Three-Dimensional Architecture and Biogenesis of Membrane Structures Associated with Hepatitis C Virus Replication

All positive strand RNA viruses are known to replicate their genomes in close association with intracellular membranes. In case of the hepatitis C virus (HCV), a member of the family Flaviviridae, infected cells contain accumulations of vesicles forming a membranous web (MW) that is thought to be the site of viral RNA replication. However, little is known about the biogenesis and three-dimensional structure of the MW. In this study we used a combination of immunofluorescence- and electron microscopy (EM)-based methods to analyze the membranous structures induced by HCV in infected cells. We found that the MW is derived primarily from the endoplasmic reticulum (ER) and contains markers of rough ER as well as markers of early and late endosomes, COP vesicles, mitochondria and lipid droplets (LDs). The main constituents of the MW are single and double membrane vesicles (DMVs). The latter predominate and the kinetic of their appearance correlates with kinetics of viral RNA replication. DMVs are induced primarily by NS5A whereas NS4B induces single membrane vesicles arguing that MW formation requires the concerted action of several HCV replicase proteins. Three-dimensional reconstructions identify DMVs as protrusions from the ER membrane into the cytosol, frequently connected to the ER membrane via a neck-like structure. In addition, late in infection multi-membrane vesicles become evident, presumably as a result of a stress-induced reaction. Thus, the morphology of the membranous rearrangements induced in HCV-infected cells resemble those of the unrelated picorna-, corona- and arteriviruses, but are clearly distinct from those of the closely related flaviviruses. These results reveal unexpected similarities between HCV and distantly related positive-strand RNA viruses presumably reflecting similarities in cellular pathways exploited by these viruses to establish their membranous replication factories.     

Germination performance of congeneric Styrax species from the Cerrado sensu lato areas and their distribution pattern in different physiognomies

When studying congeneric species, it is of reasonable importance to understand different ecophysiological performances which might determine the distribution of species in habitats with different natural resources. Styrax ferrugineus is exclusive and well adapted to the Brazilian Cerrado sensu stricto (s. str.); S. camporum is widely distributed in the Cerrado sensu lato (s. l.) areas, with young trees being observed at the edge of cerradão and other vegetation fragments; and S. pohlii occurs in permanently waterlogged soils of the Cerrado region, such as those of riparian forests. We tested the hypothesis that the higher the soil water content in the physiognomic gradient of the vegetation, the higher is the germination success of S. pohlii, but the lower is the germination success of S. ferrugineus. We also discuss whether gap conditions inside a cerradão fragment imply a high germination rates of seeds of S. camporum. Seeds from each of the three species were buried within nylon bags containing soil from the respective sites. Burial occurred in a Cerrado s. str., in understory and gap conditions of a cerradão, and in the understory of a riparian forest fragment, and lasted for 60, 120, 180 and 240 days, respectively, after the fruit dispersal time of each of the three species. After 60 days, a relationship was found showing that the percentage of germinated seeds diminished, and the percentage of damaged seeds increased as soil water content increased (Cerrado s. str. < cerradão gap < cerradão understory ? riparian forest). S. camporum still showed viable seeds 60 days after burial (DAB), and germinated seeds 120 DAB, indicating that it needed a longer time to germinate, which might be associated to its thicker seed coat, in relation to the other two species. The germination performance of each of the three species was the same in the gap and understory conditions of the cerradão. The higher concentration of adult S. camporum plants at the edge of vegetation fragments is not related to a particular high germination performance and seedling establishment.     

The endosomal protein Appl1 mediates Akt substrate specificity and cell survival in vertebrate development

During development of multicellular organisms, cells respond to extracellular cues through nonlinear signal transduction cascades whose principal components have been identified. Nevertheless, the molecular mechanisms underlying specificity of cellular responses remain poorly understood. Spatial distribution of signaling proteins may contribute to signaling specificity. Here, we tested this hypothesis by investigating the role of the Rab5 effector Appl1, an endosomal protein that interacts with transmembrane receptors and Akt. We show that in zebrafish, Appl1 regulates Akt activity and substrate specificity, controlling GSK-3beta but not TSC2. Consistent with this pattern, Appl1 is selectively required for cell survival, most critically in highly expressing tissues. Remarkably, Appl1 function requires its endosomal localization. Indeed, Akt and GSK-3beta, but not TSC2, dynamically associate with Appl1 endosomes upon growth factor stimulation. We propose that partitioning of Akt and selected effectors onto endosomal compartments represents a key mechanism contributing to the specificity of signal transduction in vertebrate development.     

Involvement of (Na+ + K+)-ATPase in binding and actions of palytoxin on human erythrocytes

Palytoxin (about 1 pM) increases the permeability of human erythrocytes. We now report its radiolabeling with 125I, followed by affinity purification on porcine kidney membranes. The resulting ligand binds fast and reversibly to intact erythrocytes. The Kd from velocity and equilibrium measurements is 2 X 10(-11) M, and the number of binding sites about 200 per cell. Binding is promoted by divalent cations (Ca2+ greater than Sr2+ greater than Ba2+) and by borate. It is inhibited by K+ (IC50 2 mM), ouabain (IC50 3 X 10(-9) M) and ouabagenin (IC50 6 X 10(-6) M). Conversely, [3H]ouabain is displaced by the substances and concentrations mentioned, and also by palytoxin (Ki 3 X 10(-11) M). Dog erythrocytes, which are known to possess a very low (Na+ + K+)-ATPase activity, are resistant to and lack specific binding sites for palytoxin. Binding of 125I-palytoxin, like that of [3H]ouabain, depends on the state of (Na+ + K+)-ATPase. ATP depletion decreases binding of both ligands to erythrocytes. Binding of 125I-palytoxin and [3H]ouabain to red cell stroma is partially restored by ATP. In contrast to [3H]ouabain, binding of 125I-palytoxin to red cell stroma is not promoted by Mg2+ and Pi. The data show that (a) all known promoters and inhibitors of palytoxin action on human red cells do so by enhancing or decreasing its binding, (b) (Na+ + K+)-ATPase serves as a receptor for palytoxin, and (c) the antagonism by ouabain is competitive at the receptor level. They support our previous hypothesis that palytoxin increases human erythrocyte permeability by formation of pores through (Na+ + K+)-ATPase or its close vicinity.     

