A molecular mechanism for Lys(49)-phospholipase A(2) activity based on ligand-induced conformational change

Agkistrodon contortrix laticinctus myotoxin is a Lys(49)- phospholipase A(2) (EC 3.1.1.4) isolated from the venom of the serpent A contortrix laticinctus (broad-banded copperhead). We present here three monomeric crystal structures of the myotoxin, obtained under different crystallization conditions. The three forms present notable structural differences and reveal that the presence of a ligand in the active site (naturally presumed to be a fatty acid) induces the exposure of a hydrophobic surface (the hydrophobic knuckle) toward the C terminus. The knuckle in A contortrix laticinctus myotoxin involves the side chains of Phe(121) and Phe(124) and is a consequence of the formation of a canonical structure for the main chain within the region of residues 118-125. Comparison with other Lys(49)-phospholipase A(2) myotoxins shows that although the knuckle is a generic structural motif common to all members of the family, it is not readily recognizable by simple sequence analyses. An activation mechanism is proposed that relates fatty acid retention at the active site to conformational changes within the C-terminal region, a part of the molecule that has long been associated with Ca2+-independent membrane damaging activity and myotoxicity. This provides, for the first time, a direct structural connection between the phospholipase active site and the C-terminal myotoxic site, justifying the otherwise enigmatic conservation of the residues of the former in supposedly catalytically inactive molecules.

FUNCTIONAL ALPHA-TROPOMYOSIN PRODUCED IN ESCHERICHIA-COLI - A DIPEPTIDE EXTENSION CAN SUBSTITUTE THE AMINO-TERMINAL ACETYL GROUP

Unlike the muscle protein, alpha-tropomyosin expressed in Escherichia coli does not bind actin, does not exhibit head-to-tail polymerization, and does not inhibit actomyosin ATPase activity in the absence of troponin. The only chemical difference between recombinant and muscle tropomyosins is that the first methionine is not acetylated in the recombinant protein (Hitchcock-DeGregori, S. E., and Heald, R. W. (1987) J. Biol. Chem. 262, 9730-9735). We expressed three fusion tropomyosins in E. coli with 2, 3, and 17 amino acids fused to its amino terminus. Ah three fusions restored actin binding, head-to-tail polymerization, and the capacity to inhibit the actomyosin ATPase to these unacetylated tropomyosins. Unlike larger fusions, the small fusions of 2 and 3 amino acids do not interfere with regulatory function. Therefore the presence of a fused dipeptide at the amino terminus of unacetylated tropomyosin is sufficient to replace the function of the N-acetyl group present in muscle tropomyosin. A structural interpretation for the function of the acetyl group, based on our results and the coiled coil structure of tropomyosin, is presented.

Correlation between the mobility of spin-labeled peptide chains and resin solvation: An approach to optimize the synthesis of aggregating sequences

Resin solvation properties affect the efficiency of the coupling reactions in solid-phase peptide synthesis. Here we report a novel approach to evaluate resin solvation properties, making use of spin label electron paramagnetic resonance (EPR) spectroscopy. The aggregating VVLGAAIV and ING sequences were assembled in benzhydrylamine-resin with different amino group contents (up to 2.6 mmol/g) to examine the extent of chain association within the beads. These model peptidyl-resins were first labeled at their N-terminus with the amino acid spin label 2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid (Toac). Their solvation properties in different solvents were estimated, either by bead swelling measurement or by assessing the dynamics of their polymeric matrixes through the analysis of Toac EPR spectra, and were correlated with the yield of the acylation reaction. In most cases the coupling rate was found to depend on bead swelling. Comparatively, the EPR approach was more effective. Line shape analysis allowed the detection of more than one peptide chain population, which influenced the reaction. The results demonstrated the unique potential of EPR spectroscopy not only for improving the yield of peptide synthesis, even in challenging conditions, but also for other relevant polymer-supported methodologies in chemistry and biology.

