Internalization of components of the host cell plasma membrane during infection by Trypanosoma cruzi

Epimastigote and trypomastigote forms of Trypanosoma cruzi attach to the macrophage surface and are internalized with the formation of a membrane bounded vacuole, known as the parasitophorous vacuole (PV). In order to determine if components of the host cell membrane are internalized during formation of the PV we labeled the macrophage surface with fluorescent probes for proteins, lipids and sialic acid residues and then allowed the labeled cells to interact with the parasites. The interaction process was interrupted after 1 hr at 37ºC and the distribution of the probes analyzed by confocal laser scanning microscopy. During attachment of the parasites to the macrophage surface an intense labeling of the attachment regions was observed. Subsequently labeling of the membrane lining the parasitophorous vacuole containing epimastigote and trypomastigote forms was seen. Labeling was not uniform, with regions of intense and light or no labeling. The results obtained show that host cell membrane lipids, proteins and sialoglycoconjugates contribute to the formation of the membrane lining the PV containing epimastigote and trypomastigote T. cruzi forms. Lysosomes of the host cell may participate in the process of PV membrane formation.

Anestésicos locais: interação com membranas de eritrócitos de sangue humano, estudada por ressonância magnética nuclear de ¹H e 31P

The literature carries many theories about the mechanism of action of local anesthetics (LA). We can highlight those focusing the direct effect of LA on the sodium channel protein and the ones that consider the interaction of anesthetic molecules with the lipid membrane phase. The interaction between local anesthetics and human erythrocyte membranes has been studied by ¹H and 31P nuclear magnetic resonance spectroscopy. It was found that lidocaine (LDC) and benzocaine (BZC) bind to the membranes, increase the mobility of the protons of the phospholipid's acyl chains, and decrease the mobility and/or change the structure of the polar head groups. The results indicate that lidocaine molecules are inserted across the polar and liquid interface of the membrane, establishing both electrostatic (charged form) and hydrophobic (neutral form) interactions. Benzocaine locates itself a little deeper in the bilayer, between the interfacial glycerol region and the hydrophobic core. These changes in mobility or conformation of membrane lipids could affect the Na+-channel protein insertion in the bilayer, stabilizing it in the inactivated state, thus causing anesthesia.

Effect of trolox C on cardiac contracture induced by hydrogen peroxide

Hydrogen peroxide (H2O2) perfused into the aorta of the isolated rat heart induces a positive inotropic effect, with cardiac arrhythmia such as extrasystolic potentiation or cardiac contractures, depending on the dose. The last effect is similar to the "stone heart" observed in reperfusion injury and may be ascribed to lipoperoxidation (LPO) of the membrane lipids, to protein damage, to reduction of the ATP level, to enzymatic alterations and to cardioactive compounds liberated by LPO. These effects may result in calcium overload of the cardiac fibers and contracture ("stone heart"). Hearts from male Wistar rats (300-350 g) were perfused at 31oC with Tyrode, 0.2 mM trolox C, 256 mM H2O2 or trolox C + H2O2. Cardiac contractures (baseline elevation of the myograms obtained) were observed when hearts were perfused with H2O2 (Tyrode: 5.9 ± 3.2; H2O2: 60.5 ± 13.9% of the initial value); perfusion with H2O2 increased the LPO of rat heart homogenates measured by chemiluminescence (Tyrode: 3,199 ± 259; H2O2: 5,304 ± 133 cps mg protein-1 60 min-1), oxygen uptake (Tyrode: 0.44 ± 0.1; H2O2: 3.2 ± 0.8 nmol min-1 mg protein-1) and malonaldehyde (TBARS) formation (Tyrode: 0.12 ± 0; H2O2: 0.37 ± 0.1 nmol/ml). Previous perfusion with 0.2 mM trolox C reduced the LPO (chemiluminescence: 4,098 ± 531), oxygen uptake (0.51 ± 0) and TBARS (0.13 ± 0) but did not prevent the H2O2-induced contractures (33.3 ± 16%). ATP (Tyrode: 2.84 ± 0; H2O2: 0.57 ± 0) and glycogen levels (Tyrode: 0.46 ± 0; H2O2: 0.26 ± 0) were reduced by H2O2. Trolox did not prevent these effects (ATP: 0.84 ± 0 and glycogen: 0.27 ± 0). Trolox C is known to be more effective than a -tocopherol or g -tocopherol in reducing LPO though it lacks the phytol portion of vitamin E to be fixed to the cell membranes. Trolox C, unlike vitamin A, did not prevent the glycogen reduction induced by H2O2. Trolox C induced a positive chronotropic effect that resulted in higher energy consumption. The reduction of energy level seemed to be more important than LPO in the mechanism of H2O2-induced contracture

