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PubMed1993

Cholesterol trafficking in steroidogenic cells. Reversible cycloheximide-dependent accumulation of cholesterol in a pre-steroidogenic pool.

Peptide hormones activate steroid hormone biosynthesis in responsive tissues by stimulating the delivery of cholesterol to a steroidogenic pool, thought to be located in the inner mitochondrial membrane. At this site, it is metabolized to pregnenolone, the precursor of the steroid hormones, by side-chain-cleaving cytochrome P-450 (cytochrome P-450scc). In the presence aminoglutethimide (an inhibitor of cytochrome P-450scc) and an activating stimulus, cholesterol accumulates in the steroidogenic pool, and increased pregnenolone generation is observed upon removal of the inhibitor. Using Y-1 adrenocortical cells and MA-10 Leydig tumor cells, we now provide evidence for a distinct, functionally relevant cholesterol pool which precedes the steroidogenic pool, which we designate the pre-steroidogenic pool. This pool was defined by activating the cells with 8-bromo-adenosine 3',5'-cyclic monophosphoric acid in the presence of cycloheximide, an inhibitor of steroidogenesis. Following a wash procedure, which removed 8-bromo-adenosine 3',5'-cyclic monophosphoric acid and cycloheximide, augmented pregnenolone synthesis was observed. Unlike synthesis from the steroidogenic pool, pregnenolone formation from pre-steroidogenic pool in Y-1 cells indicates that this pool is somewhat smaller than the steroidogenic pool. The results support a cholesterol-trafficking model in which cycloheximide-sensitive transport from the pre-steroidogenic pool to the steroidogenic pool precedes metabolism, and is regulated by cAMP.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1972

Effects of brocresine (NSD-1055) and cycloheximide on amino acid decarboxylase activities in gastric mucosa of normal and vagally denervated rats.

1. Histidine decarboxylase activity of rat stomach fluctuates depending upon the functional state of the stomach. This varying enzyme activity poses special problems in assessing the effectiveness of enzyme inhibitors. After vagal denervation gastric histidine decarboxylase is markedly activated and remains at a high, stable level, which is unaffected by the functional state of the stomach. Thus it appears that vagally denervated rats are well suited for studies on histidine decarboxylase inhibitors.2. In vivo, brocresine (NSD-1055) was found to be a more effective inhibitor of gastric DOPA decarboxylase than of gastric histidine decarboxylase. With the fairly high dose given (200 mg/kg) the inhibition of histidine decarboxylase was at most 75-85% and quite short-lasting. The DOPA decarboxylase activity, which was not affected by vagal denervation, was inhibited more than 95% by brocresine; this inhibition was longer-lasting.3. Cycloheximide, which probably lowers gastric histidine decarboxylase activity by inhibiting enzyme synthesis, was maximally effective at a dose level as low as 1 mg/kg. Gastric DOPA decarboxylase was not inhibited by cycloheximide. Vagotomized rats and control rats responded similarly.4. Combined treatment of vagally denervated rats with brocresine and cycloheximide resulted in a rapid and persistent reduction of the histidine decarboxylase activity. It is concluded that the failure of brocresine alone to induce a lasting inhibition of histidine decarboxylase is due to continuous, rapid synthesis of new enzyme.5. The calculated half-life of gastric histidine decarboxylase was 75 min in vagally denervated rats and 45 min in normal fasted rats. The results suggest that the increased enzyme activity after vagal denervation is caused by an increased rate of enzyme synthesis.

Animals↗
PubMed1977

Effects of cycloheximide on experimental epilepsy induced by daily amygdaloid stimulation in rabbits.

Daily electrical stimulation of the amygdala caused a progressive change in electrographic afterdischarge and behavioral responses that ultimately resulted in generalized convulsions (kindling). Cycloheximide blocked the completion of kindling when administered 4 hours prior to daily electrical stimulations of the amygdala, but exerted no effect when administered shortly before the stimulation. The temporal relation between administration of cycloheximide and stimulation of the amygdala revealed that the blockade of kindling by cycloheximide was due to the suppression of focal afterdischarge rather than to the persistent inhibition of cerebral protein synthesis during the interstimulus periods.

