Oxygen Availability as a Control Factor in the Density-Dependent Regulation of Protein Synthesis in Cell Culture

1974 ◽  
Vol 14 (2) ◽  
pp. 331-337
Author(s):  
DESH PAL S. VERMA ◽  
A. MARCUS
Keyword(s):  
Cell Culture ◽  
Protein Synthesis ◽  
Respiratory Rate ◽  
Initial Function ◽  
Fold Increase ◽  
Oxygen Availability ◽  
Control Factor ◽  
Atp Level ◽  
Density Dependent ◽  
Inadequate Nutrition

Dilution of a density-inhibited Arachis culture results in a 10-fold increase in capacity for protein synthesis during the first 2 h after dilution. The limitation in the density-inhibited state is not inadequate nutrition, inappropriate pH, or a diffusible inhibitor as the dilution can be carried out in medium obtained by filtration of 14-day cells. The respiratory rate of the culture increases 2-fold immediately after dilution and the ATP level increases 3-fold dunng the 2-h period subsequent to dilution. These observations suggest that the initial function activated by dilution is an increased availability of oxygen and that this increase in oxygen provides an increased level of ATP, finally resulting in an increased rate of protein synthesis. This idea is further supported by the finding that both the increase in cellular ATP and the acceleration of the rate of protein synthesis can be obtained in dense culture, in the absence of dilution, by maintaining the cells for 2 h under oxygen.

Increased Protein Synthesis by Human Platelets during Phagocytosis of Latex Particles in Vitro

1976 ◽  
Vol 35 (02) ◽  
pp. 350-357 ◽  
Author(s):  
Hana Bessler ◽  
Galila Agam ◽  
Meir Djaldetti
Keyword(s):  
Protein Synthesis ◽  
Fold Increase ◽  
Human Platelets ◽  
Polystyrene Latex ◽  
Latex Particles ◽  
The Third ◽  
Platelet Protein ◽  
Dose Dependent ◽  
Phagocytotic Activity

SummaryA three-fold increase of protein synthesis by human platelets during in vitro phagocytosis of polystyrene latex particles was detected. During the first two hours of incubation, the percentage of phagocytizing platelets and the number of latex particles per platelet increased; by the end of the third hour, the first parameter remained stable, while the number of latex particles per cell had decreased.Vincristine (20 μg/ml of cell suspension) inhibited platelet protein synthesis. This effect was both time- and dose-dependent. The drug also caused a decrease in the number of phagocytizing cells, as well as in their phagocytotic activity.

scholarly journals Albumin Enhances Caspofungin Activity against Aspergillus Species by Facilitating Drug Delivery to Germinating Hyphae

2015 ◽  
Vol 60 (3) ◽  
pp. 1226-1233 ◽  
Author(s):  
Petros Ioannou ◽  
Aggeliki Andrianaki ◽  
Tonia Akoumianaki ◽  
Irene Kyrmizi ◽  
Nathaniel Albert ◽  
...  
Keyword(s):  
Drug Delivery ◽  
Cell Culture ◽  
Antifungal Agents ◽  
Fold Increase ◽  
Culture Conditions ◽  
The Other ◽  
Related Factors ◽  
Content Type

The modestin vitroactivity of echinocandins againstAspergillusimplies that host-related factors augment the action of these antifungal agentsin vivo. We found that, in contrast to the other antifungal agents (voriconazole, amphotericin B) tested, caspofungin exhibited a profound increase in activity against variousAspergillusspecies under conditions of cell culture growth, as evidenced by a ≥4-fold decrease in minimum effective concentrations (MECs) (P= 0. 0005). Importantly, the enhanced activity of caspofungin againstAspergillusspp. under cell culture conditions was strictly dependent on serum albumin and was not observed with the other two echinocandins, micafungin and anidulafungin. Of interest, fluorescently labeled albumin bound preferentially on the surface of germinatingAspergillushyphae, and this interaction was further enhanced upon treatment with caspofungin. In addition, supplementation of cell culture medium with albumin resulted in a significant, 5-fold increase in association of fluorescently labeled caspofungin withAspergillushyphae (P< 0.0001). Collectively, we found a novel synergistic interaction between albumin and caspofungin, with albumin acting as a potential carrier molecule to facilitate antifungal drug delivery toAspergillushyphae.

scholarly journals Cell density-dependent stimulation of PAI-1 and hyaluronan synthesis by TGF-β in orbital fibroblasts

