Why,when and how does the poly(A) tail shorten during mRNA translation?

• 1.1. The length of the poly(A) tail at the 3'-end of mRNA may control protein synthesis by bringing the 3'-end in close proximity to the 5'-end of the noncoding region as well as increasing the duration of mRNA translation by its binding to the poly(A) binding protein.
• 2.2. The rate-limiting step in the decay of the body of the message is the shortening of a long poly(A) tail during mRNA translation. The shortening of the poly(A) tail occurs during pre-elongation in the protein synthesis cycle.
• 3.3. The shortening of the poly(A) tail during mRNA translation may not involve RNase activity, however poly(A) binding protein seems to play a role, at least in part, in shortening of the poly(A) tail.

     

Comparison of metabolic effects of EGF,TGF-α, and TGF-β1 in primary culture of fetal bovine myoblasts and rat L6 myoblasts

• 1.1. Comparative studies of EGF, TGF-α, and TGF-βl action on the synthesis of DNA and cellular proteins in rat L6 myogenic cells and fetal bovine myoblasts demonstrated considerable differences between particular growth factors, dependent on dose and target cells.
• 2.2. Among examined growth factors only EGF exerted mitostimulatory action, more pronounced at lower concentrations. EGF, progressively with dose, stimulated protein synthesis much more effectively in fetal bovine myoblasts than in L6 cells.
• 3.3. The dynamics of stimulation of protein synthesis by TGF-α was greater than by EGF in both examined types of cell cultures.
• 4.4. The maximal response of fetal bovine myoblasts to TGF-α in a concentration of 100 ng/ml reached 370%, whereas EGF in a 10 times higher concentration stimulated protein synthesis only to 123% of control.
• 5.5. In contrast to EGF, TGF-α significantly inhibits DNA synthesis. Inhibition of the mitogenic response with simultaneous stimulation of protein synthesis by TGF-α may indicate changes toward cell differentiation.
• 6.6. TGF-β 1 in smallest concentration inhibits both DNA and protein synthesis. The suppressive action of TGF-β 1 was more distinct in fetal bovine myoblasts than in the L6 cell line.
• 7.7. Increasing concentrations of TGF-β l diminished its inhibitory effect, even leading to stimulation of protein synthesis at higher doses in L6 myoblasts.

     

Phytochelatin synthesis and cadmium localization in wild type of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

AlthoughArabidopsis thaliana is known as a model plant, in molecular studies, as well as heavy metal tolerance of higher plants, there have been no detailed studies of its cadmium accumulation, tolerance and cellular distribution in a wild type of this species. In hydroponic experiments the wild type of A. thaliana (L.) Heynh cv. Columbia plants grew at cadmium concentrations varying from 5 to 100 M with phytotoxicity symptoms depending on the concentration and time of application. The concentration of cadmium in roots and shoots increased from 0.28 and 0.08 mg g^–1 d.wt at 5 M Cd treatment after 7 days to 0.82 and 0.85 mg g^–1 d.wt at 100 M Cd treatment after 14 days, respectively. Most of the cadmium (69–88% of its total pool) was found in shoot. Cd application induced the biosynthesis of phytochelatins (PCs) in root and shoot tissues. Studies with buthionine sulfoximine [BSO, specific inhibitor of glutathione (GSH) synthesis] supported the presence of Cd–phytochelatin complexes and their role in Cd detoxification and tolerance in wild type of A. thaliana. Cellular distribution of cadmium was examined using energy-dispersive X-ray micro-analysis. Particularly interesting was the observation of cadmium localized in the root pericycle.     

