Understanding the intracellular-to-extracellular localization switch of polyhydroxybutyrate polymerase in pseudomonas backgrounds as a microevolutionary process.

After gene duplication, paralogous genes evolve independently, and consequently, the new proteins encoded by these duplicated genes are exposed to changes in their subcellular location. Although there are increasing evidence that phylogenetically related proteins play different functions in different subcellular compartments, the number of evolutionary steps required for the emergence of a novel protein with a novel subcellular localization remains unclear. Regarding this intriguing topic, here we examine in depth our previous reports describing both intracellular and extracellular polyhydroxybutyrate polymerases (PhaC) in the Pseudomonadales group. The recapitulation of the intracellular-to-extracellular localization switch of PhaC in these strains shows a gradual evolution from a simple cytosolic PhaC form to a complex extracellular PhaC form specifically secreted via the type 1 secretion system. This gradual evolution includes several adaptive and pre-adaptive changes at the genomic, genetic and enzymatic levels, which are intimately related to the lifestyle of organisms during the evolution of protein localization. We conclude that the protein localization switch can be an extremely complex process in nature.

Purification and characterization of a novel extracellular serine-protease with collagenolytic activity from Aspergillus tamarii URM4634.

An extracellular serine-protease from Aspergillus tamarii URM4634 was purified and characterized. The possibility of using Aspergillus tamarii URM4634 protease in detergent formulations and collagenolytic activity was investigated. The protease demonstrated excellent stability at pH range 7.0-11.0, the optimum being at pH 9.0. The enzyme was stable at 40 °C for 180 min, enhanced by Mg++ and Ca++, but inhibited by Zn++, and strongly inhibited by phenylmethylsulfonyl fluoride (PMSF), suggested as serine-protease. The azocasein substrate result showed Km = 0.434 mg/mL and Vmax = 7.739 mg/mL/min. SDS-PAGE and azocasein zymography showed that the purified alkaline protease (2983.8 U/mg) had a molecular mass of 49.3 kDa. The enzyme was purified by column chromatography using Sephadex A50 resin. The proteolytic activity was activated by SDS (sodium dodecyl sulfate), Tween-80, Tween 20 and Triton-100. This study demonstrated that A. tamarii URM4634 protease has potent, stable and compatible collagenolytic activity to the desired level in local laundry detergent brands compared with similar enzymes produced by solid-state fermentation. This protease can thus be chosen as an option in both the food industry to tenderization meat and the detergent industry to washing process.

Functional roles of C-terminal extension (CTE) of salt-dependent peptidase activity of the Natrialba magadii extracellular protease (NEP).

Nep (Natrialba magadii extracellular protease) is a halolysin-like peptidase secreted by the haloalkaliphilic archaeon Natrialba magadii. Many extracellular proteases have been characterized from archaea to bacteria as adapted to hypersaline environments retaining function and stability until 4.0M NaCl. As observed in other secreted halolysins, this stability can be related to the presence of a C-terminal extension (CTE) sequence. In the present work, we compared the biochemical properties of recombinant Nep protease with the truncated form at the 134 amino acids CTE (Nep∆CTE), that was more active in 4M NaCl than the non-truncated wild type enzyme. Comparable to the wild type, Nep∆CTE protease is irreversibly inactivated at low salt solutions. The substrate specificity of the truncated Nep∆CTE was similar to that of wild type form as demonstrated by a combinatorial library of FRET substrates. The enzyme stability, the effect of different salts and the thermodynamics assays using different lengths of substrates demonstrated similarities between the two forms. Altogether, these data provide further information on the stability and structural determinants of halolysins under different salinities, especially concerning the enzymatic behavior.

An extracellular proteasome releases endostatin from human collagen XVIII.

