Role of cyclooxygenase-2 in Trypanosoma cruzisurvival in the early stages of parasite host-cell interaction

Chagas disease, caused by the intracellular protozoan Trypanosoma cruzi, is a serious health problem in Latin America. During this parasitic infection, the heart is one of the major organs affected. The pathogenesis of tissue remodelling, particularly regarding cardiomyocyte behaviour after parasite infection and the molecular mechanisms that occur immediately following parasite entry into host cells are not yet completely understood. When cells are infected with T. cruzi, they develop an inflammatory response, in which cyclooxygenase-2 (COX-2) catalyses rate-limiting steps in the arachidonic acid pathway. However, how the parasite interaction modulates COX-2 activity is poorly understood. In this study, the H9c2 cell line was used as our model and we investigated cellular and biochemical aspects during the initial 48 h of parasitic infection. Oscillatory activity of COX-2 was observed, which correlated with the control of the pro-inflammatory environment in infected cells. Interestingly, subcellular trafficking was also verified, correlated with the control of Cox-2 mRNA or the activated COX-2 protein in cells, which is directly connected with the assemble of stress granules structures. Our collective findings suggest that in the very early stage of the T. cruzi-host cell interaction, the parasite is able to modulate the cellular metabolism in order to survives.

Growth performance and body composition of giant trahira fingerlings fed diets with different protein and energy levels

The objective of this work was to determine the proper levels of protein and energy in diets of Hoplias lacerdae fingerlings. The dietary crude protein (CP) and gross energy (GE) levels for fingerlings of giant trahira were evaluated in a completely randomized 4x3 factorial design with 35, 39, 43 and 47% CP and 4,100, 4,300 and 4,500 kcal kg-1 of GE, and four replicates. The survival rate was 99.22%, and a linear improvement on the performance parameters was detected after increasing diet crude protein levels. Feed conversion ratio decreased with increasing levels of dietary protein and energy in the diets. A significant interaction between crude protein and gross energy was observed over body protein and mineral matter. Body lipid has increased linearly as gross energy in the diet increased. The retention of crude protein and energy showed a linear increasing with rising of crude protein levels in the diet. Crude protein level at 47% provides the best performance and energy retention, independently of the gross energy levels in the diet.

Biometric analysis of protein and oil contents of soybean genotypes in different environments

The objective of this work was to identify by biometric analyses the most stable soybean parents, with higher oil or protein contents, cultivated at different seasons and locations of the state of Minas Gerais, Brazil. Forty-nine genotypes were evaluated in the municipalities of Viçosa, Visconde do Rio Branco, and São Gotardo, in the state of Minas Gerais, from 2009 to 2011. Protein and oil contents were analyzed by infrared spectrometry using a FT-NIR analyzer. The effects of genotype, environment, and genotype x environment interaction were significant. The BARC-8 soybean genotype is the best parent to increase protein contents in the progenies, followed by BR 8014887 and CS 3032PTA276-3-4. Selection for high oil content is more efficient when the crossings involve the Suprema, CD 01RR8384, and A7002 genotypes, which show high mean phenotypic values, wide adaptability, and greater stability to environmental variation.

E. coli a-hemolysin: a membrane-active protein toxin

Alpha-Hemolysin is synthesized as a 1024-amino acid polypeptide, then intracellularly activated by specific fatty acylation. A second activation step takes place in the extracellular medium through binding of Ca2+ ions. Even in the absence of fatty acids and Ca2+ HlyA is an amphipathic protein, with a tendency to self-aggregation. However, Ca2+-binding appears to expose hydrophobic patches on the protein surface, facilitating both self-aggregation and irreversible insertion into membranes. The protein may somehow bind membranes in the absence of divalent cations, but only when Ca2+ (or Sr2+, or Ba2+) is bound to the toxin in aqueous suspensions, i.e., prior to its interaction with bilayers, can a-hemolysin bind irreversibly model or cell membranes in such a way that the integrity of the membrane barrier is lost, and cell or vesicle leakage ensues. Leakage is not due to the formation of proteinaceous pores, but rather to the transient disruption of the bilayer, due to the protein insertion into the outer membrane monolayer, and subsequent perturbations in the bilayer lateral tension. Protein or glycoprotein receptors for a-hemolysin may exist on the cell surface, but the toxin is also active on pure lipid bilayers.

