Low Concentrations Of Cycloheximide Research Articles

Analysis of cycloheximide in rat specimens using liquid chromatography with tandem mass spectrometry detection

The development of a selective and sensitive high-performance liquid chromatographic tandem mass spectrometric method for the determination of cycloheximide (CHX) in rat blood and plasma is described. The extraction of CHX and colchicine as internal standard from blood fluid (0.1 mL) was achieved using n-hexane: dichloromethane: isopropanol (20:10:1 v/v/v). The mobile phase, a combination of methanol:10 mM ammonium acetate (85:15, v/v), was pumped at 0.2 mL/min through a C18 analytical column with a run time of 3.5 min. Detection was carried out by electrospray positive ionization mass spectrometry in the multiple-reaction monitoring (MRM) mode. The assay exhibited excellent linearity (r2 > 0.999) in peak area response over the concentration ranges of 2–1000 ng CHX /mL blood fluid. The mean absolute recoveries for 20, 100 and 500 ng/mL CHX in blood fluid using the present extraction procedure were > 97%. The intra- and inter-day coefficients of variation in the plasma and blood and mean error were < 13% at different concentrations. Samples had limited stability at room temperature, and speedy processing is needed. After intravenous administration, rats had measurable concentrations of CHX for up to 24 h after dosing with 1 mg/kg of cycloheximide. The method displayed a high caliber of sensitivity and selectivity for detecting very low concentrations of CHX in rats.

Autoactivation of Translation Causes the Bloom of Prorocentrum donghaiense in Harmful Algal Blooms

Harmful algal blooms (HABs) are symptomatic of ecosystem imbalance, leading to major worldwide marine natural disasters, and seriously threaten the human health. Some HAB algae's exceptional genome size prohibited the genomic investigations on molecular mechanisms, for example, Prorocentrum. This study performed translatome sequencing (RNC-seq) for Prorocentrum donghaiense to assemble the translatome reference sequences on appropriate cost to enable the global molecular study at translatome and proteome levels. By analyzing the translatome and proteome of P. donghaiense in phosphor-rich, phosphor-deficient, and phosphor-restored media, we found massive up-regulation of energy and material production pathways in phosphor-rich conditions that enables autoactivation of translation, which is the key to its exponential growth in HABs. To break down the autoactivation, we demonstrated that mild translation delay using very low concentrations of cycloheximide efficiently controls the blooming without harming other aquatic organisms and humans. Our result provides a novel hint for controlling HABs and demonstrated the RNC-seq as an economic strategy on investigating functions of organisms with large and unknown genomes.

Decreasing cytosolic translation is beneficial to yeast and human Tafazzin-deficient cells.

Cardiolipin (CL) optimizes diverse mitochondrial processes, including oxidative phosphorylation (OXPHOS). To function properly, CL needs to be unsaturated, which requires the acyltransferase Tafazzin (TAZ). Loss-of-function mutations in the TAZ gene are responsible for the Barth syndrome (BTHS), a rare X-linked cardiomyopathy, presumably because of a diminished OXPHOS capacity. Herein we show that a partial inhibition of cytosolic protein synthesis, either chemically with the use of cycloheximide or by specific genetic mutations, fully restores biogenesis and the activity of the oxidative phosphorylation system in a yeast BTHS model (taz1Δ). Interestingly, the defaults in CL were not suppressed, indicating that they are not primarily responsible for the OXPHOS deficiency in taz1Δ yeast. Low concentrations of cycloheximide in the picomolar range were beneficial to TAZ-deficient HeLa cells, as evidenced by the recovery of a good proliferative capacity. These findings reveal that a diminished capacity of CL remodeling deficient cells to preserve protein homeostasis is likely an important factor contributing to the pathogenesis of BTHS. This in turn, identifies cytosolic translation as a potential therapeutic target for the treatment of this disease.

