Regulatory substances produced by lymphocytes. V. Production of inhibitor of DNA synthesis (IDS) by proliferating T lymphocytes.

The conditions neccessary for production of inhibitor of DNA synthesis (IDS) by rat lymphocytes were investigated.In concanavalin A (Con A)-stimulated lymph node cell (LNC) cultures, IDS production was not detected in the culture supernatant during the first 24 hr, and it increased gradually after that to reach a maximum at 3 to 4 days.When the cells were pretreated with mitomycin C, IDS was not produced, suggesting that DNA synthesis of LNC or a LNC subpopulation is necessary for IDS production. In contrast, Con A-stimulated spleen cells priduced a high level of IDS within 24 hr, and its production fell off sharply thereafter. Con A-stimulated rat thymocytes also produced IDS reaching a maximum at 2 to 3 dyas. However, thymus cells from rats treated with hydrocortisone 48 hr previously did not produce IDS. This finding implies that cortisol-sensitive (cortical) thymocytes are capable of producing IDS and cortisol-resistant (medullary) thymocytes are not. IDS production by lymphoblasts was proportional to cell number and unaffected eith by cell density (1 to 10 x 106/ml) or by the concomitant presence of normal cells from spleen, lymph node, or thymus. Thus Con A-stimulated cells, after becoming blasts, appear to produce IDS automatically wihtout affecting or being affected by other cells. Both spleen and thymus cells from rats injected with a large dose of antigen (ovalbumin, 100 mg, i.p.) 24 hr in advance produced substantial amounts of IDS in culture within 24 hr in the absence of mitogen or additional antigen, but not the cells from rats injected with an immunizing dose (1 mg) of the same antigen. The cells producing IDS in the spleen were shown to be adherent to glass wool, and those in the thymus were partially so. IDS production by antigen-stimulated spleen cells was abrogated by injecting rats with bromodexyuridine (BUdR) at 0 and 12 hr after the ovalbumin. These findings suggest that a subpopulation ofadherent spleen cells (possibly resembling cortical thymocytes), which begins to proliferate within a few hours after a large dose of systemic antigen, produces IDS. This may account for increased nonspecific suppressor activity observed at the same time.     

The effect of chronic and acute dietary restriction on the growth and protein turnover of fast and slow types of rat skeletal muscle

Changes in the growth and protein turnover of the anterior tibialis and soleus muscles were studied in response to acute and chronic dietary restriction (50% of ad libitum intake) between 3 and 149 weeks post partum. The effect of long-term dietary restriction from weaning to senescence was to retard the growth and normal developmental of the two types of skeletal muscle. This was evident from measurements of various parameters of growth, i.e. total protein, RNA and DNA and protein/DNA-P, which were reduced by approximately 50% when compared with age-matched controls. These decreases, however, were not accompanied by a decline in the fractional rate of synthesis (%/day) or ribosomal activity (mg protein/day per mg RNAP). The slowing down of the age-related decline in muscle growth has been attributed to a reduction in RNA capacity (RNA/protein), with similar responses in the fast- and slow-twitch skeletal muscles. The initial effects of piecemeal feeding of this restricted diet on the two types of muscle were also monitored. Short term starvation effects, i.e. 24 hr after feeding a reduced ration, were measured on the protein content and RNA/protein of both the anterior tibialis and soleus muscles; both parameters were unchanged within 24 hr. In contrast, a rapid and significant decline in the ribosomal synthetic activity (mg/d per mg RNAP), and a corresponding fall in the fractional rate of synthesis, occurred within 24 hr of feeding.     

