Regulation of the in vitro anamnestic antibody response by cyclic AMP. II. Antigen-dependent enhancement by exogenous prostaglandins of the E series.

The spleen cells from CFW/D mice injected with dimethylbenzanthracene-induced leukemia virus exhibited a progressive decline in the in vitro response to heterologous erythrocyte antigens in parallel with tumor growth. Cell transfer experiments revealed that this immunodepressed state may involve a B-cell defect rather than extrinsic factors in the cellular environment since: (i) nonresponsiveness could be transferred to irradiated non-tumor-bearing mice with spleen cells, and (ii) T cells from tumorbearing mice cooperated with normal bone marrow cells, but bone marrow from tumorbearing mice did not cooperate with normal T cells. In addition, T cells from the thymic tumor could cooperate with normal bone marrow cells upon transfer to irradiated recipients. TL 485-2 cells, a T-cell line derived from the tumor, could be specifically activated with SRBC thereby indicating that the virus transformed T cells were immunocompetent. Suppressor cells, which appeared in the spleen concomitant with immunodepression and tumor development, may directly raise B-cell thresholds for T-dependent triggering signals since the antibody response of spleen cells from tumor-bearing mice could be restored by adding agents such as LPS, 2 mercaptoethanol, or T cells exogenously preactivated in normal animals. The suppressor cell could be enriched by adherence to plastic and was removed by treatment with carbonyl iron. In addition, it was unlikely that the suppressor cell was a virus-infected cell since transformed, virus-infected cells from the tumor or TL 485-2 cells were not suppressive when added to spleen cells in vitro but rather resulted in a marked, polyclonal enhancement of the PFC response. The interaction of TL 485-2 cells and normal spleen cells resulted in the release of a stimulatory factor which increased DNA synthesis in resting cells as well as increasing PFC. The role of these enhancing factors and suppressor cells in controlling tumor growth remains to be elucidated.     

Tumor cell killing by macrophages activated in vitro with lymphocyte mediators. III. Inhibition by cytochalasins, colchicine, and vinblastine.

The effect of cytochalasin A and B, colchicine and vinblastine on tumor cell killing by macrophages activated in vitro with lymphocyte mediators was examined. Both cytochalasins reversibly inhibited the killing of tumor cells by activated macrophages. Kinetic studies with cytochalasin B suggested that this drug exerts its effect on an early step of the cytotoxic process. Additional studies revealed that the drug inhibited the binding of tumor cells by activated macrophages.Colchicine inhibited both the binding and the killing of tumor cells by activated macrophages, whereas its structural analogue, lumicolchicine, had no effect on either macrophage function.Vinblastine also inhibited the binding and killing of tumor cells. However, this drug no longer inhibited tumor cell binding at low concentrations (<10^?6M) that still inhibited tumor cell killing. Further, vinblastine inhibited tumor cell killing when added late to an ongoing cytolytic reaction.These results suggest that the cytochalasins, colchicine and vinblastine inhibit macrophage mediated cytotoxicity by preventing intimate contact between the effector macrophages and their targets. In addition, vinblastine also appears to inhibit a later step of the cytolytic process, possibly the secretion of a cytotoxic macrophage product.     

Tumor cell killing by macrophages activated in vitro with lymphocyte mediators. II. Inhibition by inhibitors of protein synthesis.

The effect of inhibitors of protein synthesis on the killing of tumor cells by in vitro activated macrophages was determined. Cytotoxicity was inhibited by concentrations of puromycin, pactamycin, and actinomycin D that almost completely inhibited protein synthesis by guinea pig macrophages, but not by concentrations of drug that inhibited protein synthesis by only ± 50%. Cytotoxicity was inhibited when the effector macrophages were pretreated with the metabolic inhibitors, but not when the drugs were added 30 to 60 min after the initiation of the reaction. Pretreatment with puromycin or pactamycin also markedly inhibited the binding of tumor cells by mediator activated macrophages. These results are consistent with the hypothesis that one possible mechanism by which inhibitors of protein synthesis inhibit macrophage mediated cytotoxicity is by inhibiting close contact between effector and target cells. The finding that pretreatment of activated macrophages with trypsin also inhibits tumor cell killing suggests that protein synthesis may be necessary to maintain an adequate number of “recognition structures” on the macrophage membrane, structures that mediate the initial contact between the activated macrophage and the target tumor.     

