Proliferation of murine thymic lymphocytes in vitro is mediated by the concanavalin A-induced release of a lymphokine (costimulator).

Mitogen-induced proliferation of lymphocytes may in theory result directly from the interaction of mitogen with the cells, or indirectly as a result of the mitogen-stimulated release of lymphokines. In the case of murine thymic lymphocytes exposed to concanavalin A (Con A) in tissue culture, we have determined that mitogenesis depends upon a lymphokine. Interaction of the thymic lymphocytes with lectin is necessary, but not sufficient, for mitogenesis. A lymphokine, or costimulator for mitogenesis, is released by normal spleen or thymus cells during the first 16 hr of their exposure to Con A, and in the presence of a phytomitogen it stimulates thymic mitogenesis. Under conditions of low costimulator levels, no mitogenesis follows the interaction of Con A with cells. The response of adult CBA/J mouse thymocytes to phytohemagglutinin (PHA) is very low, compared to their response to Con A. When costimulator is added to PHA, the cells respond as well as they do to Con A. Costimulator does not act through Con A-binding sites on thymus cells. Its production is dependent on both cells carrying omega surface antigen (T lymphocytes) and adherent cells of the macrophage-monocyte series. The adherent population, but not the T cells, may be heavily irradiated without affecting production of costimulator. Costimulator is not a mitogen on its own.     

Stimulated rat T cell-derived inhibitory factor for cellular DNA synthesis (STIF). I. Isolation and characterization

A lymphokine inhibitory for cellular DNA synthesis (termed STIF) was isolated from the culture supernatants of concanavalin A (Con A)-stimulated SD rat spleen cells. STIF inhibited the DNA synthesis of mouse bone marrow cells as well as mouse leukemia cells. STIF has an apparent m.w. of 45,000 to 50,000 and is separable from IL 2, m.w. 20,000 to 25,000, by Sephacryl S-200 gel filtration, but not from immune interferon (IFN) having the same m.w. as STIF. Con A-Sepharose chromatography of the fraction containing STIF and IFN could separate these lymphokines into Con A-unbound and Con A-bound fractions, respectively. Further fractionation of the STIF fraction by DEAE-Sephadex A-50 or Mono Q-FPLC anion exchange chromatography indicated that the STIF fraction contained two components of STIF activity, both showing the same pI value (5.1 to 5.6) on flat-bed isoelectric focusing. STIF was characterized as a sugar-free lymphokine of trypsin-sensitive protein nature.     

Purification and characterization of alpha-toxin of Clostridium oedematiens type A

The alpha-toxin of Clostridium oedematiens type A was purified from culture filtrate by two steps of column chromatography and repeated gel filtration. The purified alpha-toxin proved homogeneous in polyacrylamide gel electrophoresis and agar gel double diffusion. The molecular weight of the alpha-toxin was estimated at 280,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and at 260,000 by gel filtration on a Sephadex G-200 column. The isoelectric point determined by isoelectric focusing polyacrylamide gel electrophoresis was 6.1. No dissociation of the purified alpha-toxin into subunits was demonstrated in sodium dodecyl sulfate polyacrylamide gel electrophoresis. The 50% lethal and edematizing doses per mg protein of the purified alpha-toxin were 5.9 X 10(4) and 5.9 X 10(5), respectively. The L +/50 doses per mg protein of the toxin was 4.6 X 10(3). The purified alpha-toxin, when injected intradermally into the rabbit skin, induced increased vascular permeability. The toxin contained little or no hemolytic or lecithinase activity. These results attest that the lethal, edematizing and vascular permeability-enhancing activities elicited by C. oedematiens type A culture reside on the same protein molecule.     

Purification and characterization of a colony stimulating factor from human lung.

