Cycloheximide Is Not a Specific Inhibitor of Protein Synthesis in Vivo

ABSTRACT Pigmentation of the scrotum of the black-pelted rat, as expressed through melanocyte melanogenic activity, is controlled by androgens. Castration decreased in vitro incorporation of [14C] tyrosine into melanin. Testosterone pre-treatment for 4 days increased melanin radioactivity over castrate controls; the increment in vitro was prevented by an inhibitor of protein synthesis (cycloheximide) added to the incubation. However, cycloheximide only partially blocked melanin synthesis when added to tissue from animals hormone treated for 6 days in vivo, and was ineffective in tissue from intacts. Bulk protein synthesis in vitro (incorporation of [14C] tyrosine or -leucine) was not affected by castration or testosterone treatment but was uniformly inhibited by cycloheximide. The data suggest that new synthesis of specific protein in vitro was necessary for initial hormone-stimulation of melanogenesis, but with longer exposure to hormone sufficient protein was pre-synthetized in vivo to permit melanogenesis during incubation with the inhibitor. Radioautographs of epidermis incubated with [14C] tyrosine showed grains concentrated over macromolecular aggregates in melanocytes, a pattern not altered by cycloheximide. Though available for incorporation into general tissue protein. [14C] tyrosine was apparently incorporated preferentially into melanin by melanocytes. DOPA (3,4-dihydroxyphenylalanine) added to incubations in cofactor amounts did not affect decreased melanin synthesis after castration and appears, therefore, not to be rate limiting in that decrease. Tissue uptake of free [14C] tyrosine or — leucine during incubation was lower than normal in castrate epidermis; uptake was elevated by testosterone treatment. Concentrations appeared sufficient in all preparations to suggest that availability is not rate limiting for synthesis of melanin or protein; however, a possible influence on amino acid permeability for melanocytes remains undetermined. Tyrosinase activity was present in both particulate and cytosol fractions of epidermis but decreased significantly after castration only in the cytosol. Testosterone increased particulate activity after 4 days and soluble activity after 9 days of treatment. These and findings above are consistent with a model that tyrosinase is synthesized and incorporated into melanosome structure within 4 days testosterone treatment; with longer treatment synthesis may then exceed that required for melanosome assembly and tyrosinase appears in the soluble milieu.

Download Full-text

The use of an inhibitor of protein synthesis to investigate the roles of ecdysteroids and sex-determination genes on the expression of the genes encoding the Drosophila yolk proteins

Development ◽

10.1242/dev.101.4.931 ◽

1987 ◽

Vol 101 (4) ◽

pp. 931-941

Author(s):

M. Bownes ◽

A. Scott ◽

M. Blair

Keyword(s):

Protein Synthesis ◽

Sex Determination ◽

Fat Body ◽

Yolk Protein ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Yolk Protein Genes ◽

Fat Bodies ◽

Inhibitor Of Protein Synthesis

The three yolk-protein genes of Drosophila are normally expressed only in adult female fat bodies and ovaries. 20-hydroxyecdysone can affect the transcription of these genes in males and females, as can mutations in the sex-determining genes tra, tra-2, ix and dsx. We have asked a number of basic questions about how these genes are regulated, using an inhibitor of protein synthesis (cycloheximide), labelling RNA in vivo, a temperature-sensitive sex-determination mutant (tra-2ts1), and 20-hydroxyecdysone. We have found that the yolk-protein genes are continuously transcribed in the fat bodies of adult females and that maintenance of this transcription requires protein synthesis. Hormone induction in males is also inhibited by cycloheximide, suggesting that the products of other genes are essential both for 20-hydroxyecdysone to be able to switch on the genes, and for their continuous transcription in the female fat body. The products of the tra-2 gene are also required for continuous transcription of the yolk-protein genes, suggesting that the pathway inhibited by the cycloheximide is that of the sex-determination hierarchy. 20-hydroxyecdysone can override the sex-determination system and induce yolk protein synthesis in normal males and tra-2ts reared and maintained at the restrictive temperature.

Download Full-text

Gougerotin, a specific inhibitor of protein synthesis

Biochimica et Biophysica Acta (BBA) - Specialized Section on Nucleic Acids and Related Subjects ◽

10.1016/0926-6550(63)90092-6 ◽

1963 ◽

Vol 76 ◽

pp. 636-638 ◽

Cited By ~ 19

Author(s):

J.M. Clark ◽

Jay K. Gunther

Keyword(s):

Protein Synthesis ◽

Specific Inhibitor ◽

Inhibitor Of Protein Synthesis

Download Full-text

Respiratory energy requirements and rate of protein turnover in vivo determined by the use of an inhibitor of protein synthesis and a probe to assess its effect

Physiologia Plantarum ◽

10.1111/j.1399-3054.1994.tb03027.x ◽

1994 ◽

Vol 92 (4) ◽

pp. 585-594 ◽

Cited By ~ 63

Author(s):

T. J. Bouma ◽

R. Visser ◽

J.H.J.A. Janssen ◽

M.J. Kock ◽

P.H. Leeuwen ◽

...

