Myotoxic phospholipases A(2) isolated from Bothrops brazili snake venom and synthetic peptides derived from their C-terminal region: Cytotoxic effect on microorganism and tumor cells

This paper reports the purification and biochemical/pharmacological characterization of two myotoxic phospholipases A(2) (PLA(2)S) from Bothrops brazili venom, a native snake from Brazil. Both myotoxins (MTX-I and II) were purified by a single chromatographic step on a CM-Sepharose ion-exchange column up to a high purity level, showing M-r similar to 14,000 for the monomer and 28,000 Da for the dimer. The N-terminal and internal peptide amino acid sequences showed similarity with other myotoxic PLA2S from snake venoms, MTX-I belonging to Asp49 PLA(2) class, enzymatically active, and MTX-II to Lys49 PLA(2)S, catalytically inactive. Treatment of MTX-I with BPB and EDTA reduced drastically its PLA(2) and anticoagulant activities, corroborating the importance of residue His48 and Ca2+ ions for the enzymatic catalysis. Both PLA(2)S induced myotoxic activity and dose-time dependent edema similar to other isolated snake venom toxins from Bothrops and Crotalus genus. The results also demonstrated that MTXs and cationic synthetic peptides derived from their 115-129 C-terminal region displayed cytotoxic activity on human T-cell leukemia (JURKAT) lines and microbicidal effects against Escherichia coli, Candida albicans and Leishmania sp. Thus, these PLA(2) proteins and C-terminal synthetic peptides present multifunctional properties that might be of interest in the development of therapeutic strategies against parasites, bacteria and cancer. (C) 2008 Elsevier Inc. All rights reserved.

Antitumor effects of snake venom chemically modified Lys49 phospholipase A(2)-like BthTX-I and a synthetic peptide derived from its C-terminal region

The present work evaluates both in vitro and in vivo antitumor activity of BPB-modified BthTX-I and its cationic synthetic peptide derived from the 115-129 C-terminal region. BPB-BthTX-1 presented cytotoxicity of 10-40% on different tumor cell lines, which were also susceptible to the lytic action of the synthetic peptide. Injection of the modified protein or the peptide in mice, 5 days after transplantation of S 180 tumor cells, reduced 30 and 36% of the tumor size on day 14th and 76 and 79% on day 60th, respectively, when compared to the untreated control group. Thus, these antitumor properties might be of interest in the development of therapeutic strategies against cancer. (C) 2009 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved.

Phosphorylation of the C-terminal sites of human p53 reduces non-sequence-specific DNA binding as modeled with synthetic peptides

Phosphorylation of the tumor suppressor p53 is generally thought to modify the properties of the protein in four of its five independent domains. We used synthetic peptides to directly study the effects of phosphorylation on the non-sequence-specific DNA binding and conformation of the C-terminal, basic domain. The peptides corresponded to amino acids 361-393 and were either nonphosphorylated or phosphorylated at the protein kinase C (PKC) site, Ser378, or the casein kinase II (CKII) site, Ser392, or bis-phosphorylated on both the PKC and the CKII sites. A fluorescence polarization analysis revealed that either the recombinant p53 protein or the synthetic peptides bound to two unrelated target DNA fragments. Phosphorylation of the peptide at the PKC or the CKII sites clearly decreased DNA binding, and addition of a second phosphate group almost completely abolished binding. Circular dichroism spectroscopy showed that the peptides assumed identical unordered structures in aqueous solutions. The unmodified peptide, unlike the Ser378 phosphorylated peptide, changed conformation in the presence of DNA. The inherent ability of the peptides to form an alpha-helix could be detected when circular dichroism and nuclear magnetic resonance spectra were: taken in trifluoroethanol-water mixtures. A single or double phosphorylation destabilized the helix around the phosphorylated Ser378 residue but stabilized the helix downstream in the sequence.

Activation of beta(2)-adrenergic receptors hastens relaxation and mediates phosphorylation of phospholamban, troponin I, and C-protein in ventricular myocardium from patients with terminal heart failure

