Partial Inhibition Of Protein Synthesis Research Articles

Effects of mushroom and chicory extracts on the shape, physiology and proteome of the cariogenic bacterium Streptococcus mutans.

BackgroundDental caries is an infectious disease which results from the acidic demineralisation of the tooth enamel and dentine as a consequence of the dental plaque (a microbial biofilm) accumulation. Research showed that several foods contain some components with antibacterial and antiplaque activity. Previous studies indicated antimicrobial and antiplaque activities in a low-molecular-mass (LMM) fraction of extracts from either an edible mushroom (Lentinus edodes) or from Italian red chicory (Cichorium intybus).MethodsWe have evaluated the antimicrobial mode of action of these fractions on Streptococcus mutans, the etiological agent of human dental caries. The effects on shape, macromolecular syntheses and cell proteome were analysed.ResultsThe best antimicrobial activity has been displayed by the LMM mushroom extract with a bacteriostatic effect. At the MIC of both extracts DNA synthesis was the main macromolecular synthesis inhibited, RNA synthesis was less inhibited than that of DNA and protein synthesis was inhibited only by roughly 50%. The partial inhibition of protein synthesis is compatible with the observed significant increase in cell mass. The increase in these parameters is linked to the morphological alteration with transition from cocci of the untreated control to elongated cells. Interestingly, these modifications were also observed at sub-MIC concentrations. Finally, membrane and cytosol proteome analysis was conducted under LMM mushroom extract treatment in comparison with untreated S. mutans cells. Significant changes were observed for 31 membrane proteins and 20 of the cytosol fractions. The possible role of the changed proteins is discussed.ConclusionsThis report has shown an antibiotic-like mode of action of mushroom and chicory extracts as demonstrated by induced morphogenetic effects and inhibition of specific macromolecular synthesis. This feature as well as the safe use of this extract as result of its natural origin render the LMM both mushroom and chicory extracts suitable for the formulation into products for daily oral hygiene such as mouthwashes or toothpastes.

Induction of cell cycle arrest at G1 and S phases and cAMP-dependent differentiation in C6 glioma by low concentration of cycloheximide

BackgroundDifferentiation therapy has been shown effective in treatment of several types of cancer cells and may prove to be effective in treatment of glioblastoma multiforme, the most common and most aggressive primary brain tumor. Although extensively used as a reagent to inhibit protein synthesis in mammalian cells, whether cycloheximide treatment leads to glioma cell differentiation has not been reported.MethodsC6 glioma cell was treated with or without cycloheximide at low concentrations (0.5-1 μg/ml) for 1, 2 and 3 days. Cell proliferation rate was assessed by direct cell counting and colony formation assays. Apoptosis was assessed by Hoechst 33258 staining and FACS analysis. Changes in several cell cycle regulators such as Cyclins D1 and E, PCNA and Ki67, and several apoptosis-related regulators such as p53, p-JNK, p-AKT, and PARP were determined by Western blot analysis. C6 glioma differentiation was determined by morphological characterization, immunostaining and Western blot analysis on upregulation of GFAP and o p-STAT3 expression, and upregulation of intracellular cAMP.ResultsTreatment of C6 cell with low concentration of cycloheximide inhibited cell proliferation and depleted cells at both G2 and M phases, suggesting blockade at G1 and S phases. While no cell death was observed, cells underwent profound morphological transformation that indicated cell differentiation. Western blotting and immunostaining analyses further indicated that changes in expression of several cell cycle regulators and the differentiation marker GFAP were accompanied with cycloheximide-induced cell cycle arrest and cell differentiation. Increase in intracellular cAMP, a known promoter for C6 cell differentiation, was found to be elevated and required for cycloheximide-promoted C6 cell differentiation.ConclusionOur results suggest that partial inhibition of protein synthesis in C6 glioma by low concentration of cycloheximide induces cell cycle arrest at G1 and M phases and cAMP-dependent cell differentiation.