Neutrophil interaction with inflamed postcapillary venule endothelium alters annexin 1 expression

Annexin 1 (ANX-A1) exerts antimigratory actions in several models of acute and chronic inflammation, This is related to its ability to mimic the effect of endogenous ANX-A1 that is externalized on neutrophil adhesion to the postcapillary endothelium. In the present study we monitored ANX-A1 expression and localization in intravascular and emigrated neutrophils, using a classical model of rat peritonitis, For this purpose, a pair of antibodies raised against the ANX-A1 N-terminus tie, able to recognize intact ANX-A1) or the whole protein tie, able to interact with all ANX-A1 isoforms) was used by immunofluorescence and immunocytochemistry analyses. The majority (similar to 50%) of ANX-A1 on the plasma membrane of intravascular neutrophils was intact. Extravasation into the subendothelial matrix caused loss of this pool of intact protein (to similar to6%), concomitant with an increase in total amount of the protein; only similar to 25% of the total protein was now recognized by the antibody raised against the N-terminus tie, it was intact). In the cytoplasm of these cells, ANX-A1 was predominantly associated with large vacuoles, possibly endosomes, In situ hybridization confirmed de novo synthesis of ANX-A1 in the extravasated cells. In conclusion, biochemical pathways leading to the externalization, proteolysis, and synthesis of ANX-A1 are activated during the process of neutrophil extravasation.

INCREASED CALCIUM AFFINITY OF A FUCOSYLATED CHONDROITIN SULFATE FROM SEA-CUCUMBER

Calcium binding and charge distribution on a fucosylated chondroitin sulfate and a standard chondroitin 6-sulfate have been studied using a metallochromic indicator and conductimetric titrations. The fucosylated chondroitin sulfate has a similar to 5-fold greater affinity for calcium ions than the standard chondroitin 6-sulfate. Possibly, this increased affinity for calcium ions is due to the branches on the fucosylated chondroitin sulfate, since the calcium affinity of an unbranched, sulfated fucan is similar to that of the standard chondroitin 6-sulfate. More charged groups per disaccharide unit (and a shorter distance between these groups) also distinguish the fucosylated chondroitin sulfate from standard chondroitin 6-sulfate. Comparison between native and chemically modified (desulfated or carboxyl-reduced) polysaccharides suggests that the sulfate esters are responsible for the increased charge density of the fucosylated chondroitin sulfate and that the presence of the fucose branches does not alter the length of the repetitive units which compose the central core of chondroitin from sea cucumber. These results are consistent with the chemical studies of these two polysaccharides.

Pratylenchus jaehni sp n. from citrus in Brazil and its relationship with P-coffeae and P-loosi (Nematoda : Pratylenchidae)

A lesion nematode population infecting citrus in the state of São Paulo, Brazil is described and named Pratylenchus jaehni sp. n. Biological, molecular and morphological characteristics of this new species are compared with those of the morphologically similar P. coffeae and P. loosi. Results of mating experiments showed that R jaehni is reproductively isolated from P coffeae. Molecular (D2/D3 DNA sequences) dissimilarities among P. jaehni sp, n., P. coffeae and P. loosi were documented in a previous study. The morphology of seven R coffeae populations from tropical America and eastern Java and a P loosi population from Sri Lanka is used for comparison with the morphology of P. jaehni sp. n. Pratylenchus jaehni differs from R coffeae and P. loosi by only a few morphological ;characters of the females. The mean values of stylet length, stylet knob height, and vulva position are smaller (less than or equal to15 vs greater than or equal to15 mum, less than or equal to2.7 vs greater than or equal to2.7 mum, less than or equal to79 vs greater than or equal to79%) than those in P coffeae and P loosi. The tail terminus is usually subhemispherical and smooth in P jaehni sp. n.. whereas it is commonly truncate and indented in most P. coffeae populations and bluntly or finely pointed in P. loosi. Because of the morphological similarities among P. jaehni sp. n., P. coffeae and P. loosi, examination of at least ten specimens is required to obtain a reliable diagnosis based on morphology. Nineteen morphometric parameters for P. jaehni sp. n. and P. coffeae ranged from 0-13% smaller in fixed than in live specimens.

Effect of salt nature on structure and ionic conductivity of sodium-doped siloxane-PPO ormolytes

Siloxane-polyoxypropylene (PPO) hybrids obtained by the sol-gel process and containing short polymer chain have been doped with different sodium salts NaX (X = ClO4, BF4 or I). The effect of the counter-ion (X) on the chemical environment of the sodium ions and on the ionic conductivity of these hybrids was investigated by Na-23 NMR, small angle X-ray scattering (SAXS), complex impedance, Raman spectroscopy and differential scanning calorimetry (DSC). Results reveal that the different sodium salts have essentially the same effect on the nanoscopic structure of the hybrids. The formation of immobile Na+ cations involved in NaCl-like species could be minimized by using a low amount of HCl as hydrolytic catalyst. The differences in the ionic conductivity of hybrids doped with different sodium salts were correlated with the proportion of Na ions solvated by ether-type oxygen of the polymeric chains and by the carboxyl oxygen located in the urea groups of the PPO chain extremities. (c) 2005 Elsevier Ltd. All rights reserved.

