Antiviral and antiparasite properties of an L-amino acid oxidase from the Snake Bothrops jararaca: Cloning and identification of a complete cDNA sequence (Retracted article. See vol. 80, pg. 288, 2010)

L-Amino acid oxidases (LAAOs, EC 1.4.3.2) are flavoenzymes that catalyze the stereospecific oxidative deamination of an L-amino acid substrate to the corresponding a-ketoacid with hydrogen peroxide and ammonia production. The present work describes the first report on the antiviral (Dengue virus) and antiprotozoal (trypanocidal and leishmanicide) activities of a Bothrops jararaca L-amino acid oxidase (BjarLAAO-I) and identify its cDNA sequence. Antiparasite effects were inhibited by catalase, suggesting that they are mediated by H(2)O(2) production. Cells infected with DENV-3 virus previously treated with BjarLAAO-I, showed a decrease in viral titer (13-83-fold) when compared with cells infected with untreated viruses. Untreated and treated promastigotes (T. cruzi and L. amazonensis) were observed by transmission electron microscopy with different degrees of damage. Its complete cDNA sequence, with 1452 bp, encoded an open reading frame of 484 amino acid residues with a theoretical molecular weight and pl of 54,771.8 and 5.7, respectively. The cDNA-deduced amino acid sequence of BjarLAAO shows high identity to LAAOs from other snake venoms. Further investigations will be focused on the related molecular and functional correlation of these enzymes. Such a study should provide valuable information for the therapeutic development of new generations of microbicidal drugs. (C) 2008 Elsevier Inc. All rights reserved.

Molecular cloning and biochemical characterization of a myotoxin inhibitor from Bothrops alternatus snake plasma

Phospholipases A(2) (PLA(2)s) are important components of Bothrops snake venoms, that can induce several effects on envenomations such as myotoxicity, inhibition or induction of platelet aggregation and edema. It is known that venomous and non-venomous snakes present PLA(2) inhibitory proteins (PLIs) in their blood plasma. An inhibitory protein that neutralizes the enzymatic and toxic activities of several PLA2s from Bothrops venoms was isolated from Bothrops alternatus snake plasma by affinity chromatography using the immobilized myotoxin BthTX-I on CNBr-activated Sepharose. Biochemical characterization of this inhibitory protein, denominated alpha BaltMIP, showed it to be a glycoprotein with Mr of similar to 24,000 for the monomeric subunit. CD spectra of the PLA(2)/inhibitor complexes are considerably different from those corresponding to the individual proteins and data deconvolution suggests that the complexes had a relative gain of helical structure elements in comparison to the individual protomers, which may indicate a more compact structure upon complexation. Theoretical and experimental structural studies performed in order to obtain insights into the structural features of aBaltMIP indicated that this molecule may potentially trimerize in solution, thus strengthening the hypothesis previously raised by other authors about snake PLIs oligomerization. (C) 2010 Elsevier Masson SAS. All rights reserved.

Cloning and characterization of a gene encoding the major surface protein of the bacterial endosymbiont Wolbachia pipientis

The maternally inherited intracellular symbiont Wolbachia pipientis is well known for inducing a variety of reproductive abnormalities in the diverse arthropod hosts it infects. It has been implicated in causing cytoplasmic incompatibility, parthenogenesis, and the feminization of genetic males in different hosts. The molecular mechanisms by which this fastidious intracellular bacterium causes these reproductive and developmental abnormalities have not yet been determined. In this paper, we report on (i) the purification of one of the most abundantly expressed Wolbachia proteins from infected Drosophila eggs and (ii) the subsequent cloning and characterization of the gene (wsp) that encodes it. The functionality of the wsp promoter region was also successfully tested in Escherichia coli. Comparison of sequences of this gene from different strains of Wolbachia revealed a high level of variability. This sequence variation correlated with the ability of certain Wolbachia strains to induce or rescue the cytoplasmic incompatibility phenotype in infected insects. As such, this gene will be a very useful tool for Wolbachia strain typing and phylogenetic analysis, as well as understanding the molecular basis of the interaction of Wolbachia with its host.

Cyclic peroxides and related norterpenes from a southern Australian marine sponge, Mycale sp.

