Effect of triiodothyronine on nucleic acid synthesis of immunocytes in cell culture

A direct action of T3 on the synthesis of nucleic acids in immunocytes in a cell culture was studied. Scintillation radiometry has shown that T3 makes a regulatory effect on anabolism of nucleic acids in thymocytes and peripheral blood mononuclears at certain stimulating doses of the hormone. T- and B-splenocytes were characterized by metabolic inertness with relation to T3. Serum absence in a culture medium did not prevent the activation of RNA and DNA synthesis in thymocytes. Optimum mitogenic doses of the hormone in a serum-free medium were lower than those in a conditioned culture medium. A study of the kinetics of RNA and DNA synthesis activation by T3 has shown that a regulatory action of the hormone results in the activation of RNA synthesis "triggering". This conclusion is confirmed by evidence on inhibition of G0-G1 transition of the cellular cycle by antiserum to T3. T3 influence on DNA synthesis activation is mediated by a rise of the level of cellular RNA. It produces no direct effect on the G0-G1 phase of the cellular cycle.     

On the reversibility of some cytological ageing parameters of rat brain cells by phytohemagglutinin.

It has been shown that the thermal stability of DNA in the brain cortical cells increases with the age. At the same time, the number of perichromatin granules being connected in some way to the messenger RNA synthesis and transport of the interphase nuclei, decreases in the same cells. In vivo treatment of the old animals by mitogens like phytohemagglutinin induced a reversal of both ageing phenomena: the thermal stability of DNA returned almost completely to that of the young animals, whereas the number of perichromatin granules rised again to the young level.     

FRACTIONATION OF PHYTOHEMAGGLUTININ, I. PURIFICATION OF THE RNA AND DNA SYNTHESIS-STIMULATING SUBSTANCES AND EVIDENCE THAT THEY ARE NOT PROTEINS

We have separated the RNA synthesis-stimulating activity and the DNA synthesis-stimulating activity of extracts of Phaseolus vulgaris (phytohemagglutinin) from the erythroagglutinins, leukoagglutinins, and cytotoxic factors. We have shown that the substances stimulating the synthesis of nucleic acids are probably not proteins, since they are not significantly affected by rigorous deproteinization or treatment by trypsin and pronase. Likewise, they do not appear to be nucleic acids, since they are not precipitated by perchloric acid and resist RNase, DNase, and phosphodiesterases. They are probably not carbohydrates, since 98 per cent of the carbohydrate can be removed from a preparation with only a moderate loss in activity. The RNA and DNA synthesis-stimulating activities are probably associated with separate molecules, since they are destroyed by periodate at different rates.     

Development of Cellular Immunity in the Human Fetus: Dichotomy of Proliferative and Cytotoxic Responses of Lymphoid Cells to Phytohemagglutinin

The reactivity of cells in vitro was investigated with specimens from various lymphoid organs of seven human fetuses. Thymocytes responded to stimulation by phytohemagglutinin with significant increases in synthesis of DNA, but failed to produce destruction of xenogeneic target cells. In cells from bone marrow, precisely the converse pattern of reactivity to the mitogen was detected. Lymphocytes from spleen and peripheral blood demonstrated both phytohemagglutinin-dependent functions, while hepatic cells did not respond to phytohemagglutinin. Based on the striking dichotomy of phytohemagglutinin-dependent responses in fetal thymocytes and bone-marrow lymphoid cells, we conclude that phytohemagglutinin-dependent cytotoxicity and DNA synthesis are functions of different populations of lymphoid cells during human embryonic development.     

