Increase In Cellular Content Research Articles

Histological Analysis of Platelet-Rich Fibrin in Diabetics, Non-diabetics, and Smokers Suffering with Chronic Periodontitis: An In Vitro Study.

The present study was aimed at evaluating the variations in fibrin network patterns of the platelet-rich fibrin (PRF) procured from diabetics, non-diabetics, and smokers suffering from chronic periodontitis. Five milliliters of blood were drawn using a syringe from 60 participants (systemically healthy non-diabetics, diabetics, and smokers) who were diagnosed with chronic periodontitis. Five milliliters of blood were transferred to a dry glass tube. The clot (PRF) obtained after centrifugation from the glass tube was processed for light microscopy analysis. The PRF samples have demonstrated the presence of both dense and loose fibrin networks. There is an increase in cellular content in diabetic participants compared to systemically healthy and smoker participants. Smoker participants demonstrated a higher amount of loose fibrin content, which was arranged irregularly. A significantly greater increase in cellular content and loose fibrin network is seen in diabetics and smokers, respectively, compared to systemically healthy individuals. As compared to the systemically healthy participants, there is a variation in the fibrin network pattern and distribution of cellular structures in diabetic and smoker participants.

Ecophysiological responses of Ostreopsis towards temperature: A case study of benthic HAB facing ocean warming

Reports of the benthic dinoflagellate Ostreopsis spp. have been increasing in the last decades, especially in temperate areas. In a context of global warming, evidences of the effects of increasing sea temperatures on its physiology and its distribution are still lacking and need to be investigated. In this study, the influence of temperature on growth, ecophysiology and toxicity was assessed for several strains of O. cf. siamensis from the Bay of Biscay (NE Atlantic) and O. cf. ovata from NW Mediterranean Sea. Cultures were acclimated to temperatures ranging from 14.5 °C to 32 °C in order to study the whole range of each strain-specific thermal niche. Acclimation was successful for temperatures ranging from 14.5 °C to 25 °C for O. cf. siamensis and from 19 °C to 32 °C for O. cf. ovata, with the highest growth rates measured at 22 °C (0.54–1.06 d−1) and 28 °C (0.52–0.75 d−1), respectively. The analysis of cellular content of pigments and lipids revealed some aspects of thermal acclimation processes in Ostreopsis cells. Specific capacities of O. cf. siamensis to cope with stress of cold temperatures were linked with the activation of a xanthophyll cycle based on diadinoxanthin. Lipids (neutral reserve lipids and polar ones) also revealed species-specific variations, with increases in cellular content noted under extreme temperature conditions. Variations in toxicity were assessed through the Artemia franciscana bioassay. For both species, a decrease in toxicity was observed when temperature dropped under the optimal temperature for growth. No PLTX-like compounds were detected in O. cf. siamensis strains. Thus, the main part of the lethal effect observed on A. franciscana was dependent on currently unknown compounds. From a multiclonal approach, this work allowed for defining specificities in the thermal niche and acclimation strategies of O. cf. siamensis and O. cf. ovata towards temperature. Potential impacts of climate change on the toxic risk associated with Ostreopsis blooms in both NW Mediterranean Sea and NE Atlantic coast is further discussed, taking into account variations in the geographic distribution, growth abilities and toxicity of each species.

Rapid and Positive Effect of Bicarbonate Addition on Growth and Photosynthetic Efficiency of the Green Microalgae Chlorella Sorokiniana (Chlorophyta, Trebouxiophyceae)

Bicarbonate ions are the primary source of inorganic carbon for autotrophic organisms living in aquatic environments. In the present study, we evaluated the short-term (hours) effects of sodium bicarbonate (NaHCO3) addition on the growth and photosynthetic efficiency of the green algae Chlorella sorokiniana (211/8k). Bicarbonate was added to nonaxenic cultures at concentrations of 1, 2, and 3 g L−1 leading to a significant increase in biomass especially at the highest salt concentration (3 g L−1) and also showing a bactericidal and bacteriostatic effect that helped to keep a reduced microbial load in the algal culture. Furthermore, bicarbonate stimulated the increase in cellular content of chlorophyll a, improving the photosynthetic performance of cells. Since microalgae of genus Chlorella spp. show great industrial potential for the production of biofuels, nutraceuticals, cosmetics, health, and dietary supplements and the use of bicarbonate as a source of inorganic carbon led to short-term responses in Chlorella sorokiniana, this method represents a valid alternative not only to the insufflation of carbon dioxide for the intensive cultures but also for the production of potentially bioactive compounds in a short period.

