Proliferation of fetal brown adipocyte primary cultures: Relationship with the genetic expression of glucose 6 phosphate dehydrogenase

Fetal brown adipocyte primary cultures increase DNA synthesis; cell number; and DNA, RNA, and protein contents in response to 10% fetal calf serum, IGF-I, and EGF plus vasopressin plus bombesin when added for 64 h to quiescent cells. IGF-I is a complete growth factor in this system while EGF needs the presence of vasopressin plus bombesin for its maximal proliferative effects. These mitogens induce the genetic expression of G6P dehydrogenase, increasing its mRNA content as well as its specific activity and amount of immunoreactive protein. The presence of cAMP elevating agents prevents the stimulatory effect of EGF plus vasopressin plus bombesin on DNA synthesis, cell number, and DNA content as well as on the induction of G6P dehydrogenase expression. Thus, changes on the proliferative state of these cells are associated with the level of expression of G6P dehydrogenase.     

Activation of cation transport by lymphokines in B cells without induction of DNA synthesis or immunoglobulin gene transcription

We report on an experimental model that permitted us to evaluate the biologic relevance of membrane-associated biochemical events with respect to cell proliferation and maturation, each induced by distinct sets of signals. Antigen-affinity-enriched murine B cells cultured in the presence of a proliferative signal induced by LPS showed activation of Na+/K+ ATPase and enhanced the uptake of proline, followed by RNA, protein, and DNA synthesis, without the generation of antibody. Stimulation with both the proliferative signal(s) and the maturation signal(s) derived from lymphokines of an EL-4 thymoma induced B cells to proliferate and synthesize mRNA encoding mu-chain of IgM and to mature into IgM-secreting cells. Most important, the secretory product of EL-4, in the absence of LPS, activated Na+/K+ ATPase but failed to stimulate uptake of proline and synthesis of DNA or mu-specific mRNA. A similar response was observed in splenocytes depleted of T cells and in unfractionated spleen cells. Thus a component secreted by EL-4 can induce some of the early molecular events characteristic of the proliferative response but lacks the ability to initiate blast transformation and DNA synthesis.     

Cell growth and cell proliferation may be dissociated in the mouse uterine luminal epithelium treated with female sex steroids

The mouse uterine epithelium under various hormonal regimes is a good system to identify biochemical events associated with cell growth, DNA synthesis and cell division. This is because estradiol-17β stimulates the cells to undergo a synchronized wave of DNA synthesis and cell division. Estriol, on the other hand, also stimulates DNA synthesis but because of the rapid loss of this hormone from the tissue some of the cells abort, giving a constant epithelial cell number. Three days of progesterone pretreatment, however, completely suppresses the estradiol-17β-induced wave of DNA synthesis and cell proliferation.Using these hormonal treatments we have shown that both estradiol-17β and estriol stimulate protein and rRNA synthesis with the concomitant increase of protein and rRNA per mg of DNA. These macromolecules accumulated in direct proportion to the fraction of cell committed to DNA synthesis. Estriol, however, did not sustain the growth responses and at the peak of DNA synthesis both rRNA and protein synthesis had returned to control levels. Progesterone pretreatment, despite inhibiting the proliferative response, failed to inhibit any of the estradiol-17β-induced increases in protein and rRNA synthesis. Indeed 12 h after estradiol-17β injection the cells had identical protein and rRNA contents, regardless of whether they had been exposed to progesterone or not.The present data therefore suggests that in the uterine epithelium cell growth as defined by protein and rRNA accumulation and DNA synthesis represents two independently regulated pathways.     

Activation of Dictyostelium discoideum guanylate cyclase by ATP.

Human leucocyte cell cultures were stimulated to initiate DNA synthesis by phytohemagglutinin and mercuric chloride. Both mitogens enhanced the accumulation of β₂-microglobulin in the medium, which was synthesized by lymphocytes. Mercuric chloride promoted the accumulation of this protein optimaly with a concentration (1 × 10^?5M) to produce the maximum stimulation of DNA synthesis. Combined use of phytohemagglutinin (50 μg/ml) and mercuric chloride (1 × 10^?5M) produced additive effect on both DNA synthesis and β₂-microglobulin accumulation. These findings suggest that mercuric ion causes the proliferative response of lymphocytes by a mechanism different from that for the stimulation by phytohemagglutinin.     