DNA and RNA Synthesis in Cultures of Peripheral Blood Mononuclear Leucocytes from Normal and Lymphosarcomatous Cows

DNA and RNA synthesis was studied in bovine blood mononuclear leucocytes cultured with and without PHA.
Two of seven lymphosarcomatous cows showed a higher rate of spontaneous (i.e. without PHA) DNA and RNA synthesis, but one of these two showed no increase in DNA synthesis and a decrease in RNA synthesis in cultures with PHA. These two cows also had a large number of lymphoblasts in their peripheral blood in contrast to the other lymphosarcomatous cows and which did not show significantly different patterns of DNA and RNA synthesis from normal cows in cultures either with or without PHA, although some of the lymphosarcomatous cows had atypical lymphocytes and increased lymphocyte counts. Only in the two cows which had many lymphoblasts and a higher rate of spontaneous DNA and RNA synthesis, were there many dividing cells with abnormal chromosome complements.     

Effect of cholecystokinin and 16,16-dimethyl prostaglandin E2 on RNA and DNA of gastric and duodenal mucosa.

Fasted rats were injected with either cholecystokinin-octopeptide (CCK-OP), 20 mug per kg; 16,16-dimethyl prostaglandin E2 (16,16-dimethyl PGE2), 0.2 mg per kg; pentagastrin, 250 mug per kg, or saline every 8 hr for 48 hr. The rats were killed and the incorporation of [3H]thymidine into DNA as well as the total DNA and RNA content of the mucosa of the oxyntic gland area and the duodenum were determined. Pentagastrin increased DNA synthesis 60% (P less than 0.001) in gastric mucosa and 90% (P less than 0.001) in duodenal mucosa when compared with rates for saline controls. Neither CCK-OP nor 16,16-dimethyl PGE2 altered gastric mucosal DNA synthesis. Pentagastrin significantly increased the DNA and RNA content of both the gastric and duodenal mucosa. CCK-OP and 16,16-dimethyl PGE2 caused a slight but significant increase in duodenal DNA synthesis, CCK-OP did not significantly increase duodenal DNA content, and 16,16-dimethyl PGE 2 increased duodenal RNA but not DNA content. CCK-OP (20 mug per kg) in combination with pentagastrin did not alter the stimulation of gastric DNA synthesis but significantly decreased the effect of pentagastrin on duodenal DNA. A dose of CCK-OP (370 mug per kg) equimolar to 250 mug per kg of pentagastrin did not stimulate DNA synthesis in either tissue and significantly inhibited stimulation by pentagastrin in both tissues. Low doses of CCK-OP (2.5, 5.0, 10.0, 20.0 mug per kg) caused statistically significant increases in DNA synthesis and DNA content of the pancreas, but had no effect on either mucosa of the oxyntic gland area or duodenum. 16,16-Dimethyl PGE2 did not inhibit the stimulation of DNA synthesis or the increases in DNA and RNA content stimulated by pentagastrin. From these results it appears that: (1) moderate doses of CCK have a weak trophic effect in the duodenum but not in the stomach, (2) physiological doses of CCK-OP stimulated pancreatic DNA synthesis and increased pancreatic DNA content without affecting these parameters in the oxyntic gland area or duodenum in the same animals, (3) in the stomach and duodenum CCK is not as potent a trophic hormone as gastrin and inhibits, probably competitively, the trophic effects of gastrin, (4) 16,16-dimethyl PGE2 does not stimulate growth and does not interfere with the trophic response to gastrin even though it inhibits acid secretion, and (5) 16,16-dimethyl PGE2 increased the RNA content of duodenal mucosa indicating that it may stimulate activity resulting in hypertrophy.     

Ultraviolet B-induced DNA fragmentation (apoptosis) in activated T-lymphocytes and Jurkat cells is augmented by inhibition of RNA and protein synthesis.

