Moderate exercise improves leucocyte function and decreases inflammation in diabetes

P>The genesis and progression of diabetes occur due in part to an uncontrolled inflammation profile with insulin resistance, increased serum levels of free fatty acids (FFA), proinflammatory cytokines and leucocyte dysfunction. In this study, an investigation was made of the effect of a 3-week moderate exercise regimen on a treadmill (60% of VO(2max), 30 min/day, 6 days a week) on inflammatory markers and leucocyte functions in diabetic rats. The exercise decreased serum levels of tumour necrosis factor (TNF)-alpha (6%), cytokine-induced neutrophil chemotactic factor 2 alpha/beta (CINC-2 alpha/beta) (9%), interleukin (IL)-1 beta (34%), IL-6 (86%), C-reactive protein (CRP) (41%) and FFA (40%) in diabetic rats when compared with sedentary diabetic animals. Exercise also attenuated the increased responsiveness of leucocytes from diabetics when compared to controls, diminishing the reactive oxygen species (ROS) release by neutrophils (21%) and macrophages (28%). Exercise did not change neutrophil migration and the proportion of neutrophils and macrophages in necrosis (loss of plasma membrane integrity) and apoptosis (DNA fragmentation). Serum activities of creatine kinase (CK) and lactate dehydrogenase (LDH) were not modified in the conditions studied. Therefore, physical training did not alter the integrity of muscle cells. We conclude that moderate physical exercise has marked anti-inflammatory effects on diabetic rats. This may be an efficient strategy to protect diabetics against microorganism infection, insulin resistance and vascular complications.

Regulation of glycolysis and expression of glucose metabolism-related genes by reactive oxygen species in contracting skeletal muscle cells

Contractile activity induces a marked increase in glycolytic activity and gene expression of enzymes and transporters involved in glucose metabolism in skeletal muscle. Muscle contraction also increases the production of reactive oxygen species (ROS). In this study, the effects of treatment with N-acetylcysteine (NAC), a potent antioxidant compound, on contraction-stimulated glycolysis were investigated in electrically stimulated primary rat skeletal muscle cells. The following parameters were measured: 2-[(3)H]deoxyglucose (2-DG) uptake; activities of hexokinase, phosphofructokinase (PFK), and glucose-6-phosphate dehydrogenase (G6PDH); lactate production; and expression of the glucose transporter 4 (GLUT4), hexokinase II (HKII), and PFK genes after one bout of electrical stimulation in primary rat myotubes. NAC treatment decreased ROS signal by 49% in resting muscle cells and abolished the muscle contraction-induced increase in ROS levels. In resting cells, NAC decreased mRNA and protein contents of GLUT4, mRNA content and activity of PFK, and lactate production. NAC treatment suppressed the contraction-mediated increase in 2-DG uptake; lactate production; hexokinase, PFK, and G6PDH activities; and gene expression of GLUT4. HKII, and PFK. Similar to muscle contraction, exogenous H(2)O(2) (500 nM) administration increased 2-DG uptake; lactate production; hexokinase, PFK, and G6PDH activities; and gene expression of GLUT4. HKII, and PFK. These findings support the proposition that ROS endogenously produced play an important role in the changes in glycolytic activity and gene expression of GLUT4, HKII, and PFK induced by contraction in skeletal muscle cells. (C) 2010 Elsevier Inc. All rights reserved.

Effect of short-term creatine supplementation on markers of skeletal muscle damage after strenuous contractile activity

