A novel TIMP-insensitive type IV collagen-degrading metalloproteinase from murine metastatic sarcoma cells

A novel type IV collagen-degrading metalloproteinase was purified from the conditioned media of a murine metastatic sarcoma cell line. The molecular weight of the purified enzyme was determined to be 100 kDa by SDS-PAGE, while 700 kDa by gel filtration suggesting that the enzyme has a multimer structure. This enzyme degrades type IV collagen, but neither type I collagen nor casein. The failure of trypsin treatment to enhance the enzyme activity suggested that the purified enzyme did not require activation. Although the enzyme seems to be classified as a matrix metalloproteinase, it was inhibited by neither tissue inhibitor of metalloproteinases (TIMP) nor TIMP-2 and thus represents a novel type IV collagen-degrading metalloproteinase.     

Effect of calponin on actin-activated myosin ATPase activity

Calponin inhibited the actin-activated myosin MgATPase activity in a dose-dependent manner without affecting the phosphorylation level of myosin light chain. This inhibition was Ca2(+)-independent. The decrease in enzymatic activity of myosin was correlated with binding of calponin to actin-tropomyosin filaments. Caldesmon showed a further inhibition of the calponin-induced inhibition of MgATPase activity of the thiophosphorylated myosin. Calponin-induced inhibition of the myosin MgATPase activity was reversed by the addition of calmodulin only in the presence of Ca2+. These results suggest that calponin acts as an inhibitory component of smooth muscle thin filaments.     

Parathyroid hormone-related protein is a possible autocrine growth inhibitor for lymphocytes

Adult T-cell leukemia (ATL)-related cells have the ability to produce a newly-isolated calcium-regulating protein, parathyroid hormone-related protein (PTHrP). The present study revealed that lectin-stimulated normal lymphocytes produce immunoreactive (IR)-PTHrP. When the T-cell-enriched fraction was purified from normal lymphocytes and then treated with lectin, a similar amount of IR-PTHrP was detected, suggesting that IR-PTHrP is an actual product of T-lymphocytes. A biologically active fragment of PTHrP, PTHrP(1-34), suppressed DNA synthesis in lectin-stimulated lymphocytes at concentrations greater than 50 pg/mL; the same concentration range of IR-PTHrP detected in the cultured media of lectin-stimulated lymphocytes. Therefore, it is reasonable to postulate that PTHrP is a cytokine inhibiting the cellular growth of normal lymphocytes.     

Studies on regional blood flow of the mouse using whole-body autoradiography of 14C-iodoantipyrine

Summary In order to visualize regional blood flow in various tissues of the mouse at the same time, the distribution of radioactive carbon from ¹⁴C-iodoantipyrine was studied by whole-body autoradiography. The mice were frozen with Dry-Ice-hexane at 1, 10, 30 min, and 1 h and 3 h after intravenous injection of ¹⁴C-iodoantipyrine. Whole-sagittal sections of the frozen mouse, obtained by using a cryostat microtome, were dried in a cryostat and subjected to autoradiography. The resulting dry autoradiographs are called untreated autoradiographs in the present work. The sections were then fixed in cold 6% (w/v) HClO₄, dried at room temperature and again subjected to autoradiography. Autoradiographs thus obtained are referred to as treated autoradiographs. It was found that the method could be suitable for the estimation of regional blood flow of the renal cortex, spleen, lung, skeletal muscle, bone marrow, thymus, testes, and brain.     

Induction of sister-chromatid exchanges by indirect mutagens/carcinogens in cultured rat hepatoma and esophageal tumor cells and in Chinese hamster Don cells co-cultivated with rat cells

2 rat cell lines originated from ascites hepatoma AH66-B and esophageal tumor R1 were examined for their inducibility of sister-chromatid exchanges (SCEs) after treatment with 14 kinds of indirect mutagens/carcinogens, including 6 amine derivatives, 4 azo compounds, 3 aromatic hydrocarbons and 1 steroid. Of the 14 chemicals tested, 2-acetylaminofluorene (AAF), butylbutanolnitrosamine (BBN), dimethylnitrosamine (DMN), cyclophosphamide (CP), urethane, 2-methyl-4-dimethylaminoazobenzene (2-MeDAB), 3′-methyl-4-dimethylaminoazobenzene (3′-MeDAB), 4-o-tolylazo-o-toluidine (4-TT), benzo[a]pyrene (BP), 7,12-dimethyl-benz[a]anthracene (DMBA) and diethylstilbestrol (DES) were estimated to be effective inducers of SCEs in AH66-B and/or R1 cells, without the use of exogenous activating systems. Cell-mediated SCE tests with 6 selected chemicals, CP, 2-MeDAB, 4-TT, BP, DMBA and DES, showed a significant increase of SCEs in Chinese hamster Don-6 cells co-cultivated with AH66-B or R1 cells, depending on the number and sensitivity of AH66-B or R1 cells, as well as on the dose of chemicals tested, whereas singly cultured Don-6 cells were much less sensitive or almost insensitive to these chemicals. The above findings suggest that AH66-B and R1 cells may retain metabolic activities to convert a wide range of indirect mutagens/carcinogens into their active forms to induce SCEs, and that these cell lines provide simple and reliable screening systems in vitro, including the cell-mediated SCE assay, for detection of genotoxic agents, without the use of exogenous activation systems.     

