Capacitative pulsed electric stimulation of bone cells. Induction of cyclic-AMP changes and DNA synthesis

Pulsed electric stimulation, coupled capacitively to bone cells isolated from rat embryo calvaria, caused changes in the intracellular level of cyclic AMP and enhanced DNA synthesis. The capacitive method of electrical stimulation was characterized in terms of displacement currents (0.7-4.0 A) and voltages (10-54 V/cm) prevailing in the stimulation chamber. Changes, both in cyclic AMP and in incorporation of [3H]thymidine into DNA, were correlated with the strength of the applied electric field. Unlike the mechanical stimulation of bone cells, the electrical stimulus was not mediated by de novo synthesis of prostaglandins. The findings suggest that cyclic-AMP changes, induced by the capacitive electrical stimulation of bone cells, trigger DNA synthesis.     

Effects of various electric fields on the fusion and in vitro development of mouse two-cell embryos

This study was undertaken to examine the effects of various electric fields such as alternating current (a.c.) voltage, fusion pulse strength, pulse duration, pulse number and electrode geometry on blastomere fusion and developmental rates of mouse two-cell embryos. The a.c. voltages (6 and 12 V/mm) did not affect the fusion and developmental rates. High fusion and developmental rates were obtained when pulse strengths of 1.0 to 2.5 kV/cm, pulse durations of 30 to 90 mu sec and pulse numbers of 1 to 6 were applied using a wire chamber. Comparison of electrode geometries showed that fusion rates were similarly high (93 to 98%) when pulse strengths of 1.0 to 2.5 kV/cm were applied, regardless of the electrode geometry. However, significantly lower developmental rates were observed in a rectangular chamber compared with those in a wire chamber, except when the pulse strength was 1.0 kV/cm. It was further observed that in a rectangular chamber, the developmental rate decreased with increasing pulse strength from 1.0 to 2.0 and 2.5 kV/cm. The results of this study indicate that by using a wire chamber, electric fields can be successfully applied across a relatively wide range of pulse strength, duration and number to provide sufficiently high fusion and subsequent developmental rates. The fusion conditions did, however, vary with chambers of different electrode geometries.     

Electric stimulation of human fibroblasts causes an increase in Ca2+ influx and the exposure of additional insulin receptors

Previously we reported that treating human fibroblasts in cell culture with high-voltage, pulsed galvanic stimulation (HVPGS) can significantly increase cellular protein and DNA synthesis (Bourguignon and Bourguignon: FASEB J., 1:398-402, 1987). In this study we have identified two of the early cellular events which occur following exposure to HVPGS: 1) an increase in Ca2+ uptake from the external medium and 2) an increase in the number of insulin receptors on the fibroblast cell surface. The increase in Ca2+ uptake begins within the first minute of electric stimulation while increased insulin binding is not detected until the second minute of stimulation. The HVPGS-induced increase in insulin binding can be inhibited by bepridil, a specific Ca2+ channel blocker, suggesting that the Ca2+ influx is required for the exposure of additional insulin receptors on the cell surface. Furthermore, we have determined that the addition of insulin to electrically stimulated cultures results in 1) an immediate, second increase in Ca2+ uptake and 2) significant increases in both protein and DNA synthesis compared to cells which were not stimulated. All three of these insulin-dependent effects are also inhibited by bepridil. Based on these results, we propose that HVPGS initially triggers the opening of voltage-sensitive calcium channels in the fibroblast plasma membrane. The increased level of intracellular Ca2+ then induces the exposure of additional insulin receptors, the fibroblasts will significantly increase both protein and DNA synthesis.     

Heat shock modulates the ability of A-549 cell line from human lung adenocarcinoma to regulate the intensity of DNA and protein biosynthesis by an autocrine mechanism]