A HIF-1 Target, ATIA, Protects Cells from Apoptosis by Modulating the Mitochondrial Thioredoxin, TRX2

The regulation of apoptosis is critical for controlling tissue homeostasis and preventing tumor formation and growth. Reactive oxygen species (ROS) generation plays a key role in such regulation. Here, we describe a HIF-1 target, Vasn/ATIA (anti-TNFα-induced apoptosis), which protects cells against TNFα- and hypoxia-induced apoptosis. Through the generation of ATIA knockout mice, we show that ATIA protects cells from apoptosis through regulating the function of the mitochondrial antioxidant, thioredoxin-2, and ROS generation. ATIA is highly expressed in human glioblastoma, and ATIA knockdown in glioblastoma cells renders them sensitive to hypoxia-induced apoptosis. Therefore, ATIA is not only a HIF-1 target that regulates mitochondrial redox pathways but also a potentially diagnostic marker and therapeutic target in human glioblastoma.

A domain of the actin binding protein Abp140 is the yeast methyltransferase responsible for 3-methylcytidine modification in the tRNA anti-codon loop

The 3-methylcytidine (m³C) modification is widely found in eukaryotic species of tRNA(Ser), tRNA(Thr), and tRNA(Arg); at residue 32 in the anti-codon loop; and at residue e2 in the variable stem of tRNA(Ser). Little is known about the function of this modification or about the specificity of the corresponding methyltransferase, since the gene has not been identified. We have used a primer extension assay to screen a battery of methyltransferase candidate knockout strains in the yeast Saccharomyces cerevisiae, and find that tRNA(Thr(IGU)) from abp140-Δ strains lacks m³C. Curiously, Abp140p is composed of a poorly conserved N-terminal ORF fused by a programed +1 frameshift in budding yeasts to a C-terminal ORF containing an S-adenosylmethionine (SAM) domain that is highly conserved among eukaryotes. We show that ABP140 is required for m³C modification of substrate tRNAs, since primer extension is similarly affected for all tRNA species expected to have m³C and since quantitative analysis shows explicitly that tRNA(Thr(IGU)) from an abp140-Δ strain lacks m³C. We also show that Abp140p (now named Trm140p) purified after expression in yeast or Escherichia coli has m³C methyltransferase activity, which is specific for tRNA(Thr(IGU)) and not tRNA(Phe) and occurs specifically at C₃₂. We suggest that the C-terminal ORF of Trm140p is necessary and sufficient for activity in vivo and in vitro, based on analysis of constructs deleted for most or all of the N-terminal ORF. We also suggest that m³C has a role in translation, since trm140-Δ trm1-Δ strains (also lacking m²,²G₂₆) are sensitive to low concentrations of cycloheximide.

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.

Identification of Two SET Domain Proteins Required for Methylation of Lysine Residues in Yeast Ribosomal Protein Rpl42ab

We show that the Saccharomyces cerevisiae ribosomal protein Rpl42ab (the identical product of the RPL42A and RPL42B genes) is monomethylated at Lys-40 and Lys-55. The methylation of Lys-40 is dependent upon the Ybr030w gene product; the methylation of Lys-55 is dependent upon the Set7 gene product. Ybr030w and SET7 genes both encode SET domain containing proteins homologous to known protein lysine methyltransferases, suggesting that their products are the specific enzymes responsible for the monomethylation of the two sites in Rpl42ab. We thus designate Ybr030w as Rkm3 and Set7 as Rkm4. Yeast strains with deletions in both the Ybr030w and SET7 genes produce unmethylated Rpl42ab. A slow growth phenotype was seen for the SET7 deletion strain and the double knock-out when grown in low concentrations of the eukaryotic protein synthesis inhibitor, cycloheximide. These results suggest that modification of Rpl42ab at Lys-55 can fine-tune its structure to avoid inhibition. An intact mass fragmentation approach ("top down mass spectrometry") was used to quantitate the extent of methylation of Rpl42ab. In wild-type strains, it was found that 78% was monomethylated at both Lys-40 and Lys-55 and that 22% was a mixture of species with either Lys-40 or Lys-55 monomethylated. The top down approach was also used to reevaluate the methylation sites of Rpl12ab. We found that the yeast Rpl12ab protein is dimethylated at the N-terminal proline residue, trimethylated at Lys-3 by Rkm2, and monomethylated at Arg-66.

