Dynamics of the biosynthesis of components of the protein synthesizing apparatus of the rat liver at the stage of restoration of translation, inhibited by cycloheximide

The biosynthesis of proteins, ribosomal RNA and other components of the rat liver protein-synthesizing system during the reparation and subsequent activation of translation inhibited by a sublethal dose cycloheximide (CHI, 3 mg/kg) was studied. It was found that the incorporation of labeled precursors into proteins and ribosomal rRNA isolated from free and membrane-bound polysomes is repaired already 3 hours after CHI injection. 6-9 hours thereafter, the level of component labeling reaches control values, whereas the total protein biosynthesis is retarded. After 12-24 hours, marked stimulation of ribosome biosynthesis and the integration of ribosomes into polysomes are observed together with an asymmetric accumulation of excessive amounts of newly synthesized 40S subunits into polysomes 12 hours after CHI infection. The putative mechanisms of the activation of expression of the part of the genome responsible for protein and ribosomal rRNA synthesis as well as for the synthesis of other components of the protein-synthesizing system are discussed.     

De novo pyrimidine biosynthesis in isolated rat hepatocytes. Quantitative aspects of the regulation by UTP

Quantitative aspects of de novo pyrimidine biosynthesis in rat hepatocytes were monitored. A reduction of intracellular UTP contents by different concentrations of D-galactosamine led to a dose-dependent increase of 14CO2 incorporation into the sum of all acid-soluble uracil nucleotides. In controls the rate of de novo synthesis which was calculated from the incorporation rate of 14CO2 into the sum of all acid-soluble uracil nucleotides was 0.014 mumol X h-1 X g-1 compared to 0.056 mumol X h-1 X g-1 wet weight of liver in situations of a maximally stimulated de novo synthesis. Incubation of hepatocytes with uridine led to a dose-dependent reduction of 14CO2 incorporation to less than 25% of the amount incorporated in the controls. Alterations of the CTP content had no influence on the 14CO2 incorporation. In the presence of high D-galactosamine concentrations the increase of the total amount of acid-soluble uracil nucleotides exceeded the rate of the de novo synthesis derived from the incorporation of 14CO2 into the sum of the acid-soluble uracil nucleotide pool. It was also greater than the increase of the total amount of intra- and extracellular orotate after acidic hydrolysis--even in the presence of 6-azauridine, which stimulated de novo pyrimidine biosynthesis by itself.     

Control of RNA biosynthesis in rat liver. Some features of RNA biosynthesis during prolonged protein synthesis inhibition]

A drastic inhibition of protein biosynthesis in rat liver in vivo by cycloheximide (CHI) (0.3 mg/100 g of body weight) first caused an increase of RNA synthesis (after 1 hour), which was then followed by its decrease. Partial gradual restoration of the protein synthesis level was shown to be accompanied by a repeated increase of RNA synthesis (12 hs) and its normalisation after 24 hs. The first maximum of RNA synthesis increase in the isolated nuclei system was AU-type RNA synthesis (sensitive to alpha-amanitine), the second one was due to GC-type RNA synthesis (resistant to this toxin). Purified chromatine template activity in the system with E. coli RNA polymerase (by 14%) an hour after CHI treatment, but 3 hrs later was decreased and subsequently restored (12 hrs after CHI injection). The changes of RNA biosynthesis induced by prolonged protein synthesis inhibition suggest the existence of continuous RNA synthesis control in nuclei. This control is realized by translation system using the feed back principle.     

Expression of oncogenes in the rat liver under conditions of template biosynthesis uncoupling by sublethal doses of cycloheximide

Injection of sublethal doses of cycloheximide (CHI) to rats allowed to reveal three stages in the dynamics of protein synthesis: 1) suppression stage (0-6 hrs), 2) regeneration stage (6-12 hrs), 3) stimulation stage (6-12 hrs). RNA-polymerases are activated when protein synthesis is inhibited. The stimulation stage precedes the activation of DNA replication. This model of DNA replication induced by CHI is specified by the expression of various cell oncogenes (c-fos, c-mys, p53, c-Ha-ras, c-sis, c-src). The investigated oncogenes may be divided into 4 groups according to the character of their expression. 1. Oncogenes (c-fos, c-myc) are switched on step-by-step 1 hour after CHI injection, the superexpression of the oncogenes being comparatively short. Maximum expression of c-fos and c-myc oncogenes is registered after 2-3 hours, respectively. 2./p53 oncogene expression increases within a few hours' after CHI injection and manifests itself at all three stages of protein synthesis till DNA replication. 3. c-Ha-ras oncogene is expressed at a high level in control and experimental animals. 4. Expression of c-sis and c-src oncogenes are absent both before and after CHI injection. Sublethal doses of CHI have the same effect on oncogene expression as the lethal ones.     