The protective effect of short-term creatine supplementation (CrS) upon markers of strenuous contractile activity-induced damage in human and rat skeletal muscles was investigated. Eight Ironman triathletes were randomized into the placebo (Pl; n = 4) and creatine-supplemented (CrS; n = 4) groups. Five days prior to the Ironman competition, the CrS group received creatine monohydrate (20 g day(-1)) plus maltodextrin (50 g) divided in two equal doses. The Pl group received maltodextrin (50 g day(-1)) only. The effect of CrS (5 g day(-1)/kg body weight for 5 days) was also evaluated in a protocol of strenuous contractile activity induced by electrical stimulation in rats. Blood samples were collected before and 36 and 60 h after the competition and were used to determine plasma activities of creatine kinase (CK), lactate dehydrogenase (LDH), aldolase (ALD), glutamic oxaloacetic acid transaminase (GOT), glutamic pyruvic acid transaminase (GPT), and C-reactive protein (CRP) level. In rats, plasma activities of CK and LDH, muscle vascular permeability (MVP) using Evans blue dye, muscle force and fatigue were evaluated. Activities of CK, ALD, LDH, GOT, GTP, and levels of CRP were increased in the Pl group after the competition as compared to basal values. CrS decreased plasma activities of CK, LDH, and ALD, and prevented the rise of GOT and GPT plasma activities. In rats, CrS delayed the fatigue, preserved the force, and prevented the rise of LDH and CK plasma activities and MVP in the gastrocnemius muscle. CrS presented a protective effect on muscle injury induced by strenuous contractile activities.

Retinoic Acid and VEGF Delay Smooth Muscle Relative to Endothelial Differentiation to Coordinate Inner and Outer Coronary Vessel Wall Morphogenesis

Rationale: Major coronary vessels derive from the proepicardium, the cellular progenitor of the epicardium, coronary endothelium, and coronary smooth muscle cells (CoSMCs). CoSMCs are delayed in their differentiation relative to coronary endothelial cells (CoEs), such that CoSMCs mature only after CoEs have assembled into tubes. The mechanisms underlying this sequential CoE/CoSMC differentiation are unknown. Retinoic acid (RA) is crucial for vascular development and the main RA-synthesizing enzyme is progressively lost from epicardially derived cells as they differentiate into blood vessel types. In parallel, myocardial vascular endothelial growth factor (VEGF) expression also decreases along coronary vessel muscularization. Objective: We hypothesized that RA and VEGF act coordinately as physiological brakes to CoSMC differentiation. Methods and Results: In vitro assays (proepicardial cultures, cocultures, and RALDH2 [retinaldehyde dehydrogenase-2]/VEGF adenoviral overexpression) and in vivo inhibition of RA synthesis show that RA and VEGF act as repressors of CoSMC differentiation, whereas VEGF biases epicardially derived cell differentiation toward the endothelial phenotype. Conclusion: Experiments support a model in which early high levels of RA and VEGF prevent CoSMC differentiation from epicardially derived cells before RA and VEGF levels decline as an extensive endothelial network is established. We suggest this physiological delay guarantees the formation of a complex, hierarchical, tree of coronary vessels. (Circ Res. 2010;107:204-216.)

New Trypanosoma (Duttonella) vivax genotypes from tsetse flies in East Africa

Salivarian trypanosomes pose a substantial threat to livestock, but their full diversity is not known. To survey trypanosomes carried by tsetse in Tanzania, DNA samples from infected proboscides of Glossina pallidipes and G. swynnertoni were identified using fluorescent fragment length barcoding (FFLB), which discriminates species by size polymorphisms in multiple regions of the ribosomal RNA locus. FELLB identified the trypanosomes in 65 of 105 (61.9%) infected proboscides, revealing 9 mixed infections. Of 7 different FFLB profiles, 2 were similar but not identical to reference West African Trypanosoma vivax; 5 other profiles belonged to known species also identified in fly midguts. Phylogenetic analysis of the glycosomal glyceraldehyde phosphate dehydrogenase gene revealed that the Tanzanian T. vivax samples fell into 2 distinct groups, both outside the main chide of African and South American T. vivax. These new T. vivax genotypes were common and widespread in tsetse in Tanzania. The T. brucei-like trypanosome previously described from tsetse midguts was also found in 2 proboscides, demonstrating a salivarian transmission route. Investigation of mammalian host range and pathogenicity will reveal the importance of these new trypanosomes for the epidemiology and control of animal trypanosomiasis in East Africa.