A mechanistic view of mitochondrial death decision pores

Mitochondria increase their outer and inner membrane permeability to solutes, protons and metabolites in response to a variety of extrinsic and intrinsic signaling events. The maintenance of cellular and intraorganelle ionic homeostasis, particularly for Ca2+, can determine cell survival or death. Mitochondrial death decision is centered on two processes: inner membrane permeabilization, such as that promoted by the mitochondrial permeability transition pore, formed across inner membranes when Ca2+ reaches a critical threshold, and mitochondrial outer membrane permeabilization, in which the pro-apoptotic proteins BID, BAX, and BAK play active roles. Membrane permeabilization leads to the release of apoptogenic proteins: cytochrome c, apoptosis-inducing factor, Smac/Diablo, HtrA2/Omi, and endonuclease G. Cytochrome c initiates the proteolytic activation of caspases, which in turn cleave hundreds of proteins to produce the morphological and biochemical changes of apoptosis. Voltage-dependent anion channel, cyclophilin D, adenine nucleotide translocase, and the pro-apoptotic proteins BID, BAX, and BAK may be part of the molecular composition of membrane pores leading to mitochondrial permeabilization, but this remains a central question to be resolved. Other transporting pores and channels, including the ceramide channel, the mitochondrial apoptosis-induced channel, as well as a non-specific outer membrane rupture may also be potential release pathways for these apoptogenic factors. In this review, we discuss the mechanistic models by which reactive oxygen species and caspases, via structural and conformational changes of membrane lipids and proteins, promote conditions for inner/outer membrane permeabilization, which may be followed by either opening of pores or a rupture of the outer mitochondrial membrane.

Perimicrovillar membrane assembly: the fate of phospholipids synthesised by the midgut of Rhodnius prolixus

In this study, we describe the fate of fatty acids that are incorporated from the lumen by the posterior midgut epithelium of Rhodnius prolixus and the biosynthesis of lipids. We also demonstrate that neutral lipids (NL) are transferred to the haemolymphatic lipophorin (Lp) and that phospholipids remain in the tissue in which they are organised into perimicrovillar membranes (PMMs). 3H-palmitic acid added at the luminal side of isolated midguts of R. prolixus females was readily absorbed and was used to synthesise phospholipids (80%) and NL (20%). The highest incorporation of 3H-palmitic acid was on the first day after a blood meal. The amounts of diacylglycerol (DG) and triacylglycerol synthesised by the tissue decreased in the presence of Lp in the incubation medium. The metabolic fates of 3H-lipids synthesised by the posterior midgut were followed and it was observed that DG was the major lipid released to Lp particles. However, the majority of phospholipids were not transferred to Lp, but remained in the tissue. The phospholipids that were synthesised and accumulated in the posterior midgut were found to be associated with Rhodnius luminal contents as structural components of PMMs.

Preserved xenogenic amniotic membrane as a patch on the repair of superficial corneal ulcers in rabbits

The aim of this study was to evaluate the effects of canine amniotic membrane, previously preserved in glycerin, used as a patch on the repair of experimentally-made superficial corneal ulcers and to compare corneal epithelization between the treated and non-treated groups. Xenogeneic amniotic membranes were collected aseptically and preserved in 99% glycerin at room temperature. Each animal was anesthetized and submitted to superficial corneal keratectomy of the left eye. The treated group received a fragment of canine amniotic membrane as a patch, while the control group had no treatment. The treated group showed blepharospasm, ocular discharge and conjunctival congestion. The membrane accelerated corneal repair in the beginning of the process, however, it delayed its conclusion (p<0.05). Treated eyes showed greater vessel formation and decreased corneal transparency (p<0.05). The stroma of the control group was thicker than that of the treated group (p<0.05). We suggest that amniotic membrane used in this manner can be applied as a therapy for superficial corneal ulcers in the beginning phases of the repair process.

Comparative studies of Yersinia pestis outer membrane isolation techniques and their potential use in plague epidemiology

In the present study three techniques for obtaining outer membrane enriched fractions from Yersinia pestis were evaluated. The techniques analysed were: differential solubilization of the cytoplasmic membrane with Sarkosyl or Triton X-100, and centrifugation in sucrose density gradients. The sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of outer membrane isolated by the different methods resulted in similar protein patterns. The measurement of NADH-dehydrogenase and succinate dehydrogenase (inner membrane enzymes) indicated that the outer membrane preparations obtained by the three methods were pure enough for analytical studies. In addition, preliminary evidences on the potential use of outer membrane proteins for the identification of geographic variants of Y. pestis wild isolates are presented.

