The polyene antibiotic amphotericin B acts as a Ca2+ ionophore in sterol-containing liposomes

Amphotericin B (AmB) was shown to induce a Ca2+ influx across ergosterol- and cholesterol-containing large unilamellar liposomes, by following spectrophotometrically the formation of the Arsenazo III-Ca2+ complex. At equivalent antibiotic concentrations the Ca2+ influx was much more extensive through ergosterol-containing membranes (almost 100% with 1 microM AmB, 160 microM lipid) than through cholesterol-containing membranes (below 0.5 microM the influx of Ca2+ was negligible). In the presence of ergosterol-containing membranes the initial rate of Ca2+ influx had the same linear dependence on the ratio antibiotic/lipid whatever the lipid concentration, which was not the case in cholesterol-containing membranes. These results suggest that the channels responsible for the AmB-induced Ca2+ permeability across cholesterol- and ergosterol-containing liposomes have different structures.     

Veneza zonata (Hemiptera: Coreidae)/trypanosomatid relationship: action of hemolymph in vitro and experimental infection

The defense response of Veneza zonata (Hemiptera: Coreidae) against three different trypanosomatid infections was assessed: (1) strain 714TD, a Leptomonas which has V. zonata as vector of a plant trypanosomatid, (2) strain 563TD, a Leptomonas isolated from the digestive tract of Euchistus heros (Hemiptera: Pentatomidae), and (3) Leishmania (L.) amazonensis, a human parasite that cannot infect V. zonata. Experiments with V. zonata hemolymph showed agglutination only of L. (L.) amazonensis culture forms and hemocytic recognition was more intense with this strain. L. (L.) amazonensis also activated the prophenoloxidase system, whereas strains 714TD and 563TD did not activate this system but rather seemed to inhibit phenoloxidase activity. No flagellates were seen in the digestive tract, hemolymph, or salivary glands in insects infected with L. (L.) amazonensis. The digestive tract, the hemolymph, and the salivary glands of insects fed on tomatoes inoculated with 714TD are sequentially invaded by the flagellate, which is inoculated in plants together with saliva. Insects fed on tomatoes inoculated with 563TD exhibited culture forms in the digestive tract (6 days after) and hemocoel (three additional days); however, they died 12 to 14 days after exposure. The salivary glands in insects inoculated in the hemocoel with 714TD strain are rapidly invaded, whereas those with 563TD culture forms died approximately 24 h after infection. Bacterial proliferation in the hemocoel and hemocyte surface blebbing were seen in insects infected only with 563TD strain as the probable pathogenic mechanism of insect death.     

Dynamics of polymorphism of acidocalcisomes in<Emphasis Type="Italic"> Leishmania</Emphasis> parasites

Growth of Leishmania mexicana amazonensis promastigotes in different culture media resulted in structurally and chemically different acidocalcisomes. When grown in SDM-79 medium, the promastigotes showed large spherical acidocalcisomes of up to 1.2 m diameter distributed throughout the cell. X-ray microanalysis and elemental mapping of the organelles showed large amounts of oxygen, phosphorus, sodium, potassium, magnesium, calcium, and zinc. Immunofluorescence microscopy using antisera raised against a peptide sequence of the vacuolar-type proton pyrophosphatase of Arabidopsis thaliana that is conserved in the Leishmania enzyme, indicated localization in acidocalcisomes. When cells were transferred to Warrens medium, the acidocalcisomes transformed from spherical into branched tubular organelles. The labeling pattern of the vacuolar proton-pyrophosphatase, considered as a marker for the organelle, changed accompanying the structural changes of the acidocalcisomes, and the enzyme showed an apparently lower proton-transporting activity when measured in digitonin-permeabilized promastigotes. X-ray microanalysis and elemental mapping of these structures revealed the additional presence of iron. Together, the results reveal that the morphology and composition of acidocalcisomes are greatly influenced by the culture conditions.Electronic Supplementary Material Supplementary material is available in the online version of this article at     

Interaction of Leptomonas wallacei with the intestinal tract of its natural host Oncopeltus fasciatus (Hemiptera: Lygaeidae)

While investigating the distribution of Leptomonas wallacei in the intestine of the insect host Oncopeltus fasciatus, promastigotes and cyst-like forms of L. wallacei were observed only in the midgut ventricles V(3) and V(4) and the hindgut. In video-microscopy, once contact had occurred, the parasites remained attached to the midgut epithelium. Scanning electron microscopy revealed the adhesion of flagellates and cyst-like forms to the midgut wall and to the rectal pads of the hindgut. Using transmission electron microscopy, we observed that adhesion occurred mainly between the flagellum and the perimicrovillar membranes secreted by the midgut epithelium. No modifications were observed either in the parasite or in the epithelial cells. In the hindgut, adhesion to the superficial wax layer of the epithelial cells of the rectal pads was via flagellum. Host cell morphology appeared unaffected by L. wallacei.     

Leptomonas wallacei shows distinct morphology and surface carbohydrates composition along the intestinal tract of its host Oncopeltus fasciatus (Hemiptera: Lygaeidae) and in axenic culture

Leptomonas wallacei is a monoxenic trypanosomatid that colonizes the digestive tract of the phytophagous hemipteran Oncopeltus fasciatus. This infection was specific and took place exclusively in midgut intestinal ventricles V3 and V4, and in the hindgut. Abundances of parasites in the hindgut were 54% less than those in the hindgut. Parasites in the hindgut were more slender and had a longer flagellum than those from the hindgut, which were rounded, with a shorter flagellum. Moreover, hindgut forms expressed sugar residues on the cell surface, recognized by the lectins from Griffonia simplicifolia-I (alpha-galactose, alpha-N-acetyl-galactosamine) and Helix pomatia (N-acetyl-galactosamine); those sugar residues were not present in protozoa from the midgut. In culture, parasites were morphologically similar to midgut forms, but differed from them because they did not express sugar residues that bind to lectin (beta-galactose(1-3) N-acetyl-galactosamine) from Arachis hypogaea.     

