Activation of bone marrow cells treated with Canova in vitro

Canova is a Brazilian complex homeopathic medication produced from Aconitum, Thuya, Bryonia, Lachesis and Arsenicum. Previous studies demonstrated that Canova induces up-regulation in numbers of leukocytes. The bone marrow microenvironment is composed of growth factors, stromal cells, extracellular matrix, and progenitor cells that differentiate into mature blood cells. As it is the major site of blood cell formation, we studied in vitro Canova effects on bone marrow cells of mice. Swiss mouse femurs were dissected, cleaned, and the marrow was flushed. The cells were plated, treated or not, incubated for different times and processed for light, scanning electron, and confocal microscopy, and also flow cytometry. The treatment did not modify the expression of the analyzed surface markers or cytokine production. All microscopy techniques showed that a monocytic lineage (CD11b(+)) and stromal cells (adherent cells) were activated by treatment. Canova also increased cell clusters over adherent cells, suggesting proliferation areas.     

Study on the ability of 1,2,3,4-tetrahydropapaveroline to cause oxidative stress: Mechanisms and potential implications in relation to parkinson's disease

Tetrahydropapaveroline (THP) is a compound derived from dopamine monoamine oxidase-mediated metabolism, particularly present in the brain of parkinsonian patients receiving L-dopa therapy, and is capable of causing dopaminergic neurodegeneration. The aim of this work was to evaluate the potential of THP to cause oxidative stress on mitochondrial preparations and to gain insight into the molecular mechanisms responsible for its neurotoxicity. Our data show that THP autoxidation occurs with a continuous generation of hydroxyl radicals (*OH) and without the involvement of the Fenton reaction. The presence of ascorbate enhances this process by establishing a redox cycle, which regenerates THP from its quinolic forms. It has been shown that the production of *OH is not affected by the presence of either ferrous or ferric iron. Although THP does not affect lipid peroxidation, it is capable of reducing the high levels of thiobarbituric acid-reactive substances obtained in the presence of ascorbate and/or iron. However, THP autoxidation in the presence of ascorbate causes both an increase in protein carbonyl content and a reduction in protein-free thiol content. THP also increases protein carbonyl content when the autoxidation occurs in the presence of iron. The remarkable role played by ascorbate in the production of oxidative stress by THP autoxidation is of particular interest.     

Different signaling pathways are involved in cardiomyocyte survival induced by a Trypanosoma cruzi glycoprotein

We have recently reported that Trypanosoma cruzi infection protects cardiomyocytes against apoptosis induced by growth factor deprivation. Cruzipain, a major parasite antigen, reproduced this survival effect by a Bcl-2-dependent mechanism. In this study, we have investigated the molecular mechanisms of cruzipain-induced cardiomyocyte protection. Neonatal BALB/c mouse cardiac myocytes were cultured under minimum serum conditions in the presence of cruzipain or T. cruzi (Tulahuen strain). Some cultures were pretreated with the phosphatidylinositol 3-kinase (PI3K) inhibitor Ly294002 or specific inhibitors of the mitogen-activated protein kinase (MAPK) family members such as the mitogen-activated protein kinase kinase (MEK1) inhibitor PD098059, Jun N-terminal kinase (JNK) inhibitor SP600125, p38 MAPK inhibitor SB203580. Inhibition of PI3K and MEK1 but not JNK or p38 MAPK increased the apoptotic rate of cardiomyocytes treated with cruzipain. Phosphorylation of Akt, a major target of PI3K, and ERK1/2, MEK1-targets, was achieved at 15 min and 5 min, respectively. In parallel, these kinases were strongly phosphorylated by T. cruzi infection. In cultures treated with cruzipain, cleavage of caspase 3 was considerably diminished after serum starvation; Bcl-2 overexpression was inhibited by PD098059 but not by Ly294002, whereas Bad phosphorylation and Bcl-xL expression were increased and differentially modulated by both inhibitors. The results suggest that cruzipain exerts its anti-apoptotic property in cardiac myocytes at least by PI3K/Akt and MEK1/ERK1/2 signaling pathways. We further identified a differential modulation of Bcl-2 family members by these two signaling pathways.     

