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Porin OmpC from Escherichia coli was reconstituted in liposomes and its gating kinetics were recorded at high hydrostatic pressure, up to 90 MPa, using a development of the patch clamp technique. The composition of the recording solution influenced the results but generally high hydrostatic pressure favoured channel opening.
Normal human dermal fibroblasts were found to survive and to be active in producing interleukin (IL)-6 and IL-8 under extremely high hydrostatic pressure, up to 40 MPa (1 atm=0.101325 MPa=1.03323 kgf/cm(2)), for 20 min. An inhibitor of protein kinase C (PKC) reduced the amount of IL-6 production, whereas IL-8 production was increased following pressure application. The activation of PKC in response to exposure to the pressure stress was detected by using the PKC-specific probe Rim-1. These findings indicate that IL-6 production induced by hydrostatic pressure stresses was dependent on the PKC signaling pathway. In contrast, pressure-induced IL-8 production was inhibited by PKC activity.
High hydrostatic pressure (HHP) technique was used to evaluate a mechanism of RNA hydrolysis with RNA. We showed that hammerhead ribozyme specifically cleaves RNA substrate at HHP in the absence of Mg(2+). A deoxyribozyme "10-23" was active in the same conditions. These results pointed out that the hydrolytic activity of nucleic acid depends on proper tertiary structure of a complex with a substrate. They prove that magnesium ion is not directly involved in catalysis process. On that basis we show the mechanism of RNA hydrolysis catalyzed with nucleic acids at HHP.
High hydrostatic pressure (HHP) is an established method to inactivate biomolecules and microoganisms. It is routinely used for the sterilization of foodstuff. Recently, new applications as inactivation of microorganisms and tumour cells for bone transplants or for cancer vaccines have emerged. Characterization of the HHP-induced cellular responses are a prerequisite for its clinical use. To this end, we investigated the fate of human cells after HHP by cytofluorometry. We observed that the induction by HHP of cell death is time- and pressure-dependent. Surprisingly, an HHP-treatment of 100 MPa did not reduce viability at any time point. Pressures from 150 to 250 MPa-induced programmed cell death in most cells. However, survivors were observed in long term culture experiments under these conditions. Pressures above 300 MPa immediately induced cell death by necrosis and completely inactivated the cells. In contrast to inactivation by other necrosis inducing treatments like heat, freeze/thaw, or chemical agents, HHP avoids generation of Maillard products and disintegration and lysis of the cells. Instead HHP generates a gelatinised mixture of antigens captured in a distinct and robust particle and maintains their humoral immunogenicity. The high viscosity of the internal matrix of a pressurised cell is reflected by the slow penetration of the low molecular compound propidium iodide and limits the bleeding of antigen before uptake by antigen presenting cells. Taken together, HHP is an alternative method for the inactivation of mammalian cells in clinical settings.
Increases in renal interstitial hydrostatic pressure (RIHP) increase urinary sodium excretion (UNaV). Experimentally increasing RIHP by direct renal interstitial volume expansion (DRIVE) has been shown to decrease proximal tubule sodium reabsorption. The purpose of the present study was to investigate whether the renin-angiotensin system modulates the natriuretic response to DRIVE. Unilateral nephrectomy and implantation of two polyethylene matrices were performed 3 wk before the acute experiment. Fractional sodium excretion (FENa), RIHP, and glomerular filtration rate (GFR) were measured before and after DRIVE in control rats (n = 9) and in rats receiving the angiotensin II (ANG II) receptor antagonist, losartan potassium (10 mg/kg i.v.; n = 10). DRIVE was achieved by infusing 100 microliters of 2.5% albumin solution directly into the renal interstitium. GFR remained unchanged by DRIVE in both groups. In control animals, DRIVE significantly increased both RIHP (delta 3.8 +/- 0.5 mmHg) and FENa (delta 0.92 +/- 0.19%). In the losartan-treated group, RIHP (delta 2.8 +/- 0.4 mmHg) and FENa (delta 1.93 +/- 0.41%) also significantly increased. The natriuretic response to DRIVE was significantly enhanced during ANG II receptor blockade compared with control animals (delta UNaV/delta RIHP = 2.01 +/- 0.67 vs. 0.44 +/- 0.17 mu eq.min-1 x mmHg-1, respectively; P < 0.05). These results suggest that the blockade of angiotensin enhances the natriuretic response to increased RIHP during DRIVE.
Pork slurries inoculated with various test microorganisms were prepared and subjected to high hydrostatic pressure at 1000 to 6000 atm for 10 min at 25 degrees C to examine for the pressure effects on characteristics of the slurries and the inactivation of the microorganisms associated with meat and meat products. Pressure treatment at higher than 3000 atm caused coagulation and discoloration of the pork slurries. Harder and more white coagulants were obtained by increasing the pressure. Pressure treatment at 3000 to 6000 atm killed all the microorganisms tested