Mechanism and inhibition of delta 24-sterol methyltransferase from Candida albicans and Candida tropicalis

The S-adenosyl-L-methionine: delta 24-sterol methyltransferase from Candida albicans has been solubilized with a mixture of octyl glucoside and sodium taurodeoxycholate. The enzyme has an apparent molecular weight of approximately 150,000 as measured by gel filtration chromatography. Zymosterol is the preferred substrate for the microsomal methyltransferase. Other nuclear double bond isomers support reduced rates of methenylation, while sterols which bear methyl groups at C-4 or C-14 are not substrates. Initial velocity and product inhibition studies are consistent with a rapid equilibrium ordered kinetic mechanism. A series of novel sterol analogues which contain heteroatoms substituted for C-24 or C-25 have been kinetically characterized as dead-end inhibitors of the methyltransferase, revealing three distinct mechanisms of interaction with the enzyme. Sterols which contain positively charged moieties in these positions are particularly potent inhibitors, supporting the proposed intermediacy of C-24 and C-25 carbocations. The methyltransferase is reversibly inhibited by low concentrations of 24-thiasterols, while behavior consistent with mechanism-based enzyme inactivation is apparent at higher concentrations. Possible mechanisms for this novel inactivation reaction are discussed.     

MAJOR CLADES OF THE ANGIOSPERMS

Abstract— Our knowledge of fundamental angiosperm interrelationships is still very incomplete. The absence of a narrowly circumscribed gymnosperm outgroup, ideally the sister group, makes character evaluation, necessary for a cladistic analysis, difficult. According to current views the superorder Magnoliiflorae with a number of other groups, for example the monocotyledons, may represent a complex of families near the base of the angiosperms. Interrelationships of groups within the monocotyledons are much better understood than those between groups within the dicotyledons. A cladogram of monocotyledon orders based on earlier work by R. Dahlgren, H. T. Clifford, and F. N. Rasmussen is presented. A data matrix for a sample of the angiosperms with 61 characters for 49 taxa, mostly magnoliifloran and related families, is presented. The characters are polarized mainly according to the current view that the primitive angiosperm morphotype is a woody dicotyledon with strobiloid flowers. As an alternative the matrix is adjusted following W. C. Burger's conjecture that the primitive angiosperm was a herbaceous monocotyledon with trimerous flowers. Both matrices were run in a computerized parsimony analysis, resulting in numerous equally parsimonious solutions. This result is illustrative of the great homoplasy in the available character information, and also of how little actually is known about fundamental angiosperm interrelationships or phylogeny.     

Assignment of orthologous relationships among mammalian alpha-globin genes by examining flanking regions reveals a rapid rate of evolution

In order to study the relationships among mammalian alpha-globin genes, we have determined the sequence of the 3' flanking region of the human alpha 1 globin gene and have made pairwise comparisons between sequenced alpha-globin genes. The flanking regions were examined in detail because sequence matches in these regions could be interpreted with the least complication from the gene duplications and conversions that have occurred frequently in mammalian alpha-like globin gene clusters. We found good matches between the flanking regions of human alpha 1 and rabbit alpha 1, human psi alpha 1 and goat I alpha, human alpha 2 and goat II alpha, and horse alpha 1 and goat II alpha. These matches were used to align the alpha-globin genes in gene clusters from different mammals. This alignment shows that genes at equivalent positions in the gene clusters of different mammals can be functional or nonfunctional, depending on whether they corrected against a functional alpha-globin gene in recent evolutionary history. The number of alpha-globin genes (including pseudogenes) appears to differ among species, although highly divergent pseudogenes may not have been detected in all species examined. Although matching sequences could be found in interspecies comparisons of the flanking regions of alpha- globin genes, these matches are not as extensive as those found in the flanking regions of mammalian beta-like globin genes. This observation suggests that the noncoding sequences in the mammalian alpha-globin gene clusters are evolving at a faster rate than those in the beta-like globin gene clusters. The proposed faster rate of evolution fits with the poor conservation of the genetic linkage map around alpha-globin gene clusters when compared to that of the beta-like globin gene clusters. Analysis of the 3' flanking regions of alpha-globin genes has revealed a conserved sequence approximately 100-150 bp 3' to the polyadenylation site; this sequence may be involved in the expression or regulation of alpha-globin genes.      

P48-Triggered transmembrane signaling transduction of human monocytes:mobilization of calcium ion and activation of protein kinase C(PKC)

INTRODUCTI0NThedifferentiati0nofcelIsalongthemonocyte-macr0phagepathwayandthesig-nalsinvo1vedinthesecel1sacquiringtheabilitytokilltum0rcellsarenotfllllyundersto0d.Wehavebeenstudingamoleculewhichappearst0beanimportantmemberofthecytokinenetworkinvo1vedintheregulati0nmonocyteactivation.ThiscytokinetermedP48wasisolatedfr0mthehllmannullcellleukemiacell1ineReh.IthasbeenpurifiedtohomogeneityandfOundtobedistinctfrominterferongamma,col0nystimulatingfactors(CSFs)andTNFalphaalldbeta[1,2].Func-ti…     

Synthesis and evaluation of (+) and (-)-2,2-difluorocitrate as inhibitors of rat-liver ATP-citrate lyase and porcine-heart aconitase.

