Protein synthesis inhibition induced by dimethylnitrosamine and diethylnitrosamine on isolated rat hepatocytes

Summary Time- and dose-dependent protein synthesis inhibition takes place following exposure to high doses of dimethylnitrosamine (DMN) or diethylnitrosamine (DENA) in isolated rat hepatocytes. The ability of DENA to depress protein synthesis is 5-fold higher than that of DMN. Cells inhibited by 60 min exposure to DMN or DENA, and then incubated in a nitrosamine-free medium, regain their initial rate of protein synthesis. This recovery is faster and more complete for DENA-treated cells.     

Analysis of translation products synthesized in isolated rat hepatocytes treated with diethylnitrosamine

Isolated rat hepatocytes were labeled with 35S-methionine in the presence of 25 mM diethylnitrosamine (DENA). The intrinsically labeled proteins were analyzed by one- and two-dimensional gel electrophoresis and the fluorographic patterns were compared with those obtained from untreated hepatocytes. The results of short term experiments (2 h) show that, in the presence of 25 mM DENA, protein synthesis is inhibited by 50%. This reduction encompasses all protein species without selective inhibition of certain proteins.     

Analysis of translation products synthesized in isolated rat hepatocytes treated with diethylnitrosamine

Summary Isolated rat hepatocytes were labeled with³⁵S-methionine in the presence of 25 mM diethylnitrosamine (DENA). The intrinsically labeled proteins were analyzed by one-and two-dimensional gel electrophoresis and the fluorographic patterns were compared with those obtained from untreated hepatocytes. The results of short term experiments (2 h) show that, in the presence of 25 mM DENA, protein synthesis is inhibited by 50%. This reduction encompasses all protein species without selective inhibition of certain proteins.This work was supported by CNR (Project Control of Neoplastic Growth) grant No. 810132696 and partially by AIRC.     

Acute toxicity of dimethylnitrosamine in the presence of inhibitors of DMN demethylase.

The LD50 of DMN was determined in groups of mice in the presence of inhibitors of DMN demethylase. Piperonyl butoxide, dibutylnitrosamine and nitrososarcosine had no effect on the acute toxicity of DMN. Diethylnitrosamine and DMN were markedly synergistic. All mice treated with 100 mg/kg diethylnitrosamine and 10.7 mg/kg DMN died. These results suggest that DMN demethylase may not be involved in the acute toxicity of DMN.     

Acute toxicity of dimethylnitrosamine in the presence of inhibitors of DMN demethylase

Summary The LD₅₀ of DMN was determined in groups of mice in the presence of inhibitors of DMN demethylase. Piperonyl butoxide, dibutylnitrosamine and nitrososarcosine had no effect on the acute toxicity of DMN. Diethylnitrosamine and DMN were markedly synergistic. All mice treated with 100 mg/kg diethylnitrosamine and 10.7 mg/kg DMN died. These results suggest that DMN demethylase may not be involved in theaacute toxicity of DMN.We thank Dr.D. B. Couch, Mr.E. J. Greene and Dr.A. E. Munson for their technical assistance.     

Elimination and metabolism of dimethylnitrosamine (DMN) byXenopus laevis and other amphibians

Summary The elimination of (¹⁴C)-DMN after i.p. injection intoXenopus was measured, as was the metabolism in vitro of (¹⁴C)-DMN by liver fromXenopus and 9 other amphibian species. In view of its rapid elimination from the body and low rate of metabolism byXenopus liver in vitro, DMN is unlikely to be toxic or carcinogenic inXenopus.This work was supported by a grant from the Cancer Research Campaign. We are also grateful to Dr P. F. Swann, both for the supply of DMN and DEN and for many useful discussions.     

Elimination and metabolism of dimethylnitrosamine (DMN) by Xenopus laevis and other amphibians

The elimination of (14C)-DMN after i.p. injection into Xenopus was measured, as was the metabolism in vitro of (14C)-DMN by liver from Xenopus and 9 other amphibian species. In view of its rapid elimination from the body and low rate of metabolism by Xenopus liver in vitro, DMN is unlikely to be toxic or carcinogenic in Xenopus.     

