ABSTRACT
A new PLA2 Bj-V from Bothrops jararacussu (14039.49 Da determined by MALDI-TOF mass spectrometry) was isolated in only one chromatographic step by HPLC ion-exchange and its purity was confirmed by reverse phase. Amino acid analysis showed a high content of hydrophobic and basic amino acids as well as 14 half-cysteine residues. The N-terminal sequence (DLWQFGQMIL KETGKIPFPY YGAYGCYCGW GGRGGKPKDG TDRCCYVHD...) showed a high degree of homology with basic D49 PLA2 myotoxins from other Bothrops venoms. Bj V showed discrete sigmoidal enzymatic behavior, with maximal activity at pH 8.4 and 35-40 degrees C. Full PLA2 activity required Ca2+ (10 mM) and there was little catalytic activity in the presence of 1 mM Ca2+. The addition of Mn2+ or Mg2+ (10 mM) in the presence of low (1 mM) Ca2+ slightly increased the enzyme activity, whereas Zn2+ and Cu2+ (10 mM) diminished the activity. The substitution of Ca2+ for Mg2+ or Cu2+ also reduced the enzymatic activity. Bj V had PLA2 activity and produced cytotoxicity in murine C2C12 skeletal muscle myoblasts and myotubes. The isolation of these isoforms Bj-IV [1] and Bj-V (described herein) found in a fraction previously described as homogeneous shows us the importance of optimization in purification techniques in order to better understand their biological behavior.
Subject(s)
Bothrops/physiology , Crotalid Venoms/enzymology , Muscle Fibers, Skeletal/metabolism , Myoblasts/metabolism , Phospholipases A/physiology , Amino Acid Sequence , Animals , Cell Line , Isoenzymes/genetics , Mice , Molecular Sequence Data , Phospholipases A/genetics , Phospholipases A2ABSTRACT
Os autores estudaram retrospectivamente 72 casos de mielomeningocele tratados em um hospital pediátrico do Rio de Janeiro,65(90,2 por cento) lesöes localizavam-se nos segmentos inferiores da coluna vertebral, e 87,5 por cento dos pacientes necessitaram de derivaçöes ventriculares para controle da hidrocefalia. Infecçöes de derivaçöes ventriculares foram três vezes mais frequentes em portadores de spina bifida cística que no restante da populaçäo de hidrocéfalos tratados na instituiçäo. Houve grande incidência de complicaçöes pós-operatórias relacionadas a infecçöes e necrose de ferida cirúrgica. Onze pacientes(15,3 por cento)desenvolveram sinais e sintomas relacionados à malformaçäo de Chiari do Tipo II. A mortalidade foi de 8,3 por cento e, na maioria absoluta dos casos, associada à mal-formaçäo de Chiari.