Primary and secondary antibody responses of rabbits to bacteriophage T2: kinetic and quantitative analysis and suppression by antimacrophage globulin.

Antibody synthetic capacity of popliteal lymph node cells removed from rabbits at various times after immunization with bacteriophage T2 was assayed by radioimmunoassay of tissue culture fluid after incubation with ¹⁴C-leucine. Antibody synthesis began on day 2; IgM synthesis peaked on day 3; IgG synthesis peaked on day 5 and again on day 14. Reinjection of T2 one month later elicited an enhanced response which peaked sharply on day 2. The primary and secondary responses, but not priming for the secondary response, were suppressed by injection of goat antimacrophage globulin (AMG), but only when AMG was injected 1 to 3 days before T2. AMG reacted strongly with rabbit peritoneal macrophages and only slightly with rabbit lymphocytes or erythrocytes. Thus, macrophages appear to participate in the induction of antibody responses of rabbit lymph nodes to T2 and their function inhibited by AMG apparently operates only during the early phase of induction.     

Regulation of the in vitro anamnestic antibody response by cyclic AMP: I. Antigen-induced uptake of nucleotides and nucleosides by lymphocytes

Addition of N⁶, O²′-dibutyryl cAMP (DbcAMP) to keyhole limpet hemocyanin (KLH)-primed rabbit lymph node cells for 1 hr, followed by its removal and the addition of KLH, had no effect on the subsequent antibody response, whereas addition of KLH for 1 hr followed by DbcAMP resulted in a 100% enhancement of antibody synthesis. Addition of cholera enterotoxin (CT), which rapidly and irreversibly binds to lymphocytes and activates adenylate cyclase, either before or after the addition of antigen, elevated the antibody response by 100%. These results suggested that some antigen-induced event(s) may be required for DbcAMP to exert its enhancing effects on the antibody response. The effect of KLH on the uptake of DbcAMP by KLH-primed lymph node cells was investigated. One and one hundred micrograms of KLH, which induce optimal and supraoptimal antibody synthesis, respectively, promoted maximal uptake of DbcAMP. This induced uptake was first detectable about 12 hr after addition of KLH, and it peaked during 24–48 hr of culture. DbcAMP uptake induced by a brief exposure of KLH (0–1 hr) was equivalent to that observed with long-term KLH addition (0–24 hr). KLH-induced DbcAMP uptake required KLH-reactive lymphocytes and represented active transport. Antibody to rabbit T lymphocytes inhibited this antigen-induced uptake. The mitogens concanavalin A (Con A) (T cells) and goat anti-rabbit Fab' (anti-Fab') (B cells) also stimulated DbcAMP uptake, as did human serum albumin (HSA) and myoglobulin (Mb) when added to homologously primed cells, indicating the generality of the phenomenon. [³H]DbcAMP entered the cells as di- or monobutyryl cAMP with about 40% metabolized to 5′AMP. This uptake could be competitively inhibited by other adenine or guanine nucleotides and nucleosides.     

Proteins foretelling head or abdomen development in the embryo of Smittia spec. (Chironomidae,Diptera)