Conditioned medium prepared from human autopsy lung tissue contains high level activity of colony stimulating factor which stimulates granulocytes and macrophage colony formation in both mouse and human bone marrow. The lung colony stimulating factor has been purified about 2250-fold by methods including hydroxylapatite chromatography, preparative gel electrophoresis, preparative isoelectric focusing, and gel filtration chromatography. The final specific activity was 2.7 X 10(6) units/mg. The purified factor has a molecular weight of 41 000 as determined by gel filtration. It is stable at the pH range of 6.5--10 and 56 degrees C for 30 min but sensitive to protease digestion and periodate oxidation. On polyacrylamide gel electrophoresis, it migrates in the alpha-globulin post-albumin region. Upon isoelectrofocusing lung colony stimulating factor appears heterogeneous with isoelectric points of 3.7--4.3. Treatment with neuraminidase did not affect its activity, but caused a change in electrophoretic mobility and isoelectric point. Antibody produced by immunizing rabbits with partially purified lung colony stimulating factor exerted strong inhibitory activity on the factor from lung as well as on colony stimulating factor from other human sources including serum, urine, and placenta.     

Characterization and Partial Purification of a Novel Neuronotrophic Factor from Bovine Seminal Vesicle

Extracts from bovine seminal vesicles have been shown to contain high concentrations of nerve growth factor (NGF)-like biological activity and of the NGF protein with properties corresponding to that of NGF from other sources. We now demonstrate that a second neuronotrophic protein, termed seminal vesicle-derived neuronotrophic factor (SVNF), is present in seminal vesicle extracts (SVEs), which could not be distinguished from NGF on the basis of biological activity. SVNF has neuronotrophic activity on NGF target cells like embryonic chicken-sensory and sympathetic neurons, sympathetic neurons, and chromaffin cells from neonatal rats, but it is inactive on embryonic chicken ciliary or neonatal rat nodose ganglion neurons. It also stimulates fiber outgrowth from rat pheochromocytoma (PC 12) cells. In gel filtration chromatography on Biogel A 1.5 m, the activity is eluted with an apparent molecular weight of 40 kilodaltons, and by preparative isoelectric focusing, the isoelectric point was determined to be in the neutral range (6.8-7.8). The biological activity of SVNF, in contrast to that of NGF, is partially retained after preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and can be electrophoretically eluted with an apparent molecular weight of 16-20 kilodaltons. Electrophoretically purified SVNF is not inhibited by antisera to mouse NGF, but its activity is increased greater than 10-fold in the presence of very low concentrations of NGF. For partially purified SVNF, a specific activity of 2.9-5.8 X 10(5) biological units/mg of protein was determined in the presence of subthreshold NGF concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of costimulator on immune responses in vitro.

We recently described a factor, costimulator, that is required for the concanavalin A-induced proliferation of CBA mouse thymocytes in vitro (see Reference 1). Using the costimulator dependence of mouse thymocytes as an assay, we have now determined that spleen cells from congenitally athymic (nude) BALB/c mice do not produce costimulator in response to Con A, and spleen cells depleted of Thy 1-positive cells do not respond to it in the presence of Con A. Thus, costimulator both requires thymus-derived (Thy 1+ lymphocytes for its production and has an effect on this type of cell. (However, the costimulator-producing and responsive cells may be different.) Purified costimulator preparations are a source of the required second component for the stimulation of adult, CBA/J thymic lymphocytes by PHA, normally a poor mitogen for these cells. They also enhance the level of DNA synthesis in a mixed leukocyte reaction, and the specific generation of cytotoxic lymphocytes to allogeneic tumor cells in vitro. Costimulator is not H-2 restricted in its effects, and it is produced in mixed leukocyte reactions. Finally, it has been possible to grow normal, primary thymic lymphocytes in culture for about 20 days by adding partially purified costimulator to the cultures.     

