pH-Sensitive Binding of Cytochrome c to the Inner Mitochondrial Membrane. Implications for the Participation of the Protein in Cell Respiration and Apoptosis

Cytochrome c exhibits two positively charged sites: site A containing lysine residues with high pK(a) values and site L containing ionizable groups with pK(aobs),values around 7.0. This protein feature implies that cytochrome c can participate in the fusion of mitochondria and have its detachment from the inner membrane regulated by cell acidosis and alkalosis. In this study, We demonstrated that both horse and tuna cytochrome c exhibited two types of binding to inner mitochondrial membranes that contributed to respiration: a high-affinity and low-efficiency pi-I-independent binding (microscopic dissociation constant K(sapp2), similar to 10 nM) and a low-affinity and high-efficiency pH-dependent binding that for horse cytochrome c had a pK(a) of similar to 6.7. For tuna cytochrome c (Lys22 and His33 replaced with Asn and Trp, respectively), the effect of pH on K(sapp1), was less striking than for the horse heme protein, and both tuna and horse cytochrome c had closed K(sapp1) values at pH 7.2 and 6.2, respectively. Recombinant mutated cytochrome c H26N and H33N also restored the respiration of the cytochrome c-depleted mitoplast in a pH-dependent manner. Consistently, the detachment of cytochrome c from nondepleted mitoplasts was favored by alkalinization, suggesting that site Lionization influences the participation of cytochrome c in the respiratory chain and apoptosis.

Aminoacetone, a putative endogenous source of methylglyoxal, causes oxidative stress and death to insulin-producing RINm5f cells

Aminoacetone (AA), triose phosphates, and acetone are putative endogenous sources of potentially cytotoxic and genotoxic methylglyoxal (MG), which has been reported to be augmented in the plasma of diabetic patients. In these patients, accumulation of MG derived from aminoacetone, a threonine and glycine catabolite, is inferred from the observed concomitant endothelial overexpression of circulating semicarbazide-sensitive amine oxidases. These copper-dependent enzymes catalyze the oxidation of primary amines, such as AA and methylamine, by molecular oxygen, to the corresponding aldehydes, NH4+ ion and H2O2. We recently reported that AA aerobic oxidation to MG also takes place immediately upon addition of catalytic amounts of copper and iron ions. Taking into account that (i) MG and H2O2 are reportedly cytotoxic to insulin-producing cell lineages such as RINm5f and that (ii) the metal-catalyzed oxidation of AA is propagated by O-2(center dot-) radical anion, we decided to investigate the possible pro-oxidant action of AA on these cells taken here as a reliable model system for pancreatic beta-cells. Indeed, we show that AA (0.10-5.0 mM) administration to RINm5f cultures induces cell death. Ferrous (50-300 mu M) and Fe3+ ion (100 mu M) addition to the cell cultures had no effect, whereas Cu2+ (5.0-100 mu M) significantly increased cell death. Supplementation of the AA- and Cu2+-containing culture medium with antioxidants, such as catalase (5.0 mu M), superoxide dismutase (SOD, 50 U/mL), and N-acetylcysteine (NAC, 5.0 mM) led to partial protection. mRNA expression of MnSOD, CuZnSOD, glutathione peroxidase, and glutathione reductase, but not of catalase, is higher in cells treated with AA (0.50-1.0 mM) plus Cu2+ ions (10-50 mu M) relative to control cultures. This may imply higher activity of antioxidant enzymes C, in RINm5f AA-treated cells. In addition, we have found that AA (0.50-1.0 mM) Plus Cu2+ (100 mu M) (i) increase RINm5f cytosolic calcium; (ii) promote DNA fragmentation; and (iii) increase the pro-apoptotic (Bax)/antiapoptotic (Bcl-2) ratio at the level of mRNA expression. In conclusion, although both normal and pathological concentrations of AA are probably much lower than those used here, it is tempting to propose that excess AA in diabetic patients may drive oxidative damage and eventually the death of pancreatic beta-cells.