Keyword(s):

Protein Synthesis ◽

Protein Turnover ◽

Energy Requirements ◽

Inhibitor Of Protein Synthesis

Download Full-text

Regulation of oxytocin receptor concentration in rat uterine explants by estrogen and progesterone

Canadian Journal of Biochemistry and Cell Biology ◽

10.1139/o83-078 ◽

1983 ◽

Vol 61 (7) ◽

pp. 625-630 ◽

Cited By ~ 64

Author(s):

Melvyn S. Soloff ◽

Mats Axel Fernstrom ◽

Sumudra Periyasamy ◽

Solweig Soloff ◽

Sam Baldwin ◽

...

Keyword(s):

Protein Synthesis ◽

Oxytocin Receptor ◽

Fivefold Increase ◽

Oxytocin Receptors ◽

Immature Rats ◽

Receptor Concentration ◽

17Β Estradiol ◽

Inhibitor Of Protein Synthesis

Incubation of uterine explants from immature rats with 0.01–100 ng of 17β-estradiol/mL resulted in approximately a fivefold increase in the number of oxytocin receptors per milligram of protein in 48 h. This increase was maintained for at least an additional 48 h in the presence of estrogen. When the explants were incubated with 1 μg progesterone/mL from the outset, the concentration of oxytocin receptors was the same as initial (0 time) levels. The estrogen-induced increase in oxytocin receptor concentration was blocked by incubation with cycloheximide, an inhibitor of protein synthesis. Once increased, however, the concentration of oxytocin receptors exhibited no turnover for at least a 48-h period in the presence of estrogen. The addition of progesterone and estrogen to explants with elevated receptor levels resulted in almost a 60% reduction in oxytocin receptor concentration by 24 h, with no change in affinity of the receptor for oxytocin. The reduction in receptor concentration by progesterone was not prevented by cycloheximide. The progesterone effect may involve inactivation or degradation of oxytocin receptors or activation of substances that are inhibitory to oxytocin binding. The effects of estradiol and progesterone on oxytocin receptor concentration in uterine explants are similar to those seen when the steroids are administered in vivo. The explant system, therefore, should prove useful in clarifying factors and processes that are involved in regulation of oxytocin receptor concentration in the uterus and in the initiation of parturition in the rat.

Download Full-text

Erythroid Krüppel-Like Factor Directly Activates the Basic Krüppel-Like Factor Gene in Erythroid Cells

Molecular and Cellular Biology ◽

10.1128/mcb.01658-06 ◽

2007 ◽

Vol 27 (7) ◽

pp. 2777-2790 ◽

Cited By ~ 67

Author(s):

Alister P. W. Funnell ◽

Christopher A. Maloney ◽

Lucinda J. Thompson ◽

Janelle Keys ◽

Michael Tallack ◽

...

Keyword(s):

Protein Synthesis ◽

Chromatin Immunoprecipitation ◽

Erythroid Cells ◽

Factor Gene ◽

Upstream Promoter ◽

Murine Embryos ◽

Inhibitor Of Protein Synthesis ◽

Kinetics Of ◽

In Erythroid Cells

ABSTRACT The Sp/Krüppel-like factor (Sp/Klf) family is comprised of around 25 zinc finger transcription factors that recognize CACCC boxes and GC-rich elements. We have investigated basic Krüppel-like factor (Bklf/Klf3) and show that in erythroid tissues its expression is highly dependent on another family member, erythroid Krüppel-like factor (Eklf/Klf1). We observe that Bklf mRNA is significantly reduced in erythroid tissues from Eklf-null murine embryos. We find that Bklf is driven primarily by two promoters, a ubiquitously active GC-rich upstream promoter, 1a, and an erythroid downstream promoter, 1b. Transcripts from the two promoters encode identical proteins. Interestingly, both the ubiquitous and the erythroid promoter are dependent on Eklf in erythroid cells. Eklf also activates both promoters in transient assays. Experiments utilizing an inducible form of Eklf demonstrate activation of the endogenous Bklf gene in the presence of an inhibitor of protein synthesis. The kinetics of activation are also consistent with Bklf being a direct Eklf target. Chromatin immunoprecipitation assays confirm that Eklf associates with both Bklf promoters. Eklf is typically an activator of transcription, whereas Bklf is noted as a repressor. Our results support the hypothesis that feedback cross-regulation occurs within the Sp/Klf family in vivo.

Download Full-text