Acute nutrient regulation of the unfolded protein response and integrated stress response in cultured rat pancreatic islets

Inadequate chaperone function relative to client protein load in the endoplasmic reticulum triggers an adaptive unfolded protein response (UPR), including the integrated stress response (ISR), the latter being also activated by other types of stresses. It is well established that pancreatic beta cells, which synthesise and secrete insulin upon nutrient stimulation, are markedly affected by pathological disruption or excessive activation of the UPR. However, whether and how physiological nutrient stimulation affects the beta cell UPR has been little investigated. We compared the effects of increasing glucose concentrations and of endoplasmic reticulum Ca(2+) emptying with thapsigargin on the UPR (X-box binding protein [Xbp1] mRNA splicing and XBP1/activating transcription factor [ATF] 6-target gene expression) and ISR (eukaryotic translation initiation factor 2A phosphorylation, ATF4 protein levels and target gene expression) in isolated rat islets. Thapsigargin strongly increased both UPR and ISR. In comparison, glucose moderately increased the UPR between 5 and 30 mmol/l, but exerted complex effects on the ISR as follows: (1) marked reduction between 2 and 10 mmol/l; (2) moderate increase parallel to the UPR between 10 and 30 mmol/l. These glucose effects occurred within 2 h, were mimicked by other metabolic substrates, but were independent of changes in Ca(2+) influx or insulin secretion. Remarkably, attenuating the glucose stimulation of protein synthesis with a low concentration of cycloheximide prevented UPR activation but not ISR reduction by high glucose. Nutrient stimulation acutely activates rat islet UPR in a manner dependent on protein synthesis, while exerting complex effects on the ISR. These effects may contribute to nutrient-induced maintenance of the beta cell phenotype.

Stress-induced apoptosis in Spodoptera frugiperda (Sf9) cells: baculovirus p35 mitigates eIF2 alpha phosphorylation.

Spodoptera frugiperda (Sf9) ovarian cells, natural hosts for baculovirus, are good model systems to study apoptosis and also heterologous gene expression. We report that uninfected Sf9 cells readily undergo apoptosis and show increased phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha) in the presence of agents such as UVB light, etoposide, high concentrations of cycloheximide, and EGTA. In contrast, tunicamycin, A23187, and low concentrations of cycloheximide promoted eIF2alpha phosphorylation in Sf9 cells but without apoptosis. These findings therefore suggest that increased eIF2alpha phosphorylation does not always necessarily lead to apoptosis, but it is a characteristic hallmark of stressed cells and also of cells undergoing apoptosis. Cell death induced by the above agents was abrogated by infection of Sf9 cells with wild-type (wt) AcNPV. In contrast, Sf9 cells when infected with vAcdelta35, a virus carrying deletion of the antiapoptotic p35 gene, showed increased apoptosis and enhanced eIF2alpha phosphorylation. Further, a recombinant wt virus vAcS51D expressing human S51D, a phosphomimetic form of eIF2alpha, induced apoptosis in UVB pretreated Sf9 cells. However, infection with vAcS51A expressing a nonphosphorylatable form (S51A) of human eIF2alpha partially reduced apoptosis. Consistent with these findings, it has been observed here that caspase activation has led to increased eIF2alpha phosphorylation, while caspase inhibition by z-VAD-fmk reduced eIF2alpha phosphorylation selectively in cells exposed to proapoptotic agents. These findings therefore suggest that the stress signaling pathway determines apoptosis, and caspase activation is a prerequisite for increased eIF2alpha phosphorylation in Sf9 cells undergoing apoptosis. The findings also reinforce the conclusion for the first time that the "pancaspase inhibitor" baculovirus p35 mitigates eIF2alpha phosphorylation.

