The stability of rabbit reticulocyte polysomes at high hydrostatic pressure

The application of high hydrostatic pressure to an in vitro rabbit reticulocyte, polypeptide-synthesizing system has been shown to inhibit synthesis either partially or totally depending upon the magnitude of pressure utilized (Scheck, A.C. and Landau, J.V. (1982) Biochim. Biophys. Acta 718, 21–25). This paper shows that the total inhibition of synthesis seen at 670 atm is similar to the inhibition of elongation produced by cycloheximide in that the polysome profiles remain intact. Partial inhibition at 300 atm shows a reduced rate of ribosome run-off with elongation being affected to the same, or greater extent than initiation. In no instance was disassociation of polysomes seen as a causative factor in the inhibition of synthesis by high pressure.     

An improved sampling technique for bacterial cultures under high hydrostatic pressure

A sampling technique for bacterial cultures subjected to high hydrostatic pressure is described. A sample-receiving vessel with a motor driven interface-piston is employed. By precisely matching the pressures in the bulk culture and the sample-receiving vessel, none of the sample is subjected to the high shear forces common to other desings of high pressure sampler. The use of the technique was illustrated by the growth of an anaerobic culture at 300 bar and 75°C.     

Effect of high hydrostatic pressure on the bacterial mechanosensitive channel MscS

We have investigated the effect of high hydrostatic pressure on MscS, the bacterial mechanosensitive channel of small conductance. Pressure affected channel kinetics but not conductance. At negative pipette voltages (corresponding to membrane depolarization in the inside-out patch configuration used in our experiments) the channel exhibited a reversible reduction in activity with increasing hydrostatic pressure between 0 and 900 atm (90 MPa) at 23°C. The reduced activity was characterized by a significant reduction in the channel opening probability resulting from a shortening of the channel openings with increasing pressure. Thus high hydrostatic pressure generally favoured channel closing. Cooling the patch by approximately 10°C, intended to order the bilayer component of the patch by an amount similar to that caused by 50 MPa at 23°C, had relatively little effect. This implies that pressure does not affect channel kinetics via bilayer order. Accordingly we postulate that lateral compression of the bilayer, under high hydrostatic pressure, is responsible. These observations also have implications for our understanding of the adaptation of mechanosensitive channels in deep-sea bacteria.A Proceeding of the 28th Annual Meeting of the Australian Society for Biophysics.     

The effect of canavanine on protein synthesis and protein degradation in IMR-90 fibroblasts

Proteins of IMR-90 fibroblasts incorporating [³⁵S]methionine during a 1 h labelling period in the presence of the arginine analogue canavanine were degraded twice as rapidly in the cells as were proteins similarly made in the presence of arginine. Using both isoelectric focusing and SDS-polyacrylamide gel electrophoretic analyses, the banding patterns of proteins labelled in the presence of canavanine and arginine were found to differ. This banding difference was detected as early as 15 min after canavanine treatment. With the exception of one minor band in isoelectric focusing gel, the relative intensity of labelled protein bands for the control samples remained unchanged during the 2 h period of protein degradation being investigated. This was also true for the proteins labelled in the presence of canavanine, despite the increase in their rate of degradation. Banding difference between canavanine and arginine treatment was also detected in an in vitro reticulocyte lysate translation system dependent on fibroblast mRNA. Proteins labelled in the presence of a different analogue, p-fluorophenylalanine instead of phenylalanine, however, had similar banding patterns as the control both in the lysate system and in intact cells.     

The effect of aging on cell-free protein synthesis in the free-living nematode Turbatrix aceti

The age-related reduction in cell-free synthesis in the free-living nematode Turbatrix aceti is due to a defect in the ribosomes. Addition of young ribosomal wash or use of young medium does not improve the activity of old, run-off ribosomes in the presence of phenylalanine and poly(U). It appears that some of the old ribosomes are incapable of binding the EF-1-GTP-aminoacyl-tRNA complex. These ineffective ribosomes are present in the 80 S (monosomal) fraction. Old ribosomes obtained from polysomes appear to bind normally.     

