Effect of urethan on polyamine and DNA synthesis in the regenerating rat liver

When a single dose of urethan was injected into the peritoneal cavity of rats immediately after partial hepatectomy, DNA synthesis was delayed by 12 h. The induction of ornithine decarboxylase which was induced biphasically following partial hepatectomy was also reduced and delayed by 14–15 h by the administration of urethan. S-Adenosylmethionine decarboxylase activity in urethan-treated rat liver at 20 h and 29 h after operation was significantly lower than that of untreated animals. This enzyme activity was shown to increase thereafter, reaching a higher level than in untreated rats at 37–42 h. Hepatic spermidine content changed biphasically in a manner similar to DNA synthesis. These results suggest that the activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase may correlate with DNA synthesis and that an increase of spermidine concentration is necessary to DNA synthesis.     

Synchrony between DNA synthesis and thymidine incorporation into DNA in regenerating rat liver

DNA synthesis in regenerating liver was studied to determine whether the onset of stimulated DNA synthesis preceded the onset of increased incorporation of thymidine into DNA. Thymidine incorporation into hepatic DNA was not stimulated 15 h after operation, but was stimulated after 18 h; peak stimulation occurred 30 h after operation. Thymidine kinase activity was stimulated 24 h after operation; highest kinase activity was observed at 36 h. The onset of stimulated DNA synthesis was estimated by following the incorporation of labeled aspartic acid, sodium formate, adenine or orotic acid into appropriate DNA bases, viz., thymine, adenine, adenine or cytosine, respectively. Incorporation of adenine and orotic acid was stimulated between 15 h and 18 h after operation; incorporation of aspartic acid and sodium formate was stimulated between 18 h and 21 h after operation.The incorporation of thymidine into DNA was accelerated by stress stimulus and was inhibited by hydrocortisone. Changes in thymidine kinase activity also were correspondingly accelerated or delayed. Incorporation of labeled thymidine, adenine, formate, orotic acid or thymine into appropriate DNA bases, viz., thymine, adenine, adenine, cytosine or thymine, respectively, was stimulated by stress stimulus or was inhibited by hydrocortisone.It was concluded from these data that stimulation of DNA synthesis and of thymidine incorporation into DNA was essentially synchronized in regenerating rat liver. Results from this study were compared with results from similar studies in 2 other tissues, and the limitations, attendant with using thymidine incorporation into DNA as an indicator of stimulated DNA synthesis, were discussed.     

Induction of fumarase in resting Euglena

Exposure of dark-grown resting (carbon deficient) Euglena to light, ethanol or malate produced a transient increase in the specific activity of fumarase. Fumarase levels decreased 8–12 h after the start of induction and this decrease could not be prevented by additional inducer. During the period of fumarase accumulation, cycloheximide prevented further fumarase synthesis and enzyme levels decreased at a rate comparable to the rate of decline normally observed 8–12 h after the start of induction. Although the addition of ethanol to ethanol-induced cultures or malate to malate-induced cultures 12 or 24 h after the initial induction failed to maintain or induce additional fumarase synthesis, the addition of organic carbon to photoinduced cells 8 or 24 h after light exposure induced additional enzyme synthesis. Additional enzyme synthesis was not induced when ethanol- or malate-induced cells were exposed to light 12 or 24 h after organic carbon addition. Light exposure or ethanol addition failed to induce fumarase synthesis during balanced growth indicating that fumarse inducibility is a property of resting cells.     

The role of 20-hydroxyecdysone in housefly sex pheromone biosynthesis

Houseflies ovariectomized within 12 h after emergence do not produce (Z)-9-tricosene nor demonstrate the shift from alkene to alkane synthesis that is typcal of flies with developing ovaries. A single injection of 20-hydroxyecdysone at doses of 0.1 to 10 μg will induce the pattern in ovariectomized insects that is characteristic of flies with ovaries. Furthermore, this pattern persists for 3 days, but by 6 days after hormone injection, the synthesis of (Z)-9-tricosene stops and more alkenes are produced than alkanes. A post-hormone treatment time of 16 h was required before detectable amounts of (Z)-9-tricosene appeared on ovariectomized flies. Multiple injections of 20-hydroxyecdysone at doses of 50 ng into ovariectomized flies induced (Z)-9-tricosene synthesis and a shift in alkene to alkane synthesis. Thus, 20-hydroxyecdysone was able to act as an ovarian substitute in ovariectomized flies by stimulating pheromone synthesis.     

