In vivo incorporation of [14C]tyrosine into the C-terminal position of the α subunit of tubulin

In vitro incorporation of [¹⁴C]tyrosine into the C-terminal position of the α subunit of tubulin was not affected by 4 mm cycloheximide. This inhibitor of protein synthesis was used for in vivo experiments. The in vivo incorporation of [¹⁴C]tyrosine into soluble brain protein of cycloheximide-treated rats was 10% of that of untreated rats. Treatment with vinblastine sulfate of the soluble brain protein showed that the incorporation of [¹⁴C]tyrosine into tubulin was higher in cycloheximide-treated than in untreated rats with respect to the incorporation into the total soluble protein. In the case of cycloheximide-treated rats, about 60% of the radioactivity incorporated into protein was released by the action of carboxypeptidase A, whereas 10% was liberated from the protein of untreated rats. The radioactive compound released by the action of carboxypeptidase A was identified as [¹⁴C]tyrosine. The α and β subunits of tubulin from animals that received [¹⁴C]tyrosine were separated by polyacrylamide gel electrophoresis. The radiosactivity ratio of $\text{α}\text{β}$ subunits of tubulin from cycloheximide-treated rats was threefold higher than that of untreated rats. When a mixture of [¹⁴C]amino acids was injected, the radioactivity ratio of $\text{α}\text{β}$ subunits of tubulin was similar for cycloheximide-treated and untreated rats. The results reported are consistent with the assumption that the α subunit of tubulin can be tyrosinated in vivo.     

Premeiotic DNA synthesis in fission yeast

Sporulating and various non-sporulating strains of S. pombe, especially several mutants deficient in conjugation or meiosis, were compared with respect to DNA synthesis under sporulation conditions. Meiosis and sporulation were induced by a transfer to nitrogen-free medium. As synchronized mitotic division was observed in all the strains as a first response to the shift, reducing the DNA amount per cell from the replicated state in G2 to the unreplicated state in the G1 phase of the cell cycle. Cells of the heterothallic wild-type strains ($\text{h}{}^{\mathrm{+}}\text{h}{}^{\mathrm{+}}$ or $\text{h}{}^{\mathrm{?}}\text{h}{}^{\mathrm{?}}$) accumulated in G1 with respect to DNA synthesis when they were incubated separately. In a mixed culture of these strains a period of enhanced DNA synthesis was observed after the start of zygote formation. This period of synthesis was absent in mutant fus1, where only prezygotes accumulated. Hence we conclude that in zygotic meiosis the premeiotic DNA synthesis is confined to zygotes after conjugation has been completed. In the diploid sporulating wild-type strain ($\text{h}{}^{\mathrm{+}}\text{h}{}^{\mathrm{?}}$), capable of azygotic meiosis without prior conjugation, premeiotic DNA synthesis occurred between $\text{2}\text{1}\text{2}$ and 5 h after the shift to the sporulation medium. There was no significant premeiotic DNA synthesis observed in diploid cells of the meiosis-deficient mutants mei1 or mei3, whereas premeiotic DNA synthesis proceeded normally in mutant mei4, which is blocked at a stage after commitment to meiosis in opposition to both the other mutants.     

The influence of mitochondria on ribosomes. Cycloheximide resistance of ribosomes from petite mutants of saccharomyces cerevisiae

The influence of cycloheximide on amino acid incorporation into protein of standard strain ? + and cytoplasmic mutant ? ? of $\text{S.}\text{cerevisiae}$ was determined $\text{in}\text{vivo}$ and $\text{in}\text{vitro}$. $\text{In}\text{vivo}$ cycloheximide at the concentration which inhibits protein synthesis in ? + strain by over 60% has litle or no effect in mutant ? ? strain. $\text{In}\text{vitro}$ cycloheximide in the range of 0.05 to 0.3 ug/ml of incubation medium inhibits polyphenylalanine synthesis in ? + strain by 50% and in ? ? strain by less than 10%. Similar resistance to this antibiotic are shown in standard strain grown in anaerobic conditions. It has been found that the resistance to cycloheximide is associated with changes in cytoplasmic ribosomes and may depend on the integrity of mitochondrial system.     