Gangliosides stimulate astroglial cell proliferation in the absence of serum

Previous studies with microcultures of astroglial (AG) cells from newborn rat cerebrum had shown an ability of gangliosides to interact with AG cells cultured under defined conditions. We have now investigated the capability of gangliosides to stimulate DNA synthesis and cell number increases in similar secondary microcultures of newborn rat cerebrum AG cells. At a concentration of 6 X 10(-5)M, GM1 ganglioside stimulated DNA synthesis and increased cell numbers, with DNA synthesis leading cell increases by 12-24 hr. The ganglioside-induced AG cell proliferative response occurred with GD1a, GD1b and GT1b, GT1b being the most potent at 10(-5)M--while asialo GM1 and sialic acid were without effect. In the standard test cultures, DNA synthesis declined very steeply after the first day, with cell numbers stabilizing at the level reached after 2 days. Ganglioside was not itself responsible for the restricted proliferative response, as serum produced the same behaviors.     

Synergistic influence of epidermal growth factor, insulin and transferrin on human fetal kidney in culture

In human fetal kidneys (15-21 weeks of gestation) maintained in serum-free organ culture, protein synthesis remained relatively constant, but DNA synthesis decreased dramatically after 2 days. The addition of transferrin alone had no influence, but insulin and epidermal growth factor (EGF) both significantly stimulated DNA and protein synthesis. When supplemented in combination, transferrin strongly potentiated the insulin effect and after 5 days of culture DNA synthesis was practically restored to values observed in control uncultured renal explants (day 0). When EGF, a potent mitogen, was added as a third factor, the stimulating effectiveness of the (insulin plus transferrin) combination was significantly reduced. However, EGF had no such inhibiting influence on protein synthesis. Differentiation of brush border membranes, as evaluated by hydrolase activities, was not importantly induced nor retarded by any of the three factors supplemented either alone or in combination. The present results indicate that the individual effects of the three factors are not additive, but suggest that they rather act synergistically through a complex mechanism of receptor cross-talk. In our laboratory, there is convincing indication that the response of fetal organs varies according to age, proliferative state of tissues as well as stage of differentiation.     

The difference in the stimulation by putrescine of DNA synthesis using DNA polymerase extracts of normal rat liver or of tumour tissue or host liver from tumour-bearing rats

Putrescine biosynthesis is elevated before DNA replication, and a stimulation of DNA synthesis by 20 mM putrescine has been found using an in vitro DNA synthesizing system. Furthermore, this stimulation of DNA synthesis by putrescine involves a particular factor (factor PA). This factor PA stimulates DNA polymerases alpha, beta, and gamma, and is present in nuclei and mitochondria but not in cytoplasm. Factor PA loses about 80% of its activity by heating at 45 degrees C for 15 min or by hydrolysis with 100 mg ml(-1) Enzygel trypsin. These properties indicate that factor PA is a protein. Its size is estimated to be about 2.1 S. DNA synthesis in nuclear and mitochondrial DNA polymerase extracts from tumour tissues and host livers of tumour-bearing rats are not stimulated by 20 mM putrescine. However, the addition of excess factor PA to DNA synthesizing systems using DNA polymerase extracts from proliferative tissues again results in a stimulation of DNA synthesis by exogenous putrescine. These findings indicate that the stimulatory effect of DNA synthesis in vitro by exogenous putrescine is controlled by the ratio between factor PA and endogenously synthesized putrescine in proliferative tissues or that sent by the bloodstream from proliferative tissues. These results suggest that a non-stimulatory effect of putrescine on DNA synthesis may be diagnostic in tumour-bearing patients.     

Inhibition of the signaling pathways for macrophage proliferation by cyclic AMP. Lack of effect on early responses to colony stimulating factor-1.

Colony stimulating factor-1 (CSF-1) stimulates DNA synthesis in quiescent murine bone marrow-derived macrophages (BMM). CSF-1 action has been shown to involve activation of the CSF-1 receptor kinase. The protein kinase C activator, 12-O-tetradecanoylphorbol 13-acetate (PMA), is itself weakly mitogenic and synergises with CSF-1 for stimulation of BMM DNA synthesis suggesting a possible role for protein kinase C in the stimulation of BMM DNA synthesis. In this report we show that several agents which raise intracellular cAMP (8-bromoadenosine 3':5'-cyclic monophosphate, 3-isobutyl-1-methylxanthine, cholera toxin, and prostaglandin E2) reversibly inhibit DNA synthesis in BMM induced by CSF-1, granulocyte macrophage-colony stimulating factor, interleukin-3, and PMA. The suppressive action of cAMP elevation on the proliferative response to CSF-1 can be manifested even late in the G1 phase of the cell cycle. Several CSF-1-stimulated earlier responses, viz. protein synthesis, Na+/H+ exchange, Na+,K(+)-ATPase and c-myc-mRNA expression, were not inhibited thus showing a striking difference from some other cellular systems involving growth factor-mediated responses. c-fos-mRNA levels were raised and stabilized by the cAMP-elevating agents, and this modulation was not altered by CSF-1. Thus, the signaling pathways in the macrophages involving tyrosine kinase and protein kinase C activation are associated with increased proliferation while those involving elevation of cAMP (and presumably activation of cAMP-dependent protein kinases) appear to have an inhibitory effect.     