Gold color in onions (<Emphasis Type="Italic"> Allium cepa</Emphasis>): a natural mutation of the chalcone isomerase gene resulting in a premature stop codon

Unusual gold-colored onions were selected from a F₃ family originating from a cross between US-type yellow and Brazilian yellow onions. HPLC analysis showed that the gold onions contained a significantly reduced amount of quercetin, the most abundant flavonoid in onions. This result indicated that an early step in the flavonoid biosynthesis pathway might be abnormal in these onions. The expression of flavonoid synthesis genes isolated from onions was examined in gold onions and compared to that in onions of other colors by RT-PCR. The results showed that all genes were transcribed in gold onions as in red onions. In order to identify any critical mutations in flavonoid synthesis genes encoding enzymes involved in early steps of the pathway, the genomic sequence of chalcone isomerase (CHI) was obtained. A premature stop codon and a subsequent single base-pair addition causing a frameshift were identified in the coding region of the CHI gene in the gold onions. Co-segregation of the mutant allele of the CHI gene and the gold phenotype was investigated in the original F₂ segregating population. Genotyping of three color groups (red, yellow and gold) of F₂ onions revealed perfect co-segregation of the mutant CHI allele with the gold phenotype. All tested gold F₂ onions were homozygous for the mutant CHI allele. This perfect co-segregation implies that the presence of a premature stop codon in the gold CHI gene results in an inactive CHI. Inactivation of CHI results in a block in the flavonoid biosynthesis pathway and the accumulation of chalcone derivatives, including a yellow pigment which might be responsible for the gold color in onions.Communicated by R. Hagemann     

Expression of human papilloma virus E7 protein causes apoptosis and inhibits DNA synthesis in primary hepatocytes via increased expression of p21(Cip-1/WAF1/MDA6)

The impact of human papilloma virus (HPV16) E7 proteins and retinoblastoma (RB) antisense oligonucleotides upon mitogen-activated protein kinase (MAPK)-mediated inhibition of DNA synthesis via p21(Cip-1/WAF1/MDA6) (p21) was determined in primary hepatocytes. Prolonged activation of the MAPK pathway in p21(+/+) or p21(-/-) hepatocytes caused a large decrease and increase, respectively, in DNA synthesis. Either transfection with RB antisense oligonucleotides, expression of wild type E7, or RB binding mutant E7 (C24S) proteins increased p21 levels and reduced DNA synthesis in p21(+/+) hepatocytes. RB antisense oligonucleotides and E7 proteins increased apoptosis in p21(+/+), but not p21(-/-), hepatocytes. Expression of wild type E7 increased DNA synthesis above control levels in p21(-/-) cells, which was additive with prolonged MAPK activation. In contrast, expression of mutant E7 did not alter DNA synthesis above control levels in p21(-/-) cells and was supra-additive with prolonged MAPK activation. Antisense ablation of RB in p21(-/-) hepatocytes had a weak stimulatory effect upon DNA synthesis itself but enhanced the capacity of mutant E7 protein to stimulate DNA synthesis to the same level observed using wild type E7. The ability of prolonged MAPK activation to stimulate DNA synthesis in the presence of mutant E7 and antisense RB was additive. Collectively, the present data demonstrate that loss of RB function together with loss of p21 function plays an important role in the E7- and MAPK-dependent modulation of apoptosis and DNA synthesis in primary hepatocytes.     

