Nandrolone Decanoate associated with exercise training inhibit vascular endothelial growth factor (VEGF) mRNA expression in rat soleus muscle

Androgenic-anabolic steroids (AAS) have been used for both performance improvement and aesthetic reasons. It is well know that high doses of AAS induce serious adverse effects such as skeletal muscle injuries, including increase in the rate of muscle strains/ruptures. Vascular endothelial growth factor (VEGF) is a key factor in angiogenesis induction on both physiological and pathological conditions The aim of this study was to investigate VEGF mRNA expression in rat soleus muscle after jumping training associated with AAS administration. Wistar rats were grouped into: sedentary (S); trained without AAS (T); sedentary nandrolone decanoate (ND)-treated (AAS); and trained with AAS (AAST). The trained groups carried out jumps in water at 32°C.: 4 series of 10 jumps each, with a 30-second interval among series, for 7 weeks, with 50-80% overload of the animal corporal mass. The AAS (Decadurabolin® - 5mg/kg) was injected subcutaneously in the animal’s back twice a week. Real-time PCR analyses showed that training significantly increased VEGF mRNA expression in comparison with the S and AAS groups. When exercise training was associated with nandrolone decanoate, the VEGF mRNA expression was inhibited compared with T group. The inhibition of VEGF expression by AAS administration can decrease angiogenesis in skeletal muscle. These results suggest that the AAS may be strongly prejudicial to muscle remodeling and performance

Resistance training modulates the matrix mtalloproteinase-2 activity in different trabecular bones in aged rats

Background: Aging decreases osteogenic ability, inducing harmful effects on the bone extracellular matrix (ECM), while exercise training has been indicated as a tool to counteract bone disorders related to advancing age. The modulation of bone ECM is regulated by several types of matrix metalloproteinase (MMP); however, MMP-2 activity in different trabecular bones in response to resistance training (RT) has been neglected. Remodeling differs in different bones under the application of the same mechanical loading. Thus, we investigated the effects of 12 weeks of RT on MMP-2 activity in the lumbar vertebra (L6), tibia, and femur of young (3 months) and older rats (21 months). Methods: Twenty Wistar rats were divided into four groups (five animals per group): young sedentary or trained and older sedentary or trained. The 12-week RT consisted of climbing a 1.1-m vertical ladder three times per week with progressive weights secured to the animals’ tails. The animals were killed 48 h after the end of the experimental period. The MMP-2 activity was assessed by the zymography method. Results: The aging process induced lower MMP-2 activity in the lumbar vertebrae and tibia (p=0.01). RT upregulated pro, intermediate, and active MMP-2 activity in the tibia of young rats (p=0.001). RT also upregulated pro and active MMP-2 activity in the lumbar vertebrae and tibia with advancing age (p=0.01). There was no significant difference (p> 0.05) between groups for MMP-2 of the femur, regardless of age and RT. Conclusion: The aging process impairs MMP-2 activity, but RT is a potential therapeutic approach to minimize the deleterious effects of ECM degeneration in different aged bones. Distinct MMP-2 responses to exercise training may result in specific remodeling processes

Palladium(II) complexes with thiosemicarbazones: syntheses, characterization and cytotoxicity against breast cancer cells and anti-mycobacterium tuberculosis activity

Three PdII complexes were prepared from N(4)-substituted thiosemicarbazones: [Pd(aptsc)(PPh3)](NO3)•H2O, 1, [Pd(apmtsc)(PPh3)](NO3), 2, and [Pd(apptsc)(PPh3)](NO3)•H2O, 3, where PPh3 = triphenylphosphine; Haptsc = 2-acetylpyridine-thiosemicarbazone; Hapmtsc = 2-acetylpyridine-N(4)-methyl-thiosemicarbazone and Happtsc = 2-acetylpyridine-N(4)-phenyl-thiosemicarbazone. All complexes were characterized by elemental analysis, IR, UV-Vis, 1H and 31P{1H} NMR spectroscopies, and had their crystalline structures determined by X-ray diffractometry from single crystals. The monoanionic thiosemicarbazonate ligands act in a tridentate mode, binding to the metal through the pyridine nitrogen, the azomethine nitrogen and the sulfur atoms. The cytotoxic activity against the breast cancer cell line MDA-MB231 and the anti-Mycobacterium tuberculosis H37Rv ATCC 27294 activity were evaluated for the compounds. All PdII complexes were highly active against the studied cell line, presenting similar values of IC50, around 5 µmol L-1, while the clinically applied antitumor agent cisplatin was inactive. The compounds show remarkable anti-M. tuberculosis activities, presenting MIC values comparable or better than some commercial anti-M tuberculosis drugs.FAPESPCAPESCNPqFINE

