Comparison of the esterase and human plasminogen activator activities of various activated forms of human plasminogen and their equimolar streptokinase complexes

A comparison was made of the esterase and activator activities of the various activated forms of human plasminogen and their streptokinase complexes with Nalpha-Cbz-L-lysine-p-nitrophenyl ester as the substrate. The steady state kinetic properties of Glu- and Lys-plasmins, and Glu- and Lys-plasminogen-streptokinase complexes were identical, while the Lys-plasmin-streptokinase complex showed a 2-fold increase in Km with the same kcat and a 3-fold increase in Ki for the competitive inhibitor leupeptin. Lys-plasminogen (zymogen with an active site) was prepared which incorporated 0.7 mol of [3H]idisopropyl phosphorofluoridate and 0.43 mol of p-nitrophenyl-p'-guanidinobenzoate/mol of protein. The Km for Lys-plasminogen was 3-fold higher than that of Lys-plasmin, and its maximum velocity 10-fold lower. The steady state kinetic parameters of a plasmin-derived light (B) chain (CmCys)3, and a derived equimolar light (B) chain-streptokinase complex (CmCys)3, isolated from human plasmin and equimolar plasmin-streptokinase, or plasminogen-streptokinase, complexes, respectively, were determined. When the light (B) chain-streptokinase complex is isolated from its parent complexes, there is a complete retention of the original parent's esterase activities, with respect to Km and kcat, and interaction with the competitive inhibitors benzamidine and leupeptin. The plasmin-derived light (B) chain does not retain its parent esterase activities. This chain has very similar kinetic properties to Lys-plasminogen except that streptokinase, in an equal molar amount, does not impart full esterase activity to the light (B) chain whereas the zymogen can be completely activated by streptokinase. The kcat of the plasmin-derived light (B) chain, and its streptokinase complex can be enhanced by 50 and 30%, respectively, in the presence of 10(-4) M leupeptin, a competitive inhibitor of plasmin, attesting to the increased structural flexibility within the active site of this enzyme species. Urokinase hydrolyzes Nalpha-Cbz-L-lysine p-nitrophenyl ester efficiently with a kcat/Km of one-third that of plasmin. The human plasminogen activator activities of various activated forms of human plasminogen and their equimolar streptokinase complexes were compared in a kinetic assay. The Lys-plasmin-streptokinase complex, and streptokinase were the least active of the activator species and were approximately equal in their activator activities. Glu- and Lys-plasminogen-streptokinase complexes had approximately 1.5 times the activity of streptokinase, whereas the equimolar light (B) chain-streptokinase complexes had approximately 2- to 3-times the activator activity of streptokinase. Since the esterase activity remained unchanged, this indicates a greater degree of specificity in the active site of the equimolar light (B) chain-streptokinase activator complex. Urokinase proved to be a poor activator species...     

Indefinite integration as term rewriting: Integrals containing tangent

We describe the development of a term-rewriting system for indefinite integration; it is also called a rule-based evaluation system. The development is separated into modules, and we describe the module for a wide class of integrands containing the tangent function.     

Electrochemical Sensors for Nondestructive Evaluation of Adhesive Bonds

An in-situ corrosion sensor based on electrochemical impedance spectroscopy (EIS) has been used to detect moisture ingress into aluminum-aluminum and aluminum-composite adhesive bonds. Both wedge tests and tensile button tests (aluminum-aluminum bonds only) were performed. Upon moisture absorption, the impedance spectra change shape with the low-frequency region becoming resistive. The low-frequency impedance decreases by several orders of magnitude, depending on the adhesive and the experimental conditions. For bonds with stable interfaces, such as phosphoric acid anodized (PAA) aluminum, the absorbed moisture causes an initial weakening of the adhesive resulting in reduced strength or small crack propagation. A substantial incubation time prior to substrate hydration and bond degradation allows warning of potential joint deterioration and enables condition-based maintenance. For bonds with smooth interfaces with little or no physical bonding (mechanical interlocking), crack propagation can proceed interfacially with minimal moisture absorption. A comparison of the incubation times for Forest Products Laboratory (FPL, or sulfuric acid-sodium dichromate) etched surfaces, both bonded to epoxy adhesives and freely exposed to water or humidity at different temperatures, shows that hydration occurs with the same activation energy and, hence, the same mechanism, independent of whether or not the surface is covered with adhesive. However, the pre-exponential factor in the rate constant is dependent on the concentration of free moisture at the interface so that the hydration rate varies by several orders of magnitude.     

Effects of phentermine on responding maintained under multiple fixed-ratio schedules of food and cocaine presentation in the rhesus monkey

Drugs that decrease drug-maintained responding at doses that do not decrease other behaviors in animals may be suitable candidates for development as medications to treat drug abuse in humans. The present study examined whether this effect could be obtained with phentermine, a drug that has been reported to decrease cocaine intake in humans. Rhesus monkeys were trained under multiple fixed-ratio 30-response schedules of food and i.v. cocaine delivery. Phentermine was always given as a slow, i.v. infusion. Acute treatment with phentermine (0.3-10 mg/kg) decreased cocaine-maintained responding at doses that did not decrease, or decreased less, food-maintained responding for each of three unit doses of cocaine (10-100 microg/kg/injection). Subacute treatment with phentermine (3 or 5.6 mg/kg, daily) also decreased cocaine-maintained responding more than food-maintained responding. After subacute treatment was terminated, rates of cocaine-maintained responding generally recovered to levels comparable to those seen during untreated control sessions. Phentermine (0.3-3 mg/kg) did not generally increase responding associated with a very low (1 microg/kg/injection) unit dose of cocaine, suggesting that the decrease in cocaine-maintained responding at higher unit doses was not the result of a leftward shift in the cocaine unit dose-effect function. Phentermine (0.1-3 mg/kg) decreased responding maintained by 1-[2-[bis(4-fluorophenyl) methoxy]ethyl]-4-[3-phenylpropyl] piperazine (GBR 12909) (30 microg/kg/injection) at doses similar to those that decreased food-maintained responding. These results show that phentermine is effective in decreasing cocaine self-administration and suggest that it may be an effective medication for cocaine abuse.     

Use of poly(tetrafluoroethylene)s as a sample support for the MALDI-TOF analysis of DNA and proteins

A proline rich antigen (PRA), which protects mice against Coccidioides immitis, has been analyzed for differential expression and variation among isolates. Northern blots of RNA from different stages of growth were probed with previously cloned cDNA and showed that mRNA for PRA increased as spherules transformed and matured from mycelia. This pattern corresponds to the relative potency of whole cell vaccines from similar preparations. The PRA gene was then cloned from a genomic library of the Silveira strain of C. immitis and its sequence analyzed. Eight other coccidioidal isolates, selected for diversity in geographic origin and resulting clinical disease, demonstrated heterogeneity in Southern blots and in sequences of polymerase chain reaction products. Silveira differed from other California isolates at 33 of 555 bases, whereas it differed from non-California isolates by <=2 bases. Only one of these substitutions affected protein sequence. Thus, tests or vaccines based on this gene are likely to cover most isolates.