Shiga toxin-1 affects nitric oxide production by human glomerular endothelial and mesangial cells

Acute renal failure hallmarks the pathogenesis of the epidemic form of hemolytic uremic syndrome (D+HUS), which is caused by E. coli strains that produce Shiga-like toxin (Stx). In this study, we investigated the influence of Stx-1 on nitric oxide (NO) production by human glomerular microvascular endothelial cells (GMVEC) and human mesangial cells. NO synthesis by human mesangial cells is in the micromolar range and that of GMVEC in the picomolar range. Stx-1 reduced NO production in non-stimulated GMVEC (5 nmol/l Stx-1 required) without inhibition of protein synthesis. In non-stimulated and TNFalpha-pretreated mesangial cells, NO production was reduced with a maximal reduction at 10 fmol/l shiga toxin. The cellular iNOS antigen content in mesangial cells was reduced in a concentration-dependent way (10 fmol/l-100 pmol/l), while partial inhibition of protein synthesis required 10 nmol/l Stx-1 in these cells. Our in vitro data suggest that Stx may reduce NO synthesis during the course of HUS development, contributing to the aggravation of the thrombotic microangiopathy and renal failure as observed in HUS.

Verocytotoxin-induced apoptosis of human microvascular endothelial cells.

The pathogenesis of the epidemic form of hemolytic uremic syndrome is characterized by endothelial cell damage. In this study, the role of apoptosis in verocytotoxin (VT)-mediated endothelial cell death in human glomerular microvascular endothelial cells (GMVEC), human umbilical vein endothelial cells, and foreskin microvascular endothelial cells (FMVEC) was investigated. VT induced apoptosis in GMVEC and human umbilical vein endothelial cells when the cells were prestimulated with the inflammatory mediator tumor necrosis factor-alpha (TNF-alpha). FMVEC displayed strong binding of VT and high susceptibility to VT under basal conditions, which made them suitable for the study of VT-induced apoptosis without TNF-alpha interference. On the basis of functional (flow cytometry and immunofluorescence microscopy using FITC-conjugated annexin V and propidium iodide), morphologic (transmission electron microscopy), and molecular (agarose gel electrophoresis of cellular DNA fragments) criteria, it was documented that VT induced programmed cell death in microvascular endothelial cells in a dose- and time-dependent manner. Furthermore, whereas partial inhibition of protein synthesis by VT was associated with a considerable number of apoptotic cells, comparable inhibition of protein synthesis by cycloheximide was not. This suggests that additional pathways, independent of protein synthesis inhibition, may be involved in VT-mediated apoptosis in microvascular endothelial cells. Specific inhibition of caspases by Ac-Asp-Glu-Val-Asp-CHO, but not by Ac-Tyr-Val-Ala-Asp-CHO, was accompanied by inhibition of VT-induced apoptosis in FMVEC and TNF-alpha-treated GMVEC. These data indicate that VT can induce apoptosis in human microvascular endothelial cells.

Regulation of Wee1 kinase in response to protein synthesis inhibition

To investigate the mechanism coupling growth (protein synthesis) with cell division, we examined the relationship between the tyrosine kinase Wee1 that inhibits Cdc2-Cdc13 mitosis-inducing kinase by phosphorylating it, and protein synthesis inhibition in fission yeast. The wee1-50 mutant showed supersensitivity to protein synthesis inhibitor, cycloheximide. Wee1 was essential for the G 2 delay upon a partial inhibition of protein synthesis. Indeed, the protein synthesis inhibition caused an increase in the Wee1 protein by the Sty1/Spc1 MAPK-dependent transcriptional and the Sty1/Spc1 MAPK-independent post-transcriptional regulations. Further, the results indicated that the post-transcriptional regulation is important for the G 2 delay.

IL-6 PROTEIN PRODUCTION BY AIRWAY EPITHELIAL(-LIKE) CELLS DISABLED IN IL-6 mRNA DEGRADATION

IL-6 mRNA and protein expression in human airway epithelial-like H292 cells depends on rapid, but regulable IL-6 mRNA degradation. We restricted IL-6 mRNA degradation by partially inhibiting protein synthesis and studied the IL-6 response. Despite partial inhibition of protein synthesis, IL-6 protein production was increased and prolonged. Furthermore, the threshold concentration for stimuli of IL-6 protein production decreased and the dose–response curves became steeper. Similar findings were obtained with primary human bronchial epithelial cells. This exaggerated production may apply to other proteins encoded by labile mRNA and is likely to occur during viral infection of airway epithelial cells.