Avaliação do efeito citotóxico da lectina da esponja marinha Cliona varians contra células de leucemia mielóide crônica

In this study, a BCR-ABL expressing human chronic myelogenous leukaemia cell line (K562) was used to investigate the antitumoral potential of a novel lectin (CvL) purified from the marine sponge Cliona varians. CvL inhibited the growth of K562 cells with an IC50 value of 70 g/ml, but was ineffective to normal human peripheral blood lymphocytes in the same range of concentrations tested (180 g/ml). Cell death occurred after 72 h of exposure to the lectin and with sign of apoptosis as analysed by DAPI staining. Investigation of the possible effectors of this process showed that cell death occurred in the presence of Bcl-2 and Bax expression, and involved a caspase-independent pathway. Confocal fluorescence microscopy indicated a major role for the lysosomal protease cathepsin B in mediating cell death. Accordingly, pre-incubation of K562 cells with the cathepsin inhibitor L-trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64) abolished the cytotoxic effect of CvL. Furthermore, we found upregulation of tumor necrosis factor receptor 1 (TNFR1) and down-modulation of p65 subunit of nuclear factor kappa B (NFB) expression in CvL-treated cells. These effects were accompanied by increased levels of p21 and downmodulation of pRb, suggesting that CvL is capable of cell cycle arrest. Collectively, these findings suggest that cathepsin B acts as death mediator in CvL-induced cytotoxicity possibly in a still uncharacterized connection with the membrane death receptor pathway

Atividades Biológicas De Xilana De Sabugo De Milho

The corn cob is an agricultural by-product still little used, this in part due to the low knowledge of the biotechnological potential of their molecules. Xylan from corn cobs (XSM) is a polysaccharide present in greater quantity in the structure of plant and its biotechnology potential is little known. This study aimed to the extraction, chemical characterization and evaluation of biological activities of xylan from corn cobs. To this end, corncobs were cleaned, cut, dried and crushed, resulting in flour. This was subjected to a methodology that combines the use of alkaline conditions with waves of ultrasound. After methanol precipitation, centrifugation and drying was obtained a yield of 40% (g/g flour). Chemical analysis indicated a high percentage of polysaccharides in the sample (60%) and low contamination by protein (0.4%) and phenolic compounds (> 0.01%). Analysis of monosaccharide composition indicated the presence of xylose:glucose:arabinose:galactose:mannose:glucuronic acid in a molar ratio 50:20:15:10:2.5:2.5. The presence of xylan in the sample was confirmed by nuclear magnetic resonance (¹H and ¹³C) and infrared spectroscopy (IR). Tests were conducted to evaluate the antioxidant potential of XSM. This showed a total antioxidant capacity of 48.45 EAA/g sample. However, did not show scavenging activity of superoxide and hydroxyl radical and also reducing power. But, showing a high capacity chelating iron ions with 70% with about 2 mg/mL. The ability to XSM to influence cell proliferation in culture was also evaluated. This polymer did not influence the proliferation of normal fibroblast cells (3T3), however, decreased the rate of proliferation of tumor cells (HeLa) in a dose-dependent, reaching an inhibition of about 50% with a concentration around 2 mg/mL. Analyzing proteins related to cell death, by immunoblotting, XSM increases the amount of Bax, Bcl-2 decrease, increase cytochrome c and AIF, and reduce pro-caspase-3, indicating the induction of cell death induced apoptosis dependent and independent of caspase. XSM did not show anticoagulant activity in the PT test. However, the test of activated partial thromboplastin time (aPTT), XSM increased clotting time at about 5 times with 600 μg of sample compared with the negative control. The presence of sulfate on the XSM was discarded by agarose gel electrophoresis and IR. After carboxyl-reduction of XSM the anticoagulant activity decreased dramatically. The data of this study demonstrate that XSM has potential as antioxidant, antiproliferative and anticoagulant compound. Future studies to characterize these activities of XSM will help to increase knowledge about this molecule extracted from corn and allow their use in functional foods, pharmaceuticals and chemical industries.

