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.     

Partial purification and characterization of a factor which stimulates an increase in ornithine decarboxylase activity in the liver from tumor cell-free ascites fluid

A factor responsible for stimulating an increase in ornithine decarboxylase activity in the liver of mice was found in tumor cell-free ascites fluid of mice 3 days after inoculation of tumor cells. The factor was purified about 70-fold in 25% yield from tumor cell-free ascites fluid. As little as 1 μg of protein of purified fraction, injected intraperitoneally into normal mice, significantly increased the activity of ornithine decarboxylase in the liver. The most active preparation of the factor formed two major protein bands on analytical polyacrylamide gel electrophoresis and both these bands stained with periodic acid-Schiff's reagent. The factor was a heat-labile, alkaline-stable, acidic protein with a molecular weight of more than 300 000. It was inactivated by treatment with 10 mM dithiothreitol, 5M urea, pronase or mixed glycosidase, but was stable on treatment with DNAase, RNAase or neuraminidase.     

Production of daunorubicin by immobilized growing Streptomyces peucetius cells

Summary Streptomyces peucetius cells were immobilized by entrapment in calcium alginate and a photosensitive synthetic polymer, and used for the production of daunorubicin (daunomycin), which is known to be an antitumour reagent. The use of cultivation media removed insoluble components in a natural medium prevented rapid decrease in daunorubicin titer after maximum production. These entrapped cells could be used at least five times for repeated daunorubicin production; the total cultivation period was 45 days.     

Factors controlling induction of commitment of murine erythroleukemia (TSA8) cells to CFU-E (colony forming unit-erythroid)

On addition of DMSO, the MEL cell line TSA8 becomes committed into erythroid progenitor cells (CFU-E) which can form differentiated colonies in the presence of erythropoietin. To understand the mechanism of cellular commitment, the number and the affinity of the receptors for erythropoietin were estimated. The affinity of the receptors did not change before or after induction. The number of receptors changed depending on the growth phase, but was not dependent on the addition of the inducer. Thus, the presence of the receptors for erythropoietin may be required, but are not essential for responsiveness to erythropoietin. Further examination of the optimum conditions for commitment suggests that the concomitant actions of induced factor(s) with the receptors may control commitment of TSA8 cells to CFU-E.     

Cholecystokinin-stimulated pepsinogen secretion and cholecystokinin receptors on gastric chief cells in guinea pigs

We investigated cholecystokinin (CCK) receptors on isolated gastric chief cells from guinea pig. CCK stimulated pepsinogen secretion from chief cells at the same efficacy as that induced by carbamylcholine. Binding of 125I-labeled CCK-33 (125I-CCK) to chief cells was temperature-dependent, and was saturable and reversible at 37 degrees C. Hofstee plots of the ability of CCK-8 to inhibit binding of 125I-CCK showed a linear regression line, suggesting that CCK receptors possessed one binding site. The dissociation constant of the binding site was calculated to be 3.8 x 10(-10) M. The dose-response curve of CCK for pepsinogen secretion was superimposed on that for the binding to its receptors. These results indicated that gastric chief cells from the guinea pig possess CCK receptors that relate closely to the action of CCK involved in pepsinogen secretion.     

Involvement in DNA repair of the ruvA gene of Escherichia coli

Summary The ruv operon of Escherichia coli consists of two genes, orfl1 and ruv, which encode 22 and 37 kilodalton proteins, respectively, and are regulated by the SOS system. Although the distal gene, ruv, is known to be involved in DNA repair, the function of orf1 has not been studied. To examine whether orf1 is also involved in DNA repair, we constructed a strain with a deletion of the entire ruv operon. The strain was sensitive to UV even after introduction of low copy number plasmids carrying either orf1 or ruv, but UV resistance was restored by introduction of a plasmid carrying both orfl and ruv. These results suggest that orf1 as well as ruv is involved in DNA repair. Therefore, orf1 and ruv should be renamed ruvA and ruvB, respectively.     

Molecular cloning and nucleotide sequencing of oxyR, the positive regulatory gene of a regulon for an adaptive response to oxidative stress in Escherichia coli: homologies between OxyR protein and a family of bacterial activator proteins

Summary Treatment of Escherichia coli and Salmonella typhimurium cells with a low dose of hydrogen peroxide induces expression of a large number of genes, and confers resistance to oxidative stresses. The oxyR gene encodes a positive regulatory protein for a subset of these genes involved in the defense against oxidative damage. We cloned a DNA fragment that contains the E. coli oxyR region on a plasmid vector, and analyzed the nucleotide sequence of the gene. The amino acid sequence of OxyR protein, deduced from the nucleotide sequence, shows a high degree of homology to the sequences of a number of bacterial activator proteins including LysR, cysB, IlvY, MetR and NodD. The product of the oxyR gene identified by the maxicell procedure was a 34 kDa protein, which agrees with the size predicted from the nucleotide sequence of the gene.     

CD16+ lymphoblastic cell lines of crab-eating monkeys (Macaca fascicularis) shared U-5 antigen and expressed natural killer activity

The CD16+ lymphoblastic cell lines of crab-eating monkeys shared the U-5 antigen recognized by a monoclonal antibody. The CD16+U-5+ cell lines expressed high natural killer activity to K562 cells, whereas the CD16-U-5- control cell line had no significant natural killer activity. A possible involvement of the U-5 antigen in natural killer function was also suggested by reduction of the natural killer activity in peripheral blood mononuclear cells of Japanese monkeys after treatment with U-5 monoclonal antibody and complement.     

Development of a series of monoclonal antibodies recognizing leukocyte differentiation antigens of Japanese monkeys (Macaca fuscata)

Six monoclonal antibodies to Japanese monkey leukocytes were developed. These monoclonal antibodies, designated the U series, cover most kinds of leukocytes (pan T cells, CD8+ cells, CD8+ subset and granulocytes, CD16+ cells, monocytes/macrophages), and should be useful in the immunological analysis of primate models, such as tissue transplants and virus-related diseases, in particular, acquired immune deficiency syndrome (AIDS).