Evidence of a Ca(2+)-(center dot)NO-cGMP signaling pathway controlling zoospore biogenesis in the aquatic fungus Blastocladiella emersonii

The sporulation stage of the aquatic fungus Blastocladiella emersonii culminates with the formation and release to the medium of a number of zoospores, which are motile cells responsible for the dispersal of the fungus. The presence in the sporulation solution of 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a potent and selective inhibitor of nitric oxide-sensitive guanylyl cyclases, completely prevented biogenesis of the zoospores. In addition, this compound was able to significantly reduce cGMP levels, which increase drastically during late sporulation, suggesting the existence of a nitric oxide-dependent mechanism for cGMP synthesis. Furthermore, increased levels of nitric oxide-derived products were detected during sporulation by fluorescence assays using DAF-2 DA, whose signal was drastically reduced in the presence of the nitric oxide synthase inhibitor N omega-Nitro-L-arginine methyl ester (L-NAME). These results were confirmed by quantitative chemiluminescent determination of the intracellular levels of nitric oxide-derived products. A putative nitric oxide synthase (NOS) activity was detected throughout sporulation, and this enzyme activity decreased significantly when L-NAME and 1-[2-(Trifluoromethyl)phenyl]imidazole (TRIM) were added to the assays. NOS assays carried out in the presence of EGTA showed decreased enzyme activity, suggesting the involvement of calcium ions in enzyme activation. Additionally, expressed sequence tags (ESTs) encoding putative guanylyl cyclases and a cGMP-phosphodiesterase were found in B. emersonii EST database (http://blasto.iq.usp.br), and the mRNA levels of the corresponding genes were observed to increase during sporulation. Altogether, data presented here revealed the presence and expression of guanylyl cyclase and cGMP phosphodiesterase genes in B. emersonii and provided evidence of a Ca(2+)-(center dot)NO-cGMP signaling pathway playing a role in zoospore biogenesis. (C) 2009 Elsevier Inc. All rights reserved.

Acanthoscurrin Fragment 101-132: Total Synthesis at 60 degrees C of a Novel Difficult Sequence

Glycine-rich proteins (GRP), serve a variety of biological functions. Acanthoscurrin is an antimicrobial GRP isolated front hemocytes-of the Brazilian spider Acanthoscurria gomesiana. Aiming to contribute to the knowledge of the secondary structure and stepwise solid-phase synthesis of GRPs` glycine-rich domains, we attempted to prepare G(101)GGLGGGRGGGYG(113) GGGGYGGGYG(123)GGy(126)GGGKYK(132)-NH(2), acanthoscurrin C-terminal amidated fragment. Although a theoretical prediction did not indicate high aggregation potential for this peptide, repetitive incomplete aminoacylations were observed after incorporating Tyr(126) to the growing peptide-MBHA resin (Boc chemistry) at 60 degrees C. The problem was not solved by varying the coupling reagents or solvents, adding chaotropic salts to the reaction media or changing the resin/chemistry (Rink amide resin/Fmoc chemistry). Some improvement was mode when CLEAR amide resin (Fmoc chemistry) was 32 used, as it allowed for obtaining fragment (G(113)-K(132) NIR-FT-Raman spectra collected for samples of the growing peptide-MBHA, -Rink amide resin and -CLEAR amide resin revealed the presence of beta-sheet structures. Only the combination of CLEAR-amide resin, 60 degrees C, Fmoc-(Fmoc-Hmb)Gly-OH and LiCl (the last two used alternately) was able to inhibit the phenomenon, as proven by NIR-FT-Raman analysis of the growing peptide-resin, allowing the total synthesis of desired 132 fragment Gly(101)-K(132). In summary, this work describes a new difficult sequence, contributes to understanding stepwise solid-phase synthesis of this type of peptide and shows that, at least while protected and linked to a resin, this GRPs glycine-rich motif presents all early tendency to assume beta-sheet structures. (c) 2008 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 92: 65-75, 2009.

The mitochondrial view of Blastocladiella emersonii

The mitochondrial genome of the chytrid Blastocladiella emersonii was sequenced and annotated, revealing the complete set of oxidative phosphorylation genes and tRNAs/rRNAs necessary for the translation process. Phylogenetic reconstructions reinforce the proposal of the new phylum Blastocladiomycota. Evidences of gene duplication due to inserted elements suggest shared susceptibility to gene invasion/exchange between chytrids and zygomycetes. The gene content of B. emersonii is very similar to Allomyces macrogynus but the content of intronic and changeable elements is much lower suggesting a stronger resistance to this kind of exchange. in addition, a total of 401 potential nuclear transcripts encoding mitochondrial proteins were obtained after B. emersonii EST database scanning using Saccharomyces cerevisiae, Homo sapiens and Arabidopsis thaliana data as probes and TargetP tool to find N-terminal mitochondrial signal in translated sequences. (c) 2008 Elsevier B.V. All rights reserved.