UVB (280-320 nm) light profoundly affects cellular immunity and immunogenicity. To further characterize the interaction of UVB light with T cells, we examined UVB-induced DNA fragmentation patterns in normal T cells and the T-cell line Jurkat. Resting or preactivated peripheral blood T cells from normal donors or Jurkat cells were exposed to various doses of UVB or gamma irradiation. Cells were sampled at 0-48 h after exposure, and DNA fragmentation was analyzed by electrophoresis in agarose gels. UVB and gamma irradiation, in a dose- and time-dependent manner, induced DNA fragmentation in Jurkat cells and in T cells preactivated with phytohemagglutinin (PHA; 10 micrograms/ml) and phorbol myristate acetate (PMA; 10 ng/ml), but not in resting T cells. The presence of RNA or protein synthesis inhibitors such as actinomycin D or cycloheximide neither inhibited nor delayed DNA fragmentation; in fact, DNA fragmentation was augmented above control values. Similarly, DNA fragmentation increased in the presence of the calcium-chelating agent ethylene-glycol-bis-tetraacetic acid (EGTA) and decreased in the presence of the calcium ionophore A23187. In the presence of ethylenediaminetetraacetic acid (EDTA), DNA fragmentation decreased. In summary, these data show that UVB-induced DNA fragmentation strongly depends upon the cell activation status, and upon the presence of divalent cations other than calcium, possibly magnesium. The data indicate furthermore that in this model, inhibition of RNA or protein synthesis can induce rather than inhibit apoptosis, suggesting that the synthesis inhibitors disrupted primarily the synthesis or action of enzymes ordinarily aimed at repairing DNA fragmentation.     

Multiple initiation sites of DNA replication flanking the origin region of lambda dv genome.

Early replicative intermediates of lambda dv plasmid were prepared by an in vitro replication system in the presence of 2',3'-dideoxycytidine 5'-triphosphate, an inhibitor of DNA chain elongation. Short-chain DNAs produced from regions near the replication origin were purified from the intermediates. A fraction of the DNAs was covalently linked to primer RNA. The transition sites from primer RNA to DNA synthesis were mapped along the nucleotide sequence of the genome, by eliminating the RNA by alkaline hydrolysis and labeling the freshly exposed 5' ends of DNA with 32P. The transition sites were found to be located on both sides of the ori region, which includes four 19-base-pair repeats where one of the lambda specific initiator proteins, O, binds. No transition arose within the ori region. The transition sites are multiple on both sides of the ori region and are clustered in one of the two strands in such a way that DNA syntheses from the two sides converge. The frequency of the "leftward" DNA synthesis is several times higher than that of "rightward" synthesis, reflecting the asymmetric bidirectional replication of lambda dv DNA.     

Immunosuppressive serum factors in viral hepatitis. I. Characterization of serum inhibition factor(s) as lymphocyte antiactivator(s)

Sera from patients with acute viral hepatitis B were found to inhibit the in vitro proliferation of normal lymphocytes induced by different mitogens and antigens. In addition, an effect on concanavalin A-induced T suppressor cell activity and pokeweed mitogen-stimulated IgG and IgM synthesis was demonstrated. Studies concerning the kinetics of serum immunosuppressive effects indicated that serum immunosuppressive factor (SIF) interfered with the intermediate phase of mitogen-induced lymphocyte activation which was defined by protein and RNA synthesis. Thus, when SIF-positive sera were added to lymphocytes, which were already activated by phytohemagglutinin, for 8, 12, or 18 hr, the inhibitory effect decreased in relation to the duration of lymphocyte activation. No inhibition could be demonstrated when SIF-positive sera were added 24 hr after initiation of mitogen stimulation. Furthermore, similar inhibitory effects were found measuring either uptake of [3H]uridine (RNA synthesis) or [3H]leucine (protein synthesis) in a 24 hr culture of phytohemagglutinin-stimulated lymphocytes or [3H]thymidine uptake (DNA synthesis) after 48 hr. These results indicate that SIF act(s) like an antiactivator and may belong to immunoregulatory physiologic serum factors.     

Hyporesponsiveness of lymphocytes to virus antigens in rheumatoid arthritis.

