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1.
Electron. j. biotechnol ; 30: 1-5, nov. 2017. ilus, tab, graf
Article in English | LILACS | ID: biblio-1021034

ABSTRACT

Background: The enzymes utilized in the process of beer production are generally sensitive to higher temperatures. About 60% of them are deactivated in drying the malt that limits the utilization of starting material in the fermentation process. Gene transfer from thermophilic bacteria is a promising tool for producing barley grains harboring thermotolerant enzymes. Results: Gene for α-amylase from hydrothermal Thermococcus, optimally active at 75­85°C and pH between 5.0 and 5.5, was adapted in silico to barley codon usage. The corresponding sequence was put under control of the endosperm-specific promoter 1Dx5 and after synthesis and cloning transferred into barley by biolistics. In addition to model cultivar Golden Promise we transformed three Slovak barley cultivars Pribina, Levan and Nitran, and transgenic plants were obtained. Expression of the ~50 kDa active recombinant enzyme in grains of cvs. Pribina and Nitran resulted in retaining up to 9.39% of enzyme activity upon heating to 75°C, which is more than 4 times higher compared to non-transgenic controls. In the model cv. Golden Promise the grain α-amylase activity upon heating was above 9% either, however, the effects of the introduced enzyme were less pronounced (only 1.22 fold difference compared with non-transgenic barley). Conclusions: Expression of the synthetic gene in barley enhanced the residual α-amylase activity in grains at high temperatures.


Subject(s)
Seeds/enzymology , Hordeum/enzymology , Thermococcus/metabolism , alpha-Amylases/metabolism , Seeds/genetics , Seeds/microbiology , Transformation, Genetic , Hordeum/genetics , Hordeum/microbiology , Beer , Enzyme Stability , Plants, Genetically Modified/enzymology , Cloning, Molecular , Gene Transfer Techniques , alpha-Amylases/genetics , Fermentation , Thermotolerance , Hot Temperature , Hydrogen-Ion Concentration
2.
Rev. salud pública ; 16(3): 361-370, 2012. ilus, tab
Article in Portuguese | LILACS | ID: lil-729647

ABSTRACT

Objetivo Visando conhecer o impacto das demandas judiciais sobre a organização dos serviços públicos de saúde, realizou-se uma revisáo sistemática com enfoque na "judicialização da saúde" para fornecimento de medicamentos. Métodos Foram analisados artigos originais publicados no período de 2007 a 2011, na literatura nacional e internacional, resultando no total de 49239 artigos disponíveis nas bases de dados Science Direct e BIREME. Resultados A pesquisa indicou predominância da bibliografia proveniente do Brasil, principalmente do sudeste, bem como de estudo realizado na Colômbia. Discursáo Dentre os pleitos, configuraram-se como principais agravos relatados as doenças crônicas, podendo-se citar: diabetes, hipertensáo, cânceres e artrite reumatóide. Por serem afecções parte de programas específicos do Sistema Único de Saúde, a dificuldade de acesso a esses fármacos e consequente judicialização da saúde demonstrou a fragilidade das políticas públicas existentes. Conclusão Por fim, conclui-se que a via judicial, apesar de ser uma estratégia para garantir o acesso ao medicamento, apresenta inabilidade para lidar com o julgamento das ações e gera, dessa forma, distorções no fluxo dos sistemas públicos.


Objective A systematic review, focusing on the judicialisation of health regarding gaining access to medicines, was aimed at understanding the impact of lawsuits on the organisation of public health services. Method Original articles published between 2007 and 2011 in the pertinent national and international literature were analysed, resulting in 49,239 articles being found in Science Direct and BIREME databases. Results The survey indicated a predominance of literature from Brazil, mainly the southeast, as well as a study from Colombia. Discussion The aforementioned chronic disease-related claims involved diabetes, high blood pressure, cancer and rheumatoid arthritis. Forming part of specific Unified Healthcare System programmes highlighted the difficulty in gaining access to the appropriate medicine and consequent health judicialisation demonstrated the fragility of existing public policy. Conclusion It was concluded that the courts (despite being a strategy for ensuring access to medicine) were unable to deal with the current spate of lawsuits, thereby leading to disruption regarding the flow of public systems.


