Effect of aldehydes on polyamine metabolism. II. Methylglyoxal-induced changes in S-adenosylmethionine decarboxylase (SAMD) activity and RNA synthesis and degradation in isolated hepatocytes

In isolated rat liver cells methylglyoxal (MeG) inhibits S-adenosylmethionine decarboxylase activity and RNA and protein synthesis; MeG also stimulated RNA degradation. The changes induced by MeG in RNA metabolism are partially prevented (in the case of RNA synthesis inhibition) or totally abolished (in the case of RNA degradation stimulation) by exogenous spermidine addition. This suggests the effects of MeG on RNA metabolism are dependent, at least in part, on SAMD inhibition.     

Regulation of protein synthesis in mitotic HeLa cells.

Mitotic HeLa cells (M cells) synthesize protein at about 25% of the rate of S phase cells. This decrease in protein synthesis is due to a reduction in the rate of initiation. However, extracts prepared from M cells are almost as active in protein synthesis as S cell extracts. Both cell extracts are quite active in in vitro initiation of protein synthesis. Moreover, two steps in initiation, binding of Met-tRNAf to 40S ribosomal subunits and binding of mRNA to ribosomes, show similar activity in both extracts. The difference in protein synthesizing activity observed in vivo is largely eliminated in the preparation of cell-free systems. The ribosomes of M cells contain small mol wt RNA, which inhibits protein synthesis in vitro. This RNA, which has possibly a nuclear origin, may be a cause of the reduction in the rate of protein synthesis in M cells.     

Effects of trypsin on protein synthesis in macrophages

Treatment of rabbit alveolar macrophages with crystalline trypsin (0.04–2 mg/10⁸ cells) inhibits protein synthesis and results in increased leakage of cell proteins. Trypsinization does not significantly decrease cellular DNA content or viability, and it does not increase protein breakdown.Trypsin treatment results in decreased oxidation of [1-¹⁴C]glucose and [6-¹⁴C]glucose, and also a decrease in ATP content. Trypsinization also causes a depression of net leucine transport and a reduction in the translational activity of polyribosomes.When normal and trypsinized macrophages are preincubated at 37 °C for several hours and then pulse-labelled with radioactive leucine, protein synthesis is stimulated to approximately the same extent in both the control and the enzyme-treated cells. Since the trypsinized cells still exhibit depressed protein synthesis, this suggests that the inhibition cannot be readily reversed.Indirect evidence indicates that the inhibition of protein synthesis is not due to entry of trypsin into the cells and suggests that the inhibition is due to changes in metabolism resulting from the action of the enzyme at the cell surface.     

On the membrane translocation of diphtheria toxin: at low pH the toxin induces ion channels on cells.

Diphtheria toxin (DT) in acidic media forms ion-conducting channels across the plasma membrane and inhibits protein synthesis of both highly and poorly DT-sensitive cell lines. This results in loss of cell potassium and in entry of both sodium and protons with a concomitant rapid lowering of membrane potential. The pH dependency of the permeability changes is similar to that of the inhibition of cell protein synthesis. DT-induced ion channels close when the pH of the external medium is returned to neutrality and cells recover their normal monovalent cation content. Similar permeability changes were induced by two DT mutants defective either in enzymatic activity or in cell binding, but not with a mutant defective in membrane translocation. The implication of these findings for the mechanism of DT membrane translocation is discussed.     

The effect of inhibition of protein synthesis on some biochemical and functional activities of the brain in chick embryos]

By means of radioautographic and biochemical methods, it has been shown that cycloheximide administration to the brain in a dose of 76 mug significantly decreases protein synthesis within 30 min, the decrease being maximal after 2 hours. RNA synthesis in neuronal cytoplasm is blocked within 4 hours. Changes in biopotentials of the forebrain and midbrain structures were observed already 15-20 minutes after cycloheximide treatment. Application of cycloheximide (12.5-25 mug) to the area of vascular brannching of the foetal membranes of a 17-day embryo inhibits motor activity within 20 min.; in 28-day embryos, this effect is observed at a dose of 75 mug. Therefore, inhibition of protein synthesis in the brain results in sharp changes of the electrical activity of the brain and inhibits motor activity of the chick embryo.     

