Hydrostatic Pressure Effects on Protein Synthesis

The effects of high hydrostatic pressure on several phases of cell-free protein synthesis have been examined. The initial rate of polyuridylic acid (poly U)-directed synthesis of polyphenylalanine showed an apparent increase at 100 atm, above which the synthetic rate was reduced sharply with increased pressure up to 640 atm where 95% inhibition was observed. The magnitude of the inhibition of polyphenylalanine synthesis with increased pressure depended strongly on the magnesium salt concentration in the reaction system. Misreading of the poly U message, as measured by insertion of leucine in place of phenylalanine, dropped rapidly with increased pressure from 1 to 350 atm, above which the amount of misreading increased. Enzymatic activation of transfer RNAs (tRNAs) was reduced by increased pressure in the range 100-640 atm, where the rate of tRNA aminoacylation was 80% inhibited. Both nonenzymatic attachment of phenylalanyl-tRNA (phe-tRNA) to the poly U-ribosome complex and stability of the phe-tRNA-poly U-ribosome complex were decreased at high pressures (100-900 atm). The results of the action of pressure on the various phases of cell-free protein synthesis suggest that the major pressure-sensitive element in the protein synthetic machinery is the ribosome.     

Inhibition of Cell-Free Protein Synthesis by Hydrostatic Pressure

Pressure inhibition of cell-free polypeptide synthesis is manifested in the same manner as that observed in the intact cell: (i) starting at approximately 200 atm, there is a progressive inhibition with increasing pressures; (ii) there is complete inhibition at 680 atm; (iii) incorporation into polypeptide is instantaneously reversible after pressure release and proceeds at a rate parallel to an atmospheric control; and (iv) the volume change of activation (DeltaV*) is 100 cm(3)/mole. Peptide bond formation per se can occur at a pressure level which is totally inhibitory to polypeptide synthesis. The one investigated step in translation that is inhibited in an identical manner is the binding of aminoacyl-transfer ribonucleic acid (AA-tRNA) to the ribosome-messenger RNA (mRNA) complex. The volume change of activation (DeltaV*) calculated for the binding reaction is also 100 cm(3)/mole. Thus, the inability of AA-tRNA to bind to ribosomes and mRNA under pressure, possibly in conjunction with translocation, appears to be responsible for the observed inhibition of the translational mechanism.     

Reversal of the inhibitory effects of the pokeweed antiviral protein upon protein synthesis

Protein synthesis directed by natural mRNA is more sensitive to the inhibitory action of the pokeweed antiviral protein than synthesis directed by poly(uridylic acid). Investigations into the nature of this difference revealed that pokeweed antiviral protein does not inhibit the initiation stage of protein synthesis and that the expression of pokeweed antiviral protein inhibition is dependent upon the K⁺ and Mg²⁺ concentrations used in the protein synthesis assay. Ribosomes treated with pokeweed antiviral protein function as efficiently as untreated ribosomes if assayed at either high Mg²⁺ or low K⁺ concentrations. The influence of ionic conditions upon the individual elongation factor reactions shows that pokeweed antiviral protein inhibition of the elongation factor two translocation reaction is sensitive to ionic conditions but that the inhibition of the elongation factor one-mediated enzymatic binding is not sensitive to changes in these conditions. The results suggest that the unknown enzymatic effect of pokeweed antiviral protein produces a conformational change in ribosome, which is reversed under conditions which favor a more compact ribosomal structure.     

Effect of high hydrostatic pressure on 86Rb+ influx in the erythrocyte of the plaice (Pleuronectes platessa)

1. 86Rb+ influx in the erythrocyte of the plaice (Pleuronectes platessa) has been measured at hydrostatic pressures between 1 and 600 atm at 10 degrees C. 2. The measurements were performed with an experimental medium containing 1% (w/v) bovine serum albumin. In this medium the cells achieved a steady state level of ionic regulation. 3. At normal atmospheric pressure 46% of the 86Rb+ influx was inhibited by furosemide while 42% was inhibited by ouabain, the remainder being inhibited by neither drug. 4. It was found that all three fluxes defined by these drugs were sensitive to pressure. 5. The ouabain sensitive influx was progressively inhibited by increasing pressure, the inhibition at 600 atm being 30%. 6. The furosemide sensitive influx was inhibited by 35% between 100 and 600 atm. 7. In contrast the ouabain + furosemide insensitive influx was doubled by 400 atm. 8. This pattern of pressure inhibition and stimulation resembles that seen in comparable studies in human erythrocytes.     