Kozak序列对金黄色葡萄球菌黏附因子FnBPA-A DNA疫苗诱导小鼠免疫应答的影响

黏附因子FnbpA(纤连蛋白结合蛋白A,Fibronectin binding protein A)是金黄色葡萄球菌表面的蛋白质成分,是该菌感染早期最重要的致病因子,可促进其对寄主组织的侵入,也是一个有潜力的免疫靶标。将牛乳源金黄色葡萄球菌中FnBPA基因的A功能区克隆至真核表达载体中,分别构建含Kozak序列和不含Kozak序列的FnBPA-A基因真核表达载体,重组质粒经鉴定测序正确后,免疫C57BL/6小鼠检测其抗体水平和淋巴细胞增殖情况,并对各组小鼠进行攻毒实验。检测的结果表明Kozak修饰的重组DNA在血清抗体效价(P<0.05)和免疫保护率方面均优于不含Kozak序列的重组DNA,在刺激淋巴细胞增殖方面Kozak修饰的重组DNA的刺激效果虽然也高于不经修饰的重组DNA,但是差异不显著(P>0.05)。根据总体的免疫效果来看,Kozak序列对增强FnBPA的重组DNA疫苗诱导的免疫应答起了不容忽视的作用。     

Inhibition of DNA synthesis by phorbol esters through protein kinase C in cultured rabbit aortic smooth muscle cells

In cultured rabbit aortic smooth muscle cells (SMC), 12-O-tetradecanoylphorbol-13-acetate (TPA) induced DNA synthesis in the presence of plasma-derived serum to a small extent, but inhibited markedly the rabbit whole blood serum (WBS)-, platelet-derived growth factor (PDGF)- and epidermal growth factor-induced DNA synthesis. Phorbol-12,13-dibutyrate (PDBu) mimicked this antiproliferative action of TPA, but 4 alpha-phorbol-12,13-didecanoate was inactive in this capacity. Prolonged treatment of the cells with PDBu caused the partial down-regulation of protein kinase C. In these protein kinase C-reduced cells, WBS still induced DNA synthesis, but TPA did not inhibit the WBS-induced DNA synthesis. We have previously shown that protein kinase C is involved at least partially in the PDGF-induced DNA synthesis in rabbit aortic SMC. The present results together with this earlier observation suggest that protein kinase C has not only a proliferative but also an antiproliferative action in rabbit aortic SMC.     

Regulation of T lymphocyte differentiation and proliferation by thymus hormones, Ca2+ and chalone-T

The regulatory effect on T lymphocyte proliferation of the differentiator thymosin hormone family (thymosin fr. 5, A. L. Goldstein), Ca2+, and purified inhibitory protein fractions prepared from calf thymus was investigated in C57Bl/6 mice. 1.2 mM Ca2+ concentration was the most favourable for murine thymocyte growth in culture. Net protein synthesis was transitorily inhibited by Cu2+ concentrations higher than 2 mM. This inhibition was followed by a marked inhibition of DNA synthesis 2 hrs later. The effect of thymosin fr. 5 was slight, of short duration, and oscillatory in nature; in contrast, chalone-T preparations inhibited thymocyte DNA synthesis permanently up to 12 hrs of cultivation. When spleen cells taken from mice treated with the immunoadjuvant Bacillus Calmette-Guérin (BCG) were exposed to chalone-T in culture, then stimulated with PHA, a reduced proliferative response was measured in chalone-T pretreated cultures compared to controls or spleen cells from normal non-BCG-activated mice. This result had led us to suggest that chalone-T has a dual effect on thymocytes, viz. it inhibits cell cycle progression and induces the phenotypic conversion of suppressor T lymphocytes. The multifactorial concept of T lymphocyte production is discussed.     

