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.     

Sensitive sandwich enzyme immunoassay of human growth hormone (hGH) in serum and urine using monoclonal antibody-coated polystyrene balls

A sensitive sandwich enzyme immunoassay for human growth hormone (hGH) using monoclonal antibody is described. A monoclonal anti-hGH IgG-coated polystyrene ball was incubated with hGH and subsequently with affinity-purified rabbit anti-hGH Fab'-horseradish peroxidase conjugate. Peroxidase activity bound to the polystyrene ball was assayed by fluorimetry using 3-(4-hydroxyphenyl) propionic acid as a substrate. The detection limits of hGH in serum and urine were 1.5 ng/l using 20 microliters of serum and 0.2 ng/l using 0.15 ml of urine, respectively. The specificity and assay precision were satisfactory. hGH levels in serum and urine determined by the present sandwich enzyme immunoassay using monoclonal anti-hGH IgG-coated polystyrene balls were well correlated to those determined by the previous sandwich enzyme immunoassay using rabbit anti-hGH IgG-coated polystyrene balls. Levels of hGH in urine collected as first morning voids from healthy subjects aged 19-28 yr were 6.4 +/- 3.2 (SD) ng/g creatinine. However, the present assay gave lower hGH levels than the previous assay. This was at least partly explained by the fact that hGH in urine was less efficiently bound to monoclonal anti-hGH IgG-polystyrene balls than standard hGH, while the binding of hGH in urine and standard hGH to rabbit anti-hGH IgG-coated polystyrene balls was equally efficient. In addition, gel filtration showed that 22K hGH, a major component, in urine was less efficiently bound to monoclonal anti-hGH IgG-coated polystyrene balls than standard 22K hGH. The nature of hGH in serum and urine remains to be investigated.     

Phototropism in Hypocotyls of Radish : I. Isolation and Identification of Growth Inhibitors, cis- and trans-Raphanusanins and Raphanusamide, Involved in Phototropism of Radish Hypocotyls

Three growth inhibitors which might be involved in phototropism of Sakurajima radish (Raphanus sativus var. hortensis f. gigantissimus Makino) hypocotyls, were isolated as crystalline forms from light-exposed radish seedlings and identified as cis- and trans-raphanusanins and 6-methoxy-2,3,4,5-tetrahydro-1,3-oxazepin-2-one (designated raphanusamide). The cis- and trans-raphanusanins inhibited growth of etiolated radish hypocotyls at concentrations higher than 1.5 micromolar, raphanusamide at concentrations higher than 20 micromolar.     

Effect of adriamycin entrapped by sulfatide-containing liposomes on ovarian tumor-bearing nude mice

Sulfatide-containing liposomes showed the highest degree of adriamycin entrapment of all the liposomes tested. Adriamycin was bound to the sulfatide anions on the liposomal membrane, inserted into the membrane, and incorporated into the aqueous compartment of the vesicle. Liposome-entrapped adriamycin was maintained at a much higher blood level than free adriamycin, and reached a lower concentration in the heart than did the free drug, which might lead to lower cardiotoxicity of the drug. Incorporation of adriamycin into ovarian tumor transplanted into nude mice was increased when entrapped by the sulfatide-containing liposomes. Liposome-entrapped adriamycin did not induce the drastic loss of body weight which occurred with the free drug. The growth of ovarian tumor was inhibited by liposome-entrapped adriamycin to the same degree as free adriamycin. Having these advantages, sulfatide-containing liposomes could be useful carriers of adriamycin for cancer chemotherapy.     

Nucleotide sequence of the chromosomal gene coding for the aminoglycoside 6-adenylyltransferase from Bacillus subtilis Marburg 168

Gene aadK of Bacillus subtilis is 855 bp long and codes for aminoglycoside 6-adenylyltransferase.     

Acclimation of Respiratory Properties of Leaves of Spinacia oleracea L., a Sun Species, and of Alocasia macrorrhiza (L.) G. Don., a Shade Species, to Changes in Growth Irradiance

To clarify the way in which the light available for growth affectsrespiration in leaves of sun and shade plants, we examined therespiratory properties of mature leaves of Spinacia oleraceaL., a sun species, and of Alocasia macrorrhiza (L.) G. Don.,a shade species, that had been grown at various irradiances.In leaves of S. oleracea, the respiratory rates, on a dry massbasis, decreased with time during the night, and the higherwas the growth irradiance during the day, the higher was therespiratory rate. The marked decreases in the respiratory rateduring the night were accompanied by decreases in the concentrationof carbohydrates in the leaves. By contrast, the respiratoryrates of leaves of A. macrorrhiza were virtually constant throughoutthe night and the absolute rates were lower than those of S.oleracea even though the absolute value of the concentrationof carbohydrates and its decrease at night resembled to thosein S. oleracea. The maximum activities of respiratory enzymeswere also similar to those in S. oleracea. However, the leavesof A. macrorrhiza contained less soluble protein than thoseof S. oleracea. These results suggest that, in S. oleracea,the concentration of carbohydrates might determine the respiratoryrate while such is not the case in A. macrorrhiza. The lowerrespiratory rates in A. macrorrhiza might be due to a lowerdemand for ATP. (Received November 29, 1995; Accepted February 15, 1996)     

Formation and decomposition of vacuoles inBotryococcus in relation to the trans-Golgi network

Summary The formation and the decomposition of vacuoles in a member of Xanthophyceae,Botryococcus braunii, were examined by light and electron microscopy. Particles around the nucleus were identified as vacuoles from their stainability with neutral red. These particles disappeared during cell division. They reacted positively in an activity test for acid phosphatase. Electron microscopy revealed the presence of spherical vacuoles around the nucleus. During cell division, these vacuoles seemed to be decomposed by the ER which surrounded the vacuoles. Soon after this decomposition, many immature multivesicular bodies (MVBs) appeared to develop from the trans-Golgi network (TGN) and were pinched off from the TGN. These immature MVBs took up small vesicles in them as they grew into the mature MVBs. Mature MVBs took up and digested the surrounding cytoplasm, fused with one another, and eventually became the vacuoles.Abbreviations MVB multivesicular body - TGN trans-Golgi network