The use of confocal laser scanning microscopy to analyze the process of parasitic protozoon-host cell interaction

In this communication we review the results obtained with the confocal laser scanning microscope to characterize the interaction of epimastigote and trypomastigote forms of Trypanosoma cruzi and tachyzoites of Toxoplasma gondii with host cells. Early events of the interaction process were studied by the simultaneous localization of sites of protein phosphorylation, revealed by immunocytochemistry, and sites of actin assembly, revealed by the use of labeled phaloidin. The results obtained show that proteins localized in the interaction sites are phosphorylated. The process of formation of the parasitophorous vacuole was monitored by labeling the host cell surface with fluorescent probes for lipids (PKH26), proteins (DTAF) and sialic acid (FITC-thiosemicarbazide) before interaction with the parasites. Evidence was obtained indicating transfer of components of the host cell surface to the parasite surface in the beginning of the interaction process. We also analyzed the distribution of cytoskeletal structures (microtubules and microfilaments visualized with specific antibodies), mitochondria (visualized with rhodamine 123), the Golgi complex (visualized with C6-NBD-ceramide) and the endoplasmic reticulum (visualized with anti-reticulin antibodies and DIOC6) during the evolution of intracellular parasitism. The results obtained show that some, but not all, structures change their position during evolution of the intracellular parasitism.

Preparation and purification of Flavobacterium heparinum chondroitinases AC and B by hydrophobic interaction chromatography

Flavobacterium heparinum is a soil bacterium that produces several mucopolysaccharidases such as heparinase, heparitinases I and II, and chondroitinases AC, B, C and ABC. The purpose of the present study was to optimize the preparation of F. heparinum chondroitinases, which are very useful tools for the identification and structural characterization of chondroitin and dermatan sulfates. We observed that during the routine procedure for cell disruption (ultrasound, 100 kHz, 5 min) some of the chondroitinase B activity was lost. Using milder conditions (2 min), most of the chondroitinase B and AC protein was solubilized and the enzyme activities were preserved. Tryptic soy broth without glucose was the best culture medium both for bacterial growth and enzyme induction. Chondroitinases AC and B were separated from each other and also from glucuronidases and sulfatases by hydrophobic interaction chromatography on HP Phenyl-Sepharose. A rapid method for screening of the column fractions was also developed based on the metachromatic shift of the color of dimethylmethylene blue.

Control of the adrenocortical cell cycle: interaction between FGF2 and ACTH

FGF2 elicits a strong mitogenic response in the mouse Y-1 adrenocortical tumor cell line, that includes a rapid and transient activation of the ERK-MAPK cascade and induction of the c-Fos protein. ACTH, itself a very weak mitogen, blocks the mitogenic response effect of FGF2 in the early and middle G1 phase, keeping both ERK-MAPK activation and c-Fos induction at maximal levels. Probing the mitogenic response of Y-1 cells to FGF2 with ACTH is likely to uncover reactions underlying the effects of this hormone on adrenocortical cell growth.

A receptor for infectious and cellular prion protein

Prions are an unconventional form of infectious agents composed only of protein and involved in transmissible spongiform encephalopathies in humans and animals. The infectious particle is composed by PrPsc which is an isoform of a normal cellular glycosyl-phosphatidylinositol (GPI) anchored protein, PrPc, of unknown function. The two proteins differ only in conformation, PrPc is composed of 40% a helix while PrPsc has 60% ß-sheet and 20% a helix structure. The infection mechanism is trigged by interaction of PrPsc with cellular prion protein causing conversion of the latter's conformation. Therefore, the infection spreads because new PrPsc molecules are generated exponentially from the normal PrPc. The accumulation of insoluble PrPsc is probably one of the events that lead to neuronal death. Conflicting data in the literature showed that PrPc internalization is mediated either by clathrin-coated pits or by caveolae-like membranous domains. However, both pathways seem to require a third protein (a receptor or a prion-binding protein) either to make the connection between the GPI-anchored molecule to clathrin or to convert PrPc into PrPsc. We have recently characterized a 66-kDa membrane receptor which binds PrPc in vitro and in vivo and mediates the neurotoxicity of a human prion peptide. Therefore, the receptor should have a role in the pathogenesis of prion-related diseases and in the normal cellular process. Further work is necessary to clarify the events triggered by the association of PrPc/PrPsc with the receptor.