Bentazon influence on selected metabolic processes of isolated bean leaf cells

Time- and concentration-course studies were conducted to determine the effect of bentazon [3-isopropyl-1H-2,l,3,-benzothiadiazin-4(3H)-one 2,2-dioxide] on photosynthesis, RNA synthesis, protein synthesis, and lipid synthesis using enzymatically isolated leaf cells of red kidney bean (Phaseolus vulgaris L.). Photosynthesis and RNA synthesis were inhibited about 75% at 1 M bentazon at the 30 min treatment period. This was the lowest concentration and shortest time that significantly inhibited any of these four processes. The degree of inhibition of photosynthesis was greater than the degree of inhibition of RNA synthesis at higher concentrations and/or longer time periods. At 10 M bentazon, protein synthesis and lipid synthesis were also inhibited. Lipid synthesis was stimulated at 0.1 and 1 M at 120 min.     

Butachlor influence on selected metabolic processes of plant cells and tissues

Time- and concentration-course studies were conducted to determine the effects of butachlor (N-[butoxymethyl]-2-chloro-2,6-diethylacetanilide) on photosynthesis, protein synthesis, RNA synthesis, and lipid synthesis using isolated leaf cells of red kidney bean (Phaseolus vulgaris L.). At the 2-h incubation period, butachlor inhibited photosynthesis, protein synthesis, RNA synthesis, and lipid synthesis 99, 99, 96, and 81% respectively at 100 M, and 0, 19, 17, and 40% respectively at 10 M. At 100 M and 15-, 30-, and 60-min incubations, RNA synthesis was inhibited 20, 76 and 90% respectively, and lipid synthesis 35, 48, and 62% respectively; photosynthesis and protein synthesis were inhibited over 90% at all of these time periods. The effects of 50 M butachlor on protein and RNA synthesis in rice (Oryza sativa L.) and barnyardgrass (Echinochloa crusgalli L.) root and shoot segments were also investigated. Protein synthesis was inhibited in both species and to a greater degree in roots (81–90%) than in shoots (55–65%). RNA synthesis was inhibited 33% in barn-yardgrass roots but not significantly in barnyardgrass shoots or either organ of rice.     

Effect of calcium-binding protein regucalcin on hepatic protein synthesis: Inhibition of aminoacyl-tRNA synthetase activity

The effect of regucalcin, a calcium-binding protein isolated from rat liver cytosol, onin vitro protein synthesis in the 5500g supernatant fraction of rat liver homogenate was investigated. Addition of Ca²⁺ up to 5.0 M in the reaction mixture caused a significant decrease in protein synthesis. This decrease was saturated at 10 M Ca²⁺. The Ca²⁺ effect was not reversed by the presence of regucalcin (2.0 M); the protein caused a remarkable decrease in hepatic protein synthesis, and it enhanced significantly the Ca^2– effect. Meanwhile, calmodulin (2.5-20 g/ml), a calcium-binding protein, did not have an appreciable effect on the Ca²⁺ (10 M)-induced decrease in hepatic protein synthesis. [³H]Leucyl-tRNA synthetase activity in the 105000g supernatant fraction (cytosol) of liver homogenate was markedly decreased by addition of Ca²⁺ (1.0–50 M). This decrease was not reversed by the presence of regucalcin (2.0 M); the protein (1.0–2.0 M) caused a remarkable decrease in the enzyme activity. The present results suggest that regucalcin can regulate protein synthesis in liver cells.     

Benzyladenine induces the appearance of LHCP-mRNA and of the relevant protein in dark-grown excised watermelon cotyledons

Cotyledons were excised from imbibed watermelon seeds, grown for 4 days in darkness on water or 10 M benzyladenine (BA) and then tested for the presence of the light-harvesting chlorophyll a/b protein (LHCP) and its mRNA. LHCP was assayed immunologically by western blotting of SDS gels: the protein was present in plastids, but it was not recovered with the thylakoid fraction. Antibodies directed against LHCP precipitated a 32 kDa polypeptide from translation products of poly(A) RNA of cotyledons only if these had been grown on BA. Taken together the data suggest that in absence of light cytokinins are necessary for the maintenance of a detectable level of LHCP-mRNA as well as for synthesis of the protein.     

