Quantitative High-Resolution Imaging of Live Microbial Cells at High Hydrostatic Pressure

The majority of the Earth’s microbial biomass exists in the deep biosphere, in the deep ocean, and within the Earth’s crust. Although other physical parameters in these environments, such as temperature or pH, can differ substantially, they are all under high pressures. Beyond emerging genomic information, little is known about the molecular mechanisms underlying the ability of these organisms to survive and grow at pressures that can reach over 1000-fold the pressure on the Earth’s surface. The mechanisms of pressure adaptation are also important in food safety, with the increasing use of high-pressure food processing. Advanced imaging represents an important tool for exploring microbial adaptation and response to environmental changes. Here, we describe implementation of a high-pressure sample chamber with a two-photon scanning microscope system, allowing for the first time, to our knowledge, quantitative high-resolution two-photon imaging at 100 MPa of living microbes from all three kingdoms of life. We adapted this setup for fluorescence lifetime imaging microscopy with phasor analysis (FLIM/Phasor) and investigated metabolic responses to pressure of live cells from mesophilic yeast and bacterial strains, as well as the piezophilic archaeon Archaeoglobus fulgidus. We also monitored by fluorescence intensity fluctuation-based methods (scanning number and brightness and raster scanning imaging correlation spectroscopy) the effect of pressure on the chromosome-associated protein HU and on the ParB partition protein in Escherichia coli, revealing partially reversible dissociation of ParB foci and concomitant nucleoid condensation. These results provide a proof of principle that quantitative, high-resolution imaging of live microbial cells can be carried out at pressures equivalent to those in the deepest ocean trenches.     

Hydrostatic Pressure Does Not Cause Detectable Changes in Survival of Human Retinal Ganglion Cells

Purpose

Elevated intraocular pressure (IOP) is a major risk factor for glaucoma. One consequence of raised IOP is that ocular tissues are subjected to increased hydrostatic pressure (HP). The effect of raised HP on stress pathway signaling and retinal ganglion cell (RGC) survival in the human retina was investigated.

Methods

A chamber was designed to expose cells to increased HP (constant and fluctuating). Accurate pressure control (10-100mmHg) was achieved using mass flow controllers. Human organotypic retinal cultures (HORCs) from donor eyes (<24h post mortem) were cultured in serum-free DMEM/HamF12. Increased HP was compared to simulated ischemia (oxygen glucose deprivation, OGD). Cell death and apoptosis were measured by LDH and TUNEL assays, RGC marker expression by qRT-PCR (THY-1) and RGC number by immunohistochemistry (NeuN). Activated p38 and JNK were detected by Western blot.

Results

Exposure of HORCs to constant (60mmHg) or fluctuating (10-100mmHg; 1 cycle/min) pressure for 24 or 48h caused no loss of structural integrity, LDH release, decrease in RGC marker expression (THY-1) or loss of RGCs compared with controls. In addition, there was no increase in TUNEL-positive NeuN-labelled cells at either time-point indicating no increase in apoptosis of RGCs. OGD increased apoptosis, reduced RGC marker expression and RGC number and caused elevated LDH release at 24h. p38 and JNK phosphorylation remained unchanged in HORCs exposed to fluctuating pressure (10-100mmHg; 1 cycle/min) for 15, 30, 60 and 90min durations, whereas OGD (3h) increased activation of p38 and JNK, remaining elevated for 90min post-OGD.

Conclusions

Directly applied HP had no detectable impact on RGC survival and stress-signalling in HORCs. Simulated ischemia, however, activated stress pathways and caused RGC death. These results show that direct HP does not cause degeneration of RGCs in the ex vivo human retina.     

