Nuclear and mitochondrial DNA synthesis and energy metabolism in primary rat glial cell cultures

DNA synthesis in nuclei and mitochondria purified from serum-supplemented rat glial cell cultures at different days after plating was studied. Furthermore in mitochondria, some enzymatic activities related to energy transduction (citrate synthase, malate dehydrogenase, total NADH-cytochromec reductase, cytochrome oxidase and glutamate dehydrogenase) were measured. For DNA labeling [methyl-³H]thymidine was added to the culture medium at different days after plating. During the culture times studied the specific activity of total, nuclear, and mitochondrial DNA decreased from 8 days in vitro (DIV) to 21 DIV and increased at 30 DIV. The specific activity of nuclear DNA was always higher than that of mitochondrial DNA. The specific activity of the above mentioned mitochondrial enzymes increased from 8 DIV up to 21 DIV and decreased at 30 DIV, suggesting a relationship between the energy metabolism and the differentiation of glial cells in culture.The AA. would like to dedicate this paper to the memory of Dr. Ida Serra, Associate Professor of Biochemistry at the Medical Faculty, University of Catania, who prematurely died, after this paper was submitted for publication.     

Taxol inhibits stimulation of cell DNA synthesis by human cytomegalovirus

The microtubule (MT)-stabilizing drug, taxol, inhibited human cytomegalovirus (CMV)-initiated cell DNA synthesis by up to 100% in serum-arrested mouse embryo (ME) fibroblasts that were abortively infected by CMV. Taxol concentrations known to increase MT polymerization and to stabilize existing MTs (10 to 20 micrograms/ml) blocked CMV-stimulated cell DNA synthesis, while taxol concentrations of 2.5 micrograms/ml, or less, did not. Taxol maximally inhibited CMV initiation of cell DNA synthesis when added 3 h after virus infection and inhibited this initiation by greater than 50% when added up to 12 h after CMV infection. Control experiments suggest that taxol specifically inhibited CMV-stimulated cell DNA synthesis. Pretreatment of CMV stock with taxol did not reduce the stimulatory effect of CMV on cell DNA synthesis and taxol had no detectable effect on CMV-specific early protein synthesis. Moreover, taxol did not appear to alter thymidine pool sizes, affect cell viability, or compromise the DNA synthetic machinery in CMV-infected cells. Since taxol increases tubulin polymerization and inhibits MT disassembly, these results suggest that dynamic changes in MTs or in the pool of free tubulin subunits are necessary for CMV to stimulate cell entry into a proliferative cycle.     

Nonrequirement of continuous stimulation with MCP-1 for cell migration and determination of directional migration by initial stimulation with chemokine

Monocyte chemoattractant protein-1 (MCP-1) induces monocyte migration through interaction with the MCP-1 receptor CCR2. In this report we have examined the length of chemokine stimulation necessary for induction of cell migration and whether continuous stimulation is required for active migration. Monocytic THP-1 cells prestimulated with MCP-1 for 15 to 30 min exhibited a migration response after the chemokine was removed from the culture medium, indicating that a short exposure to chemokine stimulation is sufficient for migration of THP-1 cells and continuous stimulation is not required for active migration. A reverse gradient of MCP-1 had no effect on migration after prestimulation with MCP-1. This implies that cells are determined to directionally migrate by initial stimulation with MCP-1. Furthermore, cell migration after prestimulation with MCP-1 was inhibited by a p38 inhibitor, but not by a phosphatidylinositol 3-kinase (PI3-kinase) inhibitor, indicating that p38, but not PI3-kinase, is involved in the migration response after the determination of direction by initial chemokine stimulation.     

Studies on serum stimulation of mouse fibroblast migration

Evidence is presented that the factor present in rat serum that promotes the migration of mouse fibroblasts (3T3) is a high mol. wt molecule. Amino acids, Mg²⁺ and Ca²⁺ are essential for full expression of activity. Once exposed to serum there is an 8–10 h lag period before cell migration can be detected. Cycloheximide, velban, colchicine, actinomycin D, and cytochalasin B, all inhibit cellular migration from a wound edge.     

Stimulation of DNA synthesis by serum and amino acid in a rat neuroblastoma cell line.

A procedure for arresting B65 rat neuroblastoma cells in the G1 (or G0) phase of the cell cycle is described. This procedure is used to study the effects of serum and amino acids on the initiation of DNA synthesis in this transformed cell line. The amino acid glutamine has DNA-stimulating activity which can be increased with the addition of cystine which alone is inactive.     