2016 ◽  
Vol 229 (2) ◽  
pp. 187-196 ◽  
Author(s):  
Erika Galgoczi ◽  
Florence Jeney ◽  
Annamaria Gazdag ◽  
Annamaria Erdei ◽  
Monika Katko ◽  
...  
Keyword(s):  
Cell Density ◽  
Inverse Correlation ◽  
Fold Increase ◽  
Dermal Fibroblasts ◽  
Negative Regulator ◽  
Dependent Manner ◽  
Density Dependent ◽  
Cell Densities ◽  
Orbital Fibroblasts ◽  
Pai 1

During the course of Graves’ orbitopathy (GO), orbital fibroblasts are exposed to factors that lead to proliferation and extracellular matrix (ECM) overproduction. Increased levels of tissue plasminogen activator inhibitor type 1 (PAI-1 (SERPINE1)) might promote the accumulation of ECM components. PAI-1 expression is regulated by cell density and various cytokines and growth factors including transforming growth factorβ(TGF-β). We examined the effects of increasing cell densities and TGF-β on orbital fibroblasts obtained from GO patients and controls. Responses were evaluated by the measurement of proliferation, PAI-1 expression, and ECM production. There was an inverse correlation between cell density and the per cell production of PAI-1. GO orbital, normal orbital, and dermal fibroblasts behaved similarly in this respect. Proliferation rate also declined with increasing cell densities. Hyaluronan (HA) production was constant throughout the cell densities tested in all cell lines. In both GO and normal orbital fibroblasts, but not in dermal fibroblasts, TGF-β stimulated PAI-1 production in a cell density-dependent manner, reaching up to a five-fold increase above baseline. This has been accompanied by increased HA secretion and pericellular HA levels at high cell densities. Increasing cell density is a negative regulator of proliferation and PAI-1 secretion both in normal and GO orbital fibroblasts; these negative regulatory effects are partially reversed in the presence of TGF-β. Cell density-dependent regulation of PAI-1 expression in the orbit, together with the local cytokine environment, may have a regulatory role in the turnover of the orbital ECM and may contribute to the expansion of orbital soft tissue in GO.

scholarly journals Liposome-Embedding Silicon Microparticle for Oxaliplatin Delivery in Tumor Chemotherapy

Pharmaceutics ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 559 ◽  
Author(s):  
Armando Cevenini ◽  
Christian Celia ◽  
Stefania Orrù ◽  
Daniela Sarnataro ◽  
Maddalena Raia ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Culture ◽  
Therapeutic Agent ◽  
Fold Increase ◽  
Alternative Strategies ◽  
Mesoporous Silicon ◽  
Dimensional Changes ◽  
Liposomal Formulations ◽  
Methoxy Polyethylene Glycol

Mesoporous silicon microparticles (MSMPs) can incorporate drug-carrying nanoparticles (NPs) into their pores. An NP-loaded MSMP is a multistage vector (MSV) that forms a Matryoshka-like structure that protects the therapeutic cargo from degradation and prevents its dilution in the circulation during delivery to tumor cells. We developed an MSV constituted by 1 µm discoidal MSMPs embedded with PEGylated liposomes containing oxaliplatin (oxa) which is a therapeutic agent for colorectal cancer (CRC). To obtain extra-small liposomes able to fit the 60 nm pores of MSMP, we tested several liposomal formulations, and identified two optimal compositions, with a prevalence of the rigid lipid 1,2-distearoyl-sn-glycero-3-phosphocholine and of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. To improve the MSV assembly, we optimized the liposome-loading inside the MSMP and achieved a five-fold increase of the payload using an innovative lyophilization approach. This procedure also increased the load and limited dimensional changes of the liposomes released from the MSV in vitro. Lastly, we found that the cytotoxic efficacy of oxa-loaded liposomes and-oxa-liposome-MSV in CRC cell culture was similar to that of free oxa. This study increases knowledge about extra-small liposomes and their loading into porous materials and provides useful hints about alternative strategies for designing drug-encapsulating NPs.

Endotoxin and thrombin elevate rodent endothelial cell protein C receptor mRNA levels and increase receptor shedding in vivo

Blood ◽  
2000 ◽  
Vol 95 (5) ◽  
pp. 1687-1693 ◽  
Author(s):  
Jian-Ming Gu ◽  
Yasuhiro Katsuura ◽  
Gary L. Ferrell ◽  
Paula Grammas ◽  
Charles T. Esmon
Keyword(s):  
Cell Culture ◽  
Septic Shock ◽  
Endothelial Cell ◽  
Protein C ◽  
Fold Increase ◽  
Cell Protein ◽  
Thrombin Inhibitor ◽  
Mrna Levels ◽  
Soluble Epcr