Endostatin is a potent anti-angiogenic and anti-tumor protein capable of regressing tumors without inducing acquired resistance. Since it is a fragment of the parental molecule, collagen XVIII, its endogenous production depends on the activity of a specific proteolytic enzyme. While such an enzyme has been described in mice, a human counterpart has not been identified so far. Here, we searched for this enzyme by using a fluorescence resonance energy transfer peptide containing the cleavage site of human collagen XVIII. We found that the cleavage activity was present in various murine and human tumor cells but not in untransformed cells. It was ascribed to a large protein complex identified as an extracellular form of proteasome 20S. Since circulating proteasome 20S has recently emerged as an important marker of tumor progression, the possibility of proteasomes controlling the production of angiostatic endostatin may inspire the development of new anticancer therapies.

Extracellular enzymatic profiles and taxonomic identification of endophytic fungi isolated from four plant species.

Plants of medicinal and economic importance have been studied to investigate the presence of enzyme-producing endophytic fungi. The characterization of isolates with distinct enzyme production potential may identify suitable alternatives for specialized industry. At Universidade Estadual de Maringá Laboratory of Microbial Biotechnology, approximately 500 isolates of endophytic fungi have been studied over the last decade from various host plants, including medicinally and economically important species, such as Luehea divaricata (Martius et Zuccarini), Trichilia elegans A. Juss, Sapindus saponaria L., Piper hispidum Swartz, and Saccharum spp. However, only a fraction of these endophytes have been identified and evaluated for their biotechnological application, having been initially grouped by morphological characteristics, with at least one representative of each morphogroup tested. In the current study, several fungal strains from four plants (L. divaricata, T. elegans, S. saponaria, and Saccharum spp) were identified by ribosomal DNA typing and evaluated semi-quantitatively for their enzymatic properties, including amylase, cellulase, pectinase, and protease activity. Phylogenetic analysis revealed the presence of four genera of endophytic fungi (Diaporthe, Saccharicola, Bipolaris, and Phoma) in the plants examined. According to enzymatic tests, 62% of the isolates exhibited amylase, approximately 93% cellulase, 50% pectinase, and 64% protease activity. Our results verified that the composition and abundance of endophytic fungi differed between the plants tested, and that these endophytes are a potential enzyme production resource of commercial and biotechnological value.

Process optimization for production and purification of a thermostable, organic solvent tolerant lipase from Acinetobacter sp. AU07

ABSTRACT The purpose of this study was to isolate, purify and optimize the production conditions of an organic solvent tolerant and thermostable lipase from Acinetobacter sp. AU07 isolated from distillery waste. The lipase production was optimized by response surface methodology, and a maximum production of 14.5 U/mL was observed at 30 ºC and pH 7, using a 0.5% (v/v) inoculum, 2% (v/v) castor oil (inducer), and agitation 150 rpm. The optimized conditions from the shake flask experiments were validated in a 3 L lab scale bioreactor, and the lipase production increased to 48 U/mL. The enzyme was purified by ammonium sulfate precipitation and ion exchange chromatography and the overall yield was 36%. SDS-PAGE indicated a molecular weight of 45 kDa for the purified protein, and Matrix assisted laser desorption/ionization time of flight analysis of the purified lipase showed sequence similarity with GDSL family of lipases. The optimum temperature and pH for activity of the enzyme was found to be 50 ºC and 8.0, respectively. The lipase was completely inhibited by phenylmethylsulfonyl fluoride but minimal inhibition was observed when incubated with ethylenediaminetetraacetic acid and dithiothreitol. The enzyme was stable in the presence of non-polar hydrophobic solvents. Detergents like SDS inhibited enzyme activity; however, there was minimal loss of enzyme activity when incubated with hydrogen peroxide, Tween 80 and Triton X-100. The kinetic constants (Km and Vmax) revealed that the hydrolytic activity of the lipase was specific to moderate chain fatty acid esters. The Vmax, Km and Vmax/Km ratio of the enzyme were 16.98 U/mg, 0.51 mM, and 33.29, respectively when 4-nitrophenyl palmitate was used as a substrate.

Process optimization for production and purification of a thermostable, organic solvent tolerant lipase from Acinetobacter sp. AU07.