Purification and characterization of heat-labile toxin from Bordetella bronchiseptica

The heat-labile toxin (HLT) of Bordetella bronchiseptica was purified successively from sonic extracts of phase I organisms grown in Stainer-Scholte medium, by partition in hydrophobic interaction, sucrose density gradient centrifugation, gel filtration through Sepharose 4B and 6B, isoelectric precipitation and isoelectric focusing. The purified HLT was homogeneous by disc polyacrylamide gel electrophoresis and the gel diffusion-test, and free of detectable hemagglutinin and endotoxin activity. A 386-fold purification over the crude extract was obtained at a yield of about 28%, and a minimum dose of 0.9 ng was dermonecrotizing with a lesion 5 mm in diameter in guinea pigs and induced splenoatrophy. The mouse LD50 was 200 ng (intraperitoneal) or 70 ng (intravenous). The HLT was found to be a simple protein with an isoelectric point of pI 6.9. It has a molecular weight of 102,000 estimated by Sepharose 6B gel filtration and was found to consist of two different types of polypeptide by SDS-polyacrylamide gel electrophoresis, their molecular weights being 30,000 and 20,000. Amino acid analysis showed 15 common amino acid residues, and methionine, cysteine and tryptophan were undetectable. The HLT crystallized by methylpentanediol showed a block form. The HLT was inactivated at 56 C when heated for 10 min, and at above pH 9 and below pH 4.     

Purification and properties of a megakaryocyte stimulatory factor present both in the serum-free conditioned medium of human embryonic kidney cells and in thrombocytopenic plasma

Megakaryocyte stimulatory factor (MSF) has been purified to homogeneity (7.5 X 10(5)-fold) from serum-free conditioned medium obtained from cultured human embryonic kidney cells and to near homogeneity (1.44 X 10(7)-fold) from thrombocytopenic rabbit plasma. MSF activity from either source was assayed by its ability to enhance the rate of synthesis of platelet factor 4-like proteins in a rat promegakaryoblast cell line. The 125I-labeled factor prepared from human embryonic kidney cell conditioned medium is homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing in the presence of 9.2 M urea. MSF obtained from the above source is an acidic protein (pI = 5.1) with an Mr = 15,000 which stimulates platelet factor 4-like protein synthesis in rat promegakaryoblast cells by as much as 7-fold, and exhibits half-maximal activity at a concentration of 0.8 pM. MSF was also purified from thrombocytopenic rabbit plasma by a nearly identical isolation procedure, and 125I-labeled factor prepared from this source also possessed an Mr = 15,000. MSF exhibited no biologic activity corresponding to other known hemopoietic growth factors, and appears to be specific for the megakaryocyte lineage.     

Soluble cAMP-independent protein kinase from human spleen

A protein kinase (EC 2.7.1.37) was purified 2000-fold, from the soluble protein fraction of human spleen cells, using ion-exchange chromatography, ammonium sulfate fractionation, and gel filtration. This rapid procedure yielded 30% of the initial activity and an enzyme preparation with specific activity of 62 nmol min-1 mg-1 of protein. On the basis of disc gel electrophoresis in sodium dodecyl sulfate acrylamide gels and isoelectric focusing the enzyme preparation appears homogeneous and to consist of one polypeptide with a molecular weight of 43,000 and having a pI of 7.1. The purified enzyme activity is cyclic AMP and cGMP independent phosphorylates both alpha-casein and phosvitin, and uses Mg2+ ATP and Mg2+ GTP as phosphate donors, exhibiting an apparent Km of 2.0 and 6.6 X 10(-5)m, respectively. Furthermore, the enzyme activity is strongly inhibited by heparin (K50 = 0.1 micrograms/ml). These catalytic properties are characteristic of the enzyme casein kinase II, as described in several eukaryotic cells.     