Enhancement of B7-1 (CD80) expression on B-lymphoma cells by irradiation.

Irradiation of A20.2J mouse B-lymphoma cells enhanced their antigen-presenting ability to induce interleukin-2 (IL-2) production by 42-6A T cells specific for ovalbumin (OVA)323-339/I-Ad. Irradiated and fixed A20.2J cells were more efficient antigen-presenting cells (APC) to present OVA323-339 peptide than the unirradiated and fixed cells. Irradiation selectively increased the expression of B7-1 molecules, but not of the major histocompatibility complex class II molecules, B7-2, lymphocyte function-associated antigen-1, or intracellular adhesion molecule-1. Irradiation of A20.2J cells with 100 Gy followed by overnight incubation was optimal for the enhancement of B7-1 expression. Anti-B7-1 monoclonal antibody inhibited the irradiation-induced enhancement of APC function. Irradiation of A20.2J cells induced the accumulation of B7-1 mRNA. Thus, it was concluded that the enhancement of APC function by irradiation was due to the up-regulation of B7-1 molecules through the accumulation of its mRNA. Although partial inhibition of protein synthesis has been shown to enhance the accumulation of B7-1 mRNA and its expression, irradiation did not decrease the protein synthesis in A20.2J cells. The incubation with irradiated A20.2J cells stimulated unirradiated A20.2J cells to increase B7-1 expression, suggesting that irradiation of A20.2J cells induced expression or secretion of some molecule(s) to enhance B7-1 expression.

Synergistic enhancement of PRB-mediated RU486 and R5020 agonist activities through cyclic adenosine 3',5'-monophosphate represents a delayed primary response.

Activators of protein kinase A have been shown to affect the transactivation potential of progestins and antiprogestins. To analyze the mechanisms and factors involved, we have created HeLa and CV1 cell clones stably expressing isoform B of progesterone receptor. In the HeLa cell background, the progesterone antagonist RU486 significantly induces progesterone-regulatable reporter genes, and this agonistic effect is synergistically enhanced by elevating cAMP or through overexpression of protein kinase A catalytic subunit. In contrast, in CV1 cells containing functional progesterone receptors no agonist activity of RU486 could be detected, suggesting the involvement of cell specifically expressed factors. In a PR(B)-positive HeLa cell clone containing stably integrated copies of a thymidine kinase-luciferase reporter gene with two progesterone response elements, we observed a complete loss of RU486 antagonist potential upon cotreatment with cAMP for 25 h while partial antagonist potential was maintained in a 5-h experiment. This result shows that, particularly in the presence of protein kinase A activators, the duration of hormone treatment is a crucial parameter in the evaluation of antagonist properties of antiprogestins. A detailed analysis of the kinetics of the hormone effects on transcription revealed that the onset of cAMP/RU486 synergism is delayed relative to the responses induced by RU486 or R5020 alone. Moreover, partial inhibition of protein synthesis by cycloheximide completely abolished cAMP/RU486 synergism while R5020 and RU486 responses were not inhibited. Together, these data indicate that cAMP/RU486 synergism is a delayed primary response requiring the intermediate induction of an essential factor.

Enhancement of antigen-presenting ability of B lymphoma cells by partial inhibition of protein synthesis through inducing B7-1 expression.

During the investigation of the role of protein synthesis in antigen-presenting cell (APC) function of A20-HL B lymphoma cells, we found that partial inhibition of protein synthesis enhanced their APC function. The treatment of A20-HL cells with 0.313-2.5 microM emetine, an irreversible inhibitor of protein synthesis, decreased protein synthesis by 60-70%, and enhanced their APC function to stimulate I-Ad/OVA323-339-specific T cells to produce interleukin-2 in response to ovalbumin (OVA). The emetine-treated and paraformaldehyde-fixed A20-HL cells required only 20 nM OVA323-339 peptide to stimulate the T cells, whereas those untreated and fixed required 200 nM peptide. This enhancement of APC function was mostly because of the induction of B7-1 expression on A20-HL cells by the emetine treatment, since B7-1 molecules were detected on the emetine-treated A20-HL cells, but only negligibly, if at all, on the untreated cells, and an anti-B7-1 monoclonal antibody, 1G10, inhibited the enhanced APC function of the emetine-treated A20-HL cells. The emetine-treatment also increased B7-1 mRNA expression in A20-HL cells, suggesting that the induction of B7-1 expression was due to the increase in the accumulation of mRNA and the translation with residual ability to synthesize protein. Thus, partial inhibition of protein synthesis in A20-HL cells increases B7-1 mRNA accumulation and its expression on the cell surface, which results in the enhancement of their APC function.

