Role of Long Non-Coding RNAs and the Molecular Mechanisms Involved in Insulin Resistance.

Long non-coding RNAs (lncRNAs) are single-stranded RNA biomolecules with a length of >200 nt, and they are currently considered to be master regulators of many pathological processes. Recent publications have shown that lncRNAs play important roles in the pathogenesis and progression of insulin resistance (IR) and glucose homeostasis by regulating inflammatory and lipogenic processes. lncRNAs regulate gene expression by binding to other non-coding RNAs, mRNAs, proteins, and DNA. In recent years, several mechanisms have been reported to explain the key roles of lncRNAs in the development of IR, including metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), imprinted maternal-ly expressed transcript (H19), maternally expressed gene 3 (MEG3), myocardial infarction-associated transcript (MIAT), and steroid receptor RNA activator (SRA), HOX transcript antisense RNA (HOTAIR), and downregulated Expression-Related Hexose/Glucose Transport Enhancer (DREH). LncRNAs participate in the regulation of lipid and carbohydrate metabolism, the inflammatory process, and oxidative stress through different pathways, such as cyclic adenosine monophosphate/protein kinase A (cAMP/PKA), phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT), polypyrimidine tract-binding protein 1/element-binding transcription factor 1c (PTBP1/SREBP-1c), AKT/nitric oxide synthase (eNOS), AKT/forkhead box O1 (FoxO1), and tumor necrosis factor-alpha (TNF-α)/c-Jun-N-terminal kinases (JNK). On the other hand, the mechanisms linked to the molecular, cellular, and biochemical actions of lncRNAs vary according to the tissue, biological species, and the severity of IR. Therefore, it is essential to elucidate the role of lncRNAs in the insulin signaling pathway and glucose and lipid metabolism. This review analyzes the function and molecular mechanisms of lncRNAs involved in the development of IR.

cAMP-PKA signal transduction specificity in Saccharomyces cerevisiae.

Living cells have developed a set of complex signaling responses, which allow them to withstand different environmental challenges. Signaling pathways enable the cell to monitor external and internal states and to articulate the appropriate physiological responses. Cellular signal transmission requires the dynamic formation of spatiotemporal controlled molecular interactions. One of the most important signaling circuits in Saccharomyces cerevisiae is the one controlled by cAMP-Protein Kinase A (PKA). In budding yeast, extracellular glucose and a plethora of signals related with growth and stress conditions regulate the intracellular cAMP levels that modulate PKA activity which in turn regulates a broad range of cellular processes. The cAMP-PKA signaling output requires a controlled specificity of the PKA responses. In this review we discuss the molecular mechanisms that are involved in the establishment of the specificity in the cAMP-PKA signaling pathway in S.cerevisiae.

The Relationship of Single Nucleotide Polymorphisms in the TRPV1 Gene with Lipid Profile, Glucose, and Blood Pressure in Mexican Population.

Aim: We analyzed the frequencies of the rs222749 G>A, rs222747 G>C, rs224534 G>A, and rs8065080 C > T polymorphisms in the TRPV1 gene and their relationships with biomarkers in a Mexican population. Materials and Methods: We included 195 students from two Mexican universities (72.3% female and 27.7% male, mean age, 20.8 ± 3.3 years). The biomarkers analyzed were lipid profile, glucose levels, blood pressure (BP), and body mass index. DNA was obtained from leukocytes by the dodecyltrimethylammonium bromide and cetyltrimethylammonium bromide method and polymorphisms were determined with TaqMan single nucleotide polymorphism (SNP) genotyping assays. Results: Alterations in lipid profile were total cholesterol ≥200 mg/dL in 9.7% of participants, triglycerides (TG) ≥150 mg/dL in 9.2%, high-density lipoprotein (HDL) <35 mg/dL in 6.7%, and low-density lipoprotein (LDL) ≥130 mg/dL in 6.2% of participants. Moreover, 8.2% of the subjects had BP values consistent with hypertension. The most frequent alleles were rs222749G (89.2%), rs222747G (69.2%), rs224534G (59.7%), and rs8065080T (62.3%). An analysis of the associations between the genotypic data and the biomarkers showed that the rs222749GA and rs224534GA genotypes were associated with higher diastolic and systolic BP values, respectively; the rs222747CC genotype was associated with lower LDL levels; the rs224534AA genotype was associated with higher HDL levels and lower triglycerides and LDL. The GGGC/GCAT and GGGT/GCAT haplotypes were associated with higher systolic BP. Conclusions: This study suggests a possible association between TRPV1 gene polymorphisms and BP and lipid profiles in a Mexican population.

