Drosophila Histone Deacetylase-3 Controls Imaginal Disc Size through Suppression of Apoptosis

Histone deacetylases (HDACs) execute biological regulation through post-translational modification of chromatin and other cellular substrates. In humans, there are eleven HDACs, organized into three distinct subfamilies. This large number of HDACs raises questions about functional overlap and division of labor among paralogs. In vivo roles are simpler to address in Drosophila, where there are only five HDAC family members and only two are implicated in transcriptional control. Of these two, HDAC1 has been characterized genetically, but its most closely related paralog, HDAC3, has not. Here we describe the isolation and phenotypic characterization of hdac3 mutations. We find that both hdac3 and hdac1 mutations are dominant suppressors of position effect variegation, suggesting functional overlap in heterochromatin regulation. However, all five hdac3 loss-of-function alleles are recessive lethal during larval/pupal stages, indicating that HDAC3 is essential on its own for Drosophila development. The mutant larvae display small imaginal discs, which result from abnormally elevated levels of apoptosis. This cell death occurs as a cell-autonomous response to HDAC3 loss and is accompanied by increased expression of the pro-apoptotic gene, hid. In contrast, although HDAC1 mutants also display small imaginal discs, this appears to result from reduced proliferation rather than from elevated apoptosis. The connection between HDAC loss and apoptosis is important since HDAC inhibitors show anticancer activities in animal models through mechanisms involving apoptotic induction. However, the specific HDACs implicated in tumor cell killing have not been identified. Our results indicate that protein deacetylation by HDAC3 plays a key role in suppression of apoptosis in Drosophila imaginal tissue

Stereochemistry of the biotransformation of 1-hexene and 2-methyl-1-hexene with rat liver microsomes and purified P450s of rats and humans

The epoxidation of 1-hexene (1a) and 2-methyl-1-hexene (1b), two hydrocarbons present in the ambient air as: pollutants, is catalyzed by some human and rat P450 enzymes. The enantioselectivities of these processes, when the reactions were carried out using rat and human liver microsomal preparations, were modest and dependent on both P450 composition and substrate concentrations. Various P450 isoforms (rat P450 2B1 and human P450 2C10 and 2A6) catalyzed the double bond oxidation of 1a and 1b with different product enantioselectivities. In the case of 1a, a moderately enantioselective hydroxylation at the allylic C(3) with the formation of 1-hexen-3-ol (4a) by microsomes from control or preinduced rats was also observed. The oxidation of this metabolite was, in turn, catalyzed by rat liver microsomes and mainly by rat P450 2C11, leading exclusively to the formation of 1-hexen-3-one, with no double bond epoxidation being observed. The stereochemical course of the microsomal epoxide hydrolase-catalyzed hydrolysis of the epoxy alcohols, threo-(+/-)- and erythro-(+/-)-1,2-epoxyhexan-3-ol, theoretically expected to be formed from 4a, has been investigated

Stereochemical aspects in the 4-vinylcyclohexene biotransformation with rat liver microsomes and purified cytochrome p450s: Diepoxide formation and hydrolysis

The stereochemical course of the biotransformation of 1,2-monoepoxides of 4-vinyleyclohexene (2 and 3) by liver microsomes from control and induced rats and by purified P4502B1 and P4502E1 has been determined. The epoxidation of monoepoxides cis-4-vinylcyclohexene 1,2-epoxide (2) and trans-4-vinyleyclohexene 1,2-epoxide (3) gives the corresponding eight isomeric diepoxides cis-4-vinylcyclohexene diepoxide (9) and trans-4-vinyleyclohexene diepoxide (10). The stereoselectivity of this process is affected by P450 induction. Phenobarbital is able to enhance the yield of epoxidation to give preferentially diepoxide (1R,2S,4R,7R)-trans-10b. This enantiomer is also formed as nearly the sole product by P450-catalyzed epoxidation of (1R,2S,4R)-trans-3b, the monoepoxide that, as a consequence of the selective formation from 4-vinyleyclohexene and/or reduced elimination by epoxide hydrolase, tends to accumulate in rat. Also, the P4502B1 but not 2E1, in a reconstituted system, is able to perform the epoxidation of (1R,2S,4R)-trans-3b to produce selectively the same diepoxide. Diepoxides cis-9 and trans-10 are biotransformed by mEH catalyzed hydrolysis. Although the hydrolysis of diepoxides 9 is characterized by a lower substrate enantioselection, the reaction of diepoxides 10 occurs with a good substrate enantioselectivity favoring the hydrolysis of the epoxides (1R,2S,4R,7S)-trans-10b and (1S,2R,4S,7S)-trans-10a. Diepoxide (1R,2S,4R,7R)-trans-10b is therefore the isomer primarily formed by P450-catalyzed oxidation of monoepoxide trans-3, and it is also the compound showing the lower propensity to undergo mEH-catalyzed hydrolysis. On the basis of this result, the ovotoxicity of 4-vinylcyclohexene is expected to be due to the stereoisomer diepoxide (1R,2S,411,711)-trans-10b, whose biological reactivity, via cross-linking, may be strongly different to the other isomer diepoxides, being dependent by its specific conformation