Effect of 5-fluoro-2′-deoxyuridine and hydroxyurea on the phytohemagglutinin-induced increase of thymidine kinase,replicative DNA polymerase,deoxycytidylate deaminase and CDP reductase activities in human lymphocytes

Summary The inhibitors of DNA synthesis, 5-fluoro-2-deoxyuridine and hydroxyurea, caused an inhibition of thymidine kinase, replicative DNA polymerase and CDP reductase activities in stimulated lymphocytes when they were exposed to the inhibitors during the early transformation period (0–17 hr). However, the enzyme activities were unaffected when the inhibitors were added to cells stimulated for more than 17 hr. As opposed to these enzymes the deoxycytidylate deaminase activity was unaffected by the inhibitors during the entire transformation period (0–28 hr). This indicates a close regulatory mechanism in lymphocytes between DNA synthesis and induction of enzymes involved in DNA replication. The inhibitory mechanism exerted by the inhibitors is for the moment unknown. It might be independent of the well-known inhibition of the target enzymes, thymidylate synthetase and ribonucleoside diphosphate reductase, since there was no immediate apparent correlation in time between depletion of the pool sizes and the inhibition of the enzyme activities.     

Dissociation of differentiation and proliferation in the primary induction of cytolytic T lymphocytes by alloantigens

The requirement for DNA synthesis in the induction of cytolytic T lymphocytes (CTL) by alloantigens has been investigated. C57BL/6 splenic T cells purified by passage on nylon wool columns were stimulated in vitro in mixed leukocyte culture (MLC) and assayed for cytotoxicity against 51Cr-labeled target cells. With this system, CTL activity was detectable after 24 hr of MLC and reached high levels after 48 hr. Addition of cytosine arabinoside (ARA-C) or hydroxyurea to such cultures at concentrations that were sufficient to inhibit DNA synthesis by greater than 98% did not reduce CTL activity measured after 24 hr; however, the increase in activity that occurred between 24 and 48 hr in control cultures was strongly reduced (or abolished) by these drugs. Velocity sedimentation analysis of MLC cells activated for 48 hr in the presence of ARA-C further revealed that CTL precursor lymphocytes had enlarged into medium- to large-sized CTL under these conditions. These studies provide direct evidence that the primary induction of CTL by alloantigens can be dissociated into a differentiation step, which occurs within 24 hr in the absence of DNA synthesis and is accompanied by blast transformation, and a subsequent proliferation.     

Culture duration alters the glutathione content and sensitivity to ethacrynic acid of rat hepatocyte monolayer cultures

Many of the differentiated functions of hepatocytes are lost in culture, yet addition of certain medium supplements can aid in the retention of differentiated character. Therefore, the effect of time in monolayer culture on rat hepatocyte glutathione (GSH) synthesis and sensitivity to the GSH detoxicated xenobiotic ethacrynic acid was examined in cultures with and without medium supplementation by transferrin and sodium selenite. GSH content was found to be about 12 nmol/µg DNA at 4 hr in culture and to approximately triple by 24 hr. Intracellular GSH levels continued to increase in transferrin/sodium selenite-supplemented cultures, from 32 to 41.6 nmol/µg DNA, while GSH levels in unsupplemented cultures declined to 18 nmol/µg DNA. However, the rate of GSH synthesis after diethylmaleate depletion was found to decrease from 4.2 to 2.8 nmol/hr/µg DNA at 4 and 24 hr after inoculation, respectively. GSH repletion rate increased to 3.9 nmol/hr/µg DNA at 48 hr. The GSH accumulation rate after depletion in supplemented cultures did not vary significantly over the initial 48 hr. Incubation for 3 hr with 100 µM ethacrynic acid (EA) did not elicit an increase in LDH leakage in hepatocyte monolayers after 4 or 48 hr in culture or in cultures with supplemented medium at any time point tested. Cultures 24 hr in medium without transferrin/sodium selenite supplementation exhibited significant LDH leakage after 3 hr of EA treatment. Over the 3 hr EA treatment, intracellular GSH content was decreased in all cultures. Only in the 24 hr unsupplemented cultures did GSH depletion exceed the 90% level previously associated with depletion of the mitochondrial pool of GSH and EA toxicity in hepatocytes. The experiments show that during the redifferentiation of hepatocytes in culture, a transient period occurs when apparent GSH synthesis is depressed and enhanced sensitivity to GSH-detoxicated compounds is observed. This period of increased sensitivity is prevented or at least delayed by inclusion of supplemental transferrin and sodium selenite, suggesting that redifferentiation can be regulated by extracellular influences.Abbreviations CYSSG cysteine-glutathione mixed disulfide - DEM diethyl maleate - EA ethacrynic acid - GSH reduced glutathione - GSSG oxidized glutathione - HBS HEPES buffered saline - HWME hepatocyte Williams' Medium E (WME with insulin, corticosterone and 0.5 mM methionine) - LDH lactate dehydrogenase - TS-HWME transferrin/sodium selenite-supplemented HWME - WME Williams' Medium E     

Inhibition of growth of ASL-1w murine leukemia cells by anti-thymus leukemia antigen (TL) serum in the absence of complement.

