Biotransformation of ent-13-epi-manoyl oxides difunctionalized at C-3 and C-12 by filamentous fungi

Biotransformation of ent-3beta,12alpha-dihydroxy-13-epi-manoyl oxide with Fusarium moniliforme gave the regioselective oxidation of the hydroxyl group at C-3 and the ent-7beta-hydroxylation. The action of Gliocladium roseum in the 3,12-diketoderivative originated monohydroxylations at C-1 and C-7, both by the ent-beta face, while Rhizopus nigricans produced hydroxylation at C-7 or C-18, epoxidation of the double bond, reduction of the keto group at C-3, and combined actions as biohydroxylation at C-2/epoxidation of the double bond and hydroxylation at C-7/reduction of the keto group at C-3. In the ent-3-hydroxy-12-keto epimers, G. roseum originated monohydroxylations at C-1 and C-7 and R. nigricans originated the oxidation at C-3 as a major transformation, epoxidation of double bond and hydroxylation at C-2. Finally, in the ent-3beta-hydroxy epimer R. nigricans also originated minor hydroxylations at C-1, C-6, C-7 and C-20 and F. moniliforme produced an hydroxylation at C-7 and a dihydroxylation at C-7/C-11.     

Resource pulses,species interactions,and diversity maintenance in arid and semi-arid environments

Arid environments are characterized by limited and variable rainfall that supplies resources in pulses. Resource pulsing is a special form of environmental variation, and the general theory of coexistence in variable environments suggests specific mechanisms by which rainfall variability might contribute to the maintenance of high species diversity in arid ecosystems. In this review, we discuss physiological, morphological, and life-history traits that facilitate plant survival and growth in strongly water-limited variable environments, outlining how species differences in these traits may promote diversity. Our analysis emphasizes that the variability of pulsed environments does not reduce the importance of species interactions in structuring communities, but instead provides axes of ecological differentiation between species that facilitate their coexistence. Pulses of rainfall also influence higher trophic levels and entire food webs. Better understanding of how rainfall affects the diversity, species composition, and dynamics of arid environments can contribute to solving environmental problems stemming from land use and global climate change.     

Stent thrombosis in patients treated with thienopyridines: clinical description of 10 cases

Subacute stent thrombosis (SAST) is a major thrombotic complication of coronary stenting. Its occurrence has been substantially reduced by thienopyridine treatment. However, information on clinical profile of patients with SAST in clopidogrel era is limited. In order to define the incidence and factors predisposing to stent thrombosis, we analyzed the computerized angiographic database of three interventional cardiology centers. Out of a total number of 5903 percutaneous coronary interventions (PCIs) with stent implantation, we found 10 patients with SAST (0.17%). The indication for PCI was usually an early invasive approach (90%) during an acute coronary syndrome. All patients were treated with an apparently optimal antithrombotic regimen (90% received heparin or LMWH and 70% received IIb/IIIa receptor inhibitors and all given aspirin). In each of the patients, we could identify high-risk angiographic findings. SAST presentation was always clinically significant with definite myocardial infarction in 100% of cases. 80% of cases occurred during the first six days post PCI. Two patients had a recurrent event. Finally, despite earlier reports of atorvastatin-mediated inhibition of clopidogrel activation we did not find any patient with SAST taking both drugs. Thus, patients with stent thrombosis during thienopyridine treatment usually exhibit high-risk angiographic features. Prospective studies should be performed to elucidate drug interactions that may reduce clopidogrel efficacy and contribute to stent thrombosis.     

The not-so-humble worm

A report of the Wellcome Trust meeting "Caenorhabditis elegans past, present and future: The not-so-humble worm", Hinxton, UK, 10 September 2003.     

Modulation of adenosine receptor affinity and intrinsic efficacy in adenine nucleosides substituted at the 2-position

We studied the structural determinants of binding affinity and efficacy of adenosine receptor (AR) agonists. Substituents at the 2-position of adenosine were combined with N(6)-substitutions known to enhance human A(3)AR affinity. Selectivity of binding of the analogues and their functional effects on cAMP production were studied using recombinant human A(1), A(2A), A(2B), and A(3)ARs. Mainly sterically small substituents at the 2-position modulated both the affinity and intrinsic efficacy at all subtypes. The 2-cyano group decreased hA(3)AR affinity and efficacy in the cases of N(6)-(3-iodobenzyl) and N(6)-(trans-2-phenyl-1-cyclopropyl), for which a full A(3)AR agonist was converted into a selective antagonist; the 2-cyano-N(6)-methyl analogue was a full A(3)AR agonist. The combination of N(6)-benzyl and various 2-substitutions (chloro, trifluoromethyl, and cyano) resulted in reduced efficacy at the A(1)AR. The environment surrounding the 2-position within the putative A(3)AR binding site was explored using rhodopsin-based homology modeling and ligand docking.     

