Damage in Escherichia coli Cells Treated with a Combination of High Hydrostatic Pressure and Subzero Temperature

The relationship between membrane permeability, changes in ultrastructure, and inactivation in Escherichia coli strain K-12TG1 cells subjected to high hydrostatic pressure treatment at room and subzero temperatures was studied. Propidium iodide staining performed before and after pressure treatment made it possible to distinguish between reversible and irreversible pressure-mediated cell membrane permeabilization. Changes in cell ultrastructure were studied using transmission electron microscopy (TEM), which showed noticeable condensation of nucleoids and aggregation of cytosolic proteins in cells fixed after decompression. A novel technique used to mix fixation reagents with the cell suspension in situ under high hydrostatic pressure (HHP) and subzero-temperature conditions made it possible to show the partial reversibility of pressure-induced nucleoid condensation. However, based on visual examination of TEM micrographs, protein aggregation did not seem to be reversible. Reversible cell membrane permeabilization was noticeable, particularly for HHP treatments at subzero temperature. A correlation between membrane permeabilization and cell inactivation was established, suggesting different mechanisms at room and subzero temperatures. We propose that the inactivation of E. coli cells under combined HHP and subzero temperature occurs mainly during their transiently permeabilized state, whereas HHP inactivation at room temperature is related to a balance of transient and permanent permeabilization. The correlation between TEM results and cell inactivation was not absolute. Further work is required to elucidate the effects of pressure-induced damage on nucleoids and proteins during cell inactivation.     

超高静压协同中温对凝结芽孢杆菌芽孢灭活动力学规律的研究

研究了超高静压协同中温对凝结芽孢杆菌芽孢在磷酸缓冲液和牛奶(经超高温灭菌)中灭活的动力学规律,并对超高静压的升压过程及相应的灭活效果进行了研究.结果表明,升压过程对凝结芽孢杆菌芽孢灭活的影响不能忽略,且随压力增加这种效果越强,最高使其下降1.77个数量级;凝结芽孢杆菌芽孢在牛奶中比在磷酸缓冲液中有更高的抗性;在3种拟合模型(线性、Weibull和Log-logistic模型)中,线性模型不适合模拟这些存活曲线,而Log-logistic模型能更好地模拟这些存活曲线,其次是Weibull模型.     

超高静压协同中温对凝结芽孢杆菌芽孢灭活动力学规律的研究

研究了超高静压协同中温对凝结芽孢杆菌芽孢在磷酸缓冲液和牛奶(经超高温灭菌)中灭活的动力学规律, 并对超高静压的升压过程及相应的灭活效果进行了研究。结果表明, 升压过程对凝结芽孢杆菌芽孢灭活的影响不能忽略, 且随压力增加这种效果越强, 最高使其下降1.77个数量级; 凝结芽孢杆菌芽孢在牛奶中比在磷酸缓冲液中有更高的抗性; 在3种拟合模型(线性、Weibull和Log-logistic模型)中, 线性模型不适合模拟这些存活曲线, 而Log-logistic模型能更好地模拟这些存活曲线, 其次是Weibull模型。     

Synergistic Combination of Electrolysis and Electroporation for Tissue Ablation

Electrolysis, electrochemotherapy with reversible electroporation, nanosecond pulsed electric fields and irreversible electroporation are valuable non-thermal electricity based tissue ablation technologies. This paper reports results from the first large animal study of a new non-thermal tissue ablation technology that employs “Synergistic electrolysis and electroporation” (SEE). The goal of this pre-clinical study is to expand on earlier studies with small animals and use the pig liver to establish SEE treatment parameters of clinical utility. We examined two SEE methods. One of the methods employs multiple electrochemotherapy-type reversible electroporation magnitude pulses, designed in such a way that the charge delivered during the electroporation pulses generates the electrolytic products. The second SEE method combines the delivery of a small number of electrochemotherapy magnitude electroporation pulses with a low voltage electrolysis generating DC current in three different ways. We show that both methods can produce lesion with dimensions of clinical utility, without the need to inject drugs as in electrochemotherapy, faster than with conventional electrolysis and with lower electric fields than irreversible electroporation and nanosecond pulsed ablation.     