Serotonin,histamine and somatostatin modulation of PMA-stimulated phosphorylation of 130kDa Ca2+ pump-like protein from rat cerebellum synaptosomal membranes

• 1.1. Influence of some neurotransmitters and neuromodulators on the PMA-stimulated phosphorylation in vitro of calcium pump-like protein from rat cerebellum synaptosomal membranes was examined.
• 2.2. The prolonged time (up to 6 min) of synaptosomal membranes preincubation with 1 and 10 μM serotonin results in the increase of phosphorylation. The decrease of phosphorylation up to 80% of control value was observed for 100 μM serotonin.
• 3.3. The most stimulating effect on 130kDa protein phosphorylation was observed with 1μM of histamine (160% of control value).
• 4.4. 1 and 0.1 μM somatostatin triggered a short-time transient increase of 130 kDa phosphorylation (up to 135% of control value).

     

Induction of freezing tolerance in microspore-derived embryos of winter Brassica napus

Freezing tolerance was induced in microspore derived embryos of winter Brassica napus cv. Jet neuf by the addition of ABA or mefluidide to the culture media during embryogenesis. Survival after freezing was estimated by culture of frozen-thawed embryos to plantlets. A higher freezing tolerance (50% survival at –15°C) was induced when 50 M ABA or 3.2 M mefluidide was incorporated initially into the medium during embryogenesis at 25°C followed by culture at 2°C for 3 weeks. When embryos were induced in the absence of ABA or mefluidide and maintained at 2°C for even as long as 12 weeks a lower degree of freezing tolerance (10% survival at –15°C) was obtained. Plants regenerated from embryos hardened maximally by a combination of either ABA or MFD with low temperature did not require further vernalization for flowering.Abbreviations ABA abscisic acid - MFD mefluidide - 2,4-D 2,4-dichlorophenoxyacetic acid - LT₅₀ killing temperature for 50% of the embryos     

Contribution of de novo protein synthesis to the hypertrophic effect of IGF-1 but not of thyroid hormones in adult ventricular cardiomyocytes

Background: Enhanced expression of IGF-1 occurs in left ventricular hypertrophy (LVH) associated with systemic hypertension. Cardiac dysfunction accompanied by LVH is also observed in hyperthyroidism. Objective: to assess the relative contributions of de novo protein synthesis and attenuated protein degradation to increased protein mass associated with cardiomyocyte hypertrophy elicited by IGF-1 and thyroid hormones (tri-iodo thyronine T₃, and l-thyroxine T₄), respectively. Methods: total mass of protein, and both the incorporation, and removal of previously incorporated l-U-¹⁴C-phenylalanine, indices of protein synthesis and degradation, respectively, were assessed in quiescent adult rat ventricular cardiomyocytes maintained in short-term culture, and corrected for DNA content, as a index of cell number. Results: IGF-1 (1 pM-100 nM) increased cell protein significantly, maximally at 1 nM and by 38% above basal value after 24 h. T₃ (10 pM-2 M) and T₄ (10 pM-2 M) increased cell protein significantly maximally at 1 M and by 33.2 and 30.5%, respectively, above basal value. IGF-1 ( 10 pM), T₃ (10 pM-2 M) and T₄ (10 pM-2 M) did not increase incorporation of l-U-₁₄C-phenylalanine above basal values. IGF-1 (100 pM-100 nM) increased incorporation of radiolabel significantly maximally at 100 nM and by 56%. T₄ (100 pM) and IGF-1 (10 pM), concentrations that did not stimulate de novo protein synthesis, attenuated the degradation of radiolabelled protein by 13.6 and 11.8%, respectively, compared to control values after 48 h. Conclusion: These data indicate that the acute hypertrophic response to (i) thyroid hormones cannot be attributed to initiation of de novo protein synthesis; (ii) IGF-1 comprises two components; the response elicited by IGF-1 (< 10 pM) is independent of, while the response elicited by IGF-1 (> 100 pM) is due to de novo protein synthesis.     

