Expression of preprotachykinin-A and neuropeptide-Y messenger RNA in the thymus.

The preprotachykinin-A gene, the common gene of mRNAs encoding both substance-P (SP) and neurokinin-A (NKA), was shown to be expressed in Sprague-Dawley rat thymus by detection of specific mRNA in gel-blot analyses. In situ hybridization revealed dispersed PPT-A-labeled cells in sections from rat thymus, with a concentration of grains over a subpopulation of cells in the thymic medulla. Also, neuropeptide-Y mRNA-expressing cells were found in the rat thymus, primarily in the thymic medulla. Rat thymic extracts contained SP-like immunoreactivity (SP-LI), and the major part of the immunoreactivity coeluted with authentic SP and SP sulfoxide standards. SP-LI was also detected in human thymus, which contained between 0.09-0.88 ng SP-LI/g wet wt. Evidence for translation of preprotachykinin-A mRNA in the rat thymus was obtained from the demonstration of NKA-LI in thymic cells with an epithelial-like cell morphology. Combined with previous observations on the immunoregulatory roles of tachykinin peptides and the existence of specific receptors on immunocompetent cells, the demonstration of intrathymic synthesis of NKA suggests a role for NKA-LI peptides in T-cell differentiation in the thymus.     

Actions of exogenous histones and other proteins on [3H]-thymidine incorporation into DNA of Novikoff hepatoma cells.

The effects of exogenous proteins on the incorporation of [3H]-thymidine into DNA was studied in Novikoff hepatoma ascites cells incubated in Eagle's minimal essential medium. A liver cytosol fraction (8 mg protein/ml) caused approximately 80% inhibition of isotope incorporation. The inhibitory activity of cytosol fractions from Morris hepatomas 9618A2, 5123C, and 20 were inversely related to their growth rate. Under conditions in which there appeared to be a density dependent inhibition of growth, a mean 10-20% stimulation of isotope incorporation was observed after addition of total calf thymus histones and individual fractions in the concentration range of 100-400 microgram/ml. In experiments with lower cell concentrations, a 60% or greater increase in [3H]-thymidine incorporation could be obtained with total calf thymus histone and with F1 and arginine-rich histones from rat liver. At concentrations of 1-2 mg/ml, histones inhibited DNA synthesis. Bovine serum albumin had little effect on DNA synthesis. Polylysine caused an 80-90% inhibition at a concentration of 1 mg/ml, but stimulatory effects were detected under certain conditions at 10 microgram/ml. The results suggest critical dependence on the ratio of cell and exogenous protein concentration in the action of proteins on DNA synthesis.     

DNA primase associated with 10 S DNA polymerase alpha from calf thymus

Among multiple subspecies of DNA polymerase alpha of calf thymus, only 10 S DNA polymerase alpha had a capacity to initiate DNA synthesis on an unprimed single-stranded, circular M13 phage DNA in the presence of ribonucleoside triphosphates (DNA primase activity). The primase was copurified with 10 S DNA polymerase alpha through the purification and both activities cosedimented at 10 S through gradients of either sucrose or glycerol. Furthermore, these two activities were immunoprecipitated at a similar efficiency by a monoclonal antibody directed against calf thymus DNA polymerase alpha. These results indicate that the primase is tightly bound to 10 S DNA polymerase alpha. The RNA polymerizing activity was resistant to alpha-amanitin, required high concentration of all four ribonucleoside triphosphates (800 microM) for its maximal activity, and produced the limited length of oligonucleotides (around 10 nucleotides long) which were necessary to serve as a primer for DNA synthesis. Covalent bonding to RNA to DNA was strongly suggested by the nearest neighbour frequency analysis and the DNAase treatment. The DNA synthesis primed by the RNA oligomers may be carried out by the associating DNA polymerase alpha because it was strongly inhibited by araCTP, resistant to d2TTP, and was also inhibited by aphidicolin but at relatively high concentration. The primase preferred single-stranded DNA as a template, but it also showed an activity on the double-stranded DNA from calf thymus at an efficiency of approx. 10% of that with single-stranded DNA.     

