Purification and partial characterization of the plasma clotting protein from the pink shrimp Farfantepenaeus paulensis

A clotting protein (CP) was purified from the plasma of the pink shrimp Farfantepenaeus paulensis by sequential anion-exchange chromatography. The shrimp CP was able to form stable clots in vitro in the presence of hemocyte lysate and Ca2+, suggesting that the clotting reaction is catalyzed by a Ca2+-dependent transglutaminase present in shrimp hemocytes. Dansylcadaverine was incorporated into the shrimp CP in the presence of endogenous transglutaminase (hemocyte lysate), confirming that the shrimp purified CP is the substrate for the transglutaminase enzyme. The molecular mass of the CP was determined by gel filtration to be 341 kDa and 170 kDa by SDS-PAGE under reducing conditions. These results suggest that the shrimp CP consists of two identical subunits, covalently linked by disulphide bonds. The amino acid sequence at the N-terminus was 100% identical to that of the penaeids Litopenaeus vannamei and Penaeus monodon and 66% to 80% identical to the CPs of other decapods. This is the first report of a CP characterization in an Atlantic penaeid species. Further studies, including a molecular cloning approach would enable to detect which tissues express the gene of the clotting protein. It would be also useful to understand the mechanism by which the coagulation time is delayed in shrimps under stress conditions.     

Mild acid hydrolysis of sulfated fucans: a selective 2-desulfation reaction and an alternative approach for preparing tailored sulfated oligosaccharides

Sulfated fucans from marine invertebrates have simple, linear structures, composed of repeating units of oligosaccharides. Most of these polysaccharides contain 3-linked fucosyl units, but each species of invertebrate has a specific pattern of sulfation. No specific enzyme able to cleave or to desulfate these polysaccharides has been described yet. Therefore, we employed an alternative approach, based on mild acid hydrolysis, in an attempt to obtain low molecular-weight derivatives from sulfated fucans. Surprisingly, we observed that sulfated fucans from Lytechinus variegatus and Strongylocentrotus pallidus (but not the sulfated fucans from other species) yield by mild acid hydrolysis oligosaccharides with well-defined molecular size as shown by narrow bands in polyacrylamide gel electrophoresis (PAGE). The sulfated oligosaccharides obtained by mild acid hydrolysis were purified by gel-filtration chromatography, and their structures were identified by (1)H-nuclear magnetic resonance (NMR) spectroscopy, revealing an identical chemical composition for all oligosaccharides. When we followed the acid hydrolysis by (1)H-NMR spectroscopy, we found that a selective 2-desulfation occurs in the fucans from S. pallidus and from L. variegatus. The reaction has two stages. Initially, 2-sulfate esters at specific sites are removed. Then the desulfated units are cleaved, yielding oligosaccharides with well-defined molecular size. The apparent requirement for the selective 2-desulfation is the occurrence of an exclusively 2-sulfated fucosyl unit linked to or preceded by a 4-sulfated residue. Thus, a homofucan from Strongylocentrotus franciscanus resists desulfation by mild acid hydrolysis, because it lacks the neighboring 4-sulfated unit. Overall, our results show a new approach for desulfating sulfated fucans at specific sites and obtaining tailored sulfated oligosaccharides.     

Selective cleavage and anticoagulant activity of a sulfated fucan: stereospecific removal of a 2-sulfate ester from the polysaccharide by mild acid hydrolysis, preparation of oligosaccharides, and heparin cofactor II-dependent anticoagulant activity