DNA repair and replication in human fibroblasts treated with (±)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene

DNA repair and replication were examined in diploid human fibroblasts after treatment with (±)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-I). Unscheduled DNA synthesis exhibited a linear response to BPDE-I concentrations up to 1.5 μM and a saturation plateau after higher concentrations. Maximal unscheduled DNA synthesis was observed in the first hour after treatment with synthesis diminishing progressively thereafter. Half-maximal unscheduled DNA synthesis was seen within 4–6 h after treatment with 0.7 μM BPDE-I. DNA replication was inhibited by BPDE-I in a dose- and time-dependent fashion. The mechanisms of this inhibition were characterized by velocity sedimentation of pulse-labeled nascent DNA in alkaline sucrose gradients. Very low concentrations of BPDE-I (0.03 and 0.07 μM) were found to inhibit replicon initiation by up to 50% within 30–60 min after treatment. Recovery of initiation following these low concentrations was evident within 3 h after treatment. Higher concentrations of carcinogen inhibited DNA synthesis in active replicons. This effect was manifested by a reduction in incorporation of precursor into replication intermediates of greater than 1·10⁷ Da with the concurrent production of abnormally small nascent DNA. When viewed 45 min after treatment with 0.17 μM BPDE-I the combination of these two effects partially masked the inhibition of replicon initiation. However, even after treatment with 0.33 μM BPDE-I an effect on initiation was evident. These results reveal a pattern of response to BPDE-I that is quite similar to that produced by 254 nm radiation.     

The synthetic pathway of trypsin in the mosquito Aedes aegypti L. (Diptera: Culicidae) and in vitro stimulation in isolated midguts

Mosquito trypsin is synthesized in vivo and in vitro in two groups of forms with differing molecular sizes: one group of 32–36 KD forms is noted immediately after the blood meal, followed by the principal forms with Mr around 30 KD about 10 h later; synthesis is terminated at about 24 h after blood meal. Similar results were obtained after in vitro translation of mRNA. Trypsin precursor and trypsin mRNA were not detected by our assay of midguts of unfed females. Trypsin synthesis is induced in a dose-dependent manner by injection of either blood or sugar solutions into isolated midguts. It is concluded that the stimulus for initial trypsin synthesis is mechanical and/or osmotic stress acting independently of the nervous system. Processing of trypsin in the midgut cells involves cleavage of putative signal peptides: in vitro translation in the presence of microsomes led to a constant shift in molecular weights of 1–2 KD prior to secretion. Exposure of washed midgut epithelia from different stages to native blood in vitro inhibited final processing of the intracellular trypsin to the extracellular forms while stimulation of protein synthesis was observed. Consequently the role of the peritrophic membrane in compartmentalization of the digestive process is further emphasized.     

The effects of nutrition and methoprene treatment on ovarian ecdysteroid synthesis in Drosophila melanogaster

Radioimmunoassay of in vitro culture medium from ovaries of Drosophila melanogaster indicates that detectable ovarian ecdysteroid synthesis begins between 6 and 12 h after eclosion and reaches a peak between 24 and 30 h, when animals are reared at 25°C, 12 h photophase. Analysis of 24 and 72 h medium by a combination of high-performance liquid chromatography and radioimmunoassay demonstrates three ecdysteroid regions, two comigrating with known standards of ecdysone and 20-hydroxyecdysone and a third highly polar region containing one or more unidentified radioimmunoassay-active ecdysteroids. In 72 h medium the polar region comprises the majority of radioimmunoassay-active material while in 24 h medium the majority is in the ecdysone region. Provision of a nutritionally deficient diet to females at adult eclosion prevents the normal increase in vitellogenic-stage follicles and ovarian ecdysteroid synthesis. Methoprene treatment of such females stimulates a transient burst of ovarian ecdysteroid synthesis and the production of near normal numbers of vitellogenic oöcytes by 24 h, although by 48 h the number of vitellogenic oöcytes is less than normal.     