A promising action of riboflavin as a mediator of leukaemia cell death

Besides having a pivotal biological function as a component of coenzymes, riboflavin appears a promissing antitumoral agent, but the underlying molecular mechanism remains unclear. In this work, we demonstrate that irradiated riboflavin, when applied at μM concentrations, induces an orderly sequence of signaling events finally leading to leukemia cell death. The molecular mechanism involved is dependent on the activation of caspase 8 caused by overexpression of Fas and FasL and also on mitochondrial amplification mechanisms, involving the stimulation of ceramide production by sphingomyelinase and ceramide synthase. The activation of this cascade led to an inhibition of mitogen activated protein kinases: JNK, MEK and ERK and survival mediators (PKB and IAP1), upregulation of the proapoptotic Bcl2 member Bax and downregulation of cell cycle progression regulators. Importantly, induction of apoptosis by irradiated riboflavin was leukaemia cell specific, as normal human lymphocytes did not respond to the compound with cell death. Our data indicate that riboflavin selectively activates Fas cascade and also constitutes a death receptor-engaged drug without harmful side effects in normal cells, bolstering the case for using this compound as a novel avenue for combating cancerous disease.     

Ca(2+) and Mg(2+) binding to weak sites of TnC C-domain induces exposure of a large hydrophobic surface that leads to loss of TnC from the thin filament

The C-domain of troponin C, the Ca(2+)-binding subunit of the troponin complex, has two high-affinity sites for Ca(2+) that also bind Mg(2+) (Ca(2+)/Mg(2+) sites), whereas the N-domain has two low-affinity sites for Ca(2+). Two more sites that bind Mg(2+) with very low affinity (K(a)<10(3)M(-1)) have been detected by several laboratories but have not been localized or studied in any detail. Here we investigated the effects of Ca(2+) and Mg(2+) binding to isolated C-domain, focusing primarily on low-affinity sites. Since TnC has no Trp residues, we utilized a mutant with Phe 154 replaced by Trp (F154W/C-domain). As expected from previous reports, the changes in Trp fluorescence revealed different conformations induced by the addition of Ca(2+) or Mg(2+) (Ca(2+)/Mg(2+) sites). Exposure of hydrophobic surfaces of F154W/C-domain was monitored using the fluorescence intensity of bis-anilino naphthalene sulfonic acid. Unlike the changes reported by Trp, the increments in bis-ANS fluorescence were much greater (4.2-fold) when Ca(2+)+Mg(2+) were both present or when Ca(2+) was present at high concentration. Bis-ANS fluorescence increased as a function of [Ca(2+)] in two well-defined steps: one at low [Ca(2+)], consistent with the Ca(2+)/Mg(2+) sites (K(a) approximately 1.5 x 10(6)M(-1)), and one of much lower affinity (K(a) approximately 52.3M(-1)). Controls were performed to rule out artifacts due to aggregation, high ionic strength and formation of the bis-ANS-TnC complex itself. With a low concentration of Ca(2+) (0.6mM) to occupy the Ca(2+)/Mg(2+) sites, a large increase in bis-ANS binding also occurred as Mg(2+) occupied a class of low-affinity sites (K(a) approximately 59 M(-1)). In skinned fibers, a high concentration of Mg(2+) (10-44 mM) caused TnC to dissociate from the thin filament. These data provide new evidence for a class of weak binding sites for divalent cations. They are located in the C-domain, lead to exposure of a large hydrophobic surface, and destabilize the binding of TnC to the regulatory complex even when sites III and IV are occupied.     

Solid-state NMR triple-resonance backbone assignments in a protein

Triple-resonance solid-state NMR spectroscopy is demonstrated to sequentially assign the 13C and 15N amide backbone resonances of adjacent residues in an oriented protein sample. The observed 13C chemical shift frequency provides an orientational constraint complementary to those measured from the 1H and 15N amide resonances in double-resonance experiments.     

Differences in aerial and terrestrial visual scanning in captive black tufted-ear marmosets (Callithrix penicillata) exposed to a novel environment

Aerial and terrestrial visual scanning were investigated in captive black tufted-ear marmosets, Callithrix penicillata, exposed to a novel environment. Naive adult subjects (n=24) were individually exposed to a figure-eight maze during seven 30-min trials, 48 h apart. Habituation to the maze was observed, as indicated by the significant decrease in locomotion. The frequency of aerial scanning, however, remained elevated throughout the 7 trials, while its duration rapidly increased to high levels. Frequency and duration of terrestrial scanning persisted at constant low rates, differing significantly from aerial scanning. Males and females did not differ significantly. The different impact of aerial versus terrestrial predators could have a significant influence on vigilant behaviour in this species. Thus, visual scanning is an important and highly organized antipredation strategy in marmosets.     