Herpud1 impacts insulin-dependent glucose uptake in skeletal muscle cells by controlling the Ca2+-calcineurin-Akt axis.

Skeletal muscle plays a central role in insulin-controlled glucose homeostasis. The molecular mechanisms related to insulin resistance in this tissue are incompletely understood. Herpud1 is an endoplasmic reticulum membrane protein that maintains intracellular Ca2+ homeostasis under stress conditions. It has recently been reported that Herpud1-knockout mice display intolerance to a glucose load without showing altered insulin secretion. The functions of Herpud1 in skeletal muscle also remain unknown. Based on these findings, we propose that Herpud1 is necessary for insulin-dependent glucose disposal in skeletal muscle. Here we show that Herpud1 silencing decreased insulin-dependent glucose uptake, GLUT4 translocation to the plasma membrane, and Akt Ser473 phosphorylation in cultured L6 myotubes. A decrease in insulin-induced Akt Ser473 phosphorylation was observed in soleus but not in extensor digitorum longus muscle samples from Herpud1-knockout mice. Herpud1 knockdown increased the IP3R-dependent cytosolic Ca2+ response and the activity of Ca2+-dependent serine/threonine phosphatase calcineurin in L6 cells. Calcineurin decreased insulin-dependent Akt phosphorylation and glucose uptake. Moreover, calcineurin inhibition restored the insulin response in Herpud1-depleted L6 cells. Based on these findings, we conclude that Herpud1 is necessary for adequate insulin-induced glucose uptake due to its role in Ca2+/calcineurin regulation in L6 myotubes.

A digital volumetric tomography (DVT) study in the mandibular molar region for miniscrew placement during mixed dentition

OBJECTIVE: To assess bone thickness for miniscrew placement in the mandible during mixed dentition by using digital volumetric tomograph (DVT). MATERIAL AND METHODS: A total of 15 healthy patients aged 8-10 years old, with early exfoliated mandibular second deciduous molar, were included. DVT images of one quadrant of the mandible were obtained using Kodak extraoral imaging systems and analyzed by Kodak dental imaging software. The error of the method (EM) was calculated using Dahlberg's formula. Mean and standard deviation were calculated at 6 and 8 mm from the cementoenamel junction (CEJ).Paired t-test was used to analyze the measurements. RESULTS: Buccal cortical bone thickness, mesiodistal width and buccolingual bone depth at 6 mm were found to be 1.73 + 0.41, 2.15 + 0.49 and 13.18 + 1.22 mm, respectively; while at 8 mm measurements were 2.42 + 0.34, 2.48 + 0.33 and 13.65 + 1.25 mm, respectively. EM for buccal cortical bone thickness, mesiodistal width and buccolingual bone depth was 0.58, 0.40 and 0.48, respectively. The difference in measurement at 6 and 8 mm for buccal cortical plate thickness (P < 0.05) and buccolingual bone thickness (P < 0.05) was found to be significant, whereas for mesiodistal width it was insignificant (P > 0.05). CONCLUSION: Bone thickness measurement has shown promising evidence for safe placement of miniscrews in the mandible during mixed dentition. The use of miniscrew is the best alternative, even in younger patients. .