Actively transcribed GAL genes can be physically linked to the nuclear pore by the SAGA chromatin modifying complex

Recent work has demonstrated that some actively transcribed genes closely associate with nuclear pore complexes (NPC) at the nuclear periphery. The Saccharomyces cerevisiae Mlp1 and Mlp2 proteins are components of the inner nuclear basket of the nuclear pore that mediate interactions with these active genes. To investigate the physical link between the NPC and active loci, we identified proteins that interact with the carboxyl-terminal globular domain of Mlp1 by tandem affinity purification coupled with mass spectrometry. This analysis led to the identification of several components of the Spt-Ada-Gcn5-acetyltransferase ( SAGA) histone acetyltransferase complex, Gcn5, Ada2, and Spt7. We utilized co-immunoprecipitation and in vitro binding assays to confirm the interaction between the Mlp proteins and SAGA components. Chromatin immunoprecipitation experiments revealed that Mlp1 and SAGA components associate with the same region of the GAL promoters. Critically, this Mlp-promoter interaction depends on the integrity of the SAGA complex. These results identify a physical association between SAGA and the NPC, and support previous results that relied upon visualization of GAL loci at the nuclear periphery by microscopy ( Cabal, G. G. Genovesio, A., Rodriguez-Navarro, S., Zimmer, C., Gadal, O., Lesne, A., Buc, H., Feuerbach- Fournier, F., Olivo-Marin, J.-C., Hurt, E. C., and Nehrbass, U. ( 2006) Nature 441, 770-773). We propose that a physical interaction between nuclear pore components and the SAGA complex can link the actively transcribed GAL genes to the nuclear pore.

Mutational analyses of human eIF5A-1-identification of amino acid residues critical for eIF5A activity and hypusine modification

The eukaryotic translation initiation factor 5A (eIF5A) is the only protein that contains hypusine [N-epsilon-(4-amino-2-hydroxybutyl)lysine], which is required for its activity. Hypusine is formed by post-translational modification of one specific lysine (Lys50 for human eIF5A) by deoxyhypusine synthase and deoxyhypusine hydroxylase. To investigate the features of eIF5A required for its activity, we generated 49 mutations in human eIF5A-1, with a single amino acid substitution at the highly conserved residues or with N-terminal or C-terminal truncations, and tested mutant proteins in complementing the growth of a Saccharomyces cerevisiae eIF5A null strain. Growth-supporting activity was abolished in only a few mutant eIF5As (K47D, G49A, K50A, K50D, K50I, K50R, G52A and K55A), with substitutions at or near the hypusine modification site or with truncation of 21 amino acids from either the N-terminus or C-terminus. The inactivity of the Lys50 substitution proteins is obviously due to lack of deoxyhypusine modification. In contrast, K47D and G49A were effective substrates for deoxyhypusine synthase, yet failed to support growth, suggesting critical roles of Lys47 and Gly49 in eIF5A activity, possibly in its interaction with effector(s). By use of a UBHY-R strain harboring genetically engineered unstable eIF5A, we present evidence for the primary function of eIF5A in protein synthesis. When selected eIF5A mutant proteins were tested for their activity in protein synthesis, a close correlation was observed between their ability to enhance protein synthesis and growth, lending further support for a central role of eIF5A in translation.

Influence of HLA-DRB-1 alleles on the production of antibody against CSP, MSP-1, AMA-1, and DBP in Brazilian individuals naturally infected with Plasmodium vivax

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Soil humus characteristics in virgin and cleared areas of the paraná river basin in Brazil

A study of the characteristics and distribution of the soil humus fractions in representative ecosystems of central Brazil was carried out with special emphasis on the comparison between the soils under virgin vegetation-Cerrado-and those subjected to cultivation. In spite of the contrasted vegetation and cultural practices in the sites studied, the soil humus showed analogous characteristics: there was a negligible amount of plant residues, the humic and fulvic acids amounted to approximately 70% of the total organic carbon, and about 40% of these humic substances were in extremely stable association with the soil mineral fraction, the HCl-HF treatment being required for their extraction. The stability of such organo-mineral complexes increased slightly in the cultured sites. The study of the humic acid fraction showed increased oxidation and aromaticity in most of the cultivated sites: the lowest values for the IR alkyl vibrations and H/C atomic ratios and the highest ones for the optical density at 465 nm were observed in sites transformed into orchards, whereas the above changes were small in those used as pasture. The 14C NMR spectra confirmed that the proportion of polyalkyl structures decreased in the humic acids of soils subjected to cultivation, as opposed to that of carboxyl groups. In spite of the high stability inferred for the organic matter throughout the wide area examined, the samples from the original Cerrado as well as from those transformed into pastures showed, in laboratory conditions, higher mineralization rates than those from the sites subjected to cultivation. This is partly attributed to the decreased proportions of extractable humic substances in the latter. © 1992.