Characterization of the penicillin G acylase from Bacillus megaterium ATCC 14945

The purpose of this work was to characterize the enzyme penicillin G acylase (PGA) produced by Bacillus megaterium. Purification of the enzyme by ultra/diafiltration did not allow the detection of the PGA band by SDS-PAGE electrophoresis due to the high content of remaining proteins. However, using the DNA of the microorganism, it was possible to replicate the genes of the two B. megaterium PGA reported in literature, showing that the enzyme consisted of two sub-units, having 245 and 537 amino acids each and an average molecular mass of 26950 and 59070 Da, respectively. The parameters studied were: 1) the influence of temperature in the 25-60(0)C range, 2) pH in the 5-10 range and 3) substrate concentration, this was tested to obtain results on the Penicillin G hydrolysis reaction rate, using the initial velocities approach. The maximum hydrolysis rate was obtained at 37ºC and pH 8.0. The Michaelis-Menten model fitted well, resulting in estimated Km and Vmax parameters values of 1.83 mM and 0.165*10-3 mmol/min/UI, respectively.O objetivo deste trabalho foi caracterizar a enzima penicilina G acilase (PGA) produzida por Bacillus megaterium, uma importante enzima industrial que catalisa a hidrólise de penicilina G, para produção de antibióticos semi-sintéticos. Purificação da enzima por ultra/diafiltração não permitiu detectar a banda de PGA por eletroforese SDS-PAGE devido ao elevado conteúdo de outras proteínas remanescentes. Contudo, utilizando DNA do microrganismo que vem sendo estudado, foi possível amplificar os genes das duas sub-unidades de PGA previstas na literatura, mostrando que a enzima em estudo é também constituída de duas sub-unidades, 245 e 537 aminoácidos cada, com massas moleculares médias de 26950 e 59070 Da, respectivamente. Foram estudadas as influências da temperatura 25-60(0)C, pH 5-10, e concentração do substrato na velocidade da reação de hidrólise da penicilina G. A temperatura e pH ótimos foram de 37(0)C e 8,0, respectivamente. O modelo de Michaelis-Menten representou bem a cinética da reação, com valores de parâmetros estimados de 1,83 mM para Km e Vmáx= 0,165*10-3 mmol/min/UI

Blockage of αvβ3 integrin in 3D culture of triple-negative breast cancer and endothelial cells inhibits migration and discourages endothelial-to-mesenchymal plasticity

Breast cancer is a relevant cause of mortality in women and its triple-negative subtype (TNBC) is usually associated with poor prognosis. During tumor progression to metastasis, angiogenesis is triggered by the sprouting of endothelial cells from pre-existing vessels by a dynamic chain of events including VE-cadherin downregulation, actin protrusion, and integrin-mediated adhesion, allowing for migration and proliferation. The binding of tumoral and tumor-associated stromal cells with the extracellular matrix through integrins mediates angiogenic processes and certain integrin subtypes, such as the αvβ3 integrin, are upregulated in hypoxic TNBC models. Integrin αvβ3 inhibition by the high-affinity binding disintegrin DisBa-01 was previously demonstrated to induce anti-tumoral and anti-angiogenic responses in traditional 2D cell assays. Here, we investigate the effects of integrin αvβ3 blockage in endothelial and TNBC cells by DisBa-01 in 3D cultures under two oxygen conditions (1% and 20%). 3D cultures created using non-adhesive micromolds with Matrigel were submitted to migration assay in Boyden chambers and fluorescence analysis. DisBa-01 inhibited cell migration in normoxia and hypoxia in both MDA-MB-231 and HUVEC spheroids. Protein levels of integrin αvβ3 were overexpressed in HUVEC spheroids compared to MDA-MB-231 spheroids. In HUVEC 3D cultures, sprouting assays in collagen type I were decreased in normoxia upon DisBa-01 treatment, and VE-cadherin levels were diminished in HUVEC spheroids in hypoxia and upon DisBa-01 treatment. In conclusion, the blockage of integrin αvβ3 by DisBa-01 inhibits cell migration in 3D culture and interferes with tumor-derived responses in different oxygen settings, implicating its crucial role in angiogenesis and tumor progression

Evaluating kinetic and physiological features of rCHO-K1 cells cultured on microcarriers for production of a recombinant metalloprotease/disintegrin

We present kinetic and physiological data regarding the culturing of rCHO-K1 cells on various microcarriers, to evaluate the potential of this culture strategy for mass production of these cells and expression of a recombinant disintegrin. Cultures were performed in 500 mL spinner flasks in DMEM culture medium with 10% v/v fetal calf serum, gently shaken at 37°C, pH 7.4, in a 10% v/v CO2 atmosphere. The following values were obtained, respectively, for the adhesion time-constant Ka (h) and specific growth rate μmax (d-1) on each microcarrier: Cytodex 1 (0.91, 0.45), Cultispher S (0.28, 0.34), Immobasil FS (0.85, 0.52) and Pronectin F (5.12, 0.67). Metabolic characteristics showed some variation among the cultures with the four microcarriers, the most significant being the higher production of ammonia with microcarriers coated with adhesive molecules (Cultispher S and Pronectin F) relative to the uncoated carriers (Cytodex 1 and Immobasil FS). Experiments where the DMEM medium was gradually replaced by the serum-free medium (CHO-SFM-II) revealed important advantages over media containing serum, not only for assay purposes, but also for purification of the disintegrin. Altogether these results demonstrate that cultures on microcarriers, especially on Pronectin F, show good potential for larger scale cultures of rCHO-K1 cell

Human HSP70-escort protein 1 (hHep1) interacts with negatively charged lipid bilayers and cell membranes.