Previous studies have shown that the cell surface expression of thymus leukemia antigen (TL) on ASL-1w leukemia cells varies with the progression of the cells through the growth cycle. Expression of TL is maximal in S phase, and its quantitative expression varies directly with DNA synthesis. In the present study, the effect of anti-TL serum on the growth of ASL-1w cells was examined. The antiserum, tested in the absence of complement, affected the growth of these cells in biphasic manner. When the antiserum concentration was 0.1% or greater, there was a rapid decline in the rate of DNA synthesis, and after 5 to 7 hr, cell death. When the antiserum concentration was 0.067% or less, the decline in the rate of synthesis of DNA did not become apparent until 5 to 6 hr after treatment. Under these conditions, there was approximately a 20% increase in cell number after 24 hr of culture. The hypothesis that treatment of ASL-1w cells with the lesser concentration of anti-TL serum blocks the cells in G2 phase of the cell cycle is discussed.     

Effects of gamma radiation and hyperthermia on DNA repair synthesis and the level of NAD+ in cultured human mononuclear leukocytes

DNA repair has been investigated, estimated by unscheduled DNA synthesis (UDS) and the cellular NAD+ pool, after exposing human mononuclear leukocytes to hyperthermia and gamma radiation separately and in combination. It was found that gamma radiation induced a decline in UDS with increasing temperature through the temperature region studied (37-45 degrees C). At 42.5 degrees C the gamma-ray-induced UDS was reduced to about 70% of that at 37 degrees C. Following gamma-ray damage the NAD+ pool dropped to about 20% of control values. Without hyperthermic treatment the cells completely recovered to the original level within 5 hr. Moderate hyperthermia (42.5 degrees C for 45 min) followed by gamma-ray damage altered the kinetics so that even after 8 hr the NAD+ pool had recovered to only 70% of the original level. After heat treatment at 44 degrees C for 45 min prior to gamma radiation the cells did not recover at all, presumably because of the cytotoxic effects from the combined treatment.     

Synthesis of sterol and phospholipid induced by the interaction of phytohemagglutinin and other mitogens with human lymphocytes and their relation to blastogenesis and DNA synthesis

Human peripheral blood lymphocytes (PBL) responded to phytohemagglutinin (PHA) and a variety of other mitogens by increased synthesis of sterol and phospholipid. This activity was established within 4–7 hr of the addition of mitogen and was dependent upon the binding of the ligand to the cell membrane. Sterol and phospholipid synthesis reached a peak at approximately 24 hr in association with blastogenic expansion of the lymphocyte membrane and initiation of DNA synthesis. Lipid synthesis and blast transformation occurred independently of replication of the genome since inhibition of DNA synthesis did not reduce the degree of blast transformation and lipid synthesis observed. However, inhibition of sterol synthesis using 20α-hydroxycholesterol resulted in decreased blastogenesis and DNA synthesis, demonstrating that early synthesis of lipid is important for these subsequent events. Human thymocytes responded to T-cell mitogens in the conventional manner as regards synthesis of lipid and blast transformation; however, they did not synthesize DNA. Possible reasons for this incomplete response are discussed. Several nonmitogenic agents which agglutinate lymphocytes were also found to initiate early increases in sterol and phospholipid synthesis, and the possible significance of this observation is considered.     

Liproprotein inhibitor of bone marrow cells in tumor-bearing rats.

