SAB2 plasmid. The nucleotide sequence of the DNA region contained a long open reading frame, which turned out to encode for yeast tryptophan permease. Four putative ABF1 binding sites were found in the promoter and the structural regions of the tryptophan permease gene. Binding of ABF1 to two of the sites tested in this study was detected. Our results indicate that ABF1 may be involved in the transcriptional control of the yeast tryptophan permease gene.

Inward current generated by Na-Ca exchange during the action potential in single atrial cells of the rabbit.

To investigate the underlying ionic mechanism of the late plateau phase of the action potential in rabbit atrium the whole-cell patch-clamp technique with intracellular perfusion was used. We recorded the inward current during repolarizations following a brief 2 ms depolarizing pulse to +40 mV from a holding potential of between -70 and -80 mV. The development of this current coincides with the onset of the late plateau phase of the action potential. Peak activation of the current occurs about 10 ms from the beginning of the depolarizing pulse, and it decays spontaneously with a slow timecourse. Its voltage dependency from -40 mV to +40 mV shows very steep activation (-40 to -20 mV) and shows almost the same maximum magnitude between -10 mV and +40 mV. This behaviour is quite different from that of the calcium current. The inward current and the late plateau phase of the action potential were both abolished by the application of 5 mM EGTA, 1 microM ryanodine and by reducing the Na+ gradient. The fully activated current-voltage relation of the inward current was plotted as the difference current before and after treatment with Ryanodine, Diltiazem, 20 mM Na+ inside or 30% Na+ outside and shows an exponential voltage dependence with the largest magnitude of the current occurring at negative potentials. The current-voltage (I-V) curve was well fitted by the Na-Ca exchange equation, i = A exp (-(1 - r)EF/RT). The results suggest that the inward current contributes to the generation of the late plateau phase of the rabbit atrial action potential, and is activated by intracellular calcium released from the sarcoplasmic reticulum. Sarcoplasmic reticulum calcium release appears to be triggered both by the membrane voltage and by the calcium current. It is concluded that the inward current is generated by Na-Ca exchange.