Cell death due to ACNU-induced DNA fragmentation: Inhibition by cycloheximide

Several anticancer drugs have recently been shown to induce cell death in a manner similar to programmed cell death or apoptosis. The purpose of this study is to explore the mode of cell death caused by ACNU, a water-soluble nitrosourea. Exposure of rat glioma cell line KEG-1 to ACNU for 2 hours resulted in oligonucleosomal DNA fragmentation, creating a 'ladder' on agarose gel electrophoresis. DNA fragmentation began 18 hours after ACNU treatment, and preceded loss of membrane integrity as evaluated by the trypan blue exclusion test. The extent of DNA fragmentation increased in a dose-dependent manner, and the cell survival rate decreased reciprocally. A translational inhibitor, cycloheximide, suppressed this DNA fragmentation and enhanced cell survival rate with partial inhibition of protein synthesis. However, a transcriptional inhibitor, actinomycin D, failed to inhibit DNA fragmentation or enhance cell survival. Cycloheximide-inhibitable DNA fragmentation was also found in the KEG-1 implanted in vivo rat model following the administration of ACNU. These findings suggest that ACNU induces cell death associates with DNA fragmentation and partially with protein synthesis.

The rate of protein synthesis in needles of Norway spruce (Picea abies): Light stimulation, regulation through photophosphorylation, stress enhancement

The incorporation of 14C‐leucine into the total‐protein fraction of needles of Norway spruce (Picea abies [L.] Karst.) during short time incubation was used as a measure of protein synthesis in the light and in the dark. Light saturation curves, obtained for needles of different ages (new flush and 1 and 2 years old) or at different seasons (summer‐winter) followed the Michaelis‐Menten algorithm, exhibiting marked differences with regard to light saturation (Vmax) and the half‐saturation constant (K5. 2). The light saturation curves of ATP level (mg g−1 fresh weight) and of leucine incorporation into protein (nmol mg−1 h−1) matched each other, suggesting that photophosphorylation may be decisive for the rate of protein synthesis in the light. This is confirmed by the action spectrum of leucine incorporation. which resembled an action spectrum of leaf photosynthesis, and also by partial inhibition of protein synthesis by 3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea (DCMU), an inhibitor of non‐cyclic photophosphorylation. Light stimulated protein synthesis showed pronounced seasonal fluctuations with a summer maximum.Furmigation of 5 years old spruce trees for 3 months with SO2 in combination with O3 and/or NO2 caused a distinct enhancement of the protein synthesis rate in the light and, at a reduced absolute level, also in the dark. A similar result was obtained for 40 to 70 years old spruce stands when healthy and sick trees were compared: the latter being afflicted by the novel type of forest decline, which is characterized by yellowish bronze discolouration of sun‐exposed older needles and partial loss of older needle generations (3 to 4 years old). The 1 year old needles of the unhealthy trees showed a markedly increased 14C‐leucine incorporation rate which, in the dark, was even more pronounced than in the light. Stress‐physiological mechanisms, which could possibly explain this stimulation, are discussed.

Enhanced butanol production and reduced autolysin activity after chloramphenicol treatment of Clostridium acetobutylicum ATCC 824

Release of autolysin during the late exponential growth phase of Clostridium acetobutylicum resulted in early lysis of the culture and reduction of solvent formation. A simple and effective way of reducing autolysin activity and increasing solvent production is partial inhibition of protein synthesis with chloramphenicol (CAP). The extracellular autolytic activity in the culture, determined by following loss of turbidity of washed clostridial cells in 0.04m sodium phosphate buffer at 37° C, was decreased by 40% after CAP treatment. This caused an extension of cell viability by 12 h and an increase in butanol production by 30%. The optimal time of CAP addition was 12 h of incubation, and the optimal antibiotic concentration was 120 μg/ml. The effects of CAP on the fermentation are due to the inhibition of protein synthesis leading to a decrease in autolysin level in the culture. The results obtained provide economic advantages for industrial production of solvents by minimizing autolysin activity and maximizing solvent yield during the critical solvent-producing phase.