OBJETIVO: avaliar, por meio de tomografia volumétrica digital (TVD), a espessura óssea necessária para a instalação de mini-implante na arcada inferior durante a fase de dentição mista. MÉTODOS: um total de 15 pacientes saudáveis, com idades entre 8 e 10 anos, com segundo molar inferior decíduo irrompido recentemente, foram incluídos no presente estudo. Imagens de TVD da hemiarcada inferior foram obtidas utilizando sistemas de imagens extrabucais Kodak. As imagens foram analisadas por meio do programa de imagens Kodak. O erro do método (EM) foi calculado utilizando a fórmula de Dahlberg. Médias e desvios-padrão foram calculados de 6 a 8mm aquém da junção amelocementária. O teste t foi utilizado para a análise das medidas. RESULTADOS: a espessura do osso cortical vestibular, largura mesiodistal e profundidade óssea vestibulolingual, a 6mm, foram de 1,73 + 0,41; 2,15 + 0,49; e 13,18 + 1,22 mm, respectivamente. Já a 8mm, os valores foram de 2,42 + 0,34; 2,48 + 0,33; e 13,65 + 1,25mm. O EM para a espessura do osso cortical vestibular, largura mesiodistal e profundidade óssea vestibulolingual foi de 0,58, 0,40 e 0,48mm, respectivamente. A diferença entre as medidas a 6 e 8mm para a espessura do osso cortical vestibular (p < 0,05) e a espessura óssea vestibulolingual (p < 0,05) foi significativa, embora não tenha sido significativa para a largura mesiodistal (p < 0,05). CONCLUSÃO: a mensuração da espessura óssea demonstra evidências promissoras para a segura instalação de mini-implantes na arcada inferior e na fase de dentição mista. O uso de mini-implantes tem se mostrado a melhor alternativa, mesmo nos casos de pacientes mais jovens. .

Non-classical gluconeogenesis-dependent glucose metabolism in Rhipicephalus microplus embryonic cell line BME26.

In this work we evaluated several genes involved in gluconeogenesis, glycolysis and glycogen metabolism, the major pathways for carbohydrate catabolism and anabolism, in the BME26 Rhipicephalus microplus embryonic cell line. Genetic and catalytic control of the genes and enzymes associated with these pathways are modulated by alterations in energy resource availability (primarily glucose). BME26 cells in media were investigated using three different glucose concentrations, and changes in the transcription levels of target genes in response to carbohydrate utilization were assessed. The results indicate that several genes, such as glycogen synthase (GS), glycogen synthase kinase 3 (GSK3), phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6 phosphatase (GP) displayed mutual regulation in response to glucose treatment. Surprisingly, the transcription of gluconeogenic enzymes was found to increase alongside that of glycolytic enzymes, especially pyruvate kinase, with high glucose treatment. In addition, RNAi data from this study revealed that the transcription of gluconeogenic genes in BME26 cells is controlled by GSK-3. Collectively, these results improve our understanding of how glucose metabolism is regulated at the genetic level in tick cells.

Induction of a stringent metabolic response in intracellular stages of Leishmania mexicana leads to increased dependence on mitochondrial metabolism.

Leishmania parasites alternate between extracellular promastigote stages in the insect vector and an obligate intracellular amastigote stage that proliferates within the phagolysosomal compartment of macrophages in the mammalian host. Most enzymes involved in Leishmania central carbon metabolism are constitutively expressed and stage-specific changes in energy metabolism remain poorly defined. Using (13)C-stable isotope resolved metabolomics and (2)H2O labelling, we show that amastigote differentiation is associated with reduction in growth rate and induction of a distinct stringent metabolic state. This state is characterized by a global decrease in the uptake and utilization of glucose and amino acids, a reduced secretion of organic acids and increased fatty acid ß-oxidation. Isotopomer analysis showed that catabolism of hexose and fatty acids provide C4 dicarboxylic acids (succinate/malate) and acetyl-CoA for the synthesis of glutamate via a compartmentalized mitochondrial tricarboxylic acid (TCA) cycle. In vitro cultivated and intracellular amastigotes are acutely sensitive to inhibitors of mitochondrial aconitase and glutamine synthetase, indicating that these anabolic pathways are essential for intracellular growth and virulence. Lesion-derived amastigotes exhibit a similar metabolism to in vitro differentiated amastigotes, indicating that this stringent response is coupled to differentiation signals rather than exogenous nutrient levels. Induction of a stringent metabolic response may facilitate amastigote survival in a nutrient-poor intracellular niche and underlie the increased dependence of this stage on hexose and mitochondrial metabolism.