Simultaneous increase in cellular content and volumetric concentration of lipids in Bracteacoccus bullatus cultivated at reduced nitrogen and phosphorus concentrations

Manipulation of the nutrient concentration is an inexpensive and efficient method for increasing lipid and TAG accumulation in algal cells. However, high volumetric production requires finding a proper balance between the decrease of biomass production and the increase in the total lipid content. We isolated a strain of green microalga Bracteacoccus bullatus and increased its lipid content from 17 to 59% of biomass dry weight by manipulating of nitrogen and phosphorus content in the medium. The 10-fold reduction of the nitrogen and phosphorus concentration in the medium was the most efficient method of the lipid induction compared to nutrient deplete and high nutrient conditions. The oleic (48–64% mass of total fatty acids) and linoleic (14–24% mass of total fatty acids) acids dominated in the fatty acid profile, thus making this strain a suitable candidate for biodiesel production.

Potential of algal biofuel production in a hybrid photobioreactor

Hybrid bioreactors are equipment featuring characteristics of pneumatic bioreactors and stirred tanks. The production of lipids and carbohydrates was evaluated in a concentric draft tube stirred airlift photobioreactor. Mixotrophic cultivation was carried out using the microalgae Chlorella vulgaris grown in vegetable waste. The response surface methodology was used for the experiment optimization. A central composite rotatable design was used to evaluate the effects of light intensity, CO2 concentration and stirring (independent variables) on the lipid and carbohydrate production. The exponential phase of growth occurred mainly in the first seven days of cultivation, and the stationary phase was achieved under all experimental conditions (occurring between the 9th and 14th day). The increase in cellular concentration was obtained mainly in experiments performed with high light intensity and CO2 concentration. At a rotational speed of 800rpm, growth was affected due to excessive shearing forces caused by increased turbulence. On the other hand, in the absence or at low CO2 concentrations there was a considerable increase in lipid and carbohydrate production. Through response surfaces, we observed that higher percentages of these metabolites may be obtained without adding CO2 and operating the bioreactor with high light intensity and with rotation speed higher than 800rpm or at low rotational speeds as well as conventional airlift. From an economic point of view, stirring can cause an increase in power consumption; however, if the bioreactor is operated at low rotational speeds, the difference in power consumption with airlift bioreactors is minimal, while the productivity of lipids and carbohydrates increases, which makes the use of this equipment very attractive.

Sparse feature selection methods identify unexpected global cellular response to strontium-containing materials

Despite the increasing sophistication of biomaterials design and functional characterization studies, little is known regarding cells' global response to biomaterials. Here, we combined nontargeted holistic biological and physical science techniques to evaluate how simple strontium ion incorporation within the well-described biomaterial 45S5 bioactive glass (BG) influences the global response of human mesenchymal stem cells. Our objective analyses of whole gene-expression profiles, confirmed by standard molecular biology techniques, revealed that strontium-substituted BG up-regulated the isoprenoid pathway, suggesting an influence on both sterol metabolite synthesis and protein prenylation processes. This up-regulation was accompanied by increases in cellular and membrane cholesterol and lipid raft contents as determined by Raman spectroscopy mapping and total internal reflection fluorescence microscopy analyses and by an increase in cellular content of phosphorylated myosin II light chain. Our unexpected findings of this strong metabolic pathway regulation as a response to biomaterial composition highlight the benefits of discovery-driven nonreductionist approaches to gain a deeper understanding of global cell-material interactions and suggest alternative research routes for evaluating biomaterials to improve their design.

