Effects of Various Rates of Freezing on the Metabolism of a Drought-tolerant Plant, the Moss Tortula ruralis

The response of the drought-tolerant moss Tortula ruralis ([Hedw.] Gaertn., Meyer, Scherb.) to freezing and thawing at controlled rates has been studied. Slow freezing (at 3 C per hour to −30 C) of hydrated T. ruralis leads to only temporary, reversible changes in metabolism. These changes can be considered to result from desiccation due to extracellular ice formation. In contrast, rapid freezing in liquid N₂ and thawing in 20 C water leads to deterioration in all aspects of metabolism studied: ribosome, protein, and ATP levels decrease, and in vivo and in vitro protein synthetic activity is lost rapidly. Such changes probably result from intracellular ice formation. Following freezing and thawing at an intermediate rate (60 C per hour), only ATP levels and in vivo protein synthesis are reduced. The protein-synthesizing apparatus (the polyribosomes) remains intact and active in an in vitro protein-synthesizing system even 24 hours after one 60 C per hour freeze-thaw cycle. These metabolic responses are discussed in terms of the two-factor hypothesis of Mazur et al. (1972 Exp. Cell Res. 71: 345-355).     

Stimulation of yeast mitochondrial protein synthesis by postpolysomal supernatants from yeast,rat liver,and Escherichia coli

Isolated yeast mitochondria incubated with a protein-synthesizing mixture containing excess oxidizable substrate, amino acids, MgCl₂, an ATP-regenerating system, and optimal levels of [³H]leucine cease protein synthesis after 30 min. Postpolysomal supernatants from either yeast, rat liver, or Escherichia coli can restore protein synthetic activity to depleted yeast mitochondria; however the addition of bovine serum albumin to the incubation mixture did not restore activity. The restored incorporation activity was sensitive to chloramphenicol, insensitive to cycloheximide, and proportional to the protein concentration of the supernatants. Furthermore, addition of all three high-speed supernatants to isolated mitochondria at time zero stimulated the rate of protein synthesis to a greater extent than when these fractions were added to depleted mitochondria. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed that the translation products obtained from mitochondria labeled in vitro in the presence of supernatant fractions were identical to the proteins labeled by mitochondria in vivo; however, the synthesis of the bands corresponding to subunit III of cytochrome oxidase, cytochrome b, and VAR-3 was stimulated to the greatest extent. The stimulatory activity in the supernatants was non-dialyzable, insensitive to treatment with ribonuclease A, but completely abolished by pretreatment with trypsin suggesting that the stimulatory factor(s) is of a protein nature. The postpolysomal supernatants did not incorporate amino acids into protein when incubated without mitochondria. These results suggest that the protein synthetic capacity of mitochondria is apparently limited by extramitochondrial proteins which are present in either yeast, rat liver, or E. coli.     

Measurement of fluorescent lipid peroxidation products in biological systems and tissues

A sensitive fluorometric assay for measurement of lipid peroxidation damage to biological preparations and tissues is described. The method consists of a chloroform-methanol extract of tissue followed by measurement of fluorescence characteristics of products derived from lipid peroxidation. Fluorescence analysis has been used successfully with rats and mice in vivo with stress induced during dietary and aging experiments and in peroxidation of subcellular organelles in vitro. Replicate extractions of fluorescent tissue showed a standard error of 2%. In a test of linearity with concentration, the amount of lipid-soluble fluorescent material in extracted samples was directly proportional to that added before extraction. Specific and general applications to in vivo and in vitro lipid peroxidation experiments are diseussed.     

Plant Desiccation and Protein Synthesis: II. On the Relationship between Endogenous Adenosine Triphosphate Levels and Protein-synthesizing Capacity

Rehydration of Tortula ruralis in 2,4-dinitrophenol inhibits protein synthesis, polysome formation, and ATP production. Polysomes are conserved intact and are active in vitro in hydrated Tortula placed in this chemical, although in vivo protein synthesis is inhibited. Hydrated moss placed under nitrogen in the dark shows a reduced capacity for ATP and protein synthesis, but polysomes are conserved. During anaerobiosis in light, ATP and protein synthesis are unaffected. Rehydration of slow-dried Tortula in nitrogen in the dark results in reduced in vivo protein synthesis, but not polysome formation; this reduction is much less in the light. Slow-dried moss, but not fast-dried, has a greatly reduced ATP content in the dry state, but this rapidly returns to normal levels on rehydration. The prolonged burst in respiration observed previously on rehydration of Tortula is not paralleled by ATP accumulation. Changes in energy charge in all treatments tested follow the changes in ATP. The aquatic moss, Hygrohypnum luridum, which is intolerant to drought, loses ATP during fast drying and this is not replenished on subsequent rehydration.     

