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.     

Electron-microscopic and immunocytochemical analyses of Weibel-Palade bodies in the human umbilical vein during pregnancy

Summary The present study was done to elucidate the biological significance of the Weibel-Palade body of human umbilical vein endothelial cells. Quantitative determinations of these endothelial-specific granules throughout pregnancy revealed that their numbers and size per cell profile were maintained at low levels from 12 to 19 weeks of gestation; then both rapidly increased from 33 weeks to full term. This increase coincided with the development of the rough endoplasmic reticulum and an increase in the number of endothelial cell pinocytotic vesicles. Light-microscopic peroxidase anti-peroxidase and electron-microscopic protein A-gold techniques provided evidence that factor VIII-related antigen was localized in the Weibel-Palade bodies. Furthermore, in vitro treatment of incubated umbilical vein tissue with compound 48/80, a histamine releaser, induced degranulation of Weibel-Palade bodies from the endothelium. The present study indicates that Weibel-Palade bodies are storage sites of both histamine and factor VIII-related antigen and have an important role in the obliteration of this vessel.     

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.     

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.     

Gas-liquid Chromatographic Determination of Carbohydrates in Tobacco

An enzyme which catalyzes the degradation of polyvinyl alcohol) (PVA) oxidized by secondary alcohol oxidase, in which hydroxyl groups of PVA are partially converted to carbonyl groups, has been purified from a fraction adsorbed on DEAE-Sephadex at pH 7.0 from PVA-degrading enzyme activities produced by a bacterial symbiotic mixed culture in a minimal medium containing PVA as a sole source of carbon and energy. The purified enzyme was electrophoretically homogeneous in the absence and presence of SDS.

The enzyme is a single polypeptide with a molecular weight of about 36,000 and has an isoelectric point of 5.1. The N- and C-terminal amino acid residues are both alanine. The enzyme is most active at pH 6.5 and at 40°C and is stable between pH 6.0 and 9.0 and at temperatures below 45°C. The enzyme is inhibited by Hg²⁺ and is restored by the addition of reduced glutathione, although p-chloromercuribenzoate has no effect.

The enzyme was active on oxidized PVA, but not on PVA and on various low molecular weight carbonyl compounds examined. The enzyme reaction on oxidized PVA resulted in a rapid decrease in viscosity, a fall of pH, and production of carboxylic acids. The enzyme, therefore, is considered to be an oxidized PVA hydrolase.

The enzyme shows a common antigenicity in immunodiffusion and neutralization reactions with antisera to an oxidized PVA hydrolase previously purified from another fraction adsorbed on SP-Sephadex at pH 7.0 from the PVA-degrading enzyme activities [Agric. Biol. Chem., 45, 63 (1981)]. The relations between these two oxidized PVA hydrolases are discussed.     

Carbon cycling and sequestration in a Japanese red pine (Pinus densiflora) forest on lava flow of Mt. Fuji

Biometric-based carbon flux measurements were conducted in a pine forest on lava flow of Mt. Fuji, Japan, in order to estimate carbon cycling and sequestration. The forest consists mainly of Japanese red pine (Pinus densiflora) in a canopy layer and Japanese holly (Ilex pedunculosa) in a subtree layer. The lava remains exposed on the ground surface, and the soil on the lava flow is still immature with no mineral soil layer. The results showed that the net primary production (NPP) of the forest was 7.3 ± 0.7 t C ha^?1 year^?1, of which 1.4 ± 0.4 t C ha^?1 year^?1 was partitioned to biomass increment, 3.2 ± 0.5 t C ha^?1 year^?1 to above-ground fine litter production, 1.9 t C ha^?1 year^?1 to fine root production, and 0.8 ± 0.2 t C ha^?1 year^?1 to coarse woody debris. The total amount of annual soil surface CO₂ efflux was estimated as 6.1 ± 2.9 t C ha^?1 year^?1, using a closed chamber method. The estimated decomposition rate of soil organic matter, which subtracted annual root respiration from soil respiration, was 4.2 ± 3.1 t C ha^?1 year^?1. Biometric-based net ecosystem production (NEP) in the pine forest was estimated at 2.9 ± 3.2 t C ha^?1 year^?1, with high uncertainty due mainly to the model estimation error of annual soil respiration and root respiration. The sequestered carbon being allocated in roughly equal amounts to living biomass (1.4 t C ha^?1 year^?1) and the non-living C pool (1.5 t C ha^?1 year^?1). Our estimate of biometric-based NEP was 25 % lower than the eddy covariance-based NEP in this pine forest, due partly to the underestimation of NPP and difficulty of estimation of soil and root respiration in the pine forest on lava flows that have large heterogeneity of soil depth. However, our results indicate that the mature pine forest acted as a significant carbon sink even when established on lava flow with low nutrient content in immature soils, and that sequestration strength, both in biomass and in soil organic matter, is large.