Subtranscriptome analysis of phospholipases D in Loxosceles venom glands: Confirmation of predominance, intra-species diversity, and description of novel isoforms.

Spiders of Loxosceles genus, or Brown spiders produce a potent venom with minimal volume and protein content. Among its toxins, phospholipases D (PLDs) are notable for causing primary local and systemic manifestations observed following envenomation. They degrade cellular phospholipids, mainly sphingomyelin and lysophosphatidylcholine. We present a robust and detailed analysis of PLD transcripts from venom glands of three major clinically relevant South American species-L. intermedia, L. laeta, and L. gaucho-using next-generation sequencing. Results confirmed that PLDs are the most highly expressed toxins, accounting for 65.4 % of expression in L. intermedia, 71.8 % in L. gaucho, and 50.4 % in L. laeta. These findings further support the idea that these enzymes form a protein family both within and across species. Eighteen contigs for PLDs were found for L. gaucho, 24 for L. intermedia, and 21 for L. laeta. A detailed analysis revealed that, although all contigs display conserved amino acid residues directly involved in catalysis, magnesium coordination, and substrate affinity, they also possess distinct primary sequences with important substitutions. Such data reinforces the hypothesis that these toxins may act synergistically. Furthermore, new PLD sequences were identified within the contigs. For L. intermedia, 14 potential new isoforms were identified; 16 for L gaucho; and 16 novel sequences for L. laeta. This indicates that there is still a wealth of undisclosed information about these toxins. These data will help identify structural and functional differences among these proteins, support future functional studies, and to the comprehensive understanding of the mechanism of action of PLDs.

Agalsidase alfa long-term effect on left ventricular hypertrophy in Fabry disease.

INTRODUCTION: Fabry disease (FD) is an X-linked lysosomal storage disorder affecting glycosphingolipid metabolism. Most FD patients have cardiac involvement, mainly manifested as left ventricular hypertrophy (LVH), leading to early death due to complications (arrhythmias, valvular disease, vascular involvement). Early initiation of enzyme replacement therapy (ERT) before fibrosis development has been associated with better cardiac outcomes in terms of left ventricular mass index (LVMI) and functional parameters. METHODS: A retrospective observational study was conducted in patients with FD treated with agalsidase alfa for at least 2 years. The primary objectives were: [a] to assess the annual rate of change in LVMI; [b] to define the overall incidence of stability, regression or progression of LVMI. RESULTS: Forty-nine patients were included in the final analysis, with a median follow-up of 7 years. The overall change in LVMI was 0.38 g/m2.73/year, without significant influence of baseline LVH, gender, age at ERT initiation, LV ejection fraction, body mass index, renal disease, and classical cardiovascular risk factors. Long-term ERT with agalsidase alfa was associated with stabilization of LVMI in 98% of patients with FD and was independent of the same covariables. CONCLUSION: Our results are in line with previous literature of comparable FD populations and probably represent the first study of its kind in Argentina. We here highlight the importance of cardiac morphometric stability as a positive outcome of ERT.

Introducción: La enfermedad de Fabry (EF) es una enfermedad de almacenamiento lisosomal ligada al cromosoma X que afecta el metabolismo de glicoesfingolípidos. La mayoría de pacientes EF tienen afectación cardíaca, manifestada principalmente como hipertrofia ventricular izquierda (HVI), que conduce a muerte prematura secundaria a complicaciones (arritmias, valvulopatías, afectación vascular). El tratamiento de reemplazo enzimático (TRE) precoz, iniciado antes del desarrollo de la fibrosis, se relaciona con mejores resultados cardíacos en términos del índice de masa ventricular izquierda (IMVI) y parámetros funcionales. Métodos: Se realizó un estudio retrospectivo observacional en que se incluyeron pacientes con EF tratados con agalsidasa alfa por al menos 2 años. Los objetivos primarios fueron: [a] evaluar el cambio anual del IMVI; [b] definir la incidencia global de estabilidad, regresión o progresión del IMVI. Resultados: Se incluyeron 49 pacientes, con seguimiento (mediana) de 7 años. El cambio global en el IMVI fue 0.38 g/m2.73/año, sin influencia significativa de HVI basal, sexo, edad de inicio de TRE, fracción de eyección del VI, índice de masa corporal, insuficiencia renal y factores de riesgo cardiovascular clásicos. La TRE a largo plazo con agalsidasa alfa se relacionó con la estabilización del IMVI en el 98% de los pacientes con EF, independientemente de las mismas covariables. Conclusión: Nuestros resultados están en línea con la bibliografía previa de poblaciones comparables y, probablemente, representan el primer estudio de este tipo en Argentina. Se destaca la importancia de la estabilidad morfométrica cardíaca como resultado positivo de la TRE.

Identification of the NA+/K+-ATPase α-Isoforms in Six Species of Poison Dart Frogs and their Sensitivity to Cardiotonic Steroids.