Background-Catecholamines hasten cardiac relaxation through beta-adrenergic receptors, presumably by phosphorylation of several proteins, but it is unknown which receptor subtypes are involved in human ventricle. We assessed the role of beta(1)- and beta(2)-adrenergic receptors in phosphorylating proteins implicated in ventricular relaxation. Methods and Results-Right ventricular trabeculae, obtained from freshly explanted hearts of patients with dilated cardiomyopathy (n=5) or ischemic cardiomyopathy (n=5), were paced at 60 bpm. After measurement of the contractile and relaxant effects of epinephrine (10 mu mol/L) or zinterol (10 mu mol/L), mediated through beta(2)-adrenergic receptors, and of norepinephrine (10 mu mol/L), mediated through beta(1)-adrenergic receptors, tissues were freeze clamped. We assessed phosphorylation of phospholamban, troponin I, and C-protein, as well as specific phosphorylation of phospholamban at serine 16 and threonine 17, Data did not differ between the 2 disease groups and were therefore pooled. Epinephrine, zinterol, and norepinephrine increased contractile force to approximately the same extent, hastened the onset of relaxation by 15+/-3%, 5+/-2%, and 20+/-3%, respectively, and reduced the time to half-relaxation by 26+/-3%, 21+/-3%, and 37+/-3%. These effects of epinephrine, zinterol, and norepinephrine were associated with phosphorylation (pmol phosphate/mg protein) of phospholamban 14+/-3, 12+/-4, and 12+/-3, troponin I 40+/-7, 33+/-7, and 31+/-6; and C-protein 7.2+/-1.9, 9.3 +/- 1.4, and 7.5 +/- 2.0. Phosphorylation of phospholamban occurred at both Ser16 and Thr17 residues through both beta(1)- and beta(2)-adrenergic receptors. Conclusions-Norepinephrine and epinephrine hasten human ventricular relaxation and promote phosphorylation of implicated proteins through both beta(1)- and beta(2)-adrenergic receptors, thereby potentially improving diastolic function.

The dimerization and topological specificity functions of MinE reside in a structurally autonomous C-terminal domain

Correct placement of the division septum in Escherichia coli requires the co-ordinated action of three proteins, MinC, MinD and MinE. MinC and MinD interact to form a non-specific division inhibitor that blocks septation at all potential division sites. MinE is able to antagonize MinCD in a topologically sensitive manner, as it restricts MinCD activity to the unwanted division sites at the cell poles, Here, we show that the topological specificity function of MinE residues in a structurally autonomous, trypsin-resistant domain comprising residues 31-88, Nuclear magnetic resonance (NMR) and circular dichroic spectroscopy indicate that this domain includes both alpha and beta secondary structure, while analytical ultracentrifugation reveals that it also contains a region responsible for MinE homodimerization. While trypsin digestion indicates that the anti-MinCD domain of MinE (residues 1-22) does not form a tightly folded structural domain, NMR analysis of a peptide corresponding to MinE(1-22) indicates that this region forms a nascent helix in which the peptide rapidly interconverts between disordered (random coil) and alpha-helical conformations, This suggests that the N-terminal region of MinE may be poised to adopt an alpha-helical conformation when it interacts with the target of its anti-MinCD activity, presumably MinD.

The solution structure of the N-terminal zinc finger of GATA-1 reveals a specific binding face for the transcriptional co-factor FOG

Zinc fingers (ZnFs) are generally regarded as DNA-binding motifs. However, a number of recent reports have implicated particular ZnFs in the mediation of protein-protein interactions. The N-terminal ZnF of GATA-1 (NF) is one such finger, having been shown to interact with a number of other proteins, including the recently discovered transcriptional co-factor FOG. Here we solve the three-dimensional structure of the NF in solution using multidimensional H-1/N-15 NMR spectroscopy, and we use H-1/N-15 spin relation measurements to investigate its backbone dynamics. The structure consists of two distorted beta-hairpins and a single alpha-helix, and is similar to that of the C-terminal ZnF of chicken GATA-1. Comparisons of the NF structure with those of other C-4-type zinc binding motifs, including hormone receptor and LIM domains, also reveal substantial structural homology. Finally, we use the structure to map the spatial locations of NF residues shown by mutagenesis to be essential for FOG binding, and demonstrate that these residues all lie on a single face of the NE Notably, this face is well removed from the putative DNA-binding face of the NE an observation which is suggestive of simultaneous roles for the NF; that is, stabilisation of GATA-1 DNA complexes and recruitment of FOG to GATA-1-controlled promoter regions.

Dual C3a/C5a receptor agonism by a C5a C-terminal analogue decapeptide

No abstract

Synthesis of (Z)-tributylstannyl enynes: systematic studies of Sonogashira cross-coupling reactions between (E)-1-iodovinyl-1-tributylstannanes and terminal acetylenes using amines or tetrabutylammonium hydroxide (TBAOH) as activator

Sonogashira cross-coupling reactions involving (E)-iodo vinyl stannanes and terminal acetylenes were carried out in the presence of Pd(PPh(3))(4), Cul and several amines, affording (Z)-tributylstannyl enynes in moderate to good yields (62-91%). Utilizing the catalytic system containing Pd(PPh(3))(4) (5%), Cul (10%), and TBAOH (40% in aqueous media) as activator, better yields (72-91%) and lower reaction times were achieved. (C) 2011 Elsevier Ltd. All rights reserved.

