Changes in deoxyadenosine production during human lymphocyte mitogenesis

Immunodeficient children who lack the purine metabolic enzyme adenosine deaminase have markedly elevated plasma concentrations of 2'-deoxyadenosine and adenosine. However, little information exists concerning the magnitude of endogenous 2'-deoxyadenosine and adenosine synthesis by normal human hematopoietic cells. In the present experiments, we have used the sensitive technique of high performance liquid chromatography to quantitate changes in 2'-deoxyadenosine and adenosine production during human lymphocyte mitogenesis. In the presence of the adenosine deaminase inhibitor deoxycoformycin, human T lymphocytes stimulated with phytohemagglutinin (PHA), and non-T lymphocytes stimulated with formalinized Staphylococcus aureus Cowan strain I, excreted 2'-deoxyadenosine into the cell medium. The nucleoside was detectable as early as 20 h after addition of mitogen. The time course of 2'-deoxyadenosine excretion correlated with the uptake of [methyl-3H]thymidine into nucleic acid. Mitogen-stimulated human lymphocytes produced only minimal amounts of adenosine. The results suggest that increased 2'-deoxyadenosine synthesis and release may normally accompany human lymphocyte mitogenesis.     

Role of nuclear proteins on [H]actinomycin D binding during lymphocyte mitogenesis

The uptake of [³H]actinomycin D ([³H]AD) by ConA-stimulated lymphocytes was followed during 96 h of incubation and correlated with the level of nuclear proteins in the nucleus, DNA synthesis and the degree of AD-induced inhibition of RNA and DNA synthesis. During the first 48 h there is a parallel increase of drug binding to cells and a rising level of non-histone proteins (NHP) in the nucleus. During the next 48 h, DNA synthesis occurs, drug uptake decreases and the nuclear level of NHP continues to rise. The level of histones remains constant during 96 h. The variations in cellular [³H]AD uptake during 96 h are not due to changes in cell membrane permeability, since similar variations in drug binding are observed in isolated cell nuclei. NHP, obtained as 0.25 M NaCl extracts of cell nuclei, increase binding of [³H]AD to nuclei isolated from non-stimulated lymphocytes, while histones have no such effect. NHP extracted with phenol, after washing the nuclei with salt and acid solutions, or extracted with 0.25 M NaCl from non-stimulated and stimulated lymphocytes and Chang liver cells are equally active to bind [³H]AD to nuclei of non-stimulated lymphocytes. NHP from Chang cells, purified by DNA-cellulose chromatography using calf thymus DNA, stimulated [³H]AD binding to lymphocyte nuclei, indicating that the drug-binding activity is due to proteins binding to DNA. NHP increase binding of [³H]AD to pure DNA in the absence of histones. The degree of [³H]AD binding to ConA-stimulated lymphocytes during 96 h correlated with the degree of inhibition of RNA and DNA synthesis by AD.     

The nuclear matrix: Three-dimensional architecture and protein composition

The structural filament network of the nucleus is prepared while still connected to the cytoskeleton. The relatively gentle procedure removes about 98% of the DNA and at least 86% of the histones. The matrix is bounded by an outer nuclear lamina connected to the cytoskeletal framework, as well as the inner filaments. The filaments range in diameter from 3 to 22 nm, and are organized in a three-dimensional anastomosing network in which nucleoli are enmeshed. The nuclear matrix is separated from the cytoskeletal framework by a double detergent and then partitioned into a chromatin fraction and a matrix fraction by nuclease and high salt. Two-dimensional gel electrophoresis shows that the proteins of the cytoskeleton, chromatin and nuclear matrix are very different. A major protein found in all fractions cofocuses with actin. Vimentin is largely associated with the nuclear matrix, probably as a corona external of filaments.     

Role of nuclear proteins on [3H]actinomycin D binding during lymphocyte mitogenesis

The uptake of [³H]actinomycin D ([³H]AD) by ConA-stimulated lymphocytes was followed during 96 h of incubation and correlated with the level of nuclear proteins in the nucleus, DNA synthesis and the degree of AD-induced inhibition of RNA and DNA synthesis. During the first 48 h there is a parallel increase of drug binding to cells and a rising level of non-histone proteins (NHP) in the nucleus. During the next 48 h, DNA synthesis occurs, drug uptake decreases and the nuclear level of NHP continues to rise. The level of histones remains constant during 96 h. The variations in cellular [³H]AD uptake during 96 h are not due to changes in cell membrane permeability, since similar variations in drug binding are observed in isolated cell nuclei. NHP, obtained as 0.25 M NaCl extracts of cell nuclei, increase binding of [³H]AD to nuclei isolated from non-stimulated lymphocytes, while histones have no such effect. NHP extracted with phenol, after washing the nuclei with salt and acid solutions, or extracted with 0.25 M NaCl from non-stimulated and stimulated lymphocytes and Chang liver cells are equally active to bind [³H]AD to nuclei of non-stimulated lymphocytes. NHP from Chang cells, purified by DNA-cellulose chromatography using calf thymus DNA, stimulated [³H]AD binding to lymphocyte nuclei, indicating that the drug-binding activity is due to proteins binding to DNA. NHP increase binding of [³H]AD to pure DNA in the absence of histones. The degree of [³H]AD binding to ConA-stimulated lymphocytes during 96 h correlated with the degree of inhibition of RNA and DNA synthesis by AD.     