The endothelial cell protein C receptor (EPCR) facilitates protein C activation by the thrombin-thrombomodulin complex. Protein C activation has been shown to be critical to the host defense against septic shock. In cell culture, tumor necrosis factor- (TNF-) down-regulates EPCR expression, raising the possibility that EPCR might be down-regulated in septic shock. We examined EPCR mRNA and soluble EPCR levels in mice and rats challenged with lethal dose 95 levels of endotoxin. Toxic doses of TNF- failed to alter EPCR mRNA levels in mice. Rather than EPCR mRNA levels falling in response to endotoxin, as predicted from cell-culture experiments, they rose approximately 3-fold 6 hours after exposure to endotoxin before returning toward baseline levels at 24 hours after exposure. Soluble EPCR levels rose approximately 4-fold. Infusion of hirudin, a specific thrombin inhibitor, before endotoxin exposure almost completely blocked the increase in EPCR mRNA and soluble EPCR. Consistent with the idea that the responses were mediated by thrombin, thrombin infusion (5 U/kg of body weight for 3 hours) resulted in an approximately 2-fold increase in EPCR mRNA and soluble EPCR. Incubation of rat endothelial cells with thrombin or murine protease-activated receptor 1 agonist peptide resulted in a 2-fold increase in EPCR mRNA. These results indicate that thrombin plays a major role in up-regulating EPCR mRNA and shedding in vivo.

scholarly journals Oxygen and pH regulation of protein synthesis in mitochondria from Artemia franciscana embryos

1996 ◽  
Vol 313 (1) ◽  
pp. 207-213 ◽  
Author(s):  
Kurt E. KWAST ◽  
Steven C. HAND
Keyword(s):  
Protein Synthesis ◽  
Ph Regulation ◽  
Mitochondrial Protein ◽  
Oxygen Limitation ◽  
Oxygen Availability ◽  
Artemia Franciscana ◽  
Synthesis Rate ◽  
Protein Synthesis Rate ◽  
Mitochondrial Translation ◽  
Purine Nucleotides

To identify factors responsible for the down-regulation of mitochondrial biosynthetic processes during anoxia in encysted Artemia franciscana embryos, the effects of oxygen limitation and pH on protein synthesis were investigated in isolated mitochondria. At the optimal pH of 7.5, exposure of mitochondria to anoxia decreases the protein synthesis rate by 79%. Rates were suppressed by a further 10% at pH 6.8, the intracellular pH (pHi) measured under anoxia in vivo. Matrix pH, measured under identical conditions, was 8.43±0.01 at an extramitochondrial pH of 7.9 (mean±S.E.M., n = 3), 8.05±0.01 at pH 7.5, and 7.10±0.01 at pH 6.8. The matrix pH did not vary (P ≥0.20) as a function of oxygen availability during the 1 h assays. Intramitochondrial purine nucleotides varied little as a function of pH. In contrast, after 1 h of protein synthesis under anoxia, ATP levels decreased by up to 40%,. whereas AMP, ADP and GDP concentrations increased, and GTP and GMP concentrations remained relatively constant. The addition of 1 mM ATP at the onset of anoxia maintained the ATP/ADP ratio at the aerobic value, but did not stabilize the GTP/GDP ratio or rescue rates of protein synthesis. Thus, at present, we cannot eliminate the possibility that the decrease in the GTP/GDP ratio during anoxia may contribute to the suppression of protein synthesis. The effect of anoxia was reversible; the rate of protein synthesis upon reoxygenation after a 30 min bout of anoxia was comparable (P = 0.14) with the pre-anoxic rate (193±17 and 174±6 pmol of leucine per mg of protein respectively; mean±S.E.M., n = 3). The array of mitochondrial translation products did not differ qualitatively as a function of either oxygen availability or pH. Finally, similar pH profiles for protein synthesis were obtained with either [3H]leucine or [3H]histidine (known to use different transporters). Consequently, it is improbable that the pH-sensitivity of protein synthesis can be explained by a specific protein effect on the import of the radiolabelled amino acid used. In summary, both oxygen limitation and acidic pH suppress rates of mitochondrial protein synthesis and are likely to contribute to the arrest of mitochondrial anabolic processes during anoxia-induced quiescence in A. franciscana embryos.