Induction of 6-thioguanine resistance in synchronized human fibroblast cells treated with methyl methanesulfonate, N-acetoxy-2-acetylaminofluorene and N-ethyl-N′-nitro-N-nitrosoguanidine

Chemical induction of 6-thioguanine resistance was studied in synchronized human fibroblast cells. Cells initially grown in a medium lacking arginine and glutamine for 24 h ceased DNA synthesis and failed to enter the S phase. After introduction of complete medium, the cells progressed to the S phase after 16 h. DNA synthesis peaked 20 h after removal of nutrient stress and declined.Mutations were induced in S-phase cells by methyl methanesulfonate (MMS), N-acetoxy-2-acetylaminofluorene (NA-AAF) and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). Chemical treatments resulted in an increase in the absolute number of mutant colonies and in a dose-dependent mutation frequency. In this report, we show that NA-AAF evokes a temporal pattern of mutation in synchronized cells, with such mutations being induced only during the S phase. Evidence indicates that presence of S-phase cells in the treated cultures is a prerequisite for the induction of mutations.     

Regulation of the synthesis of lutropin-induced protein in rat testis Leydig cells

The mechanism of action of lutropin on the stimulation of the synthesis of a specific lutropin-induced protein in rat testis Leydig cells was investigated. Lutropin-induced protein has a mol.wt. of approx. 21000 and is detected by labelling the Leydig-cell proteins with [³⁵S]methionine, followed by separation by polyacrylamide-gel electrophoresis and radioautography of the dried gel. The incorporation of ³⁵S into lutropin-induced protein was used as an estimate for the synthesis of the protein. Incubation of Leydig cells with dibutyryl cyclic AMP or cholera toxin also resulted in the stimulation of synthesis of the protein. Synthesis of lutropin-induced protein, when maximally stimulated with 100ng of lutropin/ml, could not be stimulated further by addition of dibutyryl cyclic AMP. Addition of 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor, further increased synthesis of the protein in the presence of a submaximal dose of lutropin (10ng/ml) but not in the absence of lutropin or with maximal amounts of lutropin (100 and 1000ng/ml). Actinomycin D prevented the effect of lutropin on the stimulation of lutropin-induced protein synthesis when added immediately or 1h after the start of the incubation, but not when added after 5–6h. This is interpreted as reflecting that, after induction of mRNA coding for lutropin-induced protein, lutropin had no influence on the synthesis of the protein in the presence of actinomycin D. Synthesis of the protein was also stimulated in vivo by injection of choriogonadotropin into rats 1 day after hypophysectomy, and the time course of this stimulation of lutropin-induced protein synthesis in vivo was similar to that obtained by incubating Leydig cells in vitro with lutropin. From these results it is concluded that stimulation of lutropin-induced protein synthesis by lutropin is most probably mediated by cyclic AMP and involves synthesis of mRNA.     

Characterization of polyribosomes containing newly synthesized messenger RNA in preimplantation rabbit embryos

Conjugating cells of Stylonychia mytilus incorporate RNA precursors into RNA between 5–6 h after pairing. If RNA synthesis is inhibited during this period by Actinomycin D, the developmental processes are arrested immediately. After 6 h of pairing there is no detectable synthesis of RNA while protein synthesis continues for at least the next 33 h and during this time the cellular events leading to the macronuclear development are insensitive towards the action of actinomycin D. Treatment of live conjugating pairs (older than 6 h) with RNase solution digests the RNA of these cells and causes an immediate and irreversible arrest in development. On the other hand, a similar treatment of the vegetative cells, which show RNA synthesis, does not produce any irreversible effect. It is concluded that stable mRNA molecules are synthesized by the conjugants between 5–6 h following the onset of pairing.     

Nature of Ribonucleic Acid Synthesis During Early Sporulation in Saccharomyces cerevisiae

Phosphate uptake in sporulating cultures of Saccharomyces cerevisiae has been found to occur approximately 2 h after the transfer to sporulation medium. Early ribonucleic acid synthesis begins at approximately 4 h and continues to 8 h. Incorporation of phosphate into acid-extractable precursor pools parallels phosphate uptake. In triple-labeling experiments it was observed that the breakdown of vegetatively synthesized ribonucleic acid is not a significant source of precursors for ribonucleic acid synthesis during sporulation. The majority of the ribonucleic acid made in a 10-min period during sporulation does not migrate on gels with precursor or mature ribosomal ribonucleic acid.     

