Mouse lymphotoxin.

The addition of PHA to C3H mouse spleen cells in tissue culture led to the production of lymphotoxin (LT). Cytotoxicity was assayed by addition of the culture fluids to syngeneic target cells labeled with tritiated thymidine; after an incubation period of 72 hr the amount of radioactivity released into the supernatant was measured. The LT activity in unfractionated culture fluids survived lyophilization, remained unchanged for many weeks at 4 degrees C, and progressively decreased on heating at 56 degrees C for periods from 1 to 9 hr. Based on the G-200 Sephadex distribution coefficients for several preparations, the m.w. of mouse lymphotoxin was about 41,000 daltons. Lymphotoxin from three different spleen cell production runs was recovered from isoelectric focusing columns in sharply focused peaks, the pH of which ranged from 4.4 to 4.8.     

Production of soluble suppressor factors by herpes simplex virus-stimulated splenocytes from herpes simplex virus-immune mice.

Indirect evidence indicates that herpes simplex virus (HSV)-specific cytotoxic-T-lymphocyte induction is regulated by suppressor cells. To search for such suppressor effects, supernatant fluids from splenocyte cultures from normal and HSV-immune mice cultured either with or without viral stimulation were tested for their ability to inhibit HSV-specific cytotoxic-T-lymphocyte induction. Only the supernatant fluid from the HSV-stimulated, HSV-immune cultures contained a suppressor activity (HSV-SF). HSV-SF was produced by nylon-wool-purified Thy 1+ cells. HSV-SF was detectable after 3 days of culture and would only suppress cytotoxic-T-lymphocyte induction if HSV-SF was added within 24 h of initiation of the test cultures. HSV-SF was neither dialyzable nor heat stable. Molecular sieve chromatography of HSV-SF yielded multiple peaks of suppressor activity. Although most of these peaks exhibited nonspecific suppressor activity, the suppression mediated by the 90,000 to 150,000-molecular-weight fractions was antigen specific and genetically restricted. These results provide direct evidence for the regulation of HSV-cytotoxic-T-lymphocyte induction by a novel suppressor factor.     

Relationship of CIF, LT, and PIF released in vitro by activated human lymphocytes. II. A further functional comparison of LT and PIF activities on HeLa and L-929 target cells.

Lymphotoxin (LT), and proliferation inhibition factor (PIF) activities found in 5-day supernatants of mitogen-activated human lymphocytes (SAL) were further compared. In agreement with previous results, the activities could not be distinguished functionally. Quantitative differences in the amount of activity detected in the SAL could be accounted for on the basis of target cell differences, concentration of the lymphocyte effector molecules in the supernatant, and the parameter employed to assess cell function. Growth inhibitory activity detected at high supernatant dilutions was completely reversible, whereas the cytotoxic activity detected at low supernatant dilutions was irreversible. When the active medium was fractionated on DEAE, two peaks of inhibitory activity were detected. Depending upon the amount of activity and target cell, both peaks of activity were growth inhibitory or cytotoxic. Since both peaks of material affected HeLa and L-929 cells, the materials were not species specific. Thus, it appears that cloning inhibitor factor, LT, and PIF activities may actually be measures of the same stable materials found in 5-day activated lymphocyte supernatants.     

Interferon induction and prostaglandin synthetase inhibition in the in vitro and in vivo manipulation of immune response expression in an animal model of bladder cancer

Biological and physicochemical properties of keyhole limpet hemocyanin (KLH)-induced guinea pig lymphotoxin (LT) were compared to those of LT induced by another antigen, ovalbumin, or by the mitogen, phytohemagglutinin (PHA). Mitogen- and antigen-induced LT had similar colony inhibitory activities toward guinea pig and rat tumorigenic cells but none toward nontumorigenic cells. Conversely, human nontumorigenic but not tumorigenic cells were inhibited by the three LT preparations. The molecular weight of each LT was 45,000 daltons on gel filtration chromatography but sucrose density gradient ultracentrifugation indicated greater heterogeneity. The KLH-, ovalbumin-, and PHA-induced LTs were resolved into multiple peaks upon preparative column isoelectric focusing (IEF) with IpH's in the range of 4.5 to 5.3. Analytic IEF of the KLH-induced LT revealed the presence of three distinct biologically active peaks with IpH at 4.77, 5.02, and 5.20. LT activity was stabilized during preparative IEF by inclusion of 0.1% polyethylene glycol (4000 molecular weight) in the ampholine gradient. This resulted in 90% recovery of LT activity with a concomitant removal of 96% of the KLH used as the antigenic stimulus for LT production. Fractionation of LT activity during diafiltration and IEF when exogenous proteins were included as protective agents altered the number and the isoelectric characteristics of the peaks obtained suggesting that LT was being bound by the added protein. Colony inhibitory (cytostatic) and ³H-release (cytolytic) LT activities copurified during fractionation. Thus, this study demonstrated that high yields (approximately 50% of the starting material) of LT, free of antigen or exogenous serum proteins, can be obtained by a combination of diafiltration and IEF performed in the presence of polyethylene glycol.     