A Study of the Effects of Hydrostatic Pressure on Macromolecular Synthesis in Escherichia coli

In cultures of Escherichia coli 15 (thymine^-, leucine^-) which were incubated at high hydrostatic pressures, cell division occurred only at pressures below 430 atm but in a somewhat synchronous fashion at around 250 atm. The rate of leucine-¹⁴C incorporation into a macromolecular fraction of the cells diminished to a zero value at about 580 atm and that of uracil-¹⁴C incorporation to a zero value at about 770 atm. The rate of thymine-¹⁴C incorporation at pressures around 330 atm was that to be expected with a culture in which DNA synthesis is somewhat synchronous. At pressures above 500 atm, thymine-¹⁴C was incorporated only over the initial part of the pressure incubation and further incorporation under pressure was not observed no matter how long the duration of the incubation. We present evidence along several lines that the thymine incorporation kinetics reflect an effect of pressure on a locus at the origin (or termination) of a replication of the bacterial chromosome. The recovery of cell division and of the incorporation rates upon release of pressure were found to depend on the magnitude of the pressure and the duration of the pressure incubation.     

Effect of a continuously applied compressive pressure on mouse osteoblast-like cells (MC3T3-E1) in vitro

Bone metabolism is often affected by a variety of mechanical forces, but the cytological basis of their action is not known. In this study, we examined the effect of a continuously applied compressive pressure (CCP) on the growth and differentiation of clonal mouse osteoblast-like cells (MC3T3-E1) cultured in a specifically devised culture chamber. The gas phase of the chamber was maintained at a pressure of 2 atmospheres (atm) above ambient (3 atm total, 3.1 kg/cm2; 3.0 x 10(5) Pa) by continuously infusing a compressed mixed gas (O2: N2:CO2 = 7.0%:91.3%:1.7%). The pO2, pCO2, and pH in the culture medium at 37 degrees C under 3 atm were maintained at the same levels as those under 1 atm. MC3T3-E1 cells were cultured in alpha-minimal essential medium containing 10% fetal bovine serum under either 3 atm in the CCP culture chamber or 1 atm in an ordinary CO2 incubator. Alkaline phosphatase activity, a marker of osteoblasts, was greatly suppressed by the CCP treatment. The inhibition of alkaline phosphatase activity was rapidly restored when the cells were transferred to an ordinary CO2 incubator under 1 atm, indicating that the inhibition of alkaline phosphatase activity by CCP is reversible. Cell growth was not altered under CCP. The CCP treatment greatly increased the production and secretion of prostaglandin E2 (PGE2). Adding either conditioned medium from the CCP culture or exogenous PGE2 to the control culture under 1 atm suppressed alkaline phosphatase activity dose-dependently. The CCP treatment also suppressed collagen synthesis and calcification. These results suggest that CCP causes the cells to produce and secrete PGE2, which, in turn, inhibits differentiation of osteoblasts and the concomitant calcification.     

Ethanol and acetate synthesis from waste gas using batch culture of Clostridium ljungdahlii

Single inorganic carbon source was used for production of chemicals and fuels via fermentation processes. Clostridium ljungdahlii, a strictly anaerobic autotrophic bacterium, was grown on synthesis gas to produce acetate and ethanol from gaseous substrates. C. ljungdahlii was grown on a various concentrations of carbon monoxide with synthesis gas total pressures of 0.8–1.8 atm with an interval of 0.2 atm. The cell and product yields were 0.015 g cell/g CO and 0.41 g acetate/g CO, respectively. Formation of acetate was steady and the production trend was about the same for all of the gases initial pressure and at constant cell density. The ethanol concentration was enhanced by the initial presence of hydrogen and carbon dioxide in the liquid phase. There was no substrate inhibition while C. ljungdahlii was grown in the batch fermentation, even at high system pressure of 1.6 and 1.8 atm. A desired product molar ratio of ethanol:acetate (5:1) was achieved with total gas pressure of 1.6 and 1.8 atm.     

Enzymatic adaptation by bacteria under pressure.