The isolation, characterization and amino terminal sequence of the vitamin D-binding protein (group specific component) from mouse plasma

1. In order to establish a homologous system in which to study the interaction of mouse vitamin D-binding protein (MVDBP) with mouse T-cell lymphocytes, we purified MVDBP from mouse plasma. 2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that purified MVDBP had an apparent relative molecular weight of 49,000. 3. Previous work in our laboratory has shown that purified rat vitamin D-binding protein (RVDBP) has an apparent relative molecular weight of 52,000. 4. The amino terminal amino acid sequence of MVDBP is shown below and compared with that of RVDBP. MVDBP: LeuGluArgGlyArgAspTyrGluLysAspLysValCysAsnGluLeuAlaMetLeuGlyLysGlu RVDBP: LeuGluArgGlyArgAspTyrGluLysAspLysValCysGlnGluLeuSerThrLeuGlyLysAsp AspPhe AspPhe While 21 out of 24 residues (87.5%) of the amino terminus of MVDBP are the same as those in RVDBP, residues 14, 17, 18 and 22 (underlined) are different. 5. The sedimentation coefficient of the protein, determined by sucrose density gradient ultracentrifugation, is 3.8 for MVDBP and 4.1 for the rat VDBP. 6. The MVDBP purified in this study exhibits only one isoform on isoelectric focusing; the isoelectric point was 4.87 as determined on pH 4.0-6.5 isoelectric focusing gels (IEF). 7. The binding of vitamin D3, 25-hydroxyvitamin D3 and three other analogs was investigated with a charcoal dextran assay.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human leukocyte inhibitory factor (LIF): two distinct molecular species.

Human leukocyte inhibitory factor or LIF was generated in vitro by stimulating blood lymphocytes with concanavalin A (Con A). The control and Con A active supernatants were partially purified by gel filtration on Sephadex G-100. The fraction containing LIF (68,000 daltons) activity was then subjected to isoelectric focusing (pH 3 to 10 ampholines) in a sucrose gradient. Two LIF activities were reproducibly recovered by this procedure. One molecular form was found to have an isoelectric point of approximately pH 5.0 and the other approximately pH 8.5. Both molecular species were rechromatographed on Sephadex G-75 and found to have the same apparent m.w. (68 to 75,000). Furthermore, the biologic activity of both factors was destroyed after treatment with diisopropylphosphofluoridate, suggesting that they may be esterases.     

Malignancy-associated DNA-binding protein C3DP from human serum. Further characterization and purification of C3DP retaining its DNA-binding affinity.

C3DP, a malignancy-associated DNA-binding protein from human serum[1], was purified to homogeneity without loss of its DNA-binding affinity. For this purpose normal human serum was submitted to affinity chromatography on Con A-Sepharose and DNA-cellulose and to preparative polyacrylamide gel electrophoresis. The purified C3DP was identified by immunodiffusion and sodium dodecylsulfate polyacrylamide gel electrophoresis and it was shown to bind to DNA by DNA-cellulose chromatography. The isoelectric point of C3DP was determined to 4.9 by isoelectric focusing.     

Constitutive production and characterization of interferon-gamma in a human T-lymphoblastoid cell line transformed by a human retrovirus

A human T-lymphoblastoid cell line, TCL-Fuj, constitutively produced a large amount of human gamma interferon (IFN) in culture fluids and has sustained stable IFN production for more than two years. When cells were incubated in RPMI-1640 medium with 10% fetal calf serum for three days, IFN activity was detectable at a cell density of 6 X 10(4) cells/ml, whereas 2,000-16,000 units of IFN per ml were produced at 5-10 X 10(5) cells/ml. IFN production was also detected even in serumfree medium and as early as 2 hr after cultivation in fresh medium. IFN was inhibited by treatment of cells with either actinomycin D or cycloheximide, indicating the requirement of IFN-mRNA and protein for de novo synthesis. The molecular weight of the IFN was 45,000-60,000 as determined by Sephacryl S200 gel filtration. Two activity peaks corresponding to molecular weights of 22,000 and 39,000 were obtained by SDS-polyacrylamide gel electrophoresis. Analysis by isoelectric focusing revealed charge heterogeneity with four species at pIs of 6.0, 7.1, 8.6, and 9.3. Conventional IFN-gamma inducers, concanavalin A and 12-O-tetradecanoyl-phorbol-13-acetate, further enhanced the production of IFN in this cell line.     