Relationship between Sublethal Injury and Microbial Inactivation by the Combination of High Hydrostatic Pressure and Citral or tert-Butyl Hydroquinone

The aim was to investigate (i) the occurrence of sublethal injury in Listeria monocytogenes, Escherichia coli, and Saccharomyces cerevisiae after high hydrostatic pressure (HHP) treatment as a function of the treatment medium pH and composition and (ii) the relationship between the occurrence of sublethal injury and the inactivating effect of a combination of HHP and two antimicrobial compounds, tert-butyl hydroquinone (TBHQ) and citral. The three microorganisms showed a high proportion of sublethally injured cells (up to 99.99% of the surviving population) after HHP. In E. coli and L. monocytogenes, the extent of inactivation and sublethal injury depended on the pH and the composition of the treatment medium, whereas in S. cerevisiae, inactivation and sublethal injury were independent of medium pH or composition under the conditions tested. TBHQ alone was not lethal to E. coli or L. monocytogenes but acted synergistically with HHP and 24-h refrigeration, resulting in a viability decrease of >5 log₁₀ cycles of both organisms. The antimicrobial effect of citral depended on the microorganism and the treatment medium pH. Acting alone for 24 h under refrigeration, 1,000 ppm of citral caused a reduction of 5 log₁₀ cycles of E. coli at pH 7.0 and almost 3 log₁₀ cycles of L. monocytogenes at pH 4.0. The combination of citral and HHP also showed a synergistic effect. Our results have confirmed that the detection of sublethal injury after HHP may contribute to the identification of those treatment conditions under which HHP may act synergistically with other preserving processes.     

Day-Night Variations in Malate Concentration, Osmotic Pressure, and Hydrostatic Pressure in Cereus validus

Malate concentration and stem osmotic pressure concomitantly increase during nighttime CO₂ fixation and then decrease during the daytime in the obligate Crassulacean acid metabolism (CAM) plant, Cereus validus (Cactaceae). Changes in malate osmotic pressure calculated using the Van't Hoff relation match the changes in stem osmotic pressure, indicating that changes in malate level affected the water relations of the succulent stems. In contrast to stem osmotic pressure, stem water potential showed little day-night changes, suggesting that changes in cellular hydrostatic pressure occurred. This was corroborated by direct measurements of hydrostatic pressure using the Jülich pressure probe where a small oil-filled micropipette is inserted directly into chlorenchyma cells, which indicated a 4-fold increase in hydrostatic pressure from dusk to dawn. A transient increase of hydrostatic pressure at the beginning of the dark period was correlated with a short period of stomatal closing between afternoon and nighttime CO₂ fixation, suggesting that the rather complex hydrostatic pressure patterns could be explained by an interplay between the effects of transpiration and malate levels. A second CAM plant, Agave deserti, showed similar day-night changes in hydrostatic pressure in its succulent leaves. It is concluded that, in addition to the inverted stomatal rhythm, the oscillations of malate markedly affect osmotic pressures and hence water relations of CAM plants.     

Hydrostatic Pressure as an Environmental Factor in Life Processes

Hydrostatic pressure exists in all biological environments. In the deep sea the high pressure (up to approximately 100 MPa) has elicited molecular modifications in the organisms adapted to live there. Studying these is not particularly easy but a theoretical physical–chemical basis exists. Locomotor activity and behaviour are particularly sensitive to pressure and the most critical adaptations to high pressure appear to occur in the nervous system, although they are still obscure. In contrast, the effects of micro-pressures (∼kPa) manifest in the physiology of certain cells and the behaviour of many animals, which lack a gas phase, appear too small to arise from orthodox chemical reactions in solution. One particular micro-pressure sensor being studied in the crab statocyst appears to use the principle of the dilatometer, converting a bulk volume change into a linear displacement. Since some cells also respond to micro-pressure changes the question arises, is there an intracellular micro-pressure sensor and does it too work like a dilatometer?     

The Effects of Hydrostatic Pressure on Living Aquatic Organisms VIII. Behavioral and Metabolic Responses of Uca pugilator to Variations in Hydrostatic Pressure and Temperature

In systematic examination of the pressure responses of a broad spectrum of organic life, it is very important to know the range of variation exhibited by a single species. As a consequence of extensive observations on the effects of pressure and temperature on behavioral responses, lethality, and metabolic responses, it is clear that the range of variation in pressure required to induce a response diminishes as the species taxon is approached. The rate of exposure to pressure does not influence the pressure required for reversible behavioral responses. In contrast, the duration of pressure dramatically influences the pressure required to achieve death with the shorter time periods requiring much higher pressure levels than the longer time periods. Notwithstanding this relationship there appears some evidence suggesting that short term acclimation to pressure does occur.     

Hydrostatic Pressure and Proteins: Basic Concepts and New Data

In this paper we review some basic facts about the reversible and irreversible effects of high pressure on proteins. The effects include changes in intra- or intermolecular interactions (noncovalent bonds), in conformation and in solvation. Particular attention is directed to the interpretation of data where pressure-temperature dependency is an important phenomenon. Using model reactions, we have formulated a putative interpretation of physiological problems; we use these to explain how biological systems maintain activity when the temperature decreases and pressure increases, as in the case of barophilic micro-organisms in the deep sea world.     