Separation and partial characterization of two alkaline peptidases from cotyledons of resting kidney beans, Phaseolus vulagaris

Extracts prepared from cotyledons of resting kidney beans ( Phaseolus vulgaris L., cv. Processor) rapidly hydrolyzed two dipeptides, Leu-Tyr and Ala-Gly, with pH optima at 9.2 and 8.5, respectively. On ion exchange chromatography on DEAE-Sephacel the two activities eluted as separate peaks, showing that they were due to two different peptidases. The extracts also hydrolyzed Leu-β-naphthylamide optimally at pH 6.4; this activity eluted as a third peak berween the other peaks. The activity peak acting on Leu-Tyr and Ala-Gly rapidly hydrolized two tripeptides, showing that it was an aminopeptidase, whereas the Ala-Gly hydrolyzing peak acted only on dipeptides. The activities against Leu-Tyr and Ala-Gly were also separated by gel chromatography on Sephacryl S-300 with elution positions corresponding to M, values of about 360 000 and 105 000. The aminopeptidase was inhibited by bestatin, and the dipeptidase was inhibited by p-hydroxymercuribenzoate. Both enzymes were inhibited by o-phenanthroline. In most of their properties the two kidneys bean enzymes resembled the alkaline aminopeptidase and the dipeptidase earlier characterized from barley grains.     

Evidence for a surface receptor for human migration inhibitory factor on a B-lymphoid cell line.

Reception by PGLC-33H target cells for the migration inhibitory factor (MIF) produced by this established line has been investigated by pulse time and temperature dependence, MIF absorption, and abrogation by trypsinization. PGLC-33H supernatants containing MIF were concentrated 5× with Carbowax and dialyzed against serum free RPMI-1640 before use. Prior to standard capillary migration assay a minimum 30 min pulse of MIF at 37 °C is required for significant migration inhibition (MI > 20%). No significant MI is observed when cells are pulsed at 4 °C for up to 2 hr. Preincubation with PGLC-33H for 1 hr at 37 °C reduces activity of supernatants from 38 to 13% MI; at 4 °C to 27% MI. Trypsinization of target cells for 30 min at 25 °C abrogates response to MIF (43 to ?14% MI). Trypsinized cells did not reduce activity of supernatants. MIF activity is abolished (32 to 3% MI) in samples preincubated with supernatants of the trypsinized cells inactivated with serum. These data suggest that cells from the human B-lymphoid cell line PGLC-33H have a surface receptor for human MIF.     

Cloning of cDNA for natural killer cell stimulatory factor, a heterodimeric cytokine with multiple biologic effects on T and natural killer cells.

Previously we have reported the purification and characterization of a novel cytokine from an EBV-transformed B cell line, RPMI 8866. This factor, termed natural killer cell stimulatory factor (NKSF), possessed pleiotropic activities including the induction of IFN-gamma from PBL, enhancement of cytotoxicity by NK cells, and stimulation of the proliferation of PBL. Purified NKSF was found to be a disulfide-linked heterodimeric protein composed of 35-kDa and 40-kDa subunits (p35 and p40). We now report the molecular cloning of cDNA for both subunits of NKSF from RPMI 8866 cellular RNA. The cDNA sequences indicate that both genes are novel, and Southern blot analysis confirmed that both cDNA are of human genomic origin. [35S]Methionine labeling indicated that cos-1 cells transfected with either p35 or p40 cDNA produced unique protein species of appropriate size. Methionine labeling of cos-1 cells cotransfected with p35 plus p40 cDNA yielded a broad band migrating between 70 and 90 kDa on a nonreducing gel. Reduction of this high molecular weight material yielded bands correlating with p35 and p40 gene products. Only culture supernatant from cotransfected cos-1 cells had a high level of NKSF biologic activity. That the high molecular weight material was responsible for this activity was indicated by the observation that biologic activity in the culture supernatant migrated at 70 to 90 kDa in a nonreducing gel. Furthermore, anti-p40 serum was able to block the biologic activities of both recombinant and natural NKSF, which indicates that it is a component of the active protein. In contrast, no activity could be detected in the supernatants of cos-1 cells transfected with p40 or p35 cDNA alone. The spectrum of biologic activity produced by cotransfected cos-1 cells was the same as NKSF purified to homogeneity from the RPMI 8866 cell line. A synergistic augmentation of some of these responses was found by the addition of IL-2 or the co-stimulators PHA or phorbol diester. The synergistic stimulation by NKSF plus IL-2 of T and NK function supports the possibility that these cytokines might prove useful in cancer therapy.     