Human Hsp70-escort protein 1 (hHep1) is a cochaperone that assists in the function and stability of mitochondrial HSPA9. Similar to HSPA9, hHep1 is located outside the mitochondria and can interact with liposomes. In this study, we further investigated the structural and thermodynamic behavior of interactions between hHep1 and negatively charged liposomes, as well as interactions with cellular membranes. Our results showed that hHep1 interacts peripherally with liposomes formed by phosphatidylserine and cardiolipin and remains partially structured, exhibiting similar affinities for both. In addition, after being added to the cell membrane, recombinant hHep1 was incorporated by cells in a dose-dependent manner. Interestingly, the association of HSPA9 with hHep1 improved the incorporation of these proteins into the lipid bilayer. These results demonstrated that hHep1 can interact with lipids also present in the plasma membrane, indicating roles for this cochaperone outside of mitochondria

UMA REVISÃO SOBRE A PLASTICIDADE DO MÚSCULO ESQUELÉTICO: EXPRESSÃO DE ISOFORMAS DE CADEIA PESADA DE MIOSINA E CORRELAÇÃO FUNCIONAL

INTRODUÇÃO: o músculo esquelético é um tecido dinâmico com a habilidade intrínseca de se adaptar aos estímulos ambientais como resultado de mudanças qualitativas e quantitativas na expressão gênica, sendo esta capacidade definida como plasticidade. Este tecido é composto por populações de fibras rápidas e lentas que diferem fenotipicamente por expressarem diferentes isoformas de cadeias pesadas de miosina (CPM). Miosinas constituem uma família de proteínas chamadas motores moleculares com atividade ATPásica conhecidas por seu importante papel no processo de contração muscular. OBJETIVO: realizar um levantamento, por meio de dados expostos na literatura, da relação existente entre a expressão de diferentes CPM no processo de remodelamento muscular em condições fisiológicas e patológicas. METODOLOGIA: a pesquisa foi realizada por meio de levantamento de dados descritos na literatura, sendo consultados os bancos de dados internacionais Pubmed e Highwire Press e os bancos de dados nacionais Scielo e Lilacs, no período de janeiro a abril de 2008. Fisioter Mov. 2009 abr/jun; 22(2): 211-220 ISSN 0103-5150 Fisioter. Mov., Curitiba, v. 22, n. 2, p. 211-220, jan./mar. 2009 Licenciado sob uma Licença Creative Commons 212Piovesan RF, Martins MD, Fernandes KPS, Bussadori SK, Selistre-de-Araújo HS, Mesquita-Ferrari RA. RESULTADOS: A fibra muscular pode alterar suas características contráteis por meio de mudanças nas quantidades das CPM. A velocidade de encurtamento do músculo esquelético varia de acordo com a isoforma de CPM que possui, conferindo assim ao tecido muscular a capacidade de adaptação frente a estímulos fisiológicos e patológicos. CONCLUSÃO: A fibra muscular pode alterar suas propriedades contráteis por meio de mudanças nas quantidades de isoformas de CPM que a constitui

Remodeling process in bone of aged rats in response to resistance training

Aims We investigate the effects of RT on the mechanical function, gene, and protein expression of key factors involved in bone remodeling during aging. Main methods Male rats of 3 and 21 months of age were randomly allocated into four groups (8 per group): young sedentary (YS), young trained (YT), old sedentary (OS), and old trained (OT). RT was performed three times per week (12 weeks). Bone tenacity and stiffness were measured by biomechanical tests and mRNA levels of COL1A1, MEPE, SOST, OPG, BMP-2, PPAR-y, MMP-2-9-13, and TIMP-1 were evaluated by quantitative PCR. COL1A1 protein and MMP-2 activity were detected by western blotting and zymography assays. Key findings Aging increased stiffness, while BMP-2, OPG, COL1A1 and MMP-2 mRNA levels reduced (OS vs YS; p ≤ 0.05). RT increased the tenacity of the femur and reduced PPAR-γ regardless of age (YT vs. YS; OT vs. OS; p ≤ 0.05). RT downregulated SOST mRNA levels only in the OT group (vs. OS group, p ≤ 0.05). RT mitigated the age-associated increase in MMP-9 mRNA levels (p ≤ 0.05). In young animals, upregulation in MEPE, MMP-13, TIMP-1 were observed after RT, as well an increase in COL1A1 protein and MMP-2 activity (p ≤ 0.05). Significance RT improved bone tenacity independent of aging, which is relevant for mechanical function, while, at protein levels, RT upregulated MMP-2 activity and collagen 1 only in young rats. This study highlights the importance of exercise on bone health and identifies specific molecular changes in response to RT. Our findings provide insights into the mechanisms involved in age-related changes