Lipid peroxidation induced by bolesatine,a toxin ofBoletus satanas: Implication in m5dC variation in Vero cells related to inhibition of cell growth

Bolesatine, a glycoprotein fromBoletus satanas Lenz, has previously been shown to be mitogenic in rat and human lymphocytes at very low concentrations, whereas higher concentrations inhibited protein synthesisin vitro and in severalin vivo systems. The low concentrations (1–10 ng/ml) of bolesatine were shown to activate protein kinase C (PKC)in vitro (cell-free system) and in Vero cells. In the same time, Vero cells significantly proliferated when incubated with bolesatine concentrations ranging from 1 to 10 ng/ml; the DNA synthesis increased by 27–59% as referred to the control, and InsP3 release increased in a concentration-dependent manner, up to 142%. At higher concentrations, 1–10 g in cell-free systems, bolesatine inhibits protein synthesis by hydrolyzing the nucleoside triphosphates GTP and ATP.In the present work, the implication of other toxic mechanisms, such as lipid peroxidation and active radical production, was investigated in relation to inhibition of cell growth, whereas possible modifications of the ratio m⁵dC/dC+m⁵dC were determined in order to correlate with the biphasic action of bolesatine in Vero cells.Low concentrations of bolesatine up to 10 ng/ml do not increase malonaldehyde (MDA) production, while they induce hypomethylation (5.2% as compared to 7.1%). Higher concentrations (above 20 ng/ml) increase MDA production, from 58 ng/mg of cellular proteins to 113 ng/mg at a concentration of 50 ng/ml, for example, and induce hypermethylation in Vero cell DNA. It is concluded that low concentrations of bolesatine that are proliferative induce hypomethylation, which could be one of the pathways whereby bolesatine induces cell proliferation. Higher concentrations which enhance lipid peroxidation also induce hypermethylation. These mechanisms could be at least partly implicated in the pathway whereby bolesatine induces cell death.Abbreviations MDA malonaldehyde - TBA thiobarbituric acid     

Protein Synthesis during Rehydration of Rapidly Dried Tortula ruralis: Evidence for Oxidation Injury

Rapidly dried Tortula ruralis, a drought-tolerant moss, is known to synthesize proteins on rehydration at a much lower rate than the slowly dried moss. The reasons for this low rate of protein synthesis are unclear. We have found that during rehydration of rapidly dried moss, there is a negative correlation between the rate of protein synthesis and the tissue levels of oxidized glutathione (GSSG) and lipid peroxidation. When rapidly dried moss, which is known to show extensive solute leakage, is rehydrated in the presence of 100 millimolar K⁺, 5 millimolar Mg²⁺, 1 millimolar ATP, and 1 millimolar GTP, either separately or together, there is no stimulation of protein synthesis. When it is hydrated in the presence of either 5 millimolar glucose-6-phosphate or 0.1 millimolar NADPH, protein synthesis is stimulated but the stimulation is transitory. A second addition of either of these two chemicals causes a second transient stimulation of protein synthesis. A transitory decrease in the rate of GSSG accumulation is observed during rehydration in the presence of glucose-6-phosphate or NADPH. Both glucose-6-phosphate and NADPH are known to reverse GSSG-induced inhibition of protein synthesis in rabbit reticulocyte lysate. Results of the present study suggest that the rate of protein synthesis during rehydration of rapidly dried moss is not limited by the availability of ions or energy sources. Since exogenously applied GSSG has been shown to inhibit in vivo and in vitro protein synthesis and since it is known to accumulate during rehydration of rapidly dried, but not slowly dried, moss, it is suggested that the low rate of protein synthesis during rehydration of the rapidly dried moss is, at least in part, due to endogenous GSSG.     