A study of enzymic adaptation under hydrostatic pressure by moderately barotolerant bacteria that can grow at pressure up to about 500 atm revealed that some adaptive processes are relatively insensitive to pressure, whereas others are sufficiently barosensitive to compromise survival capacity in situations requiring adaptation to new substrates under pressure. Examples of the former include adaptation of Escherichia coli to arabinose catabolism for growth and adaptation of Streptococcus faecalis to catabolism of lactose, ribose, or maltose. Examples of the latter include derepression of the lac operon in Escherichia coli and induction of penicillinase synthesis by Bacillus licheniformis. For both these barosensitive systems, pressure had little effect on enzyme levels in constitutive strains or in bacteria that had previously been induced at 1 atm. Moreover, it had no detectable effect on penicillinase secretion. However, pressures of 300 to 400 atm were found to reduce markedly rates and extents of enzyme synthesis by bacteria undergoing derepression or adaptation. This inhibitory effect of pressure was reflected in greater barosensitivity with extended lag and slower growth of initially unadapted Escherichia coli cells inoculated into minimal medium with lactose as sole source of carbon and fuel, and by major reductions in the minimal inhibitory concentrations of penicillin G for unadapted B. licheniformis cells inoculated into complex, antibiotic-containing media. Cyclic adenosine 5'-monophosphate did not reverse pressure inhibition of derepression of the lac operon, and catabolite repression was complete under pressure. However, derepression of the lac operon was more sensitive to pressure at low concentrations of inducer than at high concentrations. Apparent volume changes for derepression were 94 and 60 ml/mol at inducer concentrations of about 0.5 and 5 mM, respectively. Pressure was found not to be inhibitory for uptake of beta-galactosides; in fact, it was somewhat stimulatory. Therefore, results were interpreted in terms of inducer binding and subsequent conversion of an operator-inducer-repressor complex to inactive repressor and operator. Both reactions appeared to result in an increase in volume, the former more so than the latter. We found also that 200 atm was actually stimulatory for growth of Escherichia coli in minimal media, and the bacterium was in a sense barophilic.     

The pressure tolerance of deep sea amphipods collected at their ambient high pressure

• 1.1. Deep sea benthic amphipods were collected at their normal ambient pressure of 394–442 atm from depths of approximately 4000 m (Parulicella caperesca, Orchomene sp. and other species).
• 2.2. Their activity at their normal pressure and temperature was observed and the responses to a standard pressure test were noted.
• 3.3. Contrary to a prediction derived from the responses of similar animals from lesser depths, the 4000 m amphipods did not convulse at high pressure although they exhibited mild hyperexcitability above 400 atm followed by a progressive inhibition of activity starting at approximately 700 atm.
• 4.4. In failing to convulse at high pressure the amphipods from 4000 m differ radically in their pressure tolerance from those which live at depths down to 2700 m.

     

The effect of helium and of hydrogen at high pressure on the cell division of Tetrahymena pyriformis W.

The rate of cell division of Tetrahymena growing in an observational high pressure vessel was measured at selected pressures of helium, hydrogen and at high hydrostatic pressure. Pressures greater than 100 atm reduced the rate of division, but the gases inhibited division to a lesser degree than pure hydrostatic pressure. Hydrogen's effect was distinguishable from that of hydrostatic pressure at 130 atm or more, while helium's effect appeared at 175 atm. These inert gases probably counteract the action of pressure by stabilising apolar pressure-labile targets.     

Lipid Peroxidation in Rat Brain Cortical Slices as Measured by the Thiobarbituric Acid Test

Abstract: Malonaldehyde formation by cortical brain slices from rat brain was determined as a function of incubation time and of oxygen pressure. This substance, a byproduct of lipid peroxidation, was detected by the thiobarbituric acid test. Significant amounts of malonaldehyde were formed by brain slices during incubation in the 0.2 (air) to 10 atm oxygen range, and a portion of it was released into the medium. The rate of malonaldehyde formation was the highest during the first 10 min. Elevation of oxygen pressure above 1 atm caused further increments in malonaldehyde production with kinetic properties similar to that seen at 1 atm pressure, but the increments per additional oxygen pressure were diminishing. The formation of a given amount of malonaldehyde can be expressed as a function of atm oxygen × min. This function has the shape of a saturation curve approaching a maximum at around 300 atm × min. The results indicate extensive lipid peroxidation in brain slices under standard incubation conditions.     

