A Leishmania (L.) amazonensis ATP diphosphohydrolase isoform and potato apyrase share epitopes: antigenicity and correlation with disease progression

A Leishmania (Leishmania) amazonensis ATP diphosphohydrolase isoform was partially purified from plasma membrane of promastigotes by preparative non-denaturing polyacrylamide gel electrophoresis. SDS-PAGE followed by Western blots developed with polyclonal anti-potato apyrase antibodies identified diffuse bands of about 58-63 kDa, possibly glycosylated forms of this protein. By ELISA technique, a significantly higher total IgG antibody level against potato apyrase was found in serum from promastigote-infected mice, as compared to the uninfected mice, confirming both the existence of shared epitopes between the parasite and vegetable proteins, and the parasite ATP diphosphohydrolase antigenicity. By Western blotting, serum from amastigote-infected BALB/c mice recognizes both potato apyrase and this antigenic ATP diphosphohydrolase isoform isolated from promastigotes, suggesting that it is also expressed in the amastigote stage. The infection monitored along a 90-day period in amastigote-infected mice showed reactivity of IgG2a antibody in early steps of infection, while the disappearance of the IgG2a response and elevation of IgG1 antibody serum levels against that shared epitopes were associated with the progression of experimental leishmaniasis. This is the first observation of the antigenicity of a L. (L.) amazonensis ATP diphosphohydrolase isoform, and of the ability of cross-immunoreactivity with potato apyrase to differentiate serologically stages of leishmaniasis in infected mice.     

Rhamnoarabinosyl and rhamnoarabinoarabinosyl side chains as structural features of coffee arabinogalactans

The hot water soluble green coffee arabinogalactans, representing nearly 7% of total coffee bean arabinogalactans, were characterized by (1)H and (13)C NMR and, after partial acid hydrolysis, by ESI-MS/MS. Data obtained showed that these are highly branched type II arabinogalactans covalently linked to proteins (AGP), with a protein moiety containing 10% of 4-hydroxyproline residues. They possess a beta-(1-->3)-Galp/beta-(1-->3,6)-Galp ratio of 0.80, with a sugars composition of Rha:Ara:Gal of 0.25:1.0:1.5, and containing 2mol% of glucuronic acid residues. Beyond the occurrence of single alpha-L-Araf residues and [alpha-L-Araf-(1-->5)-alpha-L-Araf-(1-->] disaccharide residues as side chains, these AGPs contain unusual side chains at O-3 position of the beta-(1-->6)-linked galactopyranosyl residues composed by [alpha-L-Rhap-(1-->5)-alpha-L-Araf-(1-->] and [alpha-L-Rhap-(1-->5)-alpha-L-Araf-(1-->5)-alpha-L-Araf-(1-->] oligosaccharides. Rhamnoarabinosyl and rhamnoarabinoarabinosyl side chains are reported for the first time as structural features of plant arabinogalactan-proteins.     

Phosphodiesterase inhibition attenuates alterations to the tight junction proteins occludin and ZO-1 in immunostimulated Caco-2 intestinal monolayers

AimsUnder normal conditions, the intestinal mucosa acts as a local barrier to prevent the influx of luminal contents. The intestinal epithelial tight junction is comprised of several membrane associated proteins, including zonula occludens-1 (ZO-1) and occludin. Disruption of this barrier can lead to the production of pro-inflammatory mediators and ultimately multiple organ failure. We have previously shown that Pentoxifylline (PTX) decreases histologic gut injury and pro-inflammatory mediator synthesis. We hypothesize that PTX prevents the breakdown of ZO-1 and occludin in an in vitro model of immunostimulated intestinal cell monolayers.Main methodsCaco-2 human enterocytes were grown as confluent monolayers and incubated under control conditions, or with PTX (2 mM), Cytomix (TNF-α, IFN-γ, IL-1), or Cytomix + PTX for 24 h. Occludin and ZO-1 protein levels were analyzed by Western blot. Confocal microscopy was used to assess the cytoplasmic localization of ZO-1 and occludin.Key findingsCytomix stimulation of Caco-2 cells resulted in a 50% decrease in both occludin and ZO-1 protein. Treatment with Cytomix + PTX restored both occludin and ZO-1 protein to control levels. Confocal microscopy images show that Cytomix caused an irregular, undulating appearance of ZO-1 and occludin at the cell junctions. Treatment with PTX prevented the Cytomix-induced changes in ZO-1 and occludin localization.SignificanceTreatment with PTX decreases the pro-inflammatory cytokine induced changes in the intestinal tight junction proteins occludin and ZO-1. Pentoxifylline may be a useful adjunct in the treatment of sepsis and shock by attenuating intestinal barrier breakdown.     

Implication of glucocorticoid in anti-inflammatory effects of Ro5-4864 in mouse pleurisy induced by carrageenan

Mouse pleurisy induced by carrageenan is used to determine the mechanism of anti-inflammatory action of 7-chloro-5-(4-chlorophenyl)-1,3-dihydro-1-methyl-2-H-1,4-benzodiazepin-2 (Ro5-4864). Pre-treatment with Ro5-4864 inhibits different inflammatory parameters, such as neutrophil influx, MPO activity and NO levels in the early phase (4 h), as well as mononuclear cells and ADA activity in the late phase (48 h) of pleurisy. dl-Aminoglutethimide, inhibitor of steroidal synthesis, reverted the effect of Ro5-4864 on these different inflammatory parameters. Our results suggest that anti-inflammatory action of Ro5-4864 may be partly due to its capacity to inhibit leukocyte migration, as well as leukocyte activation and formation of NO by a mechanism dependent on glucocorticoids.     