Translational regulation of rotavirus gene expression.

Rotavirus mRNAs are transcribed from 11 genomic dsRNA segments within a subviral particle. The mRNAs are extruded into the cytoplasm where they serve as mRNA for protein synthesis and as templates for packaging and replication into dsRNA. The molecular steps in the replication pathway that regulate the levels of viral gene expression are not well defined. We have investigated potential mechanisms of regulation of rotavirus gene expression by functional evaluation of two differentially expressed viral mRNAs. NSP1 (gene 5) and VP6 (gene 6) are expressed early in infection, and VP6 is expressed in excess over NSP1. We formulated the hypothesis that the amounts of NSP1 and VP6 were regulated by the translational efficiencies of the respective mRNAs. We measured the levels of gene 5 and gene 6 mRNA and showed that they were not significantly different, and protein analysis indicated no difference in stability of NSP1 compared with VP6. Polyribosome analysis showed that the majority of gene 6 mRNA was present on large polysomes. In contrast, sedimentation of more than half of the gene 5 mRNA was subpolysomal. The change in distribution of gene 5 mRNA in polyribosome gradients in response to treatment with low concentrations of cycloheximide suggested that gene 5 is a poor translation initiation template compared with gene 6 mRNA. These data define a regulatory mechanism for the difference in amounts of VP6 and NSP1 and provide evidence for post-transcriptional control of rotavirus gene expression mediated by the translational efficiency of individual viral mRNAs.

NF-kappaB inducers upregulate cFLIP, a cycloheximide-sensitive inhibitor of death receptor signaling.

The caspase 8 homologue FLICE-inhibitory protein (cFLIP) is a potent negative regulator of death receptor-induced apoptosis. We found that cFLIP can be upregulated in some cell lines under critical involvement of the NF-kappaB pathway, but NF-kappaB activation was clearly not sufficient for cFLIP induction in all cell lines. Treatment of SV80 cells with the proteasome inhibitor N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG-132) or geldanamycin, a drug interfering with tumor necrosis factor (TNF)-induced NF-kappaB activation, inhibited TNF-induced upregulation of cFLIP. Overexpression of a nondegradable IkappaBalpha mutant (IkappaBalpha-SR) or lack of IkappaB kinase gamma expression completely prevented phorbol myristate acetate-induced upregulation of cFLIP mRNA in Jurkat cells. These data point to an important role for NF-kappaB in the regulation of the cFLIP gene. SV80 cells normally show resistance to TNF-related apoptosis-inducing ligand (TRAIL) and TNF, as apoptosis can be induced only in the presence of low concentrations of cycloheximide (CHX). However, overexpression of IkappaBalpha-SR rendered SV80 cells sensitive to TRAIL-induced apoptosis in the absence of CHX, and cFLIP expression was able to reverse the proapoptotic effect of NF-kappaB inhibition. Western blot analysis further revealed that cFLIP, but not TRAF1, A20, and cIAP2, expression levels rapidly decrease upon CHX treatment. In conclusion, these data suggest a key role for cFLIP in the antiapoptotic response of NF-kappaB activation.

Multiple Mechanisms Regulate Expression of Low Temperature Responsive (LOT) Genes in Saccharomyces cerevisiae

Using cDNA subtraction screening, we identified five Saccharomyces cerevisiae genes whose expressions is up-regulated when culture temperature was down-shifted from 30 to 10°C. Among these LOT (low temperature-responsive) genes, three (LOT1, LOT2, and LOT3) were identical to FBA1, RPL2B, and NOP1, encoding a fructose biphosphate aldolase, a ribosomal protein L2B, and a nucleolar protein for rRNA processing, respectively. No functions were assigned for LOT5 and LOT6, which are identical to YKL183w and YLR011w, respectively. Northern hybridization analysis revealed that these genes are not uniformly regulated in response to the change of growth temperature. In addition, all the LOT genes, except for LOT1/FBA1, were induced by a low concentration of cycloheximide. The data indicate that multiple mechanisms, including translational functionality may be involved in the regulation of LOT gene expression in yeast.