The purpose of this study was to isolate, purify and optimize the production conditions of an organic solvent tolerant and thermostable lipase from Acinetobacter sp. AU07 isolated from distillery waste. The lipase production was optimized by response surface methodology, and a maximum production of 14.5U/mL was observed at 30°C and pH 7, using a 0.5% (v/v) inoculum, 2% (v/v) castor oil (inducer), and agitation 150rpm. The optimized conditions from the shake flask experiments were validated in a 3L lab scale bioreactor, and the lipase production increased to 48U/mL. The enzyme was purified by ammonium sulfate precipitation and ion exchange chromatography and the overall yield was 36%. SDS-PAGE indicated a molecular weight of 45kDa for the purified protein, and Matrix assisted laser desorption/ionization time of flight analysis of the purified lipase showed sequence similarity with GDSL family of lipases. The optimum temperature and pH for activity of the enzyme was found to be 50°C and 8.0, respectively. The lipase was completely inhibited by phenylmethylsulfonyl fluoride but minimal inhibition was observed when incubated with ethylenediaminetetraacetic acid and dithiothreitol. The enzyme was stable in the presence of non-polar hydrophobic solvents. Detergents like SDS inhibited enzyme activity; however, there was minimal loss of enzyme activity when incubated with hydrogen peroxide, Tween 80 and Triton X-100. The kinetic constants (Km and Vmax) revealed that the hydrolytic activity of the lipase was specific to moderate chain fatty acid esters. The Vmax, Km and Vmax/Km ratio of the enzyme were 16.98U/mg, 0.51mM, and 33.29, respectively when 4-nitrophenyl palmitate was used as a substrate.

Trichosporon inkin biofilms produce extracellular proteases and exhibit resistance to antifungals.

The aim of this study was to determine experimental conditions for in vitro biofilm formation of clinical isolates of Trichosporon inkin, an important opportunistic pathogen in immunocompromised patients. Biofilms were formed in microtitre plates in three different media (RPMI, Sabouraud and CLED), with inocula of 104, 105 or 106 cells ml- 1, at pH 5.5 and 7.0, and at 35 and 28 °C, under static and shaking conditions for 72 h. Growth kinetics of biofilms were evaluated at 6, 24, 48 and 72 h. Biofilm milieu analysis were assessed by counting viable cells and quantification of nucleic acids released into biofilm supernatants. Biofilms were also analysed for proteolytic activity and antifungal resistance against amphotericin B, caspofungin, fluconazole, itraconazole and voriconazole. Finally, ultrastructural characterization of biofilms formed in microtitre plates and catheter disks was performed by scanning electron microscopy. Greater biofilm formation was observed with a starter inoculum of 106 cells ml- 1, at pH 7.0 at 35 °C and 80 r.p.m., in both RPMI and Sabouraud media. Growth kinetics showed an increase in both viable cells and biomass with increasing incubation time, with maximum production at 48 h. Biofilms were able to disperse viable cells and nucleic acids into the supernatant throughout the developmental cycle. T. inkin biofilms produced more protease than planktonic cells and showed high tolerance to amphotericin B, caspofungin and azole derivatives. Mature biofilms were formed by different morphotypes, such as blastoconidia, arthroconidia and hyphae, in a strain-specific manner. The present article details the multicellular lifestyle of T. inkin and provides perspectives for further research.

Dynamics of biofilm formation and the interaction between Candida albicans and methicillin-susceptible (MSSA) and -resistant Staphylococcus aureus (MRSA).