Analysis of proteins from membrane and soluble fractions of Giardia duodenalis trophozoites of two Brazilian axenic strains

In the present study, we have analyzed by sodium docecyl sulphate - polyacrilamide gel electrophoresis (SDS-PAGE), immunoblotting and Concanavalin A blotting (Con A blotting) proteins of membrane fractions and soluble fractions obtained from Giardia duodenalis trophozoites of two axenic strains isolated in Brazil from a symptomatic (BTU-11) and an asymptomatic patient (BTU-10), as compared to the reference strain Portland 1. Both Brazilian strains showed a complex and homogeneous electrophoretic pattern of proteins, but some differences could be observed. Several glycoproteins were detected, particularly the proteins of 81, 72, 59 kDa and the protein of 62 kDa in the membrane proteins and cytosol, respectively. Many antigenic components were revealed by anti-Giardia rabbit IgG antibodies in the immunoblotting analysis. Among these components, the membrane protein of 32 kDa and the cytosol protein of 30 kDa could be related to giardin, as previously demonstrated.

MEMBRANE FRACTIONS FROM Strongyloides venezuelensis IN THE IMMUNODIAGNOSIS OF HUMAN STRONGYLOIDIASIS

Strongyloides venezuelensis is a parasitic nematode of rodents frequently used to obtain heterologous antigens for the immunological diagnosis of human strongyloidiasis. The aim of this study was to evaluate membrane fractions from S. venezuelensis for human strongyloidiasis immunodiagnosis. Soluble and membrane fractions were obtained in phosphate saline (SS and SM) and Tris-HCl (TS and TM) from filariform larvae of S. venezuelensis. Ninety-two serum samples (n = 92) were obtained from 20 strongyloidiasis patients (Group I), 32 from patients with other parasitic diseases (Group II), and 40 from healthy individuals (Group III), and were analyzed by enzyme-linked immunosorbent assay (ELISA). Soluble fractions (SS and TS) showed 90.0% sensitivity and 88.9% specificity, whereas the membrane fractions (SM and TM) showed 95.0% sensitivity and 94.4% specificity. The present results suggest the possible use of membrane fractions of S. venezuelensis as an alternative antigen for human strongyloidiasis immunodiagnosis.

Serum lipids of pemphigus foliaceus patients on long-term glucocorticoid therapy

Endemic pemphigus foliaceus, and long-term corticotherapy may affect serum lipid levels. The aim of this study was to compare serum lipids of pemphigus foliaceus patients on glucocorticoid therapy to a healthy control group. Fifteen patients receiving prednisone (0.33 ± 0.22mg/kg) for at least 12 months and 15 controls were submitted to 48-h food intake records, anthropometry, and biochemical measurements. Data were compared by chi2, Mann-Whitney and Student "t" tests. The groups were matched for gender, age, weight, body mass index, arm circumference and triceps skin fold. No differences were observed in relation to energy, fat, protein and carbohydrate daily intakes, total cholesterol, HDL, LDL, uric acid, and serum creatinine levels. Pemphigus foliaceus patients had higher triglyceride [159 (64-371) vs. 100 (45-133) mg/dl], VLDL [32 (13-74) vs. 20 (9-114) mg/dl] and ESR [44 (9-87) vs. 7 (1-30) mm/h] levels than controls, probably due to metabolic effects of inflammatory disease and corticotherapy.

Occurrence of Mansonella ozzardi diagnosed using a polycarbonate membrane in a riverside population of Lábrea in the Western Brazilian Amazon

Abstract: INTRODUCTION Mansonella ozzardi is a widely distributed filaria worm in the Amazon region. This study aimed to determine the prevalence of M. ozzardi infection in riverine communities of Lábrea municipality, Amazonas State, Brazil. METHODS A diagnostic blood filtration method in a polycarbonate membrane was used. RESULTS M. ozzardi was found in 50.3% of the sample, with the highest prevalence in farmers/fishermen (69.4%; χ 2 = -19.14, p<0.001). The prevalence was higher in longer-term residents (≥11 years; 60.2%). CONCLUSIONS M. ozzardi infection rates are high near the Purus River, much greater than those previously reported based on diagnosis using thick blood smears.

