Pharmacological Study Of Edema And Myonecrosis In Mice Induced By Venom Of The Bushmaster Snake (lachesis Muta Muta) And Its Basic Asp49 Phospholipase A(2) (lmtx-i).

Previous in vitro studies show that Lachesis muta venom and its purified Asp49 phospholipase A(2), named as LmTX-I, display potent neurotoxic and myotoxic activities. Here, an in vivo study was conducted to investigate some pharmacological effects of the venom or its LmTX-I toxin, after intra-muscular injection in tibialis anterior (TA) and following subplantar injection in hind paws of mice. Findings showed that LmTX-I increased plasma creatine kinase activity and produced strong myonecrosis and inflammatory reactions in TA muscle. In addition to these effects, the venom also induced intense local hemorrhage. Pre-treatment of the venom with EDTA (5 mM) significantly inhibited the edema and hemorrhage. Histological examination showed that L. muta venom caused inner dermal layer thickening in the pad hind paw. In addition, there was marked inflammatory cell infiltration, particularly of neutrophils, and hemorrhage. LmTX-I also demonstrated edema-forming activity, which was inhibited by pretreatment with indomethacin.27384-9

Differential effects of nitrostyrene derivatives on myelopoiesis involve regulation of C/EBPα and p38MAPK activity.

Bone marrow failure syndromes and MDS represent a heterogenous group of diseases, characterized by ineffective myelopoiesis, the risk of clonal evolution and a generally poor response to chemotherapy-based treatment regimen. Nitrostyrene derivatives have been studied as protein phosphatase inhibitors in various tumor models. Pharmacological studies have identified nitrostyrene as the structural core underlying a pro-apoptotic effect in tumor cells, yet their effects on normal cells, including those of the hematopoietic system, are largely unknown. In this study, utilizing umbilical cord blood-derived myeloid progenitor cells, patient-derived bone marrow cells, and a (BALB/c) mouse model; we investigated the effects of treatment with two nitrostyrene derivatives (NTS1 and NTS2) on myeloid development. We demonstrate that these compounds stimulate the expansion and differentiation of myeloid progenitors in vitro and improve myeloid reconstitution after chemotherapy-induced bone marrow depletion in vitro and in vivo. These effects were accompanied by increased C/EBPα expression and activity and inhibition of the p38MAPK signalling pathway. Together, our data suggest that nitrostyrenes improve myelopoiesis and represent potential new treatment strategies for patients suffering from bone marrow failure syndromes, hypocellular myelodysplastic syndrome and chemotherapy-induced aplasia

Autophagy inhibited Ehrlich ascitic tumor cells apoptosis induced by the nitrostyrene derivative compounds: Relationship with cytosolic calcium mobilization

Apoptosis induction is often associated with increased autophagy, indicating interplay between these two important cellular events in cell death and survival. in this study, the programmed cell death and autophagy induced by two nitrostyrene derivative compounds (NTS1 and NTS2) was studied using the tumorigenic Ehrlich ascitic tumor (EAT) cells. EAT cells were highly sensitive to NTS1 and NTS2 cytotoxicity in a dose-dependent manner. NTS1 and NTS2 IC50 was less than 15.0 mu M post 12 h incubation. Apoptosis was primarily induced by both compounds, as demonstrated by an increase in Annexin-V positive cells, concurrently with cytochrome c release from mitochondria to cytosol and caspase-3 activation. Although cytosolic Ca2+ mobilization is involved in autophagy as well as apoptosis in response to cellular stress in many cancer cell types, from the two nitrostyrene derivative compounds studied, mainly NTS1 mobilized this ion and disparate autophagy in EAT cells. These results suggest that EAT induced cell death by NTS1 and NTS2 involved a Ca2+-dependent and a Ca2+-independent pathways, respectively. in accordance with these results, the treatment of EAT cells with 3 methyladenine (3-MA), an autophagy inhibitor; significantly increased the number of apoptotic cells after NTS1 treatment, suggesting that pharmacological modulation of autophagy augments the NTS1 efficacy. Thus, we denote the importance of studies involving autophagy and apoptosis during pre-clinical studies of new drugs with anticancer properties. (C) 2011 Elsevier B. V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Universidade Federal de São Paulo UNIFESP, Escola Paulista Med, Dept Farmacol, São Paulo, BrazilUniv Estadual Campinas, UNICAMP, Dept Farmacol, FCM, Campinas, SP, BrazilUniversidade Federal de São Paulo UNIFESP, Escola Paulista Med, Dept Biofis, São Paulo, BrazilUniversidade Federal de São Paulo UNIFESP, Escola Paulista Med, Dept Farmacol, São Paulo, BrazilUniversidade Federal de São Paulo UNIFESP, Escola Paulista Med, Dept Biofis, São Paulo, BrazilWeb of Scienc

Biological And Biochemical Characterization Of New Basic Phospholipase A(2) Bmtx-i Isolated From Bothrops Moojeni Snake Venom.