Potencial de transdiferenciação neural das células-tronco mesenquimais da medula óssea de equino / Potential of neural transdifferentiation of mesenchymal stem cells from equine bone marrow

Os primeiros estudos demonstrando o potencial de trandiferenciação neural das células-tronco mesenquimais (CTMs) provenientes da medula óssea (MO) foram conduzidos em camundogos e humanos no início da década de 2000. Após esse período, o número de pesquisas e publicações com o mesmo propósito tem aumentado, mas com raros ou escassos estudos na espécie equina. Nesse sentindo, o objetivo desse trabalho foi avaliar o potencial in vitro da transdiferenciação neural das CTMs provenientes da MO de equinos utilizando-se dois protocolos: P1 (forksolin e ácido retinóico) e P2 (2-βmecarptoetanol). Após a confirmação das linhagens mesenquimais, pela positividade para o marcador CD90 (X=97,94%), negatividade para o marcador CD34 e resposta positiva a diferenciação osteogênica, as CTMs foram submetidas a transdiferenciação neural (P1 e P2) para avaliação morfológica e expressão dos marcadores neurais GFAP e β3 tubulina por citometria de fluxo. Os resultados revelaram mudanças morfológicas em graus variados entre os protocolos testados. No protocolo 1, vinte quatro horas após a incubação com o meio de diferenciação neural, grande proporção de células (>80%) apresentaram morfologia semelhante a células neurais, caracterizadas por retração do corpo celular e grande número de projeções protoplasmáticas (filopodia). Por outro lado, de forma comparativa, já nos primeiros 30 minutos após a exposição ao antioxidante β-mercaptoetanol (P2) as CTMs apresentaram rápida mudança morfológica caracterizada principalmente por retração do corpo celular e menor número de projeções protoplasmáticas. Também ficou evidenciado com o uso deste protocolo, menor aderência das células após tempo de exposição ao meio de diferenciação, quando comparado ao P1. Com relação a análise imunofenotípica foi observado uma maior (P<0,001) expressão dos marcadores GFAP e β3 tubulina ao término do P2 quando comparado ao P1. A habilidade das CTMs em gerar tipos celulares relacionados a linhagem neural é complexa e multifatorial, dependendo não só dos agentes indutores, mas também do ambiente no qual estas células são cultivadas. Desta forma um maior número de estudos é necessário para o melhor entendimento do processo de transdiferenciação neural a partir de CTMs de equinos.

The first studies showing the potential of neural transdifferentiation of mesenchymal stem cells (MSCs) from bone marrow (BM) were conducted in camundogos and humans in the early 2000s. After this period, the number of research and publications with the same purpose increased, but with rare or scarce studies in horses. The aim of this study was to evaluate in vitro neuronal transdifferentiation potential of MSCs from equine BM using two protocols: P1 (forksolin and retinoic acid) and P2 (2-βmecarptoetanol). After confirming the mesenchymal lineages, by positivity for the marker CD90 (X=97.94%), negative for the marker CD34 and positive response for osteogenic differentiation, MSCs were subjected to neural transdifferentiation (P1 and P2) for morphological analysis and expression of neural markers GFAP and β3 tubulin by flow cytometry. The results revealed morphological changes in varying degrees between the tested protocols. In protocol 1, twenty four hours after incubation with the media of neural differentiation, a large proportion of cells (>80%) had similar morphology to neural cells, characterized by retraction of cellular body and a large number of cytoplasmic extension (filopodia). However, comparatively, within the first 30 minutes after exposure to the antioxidant β-mercaptoethanol (P2) MSCs showed rapid morphological changes characterized mainly by retraction of cellular body and less cytoplasmic extension. It was also evidenced with the use of this protocol, lower cellular adhesion after exposure to media when compared to P1. Regarding the immunophenotyping analysis it was observed a higher (P<0.001) expression of the markers GFAP and β3 tubulin at the end of P2 compared to P1. The ability of MSCs to generate cell types related to neural lineage is complex and multifactorial, depending not only of inducing agents, but also the environment in which these cells will be cultivated. Thus a greater number of studies are necessary to better understand the process of neural transdifferentiation of MSCs from equine.