Lithium butylchalcogenolate induced Michael-aldol tandem sequence: easy and rapid access to highly functionalized organochalcogenides and unsaturated compounds

Lithium ""butylchalcogenolates are generated in situ by reacting the elements (S, Se, and Te) with (n)butyl-lithium at 0 degrees C. Reaction of the lithium alkylchalcogenolates with activated alkenes and aldehydes gives the corresponding aldol adducts. The selenium-containing products give Morita-Baylis-Hillman adducts after the oxidation/elimination of the selenoxide. The whole sequence can be performed in a one-pot procedure. (C) 2009 Elsevier Ltd. All rights reserved.

alpha-Hydroxynitrile lyase protein from Xylella fastidiosa: Cloning, expression, and characterization

Xylella fastidiosa is a xylem-restricted plant pathogen that causes a range of diseases in several and important crops. Through comparative genomic sequence analysis many genes were identified and, among them, several potentially involved in plant-pathogen interaction. The experimental determination of the primary sequence of some markedly expressed proteins for X fastidiosa and the comparison with the nucleic acids sequence of genome identified one of them as being SCJ21.16 (XFa0032) gene product. The comparative analysis of this protein against SWISSPROT database, in special, resulted in similarity with a-hydroxynitrile lyase enzyme (HNL) from Arabidopsis thaliana, causing interest for being one of the most abundant proteins both in the whole cell extract as well as in the extracellular protein fraction. It is known that HNL enzyme are involved in a process termed ""cyanogenesis"", which catalyzes the dissociation of alpha-hydroxinitrile into carbonyle and HCN when plant tissue is damaged. Although the complete genome sequences of X.fastidiosa are available and the cyanogenesis process is well known, the biological role of this protein in this organism is not yet functionally characterized. In this study we presented the cloning, expression, characterization of recombinant HNL from X fastidiosa, and its probable function in the cellular metabolism. The successful cloning and heterologous expression in Escherichia coli resulted in a satisfactory amount of the recombinant HNL expressed in a soluble, and active form giving convenient access to pure enzyme for biochemical and structural studies. Finally, our results confirmed that the product of the gene XFa0032 can be positively assigned as FAD-independent HNLs. (C) 2009 Elsevier Ltd. All rights reserved.

Fast Acquisition of (13)C NMR Spectra using the Steady-state Free Precession Sequence

In this paper, we show that the steady-state free precession sequence can be used to acquire (13)C high-resolution nuclear magnetic resonance spectra and applied to qualitative analysis. The analysis of brucine sample using this sequence with 60 degrees flip angle and time interval between pulses equal to 300 ms (acquisition time, 299.7 ms; recycle delay, 300 ms) resulted in spectrum with twofold enhancement in signal-to-noise ratio, when compared to standard (13)C sequence. This gain was better when a much shorter time interval between pulses (100 ms) was applied. The result obtained was more than fivefold enhancement in signal-to-noise ratio, equivalent to more than 20-fold reduction in total data recording time. However, this short time interval between pulses produces a spectrum with severe phase and truncation anomalies. We demonstrated that these anomalies can be minimized by applying an appropriate apodization function and plotting the spectrum in the magnitude mode.

From the road network database to a graph for localization purposes

The problems of finding best facility locations require complete and accurate road network with the corresponding population data in a specific area. However the data obtained in road network databases usually do not fit in this usage. In this paper we propose our procedure of converting the road network database to a road graph which could be used in localization problems. The road network data come from the National road data base in Sweden. The graph derived is cleaned, and reduced to a suitable level for localization problems. The population points are also processed in ordered to match with that graph. The reduction of the graph is done maintaining most of the accuracy for distance measures in the network.

All maximal-pairs in step-leap representation of melodic sequence

This paper proposes an efficient pattern extraction algorithm that can be applied on melodic sequences that are represented as strings of abstract intervallic symbols; the melodic representation introduces special “binary don’t care” symbols for intervals that may belong to two partially overlapping intervallic categories. As a special case the well established “step–leap” representation is examined. In the step–leap representation, each melodic diatonic interval is classified as a step (±s), a leap (±l) or a unison (u). Binary don’t care symbols are used to represent the possible overlapping between the various abstract categories e.g. *=s, *=l and #=-s, #=-l. We propose an O(n+d(n-d)+z)-time algorithm for computing all maximal-pairs in a given sequence x=x[1..n], where x contains d occurrences of binary don’t cares and z is the number of reported maximal-pairs.

All maximal-pairs in step-leap representation of melodic sequence

This paper proposes an efficient pattern extraction algorithm that can be applied on melodic sequences that are represented as strings of abstract intervallic symbols; the melodic representation introduces special “binary don’t care” symbols for intervals that may belong to two partially overlapping intervallic categories. As a special case the well established “step–leap” representation is examined. In the step–leap representation, each melodic diatonic interval is classified as a step (±s), a leap (±l) or a unison (u). Binary don’t care symbols are used to represent the possible overlapping between the various abstract categories e.g. *=s, *=l and #=-s, #=-l. We propose an O(n+d(n-d)+z)-time algorithm for computing all maximal-pairs in a given sequence x=x[1..n], where x contains d occurrences of binary don’t cares and z is the number of reported maximal-pairs.