A-549 cells of human lung adenocarcinoma were subjected to heat shock (30 min, 44 degrees C) which caused substantial decreases in the rates of biosynthesis of the great bulk of cellular proteins with simultaneous increases in the synthesis rates of the 70 kDa protein predominantly localized in cell cytosol. By the 6th hour after the heat shock cessation this protein synthesis reached its maximum; by the 18th hour it was no longer detectable, while the protein itself was not denatured. During the recovery after the heat shock the ability of the serum-free culture medium conditioned by A-549 cells in autocrine regulation of [3H]thymidine incorporation into DNA and [3H]leucine incorporation into proteins changed also. The conditioned medium obtained within 1-3 hours after the heat shock did not influence the intensity of DNA synthesis, while the medium obtained 4-48 hours after the heat shock stimulated this process, the maximal effect (3.3-fold stimulation) being observed in the case of the 48-hour conditioned medium. Temporary (1 hour) acidification of the conditioned media down to pH 2.0 resulted in complete inhibition of the stimulating activity. Besides, these media acquired an ability to inhibit [3H]thymidine incorporation into the DNA of tracer cells. Study of effects of conditioned media on the rate of [3H]leucine incorporation into A-549 cell proteins revealed that the media obtained 1-4 hours after the heat shock inhibited this process, while the media obtained 6-18 hours thereafter stimulated it 1.2-2.1-fold. In the test systems under study temporary acidification of the media increased their stimulating influence on [3H]leucine incorporation into cellular proteins.     

High hydrogen production rate of microbial electrolysis cell (MEC) with reduced electrode spacing

Practical applications of microbial electrolysis cells (MECs) require high hydrogen production rates and a compact reactor. These goals can be achieved by reducing electrode spacing but high surface area anodes are needed. The brush anode MEC with electrode spacing of 2 cm had a higher hydrogen production rate and energy efficiency than an MEC with a flat cathode and a 1-cm electrode spacing. The maximum hydrogen production rate with a 2 cm electrode spacing was 17.8 m(3)/m(3)d at an applied voltage of E(ap)=1 V. Reducing electrode spacing increased hydrogen production rates at the lower applied voltages, but not at the higher (>0.6 V) applied voltages. These results demonstrate that reducing electrode spacing can increase hydrogen production rate, but that the closest electrode spacing do not necessarily produce the highest possible hydrogen production rates.     

Effect of insulin at dilution endpoint concentrations on macromolecular synthesis in normal mouse mammary and human breast cancer epithelial cells

Employing defined media conditions, the insulin sensitivities of mouse mammary gland epithelial cells in primary culture and MCF-7 human mammary epithelial cells were determined. Insulin stimulated the rates of (3H] uridine incorporation into RNA and [3H] leucine incorporation into protein in both primary mouse mammary gland epithelial cell cultures and MCF-7 cell cultures at concentrations approximating the dilution endpoint of the hormone (10-21 M). Insulin stimulated the rate of [3H] thymidine incorporation into DNA in primary mouse mammary gland epithelial cells at the dilution endpoint concentrations. However, MCF-7 cells required insulin concentrations 100-1000-times that necessary in mouse mammary epithelial cultures to elicit an increased rate of [3H] thymidine incorporation into DNA. Evidence is presented which suggests that the increased rates of uptake of 3H- uridine, [3H] thymidine and [3H] leucine into their respective precursor pools is not responsible for the apparent stimulation of RNA, DNA and protein synthesis.     

Protein metabolism in senescing wheat leaves : determination of synthesis and degradation rates and their effects on protein loss

Wheat leaves (Triticum aestivum L.) at the moment of their maximum expansion were detached and put in darkness. Their protein, RNA and DNA contents, as well as their rates of protein synthesis and degradation, were measured at different times from 0 to 5 days after detachment. Rates of protein synthesis were measured by incorporation into proteins of large amounts of [³H]leucine. Fractional rates of protein degradation were estimated either from the difference between the rates of synthesis and the net protein change or by the disappearance of radioactivity from proteins previously labeled with [³H]leucine or [¹⁴C]proline.

Protein loss reached a value of 20% during the first 48 hours of the process. RNA loss paralleled that of protein, whereas DNA content proved to be almost constant during the first 3 days and decreased dramatically thereafter.

Measurements of protein synthesis and degradation indicate that, in spite of a slowdown in rate of protein synthesis, an increased rate of protein breakdown is mainly responsible for the observed rapid protein loss.

     

Molecular specificity of 2-aminopurine in Saccharomyces cerevisiae

The rates of DNA, RNA and protein synthesis were investigated by incorporation of radioactive precursors into the excised root tips of V. faba. 2-h exposure to 0.1% caffeine resulted in inhibition of protein synthesis to about 60% of the control rate. RNA synthesis was reduced in the range of 20–30%. The same concentration of caffeine did not affect the rate of DNA synthesis even during 12-h incubation, but concentrations higher than 1% caused a significant decrease in [³H]thymidine incorporation.     