Fibroblast stimulation in schistosomiasis. II. Functional and biochemical characteristics of egg granuloma-derived fibroblast-stimulating factor

The pathogenetic mechanisms that underlie hepatic fibrosis in schistosomiasis are unknown, but may be under the regulation of molecules secreted by the hepatic granulomas that encase the helminth eggs. Previous studies in mice demonstrated that isolated Schistosoma mansoni egg granulomas can elaborate in vitro substances that stimulate fibroblast proliferation and collagen synthesis. The present study provides the initial characterization of the granuloma-derived factor(s). Serum-free cell culture supernatants from isolated granulomas contained activity that stimulated the uptake of (3H)-thymidine by quiescent human dermal fibroblasts. This activity was present in fractions (Sephadex G-200 chromatography) with estimated m.w. of 30,000 to 40,000. Activity eluted with linear salt gradients from ion exchange columns (DEAE Sephadex) with 0.4 to 0.7 and 1.5 to 1.8 M NaCl (pH 8.4). Activity was present in fractions with approximate pI of 6 to 6.5 and 4, prepared by flat-bed isoelectrofocusing of crude supernatant in granular gel. Activity that stimulated thymocyte proliferation in an interleukin 1 (IL 1) assay was present in crude granuloma supernatants as well as in the partially purified fractions that contained fibroblast-stimulating activity. We conclude that the granuloma-derived fibroblast-stimulating factor has biologic activity similar to IL 1 but on the basis of m.w. and pI determination is distinct from IL 1.     

Lipoprotein lipase-like activity in the liver of mice with Sarcoma 180

The triglyceride lipase (TGL) activity of liver homogenates of mice with Sarcoma 180 was measured. The liver homogenate of normal or tumor-bearing mice was treated with 0.25% Triton X-100 and centrifuged at 100,000 g for 60 min, and the supernatant was applied to a heparin-Sepharose column. In normal mice, most of the TGL activities in the supernatant was eluted with 0.75 M NaCl from the column. In mice with Sarcoma 180, the TGL gave two peaks on heparin-Sepharose column chromatography, which were eluted with 0.75 M and 1.5 M NaCl, respectively. The activity in the first peak (0.75 M NaCl eluate) decreased; that in the second peak (1.5 M NaCl eluate) increased, and the ratio of the second peak to the first peak increased during tumor development. The livers of normal mice and mice on day 10 after tumor inoculation were perfused with heparin. The highest rate of the TGL release occurred within 1 min of heparin perfusion, and the bulk of heparin-releasable activity appeared within 2 min of perfusion in both normal and tumor-bearing mice. The TGL activity in liver perfusate of tumor-bearing mice, as well as that of liver homogenate, was resolved on a heparin-Sepharose column into two peaks, which were eluted with 0.75 M and 1.5 M NaCl, and most of the activity was eluted with 1.5 M NaCl. The nature of the TGL activity eluted from a heparin-Sepharose column was investigated. In both liver homogenates and liver perfusates, the first peak did not require serum for maximal activity and was relatively resistant to a high concentration of NaCl or protamine sulfate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heterogeneous profiles of a factor that renders neutrophils cytotoxic obtained from a concanavalin A-stimulated spleen cell culture in partial purification process