超高压处理对微生物生理特性的影响

单核增生李斯特菌(Listeriamonocytogenes)NCTC11994和大肠杆菌(Escherichiacoli)ATCC80739经高压处理,其生理特性发生了深刻的变化,主要表现是400MPa以上的压力处理10min,微生物数量下降7个对数单位,压力处理还会导致细胞内pH值的变化,使膜电位下降,细胞内钾流失,ATP浓度降低。     

The effect of hydrostatic pressure on the thermal stability of DNA hairpins

DNA hairpins consist of two distinct structural domains: a double stranded stem and a single-stranded loop that connect the two strands of the stem. Previous studies of short DNA hairpins have revealed that loop and stem sequences can significantly affect the thermodynamic stability of short DNA hairpins. In this work we present the effect of hydrostatic pressure on the helix-coil transition temperature (T_M) for 11 16-base, hairpin-forming DNA oligonucleotides. All of the samples form a hairpin with a 6-base pair stem and a four-base loop. In addition, the four base pairs at the end of the stem distal from the loop are the same for every molecule. We have varied loop sequence and identity of the two duplex base pairs adjacent to the loop. Using the change in UV absorption to monitor the conformational state of the oligonucleotide the hairpin-coil transition temperature of these molecules was studied as a function of sodium ion concentration and pressure. From these data we calculated the volume change accompanying the transition. Model-dependent (van't Hoff) transition parameters such as ΔH_vH and transition volume (ΔV) were estimated from the analysis of conformational transitions. Experiments revealed that the ΔV for denaturation of these molecules range from − 2.35 to + 6.74 cm³ mol⁻¹. The expansibility (ΔΔV/ΔT) and the pressure dependence of cation release are also presented. The difference in the volume change for this transition is related to the differences in the hydration of these molecules.     

Adaptation of Saccharomyces cerevisiae to high hydrostatic pressure causing growth inhibition

Genome-wide mRNA expression profiles of Saccharomyces cerevisiae growing under hydrostatic pressure were characterized. We selected a hydrostatic pressure of 30 MPa at 25 degrees C because yeast cells were able to grow under these conditions, while cell size and complexity were increased after decompression. Functional characterization of pressure-induced genes suggests that genes involved in protein metabolism and membrane metabolism were induced. The response to 30 MPa was significantly different from that observed under lethal conditions because protein degradation was not activated under 30 MPa pressure. Strongly induced genes those that contribute to membrane metabolism and which are also induced by detergents, oils, and membrane stabilizers.     

（超）高压对微生物的影响及其诱变效应探讨

(超)高压对微生物有多方面的影响,它不仅可使微生物细胞体积形态、细胞组分发生变化,还可使微生物的基因表达和核酸结构及其生物学功能发生改变。(超)高压的这些生物学效应,使其不仅可以应用到食品杀菌、保藏及某些加工过程,而且在微生物菌种诱变方面具有很大的应用潜力。这是因为(超)高压既然可以使核酸发生变化,那么它诱导微生物发生突变就很有可能。现从(超)高压对微生物的影响出发,并结合国内外有关实例及作者的研究工作,初步探讨其诱变育种的可行性。     

Inhibition of eukaryotic cell-free protein synthesis by thionins from wheat endosperm

Thionins are polypeptide toxins of about 5000 molecular weight, present in the endosperms of many Gramineae, which modify membrane permeability and inhibit macromolecular synthesis in cultured mammalian cells. Evidence is presented that they inhibit in vitro protein synthesis at micromolar concentrations in cell-free systems derived from wheat germ or from rabbit reticulocytes. Inhibition seems to occur by direct binding of mRNA by the toxin, as judged by the ability of thionins to mediate retention of RNA in nitrocellulose filters and by the dependence of inhibitory concentrations on the amount of exogenous RNA added to the wheat-germ translation system. Commercial preparations of wheat-germ have been found to include some endosperm contamination (up to 15%), which may result in at least partially inhibitory concentrations of the toxin in the cell-free extracts.     