Biologically active conformer of the effector region of human C5a and modulatory effects of N-terminal receptor binding determinants on activity

A conformationally biased decapeptide agonist of human C5a (C5a(65-74)Y65,F67,P69,P71,D-Ala73 or YSFKPMPLaR) was used as a functional probe of the C5a receptor (C5aR) in order to understand the conformational features in the C-terminal effector region of C5a that are important for C5aR binding and signal transduction. YSFKPMPLaR was a potent, full agonist of C5a, but at higher concentrations had a superefficacious effect compared to the natural factor. The maximal efficacy of this analogue was 216 +/- 56% that of C5a in stimulating the release of beta-glucuronidase from human neutrophils. C5aR activation and binding curves both occurred in the same concentration range with YSFKPMPLaR, characteristics not observed with natural C5a or more conformationally flexible C-terminal agonists. YSFKPMPLaR was then used as a C-terminal effector template onto which was synthesized various C5aR binding determinants from the N-terminal core domain of the natural factor. In general, the presence of N-terminal binding determinants had little effect on either potency or binding affinity when the C-terminal effector region was presented to the C5aR in this biologically active conformation. However, one peptide, C5a(12-20)-Ahx-YSFKPMPLaR, expressed a 100-fold increase in affinity for the neutrophil C5aR and a 6-fold increase in potency relative to YSFKPMPLaR. These analyses showed that the peptides used in this study have up to 25% of the potency of C5a in human fetal artery and up to 5% of the activity of C5a in the PMN enzyme release assay.

Macromolecular assembly of polycystin-2 intracytosolic C-terminal domain

Mutations in PKD2 are responsible for approximately 15% of the autosomal dominant polycystic kidney disease cases. This gene encodes polycystin-2, a calcium-permeable cation channel whose C-terminal intracytosolic tail (PC2t) plays an important role in its interaction with a number of different proteins. In the present study, we have comprehensively evaluated the macromolecular assembly of PC2t homooligomer using a series of biophysical and biochemical analyses. Our studies, based on a new delimitation of PC2t, have revealed that it is capable of assembling as a homotetramer independently of any other portion of the molecule. Our data support this tetrameric arrangement in the presence and absence of calcium. Molecular dynamics simulations performed with a modified all-atoms structure-based model supported the PC2t tetrameric assembly, as well as how different populations are disposed in solution. The simulations demonstrated, indeed, that the best-scored structures are the ones compatible with a fourfold oligomeric state. These findings clarify the structural properties of PC2t domain and strongly support a homotetramer assembly of PC2.

Cytogenetic molecular delineation of a terminal 18q deletion suggesting neo-telomere formation

Deletion of the long arm of chromosome 18 is one of the most common segmental aneusomies compatible with life and usually involves a deletion of the terminal chromosomal region. However, the mechanisms implicated in the stabilization of terminal deletions are not well understood. In this study, we analyzed a girl with moderate mental retardation who had a cytogenetically visible terminal 18q deletion. In order to characterize the breakpoint in the terminal 18q region, we used fluorescence In situ hybridization (FISH) with bacterial artificial chromosomes (BACs) and pan-telomeric probes and also the array technique based on comparative genomic hybridization (array-CGH). FISH with pan-telomeric probes revealed no signal in the terminal region of the deleted chromosome, indicating the absence of normal telomere repeat (TTAGGG)n sequences in 18q. We suggest that neo-telomere formation by chromosome healing was involved in the repair and stabilization of this terminal deletion. (C) 2010 Elsevier Masson SAS. All rights reserved.

Essential role of TM V and VI for binding the C-terminal sequences of Des-Arg-kinins

In the kallikrein-kinin and renin-angiotensin systems the main receptors, B-1 and B-2 (kinin receptors) and AT(1) and AT(2) (angiotensin receptors) respectively, are seven-transmembrane domain G-protein-coupled receptors. Considering that the B, agonists Des-Arg(9)-BK (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe), Lys-desArg(9)-BK or Des-Arg(10)-KD (Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe) and the AT, agonist (Asp-Arg-Val-Tyr-lle-His-Pro-Phe) have the same two residues at the C-terminal region (i.e. Pro-Phe), we hypothesized that TM V and TM VI of the B-1 receptor could play an essential role in agonist binding and activity, being these regions receptor sites for binding the C-terminal sequences of Des-Arg-kinins similarly to that observed to AT, receptor. To investigate this hypothesis, we replaced Arg(212) for Ala at the top of the TM V and the sequence 274-282 (CPYHFFAFL) in TM VI of the rat kinin B, receptor by the 32 receptor homologous sequence, 289-297 (FPFQISTFL) and subsequently analyzed the consequences of these mutations by competition binding and functional assays. Despite correct expression, observed at the mRNA and protein level by RT-PCR and confocal microscopy, respectively, no agonist binding and function was verified for the mutated receptors. Therefore, our results suggest an important role for Arg(212) in the TM V and a region of TM VI of rat B, receptor in the interaction with the C-terminal residues of Des-Arg-kinins, similar to that observed with AngII. (c) 2007 Elsevier B.V. All rights reserved.