Evidence that alterations in small molecule permeability are involved in the Clostridium perfringens type A enterotoxin-induced inhibition of macromolecular synthesis in Vero cells

The mechanism by which Clostridium perfringens enterotoxin (CPE) simultaneously inhibits RNA, DNA, and protein synthesis is unknown. In the current study the possible involvement of small molecule permeability alterations in CPE-induced inhibition of macromolecular synthesis was examined. Vero cells CPE-treated in minimal essential medium (MEM) completely ceased net precursor incorporation into RNA and protein within 15 minutes of CPE treatment. However, RNA and protein synthesis continued for at least 30 minutes in Vero cells CPE-treated in buffer (ICIB) approximating intracellular concentrations of most ions. Addition of intracellular concentrations of amino acids to ICIB (ICIB-AA) caused a further small but detectable increase in protein synthesis in CPE-treated cells. ICIB did not affect CPE-specific binding levels or rates. Similar small molecule permeability changes (i.e., 86Rb-release) were observed in cells CPE-treated in either ICIB or in Hanks' balanced salt solution. Collectively these findings suggest that CPE-treatment of cells in ICIB-AA ameliorates CPE-induced changes in intracellular concentrations of ions and amino acids and permits the continuation of RNA and protein synthesis. These results are consistent with and support the hypothesis that permeability alterations for small molecules are involved in the CPE-induced inhibition of precursor incorporation into macromolecules in Vero cells.     

Crosstalk between guanosine nucleotides regulates cellular heterogeneity in protein synthesis during nutrient limitation

Phenotypic heterogeneity of microbial populations can facilitate survival in dynamic environments by generating sub-populations of cells that may have differential fitness in a future environment. Bacillus subtilis cultures experiencing nutrient limitation contain distinct sub-populations of cells exhibiting either comparatively high or low protein synthesis activity. This heterogeneity requires the production of phosphorylated guanosine nucleotides (pp)pGpp by three synthases: SasA, SasB, and RelA. Here we show that these enzymes differentially affect this bimodality: RelA and SasB are necessary to generate the sub-population of cells exhibiting low protein synthesis whereas SasA is necessary to generate cells exhibiting comparatively higher protein synthesis. Previously, it was reported that a RelA product allosterically activates SasB and we find that a SasA product competitively inhibits this activation. Finally, we provide in vivo evidence that this antagonistic interaction mediates the observed heterogeneity in protein synthesis. This work therefore identifies the mechanism underlying phenotypic heterogeneity in protein synthesis.     

Effect of omeprazole on secretion,synthesis and the gene expression of pepsinogen in the guinea pig stomach mucosa

The effects of omeprazole, a proton pump inhibitor, on gene expression, protein synthesis, intracellular storage and secretion of pepsinogen in guinea pig stomach were investigated. After treatment with omeprazole for five days, acid and pepsinogen secretion into the gastric lumen was significantly reduced. Concomitant with this, there was an increase in intracellular pepsinogen as demonstrated by increased pepsin activity in the gastric mucosa, more intense immunohistochemical staining by antibodies specific of pepsinogen and accumulation of secretory granules in the cells producing pepsinogen. In these cells, the amount for pepsinogen mRNA was reduced as revealed by Northern blotting and in situ hybridization. Ultrastructurally the endoplasmic reticulum of these cells was poorly developed, the findings being consistent with a reduction in protein synthesis. It appears that omeprazole inhibits the secretion of pepsinogen, increasing the intracellular store and leading to the reduction in gene expression probably by a feedback mechanism and consequent reduction in pepsinogen synthesis. Since these changes were most evident in the acid-secreting fundic gland mucosa, as compared with other mucosae secreting only pepsinogen, namely pyloric and duodenal mucosa, it appears probable that these changes are linked with omeprazole-induced reduction in the acid secretion.     

Suppression of RNA synthesis by a specific antiviral activity in Sindbis virus-infected Aedes albopictus cells.

An antiviral protein is released by mosquito cells persistently infected with Sindbis virus. Differences in both sensitivity to and production of this virus-specific activity were apparent in three independently produced Aedes albopictus cell lines. This activity inhibits total viral RNA synthesis in a time-dependent manner. The antiviral effect is maximally realized when cells are treated with the activity 48 h before infections. These data suggest that the antiviral activity induces an antiviral state in treated cells which prevents the formation or efficient function of viral RNA-synthesizing complexes.     

Targeting endothelial cells overexpressing VEGFR-2: selective toxicity of Shiga-like toxin-VEGF fusion proteins.