Effect of histone on the nucleolar morphology of cells cultured in vitro

Summary The frequency of various types of nucleoli was investigated in tissue cultures of human embryonal lung and HeLa cells cultured in the presence of calf thymus histone. The nucleolar morphology and the frequency of various nucleolar types were dependent on the concentration of histone in the tissue cultures of the human embryonal lung cells. HeLa cells required longer cultivation with histone to manifest some effect on nucleoli. In both cases, the observed nucleolar changes suggest the depression of nucleolar RNA synthesis.     

Infradian Variation of Rat Thymic and Splenic mRNA to Novikoff’s Hepatoma

During the immune response to rat tumor cells there is participation of the thymus and the spleen via the synthesis of antibodies and immune cellular elements. During this process different mRNAs of both organs are synthesized. Here is presented the infradian variation of mRNA synthesis of immunized rat thymus and spleen to Novikoff’s Hepatoma (NH) cells. These cells were maintained and transferred in ascitic manner in the peritoneal cavity of Sprague Dawley male rats standardized with 12 hours (h) of light and 12 h of darkness. Aliquots of 1 x 10 6 NH dead cells were innoculated intraperitoneally into rats after being exposed during 30 minutes to ultraviolet light and incubated at 37°C with 50 U of neuraminidase/ml during 70 minutes. Groups of 3 controls and 3 immunized rats were killed at the same circadian timepoint (10 a.m.) under anesthesia every 3 days during a span of 18 days. At each time spleens and thymus of each group were harvested and pooled in order to isolate their mRNA. Isolation of the rRNA and mRNA of control and immunized rats was performed by affinity chromatography employing oligothymidylic acid cellulose columns. Analysis of variance demonstrated a significant (p <0.03) higher synthesis of immune (i)mRNA of rat spleen, starting on 3 days after immunization with NH cells and reaching the higher levels on 6 and 18 days after immunization and lower levels on 9, 12, 15 days after immunization. Same effect is also observed in the synthesis of imRNA rat thymus 6 days after immunization, however, there was not difference with the intact rat thymus mRNA on 3, 9 and 12 days after immunization. Interestingly, there was observed an increased synthesis of intact rat thymus mRNA 15 days after inoculation. During this cyclic synthesis of thymic and spleenic imRNAs it seems that the spleen plays the role of a possible pacemaker of the coordinated immune response to NH cells.     

DNA primase associated with 10 S DNA polymerase α from calf thymus

Among multiple subspecies of DNA polymerase α of calf thymus, only 10 S DNA polymerase α had a capacity to initiate DNA synthesis on an unprimed single-stranded, circular M13 phage DNA in the presence of ribonucleoside triphosphates (DNA primase activity). The primase was copurified with 10 S DNA polymerase α through the purification and both activities cosedimented at 10 S through gradients of either sucrose or glycerol. Furthermore, these two activities were immunoprecipitated at a similar efficiency by a monoclonal antibody directed against calf thymus DNA polymerase α. These results indicate that the primase is tightly bound to 10 S DNA polymerase α. The RNA polymerizing activity was resistant to α-amanitin, required high concentration of all four ribonucleoside triphosphates (800 μM) for its maximal activity, and produced the limited length of oligonucleotides (around 10 nucleotides long) which were necessary to serve as a primer for DNA synthesis. Covalent bonding to RNA to DNA was strongly suggested by the nearest neighbour frequency analysis and the DNAase treatment. The DNA synthesis primed by the RNA oligomers may be carried out by the associating DNA polymerase α because it was strongly inhibited by araCTP, resistant to d₂TTP, and was also inhibited by aphidicolin but at relatively high concentration. The primase preferred single-stranded DNA as a template, but it also showed an activity on the double-stranded DNA from calf thymus at an efficiency of approx. 10% of that with single-stranded DNA.     

The rat thymus--a site of atrial natriuretic peptide synthesis

The atrium of the heart has been demonstrated to represent the major site of synthesis of atrial natriuretic peptide (ANP), a potent natriuretic, diuretic and vasoactive hormone. Our recent studies revealed ANP-like material outside the heart, namely, in lymphoid follicles of the intestine and in the thymus, and now we report data demonstrating the thymus as a site of synthesis for ANP. The experimental evidence is as follows: firstly, the immunoreactive material detected corresponds chromatographically with the precursor of ANP. Secondly, the thymus contains mRNA for ANP. Thirdly, immunohistochemistry locates ANP-like material to cortical thymocytes with particularly dense staining in the subcapsular areas of the thymus. Interestingly, both ANP-like material and the mRNA coding for ANP were expressed to a larger extent in newborn rats as compared to adult animals, suggesting that ANP may be involved in the development and/or function of T-cells.     