Coordination of DNA synthesis and replicative unwinding by the S-phase checkpoint pathways

The process of DNA replication includes duplex unwinding, followed immediately by DNA synthesis. In eukaryotes, DNA synthesis is disturbed in damaged DNA regions, in replication slow zones, or as a result of insufficient nucleotide level. This review aims to discuss the mechanisms that coordinate DNA unwinding and synthesis, allowing replication to be completed even in the presence of genomic insults. There is a growing body of evidence which suggests that S-phase checkpoint pathways regulate both replicative unwinding and DNA synthesis, to synchronize the two processes, thus ensuring genome stability.     

Alpha-S-GalCer: synthesis and evaluation for iNKT cell stimulation

The synthesis and evaluation for iNKT stimulation of alpha-S-galactosylceramide is reported. Prepared by alkylation of a galactosylthiol, this analog of the potent immunostimulatory agent, KRN7000, did not stimulate iNKT cells either in vitro or in vivo.     

ErbB receptors and cell polarity: New pathways and paradigms for understanding cell migration and invasion

The ErbB family of receptor tyrosine kinases is involved in initiation and progression of a number of human cancers, and receptor activation or overexpression correlates with poor patient survival. Research over the past two decades has elucidated the molecular mechanisms underlying ErbB-induced tumorigenesis, which has resulted in the development of effective targeted therapies. ErbB-induced signal transduction cascades regulate a wide variety of cell processes, including cell proliferation, apoptosis, cell polarity, migration and invasion. Within tumors, disruption of these core processes, through cooperative oncogenic lesions, results in aggressive, metastatic disease. This review will focus on the ErbB signaling networks that regulate migration and invasion and identify a potential role for cell polarity pathways during cancer progression.     

Dermal tracts in frog skin: fibronectin pathways for cell migration

The dermis of the frog skin (Rana esculenta) displayed a remarkable organization of vertical and horizontal tracts. Vertical thick tracts connected the dermal Stratum spongiosum with the subcutaneous tissue. Horizontal thin tracts were found alongside and contiguous to them. The thick tracts were sheathed by collagen fibrils of the Stratum compactum which were vertically oriented (i.e. parallel to the axes of the tracts) according to the horizontal and orthogonal arrangement of the collagen bundles of the Stratum compactum. The thin tracts devoid of collagenous sheath were formed by clear spaces between superimposed collagen bundles of the dermal Stratum compactum. On vertical sections, the thick tracts were seen to contain fibronectin (FN), detected by indirect immunoperoxidase. Continuous vertical FN lines were centred in these tracts. On horizontal sections, a clear zone around these FN-centred lines was also sheathed by FN. The thick tracts contained flattened pigmentary cells and fibroblasts; these cells were FN-outlined. The thin tracts contained patches of FN and FN-outlined fibroblasts. In culture, in vertical thick tracts, both pigmentary cells and fibroblasts disappeared when antiserum to FN was added to the culture medium. This suggested that thick tracts were pathways allowing pigmentary cells to move upward or downward between their usual upper dermal and lower subcutaneous localizations. Fewer fibroblasts were found in the thin tracts in the presence of antiserum to FN.     

Efficient synthesis of galactosylceramide analogues for iNKT cell stimulation

Glycolipids are potential antigens for iNKT cells recognition and demonstrate important roles in both innate and adaptive immunity. However, the difficulties in the preparation of pure configuration defined glycolipids limit the exploration of their different profiles in activating iNKT cells. We report here a concise and stereospecific preparation of novel galactosylceramide analogues by oxime ligation. This strategy would provide an efficient way to generate varied glycolipid analogues with either synthetic or natural carbohydrates for biological evaluations.     

Proline is required for the stimulation of DNA synthesis in hepatocyte cultures by EGF

Summary Epidermal growth factor (EGF) has been shown to stimulate DNA synthesis in rat parenchymal hepatocytes both in vivo and in vitro (4,9). We report here that this response in vitro is dependent on the amino acids present in the media. Of all the amino acids, proline has the strongest effect. The response to EGF is absent without proline and none of the other amino acids can substitute for it. Added proline (1 mM) to the media caused the labeling index to increase from 11% to 55% in the presence of 50 ng/ml EGF and insulin. In the presence of proline, small additional increases of the EGF effect on DNA synthesis were stimulated by phenylalanine and tyrosine. This work was supported by NIH grants CA302241 and CA35373 and EPA CRA R811687010 EDITOR'S STATEMENT This paper describes an interesting, specific interaction between EGF and proline in stimulation of DNA synthesis in hepatocytes. This finding may shed light on the well-documented effect of amino acid load on hepatocyte proliferation in vivo and interactions of growth factors and nutrients in general. Wallace L. McKeehan     

The influence of calcium on the cyclic AMP-mediated stimulation of DNA synthesis and cell proliferation by prostaglandin E 1