Semaphorin 3A is a marker for disease activity and a potential immunoregulator in systemic lupus erythematosus

Introduction

Semaphorin 3A (sema3A) and neuropilin-1 (NP-1) play a regulatory role in immune responses and have a demonstrated effect on the course of collagen induced arthritis. This study was undertaken to evaluate the role of sema3A and NP-1 in the pathogenesis of systemic lupus erythematosus (SLE) and the specific effect of sema3A on the auto-reactive properties of B cells in SLE patients.

Methods

Thirty two SLE and 24 rheumatoid arthritis (RA) patients were assessed and compared with 40 normal individuals. Sema3A serum levels were measured and correlated with SLE disease activity. The in vitro effect of sema3A in reducing Toll-like receptor 9 (TLR-9) expression in B cells of SLE patients was evaluated.

Results

Sema3A serum levels in SLE patients were found to be significantly lower than in RA patients (55.04 ± 16.30 ng/ml versus 65.54 ± 14.82 ng/ml, P = 0.018) and lower yet than in normal individuals (55.04 ± 16.30 ng/ml versus 74.41 ± 17.60 ng/ml, P < 0.0001). Altered serum sema3A levels were found to be in inverse correlation with SLE disease activity, mainly with renal damage. The expression of both sema3A and NP-1 on B cells from SLE patients was significantly different in comparison with normal healthy individuals. Finally, when sema3A was co-cultured with cytosine-phosphodiester-guanine oligodeoxynucleotides (CpG-ODN)-stimulated B cells of SLE patients, their TLR-9 expression was significantly reduced, by almost 50% (P = 0.001).

Conclusions

This is the first study in which a reduced serum level of sema3A was found in association with SLE disease activity. It also raises the possibility that sema3A may have a regulatory function in SLE.     

A structural analysis of the natural egress of Toxoplasma gondii

Previous studies have analysed the process of Toxoplasma gondii egress with the aid of inducers, such as calcium ionophores. Although calcium transients have been successful in triggering T. gondii egress, the structural panorama of “natural” and artificial events should match. The present study approaches the natural egress of this parasite using super-resolution and electron microscopy and reveals lytic and non-lytic events of individual egress; this corroborates the use of calcium ionophore as a reliable tool to trigger parasite egress. Altogether, our data suggest that different signalling routes can converge to similar structural aspects in natural and induced egress.     

Spina cortica and Tapetum spinosus, two new microstructures of flight feathers: description, function and distribution in modern birds

The importance of feathers for the avian group has made them one of the most studied epidermal structures both from the morphological and evolutionary point of view. Surprisingly, our observations by Scanning Electron Microscopy detected the presence of two structures widely distributed within different avian groups and not yet described. In this paper we describe these two new structures (Spina cortica and Tapetum spinosus) and map their distribution within modern birds. The S. cortica is a thorn-like microstructure that grows on the barb cortex and the T. spinosus is the assemblage of these thorns. The distribution of these new structures among birds and their morphological diversity could be of great interest to taxonomists and evolutionary biologists interested in the origin of bird flight.     

Filtration stress-induced variations of peroxidase activity in cell suspension cultures of sycamore (Acer pseudoplatanus) cells

Filtration stress, consisting in the rapid filtration of Acer pseudoplatanus L. cell suspension cultures, resulted in significant differences between the peroxidases (EC 1.11.1.7) released during cell growth and those released after filtered cells were resuspended in fresh medium (recovery medium). These differences concerned mainly modifications of (i) the pH optimum of peroxidase activity (guaiacol as electron donor), (ii) the number and the pI values of the peroxidase isoenzymes as shown by isoelectric focusing, and (iii) the molecular weights of the different peroxidase fractions determined by gel filtration chromatography. The presence of 1 m M Li⁺ in the recovery medium inhibited the release of peroxidase and this effect was partially reversed by K⁺. The release of peroxidase by stressed cells was also strongly inhibited by Na₂CO₃ in the recovery medium. The results presented are consistent with the proposal that the characteristic isoperoxidase patterns induced by filtration stress might be used as a model to study the response of plant cells to stress.     

Spontaneous cystogenesis in vitro of a Brazilian strain of Toxoplasma gondii

Conversion of Toxoplasma gondii tachyzoites to the bradyzoite stage and tissue cyst formation in the life cycle of the parasite have crucial roles in the establishment of chronic toxoplasmosis. In this work we investigated the in vitro cystogenesis and behavior of the EGS strain, isolated from human amniotic fluid. We observed that tachyzoites of the EGS strain converted to intracellular cysts spontaneously in LLC-MK₂ epithelial cells, HSFS fibroblasts and C6 glial cell lineage. The peak of conversion occurred in the LLC-MK₂ cells after 4 days of infection, when 72.3 ± 15.9 of the infected cells contained DBA positive cysts. Using specific markers against bradyzoite, tachyzoite and cyst wall components, we confirmed stage conversion and distinguished immature from mature cysts. It was also observed that the deposition of cyst wall components occurred before the total conversion of parasites. Transmission electron microscopy confirmed the fully conversion of parasites presenting the typical characteristics of bradyzoites as the posterior position of the nucleus and the presence of amylopectin granules. A thick cyst wall was also detected. Besides, the scanning microscopy revealed that the intracyst matrix tubules were shorter than those from the parasitophorous vacuole intravacuolar network and were immersed in a granular electron dense material. The EGS strain spontaneously forms high burden of cysts in cell culture without artificial stress conditions, and constitutes a useful tool to study this stage of the T. gondii life cycle.