Amygdala↗
PubMed1982

The action of puromycin and cycloheximide on the initiation of rapid axonal transport in amphibian dorsal root neurones.

1. Amphibian dorsal root ganglia-sciatic nerve preparations were incubated in vitro and the rapid axonal transport of radioactive labels was studied with a position-sensitive detector and by conventional liquid scintillation analysis. Protein was labelled by exposure of the ganglia to [(35)S]methionine or [(3)H]leucine and lipid was labelled using [(32)P]orthophosphoric acid.2. Protein synthesis was interrupted by exposure of the ganglia to either cycloheximide or puromycin. When ganglia were exposed to either inhibitor prior to or simultaneously with a label, the somal export of both protein and lipid to the axon was reduced by two to three orders of magnitude.3. Using the position-sensitive detector, [(35)S]methionine was observed to be exported from the ninth dorsal root ganglia of Rana catesbiana 3.49+/-1.56 h (+/- S.D.) after exposure, and [(32)P]phosphate 4.46+/-1.85 h after exposure.4. Export of [(35)S]methionine or [(32)P]phosphate was disrupted 3.32+/-1.21 h (+/- S.D.) or 1.93+/-1.04 h respectively after exposure of the ganglia to cycloheximide or puromycin.5. For a given preparation the time required for [(35)S]methionine to be exported was statistically equal to the time required for cycloheximide or puromycin to disrupt export. No such correlation was found to exist for the export of [(32)P]phosphate.6. Analysis revealed that materials labelled with either [(35)S]methionine or [(32)P]phosphate continue to be exported from the ganglia for several hours after the initial disruption in outflow caused by the inhibitors.7. The results do not provide support for the hypothesis of Ambron, Goldman & Schwartz (1975) that a ;key' newly synthesized, and non-storable, polypeptide is added to an already assembled structure to allow rapid axonal transport to be initiated.

Animals↗
PubMed1972

Memory traces: experimental separation by cycloheximide and electroconvulsive shock.

Mice given cycloheximide or saline were trained with a single trial. Electroconvulsive shock was administered to both groups at various times after training. Cycloheximide led to memory that decayed with time. Cycloheximide plus electroconvulsive shock produced complete amnesia at times when neither treatment alone produced amnesia. Only two types of processes appear to support memory storage in our study.

Amnesia↗
PubMed1973

Subapical wall synthesis and wall thickening induced by cycloheximide in hyphae of Aspergillus nidulans.

Hyphae of Aspergillus nidulans continued to synthesize all the major polysaccharide components of the cell wall when cycloheximide was added to cultures. In the presence of cycloheximide, hyphae did not elongate, but electron microscopy showed that the walls became thicker around the cell. The conclusion that cycloheximide changed wall synthesis from extension at the apex to subapical thickening was supported by grain distributions on radioautograms of mutant hyphae labeled with galactose. These findings are discussed in relation to the control of hyphal wall synthesis and are compared with the effects of protein synthesis inhibitors on wall formation in gram-positive bacteria.

Aspergillus↗
PubMed1979

Nuclear accumulation of influenza viral RNA transcripts and the effects of cycloheximide, actinomycin D, and alpha-amanitin.

The use of virus-specific (32)P-labeled complementary DNA and (125)I-labeled virion RNA as hybridization probes has allowed us to quantitate the number of molecules of complementary RNA (cRNA) and progeny virion RNA in MDCK cells infected with influenza virus. We compared the distribution of cRNA between the nucleus and the cytoplasm in cycloheximide-treated cells to that found in untreated cells, beginning 1 h after infection. A greater percentage of the total cRNA was detected in the nucleus of the drug-treated cells at all times investigated. For the first 2 h after infection about 50% of the cRNA synthesized in the cycloheximide-treated cells was found in the nucleus. These nuclear cRNA molecules were characterized and shown to be polyadenylated transcripts of each of the genome virion RNA segments. Viral cRNA synthesis was not completely inhibited by the addition of actinomycin D at the beginning of infection, with or without the concomitant addition of cycloheximide. A large fraction (about 90%) of these cRNA sequences were detected in the nucleus. Characterization of these nuclear cRNA molecules showed that they contained polyadenylic acid and represented transcripts of both those segments coding for proteins synthesized predominantly early after infection ("early" proteins) and those virion RNA segments coding for "late" proteins. Also, in vitro translation of these cRNA molecules showed that they were functional virus mRNA's. In contrast to actinomycin D, alpha-amanitin completely inhibited cRNA synthesis when added at the beginning of infection, and addition of this drug after 1.5 h had no effect on further cRNA synthesis.