Phylogeographical, ecological and biological patterns shown by nuclear (ssrRNA and gGAPDH) and mitochondrial (Cyt b) genes of trypanosomes of the subgenus Schizotrypanum parasitic in Brazilian bats

The genetic diversity and phylogeographical patterns of Trypanosoma species that infect Brazilian bats were evaluated by examining 1043 bats from 63 species of seven families captured in Amazonia, the Pantanal, Cerrado and the Atlantic Forest biomes of Brazil. The prevalence of trypanosonne-infected bats, as estimated by haemoculture, was 12.9%, resulting in 77 Cultures of isolates, most morphologically identified as Trypanosoma cf. cruzi, classified by barcoding using partial sequences from ssrRNA gene into the subgenus Schizotrypanum and identified as T. cruzi (15), T cruzi marinkellei (37) or T. cf. dionisii (25). Phylogenetic analyses using nuclear ssrRNA, glycosomal glyceraldehyde 3-phosphate dehydrogenase (gGAPDH) and mitochondrial cytochrome b (Cyt b) gene sequences generated three clades, which clustered together forming the subgenus Schizotrypanum. In addition to vector association, bat trypanosomes were related by the evolutionary history, ecology and phylogeography of the bats. Tryponosoma cf. dionisii trypanosomes (32.4%) infected 12 species from four bat families captured in all biomes, from North to South Brazil, and clustered with T. dionisii from Europe despite being separated by some genetic distance. Trypanosoma cruzi marinkellei (49.3%) was restricted to phyllostomid bats from Amazonia to the Pantanal (North to Central). Trypanosoma cruzi (18.2%) was found mainly in vespertilionid and phyllostomid bats from the Pantanal/Cerrado and the Atlantic Forest (Central to Southeast), with a few isolates from Amazonia. (C) 2009 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Phylogenetic Validation of the Genera Angomonas and Strigomonas of Trypanosomatids Harboring Bacterial Endosymbionts with the Description of New Species of Trypanosomatids and of Proteobacterial Symbionts

We comparatively examined the nutritional, molecular and optical and electron microscopical characteristics of reference species and new isolates of trypanosomatids harboring bacterial endosymbionts. Sequencing of the V7V8 region of the small subunit of the ribosomal RNA (SSU rRNA) gene distinguished six major genotypes among the 13 isolates examined. The entire sequences of the SSU rRNA and glycosomal glyceraldehyde phosphate dehydrogenase (gGAPDH) genes were obtained for phylogenetic analyses. In the resulting phylogenetic trees, the symbiont-harboring species clustered as a major clade comprising two subclades that corresponded to the proposed genera Angomonas and Strigomonas. The genus Angomonas comprised 10 flagellates including former Crithidia deanei and C. desouzai plus a new species. The genus Strigomonas included former Crithidia oncopelti and Blastocrithidia cuiicis plus a new species. Sequences from the internal transcribed spacer of ribosomal DNA (ITS rDNA) and size polymorphism of kinetoplast DNA (kDNA) minicircles revealed considerable genetic heterogeneity within the genera Angomonas and Strigomonas. Phylogenetic analyses based on 16S rDNA and ITS rDNA sequences demonstrated that all of the endosymbionts belonged to the Betaproteobacteria and revealed three new species. The congruence of the phylogenetic trees of trypanosomatids and their symbionts support a co-divergent host-symbiont evolutionary history. (C) 2011 Elsevier GmbH. All rights reserved.

Exercise training enhances rat pancreatic islets anaplerotic enzymes content despite reduced insulin secretion

Endurance exercise has been shown to reduce pancreatic islets glucose-stimulated insulin secretion (GSIS). Anaplerotic/cataplerotic pathways are directly related to GSIS signaling. However, the effect of endurance training upon pancreatic islets anaplerotic enzymes is still unknown. In this sense, we tested the hypothesis that endurance exercise decreases GSIS by reducing anaplerotic/cataplerotic enzymes content. Male Wistar rats were randomly assigned to one of the four experimental groups as follows: control sedentary group (CTL), trained 1 day per week (TRE1x), trained 3 days per week (TRE3x) and trained 5 days per week (TRE5x) and submitted to an 8 weeks endurance-training protocol. After the training protocol, pancreatic islets were isolated and incubated with basal (2.8 mM) and stimulating (16.7 mM) glucose concentrations for GSIS measurement by radioimmunoassay. In addition, pyruvate carboxylase (PYC), pyruvate dehydrogenase (PDH), pyruvate dehydrogenase kinase 4 (PDK4), ATP-citrate lyase (ACL) and glutamate dehydrogenase (GDH) content were quantified by western blotting. Our data showed that 8 weeks of chronic endurance exercise reduced GSIS by 50% in a dose-response manner according to weekly exercise frequency. PYC showed significant twofold increase in TRE3x. PYC enhancement was even higher in TRE5x (p < 0.0001). PDH and PDK4 reached significant 25 and 50% enhancement, respectively compared with CTL. ACL and GDH also reported significant 50 and 75% increase, respectively. The absence of exercise-induced correlations among GSIS and anaplerotic/cataplerotic enzymes suggests that exercise may control insulin release by activating other signaling pathways. The observed anaplerotic and cataplerotic enzymes enhancement might be related to beta-cell surviving rather than insulin secretion.