A study of the interaction between Helicobacter pylori and components of the human fibrinolytic system

The interaction of plasminogen, tissue plasminogen activator (t-PA) and urokinase with a clinical strain of Helicobacter pylori was studied. Plasminogen bound to the surface of H. pylori cells in a concentration-dependent manner and could be activated to the enzymatic form, plasmin, by t-PA. Affinity chromatography assays revealed a plasminogen-binding protein of 58.9 kDa in water extracts of surface proteins. Surface-associated plasmin activity, detected with the chromogenic substrate CBS 00.65, was observed only when plasminogen and an exogenous activator were added to the cell suspension. The two physiologic plasminogen activators, t-PA and urokinase, were also shown to bind to and remain active on the surface of bacterial cells. epsilon-Aminocaproic acid caused partial inhibition of t-PA binding, suggesting that the kringle 2 structure of this activator is involved in the interaction with surface receptors. The activation of plasminogen by t-PA, but not urokinase, strongly depended on the presence of cells and a 25-fold enhancer effect on the initial velocity of activation by t-PA compared to urokinase was established. Furthermore, a relationship between cell concentration and the initial velocity of activation was demonstrated. These findings support the concept that plasminogen activation by t-PA on the bacterial surface is a surface-dependent reaction which offers catalytic advantages.

Cycle modulation of insulin-like growth factor-binding protein 1 in human endometrium

Endometrium is one of the fastest growing human tissues. Sex hormones, estrogen and progesterone, in interaction with several growth factors, control its growth and differentiation. Insulin-like growth factor 1 (IGF-1) interacts with cell surface receptors and also with specific soluble binding proteins. IGF-binding proteins (IGF-BP) have been shown to modulate IGF-1 action. Of six known isoforms, IGF-BP-1 has been characterized as a marker produced by endometrial stromal cells in the late secretory phase and in the decidua. In the current study, IGF-1-BP concentration and affinity in the proliferative and secretory phase of the menstrual cycle were measured. Endometrial samples were from patients of reproductive age with regular menstrual cycles and taking no steroid hormones. Cytosolic fractions were prepared and binding of 125I-labeled IGF-1 performed. Cross-linking reaction products were analyzed by SDS-polyacrylamide gel electrophoresis (7.5%) followed by autoradiography. 125I-IGF-1 affinity to cytosolic proteins was not statistically different between the proliferative and secretory endometrium. An approximately 35-kDa binding protein was identified when 125I-IGF-1 was cross-linked to cytosol proteins. Secretory endometrium had significantly more IGF-1-BP when compared to proliferative endometrium. The specificity of the cross-linking process was evaluated by the addition of 100 nM unlabeled IGF-1 or insulin. Unlabeled IGF-1 totally abolished the radioactivity from the band, indicating specific binding. Insulin had no apparent effect on the intensity of the labeled band. These results suggest that IGF-BP could modulate the action of IGF-1 throughout the menstrual cycle. It would be interesting to study this binding protein in other pathologic conditions of the endometrium such as adenocarcinomas and hyperplasia.

Insights into the physiological function of cellular prion protein

Prions have been extensively studied since they represent a new class of infectious agents in which a protein, PrPsc (prion scrapie), appears to be the sole component of the infectious particle. They are responsible for transmissible spongiform encephalopathies, which affect both humans and animals. The mechanism of disease propagation is well understood and involves the interaction of PrPsc with its cellular isoform (PrPc) and subsequently abnormal structural conversion of the latter. PrPc is a glycoprotein anchored on the cell surface by a glycosylphosphatidylinositol moiety and expressed in most cell types but mainly in neurons. Prion diseases have been associated with the accumulation of the abnormally folded protein and its neurotoxic effects; however, it is not known if PrPc loss of function is an important component. New efforts are addressing this question and trying to characterize the physiological function of PrPc. At least four different mouse strains in which the PrP gene was ablated were generated and the results regarding their phenotype are controversial. Localization of PrPc on the cell membrane makes it a potential candidate for a ligand uptake, cell adhesion and recognition molecule or a membrane signaling molecule. Recent data have shown a potential role for PrPc in the metabolism of copper and moreover that this metal stimulates PrPc endocytosis. Our group has recently demonstrated that PrPc is a high affinity laminin ligand and that this interaction mediates neuronal cell adhesion and neurite extension and maintenance. Moreover, PrPc-caveolin-1 dependent coupling seems to trigger the tyrosine kinase Fyn activation. These data provide the first evidence for PrPc involvement in signal transduction.