Indução de morte celular em células de adenocarcinoma cervical humano (HeLa) pela lectina da esponja Cinachyrella apion (CaL)

Cancer is a term used to represent a set of more than 100 diseases, including malignant tumors from different locations. The malignancies are the second leading cause of death in the population, representing approximately 17% of deaths of known cause. Strategies that induce differentiation have had limited success in the treatment of established cancers. In this work, a lectin purified from the marine sponge Cinachyrella apion (CaL) was evaluated due to its hemolytic, cytotoxic and antiproliferative properties, besides the ability to induce cell death via apoptosis in tumor cells. The antiproliferative activity of CaL was tested against cell lines, with the highest inhibition of tumor growth for HeLa, reducing cell growth at a dose dependent manner, with a concentration of 10 μg/mL. The hemolytic activity and toxicity against peripheral blood cells were tested using the concentration of IC50 for both trials and twice the IC50 for analysis in flow cytometry, indicating that CaL is not toxic to these cells. To assess the mechanism of cell death caused by CaL in HeLa cells, we performed flow cytometry and western blotting. The results showed the lectin probably induces cell death by apoptosis activation by pro-apoptotic protein Bax, promoting mitochondrial membrane permeabilization, cell cycle arrest in S phase, with accumulation of cells of approximately 57% in this phase, and acting as both dependent and/or independent of caspases pathway. These results suggest that CaL has the potential to be used as drug treatment against cancer.

Apoptose na infecção experimental de cães domésticos com Ehrlichia canis

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Fases e criticalidade no modelo ashkin - teller de três cores

The usual Ashkin-Teller (AT) model is obtained as a superposition of two Ising models coupled through a four-spin interaction term. In two dimension the AT model displays a line of fixed points along which the exponents vary continuously. On this line the model becomes soluble via a mapping onto the Baxter model. Such richness of multicritical behavior led Grest and Widom to introduce the N-color Ashkin-Teller model (N-AT). Those authors made an extensive analysis of the model thus introduced both in the isotropic as well as in the anisotropic cases by several analytical and computational methods. In the present work we define a more general version of the 3-color Ashkin-Teller model by introducing a 6-spin interaction term. We investigate the corresponding symmetry structure presented by our model in conjunction with an analysis of possible phase diagrams obtained by real space renormalization group techniques. The phase diagram are obtained at finite temperature in the region where the ferromagnetic behavior is predominant. Through the use of the transmissivities concepts we obtain the recursion relations in some periodical as well as aperiodic hierarchical lattices. In a first analysis we initially consider the two-color Ashkin-Teller model in order to obtain some results with could be used as a guide to our main purpose. In the anisotropic case the model was previously studied on the Wheatstone bridge by Claudionor Bezerra in his Master Degree dissertation. By using more appropriated computational resources we obtained isomorphic critical surfaces described in Bezerra's work but not properly identified. Besides, we also analyzed the isotropic version in an aperiodic hierarchical lattice, and we showed how the geometric fluctuations are affected by such aperiodicity and its consequences in the corresponding critical behavior. Those analysis were carried out by the use of appropriated definitions of transmissivities. Finally, we considered the modified 3-AT model with a 6-spin couplings. With the inclusion of such term the model becomes more attractive from the symmetry point of view. For some hierarchical lattices we derived general recursion relations in the anisotropic version of the model (3-AAT), from which case we can obtain the corresponding equations for the isotropic version (3-IAT). The 3-IAT was studied extensively in the whole region where the ferromagnetic couplings are dominant. The fixed points and the respective critical exponents were determined. By analyzing the attraction basins of such fixed points we were able to find the three-parameter phase diagram (temperature £ 4-spin coupling £ 6-spin coupling). We could identify fixed points corresponding to the universality class of Ising and 4- and 8-state Potts model. We also obtained a fixed point which seems to be a sort of reminiscence of a 6-state Potts fixed point as well as a possible indication of the existence of a Baxter line. Some unstable fixed points which do not belong to any aforementioned q-state Potts universality class was also found