Prostaglandin type E₁ (PGE₁) rapidly stimulates cyclic AMP formation and the initiation of deoxyribonucleic acid (DNA) synthesis in rat thymic lymphocytes suspended in vitro by reactions which are not affected by wide variations in the extracellular calcium concentration. On the other hand, the operation of the associated reaction(s) responsible for the subsequent progression of the stimulated cells into mitosis is profoundly affected by the extracellular calcium level. If the maximum intracellular cyclic AMP concentration is in the lower range of stimulatory values (e.g., 150 × 10^?8 picomoles per cell as produced by an exposure to 0.5 μg of PGE₁ per milliliter of medium), an extracellular calcium concentration of 0.5 to 1.0 mM is needed to obtain maximum cell proliferation, but not the maximum stimulation of DNA synthesis. Contrariwise, if the cellular cyclic AMP content is raised to a much higher level (260 × 10^?8 picomoles per cell) by exposure to a greater PGE₁ concentration (5.0 μg per millilter), cell proliferation is maximally stimulated in calcium-free medium and increasing the extracellular calcium concntration above 0.2 mM actually prevents the stimulation of cell proliferation (but does not affect the stimulation of DNA synthesis). Thus, the ultimate translation of PGE₁'s early cyclic AMP-mediated reactions into increased cell proliferation is determined by both the intracellular cyclic AMP level and the extracellular calcium concentration.     

Integrin-specific signaling pathways controlling focal adhesion formation and cell migration

The fibronectin (FN)-binding integrins alpha4beta1 and alpha5beta1 confer different cell adhesive properties, particularly with respect to focal adhesion formation and migration. After analyses of alpha4+/alpha5+ A375-SM melanoma cell adhesion to fragments of FN that interact selectively with alpha4beta1 and alpha5beta1, we now report two differences in the signals transduced by each receptor that underpin their specific adhesive properties. First, alpha5beta1 and alpha4beta1 have a differential requirement for cell surface proteoglycan engagement for focal adhesion formation and migration; alpha5beta1 requires a proteoglycan coreceptor (syndecan-4), and alpha4beta1 does not. Second, adhesion via alpha5beta1 caused an eightfold increase in protein kinase Calpha (PKCalpha) activation, but only basal PKCalpha activity was observed after adhesion via alpha4beta1. Pharmacological inhibition of PKCalpha and transient expression of dominant-negative PKCalpha, but not dominant-negative PKCdelta or PKCzeta constructs, suppressed focal adhesion formation and cell migration mediated by alpha5beta1, but had no effect on alpha4beta1. These findings demonstrate that different integrins can signal to induce focal adhesion formation and migration by different mechanisms, and they identify PKCalpha signaling as central to the functional differences between alpha4beta1 and alpha5beta1.     

Inhibition of DNA synthesis and cell division by a cell surface sialoglycopeptide

We have isolated and purified a cell surface sialoglycopeptide (SGP) from bovine cerebral cortex cells that previously was shown to be a potent inhibitor of cellular protein synthesis. The following studies were carried out to characterize the potential ability of the SGP to inhibit DNA synthesis and to arrest cell division. Treatment of exponentially proliferating Swiss 3T3 cells with the SGP inhibitor resulted in a marked inhibition of thymidine incorporation within 24 h. When the SGP was removed from inhibited cultures, a sharp rise in 3H-thymidine incorporation followed within 3-4 h that peaked well above that measured in exponentially growing cultures, suggesting that the inhibitory action of the SGP was reversible and that a significant proportion of the arrested cells was synchronized in the mitotic cycle. In addition to DNA synthesis, the inhibitory action of the SGP was monitored by direct measurement of cell number. Consistent with the thymidine incorporation data, the SGP completely inhibited 3T3 cell division 20 h after its addition to exponentially growing cultures. Upon reversal there was a delay of 15 h before cell division resumed, when the arrested cells quickly doubled. Most, if not all, of the growth-arrested cells appeared to have been synchronized by the SGP. The SGP inhibited DNA synthesis in a surprisingly wide variety of target cells, and the relative degree of their sensitivity to the inhibitor was remarkably similar. Cells sensitive to the SGP ranged from vertebrate to invertebrate cells, fibroblast and epitheliallike cells, primary cells and established cell cultures, as well as a wide range of transformed cell lines.     

Pluripotent haemopoietic stem cell entry into DNA synthesis in vitro after stimulation by Ara-C treatment in vivo

Non-cycling pluripotent bone marrow stem cells (CFU-S), taken 3 hrs after injection of 20 mg of Ara-C, have been shown to enter DNA synthesis at 1 to 3 hr after being cultured in alpha-medium. This phenomenon was observed when bone marrow was incubated as a plug, but not when incubated as a cell suspension in the present experimental conditions. These results suggest that a medullary structure is necessary in order to observe this effect and/or that accessory cells are destroyed during the process of single cell preparation.