Polymicrobial biofilms are an understudied and a clinically relevant problem. This study evaluates the interaction between C. albicans, and methicillin- susceptible (MSSA) and resistant (MRSA) S. aureus growing in single- and dual-species biofilms. Single and dual species adhesion (90 min) and biofilms (12, 24, and 48 h) were evaluated by complementary methods: counting colony-forming units (CFU mL-1), XTT-reduction, and crystal violet staining (CV). The secretion of hydrolytic enzymes by the 48 h biofilms was also evaluated using fluorimetric kits. Scanning electron microscopy (SEM) was used to assess biofilm structure. The results from quantification assays were compared using two-way ANOVAs with Tukey post-hoc tests, while data from enzymatic activities were analyzed by one-way Welch-ANOVA followed by Games-Howell post hoc test (α = 0.05). C. albicans, MSSA and MRSA were able to adhere and to form biofilm in both single or mixed cultures. In general, all microorganisms in both growth conditions showed a gradual increase in the number of cells and metabolic activity over time, reaching peak values between 12 h and 48 h (ρ<0.05). C. albicans single- and dual-biofilms had significantly higher total biomass values (ρ<0.05) than single biofilms of bacteria. Except for single MRSA biofilms, all microorganisms in both growth conditions secreted proteinase and phospholipase-C. SEM images revealed extensive adherence of bacteria to hyphal elements of C. albicans. C. albicans, MSSA, and MRSA can co-exist in biofilms without antagonism and in an apparent synergistic effect, with bacteria cells preferentially associated to C. albicans hyphal forms.

Extracellular functions of glycolytic enzymes of parasites: unpredicted use of ancient proteins.

In addition of their usual intracellular localization where they are involved in catalyzing reactions of carbohydrate and energy metabolism by glycolysis, multiple studies have shown that glycolytic enzymes of many organisms, but notably pathogens, can also be present extracellularly. In the case of parasitic protists and helminths, they can be found either secreted or attached to the surface of the parasites. At these extracellular localizations, these enzymes have been shown to perform additional, very different so-called "moonlighting" functions, such as acting as ligands for a variety of components of the host. Due to this recognition, different extracellular glycolytic enzymes participate in various important parasite-host interactions such as adherence and invasion of parasites, modulation of the host's immune and haemostatic systems, promotion of angiogenesis, and acquisition of specific nutrients by the parasites. Accordingly, extracellular glycolytic enzymes are important for the invasion of the parasites and their establishment in the host, and in determining their virulence.

Crystallization and preliminary diffraction analysis of the catalytic domain of major extracellular endoglucanase from Xanthomonas campestris pv. campestris.

Cellulases, such as endoglucanases, exoglucanases and ß-glucosidases, are important enzymes used in the process of enzymatic hydrolysis of plant biomass. The bacteria Xanthomonas campestris pv. campestris expresses a large number of hydrolases and the major endoglucanase (XccEG), a member of glycoside hydrolase family 5 (GH5), is the most strongly secreted extracellularly. In this work, the native XccEG was purified from the extracellular extract and crystallization assays were performed on its catalytic domain. A complete data set was collected on an in-house X-ray source. The crystal diffracted to 2.7 Å resolution and belonged to space group C2, with unit-cell parameters a = 174.66, b = 141.53, c = 108.00 Å, ß = 110.49°. The Matthews coefficient suggests a solvent content of 70.1% and the presence of four protein subunits in the asymmetric unit.

Different secreted phosphatase activities in Leishmania amazonensis.

Leishmania has strong acid phosphatase activity on the external surface of the plasma membrane and secreted into the extracellular milieu. Secreted acid phosphatase (sAcP), which is the most abundant secreted protein of Leishmania, is also a virulence factor that plays a role in vertebrate infection and survival in sand flies. In this study, we characterized the secreted phosphatase activities in Leishmania amazonensis. Both acidic and alkaline secreted phosphatase activities were observed with ß-glycerophosphate and p-nitrophenyl phosphate (p-NPP) hydrolysis and were inhibited with sodium tartrate and sodium orthovanadate. Cytochemical labeling revealed a significant difference in the localization of the electron-dense precipitates depending on the substrate. ß-Glycerophosphate electron-dense precipitates were concentrated on both the cell surface and flagellar pocket, whereas p-NPP labeling occurred primarily within intracellular organelles. Orthovanadate-treated metacyclic promastigotes were less infective and were confined to a tight parasitophorous vacuole (PV), which is not characteristic of this Leishmania species. Based on the results, we characterized the presence of different secreted phosphatase activities in L. amazonensis, the influence of the substrate in cytochemical labeling, and the potential involvement of secreted phosphatase activity in both PV maturation and amastigote survival.

