QUALIDADE DE SEMENTES DE MILHO (Zea mays L.) SOB CONDIÇÕES DE SECAGEM INTERMITENTE

O trabalho avaliou a remoção de água e a qualidade de sementes de milho submetidas a secagem intermitente rápida e lenta, sob temperaturas do ar de 60oC e 70oC, respectivamente. Para tanto, na safra 94/95 sementes básicas da cultivar IAPAR 26, apresentando 22% de água, foram colhidas com automotriz, em Ponta Grossa - PR, e secadas em secadores comerciais, utilizando-se como tratamento referencial a secagem à sombra até 13% de água. No transcurso das secagens a redução do teor de água das sementes foi monitorado, em intervalos de trinta minutos, para o estabelecimento das velocidades e curvas de secagem. A qualidade fisiológica foi avaliada nas sementes amostradas, antes da secagem, no estágio intermediário e ao final das secagens, através dos testes de germinação, tetrazólio (germinação e vigor) e de envelhecimento artificial. As secagens intermitentes rápida e lenta, com velocidades médias de 1,7 e 2,7% /h, respectivamente, nas temperaturas empregadas, foram eficientes na remoção de água de sementes de milho com alto grau de umidade, não afetando imediatamente, a sua qualidade fisiológica (P > 0,01). Redução da temperatura do ar pode ser recomendada no final do processo, quando o teor de água das sementes aproxima-se de 14%.<br>The work evaluated water removal and corn seed quality by "fast" and "slow" intermittent drying, under 60oC and 70oC air temperatures. In 1994/95 IAPAR 26 seeds were harvested with a combined, harvester (22% water content) in Ponta Grossa, Paraná, Brazil. They were dried in commercial dryers, using the shade drying to 13% as referential. The seed water content was monitored during drying, at intervals of thirty minutes, for the establishment of the drying speeds and curves. The physiologic quality was evaluated in the sampled seeds, before drying, at intermidiate times and at the end of drying, through the germination, tetrazolium (germination and vigor) and accelerated aging tests. The drying speed for the "fast" and "slow" dryers were 1.7% and 2.7% /hr, respectively. The dryers were efficient in the removal of corn seed water with high moisture content, not affecting immediately its physiologic quality (P> 0.01). Air temperature reduction can be recommended at the end of the process, when the seeds reach 14% water content

SECAGEM INTERMITENTE E SEUS EFEITOS NA QUALIDADE FISIOLÓGICA DE SEMENTES DE TREMOÇO AZUL

Dois modelos de secadores comerciais, um intermitente lento, com a temperatura do ar de secagem a 60oC e a 65oC e outro rápido a 50oC, foram testados para a avaliaç��o da redução do grau de umidade de 20 para 13% e sua influência na qualidade fisiológica das sementes de tremoço azul (Lupinus angustifolius L.). Foram realizados testes de germinação, envelhecimento artificial e emergência em campo, após a secagem, aos três e seis meses de armazenamento. Os testes de germinação e envelhecimento artificial não detectaram diferenças significativas entre a qualidade das sementes secadas nos secadores e das secadas à sombra. Na semeadura aos seis meses, as emergências em campo foram 81%, 89%, 90% e 83% para os secadores intermitente lento a 60oC, a 65oC, para o intermitente rápido a 50oC e para a secagem à sombra, respectivamente. O secador intermitente rápido, onde as sementes passam rapidamente pela câmara de secagem e permanecem maior período na equalização, reduziu em média 1,1 pontos porcentuais por hora o teor de água das sementes e na secagem intermitente lenta a 65oC e a 60oC obteve-se 2,3 e 2,7 pontos porcentuais por hora, portanto mais eficientes que o primeiro, quando comparadas umidades iniciais/finais semelhantes. Conclui-se que o secador intermitente lento apresenta a velocidade de secagem superior ao rápido e que a qualidade fisiológica das sementes de tremoço não é afetada pela secagem nos diferentes secadores.