Molecular cloning of the gene for plant proliferating‐cell nuclear antigen and expression of this gene during the cell cycle in synchronized cultures of Catharanthus roseus cells

A cDNA library was screened for plant proliferating-cell nuclear antigen (PCNA) from Catharanthus roseus (periwinkle). A lambda gt11 cDNA library was constructed using poly(A)-rich RNA isolated from the cells in the S phase. A cDNA clone for PCNA was isolated by using a rice genomic clone, pCJ-1, which contains PCNA-related gene sequences. The cDNA contains an open reading frame of 804 nucleotides, encoding a protein of 268 amino acids with a molecular mass of 29,765 Da. When conservative substitutions were included, a high degree of similarity (about 85%) was observed between the predicted amino acid sequence of periwinkle PCNA and that of human PCNA. Expression of mRNA for periwinkle PCNA was undetectable or very weak in quiescent cells, such as phosphate-starved cells, auxin-starved cells and cells in the stationary phase. In the synchronous progression of the cell cycle induced by the addition of phosphate or auxin, the active accumulation of periwinkle PCNA mRNA was observed preferentially in the S phase. When an inhibitor of DNA synthesis, aphidicolin, was added to the cells at the G1 phase, an increase in the level of PCNA mRNA was observed. The partial inhibition of protein synthesis at the G1 phase by a protein inhibitor, anisomycin, caused the arrest of cells in the G1 phase. No increase of the level of periwinkle PCNA mRNA was observed in cells arrested at the G1 phase by the inhibition of protein synthesis. These results indicate that the induction of mRNA for periwinkle PCNA occurred independently of the initiation of DNA replication, but that synthesis of certain proteins at the G1 phase was required for the induction of periwinkle PCNA mRNA at the S phase.

Transformed and nontransformed cells differ in stability and cell cycle regulation of a binding activity to the murine thymidine kinase promoter.

A DNA binding activity to an upstream region of the murine thymidine kinase gene is regulated differently in a transformed and nontransformed cell line pair. Differences in regulation were observed (i) after serum levels were reduced, (ii) when serum levels were returned to initial high levels, and (iii) while protein synthesis was inhibited. After reduction of serum levels, the binding activity was unstable in nontransformed BALB/c 3T3 clone A31 cells but was significantly more stable in benzo[a]pyrene-transformed BALB/c 3T3 cells. After serum concentration was returned to high levels, the kinetic pattern of the binding activity differed between nontransformed and transformed cells. While protein synthesis was inhibited, the binding activity was unstable in nontransformed cells and stable in transformed cells. Partial inhibition of protein synthesis--a more stringent condition to test instability--prevented the induction of the binding activity in nontransformed cells. Previously, the labile protein hypothesis set forth the criterion that a protein regulating the onset of DNA synthesis should be unstable in nontransformed cells and stable in transformed cells. The DNA binding activity described here satisfies this criterion.

Release of metaphase arrest by partial inhibition of protein synthesis in blue mussel oocytes

AbstractIn the blue mussel Mytilus edulis, shed oocytes are arrested at metaphase I of meiosis until fertilization. In this study, the mechanisms involved for maintaining the oocytes in metaphase were investigated. Analysis of 35‐methionine‐labelled proteins separated by 1D SDS‐PAGE reveals that two protein bands of apparent MW of 50 and 54 kDa periodically appear and disappear during meiotic and mitotic cycles. Moreover, the 50 kDa protein band, clearly detected in unfertilized oocytes, is seen to disappear within 15 min after fertilization. Unfertilized or fertilized oocytes treated with emetine have a protein synthesis rate reduced to less than half the normal rate observed in the untreated cells. Addition of this protein synthesis inhibitor at various times after fertilization inhibits the normal progression through the cell cycles. However, emetine added to unfertilized oocytes induces the completion of first meiotic maturation, polar body extrusion, and the decondensation of chromosomes which form one or two large pronuclei. This process is accompanied by DNA synthesis, and is preceded by the early disappearance of the 50 kDa protein band, seen to cycle after fertilization. These results indicate that metaphase arrest, in mussel oocytes, requires the continuous synthesis of short‐lived proteins, the destruction of which is sufficient to induce meiosis resumption followed by DNA synthesis.