Nuclear ribonucleoproteins as inhibitors of mammalian RNA polymerase.

A ribonucleoprotein complex isolated from rabbit thymus nuclear lysates was found to be an inhibitor of DNA-dependent RNA polymerase II. The inhibition appeared to be of a competitive type and was completely reversed by high concentration of DNA. Highest inhibition was observed when enzyme and complex were preincubated before addition of DNA while there was little inhibition after enzyme had started synthesis on the DNA template. The RNA isolated from the complex was equally inhibitory and was a more effective inhibitor than either tRNA or rRNA.     

Lipid metabolism in rat tissues during chronic gamma-irradiation and action of ubiquinone Q-9]

Chronic gamma-irradiation of rats with the daily dose of 0.129 Gy activates the synthesis of various classes of lipids in the thymus, spleen and bone marrow cells and induces lipid accumulation in these tissues. Feeding of rats with the antioxidant, ubiquinone Q-9, under conditions of chronic irradiation causes a considerable normalization of lipogenesis and levels of the lipid concentration in the tissues of animals irradiated with the dose of 20 Gy.     

Expression of atrial natriuretic peptide in thymic macrophages after dexamethasone treatment of rats

Summary The rat thymus represents a site of synthesis of atrial natriuretic peptide (ANP); the immunosuppressor dexamethasone strikingly increases ANP-expression in this immune organ. The presented data suggest that this increase can be attributed to macrophages. By means of immunohistochemistry and Northern blot analysis these immune cells were found to express ANP-immunoreactivity as well as mRNA coding for ANP. In contrast, macrophages of control thymi displayed only weak ANP-immunoreactivity. Thus, ANP appears to be a constituent of rat thymic macrophages, and its synthesis in the thymus is strongly elevated by acute exposure of the animals to glucocorticoids.     

Immunocytochemical localization of carbonic anhydrase isozyme III in equine thymus

Sections of equine thymus were examined for the presence of carbonic anhydrase (CA) isozymes by an immunohistochemical method. Carbonic anhydrase III, a major enzyme of skeletal muscle, was localized in some of the epithelial-reticular cells of the equine thymus. This finding suggests the presence of a new type of cell in the thymic cortex. The concentration of CA-III in the thymus was 17 micrograms/g wet tissue. CA-I and CA-II were not found in equine thymus.     

Fidelity of mammalian DNA replication and replicative DNA polymerases.

Current models suggest that two or more DNA polymerases may be required for high-fidelity semiconservative DNA replication in eukaryotic cells. In the present study, we directly compare the fidelity of SV40 origin-dependent DNA replication in human cell extracts to the fidelity of mammalian DNA polymerases alpha, delta, and epsilon using lacZ alpha of M13mp2 as a reporter gene. Their fidelity, in decreasing order, is replication greater than or equal to pol epsilon greater than pol delta greater than pol alpha. DNA sequence analysis of mutants derived from extract reactions suggests that replication is accurate when considering single-base substitutions, single-base frameshifts, and larger deletions. The exonuclease-containing calf thymus DNA polymerase epsilon is also highly accurate. When high concentrations of deoxynucleoside triphosphates and deoxyguanosine monophosphate are included in the pol epsilon reaction, both base substitution and frameshift error rates increase. This response suggests that exonucleolytic proofreading contributes to the high base substitution and frameshift fidelity. Exonuclease-containing calf thymus DNA polymerase delta, which requires proliferating cell nuclear antigen for efficient synthesis, is significantly less accurate than pol epsilon. In contrast to pol epsilon, pol delta generates errors during synthesis at a relatively modest concentration of deoxynucleoside triphosphates (100 microM), and the error rate did not increase upon addition of adenosine monophosphate. Thus, we are as yet unable to demonstrate that exonucleolytic proofreading contributes to accuracy during synthesis by DNA polymerase delta. The four-subunit DNA polymerase alpha-primase complex from both HeLa cells and calf thymus is the least accurate replicative polymerase. Fidelity is similar whether the enzyme is assayed immediately after purification or after being stored frozen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on the regulation of lymphocyte production in the murine thymus and some effects of a crude thymus extract.