Glycosylation of nascent polypeptide chains in Aspergillus niger

Polysomes were isolated from Aspergillus niger and were characterized on sucrose gradients in several ways. First, they were found to be susceptible to degradation by treatment with RNase or EDTA. Second, they were labeled after treating mycelia with short pulses of [³H]uridine or [³H]leucine prior to polysome isolation. Third, they were capable of stimulating incorporation of [³H]leucine into trichloroacetic acid-precipitable material in a chick reticulocyte cell-free protein-synthesizing system. When isolated [³H]leucine pulse-labeled polysomes were treated with either EDTA-RNase or puromycin, 80–90% of the radioactivity was released, indicating that only the nascent polypeptide chains were labeled. After exposing mycelia for 1 min to [¹⁴C]mannose, the polysomes were exclusively labeled, indicating that initial glycosylation takes place on nascent polypeptide chains. Preincubation of mycelia with 2-deoxyglucose followed by pulse-labeling with [³H]leucine and [¹⁴C]mannose showed that 2-deoxy-d-glucose inhibits both protein synthesis and glycosylation. However, similar preincubation with tunicamycin caused an 80% drop in [¹⁴C]mannose label in the polysomes, but only a 10–20% drop of [³H]leucine label, suggesting that glycosylation of nascent chains in A. niger involves an oligosaccharide-lipid intermediate, since it has been shown that tunicamycin inhibits the synthesis of such an intermediate. When isolated polysomes were placed into an in vitro glycosylating mixture containing Mn²⁺, GDP-[¹⁴C]mannose, and smooth membranes from A. niger nascent chains were labeled. This reaction was shown to be dependent on addition of polysomes to the mixture and was not inhibited by 2-deoxy-d-glucose or tunicamycin. Both in vivo and in vitro glycosylated nascent chains were found to have about the same size range, and so it is suggested that in vitro no new oligosaccharide chains were synthesized, but preexisting chains were extended.     

BIOSYNTHESIS OF COLLAGEN AND NON-COLLAGEN PROTEIN DURING DEVELOPMENT OF THE CHICK CORNEA

The relationship between the rates of increase of corneal protein fractions and incorporation of labeled precursors has been examined during embryonic and early posthatching development of the chick corneal stroma. Non-collagen protein increased gradually from 9 through 20 days of incubation. Collagen accumulated approximately logarithmically through the 19th day, the most rapid rate occurring between 13 and 20 days of incubation. The rates at which labeled amino acids are incorporated into collagen in vivo and in vitro undergo marked changes during the last week of embryonic development, corresponding closely to the rate of collagen accumulation in vivo; whereas incorporation into non-collagen protein changes much less markedly. Changes in the rate of incorporation of precursors into collagen are not due to changes in the rate of conversion of collagen from the soluble to insoluble form, or to changes in the endogenous amino acid pool size. Chick embryo corneal stroma collagen turns over very slowly, if at all. Non-collagen protein turns over more rapidly. An increase in cell number, as indicated by DNA content, does not account for the increased rate of collagen synthesis between the 9th and 16th day of incubation. It is concluded that the observed changes in collagen synthesis reflect changing activities in the individual cornea fibroblasts. These activities are comparable in the intact tissue in vivo and in isolated corneas in vitro.     

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes limitations on potential protein applications. The incorporation of noncanonical amino acids can enlarge this scope. There are two complementary approaches for the incorporation of noncanonical amino acids. For site-specific incorporation, in addition to the endogenous canonical translational machineries, an orthogonal aminoacyl-tRNA-synthetase-tRNA pair must be provided that does not interact with the canonical ones. Consequently, a codon that is not assigned to a canonical amino acid, usually a stop codon, is also required. This genetic code expansion enables the incorporation of a noncanonical amino acid at a single, given site within the protein. The here presented work describes residue-specific incorporation where the genetic code is reassigned within the endogenous translational system. The translation machinery accepts the noncanonical amino acid as a surrogate to incorporate it at canonically prescribed locations, i.e., all occurrences of a canonical amino acid in the protein are replaced by the noncanonical one. The incorporation of noncanonical amino acids can change the protein structure, causing considerably modified physical and chemical properties. Noncanonical amino acid analogs often act as cell growth inhibitors for expression hosts since they modify endogenous proteins, limiting in vivo protein production. In vivo incorporation of toxic noncanonical amino acids into proteins remains particularly challenging. Here, a cell-free approach for a complete replacement of L-arginine by the noncanonical amino acid L-canavanine is presented. It circumvents the inherent difficulties of in vivo expression. Additionally, a protocol to prepare target proteins for mass spectral analysis is included. It is shown that L-lysine can be replaced by L-hydroxy-lysine, albeit with lower efficiency. In principle, any noncanonical amino acid analog can be incorporated using the presented method as long as the endogenous in vitro translation system recognizes it.     