Amanitins↗
PubMed1983

Cycloheximide induces expression of the human interferon beta 1 gene in mouse cells transformed by bovine papillomavirus-interferon beta 1 recombinants.

Mouse cells transformed by a bovine papillomavirus recombinant vector containing the human interferon (IFN) beta 1 (IFN-beta 1) gene could be induced to produce human as well as mouse IFNs. The optimal conditions for induction of human IFN and of its mRNA in these transformants resembled those needed for mouse IFN: high concentrations of DEAE-dextran and low concentrations of polyriboinosinic acid-polyribocytidylic acid. Superinduction by inhibitors of protein synthesis which strongly stimulate IFN-beta 1 induction in human cells had only a small effect on human IFN induction in bovine papillomavirus IFN-beta 1-transformed mouse cells. In contrast, cycloheximide without double-stranded RNA could induce significant levels of human IFN in the bovine papillomavirus IFN-beta 1 mouse transformants. After cycloheximide treatment, these cells contained IFN-beta 1 mRNA whose 5' ends originated in the authentic start site of the human IFN-beta 1 gene, as shown by S1 nuclease mapping. The transferred human gene, propagated extrachromosomally in the mouse cells, was, therefore, inducible under conditions different from those in human cells. The results also confirmed that the inhibitor of protein synthesis, cycloheximide, can induce expression of a human IFN gene.

Animals↗
PubMed1986

Coronavirus minus-strand RNA synthesis and effect of cycloheximide on coronavirus RNA synthesis.

The temporal sequence of coronavirus plus-strand and minus-strand RNA synthesis was determined in 17CL1 cells infected with the A59 strain of mouse hepatitis virus (MHV). MHV-induced fusion was prevented by keeping the pH of the medium below pH 6.8. This had no effect on the MHV replication cycle, but gave 5- to 10-fold-greater titers of infectious virus and delayed the detachment of cells from the monolayer which permitted viral RNA synthesis to be studied conveniently until at least 10 h postinfection. Seven species of poly(A)-containing viral RNAs were synthesized at early and late times after infection, in nonequal but constant ratios. MHV minus-strand RNA synthesis was first detected at about 3 h after infection and was found exclusively in the viral replicative intermediates and was not detected in 60S single-stranded form in infected cells. Early in the replication cycle, from 45 to 65% of the [3H]uridine pulse-labeled RF core of purified MHV replicative intermediates was in minus-strand RNA. The rate of minus-strand synthesis peaked at 5 to 6 h postinfection and then declined to about 20% of the maximum rate. The addition of cycloheximide before 3 h postinfection prevented viral RNA synthesis, whereas the addition of cycloheximide after viral RNA synthesis had begun resulted in the inhibition of viral RNA synthesis. The synthesis of both genome and subgenomic mRNAs and of viral minus strands required continued protein synthesis, and minus-strand RNA synthesis was three- to fourfold more sensitive to inhibition by cycloheximide than was plus-strand synthesis.

Animals↗
PubMed1972

Analysis of simian virus 40-induced transformation of hamster kidney tissue in vitro. 8. Induction of infectious simian virus 40 from virogenie transformed hamster cells by amino acid deprivation or cycloheximide treatment.