<br>Two commercial intermittent dryers, one slow (60oC and 65oC air temperature) and another rapid (50oC), were tested to evaluate the water content decrease (20% to 13%) and their influence on the physiological quality of Lupinus angustifolius L. seeds. The seeds were evaluated through physiological analysis (germination, accelerated ageing and field emergence), stored for 3 and 6 months. After six months the field emergence results were 81%, 89%, 90% and 83% for the slow 60oC, 65oC, rapid (50oC) intermittent dryers and for shade drying, respectively. The drying speed of the rapid (50oC) and slow (65oC - 60oC) intermittent dryers were 1.1, 2.3 and 2.7 percentage points per hour. It was conclude that: a) the slow drier is more efficient in drying than the rapid drier; b) the lupine seed physiological quality is not affected by the tested dryers

Biochemical Characterization Of Uracil Phosphoribosyltransferase From Mycobacterium Tuberculosis

Uracil phosphoribosyltransferase (UPRT) catalyzes the conversion of uracil and 5-phosphoribosyl-α-1-pyrophosphate (PRPP) to uridine 5′-monophosphate (UMP) and pyrophosphate (PPi). UPRT plays an important role in the pyrimidine salvage pathway since UMP is a common precursor of all pyrimidine nucleotides. Here we describe cloning, expression and purification to homogeneity of upp-encoded UPRT from Mycobacterium tuberculosis (MtUPRT). Mass spectrometry and N-terminal amino acid sequencing unambiguously identified the homogeneous protein as MtUPRT. Analytical ultracentrifugation showed that native MtUPRT follows a monomer-tetramer association model. MtUPRT is specific for uracil. GTP is not a modulator of MtUPRT ativity. MtUPRT was not significantly activated or inhibited by ATP, UTP, and CTP. Initial velocity and isothermal titration calorimetry studies suggest that catalysis follows a sequential ordered mechanism, in which PRPP binding is followed by uracil, and PPi product is released first followed by UMP. The pH-rate profiles indicated that groups with pK values of 5.7 and 8.1 are important for catalysis, and a group with a pK value of 9.5 is involved in PRPP binding. The results here described provide a solid foundation on which to base upp gene knockout aiming at the development of strategies to prevent tuberculosis. © 2013 Villela et al.82(2011) Global Tuberculosis Control: WHO report 2011, , World Health OrganizationJain, A., Mondal, R., Extensively drug-resistant tuberculosis: current challenges and threats (2008) FEMS Immunol Med Microbiol, 53, pp. 145-150Velayati, A.A., Farnia, P., Masjedi, M.R., Ibrahim, T.A., Tabarsi, P., Totally drug-resistant tuberculosis strains: evidence of adaptation at the cellular level (2009) Eur Respir J, 34, pp. 1202-1203Ducati, R.G., Ruffino-Netto, A., Basso, L.A., Santos, D.S., The resumption of consumption - a review on tuberculosis (2006) Mem Inst Oswaldo Cruz, 101, pp. 697-714Pieters, J., Mycobacterium tuberculosis and the macrophage: maintaining a balance (2008) Cell Host Microbe, 3, pp. 399-407Stewart, G.R., Robertson, B.D., Young, D.B., Tuberculosis: a problem with persistence (2003) Nat Rev Microbiol, 1, pp. 97-105Cole, S.T., Brosch, R., Parkhill, J., Garnier, T., Churcher, C., Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence (1998) Nature, 393, pp. 537-544Villela, A.D., Sánchez-Quitian, Z.A., Ducati, R.G., Santos, D.S., Basso, L.A., Pyrimidine salvage pathway in Mycobacterium tuberculosis (2011) Curr Med Chem, 18, pp. 1286-1298Ducati, R.G., Breda, A., Basso, L.A., Santos, D.S., Purine Salvage Pathway in Mycobacterium tuberculosis (2011) Curr Med Chem, 18, pp. 1258-1275Ducati, R.G., Souto, A.A., Caceres, R.A., de Azevedo Jr, W.F., Basso, L.A., Purine Nucleoside Phosphorylase as a Molecular Target to Develop Active Compounds Against Mycobacterium tuberculosis (2010) Int Rev Biophys Chem, 1, pp. 34-40Ducati, R.G., Basso, L.A., Santos, D.S., de Azevedo Jr., W.F., Crystallographic and docking studies of purine nucleoside phosphorylase from Mycobacterium tuberculosis (2010) Bioorg Med Chem, 18, pp. 4769-4774Ducati, R.G., Santos, D.S., Basso, L.A., Substrate specificity and kinetic mechanism of purine nucleoside phosphorylase from Mycobacterium tuberculosis (2009) Arch Biochem Biophys, 486, pp. 155-164Ducati, R.G., Basso, L.A., Santos, D.S., Mycobacterial shikimate pathway enzymes as targets for drug design (2007) Curr Drug Targets, 8, pp. 423-435Kim, S., Park, D.H., Kim, T.H., Hwang, M., Shim, J., Functional analysis of pyrimidine biosynthesis enzymes using the anticancer drug 5-fluorouracil in Caenorhabditis elegans (2009) FEBS J, 276, pp. 4715-4726Moffatt, B.A., Ashihara, H., Purine and pyrimidine nucleotide synthesis and metabolism (2002) Arabidopsis Book, 1, pp. e0018Kadziola, A., Neuhard, J., Larsen, S., Structure of product-bound Bacillus caldolyticus uracil phosphoribosyltransferase confirms ordered sequential substrate binding (2002) Acta Crystallogr D Biol Crystallogr, 58, pp. 936-945Kantardjieff, K.A., Vasquez, C., Castro, P., Warfel, N.M., Rho, B.S., Structure of pyrR (Rv1379) from Mycobacterium tuberculosis: a persistence gene and protein drug target (2005) Acta Crystallogr D Biol Crystallogr, 61, pp. 355-364Li, J., Huang, S., Chen, J., Yang, Z., Fei, X., Identification and characterization of human uracil phosphoribosyltransferase (UPRTase) (2007) J Hum Genet, 52, pp. 415-422Renck, D., Ducati, R.G., Palma, M.S., Santos, D.S., Basso, L.A., The kinetic mechanism of human uridine phosphorylase 1: Towards the development of enzyme inhibitors for cancer chemotherapy (2010) Arch Biochem Biophys, 497, pp. 35-42Suzuki, N.N., Koizumi, K., Fukushima, M., Matsuda, A., Inagaki, F., Structural basis for the specificity, catalysis, and regulation of human uridine-cytidine kinase (2004) Structure, 12, pp. 751-764Yablonski, M.J., Pasek, D.A., Han, B.D., Jones, M.E., Traut, T.W., Intrinsic activity and stability of bifunctional human UMP synthase and its two separate catalytic domains, orotate phosphoribosyltransferase and orotidine-5′-phosphate decarboxylase (1996) J Biol Chem, 271, pp. 10704-10708Ichikawa, W., Prediction of clinical outcome of fluoropyrimidine-based chemotherapy for gastric cancer patients, in terms of the 5-fluorouracil metabolic pathway (2006) Gastric Cancer, 9, pp. 145-155Sassetti, C.M., Rubin, E.J., Genetic requirements for mycobacterial survival during infection (2003) Proc Natl Acad Sci U S A, 100, pp. 12989-12994Kelley, K.C., Huestis, K.J., Austen, D.A., Sanderson, C.T., Donoghue, M.A., Regulation of sCD4-183 gene expression from phage-T7-based vectors in Escherichia coli (1995) Gene, 156, pp. 33-36Grossman, T.H., Kawasaki, E.S., Punreddy, S.R., Osburne, M.S., Spontaneous cAMP-dependent derepression of gene expression in stationary phase plays a role in recombinant expression instability (1998) Gene, 209, pp. 95-103Sánchez-Quitian, Z.A., Schneider, C.Z., Ducati, R.G., de Azevedo Jr., W.F., Bloch Jr., C., Structural and functional analyses of Mycobacterium tuberculosis Rv3315c-encoded metal-dependent homotetrameric cytidine deaminase (2010) J Struct Biol, 169, pp. 