Pentaketide melanin biosynthesis in Aspergillus fumigatus requires chain-length shortening of a heptaketide precursor

Chain lengths and cyclization patterns of microbial polyketides are generally determined by polyketide synthases alone. Fungal polyketide melanins are often derived from a pentaketide 1,8-dihydroxynaphthalene, and pentaketide synthases are used for synthesis of the upstream pentaketide precursor, 1,3,6,8-tetrahydroxynaphthalene (1,3,6,8-THN). However, Aspergillus fumigatus, a human fungal pathogen, uses a heptaketide synthase (Alb1p) to synthesize its conidial pigment through a pentaketide pathway similar to that which produces 1,8-dihydroxynaphthalene-melanin. In this study we demonstrate that a novel protein, Ayg1p, is involved in the formation of 1,3,6,8-THN by chain-length shortening of a heptaketide precursor in A. fumigatus. Deletion of the ayg1 gene prevented the accumulation of 1,3,6,8-THN suggesting the involvement of ayg1 in 1,3,6,8-THN production. Genetic analyses of double-gene deletants suggested that Ayg1p catalyzes a novel biosynthetic step downstream of Alb1p and upstream of Arp2p (1,3,6,8-THN reductase). Further genetic and biochemical analyses of the reconstituted strains carrying alb1, ayg1, or alb1 + ayg1 indicated that Ayg1p is essential for synthesis of 1,3,6,8-THN in addition to Alb1p. Cell-free enzyme assays, using the crude Ayg1p protein extract, revealed that Ayg1p enzymatically shortened the heptaketide product of Alb1p to 1,3,6,8-THN. Thus, the protein Ayg1p facilitates the participation of a heptaketide synthase in a pentaketide pathway via a novel polyketide-shortening mechanism in A. fumigatus.     

Evidence that an imprinted gene on the X chromosome increases ovulation rate in sheep

Ovulation rate records from 1311 female progeny of 50 Coopworth rams were used to study the inheritance of ovulation rate in a screened high prolificacy sheep flock. Breeding values (BV) for ovulation rate for 33 sires used within the screened flock and ovulation rate deviations for a further 17 sires progeny tested in commercial flocks suggest that a major gene (WOODLANDS: gene) for ovulation rate with a non-Mendelian inheritance pattern is segregating in a family line. Rams assigned as carriers of the putative gene did not produce carrier sons (zero of three), and this coupled with the observation that daughters of carrier rams had ovulation rates of 0. 39 (standard error of difference [SED] = 0.06) higher than contemporaries without a significant increase in the variance of log ovulation rate strongly suggests that the gene is on the X chromosome. The evidence suggests that the gene is also maternally imprinted because ovulation rate data indicate that it is expressed where females inherit a paternal allele but is silenced when inherited on a maternal allele. Maternal granddaughters of carrier rams had mean ovulation rates that were only 0.02 (SED = 0.06) higher than noncarrier ewes from the same flock. Furthermore, carrier dams expressing the gene (paternal allele) had 24 sons, none of which had female offspring that expressed the gene, whereas carrier dams not expressing the gene (maternal allele) had 7 out of 17 sons that had female progeny expressing the gene. There is no evidence of the infertility that occurs in homozygous ewes carrying the X-linked Inverdale gene. Collectively, these results suggest the existence of a novel gene for prolificacy located on the X chromosome that is maternally imprinted. The WOODLANDS: gene was only expressed upon paternal inheritance from carrier males that were the progeny of nonexpressing carrier dams. The gene was not expressed in ewes that received it from either carrier dams (expressing or nonexpressing) or from carrier males that were the progeny of expressing carrier dams.     

Taxol biosynthesis: differential transformations of taxadien-5 alpha-ol and its acetate ester by cytochrome P450 hydroxylases from Taxus suspension cells

The biosynthesis of the diterpenoid antineoplastic drug Taxol in Taxus species involves the cyclization of the ubiquitous isoprenoid intermediate geranylgeranyl diphosphate to taxa-4(5),11(12)-diene followed by cytochrome P450-mediated hydroxylation (with allylic rearrangement) of this olefin precursor to taxa-4(20),11(12)-dien-5 alpha-ol, and further oxygenation and acylation reactions. Based on the abundances of naturally occurring taxoids, the subsequent order of oxygenation of the taxane core is considered to occur at C10, then C2 and C9, followed by C13, and finally C7 and C1. Circumstantial evidence suggests that the acetylation of taxadien-5 alpha-ol may constitute the third specific step of Taxol biosynthesis. To determine whether taxadienol or the corresponding acetate ester serves as the direct precursor of subsequent oxygenation reactions, microsomal preparations isolated from induced Taxus cells and optimized for cytochrome P450 catalysis were incubated with each potential substrate. Both taxadienol and taxadienyl acetate were oxygenated to the level of a diol and to higher polyols at comparable rates by cytochrome P450 enzymes of the microsomal preparation. Preparative-scale incubation allowed the isolation of sufficient quantities of the diol derived from taxadienol to permit the NMR-based structural elucidation of this metabolite as taxa-4(20),11(12)-dien-5 alpha,13 alpha-diol, which may represent an alternate route of taxoid metabolism in induced cells. GC-MS-based structural definition of the diol monoacetate derived in microsomes from taxadienyl acetate confirmed this metabolite as taxa-4(20),11(12)-dien-5 alpha-acetoxy-10 beta-ol, thereby indicating that acetylation at C5 of taxadienol precedes the cytochrome P450-mediated insertion of the C10-beta-hydroxyl group of Taxol.     