Regulation of Turgor Pressure by Suspension-Cultured Tobacco Cells

Turgor regulation and effects of high NaCl and water deficiton growth and internal solutes were studied after transferringtobacco cells from control culture medium (osmotic pressure= 0.13–0.15 MPa at time of transfer) to culture mediumcontaining either 82 mol m^–3 NaCl or 150 mol m^–3melibiose (osmotic pressure of media = 0.62 MPa). Followingtransfer to media with higher osmotic pressure, expansion rateand turgor pressure were reduced. Within 24 h of imposing thewater deficit, expansion rate had returned to that of cellsin control culture medium. However, by 24 h, turgor pressurehad only risen from 0.2 MPa to 0.65 MPa in the NaCl treatmentand to 0.53 MPa in the melibiose treatment, while it was 0.73MPa in the control treatment. Furthermore, turgor pressure remainedwithin 0.05 MPa of these respective values for the rest of the(75 h) experiment. These results suggest differences in bothcell wall properties (extensibility and/or threshold turgor)and the level at which turgor is maintained for cells in thevarious treatments. Solutes contributing nearly all (82–97%) of the osmoticpressure in cells were identified. The initial (up to 24 h)increases in turgor pressure were mainly due to increases insolute concentrations caused by relatively slow expansion rates.However, increased Na⁺ and Cl ^– uptake contributed toincreased turgor pressure in the NaCl treatment and caused turgorpressure of cells in this treatment to increase faster thanin the melibiose treatment. Likewise, expansion rate rose morequickly in the NaCl than in the melibiose treatment. After 24h, maximum expansion rate was reached and concentrations ofmost internal solutes began to decrease. Nevertheless, turgorpressure remained relatively constant. The constancy of turgorpressure was due to increased glucose uptake rates relativeto controls, with consequent increases in concentrations ofsucrose, glucose and fructose and, in cells in the melibiosetreatment, of organic acids. Glucose uptake was slower in theNaCl than in the melibiose treatment but higher turgor pressurewas maintained in the NaCl treatment due to high uptake of Na⁺and Cl^–. Glucose uptake appears to respond to a systemof turgor regulation, but further experiments are required toconfirm this and to determine whether Na⁺ and Cl^– uptakealso respond to a system of turgor regulation. Key words: Salinity, water deficit, growth     

Differential Regulation of Intracellular Signaling Systems by Sodium Fluoride in Rat Glioma Cells

Abstract: We investigated the rapid and slow effects of NaF on intracellular signaling systems such as Ca²⁺ homeostasis and cyclic GMP (cGMP) generation in rat glioma C6 cells, using the Ca²⁺-sensitive dye fura-2 and cGMP enzyme immunoassay. We found that the following: (a) NaF enhanced cGMP generation in a concentration-dependent manner. This enhancement was abolished by pretreatment with 100 µ M BAPTA tetraacetoxymethyl ester or in the presence of W-7 in a concentration-dependent manner. N ^G-Monomethyl- l -arginine (NMMA), a competitive inhibitor of nitric oxide synthase (NOS), also inhibited the NaF-induced generation of cGMP. These results suggest that NaF-induced cGMP generation occurs via a calcium/calmodulin- and NOS-dependent pathway. (b) The basal intracellular Ca²⁺ concentration ([Ca²⁺]_i) was transiently greater at 1 and 3 h after pretreatment with NaF. W-7 and W-13 antagonized the increase in [Ca²⁺]_i, whereas NMMA had little effect. This suggests that the NaF-induced change in basal [Ca²⁺]_i was mediated by a calmodulin-dependent pathway but was independent of a NOS-sensitive pathway. (c) The serotonin (5-HT)-induced intracellular mobilization of Ca²⁺ was reduced by pretreating the cells with NaF. The reduction in Ca²⁺ mobilization was antagonized by genistein, a tyrosine kinase inhibitor. W-7, W-5, and H-8 had no effect. Results suggest that NaF differentially regulates the cGMP generation, basal [Ca²⁺]_i, and 5-HT_2A receptor function in C6 glioma cells.     

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.     

Dye Transfer Between Cells of the Lens

Dye transfer between lens fiber cells and between lens epithelial cells and underlying fiber cells was studied using a wide dynamic range-cooled CCD camera, H₂O immersion objectives and image analysis techniques. Each lens was decapsulated by a new technique which leaves the epithelial cells adherent to the lens fiber mass. Lucifer Yellow CH was injected into either single epithelial cells or single fiber cells using the standard whole cell configuration of the patch voltage clamp technique. The results demonstrate extensive dye communication between fiber cells at the lens posterior surface, anterior surface, and equatorial surface. Dye transfer between deep fiber cells was also observed. Dye transfer between ≈10% of epithelial cells and their underlying fiber cells was apparent when care was taken to yield wide dynamic range images. This was required because the relatively high concentration of dye in the epithelial cell masks the presence of much lower dye concentrations in the underlying fiber cell. A mathematical model which includes dye concentration, time, and spatial spread suggests that those epithelial cells that are coupled to an underlying fiber cell are about as well dye coupled as the epithelial cells themselves. The relatively low dye concentration in a fiber cell is due to its larger volume and diffusion of the dye along the axis of the fiber away from the fiber/epithelial junction. Received: 14 September 1995/Revised: 13 November 1995     