Membrane lipid peroxidation, nitrogen fixation and leghemoglobin content in soybean root nodules

Membrane lipids in soybean nodules may undergo oxidative degradation resulting in the loss of membrane structural integrity and physiological activities. One of the final products of lipid peroxidation is malondialdehyde (MDA), which can react with thiobarbituric acid (TBA) in vitro to form a chromogenic adduct, a Schiff base product that can be measured spectrophotometrically. MDA formation was quantified in the nodules as well as in the adjacent root tissue. Lipid peroxidation was initially high in soybean nodules induced by Bradyrhizobium japonicum, but sharply declined following an increase in both leghemoglobin content and nitrogen fixation rate. Lipid peroxidation was 2 to 4 times higher in the nodules than in their corresponding adjoining root tissue. Malondialdehyde levels in ineffective nodules were 1.5 times higher than those in effective nodules. MDA formation was also shown to occur in the ‘leghemoglobin-free’ cytosolic fraction, the ‘leghemoglobin’ fraction, and the nodule tissue pellet. Antioxidants, such as reduced ascorbic acid, glutathione, and 8-hydroxyquinoline, caused a partial suppression of lipid peroxidation, whereas ferrous sulfate, hydrogen peroxide, iron EDTA, disodium-EDTA, and β-carotene induced MDA formation. In contrast, quenchers of oxygen free radicals such as HEPES, MES, MOPS, PIPES, phenylalanine, Tiron, thiourea, sodium azide, and sodium cyanide (uncouplers of oxidative phosphorylation) caused somewhere between a 12 to 70 percnt; reduction in MDA production. TBA-reactive products were formed despite the incorporation of superoxide dismutase, proxidase, and catalase into the reaction mixture.     

Inhibitory effects of α- and β-carotene on croton oil-induced or enzymatic lipid peroxidation and hydroperoxide production in mouse skin epidermis

• 1.1. The effects of carotenes (α- and β-) on edema, MDA contents and peroxidizability ofcroton oil-treated mouse skin epidermis, hydroperoxide production and enzymatic lipid peroxidation of epidermal homogenates were studied. Edema was determined as ear punch weight and the intensity of lipid peroxidation was measured using malondialdehyde formation.
• 2.2. Carotenes (α- and β-) significantly suppressed edema formation, hydroperoxide production, lipid peroxidation caused by croton oil, Fe + 3-ADP/NADPH or paraquat/NADPH in vivo as well as in vitro.
• 3.3. These results indicate that both α- and β-carotene have chemopreventive effects on croton oil-induced tumor promotion in skin tumorigenesis by scavenging oxygen free radicals, indirectly determined as carotene inhibition of lipid peroxidation and hydroperoxide formation.

     

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.     

The inhibition of protein synthesis in a rat brain system by ethionine in vitro and in vivo

Abstract— An amino acid incorporating system from rat brain has been used to study in vitro four aspects of protein synthesis: amino acid-AMP-enzyme complex formation; amino acid-tRNA synthesis; amino acid incorporation into protein and protein synthesis from presynthesized amino acid-tRNA. Ethionine (0.5 mm ) inhibited the system and the inhibition appeared to be in the formation of amino acid-tRNA. The inhibition in vitro was independent of the sex of the animal from which the system was derived. Pretreatment of animals in vivo with ethionine yielded in females only preparations deficient in incorporating capacity when tested in vitro. Exchange experiments demonstrated that the defect was in the pH 5 enzymes and not in the ribosomes. The inhibition in vitro was not reversed by addition of ATP and appeared to be competitive with the amino acid substrate.     