A Dual-Specificity Phosphatase Cdc25B Is an Unstable Protein and Triggers p34cdc2/Cyclin B Activation in Hamster BHK21 Cells Arrested with Hydroxyurea

By incubating at 30°C in the presence of an energy source, p34^cdc2/cyclin B was activated in the extract prepared from a temperature-sensitive mutant, tsBN2, which prematurely enters mitosis at 40°C, the nonpermissive temperature (Nishimoto, T., E. Eilen, and C. Basilico. 1978. Cell. 15:475–483), and wild-type cells of the hamster BHK21 cell line arrested in S phase, without protein synthesis. Such an in vitro activation of p34^cdc2/cyclin B, however, did not occur in the extract prepared from cells pretreated with protein synthesis inhibitor cycloheximide, although this extract still retained the ability to inhibit p34^cdc2/cyclin B activation. When tsBN2 cells arrested in S phase were incubated at 40°C in the presence of cycloheximide, Cdc25B, but not Cdc25A and C, among a family of dual-specificity phosphatases, Cdc25, was lost coincidentally with the lack of the activation of p34^cdc2/cyclin B. Consistently, the immunodepletion of Cdc25B from the extract inhibited the activation of p34^cdc2/cyclin B. Cdc25B was found to be unstable (half-life < 30 min). Cdc25B, but not Cdc25C, immunoprecipitated from the extract directly activated the p34^cdc2/cyclin B of cycloheximide-treated cells as well as that of nontreated cells, although Cdc25C immunoprecipitated from the extract of mitotic cells activated the p34^cdc2/cyclin B within the extract of cycloheximide-treated cells. Our data suggest that Cdc25B made an initial activation of p34^cdc2/cyclin B, which initiates mitosis through the activation of Cdc25C.     

Propagation of meiosis and other nuclear changes in multicellular complexes of Blepharisma

Meiosis and other nuclear changes in conjugation of Blepharisma japonicum regularly occur when cells of complementary mating types I and II unite (heterotypic union). But no nuclear changes occur if unions are induced between cells of the same mating type (homotypic union). Similarly, chains of homotypically united cells, induced by treating type II cells of a doublet strain (mutant with two attachment points) with gamone of mating type I, do not undergo nuclear changes. However, if a type I cell unites at one end of such a chain, the nuclear changes of conjugation occur not only in the doublet to which the type I cell unites but also in other doublets in the same chain. We examined the mode of propagation of nuclear activation by surgically separating all cells in the chain and observing the subsequent occurrence of nuclear changes in these isolated cells. The nuclear activation began at the site of heterotypic union and propagated from cell-to-cell without skipping. In chains of a given length, the propagation slowed down as it proceeded in the chain. If compared at the corresponding site of the chain, the propagation was slower in longer chains. We conclude that meiosis and other nuclear changes in conjugation are initiated by a substance originating at the site of heterotypic union and transferable to other cells through the united regions of the cells.     

Differential effects of cycloheximide on protein and RNA synthesis as a function of dose

The $\text{in}$$\text{vivo}$ dose response of rat liver protein and DNA synthesis to cycloheximide have been determined. Protein synthesis was quite sensitive to relatively low doses of cycloheximide being inhibited by more than 90% with 1.5 mg/kg. Maximal inhibition of 98% was achieved with 5 mg/kg. There was no inhibition of RNA synthesis with this dose of cycloheximide. Larger doses of cycloheximide did lead to quite marked inhibition of RNA synthesis without any change in the already maximally inhibited rate of protein synthesis. This differential effect of cycloheximide on protein and RNA synthesis as a function of dose indicates that the inhibition of RNA synthesis caused by the antibiotic is not a consequence of the inhibition of protein synthesis but related otherwise to the effects of large doses of cycloheximide.     