Growing endothelial cells at the sites of angiogenesis express high numbers of VEGF receptors and therefore may be particularly sensitive to VEGF-mediated drug delivery. To test this hypothesis we have constructed a protein containing the catalytic A-subunit of Shiga-like toxin I fused to VEGF121 (SLT-VEGF/L). Wild-type A-subunit is a site-specific N-glycosidase of 28S rRNA that inhibits protein synthesis after being delivered into cells by separate cell-binding B-subunits. SLT-VEGF/L retains functional activities of both SLT and VEGF121 moieties, since it inhibits protein synthesis in a cell-free translation system and induces VEGFR-2 tyrosine autophosphorylation. SLT-VEGF/L selectively inhibits growth of porcine endothelial cells expressing 2.5 x 10(5) VEGFR-2/cell with an IC50 of 0.2 nM and rapidly induces apoptosis at concentrations >1 nM. We found that sensitivity of VEGFR-2 transfected PAE cells to SLT-VEGF/L declined as the cellular VEGFR-2 density decreased; PAE cells expressing 25000 VEGFR-2/cell were as sensitive as parental cells lacking the receptor. Growth inhibition and induction of apoptosis by SLT-VEGF/L require intrinsic N-glycosidase activity of the SLT moiety, but take place without significant inhibition of protein synthesis. Selective cytotoxicity of SLT-VEGF/L against growing endothelial cells overexpressing VEGFR-2 suggests that it may be useful in targeting similar cells at the sites of angiogenesis.     

Inhibition of protein synthesis by nitric oxide correlates with cytostatic activity: nitric oxide induces phosphorylation of initiation factor eIF-2 alpha.

BACKGROUND: Nitric oxide (NO) is cytostatic for proliferating cells, inhibits microbial growth, and down-regulates the synthesis of specific proteins. Studies were undertaken to determine the mechanism by which NO inhibits total protein synthesis and whether the inhibition correlates with established cytostatic activities of NO. MATERIALS AND METHODS: In in vitro experiments, various cell types were exposed to NO using either donors or expression of inducible NO synthase (iNOS). The capacity of NO to suppress total protein synthesis, measured by incorporation of 35S-methionine into protein, was correlated with the capacity of NO to suppress cell proliferation, viral replication, or iNOS expression. Phosphorylation of eIF-2 alpha was examined as a possible mechanism for the suppressed protein synthesis by NO. RESULTS: Both NO donors and expression of the iNOS suppressed total protein synthesis in L929 cells and A2008 human ovarian tumor cells in parallel with decreased cell proliferation. Suppressed protein synthesis was also shown to correlate with decreased vaccinia virus proliferation in murine peritoneal macrophages in an iNOS-dependent manner. Furthermore, iNOS expression in pancreatic islets or RAW264.7 cells almost completely inhibited total protein synthesis, suggesting that nonspecific inhibition of protein synthesis may be the mechanism by which NO inhibited the synthesis of specific proteins such as insulin or iNOS itself. This possibility was confirmed in RAW264.7 cells where the inhibition of total protein synthesis correlated with the decreased iNOS protein. The decrease in protein levels occurred without changes in iNOS mRNA levels, implicating an inhibition of translation. Mechanistic studies revealed that iNOS expression in RAW264.7 cells resulted in the phosphorylation of eIF-2 alpha and inhibition of the 80S ribosomal complex formation. CONCLUSIONS: These results suggest that NO suppresses protein synthesis by stimulating the phosphorylation of eIF-2 alpha. Furthermore, our observations indicate that nonspecific inhibition of protein synthesis may be a generalized response of cells exposed to high levels of NO and that inhibition of protein synthesis may contribute to many of the described cytostatic actions of NO.     

A relatively low level of ribosome depurination by mutant forms of ricin toxin A chain can trigger protein synthesis inhibition,cell signaling and apoptosis in mammalian cells

The A chain of the plant toxin ricin (RTA) is an N-glycosidase that inhibits protein synthesis by removing a specific adenine from the 28S rRNA. RTA also induces ribotoxic stress, which activates stress-induced cell signaling cascades and apoptosis. However, the mechanistic relationship between depurination, protein synthesis inhibition and apoptosis remains an open question. We previously identified two RTA mutants that suggested partial independence of these processes in a yeast model. The goals of this study were to establish an endogenous RTA expression system in mammalian cells and utilize RTA mutants to examine the relationship between depurination, protein synthesis inhibition, cell signaling and apoptosis in mammalian cells. The non-transformed epithelial cell line MAC-T was transiently transfected with plasmid vectors encoding precursor (pre) or mature forms of wild-type (WT) RTA or mutants. PreRTA was glycosylated indicating that the native signal peptide targeted RTA to the ER in mammalian cells. Mature RTA was not glycosylated and thus served as a control to detect changes in catalytic activity. Both pre- and mature WT RTA induced ribosome depurination, protein synthesis inhibition, activation of cell signaling and apoptosis. Analysis of RTA mutants showed for the first time that depurination can be reduced by 40% in mammalian cells with minimal effects on inhibition of protein synthesis, activation of cell signaling and apoptosis. We further show that protein synthesis inhibition by RTA correlates more linearly with apoptosis than ribosome depurination.     