Concanavalin A (Con A)-stimulated rat spleen cells were cultured in a serum-free conditioned medium. This culture supernatant contained a certain factor(s) that renders neutrophil cytotoxic for various tumor cells. The factor was tentatively termed neutrophil-activating factor (NAF). Rat NAF was partially purified from the serum-free culture supernatant by using ion exchange chromatography of DEAE-Sephadex A-50, gel filtration of Sephadex G-100, and affinity chromatography of Con A-Sepharose 4B. NAF activity was eluted in broad fractions by the ion exchange chromatography and the gel filtration. Moreover, on the Con A column, some NAF activities were bound to the column, but other activities passed through the column. These results showed the heterogeneity or polydispersity of NAF activity in both molecular size and charge-based separation properties. Monoclonal antibodies were produced by fusing BALB/c myeloma cells (P3-X63 Ag8.653) with spleen cells from syngeneic mice immunized with partially purified NAF (pNAF) obtained from the gel filtration. Absorbent beads which were linked with one monoclonal antibody (ANAF-10) partially absorbed NAF activity from supernatants of a Con A-stimulated spleen cell culture. Further purification of pNAF was performed with the use of affinity chromatography of ANAF-10-linked Sepharose. Through these procedures, the NAF activity was concentrated about 10,000-fold. Heterogeneity of NAF activity, however, did not disappear in even this affinity chromatography. On the other hand, 125I-labeled material of the final product migrated to one major band corresponding with an m.w. of about 20,000 as determined by SDS-PAGE analysis, and NAF activity was detected in the same band.     

A "lymphokine-like" soluble product that induces proliferation and maturation of B cells appears in the serum-free supernatant of a T cell hybridoma as a consequence of mycoplasmal contamination

The serum-free supernatant of a cloned murine T cell hybridoma supports the proliferation and maturation to Ig secretion of purified B cells (mu+ cells) from BALB/c nu/nu mice, but has no effect on the proliferation of nylon wool-selected BALB/c nu/+ splenic T cells. Although the supernatant activates B cells without co-stimulation, it synergizes with anti-mu for the proliferative response. The induction of B cell proliferation and maturation to Ig secretion is directly related to contamination of the hybridoma by Mycoplasma hyorhinis. Hybridoma cells freed of mycoplasma by detergent treatment fail to produce active supernatant, and reinfection of the treated cells reconstitutes the activity. Furthermore, deliberate infection of a mycoplasma-free unrelated T cell hybridoma, as well as the monocytic cell line P388D1, results in the production of supernatants with B cell proliferating activity. Mycoplasma organisms isolated from the supernatant induce B cell proliferation without subsequent maturation to Ig secretion. Gel filtration chromatography of the supernatant from mycoplasma-contaminated hybridoma cells yields two peaks of activity. The first peak, found at the exclusion limit of the gel, results in B cell proliferation without maturation and may be attributed to mycoplasma organisms. The second peak (average m.w. 90,000) results in B cell proliferation as well as differentiation to Ig secretion. A "lymphokine-like" soluble product released by Mycoplasma hyorhinis is most likely responsible for this B cell activation, because fractionation of the supernatant from deliberately contaminated P388D1 cells gives essentially the same results, and gel filtration of mycoplasma-free supernatants does not generate any active fractions. The possibility should be considered that mycoplasma-derived soluble products may be among the many factors controlling in vitro B cell growth and maturation.     

Characterization of lymphokines mediating B cell growth and differentiation from monoclonal anti-CD3 antibody-stimulated T cells