Mechanism of glycogenolytic action of cycloheximide in rat liver

Cycloheximide (0.1 to 0.2 m$\text{M}$) increases cAMP concentration 3 to 4-fold in isolated rat liver slices $\text{in vitro}$. This increase in cAMP concentration parallels an increase in phosphorylase activity. When cycloheximide, at a concentration used to inhibit protein synthesis (1 to 2 μg/g body weight), is administered to whole animals, phosphorylase is activated up to 13-fold by 6 hours. This leads to almost complete depletion of liver glycogen (from about 40 mg/g liver to 0.4 mg/g liver).     

Preferential synthesis of yeast mitochondrial DNA in alpha factor-arrested cells

α factor is a diffusible substance produced by $\text{S. cerevisiae}$ cells of the $\text{α}$ mating type which inhibits cell division (1) and the initiation of nuclear DNA synthesis (2) in cells of the $\text{a}$ mating type. In this report, it is shown that mitochondrial DNA synthesis continues at a normal rate in $\text{a}$ cells for at least 6 hours in the presence of α factor, resulting in a 5-fold increase in the amount of mitochondrial DNA per cell. The continued synthesis of mitochondrial DNA in the absence of nuclear DNA synthesis allows specific labeling of yeast mitochondrial DNA.     

Inefficiency of the stringent response in the fungus Mucor

Removal of a required amino acid from the growth medium or addition of cycloheximide caused an immediate stoppage of growth and protein synthesis in the fungus $\text{Mucor}\text{racemosus}$. However, RNA synthesis persisted for several hours at rates that only gradually decreased under the same circumstances. An analysis of the major classes of RNA synthesized during the first hour of treatment showed that cycloheximide preferentially inhibited rRNA synthesis, whereas amino acid starvation slowed synthesis of all RNA species uniformly. Neither treatment affected the percentage of mRNA synthesized. The partial and delayed effects of amino acid starvation and cycloheximide treatment on RNA synthesis reported here suggest the absence of or the gross inefficiency of a classical stringent response in $\text{M.}\text{racemosus}$.     

Androgen-induced gene activation in the rat prostate

The effect of androgens on gene activation in the rat prostate has been investigated by examining precursor incorporation into RNA, by DNA-RNA hybridization of RNA transcribed $\text{in}\text{vitro}$ from prostate chromatin, and by thermal denaturation of prostatic chromatin. The results show a selective synthesis of nuclear RNA and a changed thermal melting profile of prostatic chromatin as a result of testosterone administration. Further, the $\text{in}\text{vitro}$ synthesized RNA transcribed from prostatic chromatin of androgen-treated rats contained new RNA species that were not transcribed from chromatin of untreated castrated controls. The data provide direct evidence for an activated state of the prostatic chromatin stimulated by androgens.     

Energy metabolism in developing Ascaris lumbricoides eggs. II, The steady state content of intermediary metabolites.

The steady state levels of intermediary metabolites were measured in freeze clamped, developing, dormant, and activated infective Ascaris lumbricoides eggs. The $\text{[ATP]}\text{[ADP]}$ ratio is low in the developmental stages and rises sharply in the dormant egg; on activation of the dormant egg the $\text{[ATP]}\text{[ADP]}$ ratio falls. The levels of the phosphorylated glycolytic intermediates of acetyl-CoA and of isocitrate do not change markedly during development, but the levels of lactate, citrate, 2-oxoglutarate, glutamate, succinate, and malate all show significant changes in the developing, dormant, and activated egg. The dormant egg also appears to be characterized by a low cytoplasmic redox potential.     