Herpes Simplex Virus Type 2 Functions Expressed During Stimulation of Human Cell DNA Synthesis

Experiments were designed to identify herpes simplex virus type 2 (HSV-2)-specific functions expressed during stimulation of human embryo fibroblast DNA synthesis. Cultures were partially arrested in DNA synthesis by pretreatment with 5-fluorouracil and maintenance in low-serum (0.2%) medium during virus infection. Results showed that continuous [methyl-(3)H]thymidine uptake into cellular DNA was ninefold greater in HSV-2-infected than in mock-infected cultures measured after 24 h of incubation at 42 degrees C. Shifting mock-infected cultures from low- to high-serum (10%) medium also caused some stimulation, but [methyl-(3)H]thymidine uptake was only twofold greater than in cells maintained with low serum. Plating efficiencies of both HSV-2-infected and mock-infected cells at 42 degrees C were essentially the same and ranged from 37 to 76% between zero time and 72 h of incubation. De novo RNA and protein syntheses were continuously required for HSV-2 stimulation of cellular DNA synthesis. HSV-2 infection markedly enhanced transport, phosphorylation, and rate of incorporation of [methyl-(3)H]thymidine into cellular DNA, starting at 3 h and reaching a maximum by 12 h; after 12 h, these processes gradually declined to low levels. In mock-infected cells these processes remained at low levels throughout the observation period. Pretreatment of cells with interferon or addition of arabinofuranosylthymine at the time of virus infection inhibited stimulation caused by HSV-2. 5-Bromodeoxyuridine density-labeled experiments revealed that HSV-2 stimulates predominantly semiconservative DNA replication and some DNA repair. Stimulation of [methyl-(3)H]thymidine into cellular DNA correlated with detection of virus-specific thymidine kinase activity. In conclusion, HSV-2 stimulation of cellular DNA synthesis appeared to involve at least four virus-specific functions: induction of thymidine transport, HSV-2 thymidine kinase activity, semiconservative replication, and repair of cellular DNA.     

Beta-adrenergic receptors and adenylate cyclase in hypertrophic and hyperplastic rat salivary glands.

Isoproterenol induces both the secretion of protein and the stimulation of DNA synthesis and growth in rat salivary glands. The specific binding of the labelled beta-adrenergic antagonist [3H]dihydroalprenolol has been used to measure the number of beta-adrenergic receptors in rat parotid glands during isoproterenol-induced growth. Isoproterenol-enlarged glands display no change in the specific binding capacity per gland for [3H]-dihydroalprenolol compared with normal tissue. Catecholamine sensitive adenylate cyclase activity varies independently of the number of specific [3H]dihydroalprenolol binding sites during isoproterenol-induced growth. Previously-described di-ferences in optimal isoproterenol doses which produce protein secretion and stimulation of DNA synthesis may reflect different responses to various rates of receptor occupancy, or may be due to the presence of more than one type of beta-adrenergic receptor.     

DNA synthesis in mouse brown adipose tissue is under beta-adrenergic control

The rate of DNA synthesis in mouse brown adipose tissue was followed with injections of [3H]thymidine. Cold exposure led to a large increase in the rate of [3H]thymidine incorporation, reaching a maximum after 8 days, whereafter the activity abruptly ceased. A series of norepinephrine injections was in itself able to increase [3H]thymidine incorporation. When norepinephrine was injected in combination with the alpha-adrenergic antagonist phentolamine or with the beta-adrenergic antagonist propranolol, the stimulation was fully blocked by propranolol. It is suggested that stimulation of DNA synthesis in brown adipose tissue is a beta-adrenergically mediated process and that the tissue is an interesting model for studies of physiological control of DNA synthesis.     

Studies on the mechanism by which prolactin regulates protein, RNA, and DNA synthesis in Nb2 node lymphoma cells

Specific aspects of the prolactin stimulation of RNA, DNA and protein synthesis in the Nb2 node lymphoma cell line were determined. In time sequence studies the onset of the prolactin stimulation of the incorporation of radiolabeled precursors into these macromolecules was found to be 0.5-1 h for [3H]uridine incorporation into RNA, 1-2 h for [3H]leucine incorporation into protein, and 4-8 h for [3H]thymidine incorporation into DNA. The total DNA content of the cell cultures was increased by 12-18 hours after addition of prolactin. Amiloride, an inhibitor of the plasma-membrane-bound Na+/H+ antiporter, was found to inhibit the mitogenic effects of prolactin. Amiloride was also found to inhibit the prolactin stimulation of DNA, RNA and protein synthesis, thus suggesting that the initial regulation of the Na+/H+ antiporter may initiate these responses as well as the mitogenic effect of prolactin. In contrast, H-7, a drug which inhibits protein kinase C, had no effect on the magnitude of the prolactin stimulation of DNA, RNA or protein synthesis at a drug concentration (100 muM) that abolished the mitogenic effect of prolactin. The early effects of prolactin on RNA, DNA and protein synthesis would therefore appear not to involve an activation of protein kinase C.     