Reaction of rats organ cells to inhibition of protein biosynthesis by sublethal doses of cycloheximide

Time-dependent responses of cellular systems in rat organs and Fe(3+)-transferrin and Cu(2+)-ceruloplasmin pools in blood to the blocking of translation by sublethal doses of cycloheximide (CHI) was studied by EPR spectroscopy and radioisotope techniques. It was shown that, within the early post-CHI-treatment time, the suppression of deoxyribonucleotide and DNA biosynthesis, the activation of catabolic enzymes, the inhibition of electron transfer in the mitochondrial electron transport chain, the activation and the following inactivation of cytochrome P-450, and an intensive production of nitrosyl complexes in rat blood and organs occur. In addition, the activation of the synthesis of steroid hormones in adrenal gland was revealed within 1-24 h after cycloheximide injection. In response to these metabolic disturbances, nonspecific compensatory recovery reactions developed, first of all, the "reprograming" of the translation process to produce new protein-synthesizing elements instead of cycloheximide-blocked ones. The activation of protein synthesis promotes the recovery of deoxyribonucleotide and DNA synthesis, the restoration of the redox state of mitochondrial and microsomal electron transport chains in organs as well as an increase of Fe(3+)-transferrin and Cu(2+)-ceruloplasmin pools in rat blood. These metabolic processes result in the full recovery of the functional ability of organs.     

Activation of nucleolar DNA and ribosome biosynthesis in hepatocytes under the effect of glucocorticoids and high density lipoproteins

The structural bases of cooperative effect of glucocorticoids and HDL brings about the activation of protein biosynthesis in hepatocytes. Using surviving rat liver it was shown that these two compounds together activate the gene expression which was indicated by increased 3H-uridine incorporation into the total RNA pool. The enhanced incorporation of 14C-leucine into proteins in these experiments confirms protein biosynthesis acceleration. With the use of liver perfusion technique it was morphologically demonstrated that the earliest changes in hepatocyte genome take place in nucleoli. The increase of nucleolar dimensions and granular component reflects the activation of ribosomal precursors synthesis. Considerable number of ribosomes in the hepatocyte perinuclear space indicates their active transport across the numerous nuclear membrane pores into the cytoplasm. In the first place and more prominently in hepatocytes the protein synthesis "for export" is stimulated, which was proved by the dynamics of ribosome accumulation on the membranes of endoplasmic reticulum according the perfusion duration. The kupffer cells play a significant role in HDL transcytosis and in the realization of their cooperative effect with glucocorticoids.     

Turnover and functions of glutathione studied with isolated hepatic and renal cells

Suspensions of freshly isolated rat hepatocytes and renal tubular cells contain high levels of reduced glutathione (GSH), which exhibits half-lives of 3-5 and 0.7-1 h, respectively. In both cells types the availability of intracellular cysteine is rate limiting for GSH biosynthesis. In hepatocytes, methionine is actively converted to cysteine via the cystathionine pathway, and hepatic glutathione biosynthesis is stimulated by the presence of methionine in the medium. In contrast, extracellular cystine can support renal glutathione synthesis; several disulfides, including cystine, are rapidly taken up by renal cells (but not by hepatocytes) and are reduced to the corresponding thiols via a GSH-linked reaction sequence catalyzed by thiol transferase and glutathione reductase (NAD(P)H). During incubation, hepatocytes release both GSH and glutathione disulfide (GSSG) into the medium; the rate of GSSG efflux is markedly enhanced during hydroperoxide metabolism by glutathione peroxidase. This may lead to GSH depletion and cell injury; the latter seems to be initiated by a perturbation of cellular calcium homeostasis occurring in the glutathione-depleted state. In contrast to hepatocytes, renal cells metabolize extracellular glutathione and glutathione S-conjugates formed during drug biotransformation to the component amino acids and N-acetyl-cysteine S-conjugates, respectively. In addition, renal cells contain a thiol oxidase acting on extracellular GSH and several other thiols. In conclusion, our findings with isolated cells mimic the physiological situation characterized by hepatic synthesis and renal degradation of plasma glutathione and glutathione S-conjugates, and elucidate some of the underlying biochemical mechanisms.     