Intraerythrocytic stages of Plasmodium falciparum biosynthesize menaquinone

Herein, we show that intraerythrocytic stages of Plasmodium falciparum have an active pathway for biosynthesis of menaquinone. Kinetic assays confirmed that plasmodial menaquinone acts at least in the electron transport. Similarly to Escherichia coli, we observed increased levels of menaquinone in parasites kept under anaerobic conditions. Additionally, the mycobacterial inhibitor of menaquinone synthesis Ro 48-8071 also suppressed menaquinone biosynthesis and growth of parasites, although off-targets may play a role in this growth-inhibitory effect. Due to its absence in humans, the menaquinone biosynthesis can be considered an important drug target for malaria. (c) 2010 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

Quantitative structure-activity relationships for a series of inhibitors of cruzain from Trypanosoma cruzi: Molecular modeling, CoMFA and CoMSIA studies

Human parasitic diseases are the foremost threat to human health and welfare around the world. Trypanosomiasis is a very serious infectious disease against which the currently available drugs are limited and not effective. Therefore, there is an urgent need for new chemotherapeutic agents. One attractive drug target is the major cysteine protease from Trypanosoma cruzi, cruzain. In the present work, comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) studies were conducted on a series of thiosemicarbazone and semicarbazone derivatives as inhibitors of cruzain. Molecular modeling studies were performed in order to identify the preferred binding mode of the inhibitors into the enzyme active site, and to generate structural alignments for the three-dimensional quantitative structure-activity relationship (3D QSAR) investigations. Statistically significant models were obtained (CoMFA. r(2) = 0.96 and q(2) = 0.78; CoMSIA, r(2) = 0.91 and q(2) = 0.73), indicating their predictive ability for untested compounds. The models were externally validated employing a test set, and the predicted values were in good agreement with the experimental results. The final QSAR models and the information gathered from the 3D CoMFA and CoMSIA contour maps provided important insights into the chemical and structural basis involved in the molecular recognition process of this family of cruzain inhibitors, and should be useful for the design of new structurally related analogs with improved potency. (C) 2009 Elsevier Inc. All rights reserved.

CoMFA and CoMSIA 3D QSAR Models for a Series of Cyclic Imides with Analgesic Activity

Three-dimensional quantitative structure-activity relationships (3D-QSAR) were performed for a series of analgesic cyclic imides using the CoMFA and CoMSIA methods. Significant correlation coefficients ( CoMFA, r(2) = 0.95 and q(2) = 0.72; CoMSIA, r(2) = 0.96 and q(2) = 0.76) were obtained, and the generated models were externally validated using test sets. The final QSAR models as well as the information gathered from 3D contour maps should be useful for the design of novel cyclic imides having improved analgesic activity.

Quantitative structure-activity studies on a series of migrastatin analogs as inhibitors of cancer cell metastasis

Migrastatin, a macrolide natural product, and its structurally related analogs are potent inhibitors of cancer cell metastasis, invasion and migration. In the present work, a specialized fragment-based method was employed to develop QSAR models for a series of migrastatin and isomigrastatin analogs. Significant correlation coefficients were obtained (best model, q(2) = 0.76 and r(2) = 0.91) indicating that the QSAR models possess high internal consistency. The best model was then used to predict the potency of an external test set, and the predicted values were in good agreement with the experimental results (R(2) (pred) = 0.85). The final model and the corresponding contribution maps, combined with molecular modeling studies, provided important insights into the key structural features for the anticancer activity of this family of synthetic compounds based on natural products.

