Verner Paetkau

Induction, suppression and superinduction of lymphokine mRNA in T lymphocytes

The expression of several lymphokine gene is characterized by a common pattern of induction, suppression and superinduction. This pattern was studied at the level of cellular mRNA in the mouse T-lymphoma cell line EL4, the human T-leukemia line Jurkat and in normal human peripheral blood lymphocytes. Lymphokine mRNA was induced by stimulating the cells with the phorbol diester PMA (TPA), with or without T-lymphocyte mitogens. The induction of Interleukin-2, Interferon gamma and the Colony Stimulating Factor for granulocytes and macrophages was suppressed by Cyclosporin A at moderate concns. Furthermore, these mRNAs accumulated to extraordinarily high levels (superinduction) if the protein synthesis inhibitor cycloheximide was added during transcription. Superinduction was not due to an increased rate of transcription. CsA interrupted ongoing transcription of IL2 by a mechanism not dependent on the induction of a new protein. The co-ordinate regulation of these genes strongly suggests that common intracellular signals mediate their expression.

[77a] Phosphofructokinase: I. Skeletal Muscle

Publisher Summary This chapter discusses the determination of phosphofructokinase from skeletal muscle. During the purification of the enzyme, phosphofructokinase (P-fructokinase) activity is determined by coupling with aldolase, triose phosphate isomerase, and α-glycerophosphate dehydrogenase. Each mole of fructose diphosphate formed by P-fructokinase leads to the oxidation of 2 moles of reduced diphosphopyridine nucleotide (DPNH). The assay is carried out at pH 8.0 where the enzyme activity is maximal. The enzyme activity may be determined with a pH-stat, or by measuring fructose diphosphate formed during a selected time of incubation. The enzyme to be assayed is diluted to a final concentration of not lower than 1 μg P-fructokinase per milliliter in 0.1 M potassium phosphate, 2 m M in dithiothreitol, pH 8.0. By definition, one unit of P-fructokinase catalyzes the conversion of 1 micromole of fructose 6-phosphate to fructose diphosphate per minute at pH 8.0 and 28°. Protein is measured by the biuret method using bovine serum albumin as standard. If Tris or ammonimn sulfate is present, the biuret reaction is carried out on the material precipitated by 5% trichloroacetic acid.

Regulation of immune responses. I. Effects of cyclic AMP and cyclic GMP on immune induction.

Abstract Agents that raise intracellular cAMP levels (dibutyryl cyclic AMP, aminophylline, adenosine and butyric acid) increase the magnitude of an in vitro primary humoral immune response when added at 10 −3 M during the first 12 hr of a 108 hr culture. Under the same conditions, cGMP has no direct effect but inhibits cAMP-mediated stimulation. DbcAMP (10 −3 M or 10 −4 M ), present from 0 to 12 hr, also increases the number of cytotoxic lymphocytes in CBA/J (H-2 k ) spleen cell cultures stimulated in a one-way mixed lymphocyte reaction with DBA/2J (H-2 d ) spleen cells. The dbcAMP effect is antigen-dependent in both humoral and cell-mediated immunity and antigen-specific in the case of humoral responses.

Cellular Origins and Targets of Costimulator (IL2)

ABBREVIATIONS: A cell cells adherent to nyton wool AEF allogeneic effect factor Con A concanavalin A CTL cytotoxic T lymphocyte IEF isoelectric focusing ILI, IL2Interleukins 1 and 2 LAF lymphocyte activating factor, same as ILI MLC mixed leukocyte culture MW molecular weight PFC plaque forming cells in an antibody response SRBC sheep red blood cells TCGF T cell growth factor, same as IL2 TMF thymocyte mitogenic factor, same as IL2 TRF T cell replacing factor TSF thymocyte stimulating factor, same as IL2

The cellular basis of cortisone-induced immunosuppression of the antibody response studied by its reversal in vitro.

Spleen cells from mice pretreated with cortisone were impaired in their ability to support an immune response in vitro to two antigens: sheep erythrocytes (SRBC) and polymeric flagellar protein of Salmonella adelaide (POL). We conclude that this immunosuppression is consistent with cortisone causing a dysfunction in accessory cells (macrophages?) and thymus-derived (T) helper cells without seriously affecting antibody forming cell precursors. Evidence for this comes from reconstitution of the response with additions of peritoneal exudate cells (PEC, irradiated, and anti- θ -treated), activated T cells, and 2-mercaptoethanol (2-Me) in culture. Additions of PEC or 2-Me restored the T cell-independent anti-POL response not only in cortisone-treated spleen cell cultures, but, contrary to previous reports, also in cultures of normal spleen cells depleted of adherent (accessory) cells by carbonyl iron treatment. The anti-SRBC response (which is dependent on T cells and accessory cells) in cortisone-treated spleen cell cultures was fully restored only by a combination of activated T cells and PEC or activated T cells and 2-Me. However, with lower doses of cortisone pretreatment, activated T cells or 2-Me alone was effective.