灯盏花chi的克隆及其生物信息学分析

查尔酮异构酶(CHI)是调控黄酮生物合成的关键酶,分离和克隆这一酶的功能基因,对利用转基因技术进行灯盏花黄酮生物合成的调控具有重要意义。本研究采用RT-PCR和RACE技术,获得了chi cDNA全序列,GenBank登录号为GU208823.1,序列全长996 bp,开放阅读框为594 bp,编码197个氨基酸,3-Race有一个多聚腺苷酸加尾信号。应用软件预测该基因编码蛋白分子量约为21.6 kD,理论等电点为4.78。该基因编码的蛋白无跨膜结构域,其二级结构的主要构件为α-螺旋和随机卷曲。对其三级结构进行了建模,表明其结构与苜蓿chi的三级结构相似。同时根据灯盏花chi N端序列变化的特征,提出了灯盏乙素的合成可能与chi在细胞亚结构的定位及其与合成代谢相关酶形成复合酶的特异性有关。研究为利用基因工程定向改变灯盏花黄酮代谢产物奠定了基础。     

Sequence of activation of template biosynthesis in normal and transformed human cells after synchronization by a double thymidine block

The sequence of matrix biosyntheses of DNA, RNA and various proteins in normal and transformed human fibroblasts in the first mitotic cycle after synchronization of cells by double thymidine block was studied. Two important regularities of synthesis of acid-soluble histone-like and acid-insoluble proteins in normal and transformed cells were established. In normal fibroblasts, the synthesis of both acid-soluble and acid-insoluble proteins is minimal before DNA replication and maximal in the G2-phase; that in transformed cells is maximal after removal of the thymidine block and decreased in the G2-phase. In normal fibroblasts, the synthesis of acid-insoluble proteins is maximal before, while that of acid-soluble ones--after the maximum of DNA synthesis. In transformed cells the situation is opposite. RNA synthesis in normal and transformed cells is stimulated at the end of the G2-phase. In normal cells, protein synthesis is coupled with the activation of RNA synthesis, whereas in transformed fibroblasts protein synthesis occurs, in all probability, in the next mitotic cycle. These differences are especially well-pronounced in the expression of some LMG proteins. It is concluded that in transformed cells the regulatory control over the coupling of matrix biosyntheses is impaired.     

Induction of ethylene biosynthesis in tobacco leaf discs by cell wall disesting enzymes

Cellulysin induces ethylene production in tobacco leaf discs by initiating the formation of 1-aminocyclopropane-1-carboxylic acid. Induction occurred within 30 to 60 min of incubation and was inhibited by aminoethoxyvinylglycine, and the antiproteases, PMSF and soybean trypsin inhibitor. Cycloheximide (CHI) at 2.8 μg/ml and chloramphenicol (CAP) at 100 μg/ml did not inhibit this induction although incorporation of the label from (3,4-¹⁴C)methionine into the acid-insoluble fraction was inhibited by 57%. At 14 μg/ml CHI, and CAP, ethylene production was inhibited by 25% while protein synthesis was inhibited by 75%. We suggest that either the low amounts of protein synthesis that appear to be insensitive to CHI is sufficient to induce ethylene biosynthesis or that Cellulysin activates a preexisting but inactive form of ACC synthase to promote ethylene biosynthesis. Also, induction of ethylene production by microbial enzymes that digests plant cell walls may be an initial protective response of plants that serves to combat microbial infection.     

Study of the ultrastructural and functional expression of the hepatocyte genome under conditions of prolonged depression of protein biosynthesis by cycloheximide. III]

Dynamics of changes in mtRNA synthesis and mitochondria ultrastructure is strictly dependent on the level of inhibition of biosynthesis of cytoplasm proteins and "soluble" proteins of mitochondria by cycloheximide in hepatocytes: 1-6 hrs later a progressive weakening of protein synthesis is accompanied by a drop in mtRNA synthesis and essential destruction of mitochondria; from 12 to 24 hrs a partial restoration of protein biosynthesis induces the processes of the above-mentioned indexes normalization.     

Synthesis of the acid-soluble proteins in early cleaving embryos of the sea urchin. Cyclic synthesis of histones.

Synthesis of the acid-soluble proteins in the early cleavage stage of the sea urichin, Hemicentrotus pulcherrimus, was investigated. As detected by the incorporation of lysine, the acid-soluble proteins were synthesized periodically even before the first cleavage, differing from the pattern of incorporation of tryptophan into the fraction. Cyclic synthesis occurred almost in parallel with DNA synthesis. However, the phase and periodicity of cyclic synthesis of the acid-soluble protein fraction were quite different from those found in the hot TCA-insoluble (acid-insoluble) protein fraction. The acid-soluble proteins were adsorbed on cation exchange resin, Amberlite CG-50, and gave an elution profile similar to that found for calf thymus histones. The migration pattern of these proteins on acrylamide gel also resembled that of histones.     