Synthesis of high mobility group proteins in regenerating rat liver.

Incorporation of [3H]lysine into the non-histone chromosomal proteins HMG1, HMG2, and HMG17 and into each of the five major classes of histones was measured in rat liver at various times after partial hepatectomy. Histone synthesis was closely coupled temporally to that of DNA, although a small amount of histone was shown to be produced before DNA replication began. In contrast, the incorporation curves for the high mobility group (HMG) proteins showed little correlation with that for DNA. At 4 h after partial hepatectomy, protein synthesis had virtually ceased. Thereafter, the rates of synthesis of the HMG proteins rose steadily so that by 12 h, well before the onset of DNA replication they had reached about two-thirds of the maximum rates attained during the first cell division cycle. Histones had only reached about one-sixth of their maximum rates at this time. The lack of coupling betweeen the synthesis of the HMG proteins and DNA was confirmed by experiments with inhibitors of DNA replication. Reduction of DNA synthesis to less than 10% of the uninhibited rate had little or no effect on incorporation into the HMG proteins, whereas, under similar conditions, the rate of synthesis of histones was reduced by more than 50%.     

Beta-adrenergic receptors and adenylate cyclase in hypertrophic and hyperplastic rat salivary glands

Isoproterenol induces both the secretion of protein and the stimulation of DNA synthesis and growth in rat salivary glands.The specific binding of the labelled beta-adrenergic antagonist [³H]dihydroalprenolol has been used to measure the number of beta-adrenergic receptors in rat parotid glands during isoproterenol-induced growth. Isoproterenol-enlarged glands display no change in the specific binding capacity per gland for [³H]-dihydroalprenolol compared with normal tissue.Catecholamine sensitive adenylate cyclase activity varies independently of the number of specific [³H]dihydroalprenolol binding sites during isoproterenol-induced growth.Previously-described differences in optimal isoproterenol doses which produce protein secretion and stimulation of DNA synthesis may reflect different responses to various rates of receptor occupancy, or may be due to the presence of more than one type of beta-adrenergic receptor.     

Formation and repair of DNA-protein crosslinks in newly replicated DNA

The production and removal of gamma-radiation-induced DNA-protein crosslinks (DPC) in nuclear matrix-associated newly replicated DNA were examined, as well as the relationship of DPC to DNA replication. In unirradiated, exponentially growing Chinese hamster V79 cells, DNA pulse labeled with [3H]thymidine was observed to be bound preferentially to protein. The pulse-labeled DNA subsequently became dissociated from protein. After a 30- to 60-min chase period, the level of labeled DNA in DPC was reduced to the same level as for bulk DNA. The radiation dose response for the formation of DPC was similar in newly replicated DNA that had been chased for various times and in mature chromatin DNA. Labeled DNA, in the DPC formed after 60 Gy, was rapidly removed from protein during the postirradiation incubation period. However, no recovery of DNA synthesis was observed, even after the majority of DPC were released. Thus either DPC are not the sole cause of the inhibition of DNA synthesis or their removal is not sufficient for DNA synthesis to resume.     

DNA and histone synthesis are uncoupled during germination of maize embryos

The rate of synthesis of DNA and histones was studied in germinating maize embryos as a function of the length of the germination period. To that end excised embryos from seeds germinated for different periods of time were pulse labelled either with [¹⁴C]protein hydrolysate or with [³H]TdR. Specific activities were determined for the total cellular proteins and the total histone fraction obtained by acid-extraction of the cellular homogenate and BioRex70 ion exchange chromatography. The results show that the early germination period is characterized by a lack of coupling between the histone synthesis and that of the nuclear DNA. The early histone synthesis peak might be necessitated by the reprogramming of the embryo genome that takes place during germination.     