HABITAT HETEROGENITY ON TROPICAL ROCKY SHORES: A SEAWEED STUDY IN SOUTHERN BRAZIL

The main concepts in coastal community ecology have been proposed to explain relatively plain substrate, specially at temperate zones. We show that, on a irregular tropical substratum (a complex system of quarry stones with diverse shapes and sizes), the classical community zonation scheme is only applicable if we simplify the tridimensional substrate arrangement to a single dimensioned universe: the topographic level. Thus, the continuous spatial distribution implicited in "zones" notions is nonexistent. Our study was conducted on a rocky shore at Ubatuba (São Paulo State, Brazil) during one year. Percentual coverage was estimated using the quadrat method located by random coordinates. The topographic level was determined for each sampling element. Analysis of spatial splitting revealed large-scale stratification of the shore into dominiums related to topographical level: 1) Upper Dominium (represented by Chthamalus sp. and Tetraclita spalactifera ); 2) Transition Level (composed of Gymnogongrus griffithsiae , Gelidium aff. pusillum and often Centroceras clavulatum );. and 3) Lower Dominium (mainly Sargassum vulgare and occasionally Padina gymnospora, Dictyopteris delicatula and Grateloupia doryphora ). These dominiums cannot be recognized as "bands" or continuous "zones" in a landscape view of the community. Otherwise, these bands could be observed on a small scale on each large quarry stone.     

Immunolocalization of arabinogalactan proteins inAmaranthus hypochondriacus L. ovules

Summary Amaranthus hypochondriacus ovules are of the crassinucellate type, having several layers of nucellus cells between the micropyle and the embryo sac through which pollen tubes have to penetrate. The ultrastructural features of the micropylar nucellus cells appear to reflect cells with high metabolic activity. With the monoclonal antibodies MAC207 and JIM8 (against arabinogalactan proteins) we have shown that the presence of the two epitopes was different in the gametophytic tissues and embryo sac. The young embryo and suspensor cells are reactive only to Mab JIM8. The selective presence and localization of these two epitopes was also demonstrated in the micropylar nucellus cells. The expression of these arabinogalactan proteins in this ovule seems to be closely aligned with the pathway of the pollen tube, possibly providing directional guides for tube growth inside the ovule.     

Physicochemical Aspects of Chitosan Dispersibility in Acidic Aqueous Media: Effects of the Food Acid Counter-Anion

Differences in formation of colloidal dispersions of chitosan in aqueous solutions of citric acid or lactic acid (25, 50 or 100 mM) were quantitatively studied. Protonation enthalpies, electrical conductivity and ζ-potential measurements were additionally undertaken, aiming at better understanding these differences at a molecular level. In dispersion kinetics assays, experimental data were well fitted (R²?>?0.9; MAPE?<?4 %) by a first-order kinetics model with two terms - one accounting for the fast, direct dispersion of biopolymers chains and another accounting for the slow dispersion of chains from lumps. In all cases, maximal dispersibility was reached after about 20?30 min of stirring. For both acids, the higher the acid concentration in the medium, the higher was the chitosan dispersibility. At a given acid concentration, chitosan showed higher dispersibility in lactic acid than in citric acid solutions. Protonation of chitosan -NH₂ groups was strongly exothermic, with ΔH values three times higher for citric acid (triprotic) than lactic acid (monoprotic) (ΔH?=??120 kJ?mol^{- 1} and ΔH?=??40 kJ?mol^{- 1}, respectively), indicating that chitosan -NH₂ protonation itself was not dependent on the type of acid. However, the electrical conductivity of suspensions of powdered chitosan in water evolved differently as these systems were titrated with citric or acid lactic. With citric acid, electrical conductivity remained virtually constant for acid concentration?<?of 15 mM, and then increased linearly as the acid concentration increased until 75 mM. Instead, with lactic acid, electrical conductivity progressively increased with increasing of acid concentration from 0 to 75 mM. The ζ-potential of chitosan dispersed particles was +28.5 mV and +52.1 mV in dispersions containing 10 mM of citric and lactic acids, respectively. The conjoint analysis of data from physicochemical analyses suggested that, contrarily to lactate anions, citrate anions bind more strongly on the electrical double layer of protonated, positively charged chains of chitosan, diminishing the inter-chains electrostatic repulsion, thus leading to a lower dispersibility of this polysaccharide in aqueous solutions of citric acid, compared to equimolar solutions of lactic acid.     

ELECTRON MICROSCOPE AUTORADIOGRAPHIC DETECTION OF SITES OF PROTEIN SYNTHESIS IN THE RABBIT RETINA MÜLLER CELLS

Rabbit retinas were incubated in medium containing 500 µCi of [³H]leucine for 3 min, and transferred to medium without isotope for another 7, 17, 37, 57, and 117 min. Retinal pieces were fixed in paraformaldehyde and osmium tetroxide and embedded in Epon. Thin sections were autoradiographed with Ilford L4 emulsion, and a quantitative study of silver grain distribution per Müller cell portion, and per Müller cell organelle, was carried out. Grain density per unit area was high over the middle cell portion at each incubation interval. Silver grains were numerous over background cytoplasm (which comprised free ribosomes) but their percentage was constant at all times and their relative concentration low. Silver grains were numerous and highly concentrated, at pulse incubation, over the rough endoplasmic reticulum (RER) and then decreased sharply, but this decline coincided with an increase over the Golgi complex, peaking at 20 min. Another peak appeared over the cell periphery at 60 min. These findings suggest the simultaneous synthesis of two types of proteins in Müller cells; structural proteins in background cytoplasm and proteins of secretory type in the RER.