An activation-dependent, T-lymphocyte-specific transcriptional activator in the mouse mammary tumor virus env gene

Transcription of the complete mouse mammary tumor virus (MMTV) proviral genome in mouse cells is controlled by a strong promoter in its long terminal repeat. In the mouse T lymphoma EL4, there is a second, activation-dependent transcriptional initiation site within the envelope (env) gene, from which a short mRNA is generated, encoding the open reading frame of the long terminal repeat. We now report the isolation of a segment of the MMTV env gene (called META, for MMTV env transcriptional activator) which has the expected transcription-activating properties seen in EL4.E1 cells. Namely, it induces activation-dependent, T-lymphocyte-specific transcription of a chloramphenicol acetyltransferase reporter gene. It is active in mouse or human T-helper lymphocyte lines when they are stimulated to transcribe lymphokine genes but is inactive in unstimulated T-helper cells, fibroblasts, a cytotoxic T-lymphocyte line, and a mastocytoma cell line. Its activity is inhibited by cyclosporin A, a specific inhibitor of lymphokine transcription. Several forms of the META have been isolated from EL4.E1 cells, a mouse T-helper cell hybridoma, and from BALB/c spleen cells. Linked to the heterologous thymidine kinase promoter, a 400-bp portion of it is an inducible, orientation-independent, and cyclosporin A-sensitive transcriptional activator in T-helper cells.

The role of bacteriophage T7 exonuclease (gene 6) in genetic recombination and production of concatemers

Abstract Genetic analysis reported here shows that bacteriophage T7 exonuclease (gene 6) is necessary for intragenic and intergenic recombination in several areas of the T7 genetic map. This supports our previous conclusion ( Lee & Miller, 1974 ) that the enzyme is necessary for T7 molecular recombination. Results of sucrose gradient analysis show that DNA concatemers are formed when both the T7 exonuclease (gene 6) and the T7 endonuclease (gene 3) are absent. Further results show that concatemers cannot be maintained in the absence of the exonuclease unless the endonuclease is also eliminated. Therefore, concatemers are formed by a process other than normal phage recombination. Selective defects in the recombination system do interfere with the stability of concatemers, however.

Human colostrum contains an activity that inhibits the production of IL-2

The effect of human colostrum on T cell immune function was investigated. Colostrum inhibited the proliferation of human T cells activated by allogeneic, concanavalin A (Con A) or phytohaemagglutinin (PHA) stimulation. Colostrum also inhibited the production of IL‐2 by Con A‐activated human peripheral blood T cells and by Con A‐activated Jurkat cells, a human T lymphoma line. Similarly, human colostrum inhibited the production of IL‐2 by EL4 cells, a murine thymoma line, when stimulated with phorbol myristate acetate. The inhibitory activity was not cytotoxic and could not be neutralized by antibody to transforming human growth factor β.

A quantitative, batch hydroxyapatite method for analyzing native and denatured DNA at room temperature

Abstract A batch hydroxyapatite method is described for separating native from denatured DNA at toom temperature. No special solvent is required, but addition of a fixed amount of native and denatured carrier DNAs is essential. The method yields quantitative recoveries, and lends itself to small scale analyses. A large number of samples can be analyzed rapidly at one time. Annealing mixtures of DNA can be analyzed directly, provided they contribute less than certain critical levels of chelating agents for divalent alkali metal cations to the binding mixture. Relatively crude samples of pulse-labeled DNA from bacteriophage T7 infected Escherichia coli were analyzed directly, with good recoveries.

Thymine-guanine base pairing during transcription of polydeoxypyrimidines in vitro.

Abstract RNA polymerase from Escherichia coli catalyses poly(rG) synthesis in the presence of defined, repeating DNAs having only thymine and cytosine residues in one strand. Poly(rG) is the only ribohomopolymer formed. The reaction is strongly inhibited by 1 μ m -dATP or 1 μ m -ATP, and to a lesser extent by dAMP, CTP or UTP. With ATP and GTP present, the ribopolymer which is complementary to the polydeoxypyrimidine strand in the strict Watson-Crick sense is synthesized, as demonstrated earlier (Nishimura, Jacob & Khorana, 1964; Nishimura, Jones & Khorana, 1965; Morgan, 1970). Mn2+ is a better divalent cation than Mg2+ for poly(rG) synthesis, and the reaction is sensitive to actinomycin D or rifampicin. At 12 °C with d(T-C-C)n as template, a 1 1 complex is formed between the single-stranded DNA template and the newly synthesized poly(rG). This complex can be isolated in Cs2SO4 density gradients. These experiments indicate that thymine · guanine base pairs can form when the thymine residues are part of polydeoxypyrimidines also containing cytosine.