Cardiotonic steroids (CTS) are a group of compounds known to be toxic due to their ability to inhibit the Na+/K+-ATPase (NKA), which is essential to maintain the balance of ions in animal cells. An evolutionary strategy of molecular adaptation to avoid self-intoxication acquired by CTS defended organisms and their predators is the structural modification of their NKA where specific amino acid substitutions confer resistant phenotypes. Several lineages of poison dart frogs (Dendrobatidae) are well known to sequester a wide variety of lipophilic alkaloids from their arthropod diet, however there is no evidence of CTS-sequestration or dietary exposure. Interestingly this study identified the presence of α-NKA isoforms (α1 and α2) with amino acid substitutions indicative of CTS-resistant phenotypes in skeletal muscle transcriptomes obtained from six species of dendrobatids: Phyllobates aurotaenia, Oophaga anchicayensis, Epipedobates boulengeri, Andinobates bombetes, Andinobates minutus, and Leucostethus brachistriatus, collected in the Valle del Cauca (Colombia). P. aurotaenia, A. minutus, and E. boulengeri presented two variants for α1-NKA, with one of them having these substitutions. In contrast, O. anchicayensis and A. bombetes have only one α1-NKA isoform with an amino acid sequence indicative of CTS susceptibility and an α2-NKA with one substitution that could confer a reduced affinity for CTS. The α1 and α2 isoforms of L. brachistriatus do not contain substitutions imparting CTS resistance. Our findings indicate that poison dart frogs express α-NKA isoforms with different affinities for CTS and the pattern of this expression might be influenced by factors related to evolutionary, physiological, ecological, and geographical burdens.

Virulence and Reproductive Fitness of Meloidogyne paranaensis Field Populations in Coffee Genotypes.

The aim of this study was to characterize Meloidogyne paranaensis populations collected from infested coffee crops. Methodologies used to characterize the 11 studied populations from municipalities in Paraná and Minas Gerais States involved the morphological analysis of perineal patterns, biochemical analysis by isozyme electrophoresis, sequencing of internal transcribes spacer 1 (ITS-1) and D2/D3 ribosomal DNA (rDNA) regions, reproductive fitness, and virulence characterization in coffee genotypes. Morphological evaluations showed the existence of variation between populations, although the majority of them showed typical perineal patterns. The biochemical identification was based on α-esterase isozyme analyses and resulted in the appearance of three distinct profiles: P1 (typical), P2 (atypical), and a nondescribed profile, P2b. BLAST of the ITS-1 and D2/D3 rDNA regions indicated homology (>95%) with other sequences deposited in GenBank. For reproductive fitness and virulence characterization, 13 coffee genotypes (5 Coffea arabica and 8 C. canephora) were inoculated with 11 M. paranaensis populations. Variation in the reproductive fitness of populations was observed for cultivar Mundo Novo, a genotype without resistance genes, and variation in the virulence of populations was observed in genotypes carrying resistance genes. Three populations exhibited virulence combined with high reproductive fitness, while one showed virulence with low reproductive fitness. Some hosts were resistant to 11 populations, while one of the hosts was resistant to only one population, indicating the presence of different resistance genes. Nevertheless, no relationship was observed between the origin of population and their variations in perineal patterns, esterase profiles, phylogeny, or reproductive fitness in coffee genotypes, or between the different characterizations, although differences were observed within each characteristic.

Niveles séricos de la isoenzima creatina quinasa-MB y lactato deshidrogenasa como indicadores de daño miocárdico en perros con enfermedad valvular degenerativa / Serum levels of the isoenzyme creatine kinase-MB and lactate dehydrogenase as indicators of myocardial damage in dogs with degenerative valve disease

RESUMEN Se analizaron los niveles séricos de creatina quinasa-MB (CK-MB) y lactato deshidrogenasa (LDH) en 10 perros diagnosticados con enfermedad valvular degenerativa y en seis perros clinicamente sanos, con el objetivo de evaluar si sus niveles séricos indican daño miocárdico. Las muestras de suero se analizaron mediante el método UV. Se utilizó la prueba de diferenciación de medias para determinar diferencias entre medias, y la prueba de correlación de Pearson para determinar si existe correlación entre los niveles séricos de ambas enzimas. Los valores de CK-MB y de LDH fueron significativamente diferentes entre los dos grupos de pacientes. Los niveles de CK-MB y LDH tuvieron correlación positiva, pero no significativa.

ABSTRACT Serum levels of creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH) were analyzed in 10 dogs diagnosed with degenerative valvular disease and in six clinically healthy dogs with the objective of evaluating whether their serum levels indicate myocardial damage. Serum samples were analyzed by UV method. The mean differentiation test was used to determine differences between means and the Pearson correlation test was performed to determine if there was a correlation between the serum levels of both enzymes. The CK-MB and LDH values were significantly different between the two groups of patients. The levels of CK-MB and LDH had a positive but not significant correlation.