Ketone-body utilization and lipid synthesis by developing rat brain—a comparison between in vivo and in vitro experiments

The distribution of ketone bodies between oxidation and lipid synthesis was analysed in homogenates of developing rat brain. The capacity for lipid synthesis of homogenized or minced brain preparations was compared with rates of lipid synthesis in vivo, assessed by incorporation of ³H from ³H₂O into fatty acids and cholesterol. Brain homogenates of suckling rats (but not those of adults) incorporated label from [3-¹⁴C]ketone bodies into lipids, but this process was slow as compared to ¹⁴CO₂ production (< 5%) and much slower than the total rate of ketone-body utilization (< 0.5%). Study of ³H₂O incorporation demonstrated that the rates of lipogenesis and cholesterogenesis are at least one order of magnitude higher in vivo than in vitro. Maximal rates of ³H incorporation into fatty acids (3 μmol/g brain . h) and into cholesterol (0.6 μmol/g brain . h) were found during the third postnatal week. Adult rats still incorporated ³H into brain fatty acids at an appreciable rate (1 μmol/g brain . h), whereas cholesterogenesis was very low. It is concluded that in vitro measurements of lipid synthesis severely underestimate the rates that occur in developing rat brain in vivo. The high rate of ³H incorporation into lipids by developing and adult rat brain as compared to the amounts of these lipids present in the brain suggests an important contribution of endogenous lipid synthesis during brain development and an appreciable rate of fatty acid turnover during brain growth, but also in the adult brain.     

Water Stress and Protein Synthesis: V. Protein Synthesis, Protein Stability, and Membrane Permeability in a Drought-sensitive and a Drought-tolerant Moss

The effects have been studied of water stress and desiccation on protein synthesis in the drought-tolerant moss Tortula ruralis and the drought-sensitive moss Hygrohypnum luridum. At any particular level of steady state water stress, the inhibition of protein synthesis was greater in H. luridum than in T. ruralis. Water stress-induced changes in the pattern of protein synthesis, as determined by the double label ratio technique, were minor in T. ruralis, but major in H. luridum. Proteins of both mosses were found to be stable during desiccation and subsequent rehydration. Changes in membrane permeability, as indicated by the leakage of amino acid, were observed during rehydration of desiccated moss and were dependent on the rate of desiccation. The leakage was small and reversible in T. ruralis but large and irreversible in H. luridum. Although H. luridum failed to recover from complete desiccation (80% loss in fresh weight), it was able to recover fully from steady state stress under conditions where a maximum loss of 55% in fresh weight was recorded.     

Plant Desiccation and Protein Synthesis : VI. Changes in Protein Synthesis Elicited by Desiccation of the Moss Tortula ruralis are Effected at the Translational Level

Upon rehydration of the moss Tortula ruralis following desiccation at a rapid or slow rate, there is increasing utilization of newly synthesized-poly(A)⁺ RNA for protein synthesis. Initially, poly(A)⁺ RNA conserved in the dry moss is associated with polysomes, but by 2 hours of rehydration there is an overwhelming recruitment of newly synthesized poly(A)⁺ RNA, at the expense of conserved messages. In rehydrated moss, there is a marked synthesis in vivo of new proteins, which are separable by two-dimensional electrophoresis, and identifiable by fluorography. These new proteins, termed rehydration proteins, are synthesized after both rapid and slow desiccation, but their synthesis persists longer after rapid desiccation. The protein patterns obtained following in vitro translation of bulk RNA from hydrated, desiccated, and rehydrated moss were qualitatively identical. Thus the differences in protein patterns observed in vivo must result from preferential selection of specific mRNAs from the same pool, which is indicative of control of protein synthesis at the translational level. The implications of these observations in relation to the response of the moss to drying in its natural environment are discussed.     

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.     

Isolation and characterization of ribosomes from thoraces of the sheep blowfly,Lucilia cuprina,and their capacity for protein synthesis at different stages of development

The isolation and characterization of ribosomal fractions from thoraces of Lucilia cuprina have been described and their capacity for in vitro protein synthesis studied at different stages of development. There are large differences in the yield of ribosomes during development, and these probably reflect differences in the number of ribosomes in vivo. The amino acid incorporation activity varied at different stages of development and this variation correlated with the percentage of polysomes present. Maximal activity was found just after adult emergence. Results obtained by the use of supernatant fractions from different stages of development suggest that control of protein synthesis in this insect takes place at the level of translation.