Induction and inhibition of meiotic maturation of follicle-enclosed porcine oocytes in vitro

Isolated porcine Graafian follicles which were explanted in vitro and maintained in organ culture were used as a test-system for the meiosis-inducing action of PMSG and hCG. The addition of either PMSG or hCG alone (10 or 20 IU/ml, respectively) to the culture medium was not effective, whereas the simultaneous administration of these hormones ($\text{15}\text{15}\text{IU/ml}$) induced resumption of meiosis in 90.3% ($\text{37}\text{41}$). The same hormone concentrations were used in a second series of experiments in which the inhibition and induction of meiosis of isolated oocytes were tested by transferring them into host follicles. In host follicles containing up to 12 foreign eggs, which were cultured in control media, meiosis was prevented in 86.0% of all oocytes ($\text{104}\text{121}$). By adding PMSG (15 IU/ml) simultaneously with hCG (15 IU/ml) to the medium, meiosis was induced in 95.0% of all oocytes ($\text{133}\text{140}$).The assumption is made that the signal initiating resumption of meiosis of the isolated and transferred oocytes is mediated by the follicular fluid, since intimate contact with the membrana granulosa of the host follicle was prevented by using a roller technique.     

The sites of synthesis and the subsequent migration of newly synthesized protein in retina

Radioautographs of rabbit retinas fixed immediately after a 1 or 2 min exposure in vitro to ³H leucine revealed high rates of protein synthesis in receptor cell inner segments, perikarya of ganglion cells, and cells of the inner nuclear layer. If these brieflly labelled retinas were returned to unlabelled medium for periods of up to 6 hr, the radioautographs revealed a progressive dispersion of the labelled proteins from their sites of synthesis. This was largely completed by $\text{1}\text{1}\text{2}$ hr and appeared, in one instance at least, to involve processes other than simple diffusion. Superimposed on the dispersive phenomenon was a process of concentration of the newly formed proteins at two sites quite distant from their synthesis, that was apparent after $\text{1}\text{1}\text{2}$hr. One of these sites was the receptor cell outer segments, as has been previously described, the other was the outer plexiform layer.     

Derepression of the high-affinity phosphate uptake in the yeast Saccharomyces cerevisiae

Phosphate starvation derepresses a high-affinity phosphate uptake system in Saccharomyces cerevisiae strain A294, while in the same time the low-affinity phosphate uptake system disappears. The protein synthesis inhibitor cycloheximide prevents the derepression, but has no effect as soon as the high-affinity system is fully derepressed. Two other protein synthesis inhibitors, lomofungin and 8-hydroxyquinoline, were found to interfere also with the low-affinity system and with Rb⁺ uptake. After incubation of the yeast cells in the presence of phosphate the high-affinity system is not derepressed, but the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ of the low-affinity system has decreased for about 35%. Phosphate supplement after derepression causes the high-affinity system to disappear to a certain extent while in the meantime the low-affinity system reappears. The results are compared with those found in the yeast Candida tropicalis for phosphate uptake.     

Presence of peptide hormones that control gonadotropin secretion in extrahypothalamic areas of the brain.

The hypocholesterolemic drug clofibrate (ethyl-α-$\text{p}$-chlorophenoxyisobutyrate) was found to strongly suppress 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34) activity in cultured mouse L cells at concentrations of 20 – 50 μg/ml. The half-life ($\text{t1}\text{2}$) of the reductase (approximately 120 min) was strongly reduced when L cells were incubated with cycloheximide plus a maximal inhibitory concentration of clofibrate (50 μg/ml), resulting in a $\text{t1}\text{2}$ value of 10 min. Preliminary kinetic analysis of the inhibition suggested that clofibrate increased the rate of inactivation (or degradation) of the reductase without affecting the rate of enzyme synthesis.     

Rapid inhibition by cycloheximide of rat hepatic nuclear free and engaged poly(A) polymerase activities

This paper reports the effect of cycloheximide on the activity of rat hepatic nuclear poly(A) polymerase. Three hours after cycloheximide treatment (3 mg/100 g body weight), total rat hepatic nuclear poly (A) polymerase activity was decreased to 50% of the normal level. This conclusion was reached when the enzyme activity was measured either in the whole nuclei $\text{in vitro}$ or with partly purified enzyme preparations. When examined at different times after a single injection of cycloheximide, it was observed that poly(A) polymerase activity decayed biphasically with an initial rapid decay phase reaching a minimum at one hour $\text{(}\text{t}\text{1}\text{2}\text{= 0.8}\text{hrs}\text{)}$, followed by a stable phase thereafter. These results have been interpreted to mean that poly(A) polymerase consists of either a mixture of two structurally distinct populations of enzymes with a different turnover rate, or of a single type of enzyme with a protein factor which is rapidly turning over and which is required for maximal enzyme activity.     

