Neurotoxic activity induced by a haemolytic substance in the extract of the marine sponge Geodia corticostylifera

In our search for marine bioactive compounds we chose a Brazilian Coast sponge, Geodia corticostylifera (Demospongiae), whose extracts showed previously antibacterial and antifungal activities. In the present work we studied the following toxic properties of G. corticostylifera extract: neurotoxic (in mouse neuromuscular junction); mouse acute toxicity (IP) and haemolytic (against mouse and frog erythrocytes). Insertion of ionic channels in planar lipid bilayers in presence of a haemolytic purified fraction of the extract was observed. The toxic activities of G. corticostylifera crude extract are related to the formation of ionic pores in the cell membrane, which induce the release of haemoglobin from erythrocytes, and depolarization of nerve and muscle membranes. These last physiological effects cause the blockade of the diaphragm contractions, leading to death through respiratory arrest.     

Mitochondrial calcium signalling in cell death

The development of targeted probes (based on the molecular engineering of luminescent or fluorescent proteins) has allowed the specific measurement of [Ca2+] in intracellular organelles or cytoplasmic subdomains. This approach gave novel information on different aspects of cellular Ca2+ homeostasis. Regarding mitochondria, it was possible to demonstrate that, upon physiological stimulation of cells, Ca2+ is rapidly accumulated in the matrix. We will discuss the basic characteristics of this process, its role in modulating physiological and pathological events, such as the regulation of aerobic metabolism and the induction of cell death, and new insight into the regulatory mechanisms operating in vivo.     

Mutational analysis of the HLA class II interaction with Epstein-Barr virus glycoprotein 42

Entry of Epstein-Barr virus (EBV) into B lymphocytes requires the binding of viral glycoprotein 42 (gp42), a C-type lectin family member, to HLA class II. Recently, the structure of the gp42:HLA-DR1 complex was determined. In order to confirm the interaction as determined in the structural study and to identify other potential interactive residues, a mutational analysis of HLA class II was performed. A secreted form of gp42 (sgp42) reacted with a conformation-specific monoclonal antibody and blocked EBV infection. The binding of sgp42 and EBV entry to two sets of HLA class II mutants were tested. The first set of mutants were based on the known interaction of the C-type lectin Ly49A with HLA class I, and the second set of mutants were based on the identified interface in the gp42:HLA-DR1 complex. As expected, none of the mutants that would be predicted to interfere with the interaction of Ly49A with class I affected the interaction of gp42 with HLA class II, whereas mutants in amino acids identified in the gp42:HLA-DR1 structure inhibited sg42 binding to class II. In general, sgp42 binding correlated with efficient entry of EBV, as demonstrated by the necessity of glutamic acid 46 or arginine 72 in class II molecules. Furthermore, other HLA class II residues buried within the interface of gp42 and HLA class II when mutated had either no effect or a decrease in both binding and entry and implicate a region of class II important in stabilizing the interaction with gp42. These studies provide insight into the entry and fusion processes of the critical interaction between gp42 and HLA class II.     

Determinants of the host range restriction of replication of bovine parainfluenza virus type 3 in rhesus monkeys are polygenic

The Kansas strain of bovine parainfluenza virus type 3 (BPIV3) is 100- to 1,000-fold restricted in replication in the respiratory tracts of nonhuman primates compared to human PIV3 (HPIV3), an important pathogen of infants and young children. BPIV3 is also restricted in replication in human infants and children, yet it is immunogenic and is currently being evaluated in clinical trials as a vaccine candidate to protect against illness caused by HPIV3. We have examined the genetic basis for the host range attenuation phenotype of BPIV3 by exchanging each open reading frame (ORF) of a recombinant wild-type HPIV3 with the analogous ORF from BPIV3, with the caveats that the multiple ORFs of the P gene were exchanged as a single unit and that the HN and F genes were exchanged as a single unit. Recombinant chimeric bovine-human PIV3s were recovered from cDNA, and the levels of viral replication in vitro and in the respiratory tract of rhesus monkeys were determined. Recombinant chimeric HPIV3s bearing the BPIV3 N or P ORF were highly attenuated in the upper and lower respiratory tracts of monkeys, whereas those bearing the BPIV3 M or L ORF or the F and HN genes were only moderately attenuated. This indicates that the genetic determinants of the host range restriction of replication of BPIV3 for primates are polygenic, with the major determinants being the N and P ORFs. Monkeys immunized with these bovine-human chimeric viruses, including the more highly attenuated ones, developed higher levels of HPIV3 hemagglutination-inhibiting serum antibodies than did monkeys immunized with BPIV3 and were protected from challenge with wild-type HPIV3. Furthermore, host range determinants could be combined with attenuating point mutations to achieve an increased level of attenuation. Thus, chimeric recombinant bovine-human PIV3 viruses that manifest different levels of attenuation in rhesus monkeys are available for evaluation as vaccine candidates to protect infants from the severe lower respiratory tract disease caused by HPIV3.     