Revisão de plantas Brasileiras com comprovado efeito hipoglicemiante no controle do diabetes mellitus

Plants have been used in the cure of diseases from the origins of the humanity. At present, in Brazil, the use is common because of the difficulty of access of part of the population to medical assistance. It is commonly believed that the medicinal plants of traditional use were already tested and ratified by the long-lasting use by the human species, being considered effective medicines, presenting no collateral effects, not needful, therefore, of evaluation. The miraculous self-medication with medicinal plants goes to the point of substituting therapies in serious diseases such as those of hypoglycemic or anti-diabetic effect. For the test of medicinal plants, it is necessary to consider the material quality to be tested, the plant component used, extraction method, dosage, and the experimental species used. Several plants have already had hypoglycemic effects proven experimentally. The objective of this paper was to accomplish a revision of Brazilian medicinal plants, used popularly, as hypoglycemics that had effects proven in animals and in humans.

Conformational basis for the biological activity of TOAC-labeled angiotensin II and bradykinin: Electron paramagnetic resonance, circular dichroism, and fluorescence studies

N-Terminally and internally labeled analogues of the hormones angiotensin (AII, DRVYIHPF) and bradykinin (BK, RPPGFSPFR) were synthesized containing the paramagnetic amino acid 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4- carboxylic acid (TOAC). TOAC replaced Asp 1 (TOAC 1-AII) and Val 3 (TOAC 3-AII) in AII and was inserted prior to Arg 1 (TOAC 0-BK) and replacing Pro 3 (TOAC 3-BK) in BK. The peptide conformational properties were examined as a function of trifluoroethanol (TFE) content and pH. Electron paramagnetic resonance spectra were sensitive to both variables and showed that internally labeled analogues yielded rotational correlation times (TC) considerably larger than N-terminally labeled ones, evincing the greater freedom of motion of the N-terminus. In TFE, τ C increased due to viscosity effects. Calculation of τ Cpeptide/τ CTOAC ratios indicated that the peptides acquired more folded conformations. Circular dichroism spectra showed that, except for TOAC 1-AII in TFE, the N-terminally labeled analogues displayed a conformational behavior similar to that of the parent peptides. In contrast, under all conditions, the TOAC 3 derivatives acquired more restricted conformations. Fluorescence spectra of All and its derivatives were especially sensitive to the ionization of Tyr 4. Fluorescence quenching by the nitroxide moiety was much more pronounced for TOAC 3-AII The conformational behavior of the TOAC derivatives bears excellent correlation with their biological activity, since, while the N-terminally labeled peptides were partially active, their internally labeled counterparts were inactive [Nakaie, C. R., et al., Peptides 2002, 23, 65-70]. The data demonstrate that insertion of TOAC in the middle of the peptide chain induces conformational restrictions that lead to loss of backbone flexibility, not allowing the peptides to acquire their receptor-bound conformation. © 2004 Wiley Periodicals, Inc.

Conformation and lytic activity of eumenine mastoparan: A new antimicrobial peptide from wasp venom

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Morphological, mechanical and thermal properties of nylon 6/ABS blends using glycidyl methacrylate-methyl methacrylate copolymers

Nylon6 is an attractive polymer for engineering applications because it has reactive functionality through amine and carboxyl end groups that are capable of reacting. For this reason, it has been used a lot in polymeric blends. Blends of nylon6/ABS (acrylonitrile-butadiene-styrene) were produced using glycidyl methacrylate-methyl methacrylate (GMA-MMA) copolymers as compatibilizer. The binary blends were immiscible and exhibited poor mechanical properties that stemmed from the unfavorable interactions among their molecular segments. This produced an unstable coarse phase morphology and weak interfaces between the phases in the solid state. The presence of the copolymer in the blends clearly led to a more efficient dispersion of the ABS phase and consequently optimized Izod impact properties. However, the compatibilized blend showed poor toughness at room temperature and failed in a brittle manner at subambient temperatures. © 2005 Springer Science + Business Media, Inc.