Spectral changes resulting from the interaction of some N-alkyl nitrosamines and rat liver microsomes

Summary Dimethyl (DMN) and diethyl nitrosamine (DEN) do not give characteristic spectral changes upon interaction with rat liver microsomes, while dipropyl (DPN) and dibutyl (DBN) nitrosamine cause type I spectral changes. The spectral binding constant is 100 mM for DPN and 1.17 mM for DBN. The maximal spectral change is 3.2×10⁶ and 1.0×10⁶ absorbance units per milligram protein for DPN and DBN respectively.Acknowledgment. This work was supported by Grants AM 13195-07 from the National Institute of Health (USA) and from the Consejo Nacional de Investigaciones Cientificas y Técnicas (Argentina).     

Eiweissvermehrung in ein- und zweikernigen systemen vonAcetabularia

Summary (1) The rate of protein synthesis was found to be different inAcetabularia crenulata andAcetabularia mediterranea the higher cytoplasmic protein synthesis inA. crenulata depending upon the diameter of the stalk.(2) In systems containing one or two nuclei, there was no difference in the rate of cytoplasmic synthesis of proteins. This corresponds to the diminution of size and efficiency of the nuclei in binucleated systems.(3) In interspecific grafts, the rate of cytoplasmic protein synthesis corresponds nearly to the rate of protein synthesis ofAcetabularia crenulata. Corresponding to morphogenetic processes, thecren-action is prevalent.     

Action of hyperoxia on hemolytic activity of the complement system]

Normobarie oxygen exposures momentarily raise by about 20% the haemolytic activity of the guinea pig complement likely by a rise of protein synthesis. About the end of this treatment the complement activity quickly decreases back to its initial value. Hyperbaric oxygen exposure immediately decreases the haemolytic activity. This shift might be result of a release in the serum of cytoplasmic elements bearing an anticomplementary activity or of an inactivation of the complement components involved in the stress reaction appearing in the treated animals. 2 days after the first exposure, during the resting phase, the complement rate increases by about 25% then decreases slowly back to normal.     

The effect of temperature and protein synthesis on the renaturation of firefly luciferase in intact H9c2 cells

A mild increase in temperature that does not exert an effect on tolerance development or synthesis of heat shock proteins (Hsps) in control cells can stimulate these processes when applied to cells that have previously been heat shocked. To study the underlying mechanism of this effect, H9c2 cells were stably transfected with the gene encoding firefly luciferase (Luc). Heat-shock-induced inactivation of Luc and its subsequent reactivation is frequently used as a model for cellular protein denaturation and renaturation. Luc reactivation was determined following a damaging heat shock (43 or 44 degrees C for 30 min) in cells that were subsequently exposed to either control temperatures (37 degrees C) or various mild hyperthermic conditions (from 38.5 to 41.5 degrees C for 1 h). To prevent changes in Luc activity consequent to new synthesis of Luc, Luc reactivation was monitored in the presence of cycloheximide, an inhibitor of protein synthesis. The results showed that reactivation of Luc was inhibited when heat-treated cells were post-treated under mild hyperthermic conditions. The observed increase in Hsp synthesis under mild hyperthermic post-heat shock conditions therefore appears to be the result of an increase in the period during which denatured proteins are present. In addition, we studied Luc reactivation in the absence of protein synthesis inhibitors. This condition led to much higher Luc activity. By estimating half-life times of Luc, the contribution of new Luc synthesis in this recovery could be determined, and only partially explained the observed increase in Luc reactivation after heat shock. Thus the synthesis of other proteins must be important for the renaturation of heat-damaged proteins.     

Kälte als Auslöser einer Glykogen-Speicherung während der Oogenese vonMusca domestica

Summary InMusca domestica reared under standard conditions (21 °C), glycogen is stored in the oocyte towards the end of egg development. By 1–3 days exposure to lower temperatures (4 °C), a glycogen deposition can be released already in young follicles. This premature glycogen synthesis is not restricted to the ooplasm. Carbohydrates are also found in the nurse cells and the follicle epithelium. Similar results were formerly obtained by inhibition of oogenetic protein synthesis.

