The antibiotic neocarzinostatin comprises a carrier protein with a well-defined cavity for accommodating an active enediyne chromophore. The protein has two disulfides, one (Cys(37)-Cys(47)) lies on the cavity bottom and the other (Cys(88)-Cys(93)) in a constrained short loop. When the chromophore is not bound to the protein, a thiol-induced cycloaromatization of the enediyne into a tetrahydroindacene derivative is responsible for the potent antitumor activity. When it is protein-bound, the protein diverts the cycloaromatization pathway to form a distinct hydroxyisochromene-type product. How the protein directs the enediyne chemistry is an interesting puzzle, and various suggestions have been proposed in the past. We screened more than fifty thiols and manipulated conditions to locate reaction features and search for factors that could influence the protein directing strength. Thiol- and oxygen-concentration-dependence studies suggested that disulfides, which maintain the steric rigidity of the protein, could play a key role in diverting the cycloaromatization pathway. For direct proofs, we made mutations at each of the two disulfides by replacing sulfur atoms with oxygen. Circular dichroism and two-dimensional NMR spectroscopy studies suggested that the mutations changed neither the protein conformation nor the ligand interactions. Analyses of the thiol-induced cycloaromatization revealed that rupture of Cys(37)-Cys(47) made the protein almost completely lose its chemical directing ability, whereas rupture of Cys(88)-Cys(93) had only a minor influence. The results demonstrated that the steric rigidity of the binding cavity, but not necessary the whole protein, played an important role in the protein-directed mechanism.

Studying the toxic effects of long-term exposing fruit flies to phenol is the object of this study. The induction of the glutathione S-transferases enzymatic activities, the change in the amount of mRNA related to phenol exposure, the change in survival rate of adult fruit flies, and the chemical interaction between phenol and benzene were the problems to be investigated. Glutathione S-transferases were separated by affinity chromatography and the mRNAs levels were quantified by reverse-transcription polymerase chain reaction. Long-term feeding phenol to wild type fruit flies had caused some toxic effects included increasing the resistance to phenol toxicity, lowering the benzene toxicity, and induction of glutathione S-transferases enzymatic activities. But no significant change in the amount of glutathione S-transferases GstD1 and GstD5 mRNAs had occurred. From these results, we concluded that fruit flies could develop resistance to phenol by decreasing its toxicity; phenol was a inducer of glutathione S-transferases; phenol could increase the glutathione S-transferases enzymatic activities by increasing the amount of proteins; phenol exposure could decrease the benzene toxicity; no new glutathione S-transferase isozyme subunit was induced; and the level of GstD1 and GstD5 mRNAs did not significantly increase in phenol-treated strain.

The BRCA1 gene has been shown to be strongly associated with the occurrence of familial breast cancer. The spectrum of BRCA1 gene mutations in breast cancer patients in various populations has been investigated. In this study, patients in Central Taiwan with breast cancer were screened for BRCA1 mutations by sequencing PCR products spanning the coding region and partial intronic regions of the BRCA1 gene. Twelve polymorphisms in four exons and three introns were found. One mutation was found in one patient with familial breast cancer. Two patients showed LOH at the locus of BRCA1. Also found in the Taiwanese population were two common haplotypes and one rare haplotype of BRCA1. These results suggest that the mutation of BRCA1 contributes little to the occurrence of breast cancer in the Taiwanese population.

Pyruvate kinase (PK; EC 2.7.1.40) is a key glycolytic enzyme of Drosophila melanogaster. It catalyzes the conversion of phosphoenolpyruvate into pyruvate with the transfer of a phosphate group to ADP to form ATP. The ATP provides energy for cell growth and metabolism, and pyruvate participates in many metabolic reactions. Therefore, PK plays an important role in cell metabolism. Southern blot analysis, PCR, and sequencing were used to determine the content of a Drosophila pyruvate kinase (Pyk) genomic clone, lambdaPK61. The results indicated that the insert of lambdaPK61 comprised 8330 bp upstream of and 7186 bp downstream of the transcription start point of the Pyk gene. The size of the insert was 15,516 bp in total, which contained six genes including Pyk. Deletion mapping was applied to identify the promoter region and cis-acting elements 5' of PyK. Ten serial deletions produced by PCR were inserted upstream of the reporter gene (LacZ) to form recombinant plasmids, which were then transfected into Drosophila S2 cells. The results revealed that the regions -1475 approximately -1033 and -1033 approximately -534 of the 5' end of PyK possessed positive regulatory function for Pyk expression; i.e., increased gene expression. There were redundant putative cis-acting elements, including ecdysone response element (EcRE), E74A, and broad complex zinc finger (BRCZ) binding sites. Both E74A and BRCZ belong to the early genes regulated by ecdysone. This result suggested that Pyk might be regulated by ecdysone, directly or indirectly. However, the results of the developmental profile of Pyk expression by Northern blot analysis suggested that the effects of ecdysone on Pyk were repressive, not inductive. In addition, it was found that in these regions, there were many cis-acting elements related to egg and embryo development. Both -258 approximately -254 and -167 approximately -163 contained a CAAT box, and deletion of these regions decreased reporter gene expression. Therefore, it is suggested that both CAAT boxes are functional and that the promoter of Pyk might be located in the region of -258 approximately +109. No TATA box or downstream promoter element were identified around the transcription start site of Pyk. Additionally, PyK might share a regulatory region with an unknown neighboring gene. It was concluded that Pyk has the characteristics of a housekeeping gene.

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