Inhibition of Acid-Enhanced Elongation of Zea mays Root Segments by Galactose

The effect of sugars and metabolic inhibitors on the elongation of Zea mays root segments was analyzed by a rhizometer which records the elongation of each of 32 root segments at the same time. Galactose suppressed the acid-enhanced rapid elongation after a lag period of 1.5 hours, but it did not inhibit the slow elongation at pH 7. Mannose was less inhibitory than galactose. Arabinose, xylose, glucose, sucrose, mannitol, and sorbitol caused no inhibition. When galactose was removed after a 1-hour treatment, the elongation was partially recovered. Cycloheximide and 2-deoxyglucose suppressed acid-enhanced elongation when these were applied at the same time as acid treatments, whereas cordycepin (3′-deoxyadenosine) inhibited elongation only if it was applied prior to acid treatment. Over the 9-hour period of elongation studied, the inhibition by galactose was comparable to that of cycloheximide. Since galactose has been reported to suppress the sugar metabolism necessary for the cell wall synthesis, the later phase of acid-enhanced elongation of root segments may at least partially depend on the synthesis or metabolism of cell wall components. The inhibition of root growth by galactose may be partially ascribed to a direct effect on the elongation process in roots, an effect that is enhanced by the acidification of the cell walls.     

Effects of hyperoxia on composition and rate of synthesis of fatty acids in Escherichia coli

Growth of and fatty acid synthesis in Escherichia coli were inhibited by oxygen at partial pressures above 1 atm and were prevented by exposure to oxygen at 4.2 atm on membranes incubated on a minimal medium. Growth and fatty acid synthesis returned to control rates when cells were removed from hyperoxia to air. The spectrum of fatty acids produced was unchanged by oxygen at pressures which reduced the rate of synthesis. In situ fatty acids were stable to oxygen at pressures which prevented growth and synthesis. Reinitiation of synthesis after complete inhibition in hyperoxia occurred without production of aberrant fatty acids. Fatty acid synthetase specific activity was virtually unchanged, compared with air controls, in cells exposed either to 3.2 or to 15.2 atm of oxygen. The spectrum of fatty acids synthesized by cell-free extracts during incubation in 4.2 atm of oxygen was not different from air-incubated controls. Synthetase assays included added NADPH, acyl carrier protein, mercaptoethanol, and malonyl coenzyme A; hence, damage, other than reversible sulfhydryl oxidation, to the apoenzymes of synthetase was ruled out.     

Cell division versus cell elongation: the control of radicle elongation during thermoinhibition of Tagetes minuta achenes

Endogenous embryo factors, which act mainly in the radicle, prevent germination in Tagetes minuta at high temperatures. These factors act to prevent cell elongation, which is critical for radicle protrusion under optimal conditions. Once the radicle has emerged both cell elongation and cell division are required for post-germination growth. Germination can be induced at high temperatures by fusicoccin, which rapidly stimulates cell elongation. In addition, priming seeds at 25 °C on polyethylene glycol (PEG) 6000 and mannitol could also induce germination on water at 36 °C, indicating that priming prevents radicle protrusion at a point subsequent to the point of control in thermoinhibited achenes. Flow cytometry studies revealed that DNA synthesis occurs during thermoinhibition and the inhibition of DNA synthesis during this process inhibits subsequent germination on water under optimal conditions, suggesting a protective role for DNA synthesis in thermoinhibited achenes of T. minuta.     

Phospholipid biosynthesis is required for stalk elongation in Caulobacter crescentus.

Membrane phospholipid synthesis was inhibited in Caulobacter crescentus by growth of a glycerol auxotroph in the absence of glycerol or by treatment with the antibiotic cerulenin. It was observed that the final step in the swarmer cell-to-stalked cell transition, stalk elongation, was inhibited under these conditions. Since an early effect of inhibiting phospholipid synthesis in C. crescentus is the termination of deoxyribonucleic acid (DNA) replication (I. Contreras, R. Bender, A. Weissborn, K. Amemiya J. D. Mansour, S. Henry, and L. Shapiro, J. Mol. Biol. 138:401-410, 1980), we questioned whether the inhibition of stalk formation was due directly to the inhibition phospholipid synthesis or secondarily to the inhibition of DNA synthesis. Under conditions which inhibited DNA synthesis but permitted phospholipid synthesis, i.e., growth of a temperature-sensitive DNA elongation mutant at the restrictive temperature or treatment with hydroxy-urea, stalk elongation occurred normally. Therefore phospholipid synthesis is required for stalk elongation in C. crescentus.     