Inhibition of hepatocytic protein degradation by methylaminopurines and inhibitors of protein synthesis

Nutritional control of protein degradation in isolated rat hepatocytes can take place in the absence of protein synthesis. Suppression of degradation by amino acids (step-up) is unaffected and the enhanced degradation seen upon amino acid deprivation (step-down) is only partially inhibited by cycloheximide at a concentration (10^?3 M) which inhibits protein synthesis virtually completely. Protein degradation per se is, however, inhibited by cycloheximide as well as by puromycin, apparently at least in part by mechanisms additional or unrelated to their effect on protein synthesis. Several puromycin analogues (methylaminopurines) are stronger inhibitors of protein degradation than of protein synthesis, most notably puromycin aminonucleoside and 6-dimethylaminopurine riboside (N⁶, N⁶-dimethyladenosine). The latter compounds appear to specifically inhibit cellular autophagy, since neither the degradation of endocytosed protein (asialofetuin) nor the extralysosoma (amino acid-, propylamine- and leupeptin-resistant) degradation are affected.     

High hydrostatic pressure inhibits the biosynthesis of eukaryotic elongation factor-2

High continuous hydrostatic pressure is known to inhibit the total cellular protein synthesis. In this study, our goal was to identify pressure-regulated proteins by using two dimensional gel electrophoresis and mass spectrometry. This analysis showed that under 30 MPa continuous hydrostatic pressure the biosynthesis of eukaryotic elongation factor-2 (eEF-2) was inhibited both in HeLa carcinoma and T/C28a4 chondrocytic cell lines. Western blot analysis of HeLa cells revealed that the cellular protein level of eEF-2 decreased by 40%-50% within 12 h of the pressure treatment. However, the steady-state mRNA level of eEF-2 was not affected by the pressure. Cycloheximide addition after 4 h-pressure treatment suggested that the half-life of eEF-2 protein was shorter in pressurized cells. eEF-2 is responsible for the translocation of ribosome along the specific mRNA during translation, and its phosphorylation prevents the ribosomal translocation. Therefore, increased phosphorylation of eEF-2 was considered as one mechanism that could explain the reduced level of protein synthesis in pressurized HeLa cell cultures. However, Western blot analysis with an antibody recognizing the Thr56-phosphorylated form of eEF-2 showed that phosphorylation of eEF-2 was not elevated in pressurized samples. In conclusion, the inhibition of protein synthesis under high pressure occurs independent of the phosphorylation of eEF-2. However, this inhibition may result from the decrease of cellular eEF-2 protein.     

A reliable method for precise initial rate determination of enzyme activity under high hydrostatic pressure with simple apparatus - Application to Aspergillus niger fructosyl-transferase activity measurements

An original procedure for initial rate accurate determination of enzyme activity under high hydrostatic pressure is reported. This method, adapted to most kinds of enzyme systems, is based on the use of the linear property of product formation during the initial phase of a reaction and does not require specific high pressure equipment. The reliability of the method was tested and illustrated with the study of Aspergillus niger fructosyl-transferase activity as a function of substrate concentration above 300 MPa. © Rapid Science Ltd. 1998     

The thermal stability of turnip yellow mosaic virus under hydrostatic pressure

The thermal stability of an isometric plant virus, Turnip Yellow Mosaic Virus (TYMV), has been investigated at low and high hydrostatic pressure, using small angle neutron scattering. Contrast variation allowed us to separately observe the structural changes of the protein capsid and the RNA core. The experiments were performed in 0.05M Tris buffer at pD = 8.0 and in 0.05M bis-Tris buffer at pD = 6.0 containing different H₂O/D₂O mixtures (40% and 70% D₂O). It was found that hydrostatic pressure enhances the stability of TYMV. The thermally induced uncoating of RNA as well as structural transitions of the protein capsid are shifted to higher temperature upon increasing the pressure from 5 × 10⁶ Pa to 2 × 10⁸ Pa.     