Purification, characterization, and specificity determination of a new serine protease secreted by Penicillium waksmanii.

The purpose of this work was to purify a protease from Penicillium waksmanii and to determine its biochemical characteristics and specificity. The extracellular protease isolated that was produced by P. waksmanii is a serine protease that is essential for the reproduction and growth of the fungus. The protease isolated showed 32 kDa, and has optimal activity at pH 8.0 and 35 °C towards the substrate Abz-KLRSSKQ-EDDnp. The protease is active in the presence of CaCl(2), KCl, and BaCl, and partially inhibited by CuCl(2), CoCl(2) and totally inhibited by AlCl(3) and LiCl. In the presence of 1 M urea, the protease remains 50 % active. The activity of the protease increases 60 % when it is exposed to 0.4 % nonionic surfactant-Triton X-100 and loses 10 % activity in the presence of 0.4 % Tween-80. Using fluorescence resonance energy transfer analysis, the protease showed the most specificity for the peptide Abz-KIRSSKQ-EDDnp with k (cat)/K (m) of 10,666 mM(-1) s(-1), followed by the peptide Abz-GLRSSKQ-EDDnp with a k (cat)/K (m) of 7,500 mM(-1) s(-1). Basic and acidic side chain-containing amino acids performed best at subsite S(1). Subsites S(2), S(3), S(') (2), and S(') (1), S(') (3) showed a preference for binding for amino acids with hydrophobic and basic amino acid side chain, respectively. High values of k (cat)/K (m) were observed for the subsites S(2), S(3), and S(') (2.) The sequence of the N-terminus (ANVVQSNVPSWGLARLSSKKTGTTDYTYD) showed high similarity to the fungi Penicillium citrinum and Penicillium chrysogenum, with 89 % of identity at the amino acid level.

A potential role for an extracellular methanol oxidase secreted by Moniliophthora perniciosa in Witches' broom disease in cacao.

The hemibiotrophic basidiomycete fungus Moniliophthora perniciosa, the causal agent of Witches' broom disease (WBD) in cacao, is able to grow on methanol as the sole carbon source. In plants, one of the main sources of methanol is the pectin present in the structure of cell walls. Pectin is composed of highly methylesterified chains of galacturonic acid. The hydrolysis between the methyl radicals and galacturonic acid in esterified pectin, mediated by a pectin methylesterase (PME), releases methanol, which may be decomposed by a methanol oxidase (MOX). The analysis of the M. pernciosa genome revealed putative mox and pme genes. Real-time quantitative RT-PCR performed with RNA from mycelia grown in the presence of methanol or pectin as the sole carbon source and with RNA from infected cacao seedlings in different stages of the progression of WBD indicate that the two genes are coregulated, suggesting that the fungus may be metabolizing the methanol released from pectin. Moreover, immunolocalization of homogalacturonan, the main pectic domain that constitutes the primary cell wall matrix, shows a reduction in the level of pectin methyl esterification in infected cacao seedlings. Although MOX has been classically classified as a peroxisomal enzyme, M. perniciosa presents an extracellular methanol oxidase. Its activity was detected in the fungus culture supernatants, and mass spectrometry analysis indicated the presence of this enzyme in the fungus secretome. Because M. pernciosa possesses all genes classically related to methanol metabolism, we propose a peroxisome-independent model for the utilization of methanol by this fungus, which begins with the extracellular oxidation of methanol derived from the demethylation of pectin and finishes in the cytosol.

Amyloid fibrils trigger the release of neutrophil extracellular traps (NETs), causing fibril fragmentation by NET-associated elastase.