Two alpha isopropylmalate synthase isozymes with similar kinetic properties are extant in the yeast Lachancea kluyveri.

The first committed step in the leucine biosynthetic pathway is catalyzed by α-isopropylmalate synthase (α-IPMS, EC 2.3.3.13), which in the Saccaromycotina subphylum of Ascomycete yeasts is frequently encoded by duplicated genes. Following a gene duplication event, the two copies may be preserved presumably because the encoded proteins diverge in either functional properties and/or cellular localization. The genome of the petite-negative budding yeast Lachancea kluyveri includes two SAKL0E10472 (LkLEU4) and SAKL0F05170 g (LkLEU4BIS) paralogous genes, which are homologous to other yeast α-IPMS sequences. Here, we investigate whether these paralogous genes encode functional α-IPMS isozymes and whether their functions have diverged. Molecular phylogeny suggested that the LkLeu4 isozyme is located in the mitochondria and LkLeu4BIS in the cytosol. Comparison of growth rates, leucine intracellular pools and mRNA levels, indicate that the LkLeu4 isozyme is the predominant α-IPMS enzyme during growth on glucose as carbon source. Determination of the kinetic parameters indicates that the isozymes have similar affinities for the substrates and for the feedback inhibitor leucine. Thus, the diversification of the physiological roles of the genes LkLEU4 and LkLEU4BIS involves preferential transcription of the LkLEU4 gene during growth on glucose and different subcellular localization, although ligand interactions have not diverged.

Effect of Fe3+ on Na,K-ATPase: Unexpected activation of ATP hydrolysis.

Iron is a key element in cell function; however, its excess in iron overload conditions can be harmful through the generation of reactive oxygen species (ROS) and cell oxidative stress. Activity of Na,K-ATPase has been shown to be implicated in cellular iron uptake and iron modulates the Na,K-ATPase function from different tissues. In this study, we determined the effect of iron overload on Na,K-ATPase activity and established the role that isoforms and conformational states of this enzyme has on this effect. Total blood and membrane preparations from erythrocytes (ghost cells), as well as pig kidney and rat brain cortex, and enterocytes cells (Caco-2) were used. In E1-related subconformations, an enzyme activation effect by iron was observed, and in the E2-related subconformations enzyme inhibition was observed. The enzyme's kinetic parameters were significantly changed only in the Na+ curve in ghost cells. In contrast to Na,K-ATPase α2 and α3 isoforms, activation was not observed for the α1 isoform. In Caco-2 cells, which only contain Na,K-ATPase α1 isoform, the FeCl3 increased the intracellular storage of iron, catalase activity, the production of H2O2 and the expression levels of the α1 isoform. In contrast, iron did not affect lipid peroxidation, GSH content, superoxide dismutase and Na,K-ATPase activities. These results suggest that iron itself modulates Na,K-ATPase and that one or more E1-related subconformations seems to be determinant for the sensitivity of iron modulation through a mechanism in which the involvement of the Na, K-ATPase α3 isoform needs to be further investigated.

Effect of B-NIPOx in Experimental Trypanosoma cruzi Infection in Mice.

Chagas disease is caused by Trypanosoma cruzi and represents a major public health problem, which is endemic in Latin America and emerging in the rest of the world. The two drugs that are currently available for its treatment, Benznidazole and Nifurtimox, are partially effective in the chronic phase of the disease. In this study, we designed and synthesized the benzyl ester of N-isopropyl oxamic acid (B-NIPOx), which is a non-polar molecule that crosses cell membranes. B-NIPOx is cleaved inside the parasite by carboxylesterases, releasing benzyl alcohol (a molecule with antimicrobial activity), and NIPOx, which is an inhibitor of α-hydroxy acid dehydrogenase isozyme II (HADH-II), a key enzyme in T. cruzi metabolism. We evaluated B-NIPOx cytotoxicity, its toxicity in mice, and its inhibitory activity on purified HADH-II and on T. cruzi homogenates. We then evaluated the trypanocidal activity of B-NIPOx in vitro and in vivo and its effect in the intestine of T. cruzi-infected mice. We found that B-NIPOx had higher trypanocidal activity on epimastigotes and trypomastigotes than Benznidazole and Nifurtimox, that it was more effective to reduce blood parasitemia and amastigote nests in infected mice, and that, in contrast to the reference drugs, it prevented the development of Chagasic enteropathy.

The Role of Celecoxib as a Potential Inhibitor in the Treatment of Inflammatory Diseases - A Review.

