Analysis of Nuclear Accumulation of Influenza Nucleoprotein Antigen in the Presence of p-Fluorophenylalanine

When p-fluorophenylalanine (FPA) was added to influenza virus RI/5+-infected cells 4 hr after infection, virus-specific proteins were synthesized but infectious progeny virus was not produced. In these cells, synthesis of viral RNA was strongly inhibited and nucleoprotein (NP) antigen was found predominantly in the nucleus in contrast to untreated cells in which NP antigen was distributed throughout the whole cell. The intracellular location and migration of NP were examined by isotope labeling followed by fractionation of infected cells. In untreated cells, a large portion of the NP was present in the cytoplasm and most of it was detected in the form of ribonucleoprotein (RNP). In contrast, in FPA-treated cells little viral RNP was detectable and NP was present predominantly in the nucleus in a nonassembled, soluble form. When FPA was removed from the culture, synthesis of viral RNA was soon restored and a large amount of viral RNP appeared in the cytoplasm; this was followed by the production of infectious virus. The results of the experiments suggest that the NP synthesized in the presence of FPA is not assembled into viral RNP because of the lack of available RNA, and such NP migrates readily into the nucleus and accumulates there.     

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.     

Identification and partial purification of a novel tumor-derived protein that induces tissue factor on cultured human endothelial cells

Conditioned medium of a human bladder carcinoma cell line (J82) was found to induce tissue factor synthesis in cultured human umbilical vein endothelial cells (HUVEC). A protein present in the J82 conditioned medium was partially purified by FPLC using a combination of MONO Q and Superose 6 columns. The bladder carcinoma-derived cytokine (BCDC) exhibited a Mr of 22 kDa by gel permeation HPLC. Polyclonal antibody against either interleukin-1, tumor necrosis factor, or transforming growth factor-beta failed to inhibit the ability of the conditioned medium to induce HUVEC tissue factor activity, suggesting that this tumor cell line secretes a novel cytokine responsible for HUVEC tissue factor induction.     

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.     

Expression in Escherichia coli of chemically synthesized gene for the human immune interferon.

A 454 base pair fragment of double stranded DNA consisting of a gene for a human immune interferon (hIFN-gamma), initiation and termination signals plus appropriate restriction endonuclease sites, was totally synthesized. The synthesis involved preparation of 62 oligodeoxyribonucleotides by rapid, solid phase procedures, and enzymatic ligation of the oligonucleotides. This synthetic gene was expressed in E. coli under the control of the lac UV5 promoter. The product has antiviral activity which was acid labile and completely neutralized by antiserum to hIFN-gamma but not by antiserum to hIFN-alpha or hIFN-beta. Molecular weight of hIFN-gamma produced by E. coli was estimated to be about 32,000 and 17,000 by gel filtration and SDS-polyacrylamide gel electrophoresis respectively.     

Electrocardiographic studies in the Japanese monkey (Macaca fuscata) with special reference to the effect of anesthesia with barbiturates

The electrocardiograms of 157 healthy Japanese monkeys (Macaca fuscata), covering a wide range of ages in both sexes, were recorded under light pentobarbital (Nembutal) anesthesia. Although results were generally similar to those reported for other macaque species, some quantitative differences were observed.The heart rate was about 160 per minute in all monkeys examined; the P-Q interval was 0.11±0.06 sec.; the duration of QRS was 0.04±0.01 sec.; the Q-T interval was 0.24±0.06 sec. The mean axis of QRS was +59° and the pattern of the QRS complex was qR type in most cases.The comparison with the human electrocardiogram shows that the heart rate ofM. fuscata is about twice that of man, while the P-Q, QRS, and Q-T intervals were about one-half of those found in human subjects. In the monkey, however, the P wave was sharp and the T wave flat.In order to estimate the effect of anesthesia on the electrocardiogram, the records of several monkeys before, during, and after intravenous administration of barbiturates were compared. Although some animals showed extrasystoles after barbiturate was administered, generally no essential changes were noted in the records, except for the retardation of the rate and proportional prolongation of intervals.This work was presented at the 10th Annual Meeting of the Primate Research Association held in Inuyama, March 13, 1966.