The accumulation of amyloid fibrils is a feature of amyloid diseases, where cell toxicity is due to soluble oligomeric species that precede fibril formation or are formed by fibril fragmentation, but the mechanism(s) of fragmentation is still unclear. Neutrophil-derived elastase and histones were found in amyloid deposits from patients with different systemic amyloidoses. Neutrophil extracellular traps (NETs) are key players in a death mechanism in which neutrophils release DNA traps decorated with proteins such as elastase and histones to entangle pathogens. Here, we asked whether NETs are triggered by amyloid fibrils, reasoning that because proteases are present in NETs, protease digestion of amyloid may generate soluble, cytotoxic species. We show that amyloid fibrils from three different sources (α-synuclein, Sup35, and transthyretin) induced NADPH oxidase-dependent NETs in vitro from human neutrophils. Surprisingly, NET-associated elastase digested amyloid fibrils into short species that were cytotoxic for BHK-21 and HepG2 cells. In tissue sections from patients with primary amyloidosis, we also observed the co-localization of NETs with amyloid deposits as well as with oligomers, which are probably derived from elastase-induced fibril degradation (amyloidolysis). These data reveal that release of NETs, so far described to be elicited by pathogens, can also be triggered by amyloid fibrils. Moreover, the involvement of NETs in amyloidoses might be crucial for the production of toxic species derived from fibril fragmentation.

Production and characterization of extracellular α-amylase produced by Wickerhamia sp. X-Fep.

A yeast isolate able to produce high levels of extracellular α-amylase was selected from a collection of 385 yeasts and identified as Wickerhamia sp. by the sequence of the D1/D2 domain of the 26 S rDNA gene. Part of the nucleotide sequence of the amy1-W gene was cloned, and a sequence of 191 amino acids deduced from this gene was analyzed. The peptide contains three characteristic well-conserved regions in the active sites of α-amylases (EC 3.2.1.1). The enzyme was purified and in situ activity showed only one band with amylolytic activity. The molecular mass of the α-amylase was estimated at 54 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Enzymatic activity on soluble starch as substrate was optimal at pH 5-6 and 50 °C. This thermostable enzyme was inhibited by EDTA-Na(2) and 1,10-phenanthroline; the activity of the dialyzed enzyme was reactivated with Ca(2+) and Mg(2+) cations, which indicates that the α-amylase is a metalloenzyme. α-Amylase production was induced by starch and maltose and repressed by glucose. The high yield and productivity found in this work makes this Wickerhamia sp. strain a promising candidate for the biotechnological production of α-amylase.

Biodegradation of a keratin waste and the concomitant production of detergent stable serine proteases from Paecilomyces lilacinus.

Paecilomyces lilacinus (LPS 876) efficiently degraded keratin in chicken feather during submerged cultivation producing extracellular proteases. Characterization of crude protease activity was done including its compatibility in commercial detergents. Optimum pH and temperature were 10.0 and 60 °C, respectively. Protease activity was enhanced by Ca²âº but was strongly inhibited by PMSF and by Hg²âº suggesting the presence of thiol-dependent serine proteases. The crude protease showed extreme stability toward non-ionic (Tween 20, Tween 85, and Triton X-100) and anionic (SDS) surfactants, and relative stability toward oxidizing agent (H2O2 and sodium perborate). In addition, it showed excellent stability and compatibility with various solid and liquid commercial detergents from 30 to 50 °C. The enzyme preparation retained more than 95% of its initial activity with solid detergents (Ariel™ and Drive™) and 97% of its original activity with a liquid detergent (Ace™) after pre-incubation at 40 °C. The protective effect of polyols (propylene glycol, PEG 4000, and glycerol) on the heat inactivation was also examined and the best results were obtained with glycerol from 50 to 60 °C. Considering its promising properties, P. lilacinus enzymatic preparation may be considered as a candidate for use in biotechnological processes (i.e., as detergent additive) and in the processing of keratinous wastes.