Investigation of a southern Australian marine sponge, Mycale sp., resulted in isolation of the known norsesterterpenes 1-3 as well as two new isomeric norsesterterpenes, mycaperoxide C methyl ester (4) and mycaperoxide D methyl ester (5), and six new norterpenes (6-11).

Molecular cloning reveals that the p160 myb-binding protein is a novel, predominantly nucleolar protein which may play a role in transactivation by Myb

We have previously detected two related murine nuclear proteins, p160 and p67, that can bind to the leucine zipper motif within the negative regulatory domain of the Myb transcription factor. We now describe the molecular cloning of cDNA corresponding to murine p160. The P160 gene is located on mouse chromosome 11, and related sequences are found on chromosomes 1 and 12. The predicted p160 protein is novel, and in agreement with previous studies, we find that the corresponding 4.5-kb mRNA is ubiquitously expressed. We showed that p67 is an N-terminal fragment of p160 which is generated by proteolytic cleavage in certain cell types. The protein encoded by the cloned p160 cDNA and an engineered protein (p67*) comprising the amino-terminal region of p160 exhibit binding specificities for the Myb and Jun leucine zipper regions identical to those of endogenous p160 and p67, respectively. This implies that the Myb-binding site of p160 lies within the N-terminal 580 residues and that the Jun-binding site is C-terminal to this position. Moreover, we show that p67* but not p160 can inhibit transactivation by Myb. Unexpectedly, immunofluorescence studies show that p160 is localized predominantly in the nucleolus. The implications of these results for possible functions of p160 are discussed.

Sigmosceptrins A-C: New norditerpenes from a southern Australian marine sponge, Sigmosceptrella sp.

A Sigmosceptrella sp. of sponge collected during trawling operations in the Great Australian Eight, Australia, has yielded a series of new norterpenes. These include a new bisnorditerpene, sigmosceptrin-A (5); two new norditerpenes, sigmosceptrin-B (14) and sigmosceptrin-C (15), isolated as their methyl esters (6) and (7) respectively; and an ethylated artefact, sigmosceptrin-B ethyl ester (8). Complete stereostructures were assigned to the sigmosceptrins by spectroscopic analysis, chemical degradation, derivatization, and by a single-crystal X-ray structural analysis. A biosynthetic pathway is proposed that requires a common biosynthetic precursor to both the sigmosceptrins and norterpene cyclic peroxides.

Molecular cloning and sequence of the ovine gastrin gene

A clone encoding ovine preprogastrin was isolated from a sheep genomic library. The deduced 104 amino acid sequence of ovine preprogastrin was 92% and 68% identical to the sequences of bovine and human preprogastrin, respectively. While the similarity was greatest in the gastrin-17 sequence, an unexpected similarity was also observed in the N-terminus of mature progastrin.

cis-3-hydroxy-N-methyl-L-proline: a new amino acid from a southern Australian marine sponge, Dendrilla sp.

A specimen of the sponge Dendrilla sp. collected during commercial trawling operations in the Great Australian Eight, Australia, analyses for a very high natural abundance of the new amino acid cis-3-hydroxy-N-methyl-L-proline (1). The complete stereostructure for (1) was determined by spectroscopic analysis and chemical derivatization.

Dragmacidins: New protein phosphatase inhibitors from a southern Australian deep-water marine sponge, Spongosorites sp.

A Spongosorites sp. collected during trawling operations off the southern coast of Australia returned the new alkaloid dragmacidin E (3), the structure of which was secured by detailed spectroscopic analysis. Dragmacidin E (3), and its co-metabolite dragmacidin D (1) have been identified as potent inhibitors of serine-threonine protein phosphatases.

Cellular origin of chlorinated diketopiperazines in the dictyoceratid sponge Dysidea herbacea (Keller)