413-423Rostirolla, D.C., Breda, A., Rosado, L.A., Palma, M.S., Basso, L.A., UMP kinase from Mycobacterium tuberculosis: Mode of action and allosteric interactions, and their likely role in pyrimidine metabolism regulation (2011) Arch Biochem Biophys, 505, pp. 202-212Nunes, J.E., Ducati, R.G., Breda, A., Rosado, L.A., de Souza, B.M., Molecular, kinetic, thermodynamic, and structural analyses of Mycobacterium tuberculosis hisD-encoded metal-dependent dimeric histidinol dehydrogenase (EC 1.1.1.23) (2011) Arch Biochem Biophys, 512, pp. 143-153Martinelli, L.K., Ducati, R.G., Rosado, L.A., Breda, A., Selbach, B.P., Recombinant Escherichia coli GMP reductase: kinetic, catalytic and chemical mechanisms, and thermodynamics of enzyme-ligand binary complex formation (2011) Mol Biosyst, 7, pp. 1289-1305de Mendonça, J.D., Adachi, O., Rosado, L.A., Ducati, R.G., Santos, D.S., Kinetic mechanism determination and analysis of metal requirement of dehydroquinate synthase from Mycobacterium tuberculosis H37Rv: an essential step in the function-based rational design of anti-TB drugs (2011) Mol Biosyst, 7, pp. 119-128Schumacher, M.A., Bashor, C.J., Song, M.H., Otsu, K., Zhu, S., The structural mechanism of GTP stabilized oligomerization and catalytic activation of the Toxoplasma gondii uracil phosphoribosyltransferase (2002) Proc Natl Acad Sci U S A, 99, pp. 78-83Jensen, K.F., Arent, S., Larsen, S., Schack, L., Allosteric properties of the GTP activated and CTP inhibited uracil phosphoribosyltransferase from the thermoacidophilic archaeon Sulfolobus solfataricus (2005) FEBS J, 272, pp. 1440-1453Linde, L., Jensen, K.F., Uracil phosphoribosyltransferase from the extreme thermoacidophilic archaebacterium Sulfolobus shibatae is an allosteric enzyme, activated by GTP and inhibited by CTP (1996) Biochim Biophys Acta, 1296, pp. 16-22Dai, Y.P., Lee, C.S., O'Sullivan, W.J., Properties of uracil phosphoribosyltransferase from Giardia intestinalis (1995) Int J Parasitol, 25, pp. 207-214Jensen, H.K., Mikkelsen, N., Neuhard, J., Recombinant uracil phosphoribosyltransferase from the thermophile Bacillus caldolyticus: expression, purification, and partial characterization (1997) Protein Expr Purif, 10, pp. 356-364Rasmussen, U.B., Mygind, B., Nygaard, P., Purification and some properties of uracil phosphoribosyltransferase from Escherichia coli K12 (1986) Biochim Biophys Acta, 881, pp. 268-275Jensen, K.F., Mygind, B., Different oligomeric states are involved in the allosteric behavior of uracil phosphoribosyltransferase from Escherichia coli (1996) Eur J Biochem, 240, pp. 637-645De Brabandere, H., Forsgard, N., Israelsson, L., Petterson, J., Rydin, E., Screening for organic phosphorus compounds in aquatic sediments by liquid chromatography coupled to ICP-AES and ESI-MS/MS (2008) Anal Chem, 80, pp. 6689-6697Henri, V., Michaelis, L., Menten, M.L., (1913) Biochem Z, 49, pp. 333-369Christoffersen, S., Kadziola, A., Johansson, E., Rasmussen, M., Willemoës, M., Structural and kinetic studies of the allosteric transition in Sulfolobus solfataricus uracil phosphoribosyltransferase: Permanent activation by engineering of the C-terminus (2009) J Mol Biol, 393, pp. 464-477Segel, I.H., (1975) Enzyme kinetics, behavior and analysis of rapid equilibrium and steady-state enzyme systems, , New York: John Wiley and Sons, IncCopeland, R.A., (2005) Evaluation of enzyme inhibitors in drug discovery, a guide for medicinal chemists and pharmacologists, , New Jersey: John Wiley and Sons, IncLadbury, J.E., Doyle, M.L., (2004) Biocalorimetry II, , London: WileyBrown, A., Analysis of cooperativity by isothermal titration calorimetry (2009) Int J Mol Sci, 10, pp. 3457-3477Lundegaard, C., Jensen, K.F., Kinetic