The immune response of peripheral blood lymphocytes to measles, rubella, parainfluenza types 1, 2, and 3 RNA virus antigens and to varicella-zoster and herpes virus type 1 DNA virus antigens was evaluated in 14 patients with rheumatoid arthritis (RA) and 14 matched controls by assessing 3H-thymidine incorporation. The results demonstrated hyporesponsivenss of lymphocytes from RA patients to virus antigens and phytohemagglutinin (PHA), which did not appear to be nonspecific because of a lack of correlation between response to virus antigens and response to mitogens. The patterns of decreased responsiveness was suggestive of a relative restriction of hyporesponsiveness to RNA virus antigens.     

Separation of T cell subpopulations capable of DNA synthesis, lymphotoxin release, and regulation of antigen and phytohemagglutinin responses on the basis of density and adherence properties.

T memory cells specifically responsive to ovalbumin and performing the diverse functions of DNA synthesis, lymphotoxin release, and regulation can be isolated in enriched numbers in the most buoyant fractions (A+B) of bovine serum albumin gradients on day 9 after sensitization. At least 20-30% of these cells are capable of mounting a blastogenic response to ovalbumin. A+B cells responding to ovalbumin with DNA synthesis have adherent properties and are further enriched on passage through glass wool. The subpopulations capable of entering into blastogenesis and DNA synthesis and of lymphotoxin release are unresponsive to T mitogens. A+B cells are capable of either potentiating or suppressing DNA synthetic responses to both phytohemagglutinin and antigen when added to 5 X 10(5) D cells in different proportions. Potentiation or suppression of phytohemagglutinin responses were observed with 1 X 10(5) A+B cells, and total suppression was observed with A+B in the range of 4 X 10(3) to 2 X 10(4). The response to antigen was sometimes inhibited in the same cell combinations that gave a potentiated response to phytohemagglutinin and vice versa. Regulatory cells in this system were not macrophages since their effect was not mimicked by addition of peritoneal macrophages, and ablation of macrophages by carrageenan affected neither the potentiation nor suppression.     

Stimulation of human fibroblast protein, ribonucleic acid, and deoxyribonucleic acid synthesis by bovine growth hormone fragments

Bovine GH and two fragments which were obtained by dissociation of a limited tryptic digest of the hormone stimulated protein, RNA, and DNA synthesis of contact-inhibited human fibroblasts. The stimulation of protein, RNA, and DNA synthesis was similar for the test substances. Maximal stimulation was noted at 10 nM. At that concentration, protein, RNA, and DNA synthesis were respectively increased 1.80, 1.42, and 1.37 times by GH; 1.93, 1.27, and 1.46 times by A-I (the larger fragment); and 1.99, 1.26, and 1.33 times by A-II (the smaller fragment). The action of GH, A-I, and A-II was similar to that of fetal calf serum, but was distinguished by the time course of stimulation and by the magnitude of the response. For example, GH, A-I, and A-II produced their earliest detectable effect at 10 h for protein synthesis, 22 h for RNA synthesis, and 26 h for DNA synthesis. On the other hand, serum stimulated protein, RNA, and DNA synthesis at 6, 10, and 16 h, respectively. These data show that human fibroblasts respond equally to GH, A-I, and A-II and suggest that there may be more than one "active site" in the GH molecule. Lastly, human fibroblasts may represent a useful system to study the actions of GH in vitro.     

Analysis of the survival of mature human eosinophils: interleukin-5 prevents apoptosis in mature human eosinophils.

We and other groups have previously shown that interleukin-5 (IL-5) maintained the viability of mature eosinophils in an in vitro liquid culture system. Mature eosinophils did not proliferate but their survival was maintained in the presence of IL-5. Using this culture system, we investigated the mechanism of IL-5-mediated survival. In the absence of human IL-5 (hIL-5) mature eosinophils succumbed after 4 days, while in the presence of hIL-5 they survived up to 10 days. When DNA extracts of cultured eosinophils were analyzed on an agar gel electrophoresis, marked DNA fragmentation was observed in the absence of hIL-5, while no significant DNA fragmentation was observed in the culture with hIL-5 for 48 hours. The DNA fragmentation appeared as early as 6 to 12 hours after hIL-5 deprivation. Concomitantly, IL-5 stimulated total RNA and protein synthesis, but did not induce DNA synthesis in mature eosinophils. Because cycloheximide or actinomycin D impeded the protection of apoptosis by hIL-5, some new RNA and protein synthesis appeared to be required in this phenomena. These findings indicate that IL-5 maintains survival of mature eosinophils with induction of new RNA and protein synthesis, thus leading to the inhibition of apoptosis.     