The effect of microwave radiation on the cell genome

Cultured V79 Chinese hamster cells were exposed to continuous radiation, frequency 7.7 GHz, power density 30 mW/cm2 for 15, 30, and 60 min. The parameters investigated were the incorporation of [3H]thymidine and the frequency of chromosome aberrations. Data obtained by 2 methods (the incorporation of [3H]thymidine into DNA and autoradiography) showed that the inhibition of [3H]thymidine incorporation took place by complete prevention of DNA from entering into the S phase. The normal rate of incorporation of [3H]thymidine was recovered within 1 generation cycle of V79 cells. Mutagenic tests performed concurrently showed that even DNA macromolecules were involved in the process. In comparison with the control samples there was a higher frequency of specific chromosome lesions in cells that had been irradiated. Results discussed in this study suggest that microwave radiation causes changes in the synthesis as well as in the structure of DNA molecules.     

The rate of protein degradation in developing brain. Methodological considerations.

Recently we reported that the rate of protein breakdown decreases during development. Breakdown rates were calculated from the rates of protein synthesis and the changes in brain protein content with age. A different study, measuring breakdown by monitoring the loss of label from brain protein after an H14CO3- pulse, came to the opposite conclusion: that the rate of breakdown is low in immature brain and increases during development. We have now investigated some of the factors (the distribution of label in protein and the potential for recycling) that might introduce errors into these measurements. The specific radioactivities of both protein-bound and free amino acids were determined in the brains of young rats several days after an intraperitoneal pulse of H14CO3-. For a number of amino acids the specific radioactivity of the free amino acid is high compared with that of the protein-bound amino acid, and therefore recycling could result in an underestimate of the degradation rate. Because glutamic acid had a relatively low specific-radioactivity ratio, [1-14C]glutamic acid was used in a pulse-labelling experiment to measure degradation. The rate so obtained, 0.6% . h-1, is twice the rate found with H14CO3- labelling (based on total protein-bound radioactivity). Insofar as recycling is a possible complication, 0.6% . h-1 may be a minimum value. Although somewhat higher degradation rates are found after labelling with an intracranial pulse, which was considered as a possible route to limit recycling, there are difficulties in interpreting these data.     

Purine nucleoside phosphorylase synthesis and turnover in human lymphoid cells

We have studied the turnover and synthesis of purine nucleoside phosphorylase by using a polyclonal rabbit antiserum to this protein. The turnover of purine nucleoside phosphorylase was studied in the B lymphoblast cell, WI-L2, by specific immunoprecipitation of [3H]leucine-labeled proteins. The half-lives for total protein and purine nucleoside phosphorylase were 14.5 and 14.1 hr, respectively. For cells cultured in the presence of inosine the half-life of purine nucleoside phosphorylase was reduced to 11.2 hr. The synthesis of purine nucleoside phosphorylase was analyzed during phytohemagglutinin-stimulated T cell transformation by pulse labeling cells with [35S]methionine. Purine nucleoside phosphorylase synthesis increased greater than 10-fold during the first 12 hr of transformation and continued to a maximum of 30-fold. The relative rate of purine nucleoside phosphorylase labeled to total proteins was 0.04% in unstimulated T cells and increased to 0.18% 12 hr after stimulation. These studies identify some preferential synthesis of purine nucleoside phosphorylase during the early stages of T cell transformation.     

Dynamics of the dATP pool in cultured mammalian cells.

Conditions for labeling the dATP pool of V79 and 3T3 cells from [3H]deoxyadenosine (salvage) or [3H]adenine (via ribonucleotide reduction) were established. With deoxyadenosine the specific radioactivity of dATP reached a constant value after 60 min. In resting 3T3 cells this value was 30 times higher than in S-phase cells. Turnover of dATP and absolute rates of DNA synthesis and excretion of breakdown products of dATP were determined from the accumulation of isotope in various compartments and the specific activity of dATP. In S-phase cells the dATP pool had a half-life of 4 min, identical to that of dTTP determined earlier. Deoxyadenosine was the major breakdown product of dATP in the presence of an inhibitor of adenosine deaminase. The rate of deoxyadenosine excretion of V79 cells amounted to 4% of the rate of dATP incorporation into DNA. Inhibition of DNA replication increased deoxyadenosine excretion 5- to 10-fold, demonstrating a continued de novo synthesis of dATP, albeit at a slightly reduced rate. Our results fit a model involving a substrate cycle between dAMP and deoxyadenosine regulating the dATP pool, similar to the model of substrate cycles involved in the regulation of pyrimidine deoxyribonucleotide pools developed earlier.