The role of a plasma inhibitor of erythropoiesis is evaluated in rats with Walker-256 carcinoma (W-256). Plasma from tumor-bearing rats was treated by gel filtration chromatography (Sephadex G-150) and fractions were combined into four pools on the basis of mol. wt. Inhibitory activity was assayed by adding an aliquot of the plasma fractions to normal rat marrow cells which were cultured for 24 hr with and without erythropoietin. 59Fe-heme synthesis, [3H]thymidine DNA synthesis, and 14C-leucine protein synthesis were studied. The results indicated that cultures containing the high mol. wt. pool (greater than 400,000 daltons) had significantly decreased heme, DNA and protein synthesis. This inhibitor also diminished the response to erythropoietin in polycythemic mice. The lower mol. wt. pool stimulated heme synthesis in vitro. To identify the inhibitor further, plasma lipoprotein classes were isolated by density gradient ultracentrifugation. The very low density lipoprotein (VLDL) and chylomicron fractions markedly inhibited DNA, protein and heme synthesis. Low density and high density lipoprotein fractions were inactive. A lipoprotein inhibitor of erythropoiesis was also identified in cancerous ascitic fluid, and to a lesser degree, in normal rat plasma. We suggest that this VLDL inhibitor of marrow erythropoiesis is a contributing factor in the anaemia of cancer.     

Acrylonitrile interaction with testicular DNA in rats.

In the present study we report the in vivo interaction of acrylonitrile (VCN) with testicular tissue in rats. Covalent binding of radioactivity to testicular tissue DNA was examined for a period of 72 hr after a single oral dose (46.5 mg/kg) of [2,3-14C] VCN. Maximal covalent binding was observed at 0.5 hr (8.9 mumol VCN equivalent/mol nucleotide). Binding decreased gradually thereafter but was still detected (2.5 mumol VCN equivalent/mol nucleotide) at 72 hr following VCN administration. Further, we examined the effects of VCN on DNA synthesis and repair in the testes of rats following a single oral dose (46.5 mg/kg) of VCN to clarify the impact of the covalent binding observed on the testicular genetic material. A significant decrease in DNA synthesis (80% of control) was observed at 0.5 hr after treatment. At 24 hr following acrylonitrile administration, testicular DNA synthesis was severely inhibited (38% of control). Testicular DNA repair was increased 1.5-fold at 0.5 hr and more than 3.3-fold at 24 hr following treatment with VCN. These results suggest that VCN can act as a multipotent genotoxic agent by alkylating DNA in testicular tissue and may affect the male reproductive function by interfering with testicular DNA synthesis and repair processes.     

Growth factor stimulated cell proliferation is accompanied by an elevated labile intracellular pool of zinc in 3T3 cells

Growth factors such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and insulin-like growth factor-I (IGF-I) are required for quiescent 3T3 cells to proliferate, but zinc deprivation impairs IGF-I-induced DNA synthesis. We recently showed that labile intracellular pool of zinc is involved in cell proliferation. Our objective was to determine whether the labile intracellular pool of zinc plays a role in growth factor (PDGF, EGF, and IGF-I)-stimulated proliferation of 3T3 cells. Quiescent 3T3 cells were cultured in DMEM with or without growth factors. Labile intracellular pool of zinc, DNA synthesis, and cell proliferation were assessed using fluorescence microscopy, 3H-thymidine incorporation, and total cell number counts, respectively. After 24 h, growth factors stimulated DNA synthesis (24%) but not cell proliferation. After 48 h, growth factors stimulated both DNA synthesis (37%) and cell proliferation (89%). In response to growth factor stimulation, the labile intracellular pool of zinc was also elevated after 24 or 48 h of treatment. In summary, growth factor (PDGF, EGF, and IGF-I)-stimulated increase in DNA synthesis and cell proliferation were accompanied by an elevated labile intracellular pool of zinc in 3T3 cells. Since elevation of the labile intracellular pool of zinc occurred along with increased DNA synthesis, but cell proliferation remained unchanged, the elevation of the labile intracellular pool of zinc likely occurred during the S phase to provide the zinc needed to support DNA synthesis and ultimately cell proliferation.     