This article aims at reviewing celecoxib as a potential inhibitor in the treatment of inflammatory diseases. The enzyme cyclooxygenase (COX) predominantly has two isoforms called cyclooxygenase 1 (COX-1) and cyclooxygenase 2 (COX-2). The former plays a constitutive role related to homeostatic effects in renal and platelets, while the latter is mainly responsible for the induction of inflammatory effects. Since COX-2 plays an important role in the pathogenesis of inflammatory diseases, it has been signaled as a target for the planning of anti-inflammatory intermediates. Many inhibitors developed and planned for COX-2 inhibition have presented side effects to humans, mainly in the gastrointestinal and/or cardiovascular tract. Therefore, it is necessary to design new potential COX-2 inhibitors, which are relatively safe and have no side effects. In this sense, celecoxib is the only potent, selective COX-2 inhibitor that is still commercially available (within the "coxib" family). Thus, celecoxib became a commercial prototype inhibitor for the development of anti-inflammatory agents for the COX-2 enzyme. This review provides inhibition highlights that should provide a structural basis for the design of promising new non-steroidal anti-inflammatory drugs (NSAIDs), which act as COX-2 inhibitors with lesser side effects on the human body.

Accelerated ageing test and enzymatic expression in the evaluation of kale seed quality / Teste de envelhecimento acelerado e expressão enzimática na avaliação da qualidade de sementes de couve

The demand for high-quality vegetable seeds and the production of vigorous seedlings has increased in recent years, as these characteristics are determining factors of production success. Vegetables are growing in national importance, and kale stands out as an important source of income for small farmers. The objective of this study was to adapt the traditional accelerated aging test methodology with a saturated NaCl solution of kale seeds and evaluate the enzymatic activity after the vigour test. Six batches of kale seeds were used, and the moisture content, weight of one thousand seeds, first germination count, germination, germination speed index, emergence, initial stand, and emergence velocity index were determined. In the accelerated ageing test, the seeds were submitted to the traditional accelerated ageing test method and the accelerated ageing test method with a saturated NaCl solution for ageing periods of 0, 24, 48, 72, and 96 hours. Electrophoretic analysis of superoxide dismutase (SOD), catalase (CAT), and alcohol dehydrogenase (ADH) isoenzymes was also performed. The accelerated ageing test, using the traditional method for 72 hours at 41 °C, is adequate for evaluating the physiological potential of kale seeds. The isoenzymatic analyses of SOD, CAT, and ADH demonstrate that the biochemical markers are efficient at differentiating kale seeds after accelerated ageing.(AU)

A demanda por sementes de hortaliças com alta qualidade e a obtenção de mudas vigorosas tem aumentado nos últimos anos, pois estas características constituem fatores determinantes do êxito da produção. As hortaliças têm uma importância nacional crescente, dentre elas se destaca a couve, por ser importante fonte de renda para pequenos agricultores. Objetivou-se adequar a metodologia do teste de envelhecimento acelerado tradicional e com solução saturada de NaCl em sementes de couve e avaliar a atividade enzimática após o teste de vigor. Foram utilizados seis lotes de sementes de couve e determinouse o grau de umidade, peso de mil sementes, primeira contagem de germinação, germinação, índice de velocidade de germinação, emergência, estande inicial e índice de velocidade de emergência. No teste de envelhecimento acelerado, as sementes foram submetidas ao método tradicional e com solução saturada de NaCl, por períodos de 0; 24; 48; 72 e 96 horas. Realizou-se também a análise eletroforética das isoenzimas superóxido dismutase (SOD), catalase (CAT) e álcool desidrogenase (ADH). O teste de envelhecimento acelerado, utilizando o método tradicional na combinação de 72 horas a 41 °C é adequado para avaliar o potencial fisiológico de sementes de couve. As análises isoenzimáticas superóxido dismutase (SOD), catalase (CAT) e álcool desidrogenase (ADH) demonstram que os marcadores bioquímicos são eficientes e promissores na diferenciação de sementes de couve após o envelhecimento acelerado.(AU)

Mitochondrial Dysfunction in Cardiorenal Syndrome 3: Renocardiac Effect of Vitamin C.

Cardiorenal syndrome (CRS) is a pathological link between the kidneys and heart, in which an insult in a kidney or heart leads the other organ to incur damage. CRS is classified into five subtypes, and type 3 (CRS3) is characterized by acute kidney injury as a precursor to subsequent cardiovascular changes. Mitochondrial dysfunction and oxidative and nitrosative stress have been reported in the pathophysiology of CRS3. It is known that vitamin C, an antioxidant, has proven protective capacity for cardiac, renal, and vascular endothelial tissues. Therefore, the present study aimed to assess whether vitamin C provides protection to heart and the kidneys in an in vivo CRS3 model. The unilateral renal ischemia and reperfusion (IR) protocol was performed for 60 min in the left kidney of adult mice, with and without vitamin C treatment, immediately after IR or 15 days after IR. Kidneys and hearts were subsequently collected, and the following analyses were conducted: renal morphometric evaluation, serum urea and creatinine levels, high-resolution respirometry, amperometry technique for NO measurement, gene expression of mitochondrial dynamic markers, and NOS. The analyses showed that the left kidney weight was reduced, urea and creatinine levels were increased, mitochondrial oxygen consumption was reduced, NO levels were elevated, and Mfn2 expression was reduced after 15 days of IR compared to the sham group. Oxygen consumption and NO levels in the heart were also reduced. The treatment with vitamin C preserved the left kidney weight, restored renal function, reduced NO levels, decreased iNOS expression, elevated constitutive NOS isoforms, and improved oxygen consumption. In the heart, oxygen consumption and NO levels were improved after vitamin C treatment, whereas the three NOS isoforms were overexpressed. These data indicate that vitamin C provides protection to the kidneys and some beneficial effects to the heart after IR, indicating it may be a preventive approach against cardiorenal insults.