Changes in nuclear non-histone protein composition during normal differentiation and carcinogenesis of intestinal epithelial cells

Nuclei from colonic epithelial cells were isolated and fractionated by centrifugation in discontinuous sucrose density gradients. Nuclei differing in buoyant density differ in size, non-histone protein to DNA ratio, and capacity for DNA synthesis in vivo. They do not differ in histone content or in proportions of the major histone classes. The distribution of cell nuclei after density gradient centrifugation corresponds functionally to their histological localization in the colonic mucosa, as judged by the nuclear capacity for DNA synthesis in both normal and tumor tissues. The nuclei of colonic epithelial cells contain a heterogeneous set of non-histone proteins which change in total amount and in relative proportions during normal differentiation. The complement of nuclear proteins differs in normal intestinal epithelial cells and in colon tumors induced by the carcinogen, 1,2-dimethylhydrazine. There is a striking increase in the nuclear content of two major protein classes (of mol. wt ca 44 000 and 62 000) during carcinogenesis. The accumulation of these proteins in the nuclei of carcinogen-treated cells follows early, selective increases in their rates of synthesis.     

Purification,properties and regulation of the level of bovine S-adenosylmethionine decarboxylase during lymphocyte mitogenesis

S-Adenosylmethionine decarboxylase was purified from the livers of calves treated with methylglyoxal bis (guanylhydrazone) to elevate the level of the enzyme. Purified bovine S-adenosylmethionine decarboxylase was similar in specific activity and subunit molecular weight (32 000) to the enzymes previously isolated from rat and mouse. The bovine liver enzyme immunologically crossreacted with S-adenosylmethionine decarboxylase from resting and mitogenically activated bovine lymphocytes. The rate of enzyme synthesis in activated lymphocytes was determined by labeling the cells with [³H]leucine and isolating the radioactive decarboxylase by affinity chromatography and sodium dodecyl sulfate gel electrophoresis. The rate of enzyme syntheis was increased 10-fold by 9 h after mitogen treatment, which accounts for the initial increase in cellular enzymatic. There was no further incraese in the rate of S-adenosylmethionine decarboxylase synthesis that correlated with a second elevation of activity occuring at approx. 24 h after mitogenic activation. It was concluded that the second increase in enzyme activity was due to lengthening the intracellular half-life of the enzyme by 2-fold.     

Effect of organ site on nuclear matrix protein composition

The nuclear matrix has been linked to several important cellular functions within cells, such as DNA organization and replication, as well as regulation of gene expression. It has been reported that the nuclear matrix protein composition is altered in cells grown on different extracellular matrices in vitro. This study examined the nuclear matrix protein composition of tumors produced by MAT-LyLu (MLL) rat prostate tumor cells implanted at different organ sites within the rat. When high resolution two-dimensional gels were utilized to compare nuclear matrix protein composition to the prostate orthotopic tumor, it was found that there were distinct protein differences depending upon where the tumor grew. In particular, there were 14 proteins found in the lung, six proteins found in intramuscular, 17 proteins is the heart, and five proteins in the tail vein tumor tissue that were not present in the prostate orthotopic tumor tissue. Therefore, this study adds evidence to support that the nuclear matrix composition of a cell is dependent, at least in part, by the extracellular matrix and/or different cellular environments and may have a role in site-specific differences in tumor properties. © 1996 Wiley-Liss, Inc.     