ANCUT2, an extracellular cutinase from Aspergillus nidulans induced by olive oil.

Cutinases are versatile carboxylic ester hydrolases with great potential in many biocatalytic processes, including biodiesel production. Genome sequence analysis of the model organism Aspergillus nidulans reveals four genes encoding putative cutinases. In this work, we purified and identified for the first time a cutinase (ANCUT2) produced by A. nidulans. ANCUT2 is a 29-kDa protein which consists of 255 amino acid residues. Comparison of the amino acid sequence of ANCUT2 with other microbial cutinase sequences revealed a high degree of homology with other fungal cutinases as well as new features, which include a serine-rich region and conserved cysteines. Cutinase production with different lipidic and carbon sources was also explored. Enzyme activity was induced by olive oil and some triacylglycerides and fatty acids, whereas it was repressed by glucose (1%) and other sugars. In some conditions, a 22-kDa post-translational processing product was also detected. The cutinase nature of the enzyme was confirmed after degradation of apple cutin.

Moderate exercise training promotes adaptations in coronary blood flow and adenosine production in normotensive rats.

OBJECTIVES: Aerobic exercise training prevents cardiovascular risks. Regular exercise promotes functional and structural adaptations that are associated with several cardiovascular benefits. The aim of this study is to investigate the effects of swimming training on coronary blood flow, adenosine production and cardiac capillaries in normotensive rats. METHODS: Wistar rats were randomly divided into two groups: control (C) and trained (T). An exercise protocol was performed for 10 weeks and 60 min/day with a tail overload of 5% bodyweight. Coronary blood flow was quantified with a color microsphere technique, and cardiac capillaries were quantified using light microscopy. Adenine nucleotide hydrolysis was evaluated by enzymatic activity, and protein expression was evaluated by western blot. The results are presented as the means ± SEMs (p<0.05). RESULTS: Exercise training increased the coronary blood flow and the myocardial capillary-to-fiber ratio. Moreover, the circulating and cardiac extracellular adenine nucleotide hydrolysis was higher in the trained rats than in the sedentary rats due to the increased activity and protein expression of enzymes, such as E-NTPDase and 59'-nucleotidase. CONCLUSIONS: Swimming training increases coronary blood flow, number of cardiac capillaries, and adenine nucleotide hydrolysis. Increased adenosine production may be an important contributor to the enhanced coronary blood flow and angiogenesis that were observed in the exercise-trained rats; collectively, these results suggest improved myocardial perfusion.

Moderate exercise training promotes adaptations in coronary blood flow and adenosine production in normotensive rats

OBJECTIVES: Aerobic exercise training prevents cardiovascular risks. Regular exercise promotes functional and structural adaptations that are associated with several cardiovascular benefits. The aim of this study is to investigate the effects of swimming training on coronary blood flow, adenosine production and cardiac capillaries in normotensive rats. METHODS: Wistar rats were randomly divided into two groups: control (C) and trained (T). An exercise protocol was performed for 10 weeks and 60 min/day with a tail overload of 5 percent bodyweight. Coronary blood flow was quantified with a color microsphere technique, and cardiac capillaries were quantified using light microscopy. Adenine nucleotide hydrolysis was evaluated by enzymatic activity, and protein expression was evaluated by western blot. The results are presented as the means ± SEMs (p<0.05). RESULTS: Exercise training increased the coronary blood flow and the myocardial capillary-to-fiber ratio. Moreover, the circulating and cardiac extracellular adenine nucleotide hydrolysis was higher in the trained rats than in the sedentary rats due to the increased activity and protein expression of enzymes, such as E-NTPDase and 59- nucleotidase. CONCLUSIONS: Swimming training increases coronary blood flow, number of cardiac capillaries, and adenine nucleotide hydrolysis. Increased adenosine production may be an important contributor to the enhanced coronary blood flow and angiogenesis that were observed in the exercise-trained rats; collectively, these results suggest improved myocardial perfusion.