Purification of an extracellular thiol-dependent hemolysin from Bacillus alvei]

Alveolysin a sulfhydryl-dependent cytolytic extracellular protein released by Bacillus alvei has been purified by salting-out by ammonium sulfate, gel filtration, isoelectric focusing on pH gradient and chromatography on DEAE-cellulose. The purified protein after reduction by thiols (active hemolytic form) proved homogeneous by disc polyacrylamide gel electrophoresis and by gel immunodiffusion. The molecular weight was 60,000 daltons, Two molecular forms of pI 5.1 and 7.0 were detected by gel isoelectrofocusing. The toxin was lethal to the mouse. Lytic activity was inhibited by cholesterol and antistreptolysin O anstisera. Immunological cross-reaction was observed between alveolysin and streptolysin O.     

Protein disulphide-isomerase of chick-embryo tendon.

Protein disulphide-isomerase can be partially purified from the high-speed-supernatant fraction of extensively disrupted chick-embryo tendon tissue. The catalytic properties of the preparation resemble those of the enzyme from mammalian liver. Gel electrophoresis and isoelectric focusing show the enzyme to be very acidic, with pI 4.4 +/- 0.3. Gel filtration indicates an Mr for the active enzyme of 140 000. The enzyme can be partially purified by preparative gel filtration or isoelectric focusing, but its limited stability has prevented purification to homogeneity; active fractions from both gel filtration and isoelectric focusing show two major polypeptide components by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The major polypeptides present in partially purified preparations have Mr 45 000 and 55 000; the latter band co-distributes with the enzyme activity in fractionations by both gel filtration and isoelectric focusing. The subcellular location of the enzyme cannot be established from work on homogenates of whole tissue, which are extensively disrupted. In homogenates from isolated tendon cells, the enzyme is located in a vesicle fraction that is excluded from Sepharose 2B but is of low density and can only be sedimented at very high speeds. This fraction is identified as deriving from the endoplasmic reticulum on the grounds of marker-enzyme studies and electron microscopy.     

The role of macrophages in thymocyte mitogenesis.

The thymic lymphocytes of CBA/J mice respond to the mitogen Concanavalin A (Con A) only in the presence of adherent cells of the monocyte-macrophage series. Depletion of adherent cells abrogates the response, and macrophage-rich population of cells restore it. The need for macrophages and mitogen is completely provided by irradiated splenic macrophages which have been exposed to Con A and washed free of the soluble mitogen. The mitogenmacrophage effect in this case is apparently not due to soluble factors. Even more striking than the effect of macrophages on fresh cultures of thymic lymphocytes is their ability to restimulate quiescent cells 72 hr after their first stimulation with Con A. The quiescent cells respond immediately and quantitatively to Con A in the presence of fresh macrophages. This stimulation, like that of fresh thymocytes, is also controlled by a lymphokine ("costimulator") produced by mixing macrophages, mitogen, ant T lymphocytes. Our data suggest a model in which two signals are required for mitogenesis. First, the interaction of macrophage, T cell, and mitogen elicits a soluble costimulator, which is itself not mitogenic. Secondly, in the presence of costimulator, the mitogen (either soluble, or, more efficiently, bound to macrophages) induces a proliferative response in the T cell.     

Purification and characterization of mouse kidney beta-glucuronidase.

Beta-Glucuronidase has been purified from mouse kidneys previously induced by gonadotrophin to a specific enzyme activity 15 times higher than the non-induced kidney. The purification procedure includes ultrasonication to solubilize the enzyme, acid and ammonium sulfate precipitations, gel filtration in Sephadex G-200, DEAE-ion exchange chromatography, and isoelectric focusing. The resulting product has a specific activity of 284,000 Fishman units/mg of protein, representing a 1,090-fold purification and is 17,000-fold higher than the level in the non-induced kidney. The purified beta-glucuronidase is apparently homogeneous by criteria of gel filtration, sodium dodecyl sulfate gel electrophoresis, and immunodiffusion. Characterization of the purified enzyme showed that it is identical with the lysosomal isoenzymic from electrophoretically, has subunit molecular weight of 74,000 (estimated by sodium dodecyl sulfate gel electrophoresis) and oligomer molecular weight of 300,000. The purified enzyme is stable at high temperature (up to 55 degrees) and at wide range of pH (from 4 to 11). It has a pH optimum for its activity at 4.7 and a Km of 1.18 times 10- minus 4 M. The purification and characterization of this enzyme from mouse kidney will have significance in the understanding of the molecular nature of the isoenzymes of beta-glucuronidase and will be useful in future studies on the mechanism of intracellular transport and distribution of this hydrolase.     

