The Disulfide Bond Arrangement in the Extracellular Domain of the Activin Type II Receptor

The initial step in the signaling cascade of the growth factor activin involves its binding to the extracellular domain of the activin type II receptor. This receptor domain contains 10 cysteine residues which are engaged in intramolecular disulfide bonds. To elucidate the structural framework of this domain we have characterized its disulfide-bonding pattern using an extracellular fragment of the receptor which binds activin A with high affinity. By combining proteolysis with mass spectroscopy and chemical sequence analysis, the disulfide connectivity was determined to be as follows: C1–C3, C2–C4, C5–C8, C6–C7, and C9–C10. A similar disulfide arrangement occurs in a family of snake toxins for which the three-dimensional structure is known.     

In vitro studies on the inhibition of colon cancer by amino acid derivatives of bromothiazole

The investment in cancer research is critical to find more and better treatments, but essentially to save lives. Here, we describe the synthesis and characterization on new bromothiazole derivatives with amino acids and with core of nitazoxanide, an FDA-approved antiprotozoal drug. Using a human adenocarcinoma-derived cell line (the Caco-2 cell line), we then investigated the antiproliferative (³H-thymidine incorporation) and cytotoxic (extracellular lactate dehydrogenase activity) effect of these derivatives. All the derivatives caused a concentration–dependent decrease in cell proliferation and viability. At their highest concentration, all compounds were able to reduce ³H-thymidine incorporation by more than 80%, corresponding to a more marked antiproliferative effect than butyrate. As to their cytotoxic effect, it was comparable to that of butyrate. The ability of bromo substituent in thiazole ring with new sequences of amino acids in inducing cell death and apoptosis in Caco-2 cells (and other cell lines) is now being studied.     

Environmental conditions modulate plasticity in the physiological responses of three plant species of the Neotropical savannah

Plasticity in plants could be changed due to abiotic factors, tending to increase fitness across environments. In the Neotropical savannah, a strong water deficit during the dry season is one of the main factors limiting the plasticity in physiological responses of plants. The present study aims to assess the plasticity in physiological responses and vegetative phenology of three plant species of the Neotropical savannah (Cerrado in Brazil) during the dry and the rainy seasons. The three species, Byrsonima verbascifolia, Roupala montana, and Solanum lycocarpum, occur in Serra do Cipó in the state of Minas Gerais, Brazil. The development and vegetative phenology of individuals of these three species were evaluated over the course of 1 year. In February 2012 (rainy season) and August 2012 (dry season), stomatal conductance (g _s), water potential (Ψ), photosynthetic quantum yield, and concentration of leaf photosynthetic pigments were measured. The relative distance among the physiological parameters of all individuals within each season was measured using the relative distance plasticity index. B. verbascifolia has pronounced senescence in July and lost leaves completely by the early September, while R. montana and S. lycocarpum have green leaves throughout the year. The three studied species had greater control of stomatal opening during the dry season. S. lycocarpum and R. montana had negative water potential values in the dry season and in the middle of the day in both seasons. In the dry season, the three species exhibited a decrease in F _v/F _m, with values between 0.7 and 0.75. The relative distance plasticity index varied from 0 to 1. R. montana demonstrated the greatest plasticity and S. lycocarpum had lower plasticity. Then, a seasonal effect on physiological response was observed in all three model-species, with lower values for leaf water potential and stomatal conductance, and increased photoinhibition, in the dry season. Ecophysiological traits, such as stomatal conductance and leaf water potential, exhibited the greatest plasticity. In addition, there was a seasonal effect on the plasticity in physiological responses of the three plants species of the Neotropical savannah. The results are contradicting the idea that water restriction in the dry season would reduce the plasticity in most species of the Neotropical savannah.     

In Silico,In Vitro and In Vivo Toxicological Assessment of BPP-BrachyNH<Subscript>2</Subscript>, A Vasoactive Proline-Rich Oligopeptide from <Emphasis Type="Italic">Brachycephalus ephippium</Emphasis>

BPP-BrachyNH₂ is a proline-rich oligopeptide (PRO) firstly identified in skin secretion of the frog Brachycephalus ephippium, which possess in vitro inhibitory activity of angiotensin-I converting enzyme (ACE) and endothelium-dependent vasorelaxant activity. Considering its potential application in the treatment of cardiovascular diseases, the present work assessed the toxicological profile of the BPP-BrachyNH₂. The in silico toxicity prediction was performed from the best model obtained through the optimization of the FASTA query peptide. This prediction study revealed that BPP-BrachyNH₂ induced high predicted LD₅₀ values for both humans and rats, and then is well-tolerated in the recommended range. The MTT assay was applied for the in vitro cytotoxic evaluation in murine macrophages. In this assay, a decrease of cell viability was not observed. The in vivo acute toxicological study was performed after the intraperitoneal administration of BPP-BrachyNH₂ at doses of 5 and 50 mg/kg. After intraperitoneal administration, no death, alterations in behavioral parameters or weight gain curve was observed, as well as none in the serum biochemical parameters, and gross pathological and histopathological analyses. These observations demonstrates an acceptable safety profile for BPP-BrachyNH₂, leading towards further studies focused on investigation of pharmacological and therapeutical applications for this peptide.     