Objetivo El estudio tiene como objetivo evaluar el impacto de las demandas judiciales sobre la organización de los servicios públicos de salud, mediante la realización de una revisión sistemática centrada en el uso de los tribunales para el suministro de medicamentos. Método Fueron identificados 49239 artículos en las bases de datos Science Direct e BIREME. Resultado El estudio indicó que la mayor parte de la bibliografía es de Brasil, con uno estudio en Colombia. Discusión Aparecen como los principales trastornos de salud relatados a las enfermedades crónicas, se pueden citar: la diabetes, la hipertensión, el cáncer y la artritis reumatoide. Debido a que son parte de los programas específicos de lo sistema de salud, la dificultad de acceso a estos fármacos y la consiguiente judicialización de la salud de manifiesto la fragilidad de las políticas públicas existentes. Conclusiones Por último, está la conclusión de que los tribunales, a pesar de ser una estrategia para garantizar el acceso a la medicina, presenta incapacidad para hacer frente al juicio de las acciones y por lo tanto genera distorsiones en el flujo de los sistemas públicos.


Subject(s)
Aspartic Acid Endopeptidases/genetics , Cacao/enzymology , Leucine/analogs & derivatives , Seeds/enzymology , Amino Acid Sequence , Aspartic Acid Endopeptidases/drug effects , Aspartic Acid Endopeptidases/metabolism , Cloning, Molecular , Cacao/genetics , Coumarins/pharmacology , DNA, Complementary/chemistry , DNA, Complementary/genetics , DNA, Complementary/isolation & purification , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Plant , Hydrogen-Ion Concentration , Isoenzymes/drug effects , Isoenzymes/genetics , Isoenzymes/metabolism , Leucine/pharmacology , Molecular Sequence Data , Phylogeny , Pepstatins/pharmacology , Plant Proteins/genetics , Plant Proteins/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Seeds/genetics , Yarrowia/genetics , Yarrowia/metabolism
3.
Indian J Biochem Biophys ; 2009 Oct; 46(5): 366-370
Article in English | IMSEAR | ID: sea-135218

ABSTRACT

-Galactosidase (-D-galactoside galactohydrolase, EC 3.2.1.22) was purified (26-fold) from the germinating seeds of lentil (Lens culinaris) by affinity precipitation with alginate. The purified enzyme gave a single band corresponding to molecular mass of 40 kDa on SDS-PAGE. The optimum temperature and pH of the enzyme were determined as 40oC and 5.5, respectively. The enzyme was very stable at a temperature range of 4-65oC and at a pH range of 4-7. The values of kinetic constants Km and Vmax using p-nitrophenyl--D-galactopyranoside (PNPG) as substrate were 0.191 mM and 0.73 U, respectively. Results suggest that affinity precipitation is an attractive process for the purification of -galactosidase.


Subject(s)
Alginates/chemistry , Chemical Precipitation , Germination , Glucuronic Acid/chemistry , Hexuronic Acids/chemistry , Hydrogen-Ion Concentration , Lens Plant/enzymology , Lens Plant/growth & development , Seeds/enzymology , Seeds/growth & development , Temperature , Time Factors , alpha-Galactosidase/chemistry , alpha-Galactosidase/isolation & purification , alpha-Galactosidase/metabolism
4.
Indian J Exp Biol ; 2003 Apr; 41(4): 373-5
Article in English | IMSEAR | ID: sea-57634

ABSTRACT

Isoproturon resistant biotype of P. minor germinates early, shows higher germination percentage and faster rate of growth as compared to the susceptible biotype. Higher amylase activity is observed in the initial hours of imbibition in the resistant biotype. In the susceptible biotype it is activated at a much later stage.


Subject(s)
Amylases/biosynthesis , Drug Resistance , Germination/drug effects , Herbicides/pharmacology , Methylurea Compounds/pharmacology , Phalaris/drug effects , Phenylurea Compounds , Seeds/enzymology
5.
Indian J Biochem Biophys ; 2001 Oct; 38(5): 335-41
Article in English | IMSEAR | ID: sea-27071

ABSTRACT

NADP+-linked isocitrate dehydrogenase (E.C.1.1.1.42) has been purified to homogeneity from germinating pea seeds. The enzyme is a tetrameric protein (mol wt, about 146,000) made up of apparently identical monomers (subunit mol wt, about 36,000). Thermal inactivation of purified enzyme at 45 degrees and 50 degrees C shows simple first order kinetics. The enzyme shows optimum activity at pH range 7.5-8. Effect of substrate [S] on enzyme activity at different pH (6.5-8) suggests that the proton behaves formally as an "uncompetitive inhibitor". A basic group of the enzyme (site) is protonated in this pH range in the presence of substrate only, with a pKa equal to 6.78. On successive dialysis against EDTA and phosphate buffer, pH 7.8 at 0 degrees C, yields an enzymatically inactive protein showing kinetics of thermal inactivation identical to the untreated (native) enzyme. Maximum enzyme activity is observed in presence of Mn2+ and Mg2+ ions (3.75 mM). Addition of Zn2+, Cd2+, Co2+ and Ca2+ ions brings about partial recovery. Other metal ions Fe2+, Cu2+ and Ni2+ are ineffective.