Characterization and expression of a Neurospora crassa ribosomal protein gene, crp-7.

We have isolated and characterized a Neurospora crassa cytoplasmic ribosomal protein gene, named crp-7, which is found upstream of the photolyase gene. The deduced amino-acid sequence of this gene is highly homologous to the YS25 ribosomal protein of Saccharomyces cerevisiae. The crp-7 ORF consists of two exons which are separated by a short intron. The deduced polypeptide contains 87 amino acid residues and has a calculated molecular weight of 9.7 kDa. RFLP mapping showed that the crp-7 gene is located on the right arm of linkage group I. Southern blot hybridization analyses indicated that there is only one copy of the crp-7 gene in the N. crassa genome. Transcriptional elements, the Dde box, the Taq box and the CG element, that have been identified in other N. crassa ribosomal protein genes are observed in the promoter region of the crp-7 gene. The crp-7 mRNA levels were low in conidia and highest in young mycelia during vegetative growth. The mRNA levels of four r-protein genes, including the crp-7 gene, as well as the tef-1 gene encoding translational elongation factor 1 alpha, were raised following the treatment of mycelia with a low concentration of cycloheximide. This indicates that the expression of r-protein genes is under the control of so-called super-induction.

Neuroprotective action of cycloheximide involves induction of bcl-2 and antioxidant pathways.

The ability of the protein synthesis inhibitor cycloheximide (CHX) to prevent neuronal death in different paradigms has been interpreted to indicate that the cell death process requires synthesis of "killer" proteins. On the other hand, data indicate that neurotrophic factors protect neurons in the same death paradigms by inducing expression of neuroprotective gene products. We now provide evidence that in embryonic rat hippocampal cell cultures, CHX protects neurons against oxidative insults by a mechanism involving induction of neuroprotective gene products including the antiapoptotic gene bcl-2 and antioxidant enzymes. Neuronal survival after exposure to glutamate, FeSO4, and amyloid beta-peptide was increased in cultures pretreated with CHX at concentrations of 50-500 nM; higher and lower concentrations were ineffective. Neuroprotective concentrations of CHX caused only a moderate (20-40%) reduction in overall protein synthesis, and induced an increase in c-fos, c-jun, and bcl-2 mRNAs and protein levels as determined by reverse transcription-PCR analysis and immunocytochemistry, respectively. At neuroprotective CHX concentrations, levels of c-fos heteronuclear RNA increased in parallel with c-fos mRNA, indicating that CHX acts by inducing transcription. Neuroprotective concentrations of CHX suppressed accumulation of H2O2 induced by FeSO4, suggesting activation of antioxidant pathways. Treatment of cultures with an antisense oligodeoxynucleotide directed against bcl-2 mRNA decreased Bcl-2 protein levels and significantly reduced the neuroprotective action of CHX, suggesting that induction of Bcl-2 expression was mechanistically involved in the neuroprotective actions of CHX. In addition, activity levels of the antioxidant enzymes Cu/Zn-superoxide dismutase, Mn-superoxide dismutase, and catalase were significantly increased in cultures exposed to neuroprotective levels of CHX. Our data suggest that low concentrations of CHX can promote neuron survival by inducing increased levels of gene products that function in antioxidant pathways, a neuroprotective mechanism similar to that used by neurotrophic factors.