Population structure and phylogenetic relationships in Brassica rapa L. subspecies by using isozyme markers / Estrutura populacional e relações filogenéticas em subespécies de Brassica rapa L. utilizando marcadores isoenzimáticos

The present study aimed to assess population structure and phylogenetic relationships of nine subspecies of Brassica rapa L. represented with thirty-five accessions cover a wide range of species distribution area using isozyme analysis in order to select more diverse accessions as supplementary resources that can be utilized for improvement of B. napus. Enzyme analysis resulted in detecting 14 putative polymorphic loci with 27 alleles. Mean allele frequency 0.04 (rare alleles) was observed in Cat4A and Cat4B in sub species Oleifera accession CR 2204/79 and in subspecies trilocularis accessions CR 2215/88 and CR 2244/88. The highest genetic diversity measures were observed in subspecies dichotoma, accession CR 1585/96 (the highest average of observed (H0) and expected heterozygosity (He), and number of alleles per locus (Ae)). These observations make this accession valuable genetic resource to be included in breeding programs for the improvement of oilseed B. napus. The average fixation index (F) is significantly higher than zero for the analysis accessions indicating a significant deficiency of heteozygosity. The divergence among subspecies indicated very great genetic differentiation (FST = 0.8972) which means that about 90% of genetic diversity is distributed among subspecies, while 10% of the diversity is distributed within subspecies. This coincides with low value of gene flow (Nm = 0.0287). B. rapa ssp. oleifera (turnip rape) and B. rapa ssp. trilocularis (sarson) were grouped under one cluster which coincides with the morphological classification.(AU)

O presente estudo teve como objetivo avaliar a estrutura populacional e as relações filogenéticas de nove subespécies de Brassica rapa L. representadas com 35 acessos, cobrindo uma ampla gama de áreas de distribuição de espécies usando análise isoenzimática, a fim de selecionar acessos mais diversos como recursos suplementares que podem ser utilizados para melhoria de B. napus. A análise enzimática resultou na detecção de 14 loci polimórficos putativos com 27 alelos. A frequência média de 0,04 alelo (alelos raros) foi observada em Cat4A e Cat4B, nas subespécies Oleifera CR 2204/79 e nas subespécies trilocularis CR 2215/88 e CR 2244/88. As maiores medidas de diversidade genética foram observadas na subespécie dicotômica CR 1585/96 (a média mais alta observada (H0) e heterozigosidade esperada (He) e número de alelos por locus (Ae). Essas observações tornam esse acesso um valioso recurso genético a ser incluído em programas de melhoramento de oleaginosas B. napus. O índice médio de fixação (F) é significativamente maior que 0 para os acessos à análise, indicando uma deficiência significativa de heterozigose. A divergência entre as subespécies indicou uma grande diferenciação genética (FST = 0,8972), o que significa que cerca de 90% da diversidade genética é distribuída entre as subespécies, enquanto 10% da diversidade é distribuída nas subespécies. Isso coincide com o baixo valor do fluxo gênico (Nm = 0,0287). B. rapa ssp. oleifera (nabo) e B. rapa ssp. trilocularis (sarson) foram agrupados conforme a classificação morfológica.(AU)

Population structure and phylogenetic relationships in Brassica rapa L. subspecies by using isozyme markers / Estrutura populacional e relações filogenéticas em subespécies de Brassica rapa L. utilizando marcadores isoenzimáticos

Abstract The present study aimed to assess population structure and phylogenetic relationships of nine subspecies of Brassica rapa L. represented with thirty-five accessions cover a wide range of species distribution area using isozyme analysis in order to select more diverse accessions as supplementary resources that can be utilized for improvement of B. napus. Enzyme analysis resulted in detecting 14 putative polymorphic loci with 27 alleles. Mean allele frequency 0.04 (rare alleles) was observed in Cat4A and Cat4B in sub species Oleifera accession CR 2204/79 and in subspecies trilocularis accessions CR 2215/88 and CR 2244/88. The highest genetic diversity measures were observed in subspecies dichotoma, accession CR 1585/96 (the highest average of observed (H0) and expected heterozygosity (He), and number of alleles per locus (Ae)). These observations make this accession valuable genetic resource to be included in breeding programs for the improvement of oilseed B. napus. The average fixation index (F) is significantly higher than zero for the analysis accessions indicating a significant deficiency of heteozygosity. The divergence among subspecies indicated very great genetic differentiation (FST = 0.8972) which means that about 90% of genetic diversity is distributed among subspecies, while 10% of the diversity is distributed within subspecies. This coincides with low value of gene flow (Nm = 0.0287). B. rapa ssp. oleifera (turnip rape) and B. rapa ssp. trilocularis (sarson) were grouped under one cluster which coincides with the morphological classification.