Nuclear-cytoplasmic "tug of war" during cloning: effects of somatic cell nuclei on culture medium preferences of preimplantation cloned mouse embryos

Cloning by somatic cell nuclear transfer is critically dependent upon early events that occur immediately after nuclear transfer, and possibly additional events that occur in the cleaving embryo. Embryo culture conditions have not been optimized for cloned embryos, and the effects of culture conditions on these early events and the successful initiation of clonal development have not been examined. To evaluate the possible effect of culture conditions on early cloned embryo development, we have compared a number of different culture media, either singly or in sequential combinations, for their ability to support preimplantation development of clones produced using cumulus cell nuclei. We find that glucose is beneficial during the 1-cell stage when CZB medium is employed. We also find that potassium simplex optimized medium (KSOM), which is optimized to support efficient early cleavage divisions in mouse embryos, does not support development during the 1-cell or 2-cell stages in the cloned embryos as well as other media. Glucose-supplemented CZB medium (CZB-G) supports initial development to the 2-cell stage very well, but does not support later cleavage stages as well as Whittten medium or KSOM. Culturing cloned embryos either entirely in Whitten medium or initially in Whittens medium and then changing to KSOM at the late 4-cell/early 8-cell stage produces consistent production of blastocysts at a greater frequency than using CZB-G medium alone. The combination of Whitten medium followed by KSOM resulted in an increased number of cells per blastocyst. Because normal embryos do not require glucose during the early cleavage stages and develop efficiently in all of the media employed, these results reveal unusual culture medium requirements that are indicative of altered physiology and metabolism in the cloned embryos. The relevance of this to understanding the kinetics and mechanisms of nuclear reprogramming and to the eventual improvement of the overall success in cloning is discussed.     

Comparative anatomical analysis of the cotyledonary region in three Mediterranean Basin Quercus (Fagaceae)

Anatomical changes at the cotyledonary node from the embryo to the seedling stage in Quercus coccifera, Q. ilex, and Q. humilis were investigated by light and scanning electron microscopy techniques. Mature embryos of Q. humilis possess 2-3 pairs of leaf primordia and a pair of cotyledonary buds, whereas in Q. coccifera and Q. ilex there are two incipient primordia, and cotyledonary buds are not observed until 1 wk after germination. In all three species the cotyledonary buds multiply, forming bud clusters, and a vascular connection is well established within 5-6 wk after germination. As development proceeds, the cotyledonary region becomes woody, but buds, which are exogenous in origin, never become embedded in the periderm. In comparison with Q. suber, another native Mediterranean Basin oak, the cotyledonary node is short and axillary buds are not present below the insertion of cotyledons. In addition, starch accumulation in the cotyledonary region is not observed from histological analysis in the three oaks. Therefore, in Q. coccifera, Q. ilex, and Q. humilis seedlings the cotyledonary node can be considered to be an important regenerative structure enabling them to resprout after the elimination of the shoot above the cotyledons, despite the absence of a lignotuberous structure.     

Sporothrix brasiliensis induces a more severe disease associated with sustained Th17 and regulatory T cells responses than Sporothrix schenckii sensu stricto in mice

Little is known about the differences in the CD4+ T-cell response induced by Sporothrix schenckii and Sporothrix brasiliensis, the most virulent species that cause sporotrichosis. Here, the helper (Th) and regulatory T cells (Tregs) responses were evaluated comparatively in a murine model of sporotrichosis on days 7, 21 and 35 after subcutaneous infection with either S. schenckii or S. brasiliensis conidia. The fungal load was measured at the site of infection, as well as in the liver and spleen. The Th1/Th17/Tregs responses were analyzed in the spleen, while the level of IL-2, IL-4, IL-6, TNF-alpha, IFN-?, IL-17A and IL-10 cytokines were measured at the local site of infection on 24 h postinfections and in sera on the indicated days. S. brasiliensis caused a longer-lasting infection in the skin and chronic systemic dissemination associated to more severe granulomatous lesions. Similar Th1/Th1-Th17/Tregs responses were induced by both S. brasiliensis and S. schenckii on 7th and 21st d.p.i but on 35 d.p.i a reduction of Th1 and Th1-Th17 cells, associated to higher values of Th17/Tregs cells was observed only in S. brasiliensis-infected mice. In summary, S. brasiliensis caused a more severe disease associated with sustained Th17/Tregs responses than S. schenckii in mice.     

Molecular interactions between Geobacter sulfurreducens triheme cytochromes and the redox active analogue for humic substances

The bacterium Geobacter sulfurreducens can transfer electrons to quinone moieties of humic substances or to anthraquinone-2,6-disulfonate (AQDS), a model for the humic acids. The reduced form of AQDS (AH₂QDS) can also be used as energy source by G. sulfurreducens. Such bidirectional utilization of humic substances confers competitive advantages to these bacteria in Fe(III) enriched environments. Previous studies have shown that the triheme cytochrome PpcA from G. sulfurreducens has a bifunctional behavior toward the humic substance analogue. It can reduce AQDS but the protein can also be reduced by AH₂QDS. Using stopped-flow kinetic measurements we were able to demonstrate that other periplasmic members of the PpcA-family in G. sulfurreducens (PpcB, PpcD and PpcE) also showed the same behavior. The extent of the electron transfer is thermodynamically controlled favoring the reduction of the cytochromes. NMR spectra recorded for ¹³C,¹⁵N-enriched samples in the presence increasing amounts of AQDS showed perturbations in the chemical shift signals of the cytochromes. The chemical shift perturbations on cytochromes backbone NH and ¹H heme methyl signals were used to map their interaction regions with AQDS, showing that each protein forms a low-affinity binding complex through well-defined positive surface regions in the vicinity of heme IV (PpcB, PpcD and PpcE) and I (PpcE). Docking calculations performed using NMR chemical shift perturbations allowed modeling the interactions between AQDS and each cytochrome at a molecular level. Overall, the results obtained provided important structural-functional relationships to rationalize the microbial respiration of humic substances in G. sulfurreducens.