The Telomerase Reverse Transcriptase Subunit from the Dimorphic Fungus Ustilago maydis

In this study, we investigated the reverse transcriptase subunit of telomerase in the dimorphic fungus Ustilago maydis. This protein (Trt1) contains 1371 amino acids and all of the characteristic TERT motifs. Mutants created by disrupting trt1 had senescent traits, such as delayed growth, low replicative potential, and reduced survival, that were reminiscent of the traits observed in est2 budding yeast mutants. Telomerase activity was observed in wild-type fungus sporidia but not those of the disruption mutant. The introduction of a self-replicating plasmid expressing Trt1 into the mutant strain restored growth proficiency and replicative potential. Analyses of trt1 crosses in planta suggested that Trt1 is necessary for teliospore formation in homozygous disrupted diploids and that telomerase is haploinsufficient in heterozygous diploids. Additionally, terminal restriction fragment analysis in the progeny hinted at alternative survival mechanisms similar to those of budding yeast

Ensamble de la secuencia genómica de la bacteria Avibacterium paragallinarum cepas CL y 2015, para analizar algunos determinantes genéticos relacionados con la virulencia

"Avibacterium paragallinarum es el agente etiológico de la coriza infecciosa, una enfermedad del tracto respiratorio superior de pollos y gallinas, caracterizada por producir descarga nasal, estornudo e inflamación facial. La distribución de esta enfermedad es mundial y causa importantes pérdidas económicas a la industria avícola debido al retraso del crecimiento, pérdida de peso, y disminución de la producción de huevo. Existen tres serogrupos (A, B y C) y 9 serotipos de esta bacteria. Debido a esta variedad de cepas, y variabilidad entre ellas, no existe un control adecuado de la enfermedad que ocasiona. En el grupo de trabajo de microbiología molecular y celular del ICUAP, consideramos que la enfermedad y la forma en que la bacteria desarrolla la patología, podrían ser comprendidas de mejor manera si se contara con la secuencia de diversos genomas de A. paragallinarum y así poder identificar factores virulencia importantes en su patogenicidad que permitiesen entender de una mejor manera el proceso patológico de esta enfermedad y que pudieran permitir un mejor control de la misma.

Comparative effect of resveratrol, carnosic acid and hernandulcin on target enzymes and biochemical markers linked to carbohydrate and lipid metabolism in mice

Resveratrol (RV), carnosic acid (CA) and hernandulcin (HE; a non-caloric sweetener) are envisioned as promising nutraceuticals to designnew functional foods for improving lipid and carbohydrate metabolism. This study aimed to investigate the in vitro inhibitory effectof these molecules on specific enzyme targets and their capacity to improve distinctive markers associated to carbohydrate and lipidmetabolism in murine model. The enzymes explored were alpha-amylase, alpha-glucosidase, and pancreatic lipase whereas ICR malemice were used for in vivo testing. Saturation curves (10-200 µM mL-1) and Lineweaver-Burk regressions suggested that RV, CA andHE exerts non-competitive inhibition on pancreatic lipase, alpha-glucosidase and alpha-amylase but, CA produced a strong competitiveactivity on alpha-amylase. RV was more effective to inhibit alpha-glucosidase (IC50, 22.1 µM) whereas CA was the most effective toinhibit both alpha-amylase (IC50, 11.7 µM) and pancreatic lipase (IC50, 31.5 µM). The effects of the oral administration of RV (300 mg/kg) HE (100 mg/kg) and CA (100 mg/kg) as well as the simultaneous administration of the three compounds at the same concentration was also explored in normoglycemic and diabetic mice. In addition, the prolonged administration of these substances combined with hypercaloric/atherogenic diet for 30 days was performed. Our results revealed a clear modulatory activity in both postprandial glucose and triglyceride levels as well an improvement in biochemical markers of mice treated with hypercaloric/atherogenic diet. The administration of HE produced a notable change (p < 0.01) in postprandial glucose assimilation at 60 min post-treatment in diabetic mice, whereas the other two compounds exerted a stronger depletion of glucose levels from 30 to 120 min post-treatment. A similar trend was recorded by RV and CA in postprandial triglyceride content, however, the latter compound was more effective (p < 0.05) at lower doses than RV. The simultaneous administration of the three compounds produced a significant improvement (p < 0.01) in biochemical parameters associated to carbohydrate (insulin and glucose) and lipid metabolism (total cholesterol, LDL-c, HDL-c, triglycerides, leptin, and adiponectin).Outstandingly, the mixture of the three compounds was more effective (p < 0.01) than the administration of sole compounds to amelioratethe side effects of the hypercaloric/atherogenic diet. Finally, the body weight of treated mice significantly decreased (from 5 to 20%; p < 0.05) in comparison with mice only fed with hypercaloric/atherogenic diet. Our results suggest that mixtures of RV, HE and CA may work better than their sole administration in mice and part of their biological activity could be associated with their inhibitory properties on the enzyme targets evaluated in this investigation.Keywords: Carnosic acid; Hernandulcin; Resveratrol; Hypoglycemic; Hypolipidemic; Murine model; Target enzyme

1H NMR Profiling of the Venom from Hylesia continua: Implications of Small Molecules for Lepidopterism