Resumo O presente estudo teve como objetivo avaliar a estrutura populacional e as relações filogenéticas de nove subespécies de Brassica rapa L. representadas com 35 acessos, cobrindo uma ampla gama de áreas de distribuição de espécies usando análise isoenzimática, a fim de selecionar acessos mais diversos como recursos suplementares que podem ser utilizados para melhoria de B. napus. A análise enzimática resultou na detecção de 14 loci polimórficos putativos com 27 alelos. A frequência média de 0,04 alelo (alelos raros) foi observada em Cat4A e Cat4B, nas subespécies Oleifera CR 2204/79 e nas subespécies trilocularis CR 2215/88 e CR 2244/88. As maiores medidas de diversidade genética foram observadas na subespécie dicotômica CR 1585/96 (a média mais alta observada (H0) e heterozigosidade esperada (He) e número de alelos por locus (Ae). Essas observações tornam esse acesso um valioso recurso genético a ser incluído em programas de melhoramento de oleaginosas B. napus. O índice médio de fixação (F) é significativamente maior que 0 para os acessos à análise, indicando uma deficiência significativa de heterozigose. A divergência entre as subespécies indicou uma grande diferenciação genética (FST = 0,8972), o que significa que cerca de 90% da diversidade genética é distribuída entre as subespécies, enquanto 10% da diversidade é distribuída nas subespécies. Isso coincide com o baixo valor do fluxo gênico (Nm = 0,0287). B. rapa ssp. oleifera (nabo) e B. rapa ssp. trilocularis (sarson) foram agrupados conforme a classificação morfológica.

Design, Synthesis, and Characterization of Novel sn-1 Heterocyclic DAG-Lactones as PKC Activators.

DAG-lactones represent useful templates for the design of potent and selective C1 domain ligands for PKC isozymes. The ester moiety at the sn-1 position, a common feature in this template, is relevant for C1 domain interactions, but it represents a labile group susceptible to endogenous esterases. An interesting challenge involves replacing the ester group of these ligands while still maintaining biological activity. Here, we present the synthesis and functional characterization of novel diacylglycerol-lactones containing heterocyclic ring substituents at the sn-1 position. Our results showed that the new compound 10B12, a DAG-lactone with an isoxazole ring, binds PKCα and PKCε with nanomolar affinity. Remarkably, 10B12 displays preferential selectivity for PKCε translocation in cells and induces a PKCε-dependent reorganization of the actin cytoskeleton into peripheral ruffles in lung cancer cells. We conclude that introducing a stable isoxazole ring as an ester surrogate in DAG-lactones emerges as a novel structural approach to achieve PKC isozyme selectivity.

Major cardiovascular adverse events in Fabry disease patients receiving agalsidase alfa / Eventos cardiovasculares mayores en pacientes con enfermedad de Fabry tratados con agalsidasa alfa

Abstract Cardiovascular mortality (CVM) has become the major contributor to overall Fabry disease (FD) mortality in the enzyme replacement therapy (ERT) era. Our objectives were to describe causes and potential predictors of mortality in FD adult patients in Argentina, and to assess risk of major adverse cardio vascular events (MACE) in the ERT era. We retrospectively studied 93 consecutive patients treated with alpha-galactosidase A (median follow up: 9.5 years from start of ERT). Mean age at ERT starting was 35±16.3 years. Prevalence of cardiomyopathy and renal disease reached 47% and 41%, respectively. Eleven subjects (11.8%, 95%CI: 5-18%) died during follow up (1.24/100 patient-years). Mean overall survival was 71 years (95%CI: 66-75 years). Seven cases were considered as CVM; main causes were sudden death and stroke. Risk of MACE was 14% (95%CI: 6.9-21.1%; 1.47 events/100 patient-years from start of ERT). All but 2 subjects had at least one comorbid cardiovascular risk factor; however, 86% of patients remained free of MACE during follow-up. CVM remained low and our study was underpowered for detection of predictors of mortality, but it is worth noting that age at diagnosis and ERT starting, left ventricular mass index and renal disease trended to correlate with CVM. Prevalence of hypertension, diabetes and dyslipidemia were lower in FD patients when compared to population level data. As in the Argentinean general population, CVM was the leading cause of mortality among this cohort of consecutive FD patients treated with agalsidase alfa.