The tropical marine sponge Dysidea herbacea (Keller) contains the filamentous unicellular cyanobacterium Oscillatoria spongeliae (Schulze) Hauck as an endosymbiont, plus numerous bacteria, both intracellular and extracellular. Archaeocytes and choanocytes are the major sponge cell types present. Density gradient centrifugation of glutaraldehyde-fixed cells with Percoll as the support medium has been used to separate the cyanobacterial symbiont from the sponge cells on the basis of their differing densities. The protocol also has the advantage of separating broken from intact cells of O. spongeliae. The lighter cell preparations contain archaeocytes and choanocytes together with damaged cyanobacterial cells, whereas heavier cell preparations contain intact cyanobacterial cells, with less than 1% contamination by sponge cells. Gas chromatography/mass spectrometry analysis has revealed that the terpene spirodysin is concentrated in preparations containing archaeocytes and choanocytes, whereas nuclear magnetic resonance analysis of the symbiont cell preparations has shown that they usually contain the chlorinated diketopiperazines, dihydrodysamide C and didechlorodihydrodysamide C, which are the characteristic metabolites of the sponge/symbiont association. However, one symbiont preparation, partitioned by a second Percoll gradient, has been found to be devoid of chlorinated diketopiperazines. The capability to synthesize secondary metabolites may depend on the physiological state of the symbiont; alternatively, there may be two closely related cyanobacterial strains within the sponge tissue.

Callyspongynes A and B: New polyacetylenic lipids from a southern Australian marine sponge, Callyspongia sp.

A Callyspongia sp. collected by SCUBA off Barwon Heads, Australia, has afforded two new polyacetylenic lipids, callyspongynes A and B, the structures of which were assigned by spectroscopic analysis and chemical derivatization.

The haliclonacyclamines, cytotoxic tertiary alkaloids from the tropical marine sponge Haliclona sp

2D-NMR spectroscopic data is reported for the haliclonacyclamines A - D (1)-(4) and for two bismethiodide adducts (5) and (6). The structures of two new alkaloids, haliclonacyclamines C (3) and D (4), which are the 15,16-dihydro analogues of the haliclonacyclamines A (1) and B (2) are described. Revised assignments deduced by 2D-INADEQUATE spectroscopy are presented for (1) and (2). The alkene substituent in the C,, spacer group of (2) and (4) is positioned between C27-C28 by NMR, and confirmed by x-ray structural analysis for (2). Metabolite (3) has a C25-C26 double bond. (C) 1998 Elsevier Science Ltd. All rights reserved.

Trunculins G-I: New norsesterterpene cyclic peroxides from a southern Australian marine sponge, Latrunculia sp.

A Latrunculia sp, collected off Port Phillip Bay, Victoria, returned three new norsesterterpene cyclic peroxides. Trunculins G (9), H (10) and I (11) were isolated as their methyl esters (12), (13) and (14) respectively. Gross structures for these new trunculins were assigned on the basis of spectroscopic analysis, while the absolute stereochemistry about the cyclic peroxide terminus was established by application of the Horeau and Mosher procedures.

A sponge/dinoflagellate association in the haplosclerid sponge Haliclona sp.: cellular origin of cytotoxic alkaloids by Percoll density gradient fractionation

Light-microscopic and electron-microscopic studies of the tropical marine sponge Haliclona sp. (Or der: Haplosclerida Family: Haliclonidae) from Heron Island, Great Barrier Reef, have revealed that this sponge is characterized by the presence of dinoflagellates and by nematocysts. The dinoflagellates are 7-10 mu m in size, intracellular, and contain a pyrenoid with a single stalk, whereas the single chloroplast is branched, curved, and lacks grana. Mitochondria are present, and the nucleus is oval and has distinct chromosomal structure. The dinoflagellates are morphologically similar to Symbiodinium microadriaticum, the common intracellular symbiont of corals, although more detailed biochemical and molecular studies are required to provide a precise taxonomic assignment. The major sponge cell types found in Haliclona sp, are spongocytes, choanocytes, and archaeocytes; groups of dinoflagellates are enclosed within large vacuoles in the archaeocytes. The occurrence of dinoflagellates in marine sponges has previously been thought to be restricted to a small group of sponges including the excavating hadromerid sponges; the dinoflagellates in these sponges are usually referred to as symbionts. The role of the dinoflagellates present in Haliclona sp. as a genuine symbiotic partner requires experimental investigation. The sponge grows on coral substrates, from which it may acquire the nematocysts, and shows features, such as mucus production, which are typical of some excavating sponges. The cytotoxic alkaloids, haliclonacyclamines A and B, associated with Haliclona sp. are shown by Percoll density gradient fractionation to be localized within the sponge cells rather than the dinoflagellates. The ability to synthesize bioactive compounds such as the haliclonacyclamines may help Haliclona sp. to preserve its remarkable ecological niche.