Diferenciação in vitro de células-tronco mesenquimais da medula óssea de cães em precursores osteogênicos / In vitro differentiation of mesenchimal stem cells of dogs into osteogenic precursors

O objetivo principal da nossa pesquisa foi avaliar o potencial de diferenciação osteogênica de células-tronco mesenquimais (MSC) obtidas da medula óssea do cão. As MSC foram separadas pelo método Ficoll e cultivadas sob duas condições distintas: DMEM baixa glicose ou DMEM/F12, ambos contendo L-glutamina, 20% de SFB e antibióticos. Marcadores de MSC foram testados, confirmando células CD44+ e CD34- através da citometria de fluxo. Para a diferenciação osteogênica, as células foram submetidas a quatro diferentes condições: Grupo 1, as mesmas condições utilizadas para a cultura de células primárias com os meios DMEM baixa glicose suplementado; Grupo 2, as mesmas condições do Grupo 1, mais os indutores de diferenciação dexametasona, ácido ascórbico e b-glicerolfosfato; Grupo 3, células cultivadas com meios DMEM/F12 suplementado; e Grupo 4, nas mesmas condições que no Grupo 3, mais indutores de diferenciação de dexametasona, ácido ascórbico e b-glicerolfosfato. A diferenciação celular foi confirmada através da coloração com alizarin red e da imunomarcação com o anticorpo SP7/Osterix. Nós observamos através da coloração com alizarin red que o depósito de cálcio foi mais evidente nas células cultivadas em DMEM/F12. Além disso, usando a imunomarcação com o anticorpo SP/7Osterix obtivemos positividade em 1:6 células para o Meio DMEM/F12 comparada com 1:12 para o meio DMEM-baixa glicose. Com base nos nossos resultados concluímos que o meio DMEM/F12 é mais eficiente para a indução da diferenciação de células-tronco mesenquimais caninas em promotores osteogênicos. Este efeito provavelmente ocorre em decorrência da maior quantidade de glicose neste meio, bem como da presença de diversos aminoácidos.

The aim of our research was to evaluate the potential for osteogenic differentiation of mesenchimal stem cells (MSC) obtained from dog bone marrow. The MSC were separated using the Ficoll method and cultured under two different conditions: DMEM low glucose or DMEM/F12, both containing L-glutamine, 20% of FBS and antibiotics. MSC markers were tested, confirming CD44+ and CD34- cells with flow cytometry. For osteogenic differentiation, cells were submitted to four different conditions: Group 1, same conditions used for primary cell culture with DMEM supplemented media; Group 2, same conditions of Group 1 plus differentiation inductors Dexametazone, ascorbic acid and β-glicerolphosphate. Group 3, Cells cultured with supplemented DMEM/F12 media, and Group 4, same conditions as in Group 3 plus differentiation inductors Dexametazone, ascorbic acid and β-glicerolphosphate. The cellular differentiation was confirmed using alizarin red and imunostaining with SP7/Osterix antibody. We observed by alizarin staining that calcium deposit was more evident in cells cultivated in DMEM/F12.Furthermore, by SP/7Osterix antibody immunostaining we obtained 1:6 positive cells when using DMEM/F12 compared with 1:12 for low-glucose DMEM. Based on our results, we conclude that the medium DMEM/F12 is more efficient for induction of differentiation of mesenchymal stem cells in canine osteogenic progenitors. This effect is probably due to the greater amount of glucose in the medium and the presence of various amino acids.

Diferenciação in vitro de células-tronco mesenquimais da medula óssea de cães em precursores osteogênicos / In vitro differentiation of mesenchimal stem cells of dogs into osteogenic precursors