Discovery of New Inhibitors of Schistosoma mansoni PNP by Pharmacophore-Based Virtual Screening

Schistosomiasis is considered the second most important tropical parasitic disease, with severe socioeconomic consequences for millions of people worldwide. Schistosoma monsoni, one of the causative agents of human schistosomiasis, is unable to synthesize purine nucleotides de novo, which makes the enzymes of the purine salvage pathway important targets for antischistosomal drug development. In the present work, we describe the development of a pharmacophore model for ligands of S. mansoni purine nucleoside phosphorylase (SmPNP) as well as a pharmacophore-based virtual screening approach, which resulted in the identification of three thioxothiazolidinones (1-3) with substantial in vitro inhibitory activity against SmPNP. Synthesis, biochemical evaluation, and structure activity relationship investigations led to the successful development of a small set of thioxothiazolidinone derivatives harboring a novel chemical scaffold as new competitive inhibitors of SmPNP at the low-micromolar range. Seven compounds were identified with IC(50) values below 100 mu M. The most potent inhibitors 7, 10, and 17 with 1050 of 2, 18, and 38 mu M, respectively, could represent new potential lead compounds for further development of the therapy of schistosomiasis.

Novel ruthenium complexes as potential drugs for Chagas`s disease: enzyme inhibition and in vitro/in vivo trypanocidal activity

Background and purpose: The discovery of the pharmacological functions of nitric oxide has led to the development of NO donor compounds as therapeutic agents. A new generation of ruthenium NO donors, cis-[Ru(NO)(bpy)(2)L]X(n) , has been developed, and our aim was to show that these complexes are able to lyse Trypanosoma cruzi in vitro and in vivo. Experimental approach: NO donors were incubated with T. cruzi and their anti-T. cruzi activities evaluated as the percentage of lysed parasites compared to the negative control. In vivo, trypanocidal activity was evaluated by observing the levels of parasitaemia, survival rate and elimination of amastigotes in mouse myocardial tissue. The inhibition of GAPDH was monitored by the biochemical reduction of NAD+ to NADH. Key results: The NO donors cis-[Ru(NO)(bpy)(2)L]X(n) presented inhibitory effects on T. cruzi GAPDH (IC(50) ranging from 89 to 153 mu M). The crystal structure of the enzyme shows that the inhibitory mechanism is compatible with S-nitrosylation of the active cysteine (cys166) site. Compounds cis-[Ru(NO)(bpy)(2)imN](PF(6))(3) and cis-[Ru(NO)(bpy)(2)SO(3)]PF(6), at a dose of 385 nmol center dot kg-1, yielded survival rates of 80 and 60%, respectively, in infected mice, and eradicated any amastigotes from their myocardial tissue. Conclusions and implications: The ruthenium compounds exhibited potent in vitro and in vivo trypanocidal activities at doses up to 1000-fold lower than the clinical dose for benznidazole. Furthermore, one mechanism of action of these compounds is via the S-nitrosylation of Cys166 of T. cruzi GAPDH. Thus, these compounds show huge potential as candidates for the development of new drugs for the treatment of Chagas`s disease. This article is commented on by Machado et al., pp. 258-259 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2010.00662.x and to view a related paper in this issue by Guedes et al. visit http://dx.doi.org/10.1111/j.1476-5381.2010.00576.x.

Structural basis for selective inhibition of purine nucleoside phosphorylase from Schistosoma mansoni: Kinetic and structural studies

Selectivity plays a crucial role in the design of enzyme inhibitors as novel antiparasitic agents, particularly in cases where the target enzyme is also present in the human host. Purine nucleoside phosphorylase from Schistosoma mansoni (SmPNP) is an attractive target for the discovery of potential antischistosomal agents. In the present work, kinetic studies were carried out in order to determine the inhibitory potency, mode of action and enzyme selectivity of a series of inhibitors of SmPNP. In addition, crystallographic studies provided important structural insights for rational inhibitor design, revealing consistent structural differences in the binding mode of the inhibitors in the active sites of the SmPNP and human PNP (HsPNP) structures. The molecular information gathered in this work should be useful for future medicinal chemistry efforts in the design of new inhibitors of SmPNP having increased affinity and selectivity. (C) 2010 Elsevier Ltd. All rights reserved.