Enhancement of the endothelial production of prostacyclin by inhibitors of protein synthesis

1. Pretreatment of bovine aortic endothelial cells with cycloheximide enhanced their capacity to release prostacyclin in response to adenosine 5'-triphosphate (ATP) and bradykinin. 2. The action of cycloheximide was time-dependent; it became detectable after a 1 h exposure to the cells and was maximal after 3 h. 3. Puromycin mimicked the effect of cycloheximide. For these two agents, the enhancement of prostacyclin release was obtained at concentrations producing a partial inhibition of protein synthesis. 4. Cycloheximide increased the mobilization of free arachidonic acid induced by ATP in bovine aortic endothelial cells. 5. In conclusion, the synthesis of new proteins is not involved in the stimulatory action of ATP and bradykinin on prostacyclin production by bovine aortic endothelial cells. Despite the short half-life of prostaglandin H synthase in endothelial cells, cycloheximide and puromycin enhanced the release of prostacyclin induced by agonists. Our data suggest that this release might be under the control of rapidly turning-over phospholipase inhibitory proteins.

Synthesis of protein and mRNA is necessary for transition of suspension‐cultured Catharanthus roseus cells from the G1 to the S phase of the cell cycle

The effects of inhibition of the synthesis of protein, mRNA or rRNA on the progression of the cell cycle have been analyzed in cultures of Catharanthus roseus in which cells were induced to divide in synchrony by the double phosphate starvation method. The partial inhibition of protein synthesis at the G1 phase by anisoniycio or cycloheximide caused the arrest of cells in the G1 phase or delayed the entry of cells into the S phase. When protein synthesis was partially inhibited at the S phase, cell division occurred to about the same extent as in the control. When asynchronously dividing cells were treated with cycloheximide, cells accumulated in the G1 phase, as shown by flow‐cytometric analysis. The partial inhibition of mRNA synthesis by α‐amanitin at the G1 phase caused the arrest of cells in the G1 phase, although partial inhibition of mRNA synthesis at the S phase had little effect on cell division. In the case of inhibition of synthesis of rRNA by actinomycin D at the G1 phase, initiation of DNA synthesis was observed, but no subsequent DNA synthesis or the division of cells occurred. However, the addition of actinomycin D during the S phase had no effect on cell division. These results suggest that specific protein(s), required for the progression of the cell cycle, are synthesized in the G1 phase, and that the mRNA(s) that encode these proteins are also synthesized at the G1 phase.

In-vitro activity of azithromycin and CP-63,956 against Treponema pallidum

The ability of the macrolide antibiotic azithromycin and its epimer CP-63,956 to inhibit protein synthesis in two pathogenic strains of Treponema pallidum was investigated. Using an in-vitro radiolabelling procedure employing treponemes freshly extracted from infected rabbit testes, our results indicate that both antibiotics were comparable with erythromycin in their ability to inhibit protein synthesis in T. pallidum Nichols strain. An erythromycin-resistant clinical isolate of T. pallidum, designated Street strain 14, exhibited only partial inhibition of protein synthesis at the highest antibiotic concentration tested.

Inducibility of rat liver cytochrome P-450IA1 (P-450c) mRNA during the partial inhibition of protein synthesis

Inducibility of rat liver cytochrome P-450IA1 (P-450c) mRNA during the partial inhibition of protein synthesis

Ubiquitln expression inNeurospora crassa: cloning and sequencing of a polyubiquitin gene

We have cloned and sequenced a polyubiquitin gene from Neurospora crassa that is organized in a four repeat-tandem array. The first repeat contains a small intron and the last is fused to an extra glutamine codon. In Northern blots, two RNA species of 1.3 kb and 0.7 kb hybridize to the isolated clone. The larger ubiquitin (UBI) transcript accumulates after partial inhibition of protein synthesis with cycloheximide, and the smaller one preferentially accumulates in conidia after germination. Unexpectedly, constitutive expression of UBI transcripts in exponentially grown mycelia is not altered by heat-shock or exposure to arsenite.