Resumen La mortalidad cardiovascular (MCV) se ha convertido en el principal contribuyente a la mortalidad general por enfermedad de Fabry (EF) en la era de la terapia de reemplazo enzimático (TRE). Nuestros objetivos fueron describir las causas y posibles predictores de mortalidad en pacientes adultos con EF en la Argentina, y evaluar el riesgo de eventos cardiovasculares mayores (MACE) en la actual era de TRE. Se estudiaron 93 pacientes consecutivos tratados con agalsidasa-alfa por una mediana de 9.5 años tras iniciar TRE. La edad al inicio de TRE fue 35 ± 16.3 años. La prevalencia de cardiomiopatía y enfermedad renal alcanzó 47% y 41%, respectivamente. Once sujetos (11.8%; IC95%: 5-18%) murieron durante el seguimiento (1.24/100 pacientes/año). La supervivencia global fue 71 años (IC95%: 66-75 años). Siete casos fueron considerados como MCV; las principales causas fueron muerte súbita e ictus. El riesgo de MACE fue 14% (IC95%: 6.9-21.1%; 1.47 eventos/100 pacientes/año desde la ERT). Todos menos 2 sujetos tenían al menos un factor de riesgo cardiovascular, pero el 86% permaneció libre de MACE. Los eventos de MCV fueron escasos. El estudio tuvo reducido poder estadístico para detectar predictores de mortalidad, pero la edad al diagnóstico y al iniciar la TRE, índice de masa ventricular izquierda y enfermedad renal tendieron a correlacionarse con MCV. La prevalencia de hipertensión, diabetes y dislipidemia fue menor en comparación con la población general. Como ocurre con la población general en Argentina, los eventos cardiovasculares fueron la principal causa de muerte en esta cohorte de pacientes consecutivos con EF tratados con agalsidasa-alfa.

IMPDH forms the cytoophidium in zebrafish.

Inosine monophosphate dehydrogenase (IMPDH) catalyzes the rate-limiting step in de novo guanine nucleotide biosynthesis. Its activity is negatively regulated by the binding of GTP. IMPDH can form a membraneless subcellular structure termed the cytoophidium in response to certain changes in the metabolic status of the cell. The polymeric form of IMPDH, which is the subunit of the cytoophidium, has been shown to be more resistant to the inhibition by GTP at physiological concentrations, implying a functional correlation between cytoophidium formation and the upregulation of GTP biosynthesis. Herein we demonstrate that zebrafish IMPDH1b and IMPDH2 isoforms can assemble abundant cytoophidium in most of cultured cells under stimuli, while zebrafish IMPDH1a shows distinctive properties of forming the cytoophidium in different cell types. Point mutations that disrupt cytoophidium structure in mammalian models also prevent the aggregation of zebrafish IMPDHs. In addition, we discover the presence of the IMPDH cytoophidium in various tissues of larval and adult fish under normal growth conditions. Our results reveal that polymerization and cytoophidium assembly of IMPDH can be a regulatory machinery conserved among vertebrates, and with specific physiological purposes.

Skeletal muscle heme oxygenase-1 activity regulates aerobic capacity.

Physical exercise has profound effects on quality of life and susceptibility to chronic disease; however, the regulation of skeletal muscle function at the molecular level after exercise remains unclear. We tested the hypothesis that the benefits of exercise on muscle function are linked partly to microtraumatic events that result in accumulation of circulating heme. Effective metabolism of heme is controlled by Heme Oxygenase-1 (HO-1, Hmox1), and we find that mouse skeletal muscle-specific HO-1 deletion (Tam-Cre-HSA-Hmox1fl/fl) shifts the proportion of muscle fibers from type IIA to type IIB concomitant with a disruption in mitochondrial content and function. In addition to a significant impairment in running performance and response to exercise training, Tam-Cre-HSA-Hmox1fl/fl mice show remarkable muscle atrophy compared to Hmox1fl/fl controls. Collectively, these data define a role for heme and HO-1 as central regulators in the physiologic response of skeletal muscle to exercise.

Major cardiovascular adverse events in Fabry disease patients receiving agalsidase alfa.

