Intensive nitrogen loss over the Omani Shelf due to anammox coupled with dissimilatory nitrite reduction to ammonium

A combination of stable isotopes (¹⁵N) and molecular ecological approaches was used to investigate the vertical distribution and mechanisms of biological N₂ production along a transect from the Omani coast to the central–northeastern (NE) Arabian Sea. The Arabian Sea harbors the thickest oxygen minimum zone (OMZ) in the world''s oceans, and is considered to be a major site of oceanic nitrogen (N) loss. Short (<48 h) anoxic incubations with ¹⁵N-labeled substrates and functional gene expression analyses showed that the anammox process was highly active, whereas denitrification was hardly detectable in the OMZ over the Omani shelf at least at the time of our sampling. Anammox was coupled with dissimilatory nitrite reduction to ammonium (DNRA), resulting in the production of double-¹⁵N-labeled N₂ from ¹⁵NO₂⁻, a signal often taken as the lone evidence for denitrification in the past. Although the central–NE Arabian Sea has conventionally been regarded as the primary N-loss region, low potential N-loss rates at sporadic depths were detected at best. N-loss activities in this region likely experience high spatiotemporal variabilities as linked to the availability of organic matter. Our finding of greater N-loss associated with the more productive Omani upwelling region is consistent with results from other major OMZs. The close reliance of anammox on DNRA also highlights the need to take into account the effects of coupling N-transformations on oceanic N-loss and subsequent N-balance estimates.     

Naphthoquinone-mediated Inhibition of Lysine Acetyltransferase KAT3B/p300, Basis for Non-toxic Inhibitor Synthesis

Hydroxynaphthoquinone-based inhibitors of the lysine acetyltransferase KAT3B (p300), such as plumbagin, are relatively toxic. Here, we report that free thiol reactivity and redox cycling properties greatly contribute to the toxicity of plumbagin. A reactive 3rd position in the naphthoquinone derivatives is essential for thiol reactivity and enhances redox cycling. Using this clue, we synthesized PTK1, harboring a methyl substitution at the 3rd position of plumbagin. This molecule loses its thiol reactivity completely and its redox cycling ability to a lesser extent. Mechanistically, non-competitive, reversible binding of the inhibitor to the lysine acetyltransferase (KAT) domain of p300 is largely responsible for the acetyltransferase inhibition. Remarkably, the modified inhibitor PTK1 was a nearly non-toxic inhibitor of p300. The present report elucidates the mechanism of acetyltransferase activity inhibition by 1,4-naphthoquinones, which involves redox cycling and nucleophilic adduct formation, and it suggests possible routes of synthesis of the non-toxic inhibitor.     

Potential risk of biologic pollution associated to the introduction of <i>Pinus radiata</i> in grassland areas

Afforestation is a recommended practice to mitigate global warming. However, their implementation may generate undesirable impacts, mostly if exotic species are used. Plantations of Pinus radiata D Don in Ventania (Bs. As., Argentina) soils showed notorious increments of extractable P (Pe), which could affect the dynamic of this element as well as the degree of phosphorus saturation (GSP_Bray). The objectives of this study were: i) to quantify the GSP_Bray in Mollisols afforested with P. radiata comparing the results with those coming from adjacent, natural grassland areas (base line); ii) to evaluate the potential environmental risk induced by afforestation through the identification of a change point (PC) in the GSP_Bray indicative of a phosphate leaching increment. Treatments included mature stands of P. radiata (TB) and adjacent areas with natural grassland vegetation (TP). Samples were taken at 0-15; 15-30 and 30-45 cm soil depth, and texture, pH, total organic carbon (COT), Pe, soluble reactive phosphorus (PSR), phosphorus sorption index (ISP) and GSP_Bray were determined. The results showed a significant acidification in TB and an increase in the COT stock, indicating an additional atmospheric CO₂ sequestration by the trees. The Pe and PSR values were notoriously higher in TB, and they were reflected in a significant increment in the GSP_Bray with respect to TP. The detection of a significant PC in the GSP_Bray-PSR regression indicates higher chances of phosphate leaching in the forest stands, which could reach water courses, lakes and artificial reservoirs promoting their eutrophication. Because of the potential environmental pollution risk of biologic origin derived from the afforestation with P. radiata in Mollisols areas, their inclusion in clean development practices must be reconsidered.     

Populations Microbiennes Des Bois

Resumé Les auteurs ont étudié la composition chimique (N, substances humiques, lignine) de copeaux de bois blanc exposés à l'air libre depuis 2 à 10 ans, ainsi que leur type de peuplement fongique. En l'absence de lignivore, le pH, les taux de lignine et d'N s'élèvent et il se forme des quantités modérées d'humus à forte capacité d'échange. Dans le cas contraire, on voit apparaître, en abondance, des substances humiques peu condensées et la matière organique subit une évolution rappelant celle du mor.Avec la collaboration technique de Melle. M. Clet.Kononova, dans sa monographie⁵, signale de Troussov (1916) une étude que nous n'avons pas eu en mains.     