Hydrocortisone regulates arylsulfatase A (cerebroside-3-sulfate-3-sulfohydrolase) by decreasing the quantity of the enzyme in cultures of cells dissociated from embryonic mouse cerebra

Previous work from our laboratory (Biochem. J. 219:689–697 (1984) had shown that hydrocortisone stimulated the net accumulation of the myelin-specific sulfolipid in cultures of cells dissociated from embryonic mouse cerebra. This accumulation caused by hydrocortisone was shown to be due to a decrease of sulfolipid degradation by arylsulfatase A (ASA) and not due to a stimulation of its synthesis by a sulfotransferase. Both ASA activity and the turnover of sulfolipid were decreased by hydrocortisone to 60–62% of untreated cells. In current work the same decrease in enzyme activity was obtained and enzyme linked immunosorbent assays demonstrate that hydrocortisone decreased the number of ASA protein molecules to 61% of untreated cells [(-)hydrocorcortisone 0.31±0.06 ng ASA/g protein; (+)hydrocortisone: 0.18±0.04 ng ASA/g protein]. This decrease in the number of ASA molecules correlates well with the decrease in both the enzyme activity and the sulfolipid turnover, which suggests that the major mode of inhibition of ASA activity by hydrocortisone involves a decrease in the concentration of ASA in the cells rather than some other mechanism of inhibition.The material in this paper has been included in a dissertation submitted by A.J.M. in partial fulfillment of the requirements for the degree of Doctor of Philosophy. Temple University.     

Regulation of urea uptake by ammonia in the cyanobacterium Anabaena doliolum

Abstract The urea uptake system was studied with regard to its repression and derepression in the cyanobacterium, Anabaena doliolum . The uptake of urea was energy-dependent and was repressed in ammonia grown cells. Repression of the urea uptake by ammonium did not require ammonium assimilation or de novo protein synthesis, suggesting that ammonium itself was the repressor signal. The derepression of the urea uptake system, however, required de novo protein synthesis and glutamine synthetase activity.     

Arginine vasopressin increases the rate of protein synthesis in isolated perfused adult rat heart via the V1 receptor

The effect of daidzein on cortical bone in vitro was investigated. Femoral-diaphyseal tissues obtained from elderly female rats were cultured for 24 h in Dulbecco's modified Eagle's medium (high glucose, 4.5%) supplementation with antibiotics and bovine serum albumin. The experimental cultures contained 10-7 to 10-5 M daidzein. The presence of daidzein (10-6 and 10-5 M) caused a significant increase of alkaline phosphatase activity, deoxyribonucleic acid (DNA) and calcium contents in bone tissues. This effect was equal to that of genistein (10-6 and 10-5 M). Daidzein (10-5 M) or genistein (10-5 M)-induced increase of calcium content and alkaline phosphatase activity in bone tissues was completely prevented by cycloheximide (10-6 M), an inhibitor of protein synthesis. Anabolic effect of daidzein and genistein on bone components was equal to that of 17-estradiol (10-8 M). The effect of isoflavohoids was not enhanced by the addition of 17-estradiol. The combination of daidzein and genistein did not have an additive effect. These findings indicate that daidzein has an anabolic effect on bone metabolism in tissue culture in vitro, and that this effect is equal to genistein effect. Isoflavonoids may stimulate bone formation and mineralization.     