Intracellular signalling of initiation of DNA synthesis: effect of cell-free extracts from anti-receptor-stimulated B lymphocytes on spleen cell nuclei

To study the relatively late intracellular signals involved in the proliferative response of B lymphocytes to antibodies specific for surface membrane immunoglobulins, extracts from antibody activated cells were mixed with Xenopus laevis splenic nuclei, and the incorporation of thymidine 5'-triphosphate into DNA was assessed. The slight incorporation observed with either nuclei or extract alone was markedly enhanced upon mixing the two entities when the extract was derived from cells cultured with but not without anti-receptor antibody. The appearance of active extract correlated well with the culture requirements necessary for the induction of B lymphocyte proliferation and, as revealed by time course studies, the active component arises relatively late in the activation process. Moreover, the appearance of active extracts is independent of DNA synthesis but is dependent on protein synthesis as judged from studies with metabolic inhibitors. Appropriate homogenization of activated cells yielded nuclei and cytoplasm with 85% of the activity confined to nuclei. In addition, purified active extracts exhibited DNA binding although the active component was readily distinguishable from polymerase alpha by chromatographic techniques. It is tentatively concluded that the active component represents either some replication protein other than polymerase or some earlier signal necessary to induce the formation or utilization of replicating proteins.     

A soluble 61-kDa protein is associated with inhibition of lectin-induced proliferation and IL-2 synthesis

An inhibitor of lectin-induced splenocyte proliferation from serum of normal chickens has been characterized. This suppressive factor, found in both serum and plasma and at concentrations as low as 3%, causes a 50% inhibition in proliferative responses to T-cell lectins of autologous and heterologous lymphoid cells. The inhibitor in serum also dramatically suppresses murine IL-2 synthesis, proliferation of murine spleen cells stimulated with PHA, and synthesis of DNA in xenogeneic-transformed mammalian lymphoblastoid cell lines. Serum does not block binding of the lectin to lymphoid cells and the suppressive activity cannot be overcome by any dose of lectin. The inhibitor of DNA synthesis is destroyed by pepsin. NH4(2)SO4 (50%) and TCA (15%) treatments both precipitate the suppressor factor, which further indicates that the suppressive factor is a protein. A 330-fold purification of the inhibitory protein from serum was obtained when boiled serum was passed over a Sepharose 6B and then a DEAE-Sephacel column which was washed at pH 5.0 and eluted with 0.2 M NaCl. SDS-PAGE with silver staining revealed a nonreduced protein with an apparent molecular weight of 61 kDa. Less than 2 micrograms of the protein thus obtained caused a 50% inhibition in the proliferation of chicken lymphoid cells to Con A. The inhibitor of DNA synthesis is therefore not cytotoxic, does not bind to Con A or to mannose or glucose residues on lymphocytes, is acid and heat stable, and is associated with a protein that has a molecular weight of 61 kDa. Since such low concentrations of this naturally occurring, proteinaceous, immunosuppressive factor cause substantial inhibition of IL-2 synthesis and proliferative activity of T cells, this protein may be a very important immunomodulator.     

Action of ketotifen on the mitogen-evoked proliferative response of human mononuclear cells

Ketotifen at low concentrations (5 and 50 microns) potentiated and at high ones (250 and 500 microM) blocked the proliferative response of human peripheral blood mononuclear cells ( NNC ) induced by PHA. The proliferative response was evaluated by 3H-thymidine incorporation into the cells. At doses inhibiting proliferative response to PHA ketotifen blocked both Con A-induced and Pokeweed mitogen-induced proliferations of MNC. At tested concentrations ketotifen inhibited and at the highest concentration (250 microM) blocked PHA-induced increase of protein synthesis in MNC evaluated by incorporation of 14C-L-leucine into the cells. The presented data showed that ketotifen acted not on the inductive step of the proliferative response but on the steps preceding the cell shift into S-phase.     

The regulatory effect of macrophages on the antigen-induced lymph node cell proliferative response

Employing an antigen-induced T cell-dependent lymph node cell (LNC) proliferative assay in the mouse we observed differences in the capacity of unstimulated and thioglycollate-activated peritoneal exudate cells (PECs) to present antigen. Antigen-primed LNCs can be induced to proliferate by a brief (2 hr) antigen exposure and the addition of various numbers of thioglycollate PECs (thio-PECs) modifies the proliferative response depending on the ratio of $\text{PEC}\text{LNC}$ in cultures. With ratios of 3–12% both DNA and protein synthesis were enhanced, but at ratios greater than 12% suppression was significant. Treatment of thio-PECs with mitomycin C, irradiation (3000 rad), anti-Thy 1.2, or anti-Ia plus complement did not alter suppression, suggesting the possibility that the Ia negative macrophages present in the PECs were involved in the suppression. An enhancing effect on the proliferative response was noted following the addition of small numbers of thio-PECs. This was comparable to that seen with an equivalent concentration of supernatant from thio-PECs suggesting that soluble factors play an important role. Two enhancing fractions (separated on a G-75 column) which were themselves mitogenic were identified which eluted with approximate molecular weights of 15,000 and 60,000.