Effects of a novel fermented soy product on the serum lipids of hypercholesterolemic rabbits

OBJECTIVE: To assess the effect of a new feed soy product fermented by Enterococcus faecium and Lactobacillus jugurti on the serum lipid levels of rabbits with induced hypercholesterolemia. METHODS: Thirty-two rabbits were divided into 4 groups as follows: 1) control (C); 2) hypercholesterolemic (H); 3) hypercholesterolemic + fermented product (HPF); and 4) control + fermented product (CPF). The H and HPF groups were fed with a diet with 0.15% (p/p) cholesterol in the first 15 days. C and CPF groups received regular food preparation. The HPF and CPF groups received 10 mL daily of the fermented 30 days. Blood samples were drawn at the beginning of the study and at the 15th and 30th days. Concentrations of total cholesterol, HDL-cholesterol, and triglycerides were analyzed. RESULTS: After 15 days, the HPF group showed a total cholesterol concentration lower (18.4%) than that of the H group (p=0.05), but this difference disappeared after 30 days. No change was observed in total cholesterol levels of C and CPF groups. After 15 days, the HDL-cholesterol was higher (17.8%) in the HPF group, but the triglyceride levels remained unchanged in all groups during the same period of time. CONCLUSION: The soy fermented product caused an 18.4% reduction in total cholesterol and a 17.8% increase in the HDL-fraction. It may, therefore, be a possible coadjutor in the treatment of hypercholesterolemia.

Lipid metabolism in rats fed diets containing different types of lipids

OBJECTIVE: To assess the effect of different types of lipid diets on the lipid metabolism of aging rats. METHODS: Fifty male Wistar rats were studied from the time of weaning to 12 and 18 months of age. Their diets were supplemented as follows: with soybean oil (S), canola oil (CA), lard and egg yolk (LE), and canola oil + lard and egg yolk (CA + LE). Blood pressure (BP) was measured every month, and the heart/body ratio (H/BR) was determined. The rats were euthanized at the age of 12 and 18 months, and blood samples were collected for lipid analysis as follows: total cholesterol (TC), LDL-C, VLDL-C, HDL-C, triglycerides (TG), and glucose. RESULTS: The type of oil ingested by the animals significantly altered BP, H/BR, and serum lipid levels in rats at 12 and 18 months. No difference was observed in the survival curve of the animals in the different groups. The LE group had the highest BP, and the CA group was the only one in which BP did not change with aging. A reduction in the H/BR was observed in the LE and CA+LE animals. At the age of 12 months, differences in TC, HDL-C, LDL-C, VLDL-C, TG, and glucose were observed. At the age of 18 months, a significant difference in TC, HDL-C, and glucose was observed. The highest TC value was found in the CA group and the lowest in the S group. CONCLUSION: No increase in BP occurred, and an improvement was evident in the lipid profile of rats fed a diet supplemented with CA, in which an elevation in HDL-C levels was observed, as compared with levels with the other types of diet.

Seasonal variations in the intermediate metabolism of the crayfish Parastacus brasiliensis (Crustacea, Decapoda, Parastacidae) in the natural environment and experimental culture

The goal of this study was to evaluate the effects of seasonal variations on energy metabolism in different tissues of the freshwater crayfish Parastacus brasiliensis (von Martens, 1869). Crayfish were collected monthly from January 2001 to January 2003 in São Francisco de Paula, Rio Grande do Sul, Brazil, in a stream and in a culture tank. Haemolymph samples were collected from each crayfish in the field with a syringe, by puncturing the membrane at the base of the chelipeds. Hepatopancreas, gills, and abdominal muscle were removed for determination of free glucose, glycogen, total lipids, and triglycerides. The haemolymph samples were used for determination of glucose, total proteins, total lipids, and triglycerides. Statistical analysis revealed significant differences in biochemical composition in crayfish collected in the stream compared to the experimental tank during the year, principally in glucose and triglycerides in haemolymph, glycogen and total lipids in all tissues study, and triglycerides only in abdominal muscle. The regular food intake partially modified these seasonal variations of the metabolic pattern. Environmental conditions (e.g., food availability and water temperature) and reproductive period appeared to be the main factors influencing the seasonal patterns of variation in energy metabolism.

The cell surface of Trypanosoma cruzi

The cell surface of trypanosomatids is formed by the plasma membrane and a layer of sub-pellicular microtubules which are connected to the plasma membrane. The plasma membrane is composed by proteins, lipids and carbohydrates which form the glycocalix. In this paper we will review briefly aspects related to the organization of the cell surface of Trypanosoma cruzi.