Biosynthesis of Host and Viral Deoxyribonucleic Acid During Hyperplastic Fowlpox Infection In Vivo

The synthesis of deoxyribonucleic acid (DNA) during in vivo infection of chick epithelium with fowlpox virus was examined by incorporation of tritiated thymidine into the acid-insoluble fraction. The proportion of precursor incorporated into host and viral DNA at various times after infection was determined by chromatography on columns of methylated albumin-kieselguhr. The first 60-hr period of infection was characterized by the synthesis of predominantly host DNA, the rate of production of which increased markedly over the control between 36 and 48 hr postinoculation (PI). Although the replication of viral DNA began between 12 and 24 hr PI, the rate of synthesis was very low during the first 60 hr. In contrast, an abrupt increase in the rate of viral DNA synthesis occurred between 60 and 72 hr PI, concomitantly with a sharp decline of host DNA synthesis. Subsequently, between 72 and 96 hr, the ratio of synthesis of viral DNA to host DNA progressively increased to a maximum of greater than 2:1. The temporal relationship of this biphasic pattern of host and viral DNA synthesis to hyperplasia and viral replication is discussed.     

Acrylonitrile interaction with testicular DNA in rats

In the present study we report the in vivo interaction of acrylonitrile (VCN) with testicular tissue in rats. Covalent binding of radioactivity to testicular tissue DNA was examined for a period of 72 hr after a single oral dose (46.5 mg/kg) of [2, 3-¹⁴C] VCN. Maximal covalent binding was observed at 0.5 hr (8.9 μmol VCN equivalent/mol nucleotide). Binding decreased gradually thereafter but was still detected (2.5 μmol VCN equivalent/mol nucleotide) at 72 hr following VCN administration. Further, we examined the effects of VCN on DNA synthesis and repair in the testes of rats following a single oral dose (46.5 mg/kg) of VCN to clarify the impact of the covalent binding observed on the testicular genetic material. A significant decrease in DNA synthesis (80% of control) was observed at 0.5 hr after treatment. At 24 hr following acrylonitrile administration, testicular DNA synthesis was severely inhibited (38% of control). Testicular DNA repair was increased 1.5-fold at 0.5 hr and more than 3.3-fold at 24 hr following treatment with VCN. These results suggest that VCN can act as a multipotent genotoxic agent by alkylating DNA in testicular tissue and may affect the male reproductive function by interfering with testicular DNA synthesis and repair processes.     

Effects of salinity on synthesis of DNA,acidic polysaccharide,and ichthyotoxin in Gymnodinium breve

A Florida red tide organism, Gymnodinium breve Davis, an unarmored dinoflagellate, was grown in enriched sea water media at salinities 20–43% and constant illumination. Use of lowest (23%) and highest (43%) salinities resulted in death within 24 hr of inoculation, though good growth was obtained at all intermediate salinities (29–39%), in accord with field observation. Rates of synthesis of DNA, acidic polysaccharide and ichthyotoxin were determined as a function of salinity and growth constant (K₁₀). The relative rate of' synthesis of DNA or polysaccharide increased linearly with growth constant. Mean cell volumes, determined during log-phase growth, showed a positive correlation with doubling time. Hemolytic activity was detected in cell extracts only at high toxin concentrations (0.35–2.05 mg of ichthyotoxin). No significant difference was noted in hemolytic activity of extracts of cells grown in high (34%) or low (26%) salinity. The rate of toxin synthesis showed a linear decrease with the rate of DNA or polysaccharide synthesis.     

Kinetics of IL-2 and interferon-gamma production, expression of IL-2 receptors, and cell proliferation in human mononuclear cells exposed to staphylococcal enterotoxin A