Template activity of chromatin during stimulation of cellular proliferation in human diploid fibroblasts

1. Contact-inhibited confluent monolayers of WI-38 human diploid fibroblasts can be stimulated to divide by replacing the medium with fresh medium containing 30% foetal calf serum. 2. Of the cells 40–75% are stimulated to divide with a peak DNA synthesis between 15 and 21h and a peak mitotic index between 28 and 30h after stimulation. 3. In the first 12h before the initiation of DNA synthesis there is a biphasic increase in the incorporation of [³H]uridine into RNA of whole cells. 4. This is paralleled by a similar biphasic stimulation of chromatin template activity measured in vitro in a system in which purified cell chromatin is incubated with an exogenous RNA polymerase isolated from Escherichia coli. 5. The changes in chromatin template activity are believed to represent activation of the genome, with more sites available for RNA synthesis, and to account almost entirely for the changes in RNA synthesis occurring in the whole cell.     

Regulation of ribonucleic acid synthesis in Escherichia coli B-r: an analysis of a shift-up. II. Fraction of RNA polymerase engaged in the synthesis of stable RNA at different steady-state growth rates

The relative rates of stable RNA synthesis (rate of stable synthesis/rate of total RNA synthesis) were determined for Escherichia coli$\text{B}\text{r}$ growing in succinate (μ = 0.69 doublings/h), glucose (μ = 1.36 doublings/h) and glucose/amino acids (μ = 2.10 doublings/h) media. The relative rates were 0.29, 0.50 and 0.66 at these growth rates. From the relative rates, the fraction of RNA polymerase engaged in the synthesis of stable RNA, ψ_s, was calculated to be 0.22, 0.36 and 0.48, respectively, by taking into account the difference between the RNA chain growth rate of stable and that of unstable RNA. The relationship between these ψ_s values and μ and our previously determined chain growth rate of stable RNA has two implications for the control of RNA synthesis during a nutritional shift-up: (1) the increase in the net rate of RNA synthesis after a shift-up results from a transfer of RNA polymerase molecules from unstable to stable RNA genes, and a concomitant increase in the stable RNA chain growth rate, but does not require an activation of RNA polymerase; (2) the synthesis of functioning RNA polymerase enzymes is subject to a growth rate-dependent control.     

Specific protein synthesis in isolated epithelium of guinea-pig seminal vesicle. Effects of castration and androgen replacement

Four intrinsic soluble secretory proteins are synthesized in vitro by isolated seminal-vesicle mucosa from sexually mature guinea pigs. Newly synthesized specific proteins labelled with [¹⁴C]glycine and [¹⁴C]lysine were precipitated by using double-antibody immunoprecipitation techniques and their radioactivity was assessed. Rates of synthesis were determined on each of 5 days after castration. By 5 days after castration the wet weight of the epithelium decreased to 42% of intact control values; the absolute amount of specific protein synthesized in vitro after 60min incubation decreased to 28% and the 27500g cytoplasmic protein content decreased to 31%. Thus androgen deprivation leads to a decrease in general protein synthesis in vivo, as well as to a decrease in specific protein synthesis in vitro. Specific protein synthesis comprised 76% of the total protein formed in isolated tissue from animals 5 days after castration as compared with 99–100% in tissue from intact animals. At 72h after an injection of testosterone or dihydrotestosterone, seminal-vesicle epithelium wet weight, cytoplasmic protein content and capability for synthesizing specific proteins in vitro were restored to approx. 70% of normal values. At 72h after onset of therapy with 3α-androstanediol, both epithelium wet weight and cytoplasmic protein content had increased significantly, but without a corresponding increase in the capability of the isolated tissue to synthesize specific proteins. The soluble labelled proteins synthesized in vitro by isolated epithelium from intact animals during 60 or 120min incubation were essentially entirely immunoprecipitable, i.e. specific. In contrast, approx. 29% of all soluble protein newly synthesized by isolated epithelium from animals 5 days after castration was acid-precipitable, but not immunoprecipitable, i.e. `non-specific'. The injection of testosterone into castrated animals inhibited the synthesis of the non-specific fraction by isolated tissue. The effects of castration on the ultrastructure of guinea-pig seminal-vesicle epithelium are also presented.     