Alcoholic Fermentation with Flocculant Saccharomyces cerevisiae in Fed-Batch Process

Studies have been conducted on selecting yeast strains for use in fermentation for ethanol production to improve the performance of industrial plants and decrease production costs. In this paper, we study alcoholic fermentation in a fed-batch process using a Saccharomyces cerevisiae yeast strain with flocculant characteristics. Central composite design (CCD) was used to determine the optimal combination of the variables involved, with the sucrose concentration of 170 g/L, a cellular concentration in the inoculum of 40% (v/v), and a filling time of 6 h, which resulted in a 92.20% yield relative to the theoretical maximum yield, a productivity of 6.01 g/L h and a residual sucrose concentration of 44.33 g/L. With some changes in the process such as recirculation of medium during the fermentation process and increase in cellular concentration in the inoculum after use of the CCD was possible to reduce the residual sucrose concentration to 2.8 g/L in 9 h of fermentation and increase yield and productivity for 92.75% and 9.26 g/L h, respectively. A model was developed to describe the inhibition of alcoholic fermentation kinetics by the substrate and the product. The maximum specific growth rate was 0.103 h(-1), with K(I) and K(s) values of 109.86 and 30.24 g/L, respectively. The experimental results from the fed-batch reactor show a good fit with the proposed model, resulting in a maximum growth rate of 0.080 h(-1).

Abstract A10: Peroxiredoxin-1 and oxidative stress: Controlling the balance between oncogenic and endocrine signalling in estrogen receptor-positive breast cancer

Abstract Introduction: Peroxiredoxin-1 (PRDX1) is a multifunctional protein, acting as a hydrogen peroxide (H2O2) scavenger, molecular chaperone and immune mediator. Differential expression of PRDX1 has been described in many tumor types, including lung and ovarian carcinoma. Despite the wealth of knowledge about PRDX1 functionality, its role in human breast cancer has not been fully elucidated. Preclinical studies suggest that PRDX1 may be protective against oncogene-induced mammary carcinogenesis, indicating that it may be an important biomarker. In this study, we describe PRDX1 as a robust prognostic biomarker in estrogen receptor α (ER)-positive breast cancer and propose a molecular mechanism which explains this observation. Materials and Methods: The anti-PRDX1 antibody was validated in breast cancer cell lines using Western blotting, immunohistochemistry and reverse phase protein array (RPPA) technology following exogenous overexpression or shRNA-mediated knockdown of PRDX1. PRDX1 protein expression was evaluated using two independent breast cancer cohorts: a 512 patient tissue microarray (TMA) and a 712 patient RPPA cohort. Increase in cellular content of H2O2 was accomplished via treatment of cell lines with exogenous (H2O2) or activation of oncogenic pathways (611CTF-HER2 overexpression). Western blotting and RPPA were used to assess changes in ERα and oncogenic signaling proteins in cell lines and a third breast cancer cohort, consisting of 410 patients. Results: High expression of PRDX1 protein was associated with a favorable outcome in ER-positive, but not ER-negative breast cancer cases across both cohorts (evaluable data for 975 patients total; log-rank p-value: TMA=0.022; RPPA=0.002). Exogenous treatment with (H2O2) or induction of oncogenic signaling suppressed ERα protein and stimulated phosphorylation/activation of Akt kinase in ER-positive cell lines. Knockdown of PRDX1 further sensitized the cells to the (H2O2)-mediated effect whilst overexpression protects against it. These observations were further validated in an additional cohort of ER-positive tumor samples, where PRDX1 protein levels correlated with ERα (positive) and pAkt-473 protein expression (negative). Conclusions: Our findings provide robust evidence of the importance of PRDX1 as a biomarker of favorable prognosis in ER-positive breast cancer. We suggest that PRDX1 acts as a shielding mechanism for ERα protein by counteracting (H2O2)-mediated suppression of this molecule in ER-positive tumors, and suppressing phosphorylation of Akt kinase under these oxidative stress conditions. This strongly implies that (H2O2) and PRDX1 are important role players in the crosstalk between oncogenic (e.g. HER2- or PI3K-induced) and ER-driven signaling pathways in breast cancer. Understanding the mechanisms underlying the regulation of oxidative stress response in ER-positive breast cancer can allow for better tailoring the management of this disease.