Inhibition of mammalian cell DNA synthesis by ionizing radiation

A semi-log plot of the inhibitory effect of ionizing radiation on the rate of DNA synthesis in normal mammalian cells yields a two-component curve. The steep component, at low doses, has a D0 of about 5 Gy and is the result of blocks to initiation of DNA replicons. The shallow component, at high doses, has a D0 of greater than or equal to 100 Gy and is the result of blocks to DNA chain elongation. The target size for the inhibition of DNA replicon initiation is about 1000 kb, and the target size for inhibition of DNA chain elongation is about 50 kb. There is evidence that the target for both components is DNA alone. Therefore, the target size for inhibition of DNA chain elongation is consistent with the idea that an effective radiation-induced lesion in front of the DNA growing point somehow blocks its advance. The target size for inhibition of DNA replicon initiation is so large that it must include many replicons, which is consistent with the concept that a single lesion anywhere within a large group (cluster) of replicons is sufficient to block the initiation of replication of all replicons within that cluster. Studies with radiosensitive human cell mutants suggest that there is an intermediary factor whose normal function is necessary for radiation-induced lesions to cause the inhibition of replicon initiation in clusters and to block chain elongation; this factor is not related to poly(ADP-ribose) synthesis. Studies with radiosensitive Chinese hamster cell mutants suggest that double-strand breaks and their repair are important in regulating the duration of radiation-induced inhibition of replicon initiation but have little to do with effects on chain elongation. There is no simple correlation between inhibition of DNA synthesis and cell killing by ionizing radiation.     

Hydrostatic Pressure Effects on Escherichia coli: Site of Inhibition of Protein Synthesis

Utilizing whole-cell preparations of Escherichia coli, it appears that 670 atm inhibits protein synthesis during elongation, while not affecting aminoacyl transfer ribonucleic acid formation, polysomal integrity, or amino acid permeability.     

Bubble formation in crustaceans following decompression from hyperbaric gas exposures

In vivo bubble formation was studied in various crustaceans equilibrated with high gas pressures and rapidly decompressed to atmospheric pressure. The species varied widely in susceptibility to bubble formation, and adults were generally more susceptible than larval stages. Bubbles did not form in early brine shrimp larvae unless equilibration pressures of at least 175 atm argon or 350 atm helium were used; for adult brine shrimp, copepods, and the larvae of crabs and shrimps, 100-125 atm argon or 175-225 atm helium were required. In contrast, bubbles formed in the leg joints of megalopa and adult crabs following decompression from only 3-10 atm argon; stimulation of limb movements increased this bubble formation, whereas inhibition of movements decreased it. High hydrostatic compressions applied before gas equilibration or slow compressions did not affect bubble formation. We concluded that circulatory systems, musculature, and storage lipids do not necessarily render organisms susceptible to bubble formation and that bubbles do not generally originate as preformed nuclei. In some cases, tribonucleation appears to be the cause of the bubbles.     

Reversal of 5-fluorodeoxyuridine-caused growth inhibition in intact and excised etiolated hypocotyls of Sinapis alba L. by thymidine

Summary The elongation growth in etiolated, intact seedlings and excised hypocotyl segments of Sinapis alba is inhibited by FdUrd in the same fashion, and in either case there is a direct correlation between FdUrd concentration and inhibition of elongation growth. Removal of the roots reduced elongation; however, the percentage inhibition by FdUrd remained the same. Therefore, the growth inhibition by FdUrd is not a consequence of root growth inhibition.The growth inhibition in excised hypocotyls cultured on synthetic media is inversely proportional to the size of the segments, and of the seedlings from which they are taken. Elongation of the smaller segments is more sensitive to FdUrd than that of the larger ones. Anatomical observations showed that the inhibition of growth elongation by FdUrd in the hypocotyl segments is due to inhibition of cell elongation, and not of cell division. Root formation is inhibited in all isolated segments.The growth inhibition by FdUrd could be reversed by dThd but not by uridine, and this reversibility depended upon the FdUrd concentration. When FdUrd inhibition is partial (up to 10^-7M) relatively high dThd concentration (up to 100 fold) are required for complete reversal; when inhibition is maximal relatively lower dThd concentrations effect a complete or near-complete reversal. Irreversible, unspecific effects of FdUrd were not found.These experiments confirm that DNA synthesis is involved in cell elongation of the hypocotyls even when the apical meristem and roots are removed, and that the growth inhibition by FdUrd is not a nonspecific, toxic effect.Abbreviations FdUrd 5-fluorodeoxyuridine - dThd thymidine