The effects of aging on phosphofructokinase induction during lymphocyte mitogenesis in relation to DNA and protein synthesis

The incorporation of radiolabeled leucine into phytohemagglutinin-stimulated human lymphocytes increases by 9 hours after mitogen addition in the young whereas this process is delayed by two-fold in the aged (18 hours). Once induced, the leucine incorporation is about 56% less in the aged as compared to the young. The induction of phosphofructokinase (PFK) catalytic activity mimics the induction of protein synthesis in both young (9 hours) and aged (18 hours) subjects also taking twice as long to induce in the aged and attaining much lower levels of induction with increasing subject age. The increase of thymidine incorporation in mitogen-stimulated cells does not occur until 12 hours after the increase in leucine incorporation in both the young (21 hours) and aged (30 hours) which also represents a 9 hour age-related delay in induction. The marked increase in protein synthesis rate occurs in a concerted manner with the induction of glycolysis and the delay and impairment in protein biosynthesis in the aged appears to relate to the similar age-related findings for glycolytic enzyme induction. The mitogen-induced increase in DNA synthesis is a later event and the age-related delay in DNA synthesis induction may be secondary to the delay in the induction of protein synthesis. Other enzyme-dependent processes besides DNA synthesis and glycolysis may also be secondary to a primary slowing of protein synthesis in the aged and related to the delayed cell cycle time frequently observed in aged subjects.     

Changes in histone H3 composition and synthesis pattern during lymphocyte activation

Freshly isolated human lymphocytes were found to synthesize histones at a significant rate even though no DNA was being synthesized. The synthesis pattern of histone variants in resting lymphocytes is similar to that found in other quiescent cells and different from that found in S-phase cells. For this reason, the histone synthesis in resting lymphocytes cannot be attributed to contamination by S-phase cells. Stimulation by the mitogen phytohemagglutinin resulted in a dramatic switch in the histone H3 variant synthesis pattern as well as a readily apparent change in the histone H3 mass pattern. Thus, the chromatin of activated lymphocytes has a different histone H3 variant composition than resting or quiescent lymphocytes. It is suggested that the proportion of H3.3 in the mass pattern of the chromatin of a cell may be related solely to how long that cell has been quiescent. Inducing resting lymphocytes to synthesize DNA by UV irradiation did not qualitatively change the histone variant synthesis pattern. No S-phase H3 variants were induced by the repair process. Furthermore, the quantity of histone synthesized neither increased nor decreased after treatment with UV light.     

Reversibly contractile nuclear matrix. Its isolation, structure, and composition

From Tetrahymena macronuclei we have isolated a reversibly contractile nucleo-skeleton, i.e., an "expanded" nuclear matrix which reversibly contracts when the total concentration of the bivalent cations, Ca and Mg (3:2), is decreased to 5 mM or increased to 125 mM. During contraction the average diameter of the expanded matrix becomes reduced by about 24%; this corresponds to a volume contraction of about 55%. The reversible contraction of the nuclear matrix does not depend on ATP and cannot be inhibited by salygran. The expanded matrix is obtained by removing carefully from the macronuclei 89.7% of the phospholipid, 99.6% of the DNA, 98.5% of the RNA, and 74.8% of the protein by treatment with Triton X-100 and digestion with DNase and RNase followed by an extraction with 2 M NaCl. Electron microscopy reveals, within the expanded matrix, residual equivalents to the structures characteristic for macronuclei: (a) a residual nuclear envelope with nuclear pore complexes; (b) residual nucleoli at the periphery; (c) a fibrillar internal network. The expanded matrix is essentially composed of proteins (96.2%) and traces of DNA (0.8%), RNA (0.5%), phospholipid (1.6%), and carbohydrates (0.9%). The last, which have been determined by gas chromatography, contain glucose, mannose, and an unidentified sugar in the ratio 1:5.4:5.7. The ratio of acidic to basic amino acids of the expanded matrix is 1.55. Sodium dodecyl sulfate (SDS) gel electrophoresis reveals a predominant protein with a mol wt of 18,000 which is apparently involved in the reversible contractile process. The mechanism of this reversible contraction of the expanded matrix remains to be elucidated, but it differs both from actin-myosin contraction systems and from the contractile spasmoneme system in vorticellids.     

Human lymphocyte potassium content during the initiation of phytohemagglutinin-induced mitogenesis.

The K+ content of human lymphocytes has been examined during the initial 24 hours after exposure of cells to phytohemagglutinin (PHA). We have reconfirmed that lymphocyte K+ exchanges rapidly for extracellular counterions during preparative washing if cells are exposed to PHA. By using a technique to measure cation content which does not require removal of cells from their culture medium, we have shown that K+ does not change for 24 hours following PHA treatment. Previous reports have demonstrated that an enhanced uptake of K+ occurs in lymphocytes treated with PHA. This increased uptake may be a compensatory change for an increased exodus, explaining the failure of K+ to change following lectin treatment.     