Studies of esterase 6 in Drosophila melanogaster. XVIII. Biochemical differences between the slow and fast allozymes

Most natural populations of Drosophila melanogaster are polymorphic for two major electrophoretic variants at the esterase-6 locus. The frequency of the EST 6F allozyme is greatest in populations in warmer latitudes, whereas the EST 6S allozyme is predominant in colder latitudes. Latitudinal clines in electromorph frequencies are found on three continents. Purified preparations of the allozymes have been characterized for their pH optimum, substrate specificity, organophosphate inhibition, alcohol activation, thermal stability, and kinetic parameters. These and previous analyses of the EST 6 allozymes reveal that the two variants have differences in their physical and kinetic properties that may provide a basis for the selective maintenance of the polymorphisms and an explanation of the clinal variation observed in natural populations.      

Application of a Newly Identified and Characterized 18-O-Acyltransferase in Chemoenzymatic Synthesis of Selected Natural and Nonnatural Bioactive Derivatives of Phoslactomycins

Phoslactomycins (PLMs) and related leustroducsins (LSNs) have been isolated from a variety of bacteria based on antifungal, anticancer, and other biological assays. Streptomyces sp. strain HK 803 produces five PLM analogs (PLM A and PLMs C to F) in which the C-18 hydroxyl substituent is esterified with a range of branched, short-alkyl-chain carboxylic acids. The proposed pathway intermediate, PLM G, in which the hydroxyl residue is not esterified has not been observed at any significant level in fermentation, and the only route to this potentially useful intermediate has been an enzymatic deacylation of other PLMs and LSNs. We report that deletion of plmS₃ from the PLM biosynthetic cluster gives rise to a mutant which accumulates the PLM G intermediate. The 921-bp plmS₃ open reading frame was cloned and expressed as an N-terminally polyhistidine-tagged protein in Escherichia coli and shown to be an 18-O acyltransferase, catalyzing conversion of PLM G to PLM A, PLM C, and PLM E using isobutyryl coenzyme A (CoA), 3-methylbutyryl-CoA, and cyclohexylcarbonyl-CoA, respectively. The efficiency of this process (k_cat of 28 ± 3 min⁻¹ and K_m of 88 ± 16 μM) represents a one-step chemoenzymatic alternative to a multistep synthetic process for selective chemical esterification of the C-18 hydroxy residue of PLM G. PlmS₃ was shown to catalyze esterification of PLM G with CoA and N-acetylcysteamine thioesters of various saturated, unsaturated, and aromatic carboxylic acids and thus also to provide an efficient chemoenzymatic route to new PLM analogs.Attachment of either short (C₂ to C₆) or medium (C₈ to C₁₂) acyl chains to both amine and alcohol moieties on polyketide and polypeptide natural products can represent a key step in generating the final biologically active molecule. This step is often, but not always, one of the later biosynthetic steps and is catalyzed by an acyltransferase. The corresponding gene is typically associated with the polyketide or polypeptide biosynthetic gene cluster. Despite the importance of this step, a relatively small number of these acyltransferases from actinomycetes have been identified, and very few have been fully characterized (2, 10, 15, 18).Studies of biosynthetic processes where there is an acylation of a polyketide chain, have indicated that the enzymes have various degrees of promiscuity for the carboxylic acid substrates. For instance, genetic evidence has shown that mdmB and acyA encode 3-O-acyltransferases which transfer either acetyl or propionyl groups to position 3 in 16-membered macrolides such as midecamycin, spiramycin, carbomycin, and tylosin (3, 10). The asm19 gene product has been identified as the 3-O-acyltransferase which catalyzes the attachment of the biologically essential acyl group in the macrocyclic ansamitocins (18). The asm19 mutant accumulates an N-demethyl-4,5-desepoxymytansinol, indicating that acylation of the macrocycle precedes N methylation and epoxidation. Escherichia coli cell extracts containing a recombinant Asm19 protein have been shown to catalyze acylation of this mytansinol intermediate using a range of short straight- and branched-chain acyl coenzyme A (CoA) thioesters (C₂ to C₅). Finally, LovD, which catalyzes the acylation of the C-8 hydroxyl group of monacolin J to yield the natural product lovastatin, a pharmaceutically important fungal polyketide product produced by Aspergillus nidulans, has been characterized (30). This enzyme is able to utilize a wide range of different acyl donors activated as CoA, N-acetylcysteamine (NAC), or methyl thioglycolate esters and thus offers an economically attractive route for generating novel lovasotatin analogs for treatment of hypercholesterolemia.One or multiple O-acyltransferases have been implied to be involved in the post-polyketide synthase tailoring steps leading to a series of natural products known as the phoslactomycins (PLMs) (Fig. ​(Fig.1)1) (6). These compounds (also known as leustroducsins [LSNs], phospholines, and phosphazomycins) have been isolated from various actinomycetes, and their structures are all identical with the exception of the acyl substituent at C-18 (4, 5, 12, 13, 20, 27, 28). Streptomyces sp. strain HK 803 produces at least five such acylated analogs (PLM A and PLMs C to F) (Fig. ​(Fig.1)1) as well as PLM B, in which the C-18 hydroxyl substituent is absent (4, 5, 27).Open in a separate windowFIG. 