Monoclonal antibodies against Pseudomonas aeruginosa elastase: a neutralizing antibody which recognizes a conformational epitope related to an active site of elastase.

We have established seven murine hybridoma cell lines which produce monoclonal antibodies (mAbs) against Pseudomonas aeruginosa elastase. The seven mAbs recognized at least six different epitopes on the elastase molecule. All mAbs inhibited both enzymatic activities of elastase and protease, in which elastin fluorescein and hide powder azure were used as substrates, respectively. One of them, mAb E-4D3, strongly neutralized enzymatic activities of peptidase in which furylacryloyl-glycyl-leucinamide was used as a substrate, as well as of elastase and protease. In contrast, the other six mAbs did not neutralize peptidase activity at all. The Ki value for furylacryloyl-glycl-leucinamide of E-4D3, as well as its Fab fragment, was comparable to those for metalloprotease inhibitors such as phosphoramidon and Zincov inhibitor. The binding of mAb E-4D3 was inhibited by phosphoramidon and Zincov inhibitor, but not by metal chelators such as EDTA and o-phenanthroline. A line of evidence suggests that mAb E-4D3 directly interacts with active site and highly neutralizes enzymatic activity of P. aeruginosa elastase. Data of Western blotting and ELISA suggest that mAb E-4D3 is likely to recognize an elastase molecule in a conformation-dependent manner as an epitope. In contrast, the neutralizing activity of the other mAbs against elastase and protease seems to be caused by a low accessibility of an enzyme to insoluble and high-molecular-mass substrates through the binding and steric hindrance of the mAbs to an enzyme.     

Naturally occurring Mycobacterium scrofulaceum infection in a laboratory mouse colony.

A contamination with Mycobacterium scrofulaceum was experienced in a colony of BALB/c-nu/nu mice. The contamination was noticed after introduction of C57BL/6 and C57BL/6. Lyt l. 1 strains into facilities that kept the colony. M. scrofulaceum seemed to be spread by oral infestation and cross-contamination of fecal excretions during handling of the mice. The organisms were shed continually or intermittently into feces of weaned nu/+ and nu/nu mice of BALB/c background, and were isolated from the mesenteric lymph nodes and spleen of some of the mice. Some of the bacillus-carrying mice developed serum antibody to M. scrofulaceum of IgG and IgA classes and gave a low degree of hypersensitivity to PPD from M. tuberculosis.     

Involvement of protein kinase C in the phosphorylation of 46 kDa proteins which are phosphorylated in parallel with activation of NADPH oxidase in intact guinea-pig polymorphonuclear leukocytes

Two proteins (Mr 46,000, pI 6.4 and 7.0), the phosphorylation of which was increased by any of the membrane-perturbing agents in parallel with activation of NADPH oxidase in intact guinea-pig polymorphonuclear leukocytes in our previous study (Okamura, N., Ohashi, S., Nagahisa, N. and Ishibashi, S. (1984) Arch. Biochem. Biophys. 228, 270-277), were also phosphorylated in a cell-free system prepared from the leukocytes. The in vitro phosphorylation of these two proteins was stimulated by the addition of phosphatidylserine in the presence of higher concentrations of Ca2+ (300-500 microM). The phosphorylation was further increased when protein kinase C partially purified from guinea-pig brain was added to the system. At a low concentration of Ca2+ (about 10 microM), stimulation of the phosphorylation was not attained by phosphatidylserine alone but required the addition of diacylglycerol or phorbol myristate acetate. On the other hand, the increase in the phosphorylation was inhibited by H-7, an inhibitor for protein kinase C. These results indicate that protein kinase C is involved in the phosphorylation of the two proteins, which may be related to the superoxide anion production stimulated by various membrane-perturbing agents.     

Studies on transfer ribonucleic acids and related compounds. XXVI. Circular dichroic properties of cyclic oligoribonucleotides and their linear counterparts

The CD spectra of cUpUp, cCpCp, and cGpGp derived from DCC-catalyzed polymerization of the relevant protected ribonucleoside 3′-phosphates are described. Similar studies on Up, U > p, and cUp, as well as cUpUpUp and cUpUpUpUp, are presented. The spectral properties of the cyclic oligomers are compared with those of the corresponding linear oligomers with terminal 3′-phosphates so as to demonstrate that disruption of normal right-handed base stacking is considerable in these RNA loops.