Mechanism of enhanced kinin release from high molecular weight kininogen by plasma kallikrein after its exposure to plasmin.

When purified high molecular weight kininogen was incubated with streptokinase-activated plasmin and kallikrein, a larger amount of kinin was released than would have been predicted from the effect of either enzyme alone. To determine the mechanism of this enhancement, high molecular weight kininogen was digested sequentially with these enzymes, and the rates of kinin release and sites of cleavage were determined. Conversion of 133 kd native high molecular weight kininogen to two-chain 112 kd or 102 kd derivatives by plasmin more than doubled the rate of kinin release by kallikrein. Conversely, digestion of high molecular weight kininogen by kallikrein and then plasmin did not enhance the rate of kinin release. The kallikrein cleavage points that provided 112 kd and 102 kd two-chain high molecular weight kininogen were after residues 437 (Arg-Lys) and 389 (Arg-Ser), whereas those for plasmin were after 438 (Lys-His) and 389 (Arg-Ser). epsilon-Aminocaproic acid, which competes for lysine residues that are critical to the binding of plasminogen or plasmin to substrates, inhibited the digestion of high molecular weight kininogen by plasmin, which is consistent with the evidence that the 438-439 Lys-His was a primary site of plasmin attack on high molecular weight kininogen. Furthermore, this cleavage was observed when plasminogen activation was induced in normal and in prekallikrein or Hageman factor-deficient plasmas. We suggest that the generation of fibrinolytic activity in blood could result in enhanced kinin release by kallikrein in regions of inflammation as a result of the collaborative actions of plasmin and kallikrein on high molecular weight kininogen.     

Validation of a recording spectrophotometric method for measurement of membrane-associated Mg- and NaK-ATPase activity.

Conventional methods for determination of membrane-associated Mg- and NaK-ATPase activity typically involve a timed incubation of enzyme with ATP followed by measurement of released Pi. They are time-consuming, require a large amount of enzyme protein, and show considerable variability induplicate samples. These problems can be largely eliminated with a coupled enzyme assay in which formation of ADP by ATPase is coupled to NADH oxidation with the intermediate enzymes PK and LDH and the intermediate substrate phosphoenolpyruvate present in excess. NADH oxidation is continuously recorded at 340 nm, and NaK-ATPase represents the ouabain suppressible fraction of total ATPase. This method allows accurate determinatin of initial reaction rates which vary linearly with respect to enzyme protein. Furthermore, it eliminates the need to measure Pi and prevents accumulation of inhibitory ADP. In over 100 preparations of LPM prepared from rats pretreated with agents known to alter ATPase activity or exposed in vitro to ATPase inhibitors. ATPase activity by the coupled-enzyme assay paralleled results obtained with the conventional method. Moreover, the coupled-enzyme assay required less membrane protein, showed less variability in duplicate samples, required half the time, and also yielded accurate values in brain, kidney, and heart tissue. This improved assay should find broad application in the study of membrane ATPase as they relate to a variety of cellular functions.     

Gram-negative folliculitis. Follow-up observations in 20 patients

Gram-negative folliculitis cleared with antibiotics effective against lactose fermenting-gram-negative rods and Proteus organisms in 19 of 20 patients with acne vulgaris. The systemic antibiotic therapy was discontinued after varying periods and the infection remained clear without further treatment for four to 48 months. Nasal cultures were negative for gram-negative organisms on follow-up repeated culture in 12 patients free of infection.     

Injury of myelin sheaths in isolated rabbit vagus nerves by alpha-toxin of Staphylococcus aureus.

S. Szmigielski, M. Blankenship, J. P. Robinson and S. Harshman. Injury of myelin sheaths in isolated rabbit vagus nerves by α-toxin of Staphylococcus aureus. Toxicon17, 363–371, 1979.—Dissected rabbit vagus nerves were incubated in vitro with 0·24-4 μg of purified staphylococcal α-toxin (17,000 H.U./mg protein) per ml. The release of ⁸⁶Rb from prelabeled nerves and the electrical activity of nerves (action potential) were measured and correlated with morphological changes observed by electron microscopy. At a concentration of α-toxin of 2 μg/ml, 30% of incorporated ⁸⁶Rb was released from the nerves after 30 min of incubation. The electrical activity was maintained over this time period and required an additional 30 min before failing completely. Concentrations of α-toxin below 2 μg/ml had no effect on either the release of ⁸⁶Rb or the electrical activity even after 60 min of incubation of the nerves. In contrast, dramatic changes in the morphology of the myelin sheaths are readily demonstrated after 30 min of incubation and can easily be observed at 0·2 μg/ml, a level of α-toxin which has no effect on ⁸⁶Rb release or electrical activity. Both non-myelinated nerve fibers and Schwann cell membranes appear to be more resistant to the action of α-toxin. We conclude that, of the parameters measured, disorganization of myelin sheaths is the first recognizable symptom of injury to peripheral nerves by staphylococcal α-toxin.     

Panrefinal Photocoagulation Influence on Vitrectomy Results for Complications of Diabetic Retinopathy

Despite panretinal photocoagulation (PRP), some diabetic eyes develop complications that are correctable with vitrectomy. The results of vitrectomy performed on 80 eyes having previtrectomy PRP are compared with 402 eyes without photocoagulation. The preoperative findings and operative procedures were almost identical, except the PRP cases had a slightly higher incidence of preoperative iris rubeosis and traction macular detachments, and more surgical membrane peeling. Six months after vitrectomy, the PRP eyes had slightly better visual acuities and fewer detached maculas, but were otherwise almost identical to the non-photocoagulated eyes. There was no evidence that pre-vitrectomy PRP prevents postoperative iris rubeosis.