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.     

Sequence-dependent hydrolysis of RNA using modified oligonucleotide splints and RNase H

To cleave RNA molecules using RNase H in a site-specific manner, a short deoxyoligonucleotide (3-5mer) joining with 2'-O-methyl oligonucleotide(s) was designed as a DNA splint to be used. Model experiments were carried out using ribooligonucleotide substrates (9 and 18 mer). It was found that the use of this type of splints (9 mer) causes a unique cleavage by RNase H. For example, when 3'm (GA)d(AGAA)m(GGU)5' was used as a hybridization strand, 32pUCUUUCUUCUUCCAGGAU was cleaved specifically between U11 and C12 to yield 32pUCUUUCUUCUU. This method will have a variety of applications for the study of RNA.     

Sequence-dependent cleavage of DNA by alkylation with antisense oligodeoxyribonucleotides containing a 2-(N-iodoacetylaminoethyl)thio-adenine.

Antisense oligodeoxyribonucleotides (15mers), containing a 2-(N-iodoacetylaminoethyl)thio-adenine, were synthesized and tested for their ability to cleave complementary DNAs (21mers). Cleavage of the target DNAs was done by alkylation followed by treatment with piperidine, and the positions of the alkylated sites were estimated by identification of the cleaved products. By using several combinations of the modified strands and their target DNAs, it was determined that alkylation occurred at adenine or guanine, depending on the torsion angle of the modified nucleoside.     

Characterization of interaction between Tb3+ and porcine intestinal brush-border membranes

Effects of ionic strength and temperature on the interaction between Tb3+ and porcine intestinal brush-border membrane vesicles were studied. When Tb3+ was added to the vesicle suspension, Tb3+ fluorescence increased with increasing concentration of Tb3+, showing a saturation. The apparent dissociation constant of one of at least two components of this binding reaction was estimated to be about 12.5 microM at 25 degrees C, pH 7.4. But the affinity of Tb3+ for the membrane vesicles was variable with changes of ionic strength and temperature. The affinity was lowered by addition of KCl to medium and by increase of temperature above 30 degrees C. In addition, temperature-induced change in the affinity of Tb3+ for the membranes was reversible over a temperature range from 13 to 46 degrees C. Temperature-dependence profiles of the excimer formation efficiency of pyrene-labeled membranes and of the harmonic mean of the rotational relaxation times of pyrene molecules in the membranes revealed that the phase transition of the membrane lipids occurs at about 30 degrees C. Based on these results, characteristics of Tb3+ binding to the membranes are discussed in relation to the nature of lipid phase and surface charges of the membranes.     

Modification of the amino acid acceptor stem of E. coli tRNAMetf by ligation of chemically synthesized ribooligonucleotides

The single-stranded region of the amino acid acceptor stem corresponding to the 3'-end of E. coli tRNAMetf was replaced by ligation of chemically synthesized ribooligonucleotides, in order to change the length of the single-stranded CCA terminus. The chemically synthesized ribooligomers, CCA, ACCA, AACCA and CAACCA, were ligated to nuclease-treated E. coli tRNAMetf, which lacked the ACCA sequence at the 3'-end. The methionine acceptor activities of these modified tRNAs were examined using E. coli methionyl-tRNA synthetase. Ligation of the chemically synthesized pentamer (AACCA) to the acceptor terminus restored the methionine acceptor activity, whereas ligation of the hexamer (CAACCA) or trimer (CCA) to the acceptor terminus did not Modification of the acceptor terminus had no effect on the formylation of accepted methionine.     

Anti-O-phosphotyrosine antibodies in human sera

Antibodies reactive with O-phosphotyrosine (PTYR) were detected in 60 out of 621 inpatients, with high frequencies in hematologic and lung malignancies, hepatic diseases, cerebrovascular diseases, and autoimmune diseases. Affinity-purified antibodies proved capable of recognizing PTYR-containing proteins in a human carcinoma cell line, A431, both by immunofluorescent staining and by immunoaffinity chromatography, but had no detectable affinity for phosphorylated serine or threonine, or for the nucleotides tested. In these respects, the antibodies observed in human sera were indistinguishable from anti-PTYR antibodies raised experimentally in rabbits or mice.     

Raman diagnosis of nucleic acid structure: sugar-puckering and glycosidic conformation in the guanosine moiety.

Observations of Raman spectra of various nucleic acids indicate that the guanine ring breathing frequency is sensitive to the internal rotation angle around the glycosidic bond and to the conformation of the five-membered ring of the ribose residue that is directly connected with the guanine residue in question. It is found that 682 cm-1 for C2'-endo-anti, at 665 cm-1 for C3'-endo-anti, and at 625 cm-1 for C3'-endo-syn. A DNA octamer d(GpGpApApTpTpCpC) shows, in its aqueous solution, a broad Raman band at 680 cm-1 with a tail at 670 cm-1. This fact suggests that the guanosine residues in this oligomer take primarily C2'-endo-anti conformation but an appreciable amount of fluctuation of the ribose ring structure towards C3'-endo is involved.