The gld genes encoding adenosylcobalamin-dependent glycerol dehydrase of Klebsiella pneumoniae were cloned by cross-hybridization with a DNA fragment of Klebsiella oxytoca diol dehydrase genes. Since the Escherichia coli clones isolated did not show appreciable enzyme activity, plasmids for high level expression of cloned genes were constructed. The enzyme expressed in E. coli was indistinguishable from the wild-type glycerol dehydrase of K. pneumoniae by the criteria of polyacrylamide gel electrophoretic, immunochemical, and catalytic properties. It was also shown that the recombinant functional enzyme consists of Mr 61,000, 22,000, and 16, 000 subunits. Sequence analysis of the genes revealed four open reading frames separated by 2-12 bases. The sequential three open reading frames from the first to the third (gldA, gldB, and gldC genes) encoded polypeptides of 555, 194, and 141 amino acid residues with predicted molecular weights of 60,659(alpha), 21,355(beta), and 16,104(gamma), respectively. High level expression of these three genes in E. coli produced more than 14-fold higher level of fully active apoenzyme than that in K. pneumoniae. It was thus concluded that these are the genes encoding the subunits of glycerol dehydrase. The deduced amino acid sequences of the three subunits were 71, 58, and 54% identical with those of the alpha, beta, and gamma subunits of diol dehydrase, respectively, but failed to show any apparent homology with other proteins.
A 79-year-old man with a cardiac pacemaker for bradycardia fell down and presented with sudden onset of right hemiplegia and aphasia. Initial computed tomography (CT) showed no cerebral infarction but angiography revealed occlusion of the left middle cerebral artery (MCA). Local intra-arterial thrombolysis with tissue plasminogen activator (tPA; tisokinase, 1,600,000 units) was performed 3 hours after the onset, and the MCA was partially recanalized. Further administration of tPA was suspended because of nosebleed. However, the patient's neurological findings did not improve. His consciousness gradually deteriorated to coma and quadriplegia with dilation of the left pupil 2.5 hours after thrombolysis. CT disclosed marked mass effect with a left acute subdural hematoma and a small intracerebral hematoma in the left frontal lobe. He underwent urgent craniotomy and removal of the subdural hematoma. The subdural hematoma originated in a frontal cerebral contusion. He died of severe brain edema 2 days after surgery. Acute subdural hematoma is a very rare complication of intra-arterial thrombolysis. Presumably he had suffered head trauma at the first onset. Evidence of head trauma should be considered a contraindication for the use of thrombolytic agents in a patient with acute stroke.
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