Pancreatic islets from dexamethasone-treated rats show alterations in global gene expression and mitochondrial pathways

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Endurance training stimulates growth and survival pathways and the redox balance in rat pancreatic islets

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cigarette smoke affects apoptosis in rat tongue mucosa: Role of bcl-2 gene family

While it has been clearly demonstrated that smoking is the most significant exogenous factor involved in oral carcinogenesis, little is known about the global molecular and cellular changes that occur prior to the appearance of clinically detectable symptoms. Thus, the aim of this study was to investigate the expressivity of bcl-2, bax and PCNA in the rat tongue mucosa exposed to cigarette smoke by means of immunohistochemistry. A total of twelve male Wistar rats were distributed into 2 groups: negative control and experimental group exposed to cigarette smoke during 75 days. After experimental period, no histopathological changes in the tongue mucosa were detected in the negative control and the experimental group. on the other hand, an overexpression of bcl-2 was detected (p < 0.01) throughout all layers of the epithelium, whereas bax did not show significant differences (p > 0.05). Also, the labeling index for bcl-2 and bax showed an increase 75 days after cigarette exposure (p < 0.01). PCNA-labeling index did not show remarkable changes between groups. Taken together, our results show that bcl-2 is overexpressed in the rat tongue keratinocytes after cigarette smoke exposure.

Modifying Effects of Lemongrass Essential Oil on Specific Tissue Response to the Carcinogen N-Methyl-N-Nitrosurea in Female BALB/c Mice

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Response of human alveolar bone-derived cells to a novel poly(vinylidene fluoride-trifluoroethylene)/barium titanate membrane

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Role of endothelial cell apoptosis in regulation of skeletal muscle angiogenesis during high and low salt intake

Angiogenesis, under normal conditions, is a tightly regulated balance between pro- and antiangiogenic factors. The goal of this study was to investigate the mechanisms involved in the control of the skeletal muscle angiogenic response induced by electrical stimulation during the suppression of plasma renin activity (PRA) with a high-salt diet. Rats fed 0.4% or 4% salt diets were exposed to electrical stimulation for 7 days. The tibialis anterior ( TA) muscles from stimulated and unstimulated hindlimbs were removed and prepared for gene expression analysis, CD31-terminal deoxynucleotide transferase-mediated dUTP nick-end labeling ( TUNEL) double-staining assay, and Bcl-2 and Bax protein expression by Western blot. Rats fed a low-salt diet showed a dramatic angiogenesis response in the stimulated limb compared with the unstimulated limb. This angiogenesis response was significantly attenuated when rats were placed on a high-salt diet. Microarray analysis showed that in the stimulated limb of rats fed a low-salt diet many genes related to angiogenesis were upregulated. In contrast, in rats fed a high-salt diet most of the genes upregulated in the stimulated limb function in apoptosis and cell cycle arrest. Endothelial cell apoptosis, as analyzed by CD31-TUNEL staining, increased by fourfold in the stimulated limb compared with the unstimulated limb. There was also a 48% decrease in the Bcl-2-to-Bax ratio in stimulated compared with unstimulated limbs of rats fed a high-salt diet, confirming severe apoptosis. This study suggests that the increase in endothelial cell apoptosis in TA muscle might contribute to the attenuation of angiogenesis response observed in rats fed a high-salt diet.

In vitro biocompatibility of poly(vinylidene fluoride-trifluoroethylene)/barium titanate composite using cultures of human periodontal ligament fibroblasts and keratinocytes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Apoptose em otorrinolaringologia e cabeça e pescoço

This paper intend to review recent advances in our understanding of programmed cell death, or apoptosis, and discuss implications of these basic science advances in the development of causes and potential treatments of a variety of diseases of the head and neck. Conclusions: apoptosis is now understood to be important in the normal development and survival of all multicellular organism. Deregulation of this normally tighly controlled process underlies a variety of disease states, including neoplasia, autoimmune disease, and disorders of the central nervous system. A better understanding of this process and regulation may help otolaryngologists better understand diseases relevant to this specialty and will lead to improved therapeutic interventions.