DNA damage-induced inhibition of rRNA synthesis by DNA-PK and PARP-1

RNA synthesis and DNA replication cease after DNA damage. We studied RNA synthesis using an in situ run-on assay and found ribosomal RNA (rRNA) synthesis was inhibited 24 h after UV light, gamma radiation or DNA cross-linking by cisplatin in human cells. Cisplatin led to accumulation of cells in S phase. Inhibition of the DNA repair proteins DNA-dependent protein kinase (DNA-PK) or poly(ADP-ribose) polymerase 1 (PARP-1) prevented the DNA damage-induced block of rRNA synthesis. However, DNA-PK and PARP-1 inhibition did not prevent the cisplatin-induced arrest of cell cycle in S phase, nor did it induce de novo BrdU incorporation. Loss of DNA-PK function prevented activation of PARP-1 and its recruitment to chromatin in damaged cells, suggesting regulation of PARP-1 by DNA-PK within a pathway of DNA repair. From these results, we propose a sequential activation of DNA-PK and PARP-1 in cells arrested in S phase by DNA damage causes the interruption of rRNA synthesis after DNA damage.     

Dose- and time-dependent synthesis of 20-hydroxyecdysone modulated polypeptides in the epidermis of Tenebrio molitor

Cellular (non-cuticular) polypeptides were isolated from mid-instar larval epidermis incubated in the presence or absence of 20-hydroxyecdysone (20HE) for 14–16 h in vitro. Polypeptides synthesized during the last few hours of incubation were labelled with [³⁵S]methionine, separated in cell lysates by two-dimensional polyacrylamide gel electrophoresis and detected fluorographically. Cells incubated with hormone incorporated 28% more label into polypeptide. The synthesis of over 250 polypeptides was detected in total cell lysates. Of these, 54 showed an altered level of synthesis in response to 20HE treatment. The synthesis of most (33) was depressed. The relative synthetic rate of most “down-regulated” 20HE-sensitive polypeptides began to drop at 10⁻³ μg/ml whereas that of most “up-regulated” polypeptides increased only at concentrations above 10⁻¹ μg/ml. An early response to 20HE, involving the de novo synthesis of a 43 kDa polypeptide, was first detectable after 2 h of exposure to hormone, and peaked after 4 h. The synthesis of this 43 kDa polypeptide was selectively enhanced by the addition of 10⁻² μg/ml cycloheximide to medium containing 20HE. The long-term effect (12–16 h) of 20HE on polypeptide synthesis in subcellular fractions of the epidermis was also examined. Polypeptide synthesis found in the nuclear, mitochondrial-lysosomal, microsomal and soluble fractions changed in response to 20HE. It appears that 20HE influences epidermal behaviour predominantly through its ability to modulate the synthetic rate of many cellular polypeptides, rather than by turning off the synthesis of a few pre-existing ones and switching on that of a few new ones.     

Magnetic chitosan beads for covalent immobilization of nucleoside 2′-deoxyribosyltransferase: application in nucleoside analogues synthesis

Cross-linked magnetic chitosan beads were prepared in presence of epichlorohydrin under alkaline conditions, and subsequently incubated with glutaraldehyde in order to obtain an activated support for covalent attachment of nucleoside 2′-deoxyribosyltransferase from Lactobacillus reuteri (LrNDT). Changing the amount of magnetite (Fe₃O₄) and epichlorohydrin (EPI) led to different macroscopic beads to be used as supports for enzyme immobilization, whose morphology and properties were characterized by scanning electron microscopy, spin electron resonance (ESR), and vibrating sample magnetometry (VSM). Once activated with glutaraldehyde, the best support was chosen after evaluation of immobilization yield and product yield in the synthesis of thymidine from 2′-deoxyuridine and thymine. In addition, optimal conditions for highest activity of immobilized LrNDT on magnetic chitosan were determined by response surface methodology (RSM). Immobilized biocatalyst retained 50 % of its maximal activity after 56.3 h at 60 °C, whereas 100 % activity was observed after storage at 40 °C for 144 h. This novel immobilized biocatalyst has been successfully employed in the enzymatic synthesis of 2′-deoxyribonucleoside analogues as well as arabinosyl-nucleosides such as vidarabine (ara-A) and cytarabine (ara-C). Furthermore, this is the first report which describes the enzymatic synthesis of these arabinosyl-nucleosides catalyzed by an immobilized nucleoside 2′-deoxyribosyltransferase. Finally, the attached enzyme to magnetic chitosan beads could be easily recovered and recycled for 30 consecutive batch reactions with negligible loss of catalytic activity in the synthesis of 2,6-diaminopurine-2′-deoxyriboside and 5-trifluorothymidine.