Addition of anti-CD3 mAb 147 (IgG1), 446 (IgG1), or 454 (IgG2a) to cultures of T plus non-T cells can result in both B cell growth and differentiation. To determine whether lymphokines mediating these activities were similar to those described from conventional mitogen-induced T cell activation, normal peripheral blood T cells were stimulated with anti-CD3 mAb for 48 h. The supernatants were assayed for factors inducing B cell growth or differentiation (BCDF). A marked increase in Ig secretion was observed when either EBV-transformed B cell lines or normal B cells, pre-activated with Staphylococcus aureus Cowan I strain, were cultured in the presence of mAb 446 (anti-CD3) stimulated T cell supernatant whereas no significant increase in Ig secretion was noted with either mAb 454- or 147-induced T cell supernatant despite equivalent T cell proliferative responses to these antibodies. In contrast, IL-2 secretion was detectable in T cell supernatants from T cells stimulated with either mAb 454 or 147 but not 446. Factors promoting B cell proliferation were detected in all antibody-stimulated T cell supernatants but, contrary to BCDF, appear to act only on non-activated B cells. To determine whether these effector activities were due to distinct lymphokines, supernatants were pooled and concentrated by ammonium sulfate precipitation. Superose 12 permeation chromatography revealed BCDF activity with an apparent Mr of approximately 30,000 Da. The growth factor activity eluted over a wider and higher molecular weight range which overlapped the differentiation factor activity. Fractions containing BCDF activity were pooled, dialyzed, applied to a Mono Q anion-exchange column, and eluted with a linear NaCl gradient. The growth factor activity came off in a single-peak while BCDF was found divided into two major areas. The growth factor eluted at an ionic strength between the two BCDF activities. BCDF has an apparent isoelectric point (pI) of 6, in contrast to the reported pI 5 for IL-6 and more acidic than the documented basic pI of IFN-gamma. Lastly, peaks with BCDF activity were not active in assays for either IL-2 or IL-4. In addition, a rabbit anti-IL-6 heteroantiserum failed to inhibit the pI 6 BCDF, suggesting non-identity between IL-6 and anti-CD3 induced BCDF. Thus, anti-CD3 activated T cells generate both growth factor activity and BCDF as separate molecular entities distinct from IFN-gamma, IL-2, IL-4, and conventional IL-6.     

Bleomycin stimulates lung fibroblasts to release neutrophil and monocyte chemotactic activity

We determined whether human lung fibroblasts might release chemotactic activity for neutrophils (NCA) and monocytes (MCA) in response to bleomycin. The human lung fibroblasts supernatant fluids were evaluated for chemotactic activity by a blind well chamber technique. Human lung fibroblasts released NCA and MCA in a dose- and time-dependent manner in response to bleomycin. Checkerboard analysis of supernatant fluids revealed that both NCA and MCA were chemotactic. Partial characterization revealed that NCA was partly heat labile, trypsin sensitive, and predominantly ethyl acetate extractable. In contrast, MCA was partly trypsin sensitive and ethyl acetate extractable. The release of chemotactic activity was inhibited by lipoxygenase inhibitors and cycloheximide. Molecular sieve column chromatography revealed that both NCA and MCA had multiple chemotactic peaks. NCA was inhibited by leukotriene B4 receptor antagonist and anti-IL-8 and G-CSF Abs. MCA was attenuated by leukotriene B4 receptor antagonist, and monocyte chemoattractant protein-1, GM-CSF, and TGF-beta Abs. Leukotriene B4 receptor antagonist and these Abs inhibited the corresponding m.w. chemotactic activity separated by column chromatography. The concentrations of IL-8, G-CSF, monocyte chemoattractant protein-1, GM-CSF, and TGF-beta in the supernatant fluids significantly increased in response to bleomycin. These data suggest that lung fibroblasts may modulate inflammatory cell recruitment into the lung by releasing NCA and MCA in response to bleomycin.     

Application of a novel PRODUCTIVTM CM column in egg-white protein separation: Effect of high flow-rate on resolution and productivity

Summary A preparative PRODUCTIV^TM CM, a new coherent ion-exchanger, column (250×10 mm ID) has been used to separate egg-white proteins without cell debris remover (CDR) pretreatment. Results showed that the productivity increased with the superficial velocity (V_s) and a very high V_s (9.2 m/h) can benefit the high productivity (87.4 g/l.h) in comparison with 6.4 and 33 g/l.h as reported by others. Despite the high flow-rate and high column loading, the resolution of the eluted peaks of lysozyme and conalbumin, was maintained at 1.16.     

Generation of alloreactive cytotoxic T lymphocytes: production of T cell and macrophage helper factors in addition to IL 1 and IL 2 by peritoneal cells from mice immunized to Listeria monocytogenes