Phagocytosis by macrophages mediated by receptors for denatured proteins - dependence on tyrosine protein kinases

Previous studies have demonstrated that some components of the leukocyte cell membrane, CR3 (Mac-1, CD11b/CD18) and p150/95, are able to bind to denatured proteins. Thus, it is of interest to know which effector functions of these cells can be triggered by these receptors when they interact with particles or surfaces covered with denatured proteins. In the present study we analyzed their possible role as mediators of phagocytosis of red cells covered with denatured bovine serum albumin (BSA) by mouse peritoneal macrophages. We observed that a) macrophages are able to recognize (bind to) these red cells, b) this interaction can be inhibited by denatured BSA in the fluid phase, c) there is no phagocytosis of these particles by normal macrophages, d) phagocytosis mediated by denatured BSA can be, however, effectively triggered in inflammatory macrophages induced by glycogen or in macrophages activated in vivo with LPS, and e) this phagocytic capacity is strongly dependent on the activity of tyrosine protein kinases in its signal transduction pathway, as demonstrated by using three kinds of enzyme inhibitors (genistein, quercetin and herbimycin A).

A calcineurin inhibitory protein overexpressed in Down's syndrome interacts with the product of a ubiquitously expressed transcript

The Down's syndrome candidate region 1 (DSCR1) protein, encoded by a gene located in the human chromosome 21, interacts with calcineurin and is overexpressed in Down's syndrome patients. As an approach to clarifying a putative function for this protein, in the present study we used the yeast two-hybrid system to identify DSCR1 partners. The two-hybrid system is a method that allows the identification of protein-protein interactions through reconstitution of the activity of the yeast GAL 4 transcriptional activator. The gene DSCR1 fused to the GAL 4 binding domain (BD) was used to screen a human fetal brain cDNA library cloned in fusion with the GAL 4 activation domain (AD). Three positive clones were found and sequence analysis revealed that all the plasmids coded for the ubiquitously expressed transcript (UXT). UXT, which is encoded in human Xp11, is a 157-amino acid protein present in both cytosol and nucleus of the cells. This positive interaction of DSCR1 and UXT was confirmed in vivo by mating the yeast strain AH109 (MATa)expressing AD-UXT with the strain Y187 (MATalpha) expressing BD-DSCR1, and in vitro by co-immunoprecipitation experiments. These results may help elucidate a new function for DSCR1 and its participation in Down's syndrome pathogenesis.

Increased expression and purification of soluble iron-regulatory protein 1 from Escherichia coli co-expressing chaperonins GroES and GroEL

Iron is an essential metal for all living organisms. However, iron homeostasis needs to be tightly controlled since iron can mediate the production of reactive oxygen species, which can damage cell components and compromise the integrity and/or cause DNA mutations, ultimately leading to cancer. In eukaryotes, iron-regulatory protein 1 (IRP1) plays a central role in the control of intracellular iron homeostasis. This occurs by interaction of IRP1 with iron-responsive element regions at 5' of ferritin mRNA and 3' of transferrin mRNA which, respectively, represses translation and increases mRNA stability. We have expressed IRP1 using the plasmid pT7-His-hIRP1, which codifies for human IRP1 attached to an NH2-terminal 6-His tag. IRP1 was expressed in Escherichia coli using the strategy of co-expressing chaperonins GroES and GroEL, in order to circumvent inclusion body formation and increase the yield of soluble protein. The protein co-expressed with these chaperonins was obtained mostly in the soluble form, which greatly increased the efficiency of protein purification. Metal affinity and FPLC ion exchange chromatography were used in order to obtain highly purified IRP1. Purified protein was biologically active, as assessed by electrophoretic mobility shift assay, and could be converted to the cytoplasmic aconitase form. These results corroborate previous studies, which suggest the use of folding catalysts as a powerful strategy to increase protein solubility when expressing heterologous proteins in E. coli.