Staphylococcal Enterotoxin A (SEA) at picogram amounts induces high levels of interleukin 2 (IL-2) and interferon in human mononuclear cells. SEA is a stronger inducer of IL-2 than phytohemagglutinin, leukoagglutinin, and concanavalin A. The IL-2 induction is very rapid with maximal levels being reached after 18 to 24 hr. The IL-2 concentration decreases rapidly and almost no IL-2 activity can be detected in supernatants of cells cultured for 3 days or more. Maximal DNA synthesis is recorded 3 days after maximal IL-2 levels have been reached in the culture medium. The DNA synthesis shows a 24 hr delay as compared to the expression of the IL-2 receptor during the initiation phase. An increase in the level of IL-2 receptor expression is apparent as early as 12 hr after stimulation with SEA and maximal expression is reached 48 to 72 hr after stimulation. The percentage of cells expressing the IL-2 receptor is maximal at 96 hr after onset of culture but the surface concentration of the receptor is lower than at 72 hr. The decline in expression of the IL-2 receptor is accompanied by a decline in mean cell size and in DNA-synthesis. The concentration of the T-cell marker T11 increases in parallel with the growing expression of the IL-2 receptor. It remains increased over a longer period than the IL-2 receptor and is still significantly augmented after 10 days' exposure to SEA.     

Inhibition by specific monosaccharides of interleukin 2-induced thymocyte proliferation

When rat thymocytes are cultured for 3 days in serum-free medium and are stimulated to divide by interleukin 2 (IL 2), concanavalin A, or sodium periodate oxidation, addition to the medium of 10–25 mMd-ribose, 2-deoxy-d-ribose, or N-acetyl-d-galactosamine inhibits by 40% or more the incorporation of [³H]thymidine. d-ribose and lectin-free IL 2 generated from sodium periodate oxidation of rat spleen cells were used to study the characteristics of this inhibition and to test possible mechanisms of inhibition. Viability of thymocytes cultured with d-ribose is similar to that of cells cultured without this sugar. In order to be inhibitory, d-ribose has to be added to the cultures within the first 24 hr, and the inhibition can be prevented if the sugar is removed 18–24 hr after the start of culture. d-Ribose does not block the absorption of IL 2 by unstimulated rat thymocytes or by concanavalin A-generated thymic or splenic blast cells. When thymocytes are cultured with d-ribose for 24 hr, inactivated with mitomycin C, and then cultured for 3 days with fresh mitogenically stimulated cells, [³H]thymidine incorporation into the latter is not altered. This suggests that the sugar does not generate suppressor cells or suppressor supernates. d-Ribose does not appear to be a general metabolic inhibitor since [³H]leucine incorporation into thymocyte proteins and the release of [³H]leucine into medium after a 2-hr. [³H]leucine pulse are not altered by d-ribose. Trivial or artifactual effects (nonspecific cytotoxicity, changes in thymidine transport, or changes in isotonicity of the culture medium) cannot explain the inhibition. A hypothetical mechanism of inhibition is discussed.     

Further studies on phosphorylation and the thiol/disulphide ratio of histones in growth and development

1. The proportion of thiol groups in the total thiol+disulphide of histone extracts from fertilized eggs from Echinus and Psammechinus was increased during periods of structural alterations in the nucleus. 2. The probable start of DNA synthesis in the fertilized eggs coincided with periods of maximum thiol content. 3. Histone extracts from rat liver and regenerating liver were predominantly in the thiol form and no significant variations could be detected during the first 30hr. after partial hepatectomy. 4. An assay system was developed to follow the phosphorylation believed to be associated with the arginine-rich histone F3. Phosphorylation increased by 50% at 1 and 2hr. after partial hepatectomy. The phosphate content also increased during the period of DNA synthesis. 5. The increased phosphorylation found 1hr. after partial hepatectomy was not prevented by actinomycin or prior irradiation. 6. The phosphate content of histone F1 was very high in livers from foetal rats and declined in neonatal rats similarly to the decline in DNA synthesis.     

The effects of gastrin, epidermal growth factor, and somatostatin on DNA synthesis in a small intestinal crypt cell line (IEC-6)