The development of the segment pattern in Smittia embryos can be manipulated experimentally. Centrifugation during intravitelline cleavage leads to a mirror image duplication of most of the head in the absence of abdominal segments (“double cephalons”). Conversely, mirror image duplications of abdominal segments in the absence of head and thorax (“double abdomens”) can be generated by UV-irradiation of the anterior pole before blastoderm formation. By subsequent exposure to blue light, UV-irradiated embryos can be reprogrammed for normal development (photoreversal). We have characterized an “anterior indicator” protein (designated AI₁; M_r ? 35,000; IEP ? 4.9). Its synthesis was restricted to anterior fragments of embryos during a late blastoderm stage (Bl_VI). This protein was synthesized, however, in both anterior and posterior fragments of prospective double cephalons. Conversely, this protein was synthesized neither in anterior nor in posterior fragments of UV-induced double abdomens. Upon photoreversal, the protein was synthesized again in anterior fragments. Thus, synthesis of this protein in a given fragment always indicated development of head and thorax there. Likewise, we have characterized a “posterior indicator protein” (designated PI₁, M_r ? 50,000, IEP ? 5.5). Its synthesis during early blastoderm stages (Bl_I and Bl_II) was restricted to posterior fragments but not to pole cells in normal embryos. In UV-induced double abdomens, PI_I was synthesized in both anterior and posterior fragments at stage Bl_II. Photoreversal again led to restriction of PI_I synthesis to posterior fragments. Thus, the synthesis of PI_I in a given fragment at stage Bl_II always foreshadowed the formation of an abdomen several hours before this can be discerned morphologically. The synthesis of two other proteins (designated a₁ and p₁) was also restricted, during certain blastoderm stages, to anterior or posterior fragments, respectively. However, UV-irradiation or centrifugation had little or no effect on the synthesis of these proteins. Conversely, programming embryos for double abdomen development by UV-irradiation caused a set of reproducible, and mostly photoreversible, changes in the pattern of proteins synthesized in anterior embryonic fragments. However, the synthesis of most of the affected proteins was not region-specific in normal embryos.     

Trypanosoma cruzi: the T-cell dependence of the primary immune response and the effects of depletion of T cells and Ig-bearing cells on immunological memory

The effects of T-cell depletion on primary infection with Trypanosoma cruzi and on immunological memory to this parasite were studied in a syngeneic mouse system. Exacerbation of T. cruzi infections occurred in thymectomized, irradiated, bone marrow-reconstituted (TX) C57BL/6J mice compared to sham thymectomized, irradiated, bone marrow-reconstituted (STX) mice. Reconstitution of TX mice with thymocytes restored the resistance to a level equivalent to that of STX mice. Immunological memory against T. cruzi present in spleen cells in mice recovered from T. cruzi infections could be ablated by treatment with rabbit anti-brain-associated theta serum but not with rabbit anti-mouse immunoglobulin serum prior to adoptive transfer of immune spleen cells into TX mice. These experiments suggest that modulation of the primary immune response and memory against T. cruzi depends largely on the thymus-derived lymphocyte. The possible implications of this T-cell regulation on previously reported effector mechanisms againt this parasite are discussed.     

Partial purification and characterization of cyclic AMP phosphodiesterases from Funaria hygrometrica

Natural-abundance ¹³C NMR spectroscopy at 15.04 MHz has been used to examine the effects of pH, calcium, and lanthanide ions on the polypeptide cardiac stimulant Anthopleurin-A in aqueous solution. The carboxyl resonance from the aspartic acid residue not observed in a previous study (R. S. Norton and T. R. Norton, 1979, J. Biol. Chem.254, 10220–10226) has been identified and an apparent pK_a of 3.4 obtained. More accurate estimates have been derived for the apparent pK_a values of the two histidine residues. Binding of Ca²⁺ ions has been found by equilibrium dialysis and ¹³C NMR to be weak (K_d > 0.1 M). The interaction with lanthanide ions is slightly stronger, but binding occurs at the C terminus as well as at a site involving one or both of the aspartate carboxylate groups. These results suggest that possible Anthopleurin-A-induced calcium translocation in the myocardial cell is a secondary effect. The interaction of Anthopleurin-A with lipid monolayers has also been examined. Binding occurs to neutral and zwitterionic lipids, but is stronger with negatively charged lipids, particularly cardiolipin. This interaction is also influenced by the presence of Ca²⁺ ions. The implications of these results for the mechanism of action of this polypeptide are discussed.     

On the function of the fluorescence quenchers in chloroplasts and their relation to the primary electron acceptor of photosystem II.