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.Open in a separate windowClick here to view.^{(73M, flv)}     

Heavy Water and Hydrostatic Pressure Effects on Tetrahymena pyriformis1

SYNOPSIS. Heat-synchronized cultures of Tetrahymena pyriformis strain GL subjected to pulses of high hydrostatic pressure (10,000 psi for 2 min) had increasing division delays during the 1st 40 min after the last heat shock (40 min after heat treatment). Pressure treatment during the subsequent 10-min interval disrupted cell synchrony. Comparable pressures applied to the cells at later stages, before the 1st synchronous division, caused negligible division delay. Continuous exposure to 10% (v/v) heavy water hardly affected division; higher concentrations delayed or blocked division. Ten-min pulses with heavy water (40%, 50%, 70%) resulted in increasing division delays depending on the stage of the cell cycle during which the heavy water was applied. Amelioration of the division-delaying effects of pressure was observed in cells treated simultaneously with pressure (3,000 psi for 30 min), and 30% D₂O. The results are consistent with the hypothesis that some of the pressure and D₂O effects could be attributed to changes in the sol-gel state of the cytoplasm.     

The Effects of Hydrostatic Pressure on Living Aquatic Organisms VII. Size and Pressure Effects

A survey of the pressure resistance of aquatic animals in different stages of their life cycle shows that adults generally are more tolerant of pressure than the egg and nauplii, but older adults appear less pressure resistant than younger adults. Data on many species of aquatic animals of different size shows no correlation between size and pressure resistance. It is concluded that size is not a special determinant in the successful deep-sea colonization of shallow-water animals and this is consonant with the fact of occasional large deep-sea species whereas the average size is quite small in comparison with littoral species.     

Effects of Hydrostatic Pressure on Energy Metabolism and Osmoregulation in Crab and Fish

This review will focus on the effects of hydrostatic pressure on the oxidative metabolism and on the energy production of the eel Anguilla anguilla, in comparison with the results of investigations conducted on the other powerful euryhaline species, the chinese crab Eriocheir sinensis. Anguilla and Eriocheir were chosen as being both aquatic ectotherms with comparable life modes, the eel being however “preadapted” to high pressure while the crab normally never encounters high levels of pressure during its life cycle. Comparison between both species should lead to better knowledge of the biological effects of hydrostatic pressure per se.Experimental evidence suggests that the oxygen consumption ṀO₂ decrease observed in both animal species during exposure to 101 ATA hydrostatic pressure and which follows a transient increase, likely results from a decrease in O₂ use at the cell level. That idea of an alteration of aerobic metabolism during the first hours under pressure is substantiated by a set of experiments on the eel. However, results indicate that, after some days under pressure, the shallow water fish is quite able to acclimate perfectly to high pressure. The hypothesis that pressure induces a state resembling histotoxic hypoxia during the first hours of exposure is put forward and discussed.The second part of the review focuses on some results showing that osmoregulation is also concerned with hydrostatic pressure. Results obtained on the freshwater eel clearly establish the occurrence of a Na⁺ balance impairment at the tissue level induced by a long-term (30 days) exposure to pressure. It is interesting to point out that this impairment occurs at the same time when a new state of energetic metabolism results from adjustments of intertissue coupling of anaerobic and aerobic metabolisms induced by pressure. It is shown that the physiological processes involved in the control of the hydromineral balance in the chinese crab (which never experiences high-pressure exposure in the course of its life cycle) are outstandingly resistant to pressure by comparison with other crustaceans like the crayfish and the shore crab. Disturbances in hydromineral balance and energetic metabolism in the chinese crab are rapidly resorbed and adjusted to a new state of activity.     

Everolimus and Sirolimus in Combination with Cyclosporine Have Different Effects on Renal Metabolism in the Rat