Regulation of Intracellular Proteolysis in Escherichia coli

Individual nitrogenous metabolites have been examined as regulating agents for the breakdown of intracellular proteins in Escherichia coli. Generally, NH(4) (+) is the most effective regulator. Its depletion progressively increases the basal proteolytic rate to maximum in most strains when the doubling time is increased to 2 h. In E. coli 9723, the rate is further increased at longer doubling times. Amino acids have individual effects on intracellular proteolysis. The basal rate in amino acid-requiring auxotrophs of E. coli 9723 is stimulated weakly by starvation for histidine, tryptophan, or tyrosine, moderately by four other amino acid depletions, and more strongly by eight others. The degree of stimulation roughly correlates with the frequency of the amino acid in the cell proteins. Amino acid analogues that incorporate extensively into protein generally slightly inhibit intracellular proteolysis, except for selenomethionine, which is slightly stimulatory. Metabolic inhibitors were studied at graded concentrations. Chloramphenicol inhibits the basal level of intracellular proteolysis when protein synthesis is slightly or moderately inhibited, and stimulates proteolysis slightly at higher levels. Graded inhibition of ribonucleic acid synthesis with rifampin progressively stimulates intracellular proteolysis. Uracil depletion is also stimulatory. Inhibition of deoxyribonucleic acid synthesis with mitomycin C or by thymine starvation slightly inhibits intracellular proteolysis. Intracellular proteolysis is postulated to be regulated primarily by active ribosomal function. At 43 to 45 C, intracellular proteolysis becomes maximally induced and unresponsive to normal regulatory control by metabolites. Most regulation is directed towards the breakdown of the more stable cell proteins. Total proteolysis in all cell proteins is no more than doubled by the most effective conditions of starvation.     

Turgor Regulation in a Brackish Charophyte, Lamprothamnium succinctum I. Artificial Modification of Intracellular Osmotic Pressure

Internodal cells of Lamprothamnium succinctum cultured in freshwater and brackish water of different salinities maintainedalmost the same turgor pressure at steady state. When the turgorpressure was increased by decreasing the external osmolality,the cells recovered their original turgor pressure within 2h. However, the recovery from decreased turgor pressure required1 day. When salts of the external medium were replaced with sorbitol,the cells still regulated the turgor pressure, indicating thatthe essential factor for the turgor regulation is not the salinitybut the osmolality. Internodal cells with osmotic pressure andion concentrations artificially modified to higher or lowervalues also regained the original turgor pressure by changingtheir intracellular osmotic pressure, whether the cells werecultured in brackish water or fresh water. These results indicate that turgor regulation is intrinsic toLamprothamnium and is initiated by a deviation of turgor pressurefrom the reference value, which is about 0.35 Osm. (Received November 28, 1983; Accepted March 14, 1984)     

Effects of Hydrostatic Pressure on Carcinogenic Properties of Epithelia

The relationship between chronic inflammation and cancer is well known. The inflammation increases the permeability of blood vessels and consequently elevates pressure in the interstitial tissues. However, there have been only a few reports on the effects of hydrostatic pressure on cultured cells, and the relationship between elevated hydrostatic pressure and cell properties related to malignant tumors is less well understood. Therefore, we investigated the effects of hydrostatic pressure on the cultured epithelial cells seeded on permeable filters. Surprisingly, hydrostatic pressure from basal to apical side induced epithelial stratification in Madin-Darby canine kidney (MDCK) I and Caco-2 cells, and cavities with microvilli and tight junctions around their surfaces were formed within the multi-layered epithelia. The hydrostatic pressure gradient also promoted cell proliferation, suppressed cell apoptosis, and increased transepithelial ion permeability. The inhibition of protein kinase A (PKA) promoted epithelial stratification by the hydrostatic pressure whereas the activation of PKA led to suppressed epithelial stratification. These results indicate the role of the hydrostatic pressure gradient in the regulation of various epithelial cell functions. The findings in this study may provide clues for the development of a novel strategy for the treatment of the carcinoma.     