Cardiovascular mortality (CVM) has become the major contributor to overall Fabry disease (FD) mortality in the enzyme replacement therapy (ERT) era. Our objectives were to describe causes and potential predictors of mortality in FD adult patients in Argentina, and to assess risk of major adverse cardiovascular events (MACE) in the ERT era. We retrospectively studied 93 consecutive patients treated with alphagalactosidase A (median follow up: 9.5 years from start of ERT). Mean age at ERT starting was 35 ± 16.3 years. Prevalence of cardiomyopathy and renal disease reached 47% and 41%, respectively. Eleven subjects (11.8%, 95% CI: 5-18%) died during follow up (1.24/100 patient-years). Mean overall survival was 71 years (95% CI: 66-75 years). Seven cases were considered as CVM; main causes were sudden death and stroke. Risk of MACE was 14% (95% CI: 6.9-21.1%; 1.47 events/100 patient-years from start of ERT). All but 2 subjects had at least one comorbid cardiovascular risk factor; however, 86% of patients remained free of MACE during follow-up. CVM remained low and our study was underpowered for detection of predictors of mortality, but it is worth noting that age at diagnosis and ERT starting, left ventricular mass index and renal disease trended to correlate with CVM. Prevalence of hypertension, diabetes and dyslipidemia were lower in FD patients when compared to population level data. As in the Argentinean general population, CVM was the leading cause of mortality among this cohort of consecutive FD patients treated with agalsidase alfa.

La mortalidad cardiovascular (MCV) se ha convertido en el principal contribuyente a la mortalidad general por enfermedad de Fabry (EF) en la era de la terapia de reemplazo enzimático (TRE). Nuestros objetivos fueron describir las causas y posibles predictores de mortalidad en pacientes adultos con EF en la Argentina, y evaluar el riesgo de eventos cardiovasculares mayores (MACE) en la actual era de TRE. Se estudiaron 93 pacientes consecutivos tratados con agalsidasa-alfa por una mediana de 9.5 años tras iniciar TRE. La edad al inicio de TRE fue 35 ± 16.3 años. La prevalencia de cardiomiopatía y enfermedad renal alcanzó 47% y 41%, respectivamente. Once sujetos (11.8%; IC95%: 5-18%) murieron durante el seguimiento (1.24/100 pacientes/año). La supervivencia global fue 71 años (IC95%: 66-75 años). Siete casos fueron considerados como MCV; las principales causas fueron muerte súbita e ictus. El riesgo de MACE fue 14% (IC95%: 6.9-21.1%; 1.47 eventos/100 pacientes/año desde la ERT). Todos menos 2 sujetos tenían al menos un factor de riesgo cardiovascular, pero el 86% permaneció libre de MACE. Los eventos de MCV fueron escasos. El estudio tuvo reducido poder estadístico para detectar predictores de mortalidad, pero la edad al diagnóstico y al iniciar la TRE, índice de masa ventricular izquierda y enfermedad renal tendieron a correlacionarse con MCV. La prevalencia de hipertensión, diabetes y dislipidemia fue menor en comparación con la población general. Como ocurre con la población general en Argentina, los eventos cardiovasculares fueron la principal causa de muerte en esta cohorte de pacientes consecutivos con EF tratados con agalsidasa-alfa.

Evaluation of biological activities, structural and conformational properties of bovine beta- and alpha-trypsin isoforms in aqueous-organic media.

The study of the biological activity of trypsin isoforms in aqueous-organic media is of great interest to various fields of knowledge and biochemistry applications. Thus enzymatic, structural, and energetic properties of bovine ß- and α-trypsin isoforms were compared in aqueous-organic media using 30 mg of each isoform. The results showed that the changes induced on the structure and activity of the same trypsin isoform occur at different concentrations. Better results for activity (ionic strength of 0.11 mol·L-1, at 37 °C and pH 8.0) were found in 0-40% of ethanolic media in which the activity for ß-trypsin was about 60% higher than ɑ-trypsin. The ethanolic system does not cause significant changes in the level of secondary structure but the ß-trypsin isoform undergoes a major rearrangement. The use of until 60% (v/v) ethanol showed that ß-trypsin presents a denaturation process 17% more cooperative. The organic solvent causes redistribution in the supramolecular arrangement of both isoforms: all concentrations used induced the ß-trypsin molecules to rearrange into agglomerates. The ɑ-trypsin rearranges into agglomerates up to 60% (v/v) of ethanol and aggregates at 80% (v/v) of ethanol. Both isoforms keep the enzymatic activity up to 60% (v/v) of ethanol.

Predictive Value of Sirtuins in Acute Myocardial Infarction - Bridging the Bench to the Clinical Practice.

Acute myocardial infarction (AMI) is a non-transmissible condition with high prevalence, morbidity, and mortality. Different strategies for the management of AMI are employed worldwide, but its early diagnosis remains a major challenge. Many molecules have been proposed in recent years as predictive agents in the early detection of AMI, including troponin (C, T, and I), creatine kinase MB isoenzyme, myoglobin, heart-type fatty acid-binding protein, and a family of histone deacetylases with enzymatic activities named sirtuins. Sirtuins may be used as predictive or complementary treatment strategies and the results of recent preclinical studies are promising. However, human clinical trials and data are scarce, and many issues have been raised regarding the predictive values of sirtuins. The present review summarizes research on the predictive value of sirtuins in AMI. We also briefly summarize relevant clinical trials and discuss future perspectives and possible clinical applications.