Interaction between aflatoxin B1 and oxytetracycline in isolated rat hepatocytes

Isolated rat hepatocytes were used as an in vitro model to investigate A possible interaction between oxytetracycline (OXT) and aflatoxin B₁ (AFB₁). LDH leakage, RNA and protein synthesis and glycogen accumulation were measured in the presence of both drugs, either separately or in combination. The evolution of LDH leakage during the incubation was identical in untreated and treated cells. AFB₁ inhibited RNA and protein synthesis at a concentration of 10^–7 M and 10^–6 M, respectively, and higher, whereas OXT did not influence RNA synthesis but inhibited protein synthesis at the highest tested concentration, 10^–3 M. As far as glycogen is concerned, rats were injected with glucagon before sacrifice in order to obtain a constant synthesis rate in isolated hepatocytes. AFB₁ inhibited the accumulation of glycogen from 10^–6 M upward. This effect was never observed before 90 min of incubation. OXT had no effect on glycogen synthesis. In the presence of both drugs, no interaction was demonstrated as far as RNA and protein synthesis were concerned, but OXT opposed the inhibition induced by AFB₁ on glycogen accumulation. If the in vivo protection, provided by OXT against AFBI-induced toxicity, is due to a direct interference in the toxic mechanisms of the mycotoxin, these results show that OXT does not influence the AFB₁-inhibition of RNA and protein synthesis. The latter are early and sensitive parameters inhibited by AFB₁. On the contrary, taking into consideration the results on glycogen accumulation, it seems more interesting to investigate further this metabolism.Abbreviations AFB₁ Aflatoxin B₁ - OXT Oxytetracycline - DMEM Dulbecco's Modified Eagle's Medium - LDH Lactate Dehydrogenase - DMSO Dimethyl Sulfoxide - BSA Bovine Serum Albumin     

Cooperation of Hepatocytes in vitro in the Rhythm of Protein Synthesis is Promoted by the GM1 Ganglioside Contained in Vesicles and Liposomes

The medium conditioned by dense, self-synchronized hepatocyte cultures was centrifuged at 150000 g to obtain two fractions. The light fraction (supernatant fluid) contained ganglioside monomers and micelles, and the heavy fraction (pellet) contained gangliosides in the vesicles shed from the cell membrane. In the test populations of hepatocytes, the rhythm of protein synthesis was used as an indicator of cell synchronization resulting from their cooperative activity. Diluted hepatocyte cultures with asynchronous fluctuations of protein synthesis proved to be synchronized by both the initial conditioned medium and its vesicular fraction. Our previous studies have shown that this occurs under the effect of GM1 monosialoganglioside, which is released from cultured cells and accumulated in the conditioned medium. Liposomes consisting of GM1 and phosphatidylcholine from egg yolk (1 : 19 mol%), compared to free exogenous GM1, synchronized the rhythm of protein synthesis more effectively: synchronization was observed at a GM1 concentration in liposome suspension of only 0.0003 M, compared to 0.06 M and higher in the case of free GM1. Thus, GM1 as a component of membranes and monolayer lipid structures proved to be much more effective than free GM1 in promoting hepatocyte cooperation with respect to the rhythm of protein synthesis.     

The effect of lactogenic hormones on protein synthesis and amino acid uptake in rat mammary acinar cell culture at various physiological stages

• 1.1. The activities of protein synthesis and amino acid uptake at various physiological stages were determined by the incorporation of radioactive materials ([³H]-lysine, [¹⁴C]-cycloleucine) in rat mammary epithelial cell cultures. The activity of protein synthesis and amino acid uptake was higher in early lactation than in virgin, pregnant and late lactation stages.
• 2.2. Lactogenic hormones (prolactin, hydrocortisone and insulin) treatment related with mammary growth and differentiation increased the activities of protein synthesis and amino acid uptake. But increase of these activities was different at each physiological stage.
• 3.3. The effect of prolactin and hydrocortisone on the activities were greater in virgin, pregnant and late lactation than in early lactation. And effect of insulin was greater in pregnant and early lactation than in virgin and weanling.

     

Studies on the dependence of chlorophyll synthesis on protein synthesis in Euglena gracilis,together with a nomogram for determination of chlorophyll concentration