Arrangement of disulfide bridges and positions of sulfhydryl groups in tetanus toxin

Tetanus toxin is a 151-kDa protein. The complete amino acid sequence is known. The mature toxin is made up of two peptide chains and contains 10 half-cystine residues. Treatment with 4-vinylpyridine in the presence of 6 M guanidine converted six of them into S-pyridylethyl cysteine residues as determined by amino acid analysis. When alkylation was preceded by mercaptolysis, all 10 half-cystine residues were recovered in the S-pyridylethylated form. It was therefore concluded that the toxin contains six sulfhydryl groups and two disulfide bonds. The positions of the residues carrying sulfhydryl groups and of those involved in disulfide bridges were determined by labelling of the toxin alternatively with 4-vinylpyridine or with 4-dimethylaminoazobenzene-4'-iodoacetamide (DABIA), directly or after mercaptolysis. The toxin derivatives were cleaved with cyanogen bromide and the elution patterns in reversed-phase HPLC compared. The chromatography components were identified by N-terminal amino acid sequence and amino acid composition. In the chromatography of the non-mercaptolysed, DABIA-treated sample four chromophore-carrying components were detected which could be demonstrated by N-terminal sequence analysis to correspond to six half-cystine-containing cyanogen bromide fragments. In the mercaptolysed, DABIA-treated sample three additional chromophore-carrying components were present, corresponding to two previously disulfide-linked cyanogen bromide fragments and one fragment which had contained an internal disulfide bridge. The HPLC patterns showed characteristic differences as the DABIA-labelled fragments were considerably more hydrophobic than the corresponding vinylpyridine-labelled fragments. It was established that the half-cystine residues in positions 26, 185, 198, 311, 868, and 1300 are present in the sulfhydryl form, that those in positions 438 and 466 are disulfide-bridged, thereby connecting the light and heavy chains of the toxin, and that those in positions 1076 and 1092 are disulfide-bridged, thereby giving rise to a loop in the heavy chain. During the progress of the investigations about 20% of the amino acid sequence previously predicted from DNA analysis was confirmed by protein-chemical methods.     

Inhibition of neurotransmitter release by botulinum neurotoxins and tetanus toxin at Aplysia synapses: role of the constituent chains

1. The effects on the release of transmitter by botulinum neurotoxins (BoNT; types A, B, E), tetanus toxin (TeTx), constituent chains or fragments were studied on identified cholinergic and non-cholinergic synapses in Aplysia. 2. Cholinergic synapses in the buccal ganglion were found to be greater than 100 fold more sensitive to extracellular application of BoNT than to TeTx whereas in non-cholinergic synapses of the cerebral ganglion the potencies of the toxins were reversed. When intracellularly applied TeTx and BoNT were found nearly equipotent. This disparity in the susceptibilities of BoNT and TeTx to inhibit transmission was attributed to differences in the toxin's acceptors or uptake systems in the two neurone types. 3. Micro-injection into cholinergic neurones of the isolated renatured toxins' chains showed that both light and heavy chains of BoNT are intracellularly required whereas the light chain of TeTx alone is sufficient. 4. The heavy chain of BoNT as well as that of TeTx were found to mediate internalization of active moieties via its amino-terminal half. Furthermore the heavy chain of one toxin could internalize the light chain of the other.     

Paracoccidioidomycosis: A comparative study of the evolutionary serologic,clinical and radiologic results for patients treated with ketoconazole or amphotericin B plus sulfonamides

A comparative study of two groups of patients with paracoccidioidomycosis was carried out with the objective of comparing the evolutionary serologic, clinical and radiologic results after 6, 12, 15 and 18 months of treatment with ketoconazole (22 patients) or amphotericin B plus sulfonamides (32 patients). The serologic data analyzed as a whole showed a tendency to sharper drops in antibody titers in the patients treated with ketoconazole. Clinically patients treated with ketoconazole fared better but the differences were not statistically significant. No statistical difference was detected between groups in terms of the results of radiologic evolution.     

GANGLIOSIDES IN NERVOUS TISSUE CULTURES AND BINDING OF 125I-LABELLED TETANUS TOXIN, A NEURONAL MARKER

Abstract— —Continuous cell lines, primary cell cultures derived from embryonic CNS, and homogenates made from adult and embryonic CNS were compared with respect to their lipid pattern and their ability to bind ¹²⁵I-labelled tetanus toxin. In parallel experiments de novo synthesis of gangliosides in the cell lines was studied, using [¹⁴C]glucosamine as precursor. Of the total lipid only gangliosides were specifically labelled by [¹⁴C]glucosamine. The patterns of the de novo synthesized gangliosides corresponded to those present in the respective cells.
Pronounced binding of ¹²⁵I-labelled toxin was only detectable in tissues containing long-chain gangliosides (ganglioside C which represents GDIb and GTI).
Accordingly, hybrid (neuroblastoma x glioma) cells, due to their lack of long-chain gangliosides, bound just-discernible amounts of labelled toxin. When previously exposed to gangliosides, their binding of tetanus toxin tremendously increased.
It was concluded that only the long-chain gangliosides in the neuronal cells are functionally involved in the binding of the tetanus toxin and that these acceptors of tetanus toxin can be transplanted.