Both alpha 1 and alpha 2-adrenoceptors mediate the cardiovascular responses to noradrenaline microinjected into the bed nucleus of the stria terminal of rats

Background and purpose: We have previously shown that noradrenaline microinjected into the bed nucleus of stria terminalis (BST) elicited pressor and bradycardiac responses in unanaesthetized rats. In the present study, we investigated the subtype of adrenoceptors that mediates the cardiovascular response to noradrenaline microinjection into the BST. Experimental approach: Cardiovascular responses following noradrenaline microinjection into the BST of male Wistar rats were studied before and after BST pretreatment with different doses of the selective alpha(1)-adrenoceptor antagonist WB4101, the alpha(2)-adrenoceptor antagonist RX821002, the combination of WB4101 and RX821002, the non-selective beta-adrenoceptor antagonist propranolol, the selective beta(1)-adrenoceptor antagonist CGP20712 or the selective beta(2)-adrenoceptor antagonist ICI118,551. Key results: Noradrenaline microinjected into the BST of unanaesthetized rats caused pressor and bradycardiac responses. Pretreatment of the BST with different doses of either WB4101 or RX821002 only partially reduced the response to noradrenaline. However, the response to noradrenaline was blocked when WB4101 and RX821002 were combined. Pretreatment with this combination also shifted the resulting dose-effect curve to the left, clearly showing a potentiating effect of this antagonist combination. Pretreatment with different doses of either propranolol or CGP20712 increased the cardiovascular responses to noradrenaline microinjected into the BST. Pretreatment with ICI118,551 did not affect cardiovascular responses to noradrenaline. Conclusion and implications: The present results indicate that alpha(1) and alpha(2)-adrenoceptors mediate the cardiovascular responses to noradrenaline microinjected into the BST. In addition, they point to an inhibitory role played by the activation of local beta(1)-adrenoceptors in the cardiovascular response to noradrenaline microinjected into the BST.

Risk stratification in neonates and infants submitted to cardiac surgery with cardiopulmonary bypass: A multimarker approach combining inflammatory mediators, N-terminal pro-B-type natriuretic peptide and troponin I

Low cardiac output syndrome (LCOS) is a common problem following cardiac surgery with cardiopulmonary bypass (CPB) in neonates and infants, and its early recognition remains a challenging task. We aimed to test whether a multimarker approach combining inflammatory and cardiac markers provides complementary information for prediction of LCOS and death in children submitted to cardiac surgery with CPB. Forty-six children younger than 18 months with congenital heart defects were prospectively enrolled. No intervention was made. Blood samples were collected pre-operatively, during CPB and post-operatively (PO) for measurement of interleukin (IL)-6, IL-8, IL-10, tumor necrosis factor (TNF)-alpha, cardiac troponin I (cTnI) and N-terminal pro-B-type natriuretic peptide (NT-proBNP). Clinical data and outcome variables were recorded. Logistic regression was used to identify predictors of LCOS and death. Multivariate logistic regression identified pre-operative NT-proBNP and IL-8 4 h PO as independent predictors of LCOS, while cTnI 4 h PO and CPB length were independent predictors of death. The use of inflammatory and cardiac markers in combination improved sensitivity, negative predictive value and accuracy of the models. In conclusion, the combined assessment of inflammatory and cardiac biochemical markers can be useful for identifying young children at increased risk for LCOS and death after heart surgery with CPB. (C) 2008 Elsevier Ltd. All rights reserved.

Bursts of transposition from non-long terminal repeat retrotransposon families of the RTE clade in Schistosoma mansoni

The genus Schistosoma is composed of blood flukes that infect vertebrates, from which three species are major causative agents of human schistosomiasis, a tropical disease that affects more than 200 million people. Current models of the recent evolution of Schistosoma indicate multiple events of migration and speciation from an Asian ancestral species. Transposable elements are important drivers of genome evolution and have been hypothesised to have an important role in speciation. In this work, we describe a comprehensive inventory of Schistosoma mansoni and Schistosoma japonicum retrotransposons, based on their recently published genomic data. We find a considerable difference in retrotransposon representation between the two species (22% and 13%, respectively). A large part of this difference can be attributed to higher representation of two previously described families of S. mansoni retrotransposons (SR2 and Perere-3/SR3), compared with the representation of their closest relative families in S. japonicum. A more detailed analysis suggests that these two S. mansoni families were the subject of recent bursts of transposition that were not paralleled by their S. japonicum counterparts. We hypothesise that these bursts could be a consequence of the evolutionary pressure resulting from migration of Schistosoma from Asia to Africa and their establishment in this new environment, helping both speciation and adaptation. (C) 2009 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.