Regulation of paracellular Na and Cl conductances by hydrostatic pressure

The effect of hydrostatic pressure on the paracellular ion conductance (Gp) composed of the Na⁺ conductance (G_Na) and the Cl⁻ conductance (G_Cl) has been Investigated. Gp, G_Na and G_Cl were time-dependently increased after applying an osmotic gradient generated by NaCl with basolateral hypotonicity. Hydrostatic pressure (1-4 cm H₂O) applied from the basolateral side enhanced the osmotic gradient-induced increase in Gp, G_Na and G_Cl in a magnitude-dependent manner, while the hydrostatic pressure applied from the apical side diminished the osmotic gradient-induced increase in Gp, G_Na and G_Cl. How the hydrostatic pressure influences Gp, G_Na and G_Cl under an isosmotic condition was also investigated. Gp, G_Na and G_Cl were stably constant under a condition with basolateral application of sucrose canceling the NaCl-generated osmotic gradient (an isotonic condition). Even under this stable condition, the basolaterally applied hydrostatic pressure drastically elevated Gp, G_Na and G_Cl, while apically applied hydrostatic pressure had little effect on Gp, G_Na or G_Cl. Taken together, these observations suggest that certain factors controlled by the basolateral osmolality and the basolaterally applied hydrostatic pressure mainly regulate the Gp, G_Na and G_Cl.     

The hormonal control of protein N-glycosylation in the developing rabbit mammary gland and its effect upon transferrin synthesis and secretion

Pregnant rabbit mammary gland explants cultured with insulin, prolactin and cortisol, synthesise and secrete transferrin radiolabelled with [³H]leucine or [³H]mannose. Omission of prolactin from the culture medium inhibited the incorporation of [³H]leucine into casein but not transferrin. Total transferrin secreted under these conditions was approx. 75% of the control (+ prolactin) value measured by rocket immunoelectrophoresis. Little incorporation of [³H]mannose into transferrin was seen in the absence of prolactin suggesting a lack of glycosylation of the protein. Dual label experiments with [³H]mannose and [¹⁴C]leucine confirmed this. The decreased incorporation of [³H]mannose into dolichol linked intermediates suggests a general effect on protein N-glycosylation in the absence of prolactin. Thus, while the synthesis of the polypeptide backbone of transferrin does not require prolactin its glycosylation does.     

Cuticle expansion during feeding in the tick Amblyomma hebraeum (Acari: Ixodidae): The role of hydrostatic pressure

Female Amblyomma hebraeum ticks (Acari: Ixodidae) increase their weight ∼10-fold during a ‘slow phase of engorgement’ (7–9 days), and a further 10-fold during the ‘rapid phase’ (12–24 h). During the rapid phase, the cuticle thins by half, with a plastic (permanent) deformation of greater than 40% in two orthogonal directions. A stress of 2.5 MPa or higher is required to achieve this degree of deformation (Flynn and Kaufman, 2015). Using a dimensional analysis of the tick body and applying the Laplace equation, we calculated that the tick must achieve high internal hydrostatic pressures in order to engorge fully: greater than 55 kPa at a fed:unfed mass ratio of ∼20:1, when cuticle thinning commences (Flynn and Kaufman, 2011). In this study we used a telemetric pressure transducer system to measure the internal hydrostatic pressure of ticks during feeding. Sustained periods of irregular high frequency (>20 Hz) pulsatile bursts of high pressure (>55 kPa) were observed in two ticks: they had been cannulated just prior to the rapid phase of engorgement, and given access to a host rabbit for completion of the feeding cycle. The pattern of periods of high pressure generation varied over the feeding cycle and between the two specimens. We believe that these pressures exceed those reported so far for any other animal.     