Expression of chalcone synthase and chalcone isomerase proteins in Arabidopsis seedlings

Antibodies have been developed against the first two enzymes of flavonoid biosynthesis in Arabidopsis thaliana. Chalcone synthase (CHS) and chalcone isomerase (CHI) were overexpressed and purified from Escherichia coli as fusion proteins with glutathione S-transferase from Schistosoma japonicum. The recombinant proteins were then used to immunize chickens and the resulting IgY fraction was purified from egg yolks. Immunoblots of crude protein extracts from Arabidopsis seedlings carrying wild-type and null alleles for CHS and CHI showed that the resulting antibody preparations provide useful tools for characterizing expression of the flavonoid pathway at the protein level. An initial analysis of expression patterns in seedlings shows that CHS and CHI proteins are present at high levels during a brief period of early seedling germination that just precedes the transient accumulation of flavonoid end-products.     

Inhibition of hepatic phosphatidylcholine synthesis by 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside is independent of AMP-activated protein kinase activation

5-Aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAr), a commonly used indirect activator of AMP-activated protein kinase (AMPK), inhibits phosphatidylcholine (PC) biosynthesis in freshly isolated hepatocytes. In all nucleated mammalian cells, PC is synthesized from choline via the Kennedy (CDP-choline) pathway. The purpose of our study was to provide direct evidence that AMPK regulates phospholipid biosynthesis and to elucidate the mechanism(s) by which AMPK inhibits hepatic PC synthesis. Incubations of hepatocytes with AICAr resulted in a dose-dependent activation of AMPK and inhibition of PC biosynthesis. Surprisingly, adenoviral delivery of constitutively active AMPK did not alter PC biosynthesis. In addition, expression of dominant negative mutants of AMPK was unable to block the AICAr-dependent inhibition of PC biosynthesis, indicating that AICAr was acting independently of AMPK activation. Determination of aqueous intermediates of the CDP-choline pathway indicated that choline kinase, the first enzyme in the pathway, was inhibited by AICAr administration. Flux through the CDP-choline pathway was directly correlated to the level of intracellular ATP concentrations. Therefore, it is possible that inhibition of PC biosynthesis is another process by which the cell can reduce ATP consumption in times of energetic stress. However, unlike cholesterol and triacylglycerol biosynthesis, PC production is not regulated by AMPK.     

Estrogen-induced synthesis of uterine proteins declines during aging

The synthesis of total cellular as well as acid-soluble nuclear proteins and estrogen receptor is high in the uteri of young (22 weeks) and decreases to half in old (104 weeks) rats. Administration of estrogen induces the synthesis of these proteins significantly in young but shows no remarkable effect in old rats. Interestingly, a specific cytosolic protein of 45 kDa is stimulated about two-fold after estrogen injection in young but not in old rats. These findings further establish the reduced responsiveness of uterus to estrogen in old age.     

Estrogen-induced synthesis of uterine proteins declines during aging.

The synthesis of total cellular as well as acid-soluble nuclear proteins and estrogen receptor is high in the uteri of young (22 weeks) and decreases to half in old (104 weeks) rats. Administration of estrogen induces the synthesis of these proteins significantly in young but shows no remarkable effect in old rats. Interestingly, a specific cytosolic protein of 45 kDa is stimulated about two-fold after estrogen injection in young but not in old rats. These findings further establish the reduced responsiveness of uterus to estrogen in old age.     

Characteristic features of protein synthesis in the extracellular matrix during tumor growth

Changes in the biosynthesis of basic extracellular proteins (e.g., collagen proteins, fibronectins, proteoglycans) in the course of neoplastic growth are reviewed. Some peculiarities of quantitative changes in the biosynthesis and modifications of the primary structure of the above macromolecules are discussed in terms of neoplastic cell differentiation. The main emphasis is laid on the mechanisms underlying the disturbances in the biosynthetic activity of extracellular matrix proteins in neoplastic cells at different steps of protein synthesis and extracellular degradation of protein molecules.