Subject(s)
Binding Sites , Chromatography, DEAE-Cellulose , Chromatography, Gel , Electrophoresis, Polyacrylamide Gel , Germination , Hot Temperature , Hydrogen-Ion Concentration , Isocitrate Dehydrogenase/chemistry , Metals/metabolism , Molecular Weight , Peas/enzymology , Seeds/enzymology
6.
Braz. j. med. biol. res ; 32(12): 1489-92, Dec. 1999. tab
Article in English | LILACS | ID: lil-249373

ABSTRACT

The aminopeptidase activity of Phaseolus vulgaris seeds was measured using L-Leu-p-nitroanilide and the L-aminoacyl-ß-naphthylamides of Leu, Ala, Arg and Met. A single peak of aminopeptidase activity on Leu-ß-naphthylamide was eluted at 750 µS after gradient elution chromatography on DEAE-cellulose of the supernatant of a crude seed extract. The effluent containing enzyme activity was applied to a Superdex 200 column and only one peak of aminopeptidase activity was obtained. SDS-polyacrylamide gel electrophoresis (10 per cent) presented only one protein band with molecular mass of 31 kDa under reducing and nonreducing conditions. The aminopeptidase has an optimum pH of 7.0 for activity on all substrates tested and the highest Vmax/KM ratio for L-Leu-ß-naphthylamide. The enzyme activity was increased 40 per cent by 0.15 M NaCl, inhibited 94 per cent by 2.0 mM Zn2+, inhibited 91 per cent by sodium p-hydroxymercuribenzoate and inhibited 45 per cent by 0.7 mM o-phenanthroline and 30 µM EDTA. Mercaptoethanol (3.3 mM), dithioerythritol (1.7 mM), Ala, Arg, Leu and Met (70 µM), p-nitroaniline (0.25 mM) and ß-naphthylamine (0.53 mM) had no effect on enzyme activity when assayed with 0.56 mM of substrate. Bestatin (20 µM) inhibited 18 per cent the enzyme activity. The aminopeptidase activity in the seeds decayed 50 per cent after two months when stored at 4oC and room temperature. The enzyme is leucyl aminopeptidase metal- and thiol group-dependent.


Subject(s)
Aminopeptidases/isolation & purification , Fabaceae/enzymology , Seeds/enzymology , Aminopeptidases/metabolism
7.
Indian J Biochem Biophys ; 1997 Jun; 34(3): 288-95
Article in English | IMSEAR | ID: sea-27091

ABSTRACT

Distribution of the enzymes of glycolytic and pentose phosphate pathways were studied in cytosolic and leucoplastic fractions of the developing seeds of Brassica. Leucoplasts were isolated using a discontinuous percoll gradient. Intactness of leucoplasts was checked by ADP-glucose pyrophosphorylase assay in presence and absence of triton X-100. No contamination by microbodies, mitochondria and cytosol was observed as assessed by measuring the activities of marker enzymes. The recovery, latency and specific activity of each enzyme in different fractions were compared. The leucoplastic fraction contained complete set of the enzymes of glycolytic and pentose phosphate pathways, indicating that the two subcellular compartments metabolize carbon independently by these pathways. However, the enzymes showed higher activities in cytosolic fraction as compared to those in the leucoplasts, suggesting the need for exchange of metabolites in the two compartments through various translocators, for acting in cooperation to produce energy, reducing power and carbon skeletons for different biosynthetic activities in the non-photosynthetic plastids. Based on these compartmentation studies, a model for carbon flow for fatty acid synthesis in leucoplasts of developing Brassica seeds has been proposed.


Subject(s)
Acetates/metabolism , Brassica/enzymology , Energy Metabolism , Fatty Acids/biosynthesis , Glucose/metabolism , Glucose-1-Phosphate Adenylyltransferase , Glycolysis/physiology , Models, Biological , Nucleotidyltransferases/metabolism , Pentose Phosphate Pathway/physiology , Seeds/enzymology
8.
Indian J Biochem Biophys ; 1995 Aug; 32(4): 213-7
Article in English | IMSEAR | ID: sea-26442

ABSTRACT

A 100,000 x g supernatant fraction prepared from developing groundnut seeds (30-35 days after flowering) catalyzed the synthesis of fatty acids from [l-14C]acetate at a rate of 120nmoles of acetate incorporated per hr per gram fresh weight of tissue. 90% of this incorporated label was associated with fatty acids. The major fatty acids formed were stearic- (77%) and palmitic acids (14%) with 4% of oleic acid. The fatty acid synthetase activity was stable when stored at 0-4 degrees C for at least fifteen days. It is concluded from these results that acetyl-coA carboxylase and all the enzymes of fatty acid synthetase from developing groundnut seeds are soluble.