Redox titrations of the fluorescence quenching components in chloroplasts indicate the presence of two components, one with E_m7.6 = + 25 mV and the second with E_m7.6 = -270 mV. These midpoint potentials are almost the same as those of two Photosystem II components previously shown to contribute to the chloroplast electrogenic reaction measured at 518 nm (R. Malkin, 1978, FEBS Lett.87, 329–333). Comparison of light-induced fluorescence yield changes with those obtained by redox titration suggests that both fluorescence quenchers are photoreduced. A direct demonstration of the photoreduction of the low-potential fluorescence quencher was observed in experiments at defined redox potentials. Fluorescence induction curves measured at low light intensity in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) also showed a contribution from both fluorescence quenchers. An additional electron acceptor, other than the two fluorescence quenchers, was also identified in the acceptor complex. These results are discussed in terms of several electron acceptors functioning in the Photosystem II reaction center complex, and the possible function of these acceptors is considered.     

Chloroplast and cytoplasmic enzymes 1, 2, 3. Subunit structure of pea leaf aldolases.

The pea leaf chloroplastic and cytoplasmic forms of aldolase are very similar in structure. The subunit molecular weights determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate are approximately 37.000. Both aldolases appear to terminate in the same sequence, SerAlaTyrCOOH, and the amino-terminal sequence H₂NGlySerTyrAla was obtained for each. The previously reported differences in kinetic properties and in isoelectric points of the two pea leaf enzymes probably are the consequence of minor differences in amino acid composition or conformation.     

The uptake of cyclic AMP by human erythrocytes and its effect on membrane phosphorylation.

The effects of time and cyclic AMP concentration on cyclic AMP uptake and membrane phosphorylation were studied using intact human erythrocytes. The rate of uptake of cyclic [³H]AMP was nearly linear with respect to cyclic AMP concentration. The amount taken up was small compared to the extracellular cyclic AMP concentration, but was sufficient to significantly increase the intracellular cyclic AMP concentration. Incubation with cyclic AMP resulted in increased incorporation of ³²P_i into several phosphorylated membrane peptides of the intact erythrocytes. Although cyclic AMP altered the distribution of radioactivity among the membrane components, the total amount of incorporation was not increased. The effect of cyclic AMP on phosphorylation of membrane peptides was observed with extracellular cyclic AMP concentrations as low as 1 μm and was most pronounced in incubations of 1 to 4 h. These results indicate that cyclic AMP can enter erythrocytes in sufficient amounts to alter the activity of cyclic AMP-dependent protein kinases, or to alter the rate of turnover of certain phosphorylated membrane peptides.     

Estrogenic regulation of uterine cyclic amp metabolism

Ovariectomized and ovariectomized, estrogen-treated (48 h) rats were injected intravenously with increasing doses of epinephrine. Uteri were frozen in situ 30 s later. Estrogen pre-treatment significantly increased the sensitivity of both cyclic AMP and phosphorylase to epinephrine. The cyclic AMP response to intravenous injection of the pure β-agonist, isoproterenol, was enhanced by estrogen pre-treatment (48 h) and the cyclic AMP response of isolated uteri treated with epinephrine in vitro was also enhanced by in vivo estrogen pre-treatment (48 h). Other groups of ovariectomized rats were treated with estrogen and cyclic AMP levels were estimated at various times after estrogen treatment. 6 h after intraperitoneal injection and 48 h after subcutaneous injection, estrogen caused 20 and 30% increases in cyclic AMP. Estrogen had no effect on cyclic AMP 30 s after intravenous injection or 15 min after intraperitoneal injection. The was also no change in uterine catecholamine sensitivity 30 s after intravenous estrogen injection.The uterine site(s) at which estrogen acts to alter uterine cyclic AMP metabolism could be uterine β-adrenergic receptors, adenyl cyclase, and/or phosphodiesterase.     

