Characterization of cloned murine cytolytic T cell lines

Murine cytolytic T lymphocytes can be kept in continuous culture apparently indefinitely by repeated passage in a concanavalin A-induced growth promoting medium. Some of these long-term cell lines maintain their cytolytic activity. Starting from three such populations, several cloned cytolytic T cell lines were derived and subsequently subcloned one or more times. Considerable variation in the levels of cytolytic activity was observed between different subclones; some initially active subclones lost activity with prolonged culture. In addition, one of the clones appeared to progressively lose the relative specificity demonstrated during the earlier passages of the parent cell line.     

Functional analysis of cloned opioid receptors in transfected cell lines

Opioids modulate numerous central and peripheral processes including pain perception, neuroendocrine secretion and the immune response. The opioid signal is transduced from receptors through G proteins to various different effectors. Heterogeneity exists at all levels of the transduction process. There are numerous endogenous ligands with differing selectivities for at least three distinct opioid receptors (μ, δ, κ). G proteins activated by opioid receptors are generally of the pertussis toxin-sensitive Gi/Go class, but there are also opioid actions that are thought to involve Gq and cholera toxin-sensitive G proteins. To further complicate the issue, the actions of opioid receptors may be mediated by G-protein α subunits and/or βγ subunits. Subsequent to G protein activation several effectors are known to orchestrate the opioid signal. For example activation of opioid receptors increases phosphatidyl inositol turnover, activates K⁺ channels and reduces adenylyl cyclase and Ca²⁺ channel activities. Each of these effectors shows considerable heterogeneity. In this review we examine the opioid signal transduction mechanism. Several important questions arise: Why do opioid ligands with similar binding affinities have different potencies in functional assays? To which Ca²⁺ channel subtypes do opioid receptors couple? Do opioid receptors couple to Ca²⁺ channels through direct G protein interactions? Does the opioid-induced inhibition of vesicular release occur through modulation of multiple effectors? We are attempting to answer these questions by expressing cloned opioid receptors in GH₃ cells. Using this well characterized system we can study the entire opioid signal transduction process from ligand-receptor interaction to G protein-effector coupling and subsequent inhibition of vesicular release.     

Simultaneous expression of mu- and gamma-chain mRNA in cloned murine B-lymphoma cell lines

The expression of immunoglobulin heavy chain genes was studied in five murine B-lymphomas known from previous studies to express either mu (38C-13), mu + delta (L10A, K46, BCL1) or gamma chains (A20). The presence of mu- and gamma-mRNAs in these tumors was determined by Northern blot analyses of the total cell poly(A)+ mRNA, using the appropriate 32P-labeled recombinant plasmid probes. In four out of the five lymphomas examined, both mu- and gamma-mRNAs were detected. The mu-mRNA appeared as multiple discrete bands of 1.9-3.0 kb. In three out of the four lymphomas, the gamma-mRNA appeared as two bands, a major one of 1.9 and a minor one of 3.9 kb. Three myelomas examined by similar methods did not contain more than one class of heavy chain mRNA. Reexamination of the Ig chains produced by the B-lymphomas which expressed both mu- and gamma-mRNAs revealed that two of them preserved their original phenotype and expressed mu (38C-13) or gamma chains only (A20). In contrast, two of the cell lines previously shown to express mu but not gamma chains (i.e., L10A and K46R) had changed during growth in culture and 'switched' to the production of gamma chains only. These results indicate that, in contrast to myelomas, B-lymphomas possess two classes of mRNA. However, the production of heavy chain mRNA in B-lymphomas is not necessarily accompanied by synthesis of the corresponding polypeptide chains. More studies are necessary to find out whether the expression of 'non-productive' heavy chain mRNA molecules in B-lymphomas is related to the phenomena of 'allelic exclusion' and/or the 'heavy chain switch' which occurs during the maturation of B-cells.     

Purification and partial characterization of ceruloplasmin receptors from rat liver endothelium

Ceruloplasmin (CP), a circulating glycoprotein, is known for its copper transport. Recently the spectrum of its activity has been increased to include numerous enzymatic functions. CP binds to the liver endothelium and is transported across the cell via a mechanism involving receptor-mediated endocytosis. To isolate CP receptors, we obtained purified preparations of liver endothelium in rats. The membrane was then isolated by ultracentrifugation and solubilized in Triton X-100. Membrane proteins were labeled with 125I and passed through an affinity column in which CP was covalently linked to Sepharose 4B. Most of the radioactivity was eluted with buffer during the first 5 days. When no more radioactivity was eluted with buffer, elution was done either competitively with cold excess CP or 1 M NaCl. By this technique, a sharp single peak of radioactivity was obtained and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions. Under both conditions receptors appeared as a single band with Mr of 35,000 containing 3% carbohydrate and an isoelectric point of 5.2.     