O objetivo principal da nossa pesquisa foi avaliar o potencial de diferenciação osteogênica de células-tronco mesenquimais (MSC) obtidas da medula óssea do cão. As MSC foram separadas pelo método Ficoll e cultivadas sob duas condições distintas: DMEM baixa glicose ou DMEM/F12, ambos contendo L-glutamina, 20% de SFB e antibióticos. Marcadores de MSC foram testados, confirmando células CD44+ e CD34- através da citometria de fluxo. Para a diferenciação osteogênica, as células foram submetidas a quatro diferentes condições: Grupo 1, as mesmas condições utilizadas para a cultura de células primárias com os meios DMEM baixa glicose suplementado; Grupo 2, as mesmas condições do Grupo 1, mais os indutores de diferenciação dexametasona, ácido ascórbico e b-glicerolfosfato; Grupo 3, células cultivadas com meios DMEM/F12 suplementado; e Grupo 4, nas mesmas condições que no Grupo 3, mais indutores de diferenciação de dexametasona, ácido ascórbico e b-glicerolfosfato. A diferenciação celular foi confirmada através da coloração com alizarin red e da imunomarcação com o anticorpo SP7/Osterix. Nós observamos através da coloração com alizarin red que o depósito de cálcio foi mais evidente nas células cultivadas em DMEM/F12. Além disso, usando a imunomarcação com o anticorpo SP/7Osterix obtivemos positividade em 1:6 células para o Meio DMEM/F12 comparada com 1:12 para o meio DMEM-baixa glicose. Com base nos nossos resultados concluímos que o meio DMEM/F12 é mais eficiente para a indução da diferenciação de células-tronco mesenquimais caninas em promotores osteogênicos. Este efeito provavelmente ocorre em decorrência da maior quantidade de glicose neste meio, bem como da presença de diversos aminoácidos.(AU)

The aim of our research was to evaluate the potential for osteogenic differentiation of mesenchimal stem cells (MSC) obtained from dog bone marrow. The MSC were separated using the Ficoll method and cultured under two different conditions: DMEM low glucose or DMEM/F12, both containing L-glutamine, 20% of FBS and antibiotics. MSC markers were tested, confirming CD44+ and CD34- cells with flow cytometry. For osteogenic differentiation, cells were submitted to four different conditions: Group 1, same conditions used for primary cell culture with DMEM supplemented media; Group 2, same conditions of Group 1 plus differentiation inductors Dexametazone, ascorbic acid and β-glicerolphosphate. Group 3, Cells cultured with supplemented DMEM/F12 media, and Group 4, same conditions as in Group 3 plus differentiation inductors Dexametazone, ascorbic acid and β-glicerolphosphate. The cellular differentiation was confirmed using alizarin red and imunostaining with SP7/Osterix antibody. We observed by alizarin staining that calcium deposit was more evident in cells cultivated in DMEM/F12.Furthermore, by SP/7Osterix antibody immunostaining we obtained 1:6 positive cells when using DMEM/F12 compared with 1:12 for low-glucose DMEM. Based on our results, we conclude that the medium DMEM/F12 is more efficient for induction of differentiation of mesenchymal stem cells in canine osteogenic progenitors. This effect is probably due to the greater amount of glucose in the medium and the presence of various amino acids.(AU)

Potencial de transdiferenciação neural das células-tronco mesenquimais da medula óssea de equino / Potential of neural transdifferentiation of mesenchymal stem cells from equine bone marrow

Os primeiros estudos demonstrando o potencial de trandiferenciação neural das células-tronco mesenquimais (CTMs) provenientes da medula óssea (MO) foram conduzidos em camundogos e humanos no início da década de 2000. Após esse período, o número de pesquisas e publicações com o mesmo propósito tem aumentado, mas com raros ou escassos estudos na espécie equina. Nesse sentindo, o objetivo desse trabalho foi avaliar o potencial in vitro da transdiferenciação neural das CTMs provenientes da MO de equinos utilizando-se dois protocolos: P1 (forksolin e ácido retinóico) e P2 (2-βmecarptoetanol). Após a confirmação das linhagens mesenquimais, pela positividade para o marcador CD90 (X=97,94%), negatividade para o marcador CD34 e resposta positiva a diferenciação osteogênica, as CTMs foram submetidas a transdiferenciação neural (P1 e P2) para avaliação morfológica e expressão dos marcadores neurais GFAP e β3 tubulina por citometria de fluxo. Os resultados revelaram mudanças morfológicas em graus variados entre os protocolos testados. No protocolo 1, vinte quatro horas após a incubação com o meio de diferenciação neural, grande proporção de células (>80%) apresentaram morfologia semelhante a células neurais, caracterizadas por retração do corpo celular e grande número de projeções protoplasmáticas (filopodia). Por outro lado, de forma comparativa, já nos primeiros 30 minutos após a exposição ao antioxidante β-mercaptoetanol (P2) as CTMs apresentaram rápida mudança morfológica caracterizada principalmente por retração do corpo celular e menor número de projeções protoplasmáticas. Também ficou evidenciado com o uso deste protocolo, menor aderência das células após tempo de exposição ao meio de diferenciação, quando comparado ao P1.(AU)