mechanism of uracil phosphoribosyltransferase from Escherichia coli and catalytic importance of the conserved proline in the PRPP binding site (1999) Biochemistry, 38, pp. 3327-3334Schumacher, M.A., Carter, D., Scott, D.M., Roos, D.S., Ullman, B., Crystal structures of Toxoplasma gondii uracil phosphoribosyltransferase reveal the atomic basis of pyrimidine discrimination and prodrug binding (1998) EMBO J, 17, pp. 3219-3232Arent, S., Harris, P., Jensen, K.F., Larsen, S., Allosteric regulation and communication between subunits in uracil phosphoribosyltransferase from Sulfolobus solfataricus (2005) Biochemistry, 44, pp. 883-892Laemmli, U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4 (1970) Nature, 227, pp. 680-685Bradford, M.M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding (1976) Anal Biochem, 72, pp. 248-254Brand, G.D., Krause, F.C., Silva, L.P., Leite, J.R., Melo, J.A., Bradykinin-related peptides from Phyllomedusa hypochondrialis (2006) Peptides, 27, pp. 2137-2146Borges, J.C., Ramos, C.H., Analysis of molecular targets of Mycobacterium tuberculosis by analytical ultracentrifugation (2011) Curr Med Chem, 18, pp. 1276-1285Natalini, P., Ruggieri, S., Santarelli, I., Vita, A., Magni, G., Baker's yeast UMP:pyrophosphate phosphoribosyltransferase. Purification, enzymatic and kinetic properties (1979) J Biol Chem, 254, pp. 1558-1563Cook, P.F., Cleland, W.W., (2007) Enzyme kinetics and mechanism, , New York: Garland Science PublishingSambandamurthy, V.K., Jacobs Jr., W.R., Live attenuated mutants of Mycobacterium tuberculosis as candidate vaccines against tuberculosis (2005) Microbes Infect, 7, pp. 955-961Kamath, A.T., Fruth, U., Brennan, M.J., Dobbelaer, R., Hubrechts, P., New live mycobacterial vaccines: the Geneva consensus on essential steps towards clinical development (2005) Vaccine, 23, pp. 3753-376

Functional assay for assessment of agonistic or antagonistic activity of angiotensin AT(2) receptor ligands reveals that EMA401 and PD123319 have agonistic properties

With the discovery of the protective arm of the renin-angiotensin system (RAS), interest has grown in protective RAS-related receptors such as the angiotensin AT(2)-receptor [AT(2)R] as potential new drug targets. While it is known that AT(2)R couple to Gi, it is also apparent that they do not signal via inhibition of adenylyl cyclase/decrease in cAMP, as do many Gi-coupled receptors. Thus, standard commercially-available assays cannot be applied to test for agonistic or antagonistic properties of AT(2)R ligands. This lack of standard assays has hampered the development of new drugs targeting the AT(2)R. Therefore, we aimed at developing a reliable, technically easy assay for the determination of intrinsic activity of AT(2)R ligands, primarily for distinguishing between AT(2)R agonists and antagonists. We found that measurement of NO release by DAF-FM fluorescence in primary human aortic endothelial cells (HAEC) or in AT(2)R-transfected CHO cells is a reliable assay for the characterization of AT(2)R ligands. While testing the assay, we made several novel findings, including: a) C21 is a full agonist at the AT(2)R (with the same efficacy as angiotensin II); b) C21 has no intrinsic activity at the receptor Mas; c) AT(2)R-transfected HEK-293 cells are unresponsive to AT(2)R stimulation; d) EMA401 and PD123319, which are commonly regarded as AT(2)R antagonists, are partial agonists at the AT(2)R. Collectively, we have developed and tested an assay based on the measurement and quantification of NO release in HAEC or in AT(2)R-CHO cells that is suitable for the characterisation of novel and established AT(2)R ligands