BmTX-I, an Asp49 phospholipase A(2), was purified from Bothrops moojeni venom after only one chromatographic step using reverse-phase HPLC on mu-Bondapak C-18 column. A molecular mass of 14238.71Da was determined by MALDI-TOF mass spectrometry. Amino acid analysis showed a high content of hydrophobic and basic amino acids as well as 14 half-cysteine residues. The BmTX-I PLA(2) had a sequence of 121 residues of amino acids: DLWQFNKMIK KEVGKLPFPF YGAYGCYCGW GGRGEKPKDG TDRCCFVHDC CYKKLTGCPK WDDRYSYSWK DITIVCGEDL PCEEICECDR AAAVCFYENL GTYNKKYMKH LKPCKKADYP C and pI value 7.84, and showed a high degree of homology with basic Asp49 PLA(2) myotoxins from other Bothrops venoms. BmTX-I presented PLA(2) activity in the presence of a synthetic substrate and showed a minimum sigmoidal behavior, reaching its maximal activity at pH 8.0 and 35-45 degrees C. Maximum PLA(2) activity required Ca(2+) and in the presence of Mg(2+), Cd(2+) and Mn(2+) it was reduced in presence or absence of Ca(2+). Crotapotin from Crotalus durissus colillineatus rattlesnake venom has significantly inhibited (P<0.05) the enzymatic activity of BmTX-I. In vitro, the whole venom and BmTX-I caused a blockade of the neuromuscular transmission in young chick biventer cervicis preparations in a similar way to other bothrops species. In mice, BmTX-I and the whole venom-induced myonecrosis and a systemic interleukin-6 response upon intramuscular injection. Edema-forming activity was also analyzed through injection of the venom and the purified BmTX-I into the subplantar region of the right footpad. Since BmTX-I exert a strong proinflammatory effect; the enzymatic phospholipids hydrolysis might be relevant for these phenomena.511509-1

Autophagy Inhibited Ehrlich Ascitic Tumor Cells Apoptosis Induced By The Nitrostyrene Derivative Compounds: Relationship With Cytosolic Calcium Mobilization.

Apoptosis induction is often associated with increased autophagy, indicating interplay between these two important cellular events in cell death and survival. In this study, the programmed cell death and autophagy induced by two nitrostyrene derivative compounds (NTS1 and NTS2) was studied using the tumorigenic Ehrlich ascitic tumor (EAT) cells. EAT cells were highly sensitive to NTS1 and NTS2 cytotoxicity in a dose-dependent manner. NTS1 and NTS2 IC(50) was less than 15.0μM post 12h incubation. Apoptosis was primarily induced by both compounds, as demonstrated by an increase in Annexin-V positive cells, concurrently with cytochrome c release from mitochondria to cytosol and caspase-3 activation. Although cytosolic Ca(2+) mobilization is involved in autophagy as well as apoptosis in response to cellular stress in many cancer cell types, from the two nitrostyrene derivative compounds studied, mainly NTS1 mobilized this ion and disparate autophagy in EAT cells. These results suggest that EAT induced cell death by NTS1 and NTS2 involved a Ca(2+)-dependent and a Ca(2+)-independent pathways, respectively. In accordance with these results, the treatment of EAT cells with 3 methyladenine (3-MA), an autophagy inhibitor; significantly increased the number of apoptotic cells after NTS1 treatment, suggesting that pharmacological modulation of autophagy augments the NTS1 efficacy. Thus, we denote the importance of studies involving autophagy and apoptosis during pre-clinical studies of new drugs with anticancer properties.6786-1

Chlorella Vulgaris Restores Bone Marrow Cellularity And Cytokine Production In Lead-exposed Mice.

Chlorella vulgaris (CV) was examined for its modulating effects on the reduction induced by lead (Pb) on the numbers of marrow hematopoietic stem cells (HSCs) (c-Kit(+)Lin(-)), granulocyte-macrophage progenitors (Gr1(+)Mac1(+)) and total bone marrow cellularity. In mice gavage-treated daily with 50mg/kg dose of CV for 10 days, concomitant to a continuous offering of 1300 ppm lead acetate in drinking water, the treatment with the algae recovered the significantly reduced numbers of these cell populations to control values. As CV may have a myelostimulating effect through the induction of cytokines, we evaluated its modulating effects on the production of IL-1α, TNF-α, IFN-γ, IL-10 and IL-6. Our results demonstrated that lead significantly impairs the production of IFN-γ, IL-1α and TNF-α and increases the production of IL-10 and IL-6 and that these effects are successfully modulated by the CV treatment. The activity of NK cells, reduced in Pb-exposed animals, was raised to levels higher than those of controls in the exposed group treated with CV. Treatment with the algae also stimulated the production of IFN-γ, IL-1α, TNF-α and NK cells activity in normal mice. In addition, zinc bone concentrations, reduced in lead-exposed mice, were partially, but significantly, reversed by the treatment with CV.492934-4

NTS treatment stimulates myeloid colony forming capacity in patient bone marrow cells.

<p>CD34+ cells were cultured in the presence of G-CSF to induce neutrophil differentiation in the absence or presence of 0.5 µM (patient 1) or 1.0 µM (patient 2–3) NTS1 and NTS2. Cells were isolated after 3 days for CFU assays in the presence of G-CSF during 11 days. Data were expressed as the number of CFU-GM (A, upper panel), CFU-G (<b>A, middle panel</b>) and CFU-M (<b>A, lower panel</b>) for each patient and for all patients together (<b>B</b>). Progenitor expansion and terminal differentiation was evaluated after 14 days, Data were expressed as fold induction (<b>C</b>) and the percentage of mature neutrophils and monocytes (<b>D</b>). Error bars represent SEM (between patients). *p = <0.05.</p

Treatment with NTS1 and NTS2 does not affect terminal neutrophil differentiation.

<p>CD34+ cells were cultured in the presence of G-CSF to induce neutrophil differentiation during 17 days. Cells were cultured either in the absence or presence of NTS1 (0.5–5 µM) or NTS2 (0.5–5 µM). After 17 days of neutrophil differentiation was determined by cytospin analysis (<b>A</b>) and FACS analysis or intracellular lactoferrin expression. Data were expressed as the percentage of mature neutrophils (banded or segmented nuclei) (N = 3) (<b>B</b>) the mean lactoferrin expression (MFI) (<b>C</b>) (N = 3), and the percentage of mature monocytes (<b>C</b>). Error bars represent SEM (between experiments). *p = <0.05, **p = <0.01.</p