The first studies showing the potential of neural transdifferentiation of mesenchymal stem cells (MSCs) from bone marrow (BM) were conducted in camundogos and humans in the early 2000s. After this period, the number of research and publications with the same purpose increased, but with rare or scarce studies in horses. The aim of this study was to evaluate in vitro neuronal transdifferentiation potential of MSCs from equine BM using two protocols: P1 (forksolin and retinoic acid) and P2 (2-βmecarptoetanol). After confirming the mesenchymal lineages, by positivity for the marker CD90 (X=97.94%), negative for the marker CD34 and positive response for osteogenic differentiation, MSCs were subjected to neural transdifferentiation (P1 and P2) for morphological analysis and expression of neural markers GFAP and β3 tubulin by flow cytometry. The results revealed morphological changes in varying degrees between the tested protocols. In protocol 1, twenty four hours after incubation with the media of neural differentiation, a large proportion of cells (>80%) had similar morphology to neural cells, characterized by retraction of cellular body and a large number of cytoplasmic extension (filopodia). However, comparatively, within the first 30 minutes after exposure to the antioxidant β-mercaptoethanol (P2) MSCs showed rapid morphological changes characterized mainly by retraction of cellular body and less cytoplasmic extension.(AU)

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.

Transcription levels of CHS5 and CHS4 genes in Paracoccidioides brasiliensis mycelial phase, respond to alterations in external osmolarity, oxidative stress and glucose concentration.

The complete sequence of Paracoccidioides brasiliensis CHS5 gene, encoding a putative chitin synthase revealed a 5583nt open reading frame, interrupted by three introns of 82, 87 and 97bp (GenBank Accession No EF654132). The deduced protein contains 1861 amino acids with a predicted molecular weight of 206.9kDa. Both its large size and the presence of a N-terminal region of approx. 800 residues with a characteristic putative myosin motor-like domain, allow us to include PbrChs5 into class V fungal chitin synthases. Sequence analysis of over 4kb from the 5' UTR region in CHS5, revealed the presence of a previously reported CHS4 gene in P. brasiliensis, arranged in a head-to-head configuration with CHS5. A motif search in this shared region showed the presence of stress response elements (STREs), three binding sites for the transcription activators Rlm1p (known to be stimulated by hypo-osmotic stress) and clusters of Adr1 (related to glucose repression). A quantitative RT-PCR analysis pointed to changes in transcription levels for both genes following oxidative stress, alteration of external osmolarity and under glucose-repressible conditions, suggesting a common regulatory mechanism of transcription.

Betaine aldehyde dehydrogenase from Pseudomonas aeruginosa: cloning, over-expression in Escherichia coli, and regulation by choline and salt.

In the human pathogen Pseudomonas aeruginosa, betaine aldehyde dehydrogenase (BADH) may play a dual role assimilating carbon and nitrogen from choline or choline precursors--abundant at infection sites--and producing glycine betaine, which protects the bacteria against the high-osmolarity stress prevalent in the infected tissues. We cloned the P. aeruginosa BADH gene and expressed the BADH protein in Escherichia coli. The recombinant protein appears identical to its native counterpart, as judged by Western blot, N-terminal amino acid sequence, tryptophan-fluorescence emission spectra, circular-dichroism spectroscopy, size-exclusion chromatography, and kinetic properties. Computational analysis indicated that the promoter sequence of the putative operon that includes the BADH gene has a consensus-binding site for the choline-sensing transcription repressor BetI, and putative boxes for ArcA and Lrp transcription factors but no known elements of response to osmotic stress. This is consistent with the strong induction of BADH expression by choline and with the lack of effect of NaCl. As there were significant amounts of BADH protein and activity in P. aeruginosa cells grown on glucose plus choline, as well as the BADH activity exhibiting tolerance to salt, it is likely that glycine betaine is synthesized in vivo and could play an important osmoprotectant role under conditions of infection.