Macrophages as effector cells of protective immunity in murine schistosomiasis: IV. Coincident induction of macrophage activation for extracellular killing of schistosomula and tumor cells

Peritoneal macrophages from Schistosoma mansoni-infected mice are activated both for nonspecific tumor cytotoxicity and for killing of skin-stage schistosomula in vitro. In the current study, mechanisms for induction of macrophage tumoricidal and schistosomulacidal activity have been compared. Examination of macrophages activated in vivo by BCG infection or C. parvum treatment, or in vitro by exposure to lymphokine prepared from antigen-stimulated BCG-immune spleen cells, showed that these effector functions were closely linked. Indeed, fractionation of lymphokine-rich supernatant fluids by Sephadex G-100 gel filtraction showed that activities responsible for induction of schistomula killing by inflammatory macrophages and for induction of tumoricidal activity cochromatographed as a single peak in the 50,000 MW region. Thus, development of macrophage-mediated cytotoxicity against these two extracellular (tumor cell or helminth) targets was coincident in several cell populations activated in vivo or in vitro. However, activation for tumoricidal and schistosomulacidal capacity appeared to be quantitatively dissociated in macrophages from mice with chronic schistosomiasis; those cells demonstrated low, yet significant, levels of larval killing ($\text{1}\text{3}$ to $\text{1}\text{5}$ those of BCG or lymphokine-activated cells) but maximal levels of tumor cell cytotoxicity. Furthermore, cytotoxicity by peritoneal cells from S. mansoni-infected mice was not increased in vitro by exposure to lymphokine. Identification of this functional alteration in S. mansoni-activated cells may help to clarify the role of macrophages in the partial immunity against challenge infection which is demonstrated by mice with chronic primary S. mansoni infection.     

Cycloheximide inhibition of insulin control of liver glycogen synthase b into a conversion.

Cycloheximide given to insulin-treated alloxan diabetic rats results in the inhibition of insulin-induced liver glycogen synthase $\text{b}\text{into}\text{a}$ conversion without affecting the level of synthase $\text{b}$. The effect of cycloheximide, believed to elevate cAMP in liver of normal rats, is independent of cAMP levels of the insulin-treated diabetic rat. The inhibition of insulin-mediated synthase $\text{b}$ to $\text{a}$ conversion by cycloheximide does not appear to be the result of a cycloheximide-induced cAMP-dependent phosphorylation of synthase $\text{a}$ to $\text{b}$ and suggests that insulin control of synthase $\text{b}$ and $\text{a}$ interconversions is dependent upon cycloheximide-sensitive protein synthesis.     

Regulation of guanylate cyclase activity during cytodifferentiation of Blastocladiella emersonii.

Levels of guanylate cyclase activity in extracts of the unicellular eukaryote $\text{Blastocladiella}$$\text{emersonii}$ differed by at least 100-fold at different stages of the cell cycle, paralleling changes in the cyclic GMP content of this organism (Proc. Natl. Acad. Sci. U.S.A. $\text{72}$, 442 (1975)). Extracts of vegetative cells lacked appreciable guanylate cyclase activity, whereas the specific activity of the enzyme in zoospore extracts was 2 nmol cyclic GMP synthesized/min/mg protein at 35°. Guanylate cyclase activity increased at least 50-fold during the period of zoospore formation when cyclic GMP begins to accumulate $\text{in}$$\text{vivo}$. Since actinomycin D or cycloheximide added at the beginning of this period blocked any increase in enzyme activity, it appears that $\text{de}$$\text{novo}$ synthesis of guanylate cyclase during sporulation is responsible for the accumulation of cyclic GMP that occurs at that time.