Use of Hydrostatic Pressure for Inactivation of Microbial Contaminants in Cheese

The objective of this study was to determine the effect of high pressure (HP) on the inactivation of microbial contaminants in Cheddar cheese (Escherichia coli K-12, Staphylococcus aureus ATCC 6538, and Penicillium roqueforti IMI 297987). Initially, cheese slurries inoculated with E. coli, S. aureus, and P. roqueforti were used as a convenient means to define the effects of a range of pressures and temperatures on the viability of these microorganisms. Cheese slurries were subjected to pressures of 50 to 800 MPa for 20 min at temperatures of 10, 20, and 30°C. At 400 MPa, the viability of P. roqueforti in cheese slurry decreased by >2-log-unit cycles at 10°C and by 6-log-unit cycles at temperatures of 20 and 30°C. S. aureus and E. coli were not detected after HP treatments in cheese slurry of >600 MPa at 20°C and >400 MPa at 30°C, respectively. In addition to cell death, the presence of sublethally injured cells in HP-treated slurries was demonstrated by differential plating using nonselective agar incorporating salt or glucose. Kinetic experiments of HP inactivation demonstrated that increasing the pressure from 300 to 400 MPa resulted in a higher degree of inactivation than increasing the pressurization time from 0 to 60 min, indicating a greater antimicrobial impact of pressure. Selected conditions were subsequently tested on Cheddar cheese by adding the isolates to cheese milk and pressure treating the resultant cheeses at 100 to 500 MPa for 20 min at 20°C. The relative sensitivities of the isolates to HP in Cheddar cheese were similar to those observed in the cheese slurry, i.e., P. roqueforti was more sensitive than E. coli, which was more sensitive than S. aureus. The organisms were more sensitive to pressure in cheese than slurry, especially with E. coli. On comparison of the sensitivities of the microorganisms in a pH 5.3 phosphate buffer, cheese slurry, and Cheddar cheese, greatest sensitivity to HP was shown in the pH 5.3 phosphate buffer by S. aureus and P. roqueforti while greatest sensitivity to HP by E. coli was exhibited in Cheddar cheese. Therefore, the medium in which the microorganisms are treated is an important determinant of the level of inactivation observed.     

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.     

Spontaneous Transformation of Bovine Lens Epithelial Cells

Bovine lens epithelial cells, in vivo, are known to perform two determined functions. First, they synthesize the lens capsule and subsequently, in the germinal region, they differentiate in fiber cells with massive production of crystallin proteins, inactivation and pyknosis of the nucleus.
Bovine lens epithelial cells from adult origin can be cultured but so far no massive crystallin production has been demonstrated in vitro. We have studied the growth and differentiation of these cells and shown that in long term culture they acquire spontaneously many characteristics of transformation: unlimited growth potential, abnormal karyotype, multilayering. Viral particles were scarcely detected. However, they retain their epithelioid character and the ability to synthesize lens capsule material. Kinetic characteristics of those cells have been determined.
When injected into nude mice, they actively proliferate and form tumors in which synthesis of α-crystallin can be demonstrated. These results show that in vitro transformation of lens epithelial cells does not affect their potential for terminal differentiation.     

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.     

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.     

Measurement of the Respiration-Dependent Component of Intracellular Pressure with an Improved Pressure Probe

We have improved a previously described pressure probe in thefollowing respect to make it suitable for continous mesurementsof intracelluar presure (p)ⁱ in higher plant cells. (1)The oil/cell-sapboundary (meniscus) at the tip of the microcapillary was moniterdphoto-electrically with a high-resolution image sensor.(2) Adjustmentof the plunger and the insertion of the probe into the targetcell were performed with specilly desinged, hydraulic manipulatorsystem. These improvements allowed us to monitor the meniscusprecisely and to maintain the meniscus at a certain positionwithout mechanical perturbation. This system has been successfullyused to measure Pⁱ continuously for more than 3 h. With this pressure probe, the dependence on respiration of Pⁱin the elongation zone of Vigna hypocotyls, which was predictedby Katou and Furumoto (1986a, b), was demonstrated directly.Changes in Pi induced by osmotic stress were also measured quitesuccessfully. The respiration-dependent component of Pi was30–40 kPa. While this component appears to be very small,the change in Pi under anoxia caused shrinkage of the segmentof hypocotyl being examined. Regulation of P^u over a range ofseveral tens of kPa or so should, therefore, make a significantcontribution to the control of elongation growth. (Received April 9, 1990; Accepted June 25, 1990)