Summary Experiments have been carried out to determine the basis for the dependence of chloroplast pigment synthesis on protein synthesis in dark-grown cells of Euglena gracilis greening in the light. The complete inhibition of chlorophyll synthesis brought about by actidione (10 g/ml) when added half way through the greening process was not relieved, even to the slightest extent, when 0.01 M -aminolaevulinic acid (ALA) was also present. The much smaller inhibition of chlorophyll synthesis brought about by chloramphenicol (2 mg/ml) was also relieved little, if at all, by the addition of ALA. It is concluded that the inhibition of chlorophyll synthesis by actidione can not be solely or primarily due to lack of ALA resulting from the decay of possibly labile enzymes of ALA synthesis, but could be due to inhibition of synthesis of the thylakoid structural protein. The results obtained with chloramphenicol are difficult to interpret because of the possibility that the drug, at high concentration, directly inhibits processes other than protein synthesis.Chlorophyll and carotenoid synthesis by E. gracilis were both markedly stimulated by the addition of ALA. It is suggested that the rate of chlorophyll synthesis in the greening cells is limited by the rate of formation of ALA. The stimulation of formation of carotenoids as well as chlorophyll may indicate that the cells have a mechanism for ensuring that the rate of carotenoid synthesis does not fall below a certain proportion of the rate of chlorophyll synthesis.A nomogram has been devised from which the concentrations of chlorophylls a and b, and total chlorophyll can be read off once the absorbances of an 80% acetone extract at 663 and 645 m have been determined.     

The effect of triiodothyronine on glucose utilization in adipocytes from obese rats

• 1.1. In the presence of insulin, 10⁻⁵ M 3,3',5-triiodothyronine (T₃) treatment for 1/2 hr decreased fatty acid synthesis 35% only in adipocytes from lean rats, whereas at 10⁻¹¹ M through 10⁻⁷M T₃ the obese adipocytes had nearly a 20% increase in fatty acid synthesis.
• 2.2. A 2 hr pretreatment of adipocytes with 10⁻⁹ and 10⁻⁷ M T₃ decreased insulin-stimulated fatty acid synthesis by nearly 20% in both lean and obese adipocytes.
• 3.3. In the absence of insulin, the 2 hr pretreatment with 10⁻⁹ M T₃ resulted in a 45% increase in lean adipocyte fatty acid synthesis, though the obese adipocytes required at least 10⁻⁷ M T₃ for 2 hr to increase the non-insulin-stimulated fatty acid synthesis by 50%.
• 4.4. At 10⁻⁹M T₃ concentrations non-insulin-stimulated fatty acid synthesis was increased by 200% in lean adipose tissue explants, but obese adipose expiants were not significantly affected under these conditions.
• 5.5. The addition of 10⁻⁹ M T₃ plus insulin to the explant media decreased fatty acid synthesis by 35% in both the lean and obese tissues.
• 6.6. The results also imply that the low T₃ status of the obese rat may be contributory to the elevated fatty acid synthesis observed in obese adipocytes.

     

Program of early development in the mammal: Synthesis and intracellular migration of histone H4 during oogenesis in the mouse

Synthesis of histone H4 by mouse oocytes and unfertilized eggs has been examined by using a modified high-resolution two-dimensional gel electrophoresis procedure capable of resolving basic proteins (M. J. LaMarca and P. M. Wassarman, 1979, Develop. Biol.73, 103–119). Histones were separated on such gels and observed rates of incorporation of [³⁵S]methionine into histone H4 were converted into absolute rates of synthesis by using previously determined values for the absolute rates of total protein synthesis in mouse oocytes and unfertilized eggs Schultz et al., 1979a, Schultz et al., 1979b. Histone H4 was synthesized at all stages of oogenesis examined, and accounted for 0.07, 0.05, and 0.04% of total protein synthesis in growing oocytes, fully grown oocytes, and unfertilized eggs, respectively. During oocyte maturation the absolute rate of histone H4 synthesis decreased by about 40%, as compared to a 23% decrease in the rate of total protein synthesis during the same period. These measurements indicate that enough histone is synthesized during oogenesis in the mouse to support two to three cell divisions. Examination of the intracellular location of newly synthesized proteins in fully grown oocytes revealed that histone H4 was highly concentrated in the nucleus (germinal vesicle), whereas total protein and tubulin were not. Nearly 50% of the histone H4 synthesized during a 5-hr period was located in the oocyte's germinal vesicle, as compared to 1.9 and 0.9% for total protein and tubulin, respectively. These results are compared with those obtained using oocytes and eggs from nonmammalian animal species.