We investigate the production and biological activity of soluble helper factors produced by peritoneal T cells and macrophage derived from mice primed in vivo with Listeria monocytogenes. Supernatant fluids from co-cultures of these immune T cells and activated macrophages contained Interleukin 1 (IL 1) and Interleukin 2 (IL 2), and had the ability to assist the generation of cytotoxic T lymphocytes (CTL) from a population of nylon wool nonadherent spleen cells sensitized to allogeneic heat-treated thymocytes. The ability to assist CTL development involved T cell and macrophage factors in addition to IL 1 and IL 2. Immune T cells cultured alone produced a factor, devoid of significant IL 2 activity, that assisted CTL development only if adherent cells were present in the responding population. Activated macrophage produced a 38,000 dalton component, distinct from IL 1 on the basis of m.w., that assisted the development of CTL from nylon wool nonadherent splenic cells. Supernatants fluids from co-cultures of immune T cells and allogeneic, nonactivated macrophage contained a CTL helper factor but did not contain IL 1 or IL 2 activities. In contrast, supernatant fluids from co-cultures of immune T cells and syngeneic, nonactivated macrophage contained all 3 activities. This suggests a genetic restriction for the production of IL 1 and IL 2 that does not restrict the production of a CTL helper factor. These results demonstrate that T cell- and macrophage-derived helper factors distinct from IL 1 and IL 2 participate in the development of CTL.     

Histamine-releasing activity. IV. Molecular heterogeneity of the activity from stimulated human thoracic duct lymphocytes

Previous studies with the lymphokine, histamine-releasing activity (HRA), showed that HRA consisted of a heterogeneous group of molecules. The possibility of using thoracic duct lymphocytes (TDL) as a source of large quantities of HRA has been investigated. Antigen-stimulated TDL synthesize and release HRA in quantities similar to an equivalent number of peripheral blood lymphocytes (PBL). Streptokinase (SK) antigen routinely caused TDL to produce HRA approximately 15,000 Da. In contrast, staphylococcus enterotoxin B (SEB) induced the formation of a heterogeneous mixture of HRAs with apparent molecular weights of 50,000 and 15,000. Two peaks of activity (HRA I and II) were recovered when the supernatant from SK-stimulated TDL was subjected to ion-exchange chromatography. Interestingly, basophil chemotactic activity (BCA) was also eluted in these two peaks. Although interferon (IFN) is also released by antigen-stimulated TDL, the nonidentity of IFN and HRA was established by fundamental differences in chromatographic properties and specific antisera to IFN. In contrast, these studies suggest that HRA and BCA may be present on the same molecular entity.     

Biological expressions of lymphocyte activation. V. Characterization of a soluble immune response suppressor (SIRS) produced by concanavalin A-activated spleen cells.

Supernatant fluids from murine spleen cell cultures incubated with concanavalin A for 48 hr contain a factor(s), soluble immune response suppressor (SIRS), which suppresses plaque-forming cell responses to sheep erythrocytes by murine spleen cells in vitro. In the present studies, some of the biochemical and biophysical properties of SIRS were investigated. SIRS was non-dialysable; the suppressive activity was stable at 56 degrees C for 30 min, but was destroyed by treatment at 70 degrees C for 30 min, 80 degrees C for 10 min, or at pH 2. The suppressive activity was not absorbed by the stimulating antigen, SRBC, or antisera against murine IgG or mu-chain, suggesting that SIRS does not contain immunoglobulin determinants. Murine spleen and thymus, but not kidney cells, however, absorbed SIRS activity. Enzyme treatments revealed that SIRS was resistant to DNase and RNase, but was destroyed by trypsin and chymotrypsin. In gel filtration with Sephadex G-100, SIRS activity eluted in the fraction corresponding to m.w. in the range between 48,000 and 67,000. With polyacrylamide gel electrophoresis, SIRS activity migrated in the region cathodal to albumin. Isopycnic centrifugation in a cesium chloride gradient suggested that SIRS is a glycoprotein. These supernatant fluids with SIRS activity were also found to contain macrophage migration inhibitory factor (MIF). In the experiments using gel filtration, polyacrylamide gel electrophoresis, and isopycnic centrifugation to fractionate supernatant fluids, SIRS and MIF activity were found in the same fractions, and to date we have been unable to dissociate definitively SIRS activity from MIF activity.     

Standardization of a sensitive and rapid assay for lymphotoxin.

Actinomycin-treated mouse L cells and human HeLa cells are sensitive indicators of lymphotoxin activity in supernatant fluids of mitogen-stimulated lymphocytes. Without actinomycin, various strains of these cells are 10–200 times less sensitive. The concentration of actinomycin used amplifies the toxic effect of LT but is not itself cytotoxic. Actinomycin-treated indicator cells permit detection of LT activity where toxicity is not often found, as in supernatants of mixed lymphocyte cultures and of cell-mediated cytotoxicity reactions. This assay makes available to any investigator a sensitive indicator of LT activity.     