Comparison of anti-tumor efficacy of paclitaxel delivered in nano- and microparticles

This research compares the anti-tumor efficacy of paclitaxel delivered intratumorally in PLGA nanoparticles, microparticles, or the commercial Paclitaxel Injection ®. The hypothesis of the research is that larger PLGA microparticles adhere to mucus on the cell surface, release paclitaxel locally, and enhance cellular association of paclitaxel. PLGA-paclitaxel particles of mean diameters 315 nm, 1 μm, and 10 μm were prepared and their drug content, in vitro release, and cellular association of paclitaxel into 4T1 cells quantified. These particles were injected intratumorally into tumor xenografts, and the tumor volumes monitored over 13 days. Mean tumor volumes of the groups that received placebo and the 315 nm nanoparticles increased 2 and 1.5 times, respectively. Tumor growth was arrested in groups that received 1 μm and 10 μm microparticles. Additional cell culture studies were performed to test the hypothesis. The size-dependent increase in cellular concentration of paclitaxel was independent of duration of incubation of PLGA particles with 4T1 cells, and was enhanced 1.5 times by coating the particles or 4T1 cells with mucin. These particles were not internalized by clathrin-mediated endocytosis or macropinocytosis. In conclusion, PLGA microparticles sustained drug release, increased cellular concentration, and enhanced anti-tumor efficacy of paclitaxel compared to nanoparticles and Paclitaxel Injection ®.

Prevention of Alloxan-induced Diabetes by Se-Methylselenocysteine Pretreatment in Rats: The Effect on Antioxidant System in Pancreas

In this study, we assessed the effects of Se-methylselenocysteine (MSC) pretreatment on the antioxidant system in the pancreas and the development of alloxan-induced diabetes in rats. The rats were treated with MSC at a dose of 0.75 ㎎/rat/day for 2 weeks. The MSC-treated rats evidenced significantly increased glutathione content, GSH/GSSG ratio, and glutathione peroxidase (GPx) and glutathione reductase (GRd) activities in the pancreas. Diabetes was induced via alloxan injection. The alloxan-diabetic rats evidenced significantly reduced glutathione content and glucose 6-phosphate dehydrogenase (G6PD) activity and increased catalase activity in the pancreas, when measured 3 days after the alloxan injection. 2-week MSC pretreatment was shown to prevent the alloxan-induced hyperglycemia as well as changes in glutathione content, G6PD activity, and catalase activity. The results of this study indicate that the prevention of alloxan-diabetes by MSC pretreatment is associated with its effects on antioxidants in the pancreas, namely, the increase in cellular content and the reduction of glutathione by the facilitation of glutathione recycling induced via increased GPx, GRd, and G6PD activities.

NITZSCHIA OVALIS (BACILLARIOPHYCEAE) MONO LAKE STRAIN ACCUMULATES 1,4/2,5 CYCLOHEXANETETROL IN RESPONSE TO INCREASED SALINITY(1).

The growth of microalgae in hypersaline conditions requires that cells accumulate osmoprotectants. In many instances, these are polyols. We isolated the diatom Nitzschia ovalis H. J. Arn. from the saline and alkaline water body Mono Lake (CA, USA). This isolate can grow in salinities ranging from 5 to 120 parts per thousand (ppt) of salt but normally at 90 ppt salinity. In this report, we identified the major polyol osmoprotectant as 1,4/2,5 cyclohexanetetrol by electron ionization-mass spectrometry (EI-MS), (1) H, (13) C nuclear magnetic resonance spectroscopy (NMR), and infrared (IR) and showed an increase in cellular concentration in response to rising salinity. This increase in the cyclitol concentration was evaluated by gas chromatography of the derived tetraacetylated cyclohexanetetrol obtaining an average of 0.7 fmol · cell(-1) at 5 ppt and rising to 22.5 fmol · cell(-1) at 120 ppt. The 1,4/2,5 cyclohexanetetrol was also detected in the red alga Porphyridium purpureum. Analysis of the free amino acid content in N.ovalis cultures exposed to changes in salinity showed that proline and lysine also accumulate with increased salinity, but the cellular concentration of these amino acids is about 10-fold lower than the concentration of 1,4/2,5 cyclohexanetetrol. The comparison of amino acid concentration per cell with cyclitol suggests that this polyol is important in compensating the cellular osmotic pressure due to increased salinity, but other physiological functions could also be considered.