The purification and characterization of a necrotoxin from tarantula, Dugesiella hentzi (Girard), venom

The major toxin, a necrotoxin, of the venom of Dugesiella hentzi (Girard) has been purified by gel filtration. The purified toxin was homogeneous by gel filtration, polyacrylamide gel electrophoresis, and an isoelectric focusing procedure. The molecular weight estimation was 6700 and the isoelectric pH was 10.0. The amino acid composition shows 16 lysine, 8 cysteine, and one tryptophan residues, with no tyrosine, methionine, alanine, arginine, or histidine residues. The purified protein is toxic to certain insects and mice with the primary site of action being muscle tissue in the mouse. Modification of the single tryptophan residue resulted in a loss of toxicity.A significant increase of serum creatine phosphokinase activity was observed in mice injected with the necrotoxin. Histological examination showed the primary lesions were acute focal areas of myocardial necrosis, and no histological differences in myocardial lesions were seen between mice injected with the purified necrotoxin or with the whole venom.     

Regulated phosphorylation and low abundance of HeLa cell initiation factor eIF-4F suggest a role in translational control. Heat shock effects on eIF-4F

Initiation factor eIF-4F, a multiprotein cap binding protein complex, was purified from HeLa cells by m7G affinity chromatography and independently by phosphocellulose column chromatography. The m7G affinity-purified sample contains three major proteins, p220, eIF-4A, and p28 (also known as CBP-I or eIF-4E). The abundancies of these proteins are roughly 2, 10, and 0.8 X 10(6) molecules/cell, respectively. Two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the eIF-4F samples shows that p28 comprises two isoelectric variants, one of which labels with phosphate and disappears when samples are treated with alkaline phosphatase. The 45,000-dalton protein in eIF-4F appears to be identical to eIF-4A. The p220 subunit rarely produces discrete spots on two-dimensional gel electrophoresis; in purified samples it usually forms 3 closely spaced streaks. eIF-4F fractionated by phosphocellulose chromatography separates into forms containing either phosphorylated or unphosphorylated p28. However, both fractions possess similar specific activities in in vitro translation assays for eIF-4F activity. The phosphorylation of p28 decreases upon heat shock when protein synthesis is repressed. The correlation of dephosphorylation of p28 with the inhibition of protein synthesis and the relatively low abundance of the eIF-4F complex suggest that eIF-4F plays a role in the translational control of mRNA binding. Limitations of the in vitro assay system may account for the failure to detect phosphorylation-dependent activity differences.     

Immunocytology of cultured IgM-forming cells of mouse. II. Purification of phagocytic cell factor and its role in antibody formation

A factor required for the proliferation of IgM-forming tumor cells of mouse was purified approximately 1500-fold from culture supernatant of phagocytic cells through conventional protein fractionation procedures. The isoelectric point of the factor was pH 6.1 ± 0.1 as measured by isoelectric focusing. Its molecular weight was estimated to be approximately 5 × 10⁴ daltons. These characteristics of the factor were not identical with those of the stimulating factor for granulocyte and macrophage colony formation. Antiserum against the purified factor inhibited the growth-promoting activity of the factor. The effect of the antiserum on the normal antibody response of mouse to sheep red blood cells was examined in the tissue culture system. The antiserum inhibited only the late stage in the formation of direct plaque-forming cells.