Inhibitors of animal cell-free protein synthesis from grains

We described earlier the purification and properties of a protein (tritin) from wheat that enzymatically inhibits translation in cell-free systems from animals but not plants. In this report, we have examined 11 additional grains (Family Gramineae) and three other seeds for the presence of tritin-like proteins. In addition to wheat species, barley, oats, rye, triticale and corn were found to be sources of inhibitor; no inhibitor could be detected in rice, millet, sesame, alfalfa, mung bean or common bean seeds. The inhibitors from barley and rye were purified and found to differ from tritin with respect to heat inactivation, although they are similar to tritin with respect to molecular weight, behavior during purification and specific activity. The inhibitor from corn was purified and found to differ from tritin with respect to heat inactivation and molecular weight, although it is similar to tritin in behavior during purification and specific activity. These inhibitors constitute 2–17% of the total extractable protein in these grain s. Thus, wheat, barley, rye and corn can serve as convenient sources of a family of closely related inhibitors of protein synthesis which, when conjugated with lectins, antibodies, or hormones, could prove useful as chimeric toxins.     

Transport protein synthesis in non-growing yeast cells

Addition of a metabolizable substrate (glucose, ethanol and, to a degree, trehalose) to non-growing baker's yeast cells causes a boost of protein synthesis, reaching maximum rate 20 min after addition of glucose and 40–50 min after ethanol or trehalose addition. The synthesis involves that of transport proteins for various solutes which appear in the following sequence: H⁺, l-proline, sulfate, l-leucine, phosphate, α-methyl-d-glucoside, 2-aminoisobutyrate. With the exception of the phosphate transport system, the K_t of the synthesized systems is the same as before stimulation. Glucose is usually the best stimulant, but ethanol matches it in the case of sulfate and exceeds it in the case of proline. This may be connected with ethanol's stimulating the synthesis of transport proteins both in mitochondria and in the cytosol while glucose acts on cytosolic synthesis alone. The stimulation is often repressed by ammonium ions (leucine, proline, sulfate, H⁺), by antimycin (proline, trehalose, sulfate, H⁺), by iodoacetamide (all systems tested), and by anaerobic preincubation (leucine, proline, trehalose, sulfate). It is practically absent in a respiration-deficient petite mutant, only little depressed in the op₁ mutant lacking ADP/ATP exchange in mitochondria, but totally suppressed (with the exception of transport of phosphate) in a low-phosphorus strain. The addition of glucose causes a drop in intracellular inorganic monophosphate by 30%, diphosphate by 45%, ATP by 70%, in total amino acids by nearly 50%, in transmembrane potential (absolute value) by about 50%, an increase of high-molecular-weight polyphosphate by 65%, of total cAMP by more than 100%, in the endogenous respiration rate by more than 100%, and a change of intracellular pH from 6.80 to 7.05. Ethanol caused practically no change in ATP, total amino acids, endogenous respiration, intracellular pH or transmembrane potential; a slight decrease in inorganic monophosphate and diphosphate and a sizeable increase in high-molecular-weight polyphosphate. The synthesis of the various transport proteins thus appears to draw its energy from different sources and with different susceptibility to inhibitors. It is much more stimulated in facultatively aerobic species (Saccharomyces cerevisiae, Endomyces magnusii) than in strictly aerobic ones (Rhodotorula glutinis, Candida parapsilosis) where an inhibition of transport activity is often observed after preincubation with metabolizable substrates.     

The effect of nisin on murein synthesis

Nisin inhibits murein synthesis with concomitant accumulation of undecaprenyl-pyrophospho-MurNAc(pentapeptide) (lipid intermediate I). This inhibition is caused by the formation of a complex between the antibiotic and lipid intermediate I. Undecaprenyl-pyrophospho-MurNAc(pentapeptide)-GlcNAc (lipid intermediate II) also forms a complex with nisin. However, when murein synthesis is inhibited by nisin, this latter complex is not formed since lipid intermediate II is no longer synthesized.Abbreviations GlcNAc N-acetylglucosamine - MurNAc N-acetylmuramyl - Pentapeptide Ala--DGlu-Lys-DAla-DAla - C₅₅ undecaprenol Dedicated to Professor Otto Kandler on occasion of his 60th birthday