Studies with a human plasma-derived immunosuppressive,anti-lymphoma factor

Summary A low-molecular-weight (1,400) factor isolated from a human plasma -globulin concentrate by acid-salt dissociation and ultrafiltration inhibits proliferation of mitogen-stimulated T cells and L1210 leukemia cells. The factor (UM05R) inhibits DNA, RNA, and protein synthesis in sensitive cells, acts in G1 of the cell cycle, and appears to suppress mitogen-responsive T cells without an accessory cell requirement. UM05R activity is enhanced by known cAMP-elevating agents and by sulfhydryl compounds. The results of the present study are consistent with the hypothesis that the plasma-derived agent inhibits lympho-proliferation as a result of elevation of intracellular cAMP.     

体细胞胚发生的生化基础

在胚性细胞分化和分裂过程中ATP酶活性和分布的动态变化表明,这些胚性细胞进行着旺盛的主动物质吸收和活跃的新陈代谢过程。在多种植物的体细胞胚发生中过氧化物酶的活性与同工酶的种类都高于对照,而且在大麦中发现过氧化物酶、酯酶和酸性磷酸酶同工酶的结合应用可以作为体细胞胚发生的标志酶。胚性愈伤组织中可溶性蛋白质含量与组分远高于或多于非胚性愈伤组织。大多数材料中都存在45kD-55kD的胚胎发生特异性蛋白质组分。而且在体细胞胚发生中蛋白质和核酸代谢动态呈规律性变化,首先是RNA合成速率增加,继而是蛋白质的迅速合成,并在胚性细胞分化和发育过程中一直保持相对较高水平,其中mRNA种类丰富,不同发育时期mRNA种类不同,因此转译形成多种蛋白质。DNA的代谢相对较稳定,但在胚性细胞系中DNA合成量仍高于非胚性细胞系。加入蛋白质或核酸合成抑制剂,不仅抑制了蛋白质和核酸的合成,同时也抑制了体细胞胚的发生与发育,而且抑制剂加和时间愈早,影响愈严重。由此表明,蛋白质与核酸的合成为体细胞胚的分化和发育奠定了分子基础。     

Cyclin D1 and c-myc internal ribosome entry site (IRES)-dependent translation is regulated by AKT activity and enhanced by rapamycin through a p38 MAPK- and ERK-dependent pathway

The macrolide antibiotic rapamycin inhibits the mammalian target of rapamycin protein (mTOR) kinase resulting in the global inhibition of cap-dependent protein synthesis, a blockade in ribosome component biosynthesis, and G1 cell cycle arrest. G1 arrest may occur by inhibiting the protein synthesis of critical factors required for cell cycle progression. Hypersensitivity to mTOR inhibitors has been demonstrated in cells having elevated levels of AKT kinase activity, whereas cells containing quiescent AKT activity are relatively resistant. Our previous data suggest that low AKT activity induces resistance by allowing continued cap-independent protein synthesis of cyclin D1 and c-Myc proteins. In support of this notion, the current study demonstrates that the human cyclin D1 mRNA 5' untranslated region contains an internal ribosome entry site (IRES) and that both this IRES and the c-myc IRES are negatively regulated by AKT activity. Furthermore, we show that cyclin D1 and c-myc IRES function is enhanced following exposure to rapamycin and requires both p38 MAPK and RAF/MEK/ERK signaling, as specific inhibitors of these pathways reduce IRES-mediated translation and protein levels under conditions of quiescent AKT activity. Thus, continued IRES-mediated translation initiation may permit cell cycle progression upon mTOR inactivation in cells in which AKT kinase activity is relatively low.     

Cytochalasin D inhibits nuclear translocation of nonhistone proteins induced by lectin and other agents in lymphocytes.

Cytochalasin D (CD), known to interfere with microfilament activity, inhibits RNA and protein synthesis and the cellular uptake of [3H]actinomycin D in concanavalin A-stimulated lymphocytes. It also inhibits the nuclear translocation of nonhistone proteins (NHP) induced by the lectin. Since NHP mediate RNA and protein synthesis and [3H]actinomycin D binding, inhibited nuclear translocation of NHP can explain the inhibition of the former events. CD also inhibits nuclear translocation of NHP caused by NaF or chloroquine. The mechanism of how CD inhibits nuclear translocation of the NHP is obscure. Since NaF and chloroquine enter cells by diffusion, it would appear that CD acts on an intracellular target, rather than abrogating a cell surface-mediated signal generated by binding of the lectin to a cell surface receptor.