Solubilization and partial characterization of cell membrane Fc receptors

We assayed detergent cell lysates and culture supernatant fluid of mouse cells for the presence of soluble FcR by means of a radioligand bioassay. Strict correlation was found between the amount of FcR present in soluble form and that found on intact cells. The soluble FcR of all cells tested, except mouse macrophages, behaved as lipoprotein by buoyant density centrifugation analysis and enzyme treatment. In the absence of detergent, soluble FcR sedimented as a 20S macromolecule; in detergent the material was 7 to 9S. It was readily bound by insolubilized IgG but not by insolubilized BSA or F(ab')2 fragments of IgG.     

Insulin receptors on cultured murine lymphoid tumor cell lines

Summary Eight murine lymphoid tumor cell lines have been examined for the presence of high-affinity insulin receptors. The eight cell lines included two Abelson murine leukemia virus-transformed pre-B cell lines, three plasmacytoma cell lines, and three spontaneous T-cell lymphomas from AKR mice. All of the cell lines in the B-cell series had high-affinity insulin binding sites. The apparent equilibrium association constant (K_a) for the high-affinity binding sites on these cells was 1.3–3.3 × 109 M^–1. Two of the T-cell lymphomas had high-affinity receptor levels so low as to be undetectable in the whole cell binding assay under the conditions used for assaying the other cell lines, although in binding assays performed at very high cell densities, these two cell lines did appear to have a small number of high-affinity insulin binding sites. These results indicate that the growth stimulus provided by the tumor virus in neoplastic transformation of the AKR thymic lymphocytes differs from that provided by lectins in blast transformation of lymphocytes in that the neoplastic transforming event does not always result in the emergence of large numbers of high-affinity insulin receptors. In addition, the existence of cell lines such as the T-cell lymphomas that have nearly exclusively low-affinity binding sites suggests that the low-affinity sites may represent a distinct receptor that is not freely interconvertible with the high-affinity receptor.     

Purification and partial characterization of two extracellular keratinases of Scopulariopsis brevicaulis

Two extracellular keratinases of Scopulariopsis brevicaulis were purified and partially characterized. The enzymes were isolated by the techniques of gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). These keratinases (K I & K II) were purified approximately 33 and 29 fold, respectively. SDS-PAGE of the products of gel filtration chromatography (K I & II) produced only one band each, suggesting homogeneity. The optimum pH for both keratinases was 7.8, while the optimum temperatures were 40°C (K I) and 35°C (K II). Estimated molecular weights were 40–45 KDa and 24–29 KDa for K I & K II respectively. Both keratinases were inhibited by phenylmethylsulfonyl fluoride which suggests a serine residue at or near an active site.     

Purification and partial characterization of two forms of urinary trypsin inhibitor

The activation of bovine thyroid adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) by Gpp(NH)p has been studied using steady-state kinetic methods. This activation is complex and may be characterized by two Gpp(NH)p binding sites of different affinities with measured constants: Ka1 = 0.1 micro M and Ka2 = 2.9 micro M. GDP beta S does not completely inhibit the Gpp(NH)p activation: analysis of the data is consistent with a single GDP beta S inhibitory site which is competitive with the weaker Gpp(NH)p site. Guanine nucleotide effects upon F- activation of adenylate cyclase have been studied. When App(NH)p is the substrate, 10 micro M GTP along with 10 mM NaF gives higher activity than NaF alone, while GDP together with NaF inhibits the activity by 50% relative to NaF. These features are not observed when the complex is assayed with ATP in the presence of a nucleotide regenerating system or when analogs Gpp)NH)p or GDP beta S are used along with NaF. These effects were studied in three other membrane systems using App(NH)p as substrate: rat liver, rat ovary and turkey erythrocyte. No consistent pattern of guanine nucleotide effects upon fluoride activation could be observed in the different membrane preparations. Previous experiments showed that the size of soluble thyroid adenylate cyclase changed whether membranes were preincubated with Gpp(NH)p or NaF. This size change roughly corresponded to the molecular weight of the nucleotide regulatory protein. This finding, coupled with the present data, suggests that two guanine nucleotide binding sites may be involved in regulating thyroid cyclase and that these sites may be on different protein chains.     