Clones of virogenic simian virus 40 (SV40)-transformed hamster kidney cells were exposed to medium deficient in the essential amino acids leucine, arginine, or methionine. Infectious virus was induced after deprivation periods of from 24 to 32 hr. The highest yields of infectious SV40 were obtained from cultures deprived for 3 to 4 days. Infectious virus was also induced in cells that were treated with the metabolic inhibitor cycloheximide. Pulse labeling experiments revealed that both protein synthesis and deoxyribonucleic acid (DNA) synthesis were inhibited by concentrations of cycloheximide which were effective for virus induction. It is suggested that inhibition of protein synthesis by either amino acid deprivation or by cycloheximide was responsible for the induction of infectious virus from virogenic cells. We postulate that the inhibition of protein synthesis caused a temporary inhibition of DNA synthesis which resulted in the induction of infectious virus.

Animals↗
PubMed1983

Characterization of a cycloheximide-resistant Tetrahymena thermophila mutant which also displays altered growth properties.

A cycloheximide-resistant strain of Tetrahymena thermophila, expressing a mutant chx-B gene (Ares and Bruns, Genetics 90:463-474, 1978), displayed very different temperature-dependent growth characteristics than either wild-type cells or another cycloheximide-resistant strain expressing a different mutant gene. Whereas wild-type cells showed an immediate decline in ribosome translocation rates when shifted from 30 to 38 or 40 degrees C, this mutant strain (X-8) showed no such decline. These results directly correlated with the growth rate differences we found for these cells at these temperatures. By genetic analysis, we showed that the phenotype of cycloheximide resistance cosegregated with the ability to grow rapidly at 40 degrees C. Analyses, both direct and indirect, suggested that a number of functional and structural characteristics of the ribosomes from strain X-8 cells are most likely conformationally different from those of wild-type ribosomes.

Animals↗
PubMedDate not supplied

Effect of cycloheximide and tunicamycin on the gonadotrophin-releasing hormone stimulated distal glycosylation of luteinizing hormone by rat pituitary cells.

We have previously demonstrated that gonadotrophin-releasing hormone (GnRH) induces not only changes in quantity but also in quality on secreted luteinizing hormone (LH), by increasing [14C]Leu (translation) and [3H]Gal (distal glycosylation) incorporation into newly synthesized hormone. In the present report, we have further examined the GnRH-induced [3H]Gal-LH synthesis and release by treating anterior pituitary cells with polypeptide synthesis and glycosylation inhibitors (cycloheximide and tunicamycin, respectively). Pituitary cells from ovariectomized adult rats were cultured for 4 days and then incubated for different periods (0-5 h) in medium containing [14C]Leu plus [3H]Man or [14C]Leu plus [3H]Gal in the absence (basal) or presence of 10 nmol/L GnRH with or without (control) cycloheximide (1.0 and 4.0 microg/mL) or tunicamycin (0.5 and 2.0 microg/mL). At the end of each incubation period, the cells and the medium were separated and processed for DNA uptake and newly synthesized LH (labeled LH, by immunoprecipitation with a-betaLH) determinations. The velocity of synthesis and release (between 0 and 2 h, and between 2 and 5 h) was calculated by regression analysis and the statistical significance of differences was determined by the slope test. GnRH enhanced the rates of synthesis and release of [14C]Leu-, [3H]Man-, and [3H]Gal-LH to 157 and 237; 164 and 190; and 272 and 508% of basal values, respectively. Cycloheximide totally blocked synthesis and release of [14C]Leu-LH and greatly reduced those of [3H]Man-LH, resulting in the loss of cellular responsiveness to GnRH. Addition of tunicamycin to the pituitary cells inhibited the rates of synthesis and release of [3H]Man-LH which had been induced by GnRH, without altering those of [14C]Leu-LH. These findings indicate that glycosylation is not a condition for GnRH-stimulated LH translation. The GnRH-increased [3H]Gal-LH rates of synthesis and release were affected to a lesser extent by the inhibitors. Thus, GnRH stimulation of distal glycosylation can occur, albeit at a reduced rate, even though protein synthesis and glycosylation are blocked. In conclusion, the present results corroborate that GnRH stimulates the addition of galactose residues into LH molecule. This effect is not simply the consequence of stimulating LH polypeptide chain synthesis. In addition, it is shown that GnRH-increased LH translation is independent of glycosylation.