Enhancement of calcineurin inhibitor nephrotoxicity by sirolimus (SRL) is limiting the clinical use of this drug combination. We compared the dose-dependent effects of the structurally related everolimus (EVL) and sirolimus (SRL) alone, and in combination with cyclosporine (CsA), on the rat kidney. Lewis rats were treated by oral gavage for 28 days using a checkerboard dosing format (0, 3.0, 6.0 and 10.0 CsA and 0, 0.5, 1.5 and 3.0 mg/kg/day SRL or EVL, n = 4/dose combination). After 28 days, oxidative stress, energy charge, kidney histologies, glomerular filtration rates, and concentrations of the immunosuppressants were measured along with ¹H-magnetic resonance spectroscopy (MRS) and gas chromatography- mass spectrometry profiles of cellular metabolites in urine. The combination of CsA with SRL led to higher urinary glucose concentrations and decreased levels of urinary Krebs cycle metabolites when compared to controls, suggesting that CsA+SRL negatively impacted proximal tubule metabolism. Unsupervised principal component analysis of MRS spectra distinguished unique urine metabolite patterns of rats treated with CsA+SRL from those treated with CsA+EVL and the controls. SRL, but not EVL blood concentrations were inversely correlated with urine Krebs cycle metabolite concentrations. Interestingly, the higher the EVL concentration, the closer urine metabolite patterns resembled those of controls, while in contrast, the combination of the highest doses of CsA+SRL showed the most significant differences in metabolite patterns. Surprisingly in this rat model, EVL and SRL in combination with CsA had different effects on kidney biochemistry, suggesting that further exploration of EVL in combination with low dose calcineurin inhibitors may be of potential benefit.     

Plasmids and Bacteriocins in Caulobacter Species

A survey of wild-type Caulobacter strains revealed naturally occurring plasmids in three species. Further analysis showed instances of naturally occurring antibiotic resistance and bacteriocin production.     

生物科学中一个崭露头角的领域：高静压力研究

通过介绍高静压力作用于生物大分子的机制,提出一些有关高压力生物学的基本概念。此外,还介绍了高静压力在研究蛋白质构象,蛋白质－蛋白质、蛋白质－核酸、蛋白质－配基等相互作用,酶活性的调制以及在生物技术中的应用,表明高压力是一很好的研究手段。     

Induction of Oxidative Stress by High Hydrostatic Pressure in Escherichia coli

Using leaderless alkaline phosphatase as a probe, it was demonstrated that pressure treatment induces endogenous intracellular oxidative stress in Escherichia coli MG1655. In stationary-phase cells, this oxidative stress increased with the applied pressure at least up to 400 MPa, which is well beyond the pressure at which the cells started to become inactivated (200 MPa). In exponential-phase cells, in contrast, oxidative stress increased with pressure treatment up to 150 MPa and then decreased again, together with the cell counts. Anaerobic incubation after pressure treatment significantly supported the recovery of MG1655, while mutants with increased intrinsic sensitivity toward oxidative stress (katE, katF, oxyR, sodAB, and soxS) were found to be more pressure sensitive than wild-type MG1655. Furthermore, mild pressure treatment strongly sensitized E. coli toward t-butylhydroperoxide and the superoxide generator plumbagin. Finally, previously described pressure-resistant mutants of E. coli MG1655 displayed enhanced resistance toward plumbagin. In one of these mutants, the induction of endogenous oxidative stress upon high hydrostatic pressure treatment was also investigated and found to be much lower than in MG1655. These results suggest that, at least under some conditions, the inactivation of E. coli by high hydrostatic pressure treatment is the consequence of a suicide mechanism involving the induction of an endogenous oxidative burst.     

Variation in Resistance to Hydrostatic Pressure among Strains of Food-Borne Pathogens

Among food-borne pathogens, some strains could be resistant to hydrostatic pressure treatment. This information is necessary to establish processing parameters to ensure safety of pressure-pasteurized foods (N. Kalchayanand, A. Sikes, C. P. Dunne, and B. Ray, J. Food Prot. 61:425–431, 1998). We studied variation in pressure resistance among strains of Listeria monocytogenes, Staphylococcus aureus, Escherichia coli O157:H7, and Salmonella species at two temperatures of pressurization. Early-stationary-phase cells in 1% peptone solution were pressurized at 345 MPa either for 5 min at 25°C or for 5, 10, or 15 min at 50°C. The viability loss (in log cycles) following pressurization at 25°C ranged from 0.9 to 3.5 among nine L. monocytogenes strains, 0.7 to 7.8 among seven S. aureus strains, 2.8 to 5.6 among six E. coli O157:H7 strains, and 5.5 to 8.3 among six Salmonella strains. The results show that at 25°C some strains of each species are more resistant to pressure than the others. However, when one resistant and one sensitive strain from each species were pressurized at 345 MPa and 50°C, the population of all except the resistant S. aureus strain was reduced by more than 8 log cycles within 5 min. Viability loss of the resistant S. aureus strain was 6.3 log cycles even after 15 min of pressurization. This shows that strains of food-borne pathogens differ in resistance to hydrostatic pressure (345 MPa) at 25°C, but this difference is greatly reduced at 50°C. Pressurization at 50°C, in place of 25°C, will ensure greater safety of foods.