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Herrn Prof. Dr. Dr. h.c.Bernhard Rensch zum 70. Geburtstag gewidmet.     

Amino acid incorporation by human milk fat globules membranes (author's transl)]

Human milk fat globule membranes (MFGM) can incorporate radioactive 14C amino acids in a hot trichloracetic acid-insoluble material. Aspecific adsorption and bacterial contamination are unlikely. The products of protein synthesis were analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate or by action of proteolytic enzymes. Various inhibitors of protein synthesis were assayed. Fragments of rough endoplasmic reticulum or mitochondria could be involved in this incorporation.     

Protein synthesis in vivo in muscles of the growing lamb]

The relationship between incorporation of intravenously injected 14C lysine and specific radio-activity of precursor was used to estimate protein synthesis in muscle of growing lambs. The rate of protein synthesis per unit of muscle weight in Supraspinatus and Extensor digitorum longus decreased strongly from one week of age to puberty (10 weeks); afterwards it decreased in supraspinatus and increased slightly in Extensor digitorum longus. The rate of protein synthesis increase in muscle protein weight was constant during the whole experiment (1 week-16 weeks). In preruminant Lambs )1 week-5 weeks) the rate of protein synthesis per unit of muscle weight decreased; however, due to the increase in muscle weight, the rate of protein synthesis in whole muscle remained relatively constant. In order Lambs the rate of protein synthesis in whole muscle decreased. The turnover time of protein increased with age. These results give some explanation on muscular development of Lambs.     

Incorporation d'acides aminés radioactifs dans les membranes des globules lipidiques du lait humain

Summary Human milk fat globule membranes (MFGM) can incorporate radioactive¹⁴C amino acids in a hot trichloracetic acid-insoluble material. Aspecific adsorption and bacterial contamination are unlikely. The products of protein synthesis were analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate or by action of proteolytic enzymes. Various inhibitors of protein synthesis were assayed. Fragments of rough endoplasmic reticulum or mitochondria could be involved in this incorporation.     

Effect of acute administration of triiodothyronine in chicken. Liver glycogen depletion and amino acid incorporation to proteins

Summary Single injections of thyroid hormone (T₃) produce liver glycogen depletion in chickens. This effect cannot be suppressed by protein synthesis inhibitors and is previous to the hormone-induced increase in protein synthesis.     

Ureido-ethyl-imidazolines active against autochthonous diethylnitrosamine-induced epidermoid,papillary and adenocarcinomatous tumors of the respiratory tract of Syrian hamsters and against human bronchogenic carcinomas in nu/nu mice

Summary The detection of a new class of tumor inhibiting substances is described. Employing a chemical reaction discovered several years ago, a series of imidazolinylureas were prepared. It was found that some compounds of this group were active against diethylnitrosamine (DENA)-induced tumours in hamsters. CGP 15 720 A (1-{2-[2-(4-pyridyl)-2-imidazoline-l-yl]-ethyl}-3-(4-carboxy-phenyl)urea,Xb), the most active compound at present, was developed through a series of structural variations. CGP 15 720 A inhibits significantly in oral or parenteral treatment with well tolerated doses (10–30 mg/kg) the progressive growth of autochthonous, DENA-induced papillary, epidermoid and adenocarcinomatous tumors of the respiratory system in Syrian hamsters and prolongs significantly the survival. The substance also inhibits significantly the growth of 2 poorly differentiated human epidermoid or anaplastic bronchogenic carcinomas in nu/nu Balb/c mice and prolongs the mean survival time. In these mice, the substance is also active against the rodent ascites tumors Ehrlich carcinoma, CrSa 180 and Yoshida Sa AH 66, although it is only marginally active or inactive against these tumors in normal mice or rats. — In the therapeutic trials, hamsters tolerated the highest dose administered for 4 weeks, 1000 mg/kg p.o., without signs or symptoms of toxicity.Editorial remarks. There is still an urgent medical need for effective and welltolerated drugs for the treatment of the most common forms of cancer, such as bronchial carcinoma, or for post-operative prophylaxis against relapse and metastasis. — The old-established screening method based on rapidly proliferating acute transplantable lymphatic leukemias in the mouse that is applied in the major cancer research centers has certainly achieved some measure of clinical success, inasmuch as the mean duration of survival of patients with acute lymphatic leukemia has increased from 3 months to about 6 years and similar activity has been found in some rapidly proliferating lymphomas, sarcomas and teratomas.The authors were convinced, however, that chemotherapeutic agents effective against lung cancer could only be found with the help of new specific animal models. They developed a model of an autochthonous tumor in the hamster, applied it in extensive series of experiments, and succeeded in synthesizing and identifying a group of compounds that were both effective and well tolerated. They describe the synthesis and biological activity of CGP 15 720, the compound with the highest therapeutic index and an apparently non-cytotoxic mode of action.     