Changes in distribution of nuclear matrix antigens during the mitotic cell cycle

We examined the distribution of nonlamin nuclear matrix antigens during the mitotic cell cycle in mouse 3T3 fibroblasts. Four monoclonal antibodies produced against isolated nuclear matrices were used to characterize antigens by the immunoblotting of isolated nuclear matrix preparations, and were used to localize the antigens by indirect immunofluorescence. For comparison, lamins and histones were localized using human autoimmune antibodies. At interphase, the monoclonal antibodies recognized non-nucleolar and nonheterochromatin nuclear components. Antibody P1 stained the nuclear periphery homogeneously, with some small invaginations toward the interior of the nucleus. Antibody I1 detected an antigen distributed as fine granules throughout the nuclear interior. Monoclonals PI1 and PI2 stained both the nuclear periphery and interior, with some characteristic differences. During mitosis, P1 and I1 were chromosome-associated, whereas PI1 and PI2 dispersed in the cytoplasm. Antibody P1 heavily stained the periphery of the chromosome mass, and we suggest that the antigen may play a role in maintaining interphase and mitotic chromosome order. With antibody I1, bright granules were distributed along the chromosomes and there was also some diffuse internal staining. The antigen to I1 may be involved in chromatin/chromosome higher-order organization throughout the cell cycle. Antibodies PI1 and PI2 were redistributed independently during prophase, and dispersed into the cytoplasm during prometaphase. Antibody PI2 also detected antigen associated with the spindle poles.     

Changes in nuclear structure during eupyrene spermatogenesis in Murex brandaris

Changes in nuclear structure during eupyrene spermatogenesis of Murex brandaris have been studied using light and electron microscopy. In the first phases, spermatogonia show round nuclei, with several electrodense masses of chromatin and a thin layer of heterochromatin associated with the nuclear membrane. Primary spermatocytes possess larger nucleii, with less condensed chromatin, and the synaptonemal complexes are apparent. During spermiogenesis, chromatin becomes lamellar, and the nucleus twists about its principal axis while it elongates. The nuclear twisting is accompanied by a progressive chromatin condensation, which causes a highly electrodense nucleus at the end of the process.     

Induction of a substrate for casein kinase II during lymphocyte mitogenesis

Particulate fractions prepared from concanavalin A-activated murine T lymphocytes contain an endogenous protein kinase that phosphorylates an endogenous protein substrate of Mr 112 000. The phosphorylation of 112 kDa protein is greatly reduced or absent in unstimulated T cells. Phosphoamino acid analysis indicates that 112 kDa protein is labeled on a serine. Add-back experiments using purified protein kinases indicate that 112 kDa protein serves as a substrate for casein kinase II. Phosphorylation of 112 kDa protein by the endogenous kinase is inhibited by heparin, a known casein kinase II inhibitor. The site or sites modified by the endogenous kinase and exogenous casein kinase II appear identical by peptide-mapping experiments. A time-course of the appearance of phosphorylated 112 kDa protein following stimulation with concanavalin A, measured in the presence or absence of added casein kinase II, suggests that 112 kDa protein is induced in activated T cells. Subcellular localization studies suggest that 112 kDa protein is a nuclear protein. Silver-binding and purification studies suggest that 112 kDa protein is of the nucleolar organizing region.     

Induction of a calcium/calmodulin-dependent phosphodiesterase during phytohemagglutinin-stimulated lymphocyte mitogenesis

A calmodulin (CaM)-dependent phosphodiesterase activity that hydrolyzes both cGMP and cAMP was observed in anion exchange high performance liquid chromatography (HPLC) profiles from phytohemagglutinin-stimulated mononuclear cells but not in profiles from unstimulated cells. A single polypeptide was detected by an antibody to the calmodulin-dependent phosphodiesterases on a Western blot of homogenates of stimulated mononuclear cells. The phosphodiesterase activity was immunoadsorbed in a calcium-dependent manner by an antibody to calmodulin but not by an antibody to the 61-kDa bovine brain phosphodiesterase. The mononuclear cell enzyme eluted from the HPLC column in the same fractions as the 63-kDa calmodulin-dependent isozyme from bovine brain and appeared to have the same subunit molecular weight when probed on a Western blot. The electrophoretic mobility of proteolytic fragments derived from the mononuclear cell phosphodiesterase were identical to those from the 63-kDa brain isozyme. The enzyme could be detected in mononuclear cells by activity assays and on a Western blot 14 h after stimulation with mitogen. The enzyme remained elevated for at least 100 h after stimulation. A dose-response experiment with phytohemagglutinin demonstrated that similar concentrations of mitogen could induce both mitogenesis and the phosphodiesterase. The induction of this enzyme requires mRNA as well as protein synthesis but not DNA synthesis. An enzyme similar to the 63-kDa phosphodiesterase found in brain seems to demonstrate a regulatory interface for the metabolism of calcium and cyclic nucleotides during lymphocyte mitogenesis.