Controlling the mass action of α‐synuclein in Parkinson’s disease

Parkinson's disease (PD) is an age-related neurodegenerative disease with unknown etiology. Growing evidence from genetic, pathologic, animal modeling, and biochemical studies strongly support the theory that abnormal aggregation of alpha-synuclein plays a critical role in the pathogenesis of PD. Protein aggregation is an alternative folding process that competes with the native folding pathway. Whether or not a protein is subject to the aggregation process is determined by the concentration of the protein as well as thermodynamic properties inherent to each polypeptide. An increase in cellular concentration of alpha-synuclein has been associated with the disease in both familial and sporadic forms of PD. Thus, maintenance of the intraneuronal steady state levels of alpha-synuclein below the critical concentration is a key challenge neuronal cells are facing. Expression of the alpha-synuclein gene is under the control of environmental factors and aging, the two best-established risk factors for PD. Studies also suggest that the degradation of this protein is mediated by proteasomal and autophagic pathways, which are two mechanisms that are related to the pathogenesis of PD. Recently, vesicle-mediated exocytosis has been suggested as a novel mechanism for disposal of neuronal alpha-synuclein. Relocalization of the protein to specific compartments may be another method for increasing its local concentration. Regulation of the neuronal steady state levels of alpha-synuclein has significant implications in the development of PD, and understanding the mechanism may disclose potential therapeutic targets for PD and other related diseases.

Socs3 maintains the specificity of biological responses to cytokine signals during granulocyte and macrophage differentiation

Granulocyte colony-stimulating factor (G-CSF) and interleukin-6 (IL-6) play key roles in regulating emergency granulopoiesis and inflammation, and are both negatively regulated by the inducible intracellular protein suppressor of cytokine signaling-3 (Socs3). Mice with Socs3 deleted specifically in hematopoietic cells succumb to severe neutrophil and macrophage-driven inflammation by 1 year of age, and responses to G-CSF are grossly exacerbated. In order to determine which elements of cellular responses to cytokines require Socs3, we have examined the differentiative and proliferative capacity of hematopoietic progenitor cells stimulated by G-CSF and IL-6. The differentiation of Socs3-deficient progenitor cells is skewed toward macrophage production in response to G-CSF or IL-6, whereas wild-type progenitor cells produce mainly neutrophils. The proliferative capacity of Socs3-deficient progenitor cells is greatly enhanced in response to G-CSF at all concentrations, but only at low concentrations for IL-6. Strikingly, synergistic responses to costimulation with stem cell factor and IL-6 (but not G-CSF) are lost at higher concentrations in Socs3-deficient progenitor cells. Cytokine-induced expression of transcriptional regulators including cebpb, Ets2, Bcl3, c-Myc, Jun, and Fosl2 are differentially regulated in Socs3-deficient cells. The tight regulation by Socs3 of signal transducer and activator of transcription 3 phosphorylation and gene transcription after cytokine receptor ligation significantly influences the fate of myeloid progenitor cells.

Role of P-glycoprotein in cyclosporine cytotoxicity in the cyclosporine–sirolimus interaction

Cyclosporine nephrotoxicity remains a major side effect in solid organ transplantation, and can be exacerbated by concomitant administration of sirolimus. Cyclosporine and sirolimus are P-glycoprotein (Pgp) substrates. We hypothesized that the Pgp activity level may affect cyclosporine cytotoxicity by interfering with the ability of Pgp to remove cyclosporine from within tubular cells, and that an interaction between cyclosporine and sirolimus on Pgp function may explain the enhancement of cyclosporine nephrotoxicity by sirolimus. Cyclosporine cytotoxicity was evaluated in primary cultures of normal human renal epithelial cells (HRECs) by cell viability and cytotoxicity assays. Verapamil, quinine, PSC833, and PGP-4008 were used as Pgp inhibitors. Rhodamine-123 (R-123), a fluorescent substrate of Pgp, was used to assess Pgp-mediated transport. Cellular cyclosporine concentration was measured by high-performance liquid chromatography coupled to tandem mass spectrometry. Pgp expression and function were confirmed in HRECs and cyclosporine and sirolimus were shown to be Pgp inhibitors in this model. Verapamil-induced inhibition of Pgp led to a significant increase in cellular concentration of cyclosporine (P<0.05). Cyclosporine exerted a concentration-dependent cytotoxic effect on HRECs that was significantly increased by inhibition of Pgp activity. Sirolimus exerted an inhibitory effect on R-123 efflux in HRECs and increased cellular cyclosporine concentrations in a dose-dependent manner. These data demonstrate that Pgp plays a critical role in protecting renal epithelial cells from cyclosporine toxicity. The inhibitory effect of sirolimus on Pgp-mediated efflux and the cellular concentration of cyclosporine could explain the exacerbation of cyclosporine nephrotoxicity observed clinically.