Exposure of IEC-6 cells for 24 hr to either gastrin (50-500 ng/ml) or EGF (100-500 ng/ml) significantly stimulated (100-165%) the rate of [3H]thymidine incorporation into DNA (referred to as DNA synthesis) when compared with the corresponding basal levels. Somatostatin (10-500 ng/ml) produced no apparent change in DNA synthesis in IEC cells. On the other hand, somatostatin completely inhibited the EGF-induced rise in DNA synthesis. The gastrin-mediated stimulation in DNA synthesis was not affected by somatostatin. The rate of DNA synthesis in IEC cells in the presence of both gastrin and EGF was found to be greater (additive) than that caused by either of the peptides alone. A similar but less dramatic change in the actual number of cells (assessment of cell replication) was observed when the IEC cells were exposed for 24 hr to gastrin, EGF, and somatostatin, either alone or in combination. Whereas gastrin (250 ng/ml) and EGF (250 ng/ml) by themselves increased the number of cells significantly by 29 and 37%, respectively, together they caused a 72% stimulation, when compared with the basal levels. Somatostatin by itself caused no apparent change in IEC cell population, but it significantly inhibited the EGF- but not the gastrin-induced stimulation in IEC cell replication. It is concluded that both gastrin and EGF exert a direct proliferative effect on IEC cells, and the EGF action is regulated by somatostatin.     

Mechanisms of lymphocyte "deletion" by high concentrations of ligand. I. Cyclic AMP levels and cell death in T-lymphocytes exposed to high concentrations of concanavalin A

DNA synthesis, cell survival, and cyclic AMP (cAMP) levels were compared in whole and purified lymph node cells (LNC) cultured with optimal (5 μg/ml) and excess (200 μg/ml) concentrations of native (N) or succinyl (S) concanavalin A (Con A) as possible models for antigen-induced lymphocyte activation and “high-dose” tolerance. Whole LNC cultured with optimal N-Con A or S-Con A showed continuing DNA synthesis and cell viability between 30 and 50% at 48 hr. In contrast, with excess N-Con A, they showed virtually no [³H]TdR uptake at this time and there was progressive loss of cell viability beginning at 8 hr; by 48 hr almost no viable cells remained. Excess S-Con A induced little cell death up to 24 hr, but by 48 hr only 20% of the cells initially placed in culture remained alive and sythesized DNA. Intracellular cAMP showed a transient rise in cultures stimulated with optimal N-Con A, peaking at 15 min, then returning to normal levels, to rise again between 24 and 48 hr. With excess N-Con A, cAMP rose within 15 min and continued to increase to a peak at 24 hr. cAMP levels in the presence of excess S-Con A remained at control levels for the first 24 hr and increased between 24 and 48 hr. LNC depleted of macrophages and B cells, when cultured in excess N-Con A, had an inhibition of DNA synthesis, elevated cAMP levels, and cell death comparable to whole LNC. It seems unlikely, however, that the increase in cAMP mediates cell killing since cAMP was not elevated yet cell death occurred in nylon wool-purified T cells exposed to excess N-Con A. Dibutyryl cAMP, and prostaglandin E₁, which markedly increase cAMP levels, failed to kill LNC at doses which totally inhibited DNA synthesis, and cells of the mouse T-lymphoma S49 and its cAMP-dependent protein kinase-deficient variant were killed equally by excess N-Con A. It is suggested that a sustained elevation of either cAMP or Ca²⁺ after early commitment may provide a significant mechanism of tolerogenesis.     

Cytomegalovirus-induced membrane antigens in productively infected cells.

Adsorbed but not penetrated virus can be removed from the CMV-infected cell membrane by digestion with cystine-activated papain. Membrane antigens appear on 80-90% of the infected cells 14-20 hr after infection as a result of de novo protein synthesis. Antigen synthesis can be blocked with inhibitors of protein synthesis, but not with DNA inhibitors. In the early stage of infection, pooled human convalescent serum reacted well with the membrane antigen, whereas pooled antiserum of rabbits immunized with CMV virion suspension gave a positive reaction with a small proportion of the cells. After the 48th hr, both the human and the rabbit serum pool reacted with the membrane of the infected cells. Absorption with cell cultured for 24 hr after CMV infection reduced the neutralization titres of the antisera only slightly but the titre reduction was considerable when absorption was performed with cells cultured for more than 48 hr after infection. It is concluded that on the membrane of cells productively infected by CMV at least two membrane antigens are present, one coded for by the DNA of the parent virus and another which is the product of the DNA of the virus progeny. The two antigens can be differentiated serologically.