Cell proliferation in the murine thymus was studied in vivo under normal conditions and from 0 to 24 hr after a single injection of a water-soluble extract from mouse thymus, mouse spleen, and mouse skin. The thymus extract reduced during the first 24 hr the mitiotic activity 40%; the spleen extract had a weaker inhibitory effect. The skin extract had no such effect. The thymus extract and spleen extract inhibited the flux of cells into the S phase 0-8 hr after the injection of the extract. Initial labelling index was also reduced in this period. Eight hours after injection of the thymus or spleen extracts the inhibited cells initiated DNA synthesis. The rate of progression of blast cells through the cell cycle was normal 24 hr after the injection of the extracts. It was deduced from the analysis that the thymus extract inhibits processes triggering G0/G1 cells into DNA synthesis, the inhibition of G2 efflux being of minor importance. Finally a model for the regulation of proliferating thymic blast cells and the emigration of small lymphocytes from the thymus is proposed.     

Changes in the glycolipid composition and characteristic activation of GM3 synthase in the thymus of mouse after administration of dexamethasone

Glycolipids in the thymus of mice after administration of dexamethasone were compared with those in control mice. In parallel with a decrease in the tissue weight due to the disappearance of immature thymocytes in the cortex, the amounts of GlcCer, Gg₄Cer and GM1 decreased from 18 h after intraperitoneal administration of dexamethasone, but those of Gb₄Cer and Forssman glycolipid did not change, indicating the differential distribution of ganglio- and globo-series glycolipids in the thymus, GlcCer, Gg₄Cer and GM1 being on dexamethasone-sensitive cortical thymocytes, and Gb₄Cer and Forssman glycolipid on dexamethasone-resistant cells including thymic stromal cells, respectively. At the same time, a characteristic increase in GM3, whose amount per thymus and concentration per mg of thymus were increased 4-fold and 13-fold compared to those in the control mice, respectively, was observed at the onset of the decrease in tissue weight and was due to the increased activity of LacCer sialyltransferase with the enhanced expression of its gene and the concomitant decrease in cytosolic sialidase activity. One can suggest that endogenous accumulation of GM3 is involved in the dexamethasone-induced apoptosis of cortical thymocytes. On radiolabeling of the thymus with CMP-[¹⁴C]-NeuAc, the incorporation of radioactivity into GM3 was preferentially observed in the thymuses of dexamethasone-administered mice, but not in those of control mice, suggesting the possible involvement of plasma membrane-associated sialytransferase in GM3 synthesis in the thymuses of dexamethasone-administered mice. Published in 2005.     

Convolution analysis of transcription by yeast DNA-dependent ribonucleic acid polymerase A. A mathematical method for studying ribonucleic acid chain elongation.

The rate of initiation of RNA synthesis catalysed by yeast RNA polymerase A on native calf thymus DNA decayed exponentially with a half-life of about 4.3 min. The rate constant for initiation was unaffected by preincubating the enzyme with DNA, or by decreasing the concentration of GTP 4-fold. The rate of RNA synthesis was constant for 15--20 min and then decreased. Each enzyme molecule made no more than one RNA molecule. In this situation, initiation, elongation and total RNA synthesis are related by a convolution integral. Solution of the convolution integral revealed that the rate of elongation was apparently biphasic. Analysis of the size of the RNA product showed that this biphasic profile arose because most but not all of the enzyme stopped RNA synthesis soon after initiation.     

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.     

The effect of adriamycin on Z-DNA formation and DNA synthesis.

The ability of adriamycin to inhibit Z-DNA formation induced by a high-salt environment was investigated. ADM inhibited this conversion, such that in poly (dG-dC) total inhibition was observed at 1 ADM: 9 base pairs and in eukaryotic DNA (calf thymus) at 1 ADM: 11,5 base pairs. Even at low ADM concentration, 1 ADM: 160 base pairs, some inhibition was observed. At similar ADM:DNA concentrations, an inhibition in DNA synthesis in cells in culture was observed, which showed some parallel with the inhibition of Z-DNA formation. A model is proposed where Z-DNA formation precedes DNA synthesis and where inhibition of the former could explain the antineoplastic nature of adriamycin.