DNA-protein cross-linking via guanine oxidation: dependence upon protein and photosensitizer

DNA-protein cross-links form when guanine undergoes a 1-electron oxidation in a flash-quench experiment, and the importance of reactive oxygen species, protein, and photosensitizer is examined here. In these experiments, a strong oxidant produced by oxidative quenching of a DNA-bound photosensitizer generates an oxidized guanine base that reacts with protein to form the covalent adduct. These cross-links are cleaved by hot piperidine and are not the result of reactive oxygen species, since neither a hydroxyl radical scavenger (mannitol) nor oxygen affects the yield of DNA-histone cross-linking, as determined via a chloroform extraction assay. The cross-linking yield depends on protein, decreasing as histone > cytochrome c > bovine serum albumin. The yield does not depend on the cytochrome oxidation state, suggesting that reduction of the guanine radical by ferrocytochrome c does not compete effectively with cross-linking. The photosensitizer strongly influences the cross-linking yield, which decreases in the order Ru(phen)(2)dppz(2+) [phen = 1,10-phenanthroline; dppz = dipyridophenazine] > Ru(bpy)(3)(2+) [bpy = 2,2'-bipyridine] > acridine orange > ethidium, in accordance with measured oxidation potentials. A long-lived transient absorption signal for ethidium dication in poly(dG-dC) confirms that guanine oxidation is inefficient for this photosensitizer. From a polyacrylamide sequencing gel of a (32)P-labeled 40-mer, all of these photosensitizers are shown to damage guanines preferentially at the 5' G of 5'-GG-3' steps, consistent with a 1-electron oxidation. Additional examination of ethidium shows that it can generate cross-links between histone and plasmid DNA (pUC19) and that the yield depends on the quencher. Altogether, these results illustrate the versatility of the flash-quench technique as a way to generate physiologically relevant DNA-protein adducts via the oxidation of guanine and expand the scope of such cross-linking reactions to include proteins that may associate only transiently with DNA.     

Fluorescent labeling of endothelial cells allows in vivo,continuous characterization of the vascular development of Xenopus laevis

Appropriate blood supply and vascular development are necessary in development and in cancer, heart disease, and diabetes. Here, we report the use of DiI-labeled acetylated low-density lipoprotein (DiI-Ac-LDL) to label endothelial cells and characterize the vasculature of live Xenopus embryos. The atlas we have created provides a detailed map of normal vascular development against which perturbations of normal patterning can be compared. By following the development of the intersomitic vessels in real-time, we show that, while rostrocaudal gradient of maturing intersomitic vessels occurs, it is not absolute. In addition, the comparative study of the ontogeny of nerve bundles from the spinal cord of transgenic Xenopus embryos expressing green fluorescent protein in the nervous system and blood vessels demonstrates a strong anatomical correlation in neurovascular development. These studies provide the basis for understanding how the vascular system forms and assumes its complicated stereotypical pattern in normal development and in disease.     

Type III protein translocase: HrcN is a peripheral ATPase that is activated by oligomerization

Type III protein secretion (TTS) is catalyzed by translocases that span both membranes of Gram-negative bacteria. A hydrophilic TTS component homologous to F1/V1-ATPases is ubiquitous and essential for secretion. We show that hrcN encodes the putative TTS ATPase of Pseudomonas syringae pathovar phaseolicola and that HrcN is a peripheral protein that assembles in clusters at the membrane. A decahistidinyl HrcN derivative was overexpressed in Escherichia coli and purified to homogeneity in a folded state. Hydrodynamic analysis, cross-linking, and electron microscopy revealed four distinct HrcN forms: I, 48 kDa (monomer); II, approximately 300 kDa (putative hexamer); III, 575 kDa (dodecamer); and IV, approximately 3.5 MDa. Form III is the predominant form of HrcN at the membrane, and its ATPase activity is dramatically stimulated (>700-fold) over the basal activity of Form I. We propose that TTS ATPases catalyze protein translocation as activated homo-oligomers at the plasma membrane.