Glutamic acid residues of bacteriorhodopsin at the extracellular surface as determinants for conformation and dynamics as revealed by site-directed solid-state 13C NMR

We recorded (13)C NMR spectra of [3-(13)C]Ala- and [1-(13)C]Val-labeled bacteriorhodopsin (bR) and a variety of its mutants, E9Q, E74Q, E194Q/E204Q (2Glu), E9Q/E194Q/E204Q (3Glu), and E9Q/E74Q/E194Q/E204Q (4Glu), to clarify contributions of the extracellular (EC) Glu residues to the conformation and dynamics of bR. Replacement of Glu-9 or Glu-74 and Glu-194/204 at the EC surface by glutamine(s) induced significant conformational changes in the cytoplasmic (CP) surface structure. These changes occurred in the C-terminal alpha-helix and loops, and also those of the EC surface, as viewed from (13)C NMR spectra of [3-(13)C]Ala- and [1-(13)C]Val-labeled proteins. Additional conformational changes in the transmembrane alpha-helices were induced as modified retinal-protein interactions for multiple mutants involving the E194Q/E204Q pair. Significant dynamic changes were induced for the triple or quadruple mutants, as shown by broadened (13)C NMR peaks of [1-(13)C]Val-labeled proteins. These changes were due to acquired global fluctuation motions of the order of 10(-4)-10(-5) s as a result of disorganized trimeric form. In such mutants (13)C NMR signals from Val residues of [1-(13)C]Val-labeled triple and quadruple mutants near the CP and EC surfaces (including 8.7-A depth from the surface) were substantially suppressed, as shown by comparative (13)C NMR studies with and without 40 micro M Mn(2+) ion. We conclude that these Glu residues at the EC surface play an important role in maintaining the native secondary structure of bR in the purple membrane.     

Unfolding events in the water-soluble monomeric Cry1Ab toxin during transition to oligomeric pre-pore and membrane-inserted pore channel

The insecticidal crystal (Cry) proteins produced by Bacillus thuringiensis undergo several conformational changes from crystal inclusion protoxins to membrane-inserted channels in the midgut epithelial cells of the target insect. Here we analyzed the stability of the different forms of Cry1Ab toxin, monomeric toxin, pre-pore complex, and membrane-inserted channel, after urea and thermal denaturation by monitoring intrinsic tryptophan fluorescence of the protein and 1-anilinonaphthalene-8-sulfonic acid binding to partially unfolded proteins. Our results showed that flexibility of the monomeric toxin was dramatically enhanced upon oligomerization and was even further increased by insertion of the pre-pore into the membrane as shown by the lower concentration of chaotropic agents needed to achieve unfolding of the oligomeric species. The flexibility of the toxin structures is further increased by alkaline pH. We found that the monomer-monomer interaction in the pre-pore is highly stable because urea promotes oligomer denaturation without disassembly. Partial unfolding and limited proteolysis studies demonstrated that domains II and III were less stable and unfold first, followed by unfolding of the most stable domain I, and also that domain I is involved in monomer-monomer interaction. The thermal-induced unfolding and analysis of energy transfer from Trp residues to bound 1-anilinonaphthalene-8-sulfonic acid dye showed that in the membrane-inserted pore domains II and III are particularly sensitive to heat denaturation, in contrast to domain I, suggesting that only domain I may be inserted into the membrane. Finally, the insertion into the membrane of the oligomeric pre-pore structure was not affected by pH. However, a looser conformation of the membrane-inserted domain I induced by neutral or alkaline pH correlates with active channel formation. Our studies suggest for the first time that a more flexible conformation of Cry toxin could be necessary for membrane insertion, and this flexible structure is induced by toxin oligomerization. Finally the alkaline pH found in the midgut lumen of lepidopteran insects could increase the flexibility of membrane-inserted domain I necessary for pore formation.