Characteristics and behavior during partial purification of estrogen sulfotransferase of guinea pig liver and chorion

Some characteristics of estrogen sulfotransferases from guinea pig liver and chorion were compared. Liver cytosolic activity was stimulated 10-fold by 25 mM monothiolglycerol and 2-fold by 15 mM MgCl2 or CaCl2, similar to that found previously for chorion. Liver and chorion activities were each eluted as a single peak from fast protein liquid chromatography (FPLC) gel filtration columns at apparent molecular weights of 52,300 and 50,000, respectively. Each was eluted during FPLC anion exchange under single, wide peaks with low recoveries. Liver sulfotransferase activity was eluted from Affi-gel Blue columns in the form of several peaks whereas the chorion activity behaved as a single species. The enzymes from both tissues, when partially purified by gel filtration followed by anion exchange, acted upon estrone and estradiol at the 3-position but activity toward dehydroepiandrosterone and testosterone was minimal or undetectable. Affi-gel Blue chromatography followed by FPLC gel filtration resulted in increases in specific activity of 26- and 90-fold for liver and chorion, respectively. Both enzymes were eluted from agarose-hexane-adenosine 3',5'-diphosphate (PAP-agarose) columns as single peaks. Average increases in specific activity for this column step were 40-fold and 96-fold for the entire eluted peaks of liver and chorion enzyme, respectively. Individual fractions from the PAP-agarose column indicated a specific activity increase of as much as 60-fold for liver and 208-fold for chorion. These latter were markedly unstable and it was not possible to obtain further purification by additional steps. Velocity versus substrate concentration curves for the partially purified enzymes showed complex kinetics, particularly with estradiol as substrate.     

Dissimilar cyclic nucleotide phosphodiesterase activities in subcellular fractions from normal and SV40-transformed WI-38 fibroblasts.

Broken cell preparations of WI-38 and SV40-transformed WI-38 (VA13) fibroblasts were used to compare the cyclic nucleotide phosphodiesterase activities of the two cell strains. The bulk of the cAMP or cGMP phosphodiesterase activity of WI-38 and VA13 homogenates was found in the 100,000 x g fibroblast supernatant fractions. WI-38 and VA13 soluble phosphodiesterase activities showed anomalous kinetic behavior with either cAMP or cGMP as the substrate. At low substrate concentrations, e.g., 0.1 muM, WI-38 supernatant fractions hydrolyzed cGMP much more rapidly than cAMP. At high substrate concentrations, e.g., 100muM, the same enzyme preparations degraded cAMP more than twice as fast as cGMP. In contrast, VA13 soluble phosphodiesterase activity catalyzed the hydrolysis of a wide range of cAMP and cGMP concentrations at similar rates. Phosphodiesterase activity in WI-38 supernatant fractions was generally more sensitive than that of the comparable VA13 enzyme activity to inhibition by MIX and papaverine. The cAMP phosphodiesterase activity of both WI-38 and VA13 supernatant preparations was decreased by cGMP in a concentration-dependent manner. cAMP was an effective inhibitor of cGMP hydrolysis by VA13 soluble phosphodiesterase activity. Yet, the cGMP phosphodiesterase activity of WI-38 supernatant fractions was only slightly reduced in the presence of cAMP. DEAE-cellulose chromatography of WI-38 and VA13 supernatant preparations revealed two major peaks of phosphodiesterase activity for each cell type. WI-38 peak I showed much greater activity with 1muM cGMP than with 1muM cAMP and appeared to be composed of two different phosphodiesterase activities. WI-38 peak Ia included phosphodiesterase activity which could be stimulated by boiled, dialyzed fibroblast homogenates while WI-38 peak Ib coincided with column fractions which contained most of the cyclic GMP hydrolytic activity. VA13 peak I phosphodiesterase activity was eluted from DEAE cellulose columns at the same ionic strength as WI-38 peak Ia and hydrolyzed these two substrates at nearly identical rates. This enzyme activity was also increased in the presence of boiled, dialyzed fibroblast preparations. Peak II phosphodiesterase activities from both WI-38 and VA13 fibroblasts were relatively specific for cAMP as the substrate. Phosphodiesterase activity with the properties of WI-38 peak Ib was not isolated from VA13 supernatant fractions. These results suggested that the dissimilar patterns of cAMP accumulation in WI-38 and VA13 cultures may be at least partially related to different phosphodiesterase activities in the normal and the transformed fibroblasts.