Stereochemical aspects in the 4-vinylcyclohexene biotransformation with rat liver microsomes and purified P450s. Monoepoxides and diols

The stereochemical course of the biotransformation of 4-vinylcyclohexene (VCH, 1) by liver microsomes from male and female control and induced rats and purified rat F450 2B1 and 2E1 has been determined. The epoxidation of 1, catalyzed by male microsomes, occurs on both the endo- and exocyclic double bond to give four isomeric epoxides, cis-4-vinylcyclohexene 1,2-epoxide (2), trans-4-vinylcyclohexene 1,2-epoxide (3), (4R*,7S*)-4-vinylcyclohexene 7,8-epoxide (4), and (4R*,7R*)-4-vinylcyclohexene 7,8-epoxide (5). On the other hand, microsomes from female rats catalyzed primarily the endocyclic epoxidation. The stereoselectivity of this process was strongly dependent on gender and P450 induction. Only the phenobarbital and pyrazole, at lower levels, were able to enhance the epoxidation of 1 and mostly on the endocyclic double bond. Also, P450 2E1 and 2B1 in a reconstituted system were able to perform the epoxidation of 1 primarily on its endocyclic double bond. The metabolites, cis- and trans-4-vinylcyclohexene 1,2-epoxide (2 and 3, respectively) and the isomeric 4-vinylcyclohexene 7,8-epoxides (4 and 5), were rapidly biotransformed into the corresponding vicinal diols by mEH-catalyzed hydrolysis. The reaction of the endocyclic epoxides occurred with good substrate diastereo- and enantioselectivity favoring the hydrolysis of epoxides (1S,2R,4S)-3 and (1R,2S,4S)-2 to give, before 50% conversion, selectively (1R,2R,4S)-diol (6). At variance, the hydrolysis of the exocyclic epoxides was characterized by a high level of substrate enantioselection associated with a very low, if any, level of substrate diastereoselection, the two epoxides, (4R,7S)-4 and (4R,7R)-5, being hydrolyzed practically with the same rate. On the basis of the major resistance to mEH hydrolysis, the endocyclic epoxides, (1R,2S,4R)-3 and (1S,2R,4R)-2, are expected to be further oxidized, in a stereochemical manner, to the specific mutagenic diepoxides which are thought to play a crucial role in VCH ovotoxicity. Thus, VCH ovotoxicity may be markedly affected by the reactivity of the diepoxidic stereoisomers formed and detoxicated

Stereochemical course of the biotransformation of isoprene monoepoxides and of the corresponding diols with liver microsomes from control and induced rats

The stereochemical course of the biotransformation of isoprene by liver enzymes from control and induced rats has been determined. Between the two primarily formed metabolites, 2-methyl-2-vinyloxirane (2) and isopropenyloxirane (3), epoxide 2 is rapidly transformed into the corresponding vicinal racemic diol 4, predominantly through a nonenzymatic hydrolysis reaction. At variance, epoxide 3 is mainly biotransformed into the diol 5 by microsomal epoxide hydrolase (mEH) to give, before 50% conversion, selectively (R)-3-methyl-3-butene-1,2-diol, 5. The hydrolysis competes with the oxidation of the monoepoxide 3 to the corresponding diepoxides 6. Epoxidation of 3 catalyzed by P450 is characterized by a moderate stereoselectivity which, however, was strongly dependent on P450 induction. Treatment of rats with phenobarbital (PB) (an inducer of P450 2B1 and 3A) leads to threo-(2R,2'R)-6 with a high selectivity, while with pyrazole (Pyr) (an inducer of P450 2E1), the formation of both erythro-(2S,2'R)- and threo-(2R,2'R)-6 is favored. The mEH-catalyzed hydrolysis of diepoxides 6 proceeds, although with a moderate turnover rate, with substrate and product diastereo- and enantioselection by nucleophilic attack on the more substituted oxirane ring to give selectively (2R,3S)-3,4-epoxy-2-methyl-1,2-diol (7). Both diols 4 and 5 may be further oxidized on their double bond by P450. These reactions, which occur at a slow rate and are dependent on P450 induction with PB and Pyr, may be negligible in the overall isoprene biotransformation. On the other hand, the epoxydiol 7, which is formed by hydrolysis of diepoxides 6 but it is itself not hydrolyzable, may play an important role in the isoprene toxicity

Construction of a library of bovine genomic fragments enriched in CpG islands

A procedure is described to isolate DNA probes from the bovine genome that are enriched in sites for the so-called rare cutter restriction endo- nucleases. A collection of Sac11(C"CGCGG)-Hin- dII1 fragments from bovine sperm was established in the plasmid Bluescript. 180 clones were picked at random and analysed for the presence of inserts with sites for the following rare cutters:EagI,BsshII,NarI,MluI,NruI,NaeI: 70%oftheclonescontainedatleast1siteand5% contained four different such sites. 22.8% had multiple sites for one or more of the rare cutters tested. Sequence analysis for 16 clones con- firmed the cloning of DNA with a G+C content and a proportion of CpG vs GpCs indicative of CpG islands