RESUMO
The interaction of acute lymphoblastic leukemia (ALL) blasts with bone marrow (BM) stromal cells (BMSCs) has a positive impact on ALL resistance to chemotherapy. We investigated the modulation of a series of putative asparaginase-resistance/sensitivity genes in B-precursor ALL cells upon coculture with BMSCs. Coculture with stromal cells resulted in increased insulin-like growth factor (IGF)-binding protein 7 (IGFBP7) expression by ALL cells. Assays with IGFBP7 knockdown ALL and stromal cell lines, or with addition of recombinant rIGFBP7 (rIGFBP7) to the culture medium, showed that IGFBP7 acts as a positive regulator of ALL and stromal cells growth, and significantly enhances in-vitro resistance of ALL to asparaginase. In these assays, IGFBP7 function occurred mainly in an insulin- and stromal-dependent manner. ALL cells were found to contribute substantially to extracellular IGFBP7 levels in the conditioned coculture medium. Diagnostic BM plasma from children with ALL had higher levels of IGFBP7 than controls. IGFBP7, in an insulin/IGF-dependent manner, enhanced asparagine synthetase expression and asparagine secretion by BMSCs, thus providing a stromal-dependent mechanism by which IGFBP7 protects ALL cells against asparaginase in this coculture system. Importantly, higher IGFBP7 mRNA levels were associated with lower leukemia-free survival (Cox regression model, P=0.003) in precursor B-cell Ph(-) ALL patients (n=147) treated with a contemporary polychemotherapy protocol.
Assuntos
Asparaginase/farmacologia , Células da Medula Óssea/patologia , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/fisiologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Células Estromais/patologia , Estudos de Casos e Controles , Criança , Pré-Escolar , Técnicas de Cocultura , Meios de Cultivo Condicionados , Resistencia a Medicamentos Antineoplásicos , Feminino , Citometria de Fluxo , Técnicas de Silenciamento de Genes , Humanos , Lactente , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/genética , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/metabolismo , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , RNA Interferente Pequeno , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
BACKGROUND AND AIMS: Flooding results in hypoxia of the root system to which N2 fixation of nodulated roots can be especially sensitive. Morphological adaptions, such as aerenchyma formation, can facilitate the diffusion of oxygen to the hypoxic tissues. Using soybean, the aim of the study was to characterize the morphological response of the nodulated root system to flooding and obtain evidence for the recovery of N metabolism. METHODS: Sections from submerged tissues were observed by light microscopy, while sap bleeding from the xylem was analysed for nitrogenous components. KEY RESULTS: Flooding resulted in the rapid formation of adventitious roots and aerenchyma between the stem (immediately above the water line), roots and nodules. In the submerged stem, taproot, lateral roots and adventitious roots, lysigenous aerenchyma arose initially in the cortex and was gradually substituted by secondary aerenchyma arising from cells derived from the pericycle. Nodules developed aerenchyma from cells originating in the phellogen but nodules situated at depths greater than 7-8 cm showed little or no aerenchyma formation. As a result of aerenchyma formation, porosity of the taproot increased substantially between the 4th and 7th days of flooding, coinciding with the recovery of certain nitrogenous products of N metabolism of roots and nodules transported in the xylem. Thus, on the first day of flooding there was a sharp decline in xylem ureides and glutamine (products of N2 fixation), together with a sharp rise in alanine (product of anaerobic metabolism). Between days 7 and 10, recovery of ureides and glutamine to near initial levels was recorded while recovery of alanine was partial. CONCLUSIONS: N metabolism of the nodulated soybean root system can recover at least partially during a prolonged period of flooding, a process associated with aerenchyma formation.
Assuntos
Glycine max/anatomia & histologia , Glycine max/metabolismo , Oxigênio/metabolismo , Raízes de Plantas/anatomia & histologia , Raízes de Plantas/metabolismo , Fixação de NitrogênioRESUMO
The PLA2 and crotapotin subunits of crotoxin from Crotalus durissus cascavella venom were purified by a combination of high-performance liquid chromatography (HPLC) molecular exclusion (Protein Pack 300SW column) and reverse-phase HPLC (RP-HPLC). Tricine SDS-PAGE showed that the PLA2 and crotapotins migrated as single bands with estimated molecular masses of 15 and 9 kDa, respectively. The amino acid composition of the PLA2 showed the presence of 14 half-cysteines and a high content of basic residues (Lys, Arg, His), whereas the crotapotins were rich in hydrophobic, negatively charged residues and half-cysteines. The PLA2 showed allosteric behavior, with maximal activity at pH 8.3 and 35-40 degrees C. C. d. cascavella PLA2 required Ca2+ for activity but was inhibited by Cu2+ and Zn2+ and by Cu2+ and Mg2+ in the presence and absence of Ca2+, respectively. Crotapotin (F3) and heparin inhibited the catalytic activity of the PLA2 by acting as allosteric inhibitors.