Differential effects of cysteine on protein and coenzyme A synthesis in rat heart

1984 ◽  
Vol 247 (1) ◽  
pp. C99-C106 ◽  
Author(s):  
B. H. Chua ◽  
K. E. Giger ◽  
B. J. Kleinhans ◽  
J. D. Robishaw ◽  
H. E. Morgan
Keyword(s):  
Protein Synthesis ◽  
Rat Heart ◽  
Coenzyme A ◽  
Pantothenic Acid ◽  
Fold Increase ◽  
Creatine Phosphate ◽  
Aortic Pressure ◽  
Protective Agent ◽  
Bicarbonate Buffer ◽  
Perfused Rat Heart

The effect of cysteine availability on protein and coenzyme A (CoA) synthesis in perfused rat heart was incompletely evaluated in earlier experiments because rapid conversion of cysteine to cystine occurred when the perfusion buffer was oxygenated. This conversion was minimized by addition of an excess of reducing agents such as dithiothreitol or mercaptodextran or by provision of bathocuproine disulfonate, a copper chelator. Dithiothreitol was not a suitable protective agent because it reduced ATP and creatine phosphate contents. Perfusion of hearts with [35S]cystine or [35S]cysteine in the presence of mercaptodextran resulted in a 22-fold or 5-fold increase, respectively, in incorporation of [35S] into protein and a 5-fold or 8-fold increase, respectively, in incorporation into CoA compared with hearts supplied [35S]cystine or [35S]cysteine without the reducing agent. When compared with hearts perfused at an aortic pressure of 90 mmHg with bicarbonate buffer that contained 15 mM glucose, 25 mU insulin/ml, 0.4 mM [14C]phenylalanine, no cysteine and plasma levels of other amino acids, provision of 0.09 or 0.2 mM cysteine alone or in the presence of mercaptodextran, or bathocuproine disulfonate enhanced rates of protein synthesis 16-35%. When 0.2 mM cysteine was added to bicarbonate buffer containing 7 microM pantothenic acid, supplementation with mercaptodextran or bathocuproine disulfonate was required to raise CoA content. These results indicated that an exogenous supply of cysteine was needed to maintain maximal rates of protein and CoA synthesis in the perfused rat heart. Protective compounds were required to obtain the cysteine effect on CoA but not on protein synthesis.

scholarly journals Hormonal stimulation in the exocrine pancreas results in coordinate and anticoordinate regulation of protein synthesis.

1984 ◽  
Vol 99 (5) ◽  
pp. 1569-1574 ◽  
Author(s):  
J Schick ◽  
H Kern ◽  
G Scheele
Keyword(s):  
Protein Synthesis ◽  
Fold Increase ◽  
Response Patterns ◽  
Hormonal Stimulation ◽  
Group Iii ◽  
Hormone Stimulation ◽  
Control Value ◽  
Group I ◽  
Separation Of Proteins

24-h intravenous caerulein infusion studies in the rat were combined with in vitro amino acid incorporation studies followed by high-resolution separation of proteins by two-dimensional isoelectric focusing and SDS gel electrophoresis to study the extent to which persistent changes in the biosynthesis of exocrine pancreatic proteins are regulated by cholecystokinin-like peptides. Beginning in the third hour of optimal hormone infusion at 0.25 microgram kg-1 h-1, changes were observed in the synthetic rates of 12 proteins, which progressed over the course of the 24-h study. Based on coordinate response patterns, exocrine proteins could be classified into four distinct groups. Group I (trypsinogen forms 1 and 2) showed progressive increases in synthetic rates reaching a combined 4.3-fold increase over control levels. Group II (amylase forms 1 and 2) showed progressive decreases in synthesis to levels 7.1- and 14.3-fold lower than control levels, respectively. Group III proteins (ribonuclease, chymotrypsinogen forms 1 and 2, procarboxypeptidase forms A and B, and proelastase 1) showed moderate increases in synthesis, 1.4-2.8-fold, and group IV proteins (trypsinogen 3, lipase, proelastase 2, and unidentified proteins 1-4) did not show changes in synthesis with hormone stimulation. Regulation of protein synthesis in response to caerulein infusion was specific for individual isoenzymic forms in the case of both trypsinogen and proelastase. The ratio of biosynthetic rates of trypsinogen forms 1 + 2 to amylase forms 1 + 2 increased from a control value of 0.56 to 24.4 after 24 h of hormonal stimulation (43.5-fold increase). Biosynthetic rates for an unidentified protein (P23) with an Mr = 23,000 and isoelectric point of 6.2 increased 14.2-fold, and the ratio of synthesis of P23 to amylase 2 increased 200-fold during caerulein infusion. During hormone stimulation the anticoordinate response in the synthesis of pancreatic glycosidases (decreased synthesis) and serine protease zymogens (increased synthesis) explain previous observations that showed little change in rates of total protein synthesis under similar conditions.

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