Osmotic regulation of ATA2 mRNA expression and amino acid transport System A activity

When porcine endothelial cells were exposed to hypertonicity, both the level of ATA2 (amino acid transporter 2) mRNA and activity of amino acid transport System A increased transiently, peaking after about 6 and 9 h, respectively. Cycloheximide, like actinomycin D, prevented both responses, showing that an earlier step also involves protein synthesis. Withdrawal of hypertonicity after 6 h increased the rate of down regulation. These findings confirm that ATA2 is a major isoform of System A and show that changes in the expression of ATA2 mRNA precede both the induction and subsequent down regulation of transport activity.     

Antifeedant and toxic activity of Trichilia americana extract against the larvae of Spodoptera litura

The biological activity of a crude methanolic extract of Trichilia americana (Sesse and Mocino) Pennington (Meliaceae) was assessed using the Asian armyworm, Spodoptera litura (Fabr.) (Lepidoptera: Noctuidae). The extract exhibited strong antifeedant activity in a choice leaf disc bioassay with 0.18 g cm^–2 extract deterring feeding by 50%. In nutritional assays, the crude extract reduced growth, consumption and the utilisation of ingested and digested food in a dose-dependent manner when fed to larvae, suggesting both antifeedant and toxic activities. When relative growth rates were plotted against relative consumption rates, the growth efficiency of the S. litura fed on diet containing T. americana crude extract was significantly less than that of control larvae. This result further indicates that the extract acts as both an antifeedant and chronic toxin. Toxicity is only seen following ingestion and was not observed following topical application or injection into the hemocoel. Larvae reared initially on extract-containing diet then transferred to control diet showed nutritional indices comparable to those of larvae fed continuously on control diet. This suggests that the extract is not permanently damaging the insect's digestive tract. The mode-of-action of the extract as a chronic toxin remains unknown.     

Differential localization of two histidine kinases controlling bacterial cell differentiation

The bacterium C. crescentus coordinates cellular differentiation and cell cycle progression via a network of signal transduction proteins. Here, we demonstrate that the antagonistic DivJ and PleC histidine kinases that regulate polar differentiation are differentially localized as a function of the cell cycle. The DivJ kinase localizes to the stalked pole in response to a signal at the G1-to-S transition, while the PleC kinase is localized to the flagellar pole in swarmer and predivisional cells but is dispersed throughout the cell in the stalked cell. PleC, which is required for DivJ localization, may provide the cue at the G1-to-S transition that directs the polar positioning of DivJ. The dynamic positioning of signal transduction proteins may contribute to the regulation of polar differentiation at specific times during the bacterial cell cycle.     

Recent advances in alcoholic liver disease III. Role of the innate immune response in alcoholic hepatitis

Ethanol consumption is known to cause significant acute liver damage resulting in hepatic fibrosis and eventual cirrhosis when consumed chronically. The mechanism(s) by which ethanol exerts its damaging effects on the liver are not well understood; however, recent scientific investigation has begun to delineate the earliest events in alcoholic liver disease. From these studies, it is apparent that components of the innate immune system and, in particular, Kupffer cells, play a significant role in this process. It is also becoming clear that other parts of the immune system including T cells may also be responsible for mediating the devastating effects of chronic alcohol consumption on the liver. This review will highlight recent experiments demonstrating a role for the innate immune response in the initiation and progression of alcohol-induced liver hepatitis and subsequent organ damage.     

Synthesis and biological activity of some known and putative duloxetine metabolites

Several putative phase I duloxetine metabolites, 4-hydroxy-, 5-hydroxy-, 6-hydroxy-, 5-hydroxy-6-methoxy-, 6-hydroxy-5-methoxy-, 5,6-dihydroxy-, and 4,6-dihydroxyduloxetine were synthesized, and their phase II metabolite as glucuronide or sulfate conjugates were also synthesized. Their in vitro binding activities were compared to that of parent compound duloxetine.