The enantiomers (+) and (-)-2,2-difluorocitrate have been synthesized. Both are good inhibitors of ATP-citrate lyase, showing competitive inhibition against citrate, with Kis = 0.7 microM for (+)-2,2-difluorocitrate and 3.2 microM for (-)-2,2-difluorocitrate. The inhibition patterns with either ATP or CoA as the varied substrate were uncompetitive and mixed, respectively, but with much weaker inhibition constants. Neither isomer undergoes carbon-carbon bond cleavage as a substrate and there is no evidence of irreversible time-dependent inactivation. When ATP-citrate lyase is incubated with CoA and difluorocitrate, the maximal intrinsic ATPase rate is 10% of the citrate-induced rate for the (+)-enantiomer and 2% for the (-)-enantiomer. 19F-NMR studies confirm that only the (+)-enantiomer is chemically processed. The effects of the difluorocitrate enantiomers on the reaction catalysed by aconitase were examined. (-)-2,2-Difluorocitrate is a competitive inhibitor against citrate (Kis = 1.5 microM), whereas the (+)-enantiomer is a relatively poor mixed inhibitor (Ki greater than 300 microM). The (-)-enantiomer irreversibly inactivates aconitase at 1.1 min-1.mM-1 at 25 degrees C and pH 7.4, whereas no irreversible inhibition is seen with the (+)-enantiomer. Therefore, it would be expected that the (+)-enantiomer would slow the rate of acetyl-CoA synthesis in vivo, without inhibiting the citric acid cycle.     

Performance of a new dynamic model for predicting diurnal time courses of stomatal conductance at the leaf level

Under natural conditions, plants are subjected to continuous changes of irradiance that drive variations of stomatal conductance to water vapour (g_s). We propose a dynamic model to predict the temporal response of g_s at the leaf level using an asymmetric sigmoid function with a unique parameter describing time constants for increasing and decreasing g_s. The model parameters were adjusted to observed data using Approximate Bayesian Computation. We tested the model performance for (1) instant changes of irradiance; or (2) continuous and controlled variations of irradiance simulating diurnal time courses. Compared with the two mostly used steady‐state models, our dynamic model described daily time courses of g_s with a higher accuracy. In particular, it was able to describe the hysteresis of g_s responses to increasing/decreasing irradiance and the resulting rapid variations of intrinsic water‐use efficiency. Compared to the mechanistic model of temporal responses of g_s by Kirschbaum, Gross & Pearcy, for which time constants were estimated with a large variance, our model estimated time constants with a higher precision. It is expected to improve predictions of water loss and water‐use efficiency in higher scale models by using a small number of parameters.     

CYP26A1 and CYP26C1 cooperatively regulate anterior-posterior patterning of the developing brain and the production of migratory cranial neural crest cells in the mouse

The appropriate regulation of retinoic acid signaling is indispensable for patterning of the vertebrate central nervous system along the anteroposterior (A-P) axis. Although both CYP26A1 and CYP26C1, retinoic acid-degrading enzymes that are expressed at the anterior end of the gastrulating mouse embryo, have been thought to play an important role in central nervous system patterning, the detailed mechanism of their contribution has remained largely unknown. We have now analyzed CYP26A1 and CYP26C1 function by generating knockout mice. Loss of CYP26C1 did not appear to affect embryonic development, suggesting that CYP26A1 and CYP26C1 are functionally redundant. In contrast, mice lacking both CYP26A1 and CYP26C1 were found to manifest a pronounced anterior truncation of the brain associated with A-P patterning defects that reflect expansion of posterior identity at the expense of anterior identity. Furthermore, Cyp26a1-/-Cyp26c1-/- mice fail to produce migratory cranial neural crest cells in the forebrain and midbrain. These observations, together with a reevaluation of Cyp26a1 mutant mice, suggest that the activity of CYP26A1 and CYP26C1 is required for correct A-P patterning and production of migratory cranial neural crest cells in the developing mammalian brain.     

Ecological mechanisms of population change during outbreaks of the spruce budworm

Abstract.  1. Stage-specific survival and recruitment of spruce budworm were measured by frequent sampling of foliage in four outbreak populations over a 15-year period in Ontario and Quebec, Canada.
2. Patterns of change in population density during the outbreak collapse phase were closely linked to changes in survival of the late immature stages, and were determined largely by the impact of natural enemies.
3. Host-plant feedback also contributed significantly to survival patterns throughout the outbreak: annual defoliation influenced survival of fourth and fifth instars and fecundity while cumulative defoliation influenced survival of the very early larval stages (first and second) via impacts on stand condition.
4. Inclusion of this host-plant feedback reveals spruce budworm population dynamics as a function of density-related trophic interactions that vary in their order and strength of influence over time. This view re-introduces the importance of forest interactions as a component of dynamics of the spruce budworm.