Secondary necrosis in multicellular animals: an outcome of apoptosis with pathogenic implications

In metazoans apoptosis is a major physiological process of cell elimination during development and in tissue homeostasis and can be involved in pathological situations. In vitro, apoptosis proceeds through an execution phase during which cell dismantling is initiated, with or without fragmentation into apoptotic bodies, but with maintenance of a near-to-intact cytoplasmic membrane, followed by a transition to a necrotic cell elimination traditionally called “secondary necrosis”. Secondary necrosis involves activation of self-hydrolytic enzymes, and swelling of the cell or of the apoptotic bodies, generalized and irreparable damage to the cytoplasmic membrane, and culminates with cell disruption. In vivo, under normal conditions, the elimination of apoptosing cells or apoptotic bodies is by removal through engulfment by scavengers prompted by the exposure of engulfment signals during the execution phase of apoptosis; if this removal fails progression to secondary necrosis ensues as in the in vitro situation. In vivo secondary necrosis occurs when massive apoptosis overwhelms the available scavenging capacity, or when the scavenger mechanism is directly impaired, and may result in leakage of the cell contents with induction of tissue injury and inflammatory and autoimmune responses. Several disorders where secondary necrosis has been implicated as a pathogenic mechanism will be reviewed.     

Genotoxic damage in Solea senegalensis exposed to sediments from the Sado Estuary (Portugal): Effects of metallic and organic contaminants

Juvenile Solea senegalensis (Senegalese sole) were exposed to freshly collected sediments from three sites of the Sado Estuary (West-Portuguese coast) in 28-day laboratory assays in order to assess the ecological risk from sediment contaminants, by measuring two genotoxicity biomarkers in peripheral blood: the percentage of Erythrocyte Nuclear Abnormalities (ENA) by use of an adaptation of the micronucleus test, and the percentage of DNA strand-breakage (DNA-SB) with the Comet assay. Sediments were surveyed for metallic (Cr, Ni, Cu, Zn, As, Cd and Pb) and organic (PAHs (polycyclic aromatic hydrocarbons), PCBs (polychlorinated biphenyls) and DDTs (dichloro-diphenyl-trichloroethane)) contaminants. Sediments from site A (farthest from hotspots of contamination) were found to be the least contaminated and weaker inducers of genotoxic damage, whereas sediments from sites B (urban influence) and C (affected by industrial effluents and agricultural runoffs) were responsible for a very significant increase in both ENA and DNA-SB, site B being most contaminated with metals and site C mainly with organic pollutants, especially PAHs and PCBs . Analysis of genotoxic effects showed a strong correlation between the concentrations of PAHs and PCBs and both biomarkers at sampling times T₁₄ and T₂₈, while the amounts of Cu, As, Cd and Pb were less strongly correlated, and at T₂₈ only, with ENA and DNA-SB. These results show that organic contaminants in sediment are stronger and faster acting genotoxic stressors. The results also suggest that metals may have an inhibitory effect on genotoxicity when interacting with organic contaminants, at least during early exposure. ENA and DNA-SB do not show a linear relationship, but a strong correlation exists between the overall increase in genotoxicity caused by exposure to sediment, confirming that they are different, and possibly non-linked effects that respond similarly to exposure. Although the Comet assay showed enhanced sensitivity, the two analyses are complementary and suitable for the biomonitoring of sediment contaminants in a benthic species like S. senegalensis.     

One-step refolding and purification of disulfide-containing proteins with a C-terminal MESNA thioester

Background  

Expression systems based on self-cleavable intein domains allow the generation of recombinant proteins with a C-terminal thioester. This uniquely reactive C-terminus can be used in native chemical ligation reactions to introduce synthetic groups or to immobilize proteins on surfaces and nanoparticles. Unfortunately, common refolding procedures for recombinant proteins that contain disulfide bonds do not preserve the thioester functionality and therefore novel refolding procedures need to be developed.     

Anti-Pneumocystis carinii and antiplasmodial activities of primaquine-derived imidazolidin-4-ones

A series of primaquine-derived imidazolidin-4-ones were screened for their in vitro activity against Pneumocystis carinii and Plasmodium falciparum W2 strain. Most compounds were active against P. carinii above 10 microg/mL and displayed slight to marked activity. The imidazolidin-4-ones most active against P. carinii were also those most active antiplasmodial agents, in the muM range. One of the tested imidazolidin-4-ones was slightly more active than the parent primaquine and may represent a lead compound for the development of novel anti-P. carinii 8-aminoquinolines with increased stability and resistance to metabolic inactivation.     

Dynamics of myosin,microtubules, and Kinesin-6 at the cortex during cytokinesis in Drosophila S2 cells

Signals from the mitotic spindle during anaphase specify the location of the actomyosin contractile ring during cytokinesis, but the detailed mechanism remains unresolved. Here, we have imaged the dynamics of green fluorescent protein–tagged myosin filaments, microtubules, and Kinesin-6 (which carries activators of Rho guanosine triphosphatase) at the cell cortex using total internal reflection fluorescence microscopy in flattened Drosophila S2 cells. At anaphase onset, Kinesin-6 relocalizes to microtubule plus ends that grow toward the cortex, but refines its localization over time so that it concentrates on a subset of stable microtubules and along a diffuse cortical band at the equator. The pattern of Kinesin-6 localization closely resembles where new myosin filaments appear at the cortex by de novo assembly. While accumulating at the equator, myosin filaments disappear from the poles of the cell, a process that also requires Kinesin-6 as well as possibly other signals that emanate from the elongating spindle. These results suggest models for how Kinesin-6 might define the position of cortical myosin during cytokinesis.