1.Proposed biosynthetic relationship between PLM products made by Streptomyces sp. strain HK 803. A cytochrome P450 monooxygenase (PlmS₂) catalyzes C-18 hydroxylation of PLM B to generate PLM G, which is subsequently 18-O acylated by PlmS₃.These natural products have been isolated based on their potent activity (as low as 0.008 μg/ml) against some phytopathogenic fungi (27, 28). The compounds also have relatively weak antitumor activity (50% inhibitory concentration of 2 to 3 μg/ml against L1210, P38,8, and El-4 cell lines) (19) which may arise from their activity as selective inhibitors of protein phosphatase 2A. (26). These natural products also show induction of a colony-stimulating factor (12) via NF-κB activation and thrombopoiesis (14). This array of promising biological activities has stimulated research into the field of PLMs for treatments of various diseases. Low yields and the presence of multiple acylated products from fermentations have provided a barrier to this work, and circuitous routes to obtaining individual compounds have been described. For instance, there have been no reports of any actinomycetes which produce PLM G (LSN H) (Fig. ​(Fig.1),1), in which the C-18 hydroxyl residue is present but not acylated, and this compound has been reported to be obtained only by cleavage of the acyl groups of a mixture of other PLMs and LSNs using porcine liver esterase (24). A multistep synthetic route to selectively acylate PLM G with 6-methyloctanoic acid (producing LSN B) has also been described (17).Recently the entire 75-kbp Plm biosynthetic gene cluster has been cloned, sequenced, and analyzed (21) and has provided an opportunity to study the enzymatic hydroxylation and acylation processes which give rise to the range of PLM products. Deletion of the plmS₂ open reading frame (ORF), showing high sequence similarity to bacterial cytochrome P450 monooxygenases, has resulted in an NP1 mutant producing only PLM B (Fig. ​(Fig.1)1) (21). The plmS₂ ORF has been expressed as an N-terminally polyhistidine-tagged protein in Streptomyces coelicolor, and the purified protein has been shown to catalyze conversion of PLM B to PLM G (7). This work in conjunction with other studies (1, 23) has led to a proposal that the final two biosynthetic steps involve hydroxylation of PLM B (to give PLM G) and subsequent acylation with a broad range of acyl-CoA substrates (to give PLM A and PLMs C to F). The acylation step is required for potent antifungal activity of the PLMs. Initial analysis of the PLM biosynthetic gene cluster (21) did not reveal a candidate gene or genes whose products might be responsible for this acylation.Here we identify the plmS₃ gene product as the singular acyltransferase in Streptomyces sp. strain HK 803 responsible for C-18 acylation of PLM G. Generation of a plmS₃ deletion mutant results in selective production of PLM G, supporting the proposed role of this gene product and providing the first direct fermentation method to access this intermediate. The 921-bp plmS₃ ORF was cloned and expressed as an N-terminally polyhistidine-tagged protein in E. coli, and the recombinant purified protein was shown to catalyze acylation of PLM G with isobutyryl-CoA, 3-methylbutyryl-CoA, and cyclohexylcarbonyl-CoA to give PLM A, PLM C, and PLM E, respectively. This efficient one-step enzymatic process offers an attractive alternative to the multistep synthetic process for selective acylation of PLM G. PlmS₃ was also shown to catalyze esterification of PLM G with CoA and NAC thioesters with a remarkably wide range of various saturated, unsaturated, and aromatic carboxylic acids and thus provides an efficient chemoenzymatic route to new PLM analogs.     

Effects of cyanobacterial extracellular products and gibberellic acid on salinity tolerance in Oryza sativa L

The XI International Rotifer Symposium was held during 11–18 March, 2006 at the National Autonomous University of Mexico Campus Iztacala located at the North Mexico City (Mexico). These triennial international meetings, first organized in Austria by Late Ruttner-Kolisko in September 1976, are gradually becoming the focal point of discussion and collaboration from rotifer workers across the world. The present XI symposium was attended by 125 participants from 20 nations. During this meeting, different themes of rotifer research from morphology to molecular biology were considered. In addition, there were four invited lectures and four workshops covering different themes of the symposium. During the last 30 years, rotifer research has witnessed gradual shift from the conventional morphological taxonomy to molecular and evolutionary systematics. While the basic rotifer ecological studies continue today, applied areas such as ecotoxicology and aquaculture have taken key roles in the recent meetings. The international rotifer meetings provide ample opportunities not only for exchange of ideas and recent research, but also for material and in establishing inter-personal relationships. Over the last 30 years, the number of participants attending the rotifer meetings has increased.