Subject(s)
Arachis/enzymology , Catalysis , Fatty Acids/chemical synthesis , Seeds/enzymology , Solubility
9.
Braz. j. med. biol. res ; 25(11): 1103-6, 1992. tab, graf
Article in English | LILACS | ID: lil-134605

ABSTRACT

The effect of 2-naphthylamine, p-nitroaniline, o-phenanthroline, sodium deoxycholate and hydrocortisone succinate on the activity of human urine aminopeptidase, rat kidney methionyl and arginyl aminopeptidase, soybean and Enterolobium contortisiliquum seed aminopeptidase was studied using aminoacyl-2-naphthylamide and L-Leu-p-nitroanilide as substrates. Ki values ranged from 10 microM to 2.7 mM. On the basis of Ki and Km values, and catalytic efficiency for each enzyme, it is clear that the aminopeptidases from human urine and from soybean seed should be assayed with both substrates, whereas L-Leu-p-nitroaniline is a more appropriate substrate for the rat kidney aminopeptidases. Sodium deoxycholate is a better inhibitor than hydrocortisone succinate. Non-competitive inhibition was observed in all cases except for E. contortisiliquum seed aminopeptidase


Subject(s)
Animals , Humans , Aminopeptidases/antagonists & inhibitors , Hydrocarbons, Cyclic/pharmacology , Aminopeptidases/drug effects , Aminopeptidases/urine , Dose-Response Relationship, Drug , Kidney/enzymology , Rats , Seeds/enzymology , Soybeans/enzymology , Substrate Specificity/drug effects , Trees/enzymology
10.
Braz. j. med. biol. res ; 24(4): 337-44, 1991. tab
Article in English | LILACS | ID: lil-99462

ABSTRACT

Arylamidase activity was isolated from Enterolobium contortisiliquum seeds (2 U/g) using L-Leu-2-naphthlamide as substrate to monitor the prification. The enzyme preparation was purified 733-fold by ammonium sulfate precipitation, and by ion eschange and gel filtration chromatography, in 6,6% yield. SDS-Polyacrylamide gel electrophoresis after fast protein liquid chromatography on a Mono Q column, showed only one protein band with a molecular weight of 35 kDa. The optimum pH for arylamidase activity was 6.5. Taking the hydrolysis rate of Lys-2-naphthylamide as one, the relative rates at which the other substrates were hydrolyzed were: Leu-2-naphthlamide, 30, Met-2-naphthlamide, 18, Arg-2-naphthlamide, 2, Ala-2-naphthylamide, 1.5, and L-Leu-p-nitroanilide, 26. The arylamidase activity was inhibited 50% by 0.1 mM HgCl2, 0.1 mM ZnCl2, 0.13 mM NiCl2, 0.2 mM o-phenanthroline and 1 * M soidum p-hydroxymercuribenzoate, and activated 35% by 5.0 * M EDTA. Iodoacetate (0.067 mM), dithioerythritol and 2-mercaptoethanol (3.3 mM), and chloride ions (0.2 M) had no effect on the enzyme activity


Subject(s)
Aminopeptidases/metabolism , Plant Proteins/isolation & purification , Seeds , Aminopeptidases/antagonists & inhibitors , Aminopeptidases/drug effects , Aminopeptidases/isolation & purification , Chromatography, Gel , Chromatography, Ion Exchange , Electrophoresis, Polyacrylamide Gel , Hydrolysis , Molecular Weight , Plant Proteins/metabolism , Seeds/enzymology
11.
Indian J Biochem Biophys ; 1989 Dec; 26(6): 386-9
Article in English | IMSEAR | ID: sea-29020

ABSTRACT

Inactivation of isocitrate lyase (native and EDTA-dialysed) by excess tetranitromethane (TNM) exhibits, biphasic kinetics, in which half of the initial activity is lost in a fast and the remaining half in a slow phase each following the pseudo-first order kinetics. Rate constants of the two phases are proportional to the TNM concentration. High succinate concentration protects the enzyme against TNM inactivation only in the slow phase without any effect on the fast phase. With the EDTA-dialysed enzyme, no such protection (against inactivation by TNM) is observed in the presence of succinate or Mg2+ ions. Addition of both these ligands together brings about protection against the slow phase (as with the native enzyme). It has been proposed that the site-site heterogeneity of isocitrate lyase is a consequence of its quaternary structure constraints.


Subject(s)
Castor Bean/enzymology , Isocitrate Lyase/antagonists & inhibitors , Kinetics , Methane/analogs & derivatives , Oxo-Acid-Lyases/antagonists & inhibitors , Plants, Toxic , Ricinus/enzymology , Seeds/enzymology , Tetranitromethane/pharmacology
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