Initiator RNA in Discontinuous Polyoma DNA Synthesis

During replication of polyoma DNA in isolated nuclei, RNA was found attached to the 5' ends of growing progeny strands. This RNA starts with either ATP or GTP and can be labeled at its 5' end with (32)P from beta-labeled nucleotides. Digestion of progeny strands with pancreatic DNase released (32)P-labeled RNA that, on gel electrophoresis, gave a distinct peak in the position expected for a decanucleotide. We believe that this short RNA is involved in the initiation of the discontinuous synthesis of DNA and propose the name "initiator RNA" for it. The covalent linkage of initiator RNA to 5' ends of growing DNA chains was substantiated by the finding that (32)P was transferred to ribonucleotides by alkaline hydrolysis of purified initiator RNA obtained by DNase digestion of polyoma progeny strands synthesized from [alpha-(32)P]dTTP. While initiator RNA was quite homogeneous in size, it had no unique base sequence since digestion with pancreatic RNase of initiator RNA labeled at its 5' end with (32)P released a variety of different [(32)P]oligonucleotides. The switch from RNA to DNA synthesis during strand elongation may thus depend on the size of initiator RNA rather than on a specific base sequence.     

Coupling of replication to transcription in vitro

In a coupled system consisting of RNA polymerase and DNA polymerase I of Escherichia coli, the four deoxyribo- and the four ribonucleoside triphosphates, and DNA of bacteriophage f1 as template, DNA synthesis depends on the concomitant synthesis of RNA. Over a wide range of concentrations of the two polymerases, RNA synthesis was unaffected by the simultaneous synthesis of DNA, whereas the rate of DNA synthesis depended on the level of RNA synthesis. In the coupled reaction, RNA synthesis starts immediately at a high rate, which subsequently decreases, whereas DNA synthesis starts after a lag and its rate increases as the reaction proceeds. Upon addition of rifampicin, the rate of RNA synthesis falls abruptly, while that of DNA declines only gradually. The base composition of the DNA synthesized in the coupled reaction is complementary to that of f1 DNA template. It is suggested that the RNA synthesized by the RNA polymerase serves as a primer rather than as a template for the DNA polymerase.     

RNA, protein and DNA synthesis stimulated by testosterone, insulin and prolactin in the rat ventral prostate cultured in chemically defined medium.

In an organ type tissue culture of the rat ventral prostate in a chemically defined medium insulin (0.08 IU/ml) stimulated the synthesis of RNA within 6-12 h, the synthesis of protein within 6-12 h and the synthesis of DNA within 2-4 days. Testosterone (10(-8)m) stimulated these synthetic processes somewhat more slowly: the synthesis of RNA within 12-24 h, protein within 12-24 h and DNA at 4 days. Rather high concentrations of insulin were needed while testosterone was effective at a physiological concentration. Prolactin (1000 ng/ml) stimulated the synthesis of RNA and protein, but not DNA, when added together with either testosterone or insulin, but was completely ineffective when added alone. The response times resembled those of insulin. The lower concentrations of prolactin were ineffective. Growth hormone, luteinizing hormone and follicle stimulating hormone did not stimulate the synthesis of RNA, protein or DNA even when added with testosterone. The results confirm the findings of the numerous in vivo experiments that the hypophyseal hormone prolactin has a direct effect on the ventral prostate.