Purification and characterization of human lung calmodulin-independent cyclic AMP phosphodiesterase

A high-affinity calmodulin-independent cyclic AMP phosphodiesterase was purified to homogeneity from human lung tissue. This enzyme has a molecular weight of 60,000, a sedimentation coefficient of 3.2–3.4 S, and an isoelectric pH of 4.6–4.8. Neither Ca²⁺ nor calmodulin (in the presence or absence of added Ca²⁺) stimulates the enzymatic activity. This enzyme appears to be very similar to that described previously from dog kidney (W. J. Thompson, P. M. Epstein, and S. J. Strada, (1979) Biochemistry18, 5228–5237). Hydrolysis of cyclic AMP is greatly enhanced by Mg²⁺ (25–30× at 10 mm Mg²⁺) and Mn²⁺ (20× at 10 mm Mn²⁺). Zn²⁺, Cu²⁺, and Co²⁺ are ineffective at these concentrations. Cyclic AMP is the exclusive substrate with a K_m of 0.7–0.8 μm. The I₅₀ of cyclic GMP is 1 mm using 1 μm cyclic AMP as substrate. In contrast, aminophylline, MIX, and SQ 20009 have I₅₀s of 0.28, 0.021, and 0.001 mm, respectively). The purified enzyme is susceptible to temperature inactivation and protease degradation. Significant (10%) inhibition is seen at 37 °C for 20 min. Trypsin, at 0.1 μg/ml, destroys 50% of the activity in 30 min at 25 °C. Our observations concerning its lability to temperature and proteases coupled with its lack of response to calmodulin suggest this enzyme is a basic catalytic subunit of other cyclic AMP phosphodiesterases present within human lung tissue.     

Amylase secretion by rabbit parotid gland role of cyclic AMP and cyclic GMP

Amylase secretion and changes in the levels of cyclic AMP and GMP were studied in rabbit parotid gland slices incubated in vitro with a variety of neurohumoral transmitters, their analogs and inhibitors. Cyclic GMP levels increased 8-fold 5 min after exposure to carbachol (10⁻⁴ M), without a change in cyclic AMP levels; amylase output also rose. These effects were completely inhibited by muscarinic blockade with atropine, but were unaffected by α-adrenergic blockade with phenoxybenzamine. Epinephrine (4 · 10⁻⁵ M) produced a rapid increase in the levels of both cyclic nucleotides and in amylase release. The increase in cyclic GMP level was inhibited by previous exposure of the slices to phenoxybenzamine, while the cyclic AMP rise was prevented by the β-blocking agent, propranolol. Pure α-adrenergic stimulation with methoxamine (4 · 10⁻⁴ M) produced modest elevations in cyclic GMP content and amylase output, effects blocked by pre-treatment of slices with either atropine or phenoxybenzamine. At a concentration of 4 · 10^{−6 M}, isoproterenol (a β-agonist) failed to affect cyclic GMP levels, but promptly stimulated increases in cyclic AMP levels, and after a short lag, amylase secretion. At a higher dose (4 · 10⁻⁵ M) isoproterenol produced elevations in the levels of both nucleotides. The carbachol-induced effects on cylcic GMP content and amylase release were greatly potentiated by the addition of isoproterenol (4 · 10⁻⁶ M).These data strongly suggest that cholinergic muscarinic agonists and α-adrenergic agonist stimulate amylase output in rabbit parotid gland by mechanisms involving cyclic GMP. The atropine-sensitive intracellular events effected by α-stimulation may be dependent upon endogenous generation of acetylcholine. Both cyclic nucleotides seem to be required for the early rapid secretion of amylase. The unique responses achieved by the combination of carbachol and isoproterenol suggest that isoproterenol may increase the sensitivity of this issue to the effects of cholinergic stimuli.     