EGCG Attenuates AMPA-Induced Intracellular Calcium Increase in Hippocampal Neurons

We have investigated the protective effect of (−)-epigallocatechin gallate (EGCG) on α-amino-3-hydroxy-5-methyl-4-isoxazolo propionate (AMPA)-induced toxicity in cultured rat hippocampal neurons. Treatment of 24 h AMPA (10 μM) reduced the neuronal viability in both survival neuron counting and MTT reduction assay compared with control, with increase in cellular concentrations of hydrogen peroxide and malondialdehyde. These responses to AMPA were significantly blocked by co-treatments with EGCG (10 μM), which effect was very similar to the protective ability of a known antioxidant catalase (2000 U/ml). AMPA (50 μM) elicited the increase in intracellular calcium concentration ([Ca2+]i) on which EGCG significantly attenuated both peak amplitude and sustained nature of that [Ca2+]i increase in a dose-dependent manner. These data suggest that EGCG has a neuroprotective effect against AMPA through inhibition of AMPA-induced [Ca2+]i increase and consequent attenuation of reactive oxygen species production and lipid peroxidation as an antioxidant and a radical scavenger.

Anti-leptin receptor antibody mimics the stimulation of lipolysis induced by leptin in isolated mouse fat pads

An anti-leptin receptor polyclonal antibody (receptor antibody), as well as leptin, stimulated the release of free fatty acids from isolated mouse fat pads in a time-dependent manner. Following a 90-min incubation, maximal lipolysis was observed at 6 μg/ml receptor antibody and 0.1 nM leptin. The receptor antibody did not show any additive effect to the stimulation of lipolysis induced by leptin, suggesting that they exert their actions through a similar mechanism involving the leptin receptor. N-[2-(p-bromocinnamylamino) ethyl]-5-isoquinolinesulfonamide (H-89), quin 2-AM, N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7), and neomycin sulfate (neomycin) all potently inhibited the stimulation of lipolysis by the receptor antibody and leptin. Short-term incubation of the fat pads with the receptor antibody or leptin showed a transient increase in the cellular content of cAMP and myo-inositol 1,4,5-trisphosphate (IP3) in similar concentrations to the free fatty acid release. Quin 2-AM and W-7 also inhibited the increase in cAMP content, suggesting that a Ca2+/calmodulin-dependent process may be involved in a part of the mechanism in which the receptor antibody and leptin exert their effects.The increase in cellular IP3 content via phosphoinositide-specific phospholipase C (PLC) sensitive to neomycin appears to be a primary step to initiate intracellular events. Both the receptor antibody and leptin may stimulate the lipolysis through mechanisms involving a transient increase in the cellular IP3 content followed by cAMP production, which leads to the activation of cAMP-dependent protein kinase.

Down-regulation of fibronectin in rainbow trout gonadal cells exposed to retinoic acid

Exposure of fish to some environmental contaminants results in alterations to the levels of retinoid (Vitamin A) stores, which could result in an increase in cellular concentrations of biologically active metabolites such as retinoic acid (RA). However, a link has not been established between changes in retinoid metabolism and impacts on the health of biota. In vitro studies with mammalian cells have demonstrated a relationship between exposure to RA and expression of the extracellular matrix protein, fibronectin (FN); a protein critical for cell migration, adhesion, and transformation. In this study, in vitro exposures of rainbow trout gonadal cells (RTG-2) to RA reduced levels of FN in culture medium; as measured using SDS-PAGE and immunoblot analysis with antisera prepared against RTG-2 cellular fibronectin. This apparent down-regulation of FN secretion occurred in a dose-dependent manner over a range of RA concentrations (10 −10–10 −6 M). FN down-regulation was not accompanied by changes in the morphology of RTG-2. Future studies should be directed at determining the relationships between retinoid metabolism and FN expression and the potential effects of contaminant-induced changes to vitamin A metabolism on the health of fish.