Purification and partial characterization of two extracellular endoglucanases from Cellulomonas fermentans

Avicelase assay of gel slices after non-denaturing polyacrylamide gel electrophoresis of concentrated supernatants from Cellulomonas fermentans revealed four active bands. One of them corresponded to the principal active band on CM-cellulose. Among the three others, at least one did not correspond to any active band on CM-cellulose and might reflect the presence of an exoglucanase (EC 3.2.1.91). The active band on CM-cellulose was composed of two endoglucanases (EC 3.2.1.4), called CFA and CFB, which we purified by the means of DEAE-Trisacryl chromatography and high performance liquid chromatography (anion exchange chromatography and gel chromatography). These two monomeric enzymes differ in their molecular weights (40,000 and 57,000 for CFA and CFB, respectively) and in their catalytic constants in the reaction with CM-cellulose (Km were 1.5 g/l and 59 g/l for CFA and CFB, respectively), but have similar modes of action on this substrate and similar substrate specificities.     

Estrogen and progesterone receptors in some human myeloma cell lines and murine hybridomas

The control of immune responses by sex hormones is well documented but the effect of sex hormones on lymphoid cell subsets is poorly understood. We have investigated the expression of receptors for androgens (AR), estradiol (ER) and progesterone (PR) by human cell lines of the B lymphocyte lineage and by murine myeloma or hybridomas. AR, ER and PR were determined by cytosol and nuclear binding assays. Eleven human lymphoblastoid cell lines obtained by in vitro infection of blood or tonsil B cells with Epstein-Barr Virus (EBV) B95, did not express AR or ER. Similarly, 10 Burkitt's lymphoma cell lines were AR, ER and PR negative with the exception of the pre-B RAJI cells which bear AR. Among 13 cell lines derived from patients with multiple myeloma none expressed AR but five were found to bear ER (20-164 fmol/mg DNA or 5-10 fmol/mg protein). Four of the latter group also bear PR (86-450 fmol/mg DNA). Two mouse hybridomas out of seven tested were ER and PR positive. The MOPC 315 myeloma expressed ER but not PR. The possible functional role of these sex hormone binding sites in cell proliferation and immunoglobulin secretion deserves further investigation.     

Inhibition of early murine hemopoietic progenitor cell proliferation after in vivo locoregional administration of transforming growth factor-beta 1

Transforming growth factor-beta 1 (TGF beta 1) has been shown in vitro to be a potent negative regulator of growth and differentiation of early hemopoietic progenitor cells, but not of more mature progenitors. However, little information is yet available regarding similar effects in vivo. We have developed an approach whereby TGF beta 1 can be administered locoregionally to the bone marrow via direct injection into the femoral artery. Our studies show that intrafemoral administration of a single bolus dose of TGF beta 1 potently inhibits the baseline and IL-3-driven proliferation of bone marrow cells. This inhibition is relatively selective for the earlier multipotential granulocyte, erythroid, megakaryocyte, and macrophage CFU progenitor cells since these are completely inhibited while the more differentiated CFU assayed in culture colonies are inhibited by about 50%. The inhibition of hemopoietic progenitor growth and differentiation is both time and dose dependent with the maximal effect on the marrow observed at 24 h with doses greater than or equal to 5 micrograms/mouse, and the effect is reversed at later times. A possible practical implication of these in vivo results could be the use of TGF beta 1 to protect stem cells in the bone marrow from the myelotoxic effects of chemotherapeutic drugs.     

Purification and partial characterization of two genetic variants of placental alkaline phosphatase

The two most common variants of placental alkaline phosphatase, the F and S variants, were purified to homogeneity and characterized. Their molecular weights were determined by equilibrium ultracentrifugation and sodium dodecylsulfate polyacrylamide gel electrophoresis, which gave almost identical values for the two variants, 118,000 (F) and 119,000 (S). The amino acid compositions of the F and S variants presented here are found to be very similar. Differences between the two variants were found in specific activity (160 U/mg for F and 250 U/mg for S), isoelectric point (IP=4.5 for F and 4.7 for S), sedimentation coefficient (6.5×10^?13 sec for F and 6.4×10^?13 sec for S). Thus the structural differences observed for these enzyme variants seem to affect both the active site and the protein conformation.     