Developmental changes in the sensitivity of embryonic heart cells to tetrodotoxin and D600

During Days 4 to 7 in ovo, beating of embryonic chick hearts becomes progressively more sensitive to inhibition by tetrodotoxin, an inhibitor of fast Na⁺ channels, and progressively less sensitive to inhibition by D600, an inhibitor of slow Ca2+/Na+ channels. The developmental change in tetrodotoxin sensitivity is not retained in heart cells cultured in monolayer. In contrast, the developmental change in D600 sensitivity is retained. Veratridine-stimulated ²²Na⁺ influx mediated by fast Na⁺ channels is inhibited by tetrodotoxin (K_i = 1.6 nM) in cells prepared from either 3-day or 12-day embryos. These results suggest that young embryonic hearts contain physiologically inactive Na⁺ channels. ²²Na⁺ influx mediated by slow Ca2+/Na+ channels is inhibited by D600 with a K_i of 40 nM for cells from 3-day hearts and 8 μM for cells from 12-day hearts. Beating of heart cells in aggregate cultures is also inhibited by D600. Aggregates which have reactivated after inhibition by tetrodotoxin are 10-fold more sensitive to inhibition by D600 than untreated controls. The results suggest that the primary developmental event is a change in slow Ca2+/Na+ channels which reduces their sensitivity to D600 and diminishes their ability to support beating without the activity of the fast Na⁺ channel.     

Effects of indomethacin on intestinal secretion, prostaglandin E and cyclic AMP: Evidence against a role for prostaglandins in cholera toxin-induced secretion

The effects of indomethacin on intestine mucosal cAMP, intestinal fluid secretion, and mucosal and fluid PGE were studied in rabbits in vivo following challenge with cholera toxin. Indomethacin had no effect on cholera toxin-induced fluid secretion or cAMP accumulation. Inhibition of PGE synthesis was achieved by the administration of two but not one injection of indomethacin. These studies provide evidence against a role for PGE in mediating cholera toxin-induced secretion and point out the need to measure prostaglandin levels when using prostaglandin synthetase inhibitors in vivo.     

Estradiol binding by intact cells isolated from DMBA-induced mammary tumors of the rat

Study of hormone binding in intact cells enables one to examine binding under conditions which elicit a biological response. Cells from 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary tumors of the rat were enzymatically dispersed. More than 80% of these cells excluded trypan blue and were used to study binding of [3H] estradiol-17 beta. Specific binding was determined by subtracting the amount of [3H]estradiol bound in the absence and presence of 200-fold excess unlabeled estradiol. Specific binding at 37 degrees was maximal after 15 min. Steroid competition studies indicated that [3H]estradiol binding sites were relatively specific for estrogens, although there was a 9-18% inhibition of binding by androgens and progestins when present at 150-fold molar excess. Scatchard analyses of [3H]estradiol (0.15-5.0 nM) binding by whole cells suggest a single, high-affinity binding site (Kd = 7.5 x 10-10M) of low capacity (6.1 fmol/10(6) cells). More [3H]estradiol was translocated to the nucleus after 1 hr at 37 degrees than at 0 degrees. Preliminary studies indicated that incubations at 37 degrees result in appreciable metabolism of [3H]estradiol to other steroids and/or conjugates when examined by silica gel thin layer chromatography.     

Lymphokines in sensitized rats. III. Influence of prednisolone treatment of lymph node and spleen architecture in sensitized rats and upon MIF release in vitro.

The influence of prednisolone (corticosteroid, C.S.) treatment upon established cell-mediated immunity (CMI) induced by complete Freund's adjuvant (CFA) has been studied in rats by using in vitro migration inhibitory factor (MIF) assays, type IV skin reactions, and regional lymph node and spleen histology. Additionally, changes in the mononuclear-polymorphonuclear ratio of peripheral blood and T-cell accumulation in bone marrow in response to C.S. treatment have been determined. These results have been evaluated by comparison with equivalent experiments upon animals treated with anti-rat lymphocyte serum (ALS), oxisuran, or 2-[(methylsulfinyl)-acetyl]pyridine, which selectively suppresses CMI. The results suggest the existence of a population of “educated” T-cells in the thymic cortex of sensitized rats, and they suggest that prolonged C.S. administration does not suppress T-effector cells involved in established CMI but, rather, affects lymphocyte and monocyte migration patterns, including the migration of educated T-cells from the thymic cortex into other tissue compartments.