Regulation of ribosomal DNA transcription by insulin.

The experiments reported here used 3T6-Swiss albino mouse fibroblasts and H4-II-E-C3 rat hepatoma cells as model systems to examine the mechanism(s) through which insulin regulates rDNA transcription. Serum starvation of 3T6 cells for 72 h resulted in a marked reduction in rDNA transcription. Treatment of serum-deprived cells with insulin was sufficient to restore rDNA transcription to control values. In addition, treatment of exponentially growing H4-II-E-C3 with insulin stimulated rDNA transcription. However, for both cell types, the stimulation of rDNA transcription in response to insulin was not associated with a change in the cellular content of RNA polymerase I. Thus we conclude that insulin must cause alterations in formation of the active RNA polymerase I initiation complex and/or the activities of auxiliary rDNA transcription factors. In support of this conclusion, insulin treatment of both cell types was found to increase the nuclear content of upstream binding factor (UBF) and RNA polymerase I-associated factor 53. Both of these factors are thought to be involved in recruitment of RNA polymerase I to the rDNA promoter. Nuclear run-on experiments demonstrated that the increase in cellular content of UBF was due to elevated transcription of the UBF gene. In addition, overexpression of UBF was sufficient to directly stimulate rDNA transcription from a reporter construct. The results demonstrate that insulin is capable of stimulating rDNA transcription in both 3T6 and H4-II-E-C3 cells, at least in part by increasing the cellular content of components required for assembly of RNA polymerase I into an active complex.

Basic fibroblast growth factor increases nitric oxide synthase production in bovine endothelial cells.

Basic fibroblast growth factor (bFGF) and nitric oxide (NO) are expressed by endothelial cells (EC) and are involved in regulation of endothelial functions. In vivo, bFGF has a hypotensive effect which is mediated, in part, through activation of nitric oxide synthase (NOS) and the subsequent generation of NO. Thus we hypothesized that regulation of NOS in EC might be modulated by bFGF. bFGF treatment of EC in vitro resulted in increased NADPH diaphorase staining, a histochemical marker associated with the presence of NOS. Using cGMP generation in a reporter cell as a bioassay for NO release, we demonstrated that bFGF treatment of EC leads to increased production of biologically active NO. Furthermore, bFGF treatment of EC resulted in an increase in cellular content of the endothelial form of NOS as shown by Western blot analysis. Finally, Northern blot analysis was used to demonstrate that message levels of the constitutive, calcium-dependent, endothelial form of NOS is increased in EC by treatment with bFGF in vitro. These results suggest that bFGF has potential to regulate vascular tone through the modulation of levels of endothelial NOS.

Mechanical strain increases contractile enzyme activity in cultured airway smooth muscle cells

Smooth muscle hypertrophy is often found in tissue subjected to abnormal physical stress. To determine if physical stress (strain) per se could increase the contractile potential of airway smooth muscle (ASM), we compared cultured ASM cells subjected to strain to control cells (no strain) for rates of 1) myosin light chain kinase (MLCK)-mediated myosin light chain (LC20) phosphorylation, 2) actin-activated myosin ATPase, and 3) myosin light chain phosphatase-mediated myosin dephosphorylation. Lysates from strained cells showed increases in both LC20 phosphorylation activity and actomyosin ATPase activity but decreased rates of phosphatase-dependent myosin dephosphorylation. The increased LC20 phosphorylation activity and ATPase activity of the strained cells were accompanied by increases in cellular content of MLCK and myosin, respectively, compared with control. Because the cultured ASM cells exposed to strain expressed higher MLCK activity and actomyosin ATPase activity but lower myosin light chain phosphatase activity, these data suggest that physical stress in part determines ASM potential for contractile state.