Induction of a neutral protease from rat intestine by a dietary manipulation

The activity of a neutral protease is increased in soluble fractions from the intestine of rats after a shift from a protein-free to a casein mash and an injection of triiodothyronine. The increased activity is prevented by the administration of cycloheximide. The time curve of the response of the protease activity in intestine is similar to that of liver DNA synthesis after a shift to 50% casein and triiodothyronine.     

Interrelation of Two Forms of Ferredoxin-NADP+ Reductase with Different Molecular Weights

The heavier form of ferredoxin-NADP⁺ reductase was extractedfrom spinach leaves or chloroplasts and isolated as the majorfraction at high ionic strengths with ammonium sulfate or sodiumchloride. At low ionic strengths, the form with the higher molecularweight was relatively unstable and was converted gradually intothe lower form. We concluded that the enzyme exists in vivoas the form with the higher molecular weight. ¹Present address: Market Development Department, Shionogi &Co. LTD., 5-Sagisu, Fukushima-ku, Osaka 553, Japan. (Received December 16, 1980; Accepted January 19, 1981)     

Immune cytolysis of human malignant melanoma by antibody to oncofetal antigen-I (OFA-I)

Summary IgG anti-OFA-I found in melanoma patients was tested for its ability to lyse human tumor cells in antibody-dependent cell-mediated cytotoxicity (ADCC). Sera from 89 stage II melanoma patients which contained non-HLA-related IgG antibody to an OFA-I-positive melanoma cell line (M14) as tested by indirect membrane immunofluorescence (IMI) were originally chosen as possible sources of IgG anti-OFA-I. Of those tested for specific IgG activity to OFA-I by IMI, anti-OFA-I was found only in those patients immunized with OFA-I-positive tumor cells. When the same sera were tested in ADCC, no non-HLA-related activity could be demonstrated. This result was confirmed with purified IgG fractions that could, nevertheless, show anti-OFA-I reactivity in a complement-dependent cytotoxicity assay. The fact that naturally occurring IgG anti-OFA-I antibody was not readily detectable in patients' sera and that induced IgG anti-OFA-I did not participate in ADCC indicates that OFA-I-related tumor cell lysis via ADCC is an unlikely phenomenon in cancer patients.     

The shortening and action potential of the cortex in the main pulvinus ofMimosa pudica

The shortening and action potential of the upper and lower cortices of the main pulvinus ofMimosa pudica were recorded simultaneously. The shortening and action potential were observed only in the lower cortex. The extensibility and the excitability of the cortex are discussed.     

Macrophage activation for tumor cytotoxicity: Control of cytotoxic activity by the time interval effector and target cells are exposed to lymphokines

Macrophages continuously exposed to lymphokines (LK) and target cells throughout a 48-hr cytotoxicity assay exhibit 3-fold more tumoricidal activity than do cells optimally treated with LK before addition of tumor cells. Increased cytotoxic activity induced by continuous LK treatment was not due to direct toxic effects of LK on tumor target cells or to alterations in target cell susceptibility to cytopathic effects of LK-activated macrophages. Moreover, sensitivities of responsive macrophages to LK activation signals and time courses for onset and loss of tumoricidal activity during continuous exposure or LK pulse were identical. Analysis of macrophage or LK dose responses and time courses for development of cytotoxicity each suggest that differences in tumoricidal activity between macrophages continuously exposed or pulsed with LK were quantitative: the number of cytotoxic events was increased 2.7 ± 0.2-fold (mean ± SEM for 11 experiments) during continuous LK treatment. Optimal levels of macrophage tumoricidal activity then occur only if effector cells, target cells and activation stimuli are simultaneously present for a defined time interval: tumor cells need not be present during the initial 2 to 3 hr of culture; LK can be removed after 8 hr with little or no loss of cytotoxic activity. However, removal of LK or target cells during the critical 4- to 8-hr interval decreased levels of cytotoxicity 3-fold. Thus, nonspecific effector function by LK-activated macrophages in controlled by both the physicochemical nature of the LK mediator and the time interval effector and target cells are exposed to LK.