Assuntos
Venenos de Crotalídeos/enzimologia , Fosfolipases A/metabolismo , Aminoácidos/análise , Animais , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Crotalus , Crotoxina/farmacologia , Eletroforese em Gel de Poliacrilamida , Inibidores Enzimáticos/farmacologia , Fosfolipases A/antagonistas & inibidores , Fosfolipases A/química , Fosfolipases A2RESUMO
The PLA2 and crotapotin subunits of crotoxin from Crotalus durissus cascavella venom were purified by a combination of HPLC molecular exclusion (Protein Pack 300SW column) and reverse-phase HPLC (RP-HPLC). Tricine SDS-PAGE showed that the PLA2 and crotapotins migrated as single bands with estimated molecular masses of 15 and 9 kDa, respectively. The amino acid composition of the PLA2 showed the presence of 14 half-cysteines and a high content of basic residues (Lys, Arg, His), whereas the crotapotins were rich in hydrophobic, negatively charged residues and half-cysteines. The PLA2 showed allosteric behavior, with maximal activity at pH 8.3 and 35-40 degrees C. The C. d. cascavella PLA2 required Ca2+ for activity, but was inhibited by Cu2+ and Zn2+ and by Cu2+ and Mg2+ in the presence and absence of Ca2+, respectively. Crotapotin (F3) and heparin inhibited the catalytic activity of the PLA2 by acting as allosteric inhibitors.
Assuntos
Venenos de Crotalídeos/enzimologia , Fosfolipases A/química , Regulação Alostérica/efeitos dos fármacos , Aminoácidos/análise , Animais , Cromatografia Líquida de Alta Pressão , Crotoxina/química , Crotoxina/isolamento & purificação , Crotoxina/farmacologia , Eletroforese em Gel de Poliacrilamida , Heparina/farmacologia , Concentração de Íons de Hidrogênio , Metais Pesados/farmacologia , Peso Molecular , Fosfolipases A/antagonistas & inibidores , Fosfolipases A/metabolismo , Fosfolipases A2 , Subunidades Proteicas , TemperaturaRESUMO
In plant, the catabolism of lysine has only been studied in some detail in maize. The enzymes lysine 2-oxoglutarate reductase (also known as lysine alpha-ketoglutarate reductase; LOR) and saccharopine dehydrogenase (SDH), which convert lysine into saccharopine, and saccharopine into glutamic acid and 2-aminoadipate 6-semialdehyde, respectively, were isolated from immature rice seeds and partially purified through a three-step purification procedure involving ammonium sulphate precipitation, and anion-exchange and gel-filtration chromatographies, leading to a final yield of 30% for LOR and 24% for SDH. The molecular masses estimated by gel-filtration chromatography on a Sephacryl S200 column and by native non-denaturing PAGE using Ferguson plots were 203 kDa for both enzymes by gel-filtration and 202 kDa for both enzymes by native non-denaturing PAGE. A second band of LOR and SDH activities on native gels was observed for both enzymes with an estimated molecular mass of 396 kDa, which indicated a multimeric structure. Kinetic studies were consistent with an ordered sequence mechanism for LOR, where 2-oxoglutarate is the first substrate and saccharopine is the last product. The results observed for the LOR/SDH activity ratios during purification, the copurification in all three steps, the molecular masses, the relative mobilities on native non-denaturing gels and the pI estimated for LOR and SDH suggest the existence of a bifunctional polypeptide containing LOR and SDH activities.
Assuntos
Lisina/metabolismo , Oryza/metabolismo , Sacaropina Desidrogenases/isolamento & purificação , Cinética , Peso Molecular , Sacaropina Desidrogenases/metabolismo , Sementes/metabolismoRESUMO
Lysine-ketoglutarate reductase activity was detected and characterized in the developing endosperm of maize (Zea mays L.). The enzyme showed specificity for its substrates: lysine, alpha-ketoglutarate, and NADPH. Formation of the reaction product saccharopine was demonstrated. The pH optimum of the enzyme was close to 7, and the K(m) for lysine and alpha-ketoglutarate were 5.2 and 1.8 millimolar, respectively.
RESUMO
Asparaginase (EC 3.5.1.1) was isolated from the developing seed of Pisum sativum. The enzyme is dependent upon the presence of K(+) for activity, although Na(+) and Rb(+) may substitute to a lesser extent. Maximum activity was obtained at K(+) concentrations above 20 millimolar. Potassium ions protected the enzyme against heat denaturation. The enzyme has a molecular weight of 68,300.Asparaginase activity developed initially in the testa, with maximum activity (3.6 micromoles per hour per seed) being present 13 days after flowering. Maximum activity (1.2 micromoles per hour per seed) did not develop in the cotyledon until 21 days after flowering. Glutamine synthetase and glutamate dehydrogenase were also present in the testae and cotyledons but maximum activity developed later than that of asparaginase.Potassium-dependent asparaginase activity was also detected in the developing seeds of Vicia faba, Phaseolus multiflorus, Zea mays, Hordeum vulgare, and two Lupinus varieties. No stimulation of activity was detected with the enzyme isolated from Lupinus polyphyllus, which has previously been shown to contain a K(+)-independent enzyme.