Purification and partial characterization of two glycoproteins in bovine peripheral nerve myelin membrane

Two major glycoproteins of bovine peripheral nerve myelin were isolated from the acid-insoluble residue of the myelin by a procedure involving delipidation with chloroform/methanol (2:1, v/v) and chromatography on Sephadex G-200 column with a buffer containing sodium dodecyl sulfate. The separation patterns of the proteins on the gel were affected considerably by the dodecyl sulfate concentration in the elution buffer. At above 2% dodecyl sulfate concentration in the elution buffer, the glycoproteins could be separated clearly on the gel and were purified. The purified proteins, the BR protein (mol. wt. 28 000) and the PAS-II protein (mol. wt. 13 000), were homogeneous on dodecyl sulfate-polyacrylamide gel electrophoresis. The NH₂-terminal amino acids of the BR and the PAS-II proteins were isoleucine and methionine, respectively. The BR protein contained glucosamine, mannose, galactose, fucose and sialic acids and the PAS-II protein contained glucosamine, mannose, galactose, fucose and glucose. Neither the BR protein nor the PAS-II were a glycosylated derivative of a basic protein of bovine peripheral nerve myelin, a deduction based on the results of amino acid analysis. The two major glycoproteins were observed commonly in the peripheral nerve myelin of cows, pigs, rabbits and guinea pigs, using dodecyl sulfate-polyacrylamide gel electrophoresis.     

Double immunoglobulin production in cloned somatic cell hybrids between two human lymphoid cell lines.

Several clones of independently established somatic cell hybrids between two human lymphoid cell lines, Raji and Namalwa, were examined for surface immunoglobulin expression. Double-antibody radioimmunoassays were established for kappa and lambda light chains. Immunoglobulins were detergent-extrated by Triton X-100 and quantified by radioimmunoassay. The Raji parent expressed small amounts of kappa chains on its surface, and the Namalwa parent a 10 fold greater amount of lambda chains. We show that the majority of the hybrid clones co-express both parental phenotypes.     

Purification and partial characterization of two lectins from the cactus Machaerocereus eruca

Two lectins (MEAI and MEAII) were isolated from the cactus Machaerocereus eruca by affinity chromatography on mucin-Sepharose and partially characterized with respect to their biochemical and carbohydrate binding properties. Both are oligomeric glycoproteins consisting of 35 kDa monomers. Amino acid analysis indicates that both lectins have similar composition with high amounts of glycine, glutamic acid and serine. MEAI and MEAII contain approximately 36 and 24% (w/w) of carbohydrates, respectively. They agglutinate erythrocytes from several animal species. Binding specificity was directed to galactose-containing oligosaccharides and glycopeptides. The M. eruca lectins are the first lectins to be isolated from a species belonging to the plant family of Cactaceae.     

Purification and partial characterization of two azoreductases from Shigella dysenteriae Type 1

Two azoreductases (I and II) were purified to homogeneity from extracts of Shigella dysenteriae (type 1). Azoreductase I was a dimer of identical subunits of M(r) 28,000, whereas azoreductase II was a monomer of 11,000 M(r). Both were flavoproteins, each containing 1 mol of FMN per mol enzyme. Both NADH and NADPH functioned as electron donors for the azoreductases. Azoreductase I used Ponceau SX, Tartrazine, Amaranth and Orange II as substrates. Azoreductase II utilized all the dyes except Amaranth.     

Expression and characterization of melanin-concentrating hormone receptors on mammalian cell lines

The neuropeptide melanin-concentrating hormone (MCH) is expressed in central and peripheral tissues where it participates in the complex network regulating energy homeostasis as well as in other physiologically important functions. Two MCH receptor subtypes, MCH-R1 and MCH-R2, have been cloned which signal through activation of Gi/o/q proteins and hence regulate different intracellular signals, such as inhibition of cAMP formation, stimulation of IP3 production, increase in intracellular free Ca2+ and/or activation of MAP kinases. Most of the data were obtained with cell systems heterologously expressing either of the MCH receptors. Fewer reports exist on studies with cell lines which endogenously express MCH receptors. Here, we describe human and other mammalian cell lines with which MCH receptor activation can be studied under "natural" conditions and we summarize the characteristics and signaling pathways of the MCH receptors in the different cell systems.