Stimulation of Sendai virus C′ protein synthesis by cycloheximide

The polycistronic Sendai virus P/C mRNA is translated into five proteins (P, C', C, Y1 and Y2) from distinct start sites in virus-infected cells. The translation mechanism(s) of these proteins from two overlapping open reading frames in the P/C mRNA are poorly understood [Gupta, Ono and Xu (1996) Biochemistry 35, 1223-1231]. While investigating the initiation mechanism of C' from an ACG start site, we found that C' synthesis was resistant to inhibitors of peptide chain elongation such as cycloheximide (CHX) and anisomycin, but not to pactamycin (an inhibitor of chain initiation) or puromycin (a peptide chain terminator). Moreover, low levels (less than 30 microg/ml) of CHX significantly stimulated C' synthesis. Whereas C' synthesis was stimulated, synthesis of the P and C proteins, which are translated from the same mRNA, decreased by more than 95%. Stimulation of C' synthesis by CHX is not related to its initiation at an ACG codon. Mutation of ACG to alternative start sites had no effect on the CHX-stimulated C' synthesis. Similarly, C' synthesis was preferentially stimulated when Sendai virus-infected cells were exposed to hypotonic growth medium. These results suggest that the P/C mRNA may exist in at least two reversible conformations: whereas one conformation allows synthesis of the P and C proteins, the alternative conformation allows synthesis of the C' protein. It might be that low concentrations of CHX somehow increase the alternative conformation, which increases C' synthesis. The C' protein synthesis is reminiscent of the synthesis of stress-related proteins. Perhaps Sendai virus has evolved a novel mechanism to express both non-stress-related and stress-related proteins from the same mRNA.

Inhibition by bacterial lipopolysaccharide of spontaneous and TNF-alpha-induced human neutrophil apoptosis in vitro.

In the previous paper (Takeda et al, Int. Immunol., 5, 691-694, 1993), we demonstrated that tumor necrosis factor-alpha (TNF-alpha) promptly accelerates apoptosis of human neutrophils in vitro. In order to determine the role of neutrophil apoptosis in defending against bacterial infection, we studied the effect of bacterial lipopolysaccharide (LPS) on this process. LPS inhibited spontaneous and TNF-alpha-induced human neutrophil apoptosis in vitro, as determined by 1) light and electron microscopy, 2) flow cytometry, and 3) agarose gel electrophoresis of DNA. Low concentrations of cycloheximide, a protein synthesis inhibitor, which alone did not affect neutrophil apoptosis, were able to reduce spontaneous apoptosis inhibition by LPS, suggesting the involvement of newly synthesized protein in this phenomenon.

Translation and M1 double-stranded RNA propagation: MAK18 = RPL41B and cycloheximide curing

MAK18 is one of nearly 30 chromosomal genes of Saccharomyces cerevisiae necessary for propagation of the killer toxin-encoding M1 double-stranded RNA satellite of the L-A double-stranded RNA virus. We have cloned and sequenced MAK18 and find that it is identical to RPL41B, one of the two genes encoding large ribosomal subunit protein L41. The mak18-1 mutant is deficient in 60S subunits, which we suggest results in a preferential decrease in translation of viral poly(A)-deficient mRNA. We have reexamined the curing of M1 by low concentrations of cycloheximide (G. R. Fink and C. A. Styles, Proc. Natl. Acad. Sci. USA 69:2846-2849, 1972), which is known to act on ribosomal large subunit protein L29. We find that when M1 is supported by L-A proteins made from the poly(A)+ mRNA of a cDNA clone of L-A, cycloheximide does not decrease the M1 copy number, consistent with our hypothesis.