A linear sulfated fucan with a regular repeating sequence of [3)-alpha-L-Fucp-(2SO4)-(1-->3)-alpha-L-Fucp-(4SO4)-(1-->3)-alpha-L-Fucp-(2,4SO4)-(1-->3)-alpha-L-Fucp-(2SO4)-(1-->]n is an anticoagulant polysaccharide mainly due to thrombin inhibition mediated by heparin cofactor II. No specific enzymatic or chemical method is available for the preparation of tailored oligosaccharides from sulfated fucans. We employ an apparently nonspecific approach to cleave this polysaccharide based on mild hydrolysis with acid. Surprisingly, the linear sulfated fucan was cleaved by mild acid hydrolysis on an ordered sequence. Initially a 2-sulfate ester of the first fucose unit is selectively removed. Thereafter the glycosidic linkage between the nonsulfated fucose residue and the subsequent 4-sulfated residue is preferentially cleaved by acid hydrolysis, forming oligosaccharides with well-defined size. The low-molecular-weight derivatives obtained from the sulfated fucan were employed to determine the requirement for interaction of this polysaccharide with heparin cofactor II and to achieve complete thrombin inhibition. The linear sulfated fucan requires significantly longer chains than mammalian glycosaminoglycans to achieve anticoagulant activity. A slight decrease in the molecular size of the sulfated fucan dramatically reduces its effect on thrombin inactivation mediated by heparin cofactor II. Sulfated fucan with approximately 45 tetrasaccharide repeating units binds to heparin cofactor II but is unable to link efficiently the plasma inhibitor and thrombin. This last effect requires chains with approximately 100 or more tetrasaccharide repeating units. We speculate that the template mechanism may predominate over the allosteric effect in the case of the linear sulfated fucan inactivation of thrombin in the presence of heparin cofactor II.     

Overlapping binding sites for trypsin and papain on a Kunitz-type proteinase inhibitor from Prosopis juliflora

Proteinase inhibitors are among the most promising candidates for expression by transgenic plants and consequent protection against insect predation. However, some insects can respond to the threat of the proteinase inhibitor by the production of enzymes insensitive to inhibition. Inhibitors combining more than one favorable activity are therefore strongly favored. Recently, a known small Kunitz trypsin inhibitor from Prosopis juliflora (PTPKI) has been shown to possess unexpected potent cysteine proteinase inhibitory activity. Here we show, by enzyme assay and gel filtration, that, unlike other Kunitz inhibitors with dual activities, this inhibitor is incapable of simultaneous inhibition of trypsin and papain. These data are most readily interpreted by proposing overlapping binding sites for the two enzymes. Molecular modeling and docking experiments favor an interaction mode in which the same inhibitor loop that interacts in a canonical fashion with trypsin can also bind into the papain catalytic site cleft. Unusual residue substitutions at the proposed interface can explain the relative rarity of twin trypsin/papain inhibition. Other changes seem responsible for the relative low affinity of PTPKI for trypsin. The predicted coincidence of trypsin and papain binding sites, once confirmed, would facilitate the search, by phage display for example, for mutants highly active against both proteinases.     

Human venous and arterial glycosaminoglycans have similar affinity for plasma low-density lipoproteins

We compared the glycosaminoglycan content of human venous and arterial walls. The most abundant glycosaminoglycan in human veins is dermatan sulfate whereas chondroitin 4/6-sulfate is preponderant in arteries. The concentrations of chondroitin 4/6-sulfate and heparan sulfate are approximately 4.8- and approximately 2.5-fold higher in arteries than in veins whereas dermatan sulfate contents are similar in the two types of blood vessels. Normal and varicose saphenous veins do not differ in their glycosaminoglycan contents. It is known that certain glycosaminoglycan species from the arterial wall, mainly high-molecular-weight fractions of dermatan sulfate+chondroitin 4/6-sulfate have greater affinity for plasma LDL. These types of glycosaminoglycans can be identified on a LDL-affinity column. We now demonstrated that a similar population of glycosaminoglycan also occurs in veins, although with a lower concentration than in the arteries due to less chondroitin 4/6-sulfate with affinity for LDL. The concentrations of dermatan sulfate species, which interact with LDL, are similar in arteries and veins. The presence of these glycosaminoglycans with affinity to plasma LDL in veins raises interesting questions concerning the role of these molecules in the pathogenesis of atherosclerosis. Possibly, the presence of these glycosaminoglycans in the vessel wall are not sufficient to cause retention of LDL and consequently endothelial dysfunction, but may require additional intrinsic factors and/or the hydrodynamic of the blood under the arterial pressure.     