Calcium activity and post-ischemic suppression of protein synthesis

Increase in intracellular calcium concentration is a prominent feature of ischemia and has been considered a major factor in the initiation of ischemic pathology, which involves inhibition of protein synthesis. A reduction of calcium ion activity during and immediately after in vitro ischemia did not prevent inhibition of protein synthesis in hippocampae slices. When slices were overloaded with calcium by NMDA receptor activation or by the calcium ionophore A23187, no significant inhibition of protein synthesis was observed. We conclude that calcium overload plays only a limited role in ischemic inhibition of protein synthesis.     

Cellular regulation and molecular interactions of the ferritins

Controlling iron/oxygen chemistry in biology depends on multiple genes, regulatory messenger RNA (mRNA) structures, signaling pathways and protein catalysts. Ferritin, a protein nanocage around an iron/oxy mineral, centralizes the control. Complementary DNA (antioxidant responsive element/Maf recognition element) and mRNA (iron responsive element) responses regulate ferritin synthesis rates. Multiple iron-protein interactions control iron and oxygen substrate movement through the protein cage, from dynamic gated pores to catalytic sites related to di-iron oxygenase cofactor sites. Maxi-ferritins concentrate iron for the bio-synthesis of iron/heme proteins, trapping oxygen; bacterial mini-ferritins, DNA protection during starvation proteins, reverse the substrate roles, destroying oxidants, trapping iron and protecting DNA. Ferritin is nature’s unique and conserved approach to controlled, safe use of iron and oxygen, with protein synthesis in animals adjusted by dual, genetic DNA and mRNA sequences that selectively respond to iron or oxidant signals and link ferritin to proteins of iron, oxygen and antioxidant metabolism. Received 25 June 2005; received after revision 17 October 2005; accepted 25 November 2005     

Memory formation and the regulation of gene expression

On a cellular level, formation of memory is based on a selective change in synaptic efficacy that is both fast and, in case of important information, long-lasting. Rapidity of cellular changes is achieved by modifying preexisting synaptic molecules (receptors, ion channels), which instantaneously alters the efficacy of synaptic transmission. Endurance, that is the formation of long-term memory (LTM), is based on transient and perhaps also long-lasting changes in protein synthesis. A number of different methods exist to interfere with the synthesis of specific proteins or proteins in general. Other methods, in turn, help to identify proteins whose synthesis is changed following learning. These mostly molecular methods are briefly described in the present review. Their successful application in a variety of memory paradigms in invertebrates and vertebrates is illustrated. The data support the importance of selective changes in gene expression for LTM. Proteins newly synthesized during memory consolidation are likely to contribute to restructuring processes at the synapse, altering the efficiency of transmission beyond the scope of STM. Increased or, less often, decreased synthesis of proteins appears during specific time windows following learning. Recent evidence supports older data suggesting that two or even more waves of protein synthesis exist during the consolidation period. It is expected that the new molecular methods will help to identify and characterize molecules whose expression changes during LTM formation even in complex vertebrate learning paradigms.