Multiple interacting elements delineate an ecdysone-dependent regulatory region with secondary responsive character

Within the 2.2 kb region between hsp23 and gene 1 of the small heat shock gene locus 67B1 of Drosophila melanogaster, an approximately 1 kb perturbation of the chromatin architecture has previously been observed to occur in response to the steroid hormone ecdysone. Transient expression assays in hormonally-responsive Drosophila tissue culture cells utilizing hsp70-lacZ chimeric reporter constructs revealed the presence of ecdysone-dependent regulatory sequences in this hsp23-gene 1 intergenic region. The analysis delimited five functional segments: three core regions which were completely encompassed within the region of chromatin perturbation, and two gene-proximal regions which appear to be functionally equivalent under some circumstances. None of the delineated regions was capable of stimulating expression independently, while sub-maximal expression was obtained when combinations of two or three regions were monitored. This requirement for multiple DNA segments to drive maximal transcription suggested that cooperative interactions between the regions were essential for full hormonal responsiveness. Unexpectedly, no binding of the ecdysone receptor was detectable within any of the delineated regions, implying the involvement of multiple non-receptor factors in the observed hormonal responsiveness. The ecdysone-dependent activation of reporter constructs driven by these sequences showed a significant time lag and was coupled with a marked sensitivity to low concentrations of cycloheximide. The data obtained strongly suggest that the cis-acting elements delimited within the hsp23-gene 1 intergenic region respond to ecdysone in a secondary manner, presumably by requiring interaction with the product(s) of primary ecdysone-responsive genes.

Differential polysomal localization of human insulin-like-growth-factor-2 mRNAs in cell lines and foetal liver.

Examination of the association of insulin-like-growth-factor-2 mRNAs with polyribosomes in five cell lines revealed that greater than 50% of the total mRNA population was present in the untranslated free mRNP fraction for each cell line. Of the different subtypes of insulin-like-growth-factor-2 messengers, the least abundant mRNAs, starting with exon 4 (leader 2, 5.0 kb) and exon 6 (leader 4, 4.8 kb), were found in the polysomes only, while the most abundant transcript, starting with exon 5 (leader 3, 6.0 kb and 2.1 kb) was found predominantly in the untranslated fractions. 20-30% of leader 3 mRNAs, however, were in the larger polysomes (four or more ribosomes), indicating that a subpopulation of this mRNA can be translated efficiently. The peak fraction for the leader 4 insulin-like-growth-factor-2 mRNA (4.8 kb) in the polysomes was migrating faster in the sucrose gradients than the peak fractions of leader 2 and 3 mRNAs (5.0 kb and 6.0 kb), implying that more ribosomes were associated with this type of mRNA. In foetal liver, the situation was similar, though in this case the leader 2 mRNA was most heavily loaded with polysomes. Treatment of cells with low concentrations of cycloheximide caused the polysomal RNAs to shift to even larger polysomes while the untranslated fraction of the leader 3 mRNAs stayed in the untranslated fractions. These results indicate that, both in established cell lines and in foetal liver, insulin-like-growth-factor-2 translation is influenced both by mRNP sequestration and differential translation initiation efficiency of the insulin-like-growth-factor-2 mRNAs.

Enhancement of inducible-type NO synthase gene transcription by protein synthesis inhibitors. Activation of an intracellular signal transduction pathway by low concentrations of cycloheximide.

Treatment of mouse macrophage-like RAW 264.7 cells with certain protein synthesis inhibitors is followed by accumulation of the mRNA for the inducible isoform of nitric oxide synthase (i-NOS). The activity of these compounds on the i-NOS gene in RAW 264.7 cells was analyzed here in detail. Results show that both cycloheximide and anisomycin can efficiently induce i-NOS mRNA, even when used at concentrations so low (0.25 microgram/ml) to have only negligible effects on protein synthesis; puromycin, on the other hand, shows only a limited effect on i-NOS mRNA expression, detectable only when cells are treated with higher concentrations of inhibitor (25 micrograms/ml). In RAW 264.7 cells, low concentrations of cycloheximide trigger an immediate-early gene response, as indicated by induction of c-fos and JE mRNAs, and can efficiently activate transcription of transiently transfected recombinant reporter genes including either the i-NOS or the c-fos gene promoters.