Electrophysiological studies and identification of possible sex pheromone components of Brazilian populations of the sugarcane borer,Diatraea saccharalis

Virgin female gland extracts of sugarcane moth Diatraea saccharalis (Fabricius) (Lepidoptera: Pyralidae), from three locations in Brazil, have been analyzed. By GC-MS analysis and comparison of the chromatographic retention time of the components of the pheromone gland with those retention times of synthetic standards, we observed the presence of (Z)-hexadec-11-enal (1), hexadecanal (2), (9E,11Z)-hexadecadienal (4), (9Z,11Z)-hexadecadienal (5) and (9E,11E)-hexadecadienal (6), as minor components besides the major constituent (9Z,11E)-hexadecadienal (3) already reported. We found no variations in the composition of the gland extracts deriving from the three Brazilian populations and only two compounds, (Z)-hexadec-11-enal (1) and (9Z,11E)-hexadecadienal (3), elicited antennal responses (GC-EAD). In electroantennography (EAG), however, pure compounds 1 and 3, a binary mixture containing 1 and 3, and a mixture containing all of the six synthetic compounds 1-6 elicited a depolarization in male antennae of D. saccharalis, without any statistically different delay. The EAG responses to the other isomers of 9,11-hexadecadienal were small and not significantly different from the control, except for the (9Z,11Z)-isomer (5) which showed an relatively strong electroantennal activity.     

Pseudempleurosoma gibsoni n. sp., a new ancyrocephalid monogenean from Paralonchurus brasiliensis (Sciaenidae) from off the southeastern coast of Brazil

Pseudempleurosoma gibsoni n. sp. (Monogenea: Ancyrocephalidae) is described from the oesophagus of Paralonchurus brasiliensis (Steindachner) from off the coast of Brazil. The type-species of Pseudempleurosoma Yamaguti, 1965, P. carangis Yamaguti, 1965, is redescribed and the diagnosis of the genus is amended. Metadiplectanotrema Gerasev et al. 1987 is considered synonym of Pseudempleurosoma. This genus now contains four species, including P. carangis, P. caranxi Gerasev et al., 1987 n. comb., P. myripristi Gerasev et al., 1987 n. comb. and the one new species.     

Afforestation effects on vegetation structure and diversity of grasslands in southern Brazil: The first years

We evaluated the effects of afforestation on the composition and structure of southern Brazilian subtropical grasslands directly beneath tree plantations, in pastures, and in permanent protection areas near watercourses. Cover of plants was registered in 120 plots of 1 m² in grasslands located at two distances from small watercourses in pasture and eucalypt plantation areas within a livestock ranching landscape. Livestock density is reduced in plantation areas. Species composition beneath tree plantations was significantly different from both pasture and nearby permanent protection areas. Permanent protection areas near plantations were also compositionally different from pastures. Eucalypt plantation was the environment type where ruderals were most abundant. Growth form cover distribution in pasture areas showed a consistent pattern, with graminous herbs being the most common growth form followed by prostrate herbs, prostrate subshrubs, and standing subshrubs. In plantation areas, the dominance of the graminoid herb growth form increased markedly, as well as standing herbs. These increases occurred mainly at the expense of prostrate herbs and subshrubs, which were significantly reduced. Plot-level species density was significantly affected by distance from watercourse and, more importantly, was significantly influenced by the interaction between management regime and distance to watercourse. It was reduced in eucalypt plantations relative to permanent protected areas. Current management of eucalypt plantations in southern Brazil produce unsuitable conditions for grassland species to thrive within plantations. Livestock densities <1.0 animal unit ha^?1 in eucalypt plantations undermines the ability of surrounding permanent protection areas to act as ecological networks for biodiversity conservation.     

Comparative environmental performance of semi-trailer load boxes for grain transport made of different materials

Background, aim, and scope  

Semi-trailers with load boxes are the most important mode of grain transport by land in the world. Load boxes can be produced with different materials such as: wood, steel, and synthetic material. They are responsible for effectiveness retention and quality of grains during the transport. Among the main aspects to be considered and valued when selecting materials for load boxes are the final mass of the semi-trailer, loss of grains, and mechanical properties. Environmental performance is another important aspect to be taken into account for developing and selecting new materials in this kind of application. This study presents a comparative environmental evaluation of load boxes built from two different materials (a wood panel and a three-layer synthetic (TLS) panel). Mass balance and life cycle assessment (LCA) were used in this study.