Lepidopterism caused by caterpillar contact is considered a public health problem around the world. The local and systemic responses of this pathology include short- and long-term inflammatory events. Although the proteolytic activity of the venoms from caterpillars is strongly associated with an inflammatory response in humans and murine models, fast and acute symptoms such as a burning sensation, itching, and pain should be related to the presence of low-weight hydrophilic molecules which easily influence cell metabolism. This investigation reports on the 1H-Nuclear Magnetic Resonance (NMR) profiling of the venom from the larva of Hylesia continua, a caterpillar linked to frequent cases of lepidopterism in the northern highlands of Puebla, Mexico. According to one-dimensional (1D) and two-dimensional (2D) NMR data, the venom of H. continua contained 19 compounds with proven pain-inducing activity (i.e., acetic acid, lactic acid, formic acid, succinic acid, 2-hydroxyglutaric acid, ethanol, and glutamate), inflammatory activity (i.e., cadaverine, putrescine, and acetoin), as well as natural immunosuppressive activity (i.e., O-phosphocholine and urocanic acid). The levels of the 19 compounds were calculated using quantitative-NMR (qNMR) and extensively discussed on the basis of their toxic properties which partially explain typical symptoms of lepidopterism caused by the larvae of H. continua. To the best of our knowledge, this is the first investigation reporting a complex mixture of small molecules with inflammatory properties dissolved in the venom of a lepidopteran larva

Comparative effect of resveratrol, carnosic acid and hernandulcin on target enzymes and biochemical markers linked to carbohydrate and lipid metabolism in mice

Resveratrol (RV), carnosic acid (CA) and hernandulcin (HE; a non-caloric sweetener) are envisioned as promising nutraceuticals to designnew functional foods for improving lipid and carbohydrate metabolism. This study aimed to investigate the in vitro inhibitory effectof these molecules on specific enzyme targets and their capacity to improve distinctive markers associated to carbohydrate and lipidmetabolism in murine model. The enzymes explored were alpha-amylase, alpha-glucosidase, and pancreatic lipase whereas ICR malemice were used for in vivo testing. Saturation curves (10-200 µM mL-1) and Lineweaver-Burk regressions suggested that RV, CA andHE exerts non-competitive inhibition on pancreatic lipase, alpha-glucosidase and alpha-amylase but, CA produced a strong competitiveactivity on alpha-amylase. RV was more effective to inhibit alpha-glucosidase (IC50, 22.1 µM) whereas CA was the most effective toinhibit both alpha-amylase (IC50, 11.7 µM) and pancreatic lipase (IC50, 31.5 µM). The effects of the oral administration of RV (300 mg/kg) HE (100 mg/kg) and CA (100 mg/kg) as well as the simultaneous administration of the three compounds at the same concentration was also explored in normoglycemic and diabetic mice. In addition, the prolonged administration of these substances combined with hypercaloric/atherogenic diet for 30 days was performed. Our results revealed a clear modulatory activity in both postprandial glucose and triglyceride levels as well an improvement in biochemical markers of mice treated with hypercaloric/atherogenic diet. The administration of HE produced a notable change (p < 0.01) in postprandial glucose assimilation at 60 min post-treatment in diabetic mice, whereas the other two compounds exerted a stronger depletion of glucose levels from 30 to 120 min post-treatment. A similar trend was recorded by RV and CA in postprandial triglyceride content, however, the latter compound was more effective (p < 0.05) at lower doses than RV. The simultaneous administration of the three compounds produced a significant improvement (p < 0.01) in biochemical parameters associated to carbohydrate (insulin and glucose) and lipid metabolism (total cholesterol, LDL-c, HDL-c, triglycerides, leptin, and adiponectin).Outstandingly, the mixture of the three compounds was more effective (p < 0.01) than the administration of sole compounds to amelioratethe side effects of the hypercaloric/atherogenic diet. Finally, the body weight of treated mice significantly decreased (from 5 to 20%; p < 0.05) in comparison with mice only fed with hypercaloric/atherogenic diet. Our results suggest that mixtures of RV, HE and CA may work better than their sole administration in mice and part of their biological activity could be associated with their inhibitory properties on the enzyme targets evaluated in this investigation.Keywords: Carnosic acid; Hernandulcin; Resveratrol; Hypoglycemic; Hypolipidemic; Murine model; Target enzyme

The Telomerase Reverse Transcriptase Subunit from the Dimorphic Fungus <i>Ustilago maydis</i>

<div><p>In this study, we investigated the reverse transcriptase subunit of telomerase in the dimorphic fungus <i>Ustilago maydis</i>. This protein (Trt1) contains 1371 amino acids and all of the characteristic TERT motifs. Mutants created by disrupting <i>trt1</i> had senescent traits, such as delayed growth, low replicative potential, and reduced survival, that were reminiscent of the traits observed in <i>est2</i> budding yeast mutants. Telomerase activity was observed in wild-type fungus sporidia but not those of the disruption mutant. The introduction of a self-replicating plasmid expressing Trt1 into the mutant strain restored growth proficiency and replicative potential. Analyses of <i>trt1</i> crosses <i>in planta</i> suggested that Trt1 is necessary for teliospore formation in homozygous disrupted diploids and that telomerase is haploinsufficient in heterozygous diploids. Additionally, terminal restriction fragment analysis in the progeny hinted at alternative survival mechanisms similar to those of budding yeast.</p></div

Telomere repeat amplification protocol (TRAP) analysis in <i>U. maydis</i>.

<p>Telomerase activity in wild-type and mutant strains was determined. The absorbance data were used to construct a graphical representation of the telomerase activity for the sporidia of <i>U. maydis</i> strains (either wild-type or <i>trt</i>^-). Tumor cells derived from the 521×520 cross and a plant control were included to evaluate and detect telomerase activity. The medians of the telomerase-positive control cells (HEK293) and the 521 wild-type strain were significantly different from the median of the treated negative controls (P<0.05); however, no significant differences were detected between the negative controls and the <i>trt1</i>-disrupted mutants. The samples heated to 85°C are indicated with Δ, and the RNase-treated samples are designated as RNase. Telomerase activity was also determined in tumors and maize leaves.</p