Animals↗
PubMed1975

The effect of chloramphenicol and cycloheximide on meiotic zoosporogenesis and mitotic gametogenesis in Ulva mutabilis.

Ulva mutabilis cells induced to form zoospores or gametes were exposed to chloramphenicol or cycloheximide during the 48 hours preparatory period. Chloramphenicol inhibited protein and RNA accumulation to the same degree at all concentrations, and appeared to have a general toxic effect. Cycloheximide at low concentrations inhibited primarily protein accumulation, while at higher concentrations it had also an effect on the net synthesis of RNA. Most of the inhibited cells were found in interphase, but a small percent of vegetative mitoses were observed and may indicate a return to this type of division. The gametophyte appeared to be more sensitive to chloramphenicol than the sporophyte. The effect of cycloheximide on the two processes seems to be more or less the same. A transition period, after which the sporulation process could no longer be inhibited, was found about 10 hours before the cells entered meiotic prophase in zoosporogenesis.

Chloramphenicol↗
PubMed2006

Degradation of STAT5 proteins in 3T3-L1 adipocytes is induced by TNF-{alpha} and cycloheximide in a manner independent of STAT5A activation.

Tumor necrosis factor-alpha (TNF-alpha) is a multifunctional cytokine that has been implicated as a causative factor in obesity-linked insulin resistance. It is commonly accepted that macrophage-derived TNF-alpha acts in a paracrine manner on adjacent adipocytes to inhibit the expression of various adipocyte genes and to attenuate insulin signaling. Several studies have revealed that signal transducer and activator of transcription (STAT)5 proteins are modulated during adipogenesis and can modulate the transcription of some adipocyte genes. In this study, we demonstrate that TNF-alpha treatment, in the presence of cycloheximide, also results in the rapid turnover of STAT5A and STAT5B in a process that is independent of STAT5 activation by tyrosine phosphorylation. In addition, STAT5B is more labile than STAT5A under these conditions, suggesting that the COOH terminus of STAT5 may be involved in the turnover of each protein. Initial characterization of the TNF-alpha and cycloheximide-mediated degradation of STAT5 indicates that inhibition of the proteasome stabilizes both forms of STAT5 in the presence of TNF-alpha. In addition, the use of an NF-kappaB inhibitor results in the stabilization of STAT5A in the presence of TNF-alpha and cycloheximide, indicating that the degradation of STAT5 proteins under these conditions may involve the NF-kappaB pathway. STAT5 proteins are abundantly expressed in mature adipocytes and are normally extremely stable proteins under a wide range of conditions. However, our results demonstrate that the potentiation of TNF-alpha-mediated signaling in the presence of cyclohexmide is associated with a significant increase in the degradation of STAT5 proteins in 3T3-L1 adipocytes.

3T3-L1 Cells↗
PubMedDate not supplied

Effects of cycloheximide on chromatin-bound and -unbound PCNA in HeLa cells.

We examined the effects of cycloheximide on the amount of chromatin-bound and -unbound PCNA subpopulations in HeLa cells. The chromatin-bound PCNA content decreased rapidly in the presence of cycloheximide, whereas the reduction of the total amount of PCNA was minimal even 24 h after the treatment with cycloheximide. From these data, the half-life of PCNA was estimated to be 31.0 h for chromatin-bound PCNA and 276.8 h for the total.

Chromatin↗
PubMed1989

Cycloheximide increases endogenous retroviral RNA levels in murine liver and lung.

The expression of moloney murine leukemia virus (Mo-MuLV), intracisternal A particle (IAP), virus-like 30S sequence (VL30), early transposon (ET) endogenous retroviral sequences was analysed in the liver of untreated C3Hf, C57BL/6J and AKR mice, and in the lungs of untreated A/J, BALB/c, C3Hf and C57BL/6J mice. C3Hf mice are genetically susceptible to hepatocarcinogenesis, whereas the other strains are resistant. A/J and BALB/c mice are genetically susceptible to lung carcinogenesis, whereas the other strains are resistant. Both in liver and lung tissues we found differences between murine strains in the pattern of basal retrovirus expression. The effect of inhibition of protein synthesis on the levels of retroviral mRNAs was studied in the same tissues and strains 3 h following in vivo cycloheximide treatment. Cycloheximide treatment increased the liver and lung levels of virus Mo-MuLV, IAP, ET related transcripts in a strain dependent way, whereas VL30 mRNA levels increased in both tissues of all strains examined. These results suggested the existence of labile proteins that regulate the abundance of specific retroviral mRNAs in murine liver and lung in a strain specific fashion. No clear relationships between pattern of retrovirus expression and genetical susceptibility to hepatocarcinogenesis was found. The strains genetically resistant to lung carcinogenesis (C3Hf, C57BL/6J) showed higher lung basal levels and higher cycloheximide inducibility of mRNAs homologous to Mo-MuLV than the susceptible strains (A/J, BALB/c).

Animals↗
PubMed1980

Polyphosphoinositides:stimulator of mitochondrial cholesterol side chain cleavage and possible identification as an adrenocorticotropin-induced, cycloheximide-sensitive, cytosolic, steroidogenic factor.

ACTH treatment in vivo enhances the activity of adrenal cytosol for supporting pregnenolone synthesis in adrenal mitochondria. Upon fractionation of adrenal cytosol by gel filtration through Sephadex G200, pregnenolone synthesis-stimulating activity is eluted just after 3H2O(Kd = 1.1--2.1). This late eluting steroidogenic activity is enhanced by ACTH treatment and diminished by cycloheximide treatment, which also inhibits adrenal protein synthesis and ACTH-induced steroidogenesis. Chromatography on Sephadex G-10 yields even further separation of steroidogenic factor(s) from inactive substances; it appears that the active factor(s) adsorbs to Sephadex and that such adsorption is enhanced by a greater degree of cross-linking in the gel. The phospholipids, cardiolipin, diphosphoinositide, and triphosphoinositide, are recovered partly in adrenal cytosol and, by virtue of their polyphosphorylated head group, specifically stimulate mitochondrial pregnenolone synthesis. It seems likely that the ACTH effect on cytosolic steroidogenic activity is due to changes in polyphosphoinositides, since these phospholipids are specifically stimulated by ACTH, cycloheximide blocks the ACTH-induced increase in polyphosphoinositides, and these phospholipids comigrate with steroidogenic activity during gel filtration of adrenal cytosol. These findings along with other findings from our laboratory suggest that polyphosphoinositides may function as a cycloheximide-sensitive mediator in the steroidogenic action of ACTH.

Adrenal Glands↗
PubMed1981

Kinetic aspects of cycloheximide-induced reversal of adrenocorticotropin effects on steroidogenesis and phospholipid metabolism in rat adrenal sections in vitro.

We examined the rate of cycloheximide-induced reversal of ACTH effects on steroidogenesis and phospholipid metabolism in adrenal sections in vitro. In the absence of cycloheximide, ACTH treatment elicited sustained increases in steroidogenesis (approximately 3- to 5-fold) and adrenal concentrations (approximately 2-fold) of phosphatidic acid, phosphatidylinositol, and polyphosphoinositides. These increases in phospholipids were not dependent on steroidogenesis, since they were also apparent in aminoglutethimide-blocked adrenal sections. The addition of cycloheximide 30 min after ACTH treatment reversed the stimulatory effects of ACTH on steroidogenesis and phospholipids, and the rates of decay for both processes were identical, viz 28 min. This relatively slow turnover in adrenal sections in vitro contrasts with a much more rapid turnover observed previously in vivo and presently in dispersed adrenal cells in vitro; thus, it appears that an artefact of the in vitro adrenal section system stabilizes the putative labile protein. More importantly, the identical rates of decay for steroidogenesis and phospholipids suggest that the same labile protein is required for both phospholipid metabolism and steroidogenesis. These findings are in keeping with our postulate that the labile protein is required for phospholipid metabolism, which, in turn, is required for steroidogenesis.

Adrenal Glands↗