Temporal analysis of cellular cytotoxicity and humoral factors during progession and regression of Rous sarcomas in Japanese quails.

Temporal appearance of cellular cytotoxicity and humoral activities including blocking and arming activities during the entire course of Rous sarcoma development in Japanese quails was examined by microcytotoxicity assay with comparison of animals bearing regressing tumors induced by a moderate dose of virus (regressors) and animals bearing growing tumors induced by a large dose of virus (progressors). Cellular cytotoxicity of the spleen cells in regressors was detected in a biphasic pattern; the first phase being observed as early as 3-5 days post inoculation (p.i.), followed by an eclipse period between 7-10 days p.i. which was the time of active tumor growth, and the second phase occurring after 12 days p.i. when the tumor had attained the maximum size. In progressors, only the first phase was observed. Instead, a stimulatory effect of the spleen cells on growth of target cells was noticed. Arming activity which confers cytotoxic activity on the normal spleen cells was demonstrated in the sera of regressors in the similar biphasic pattern as the cellular cytotoxicity; the early activity being present at 3 days p.i., and the late one after 19 days p.i. The former was detected by pre-incubation of serum with effector cells in microcytotoxicity assay and the latter by pre-incubation with target cells. In progressors, only the early arming activity which reacts with effector cells was demonstrated. Blocking activity which abrogates cellular cytotoxicity was demonstrated in both regressors and progressors but in different patterns of appearance, that is, blocking activity in regressors was only transiently demonstrated only by pre-incubation with effector cells at the time of maximum tumor growth, while the activity in progressors seemed to persist after the tumor reached the maximum size. Since the earlier activity was found to be effective at effector cell level, and the later one at both effector and target cell levels, participation of blocking factors of different types in progressors was also suggested.     

Beta-adrenergic agonists and cyclic AMP decrease intracellular resting free-calcium concentration in ileum smooth muscle

Intracellular free-calcium levels were measured in strips of longitudinal smooth muscle from guinea-pig ileum; fura-2 was used as a calcium monitor. At rest the calcium concentration was about 180 nM, and this rose to 300-400 nM following electrical stimulation and during spontaneous calcium transients (all measurements at 23-25 degrees C). Isoprenaline suppressed the spontaneous calcium transients, and reduced the resting calcium level to about 130 nM. This fall in resting calcium concentration was seen even in muscle strips which did not have spontaneous activity. Elevation of intracellular cyclic AMP levels, produced by forskolin or dibutyryl cyclic AMP, mimicked the actions of isoprenaline. We conclude that the relaxant effects of beta-adrenergic agonists of visceral smooth muscle may be explained partly by a fall in intracellular resting free-calcium level, mediated via an increase in cyclic AMP.     

The isolation and culture of lily pollen protoplasts

Methods for the enzymatic isolation of lily protoplasts and their successful culture are described. When pre-anthesis binucleate pollen (immature pollen grains) was treated in enzyme solution containing macerozyme and cellulase, up to 80% lost their exine and gave rise to intact protoplasts within 1 h. These pollen protoplasts were uniform in size and densely cytoplasmic with two prominent generative and vegetative nuclei. The isolated pollen protoplasts regenerated a cell wall within 1 day of culture and produced a structure resembling a pollen tube after 10–12 days of culture. During this culture period, dividing generative nuclei or 2 sperm nuclei were observed in many protoplasts with regenerated cell walls.     

Rate of DNA Synthesis during Meiotic Prophase in Lily Microsporocytes in the Presence of Deoxyadenosine and Nalidixic Acid

The rate and period of DNA synthesis during meiotic prophasewere examined using lily microsporocytes. Meiocytes at the earlyleptotene stage were cultured for discrete periods in the presenceof inhibitors of DNA synthesis, deoxyadenosine and nalidixicacid. Deoxyadenosine, which arrests meiotic development at theearly zygotene stage, markedly suppressed DNA synthesis to 35%of control at 2 mM. Nalidixic acid simply reduced the rate ofDNA synthesis, resulting in prolongation of the synthetic period.The relevance of DNA synthesis to meiotic development is discussed. (Received January 12, 1987; Accepted May 7, 1987)     

Estimation of interaction function gamma(x) with sparsely ionizing radiation

The interaction function gamma(chi), which was introduced in the theory of dual radiation action as the probability that two energy transfers separated by distance chi combine with each other to produce a lesion, was estimated with sparsely ionizing radiation (60Co gamma rays and 40 kV X rays). Gamma(chi) was deduced on the assumption that the sensitive matrix is made up of small spherical flocculi distributed over the cell nucleus. The diameter of a flocculus was estimated at (4.0-11.2) X 10(-8) m when the diameter of the cell nucleus d was assumed to be 5 microns, and (4.0-11.4) X 10(-8) m when d was assumed to be 10 microns. It seems reasonable to hypothesize that the flocculus corresponds to the linker DNA in the chromatin structure of DNA, because the size of the linker DNA as a target (about 40 nm) is consistent with the diameter of flocculi obtained in this study.     

Effect of the recombinant vaccinia viruses that express HTLV-I envelope gene on HTLV-I infection.

The human T-lymphotropic virus type I (HTLV-I) is etiologically linked to adult T-cell leukemia (ATL). To develop a vaccine against ATL, we constructed recombinant vaccinia viruses containing the envelope gene of HTLV-I in the vaccinia virus hemagglutinin (HA) gene, a new site where foreign genes can be inserted. A single inoculation of the recombinant virus induced antibodies to the env proteins of HTLV-I in rabbits and had a protective effect against HTLV-I infection.     

Measurement of human urokinase activity in plasma by using mono-specific antibody-conjugated paper disk

Human urokinase (HUK) was purified from commercial product by high performance liquid chromatography on TSK GEL-G3000SW and affinity chromatography on benzamidine-Sepharose 4B. The purified enzyme was of a high molecular weight form (molecular weight of 53,000). This preparation was utilized as an antigen to immunize rabbits; the obtained antibody showed a high specificity against HUK. The antibody was conjugated to CNBr-activated paper disks. The antibody-conjugated paper disk and a fluorogenic peptide substrate, glutaryl-Gly-Arg-4-methyl-coumarine-7-amide, were used to measure urokinase (UK) activity in plasma. The calibration curve obtained by the proposed method passed through the origin and was linear in the range of 0-0.16 IU of HUK. The incubation of HUK with an excess amount of alpha 2-macroglobulin at 37 degrees C for 3 h gave only about a 30% decrease of the activity assayed by the proposed method. After incubations of HUK with alpha 1-antitrypsin and antithrombin III, the activity was completely inhibited. The incubation of HUK with plasma at 37 degrees C decreased the activity as a function of time. However, when the antibody-conjugated paper disk was used for the immunoreaction to HUK in plasma at 4 degrees C, no decrease of UK activity was observed. The plasma decay curve of UK activity after a single intravenous (i.v.) injection of HUK into a rabbit (12,000 IU/kg) indicated bi-exponential kinetics by using this assay method. The rate constants of the alpha and beta phases were 0.120 +/- 0.020 and 0.021 +/- 0.002 min-1, respectively. These result suggest that the proposed method is useful for measuring UK activity in plasma of patients with intravascular coagulation after i.v. administration of UK.     

Glutamine Synthetase of the Human Brain: Purification and Characterization

Glutamine synthetase (GS) isolated from human brain formed a single band on sodium dodecyl sulfate-polyacrylamide gel with a molecular weight of 44,000. The enzyme had a specific activity of 179.2 U/mg protein when assayed by measuring the rate of the formation of gamma-glutamylhydroxamate using hydroxylamine as a substrate. In the presence of manganese ions, the relative activity of human brain GS was much lower than that of the sheep brain enzyme. The suppression of activity by increasing the ADP concentration, however, was less marked in the human enzyme than that in the sheep enzyme. Antibodies were raised in rabbits against the purified enzyme. The double-immunodiffusion technique disclosed cross-reactivities among GSs isolated from human, sheep, and rat brains, but the enzymes were not immunologically identical. Immunohistochemically, GS was localized in the cytoplasm of astrocytes in the human and rat brains and in pericentral hepatocytes of the liver.     

Optimum conditions for electric pulse-mediated gene transfer to mammalian cells in suspension

A pulse-generating machine which delivers exponentially decaying pulses over broad range of pulse lengths was used to determine the optimum pulse conditions for gene transfer to FM3A cells. In the transformation of tk- cells with pTK1, a single pulse of 100-2000 microseconds gave a high transformation frequency at 1.5-6 kV/cm and room temperature, the highest transformation frequency obtained being 3 X 10(-3). As the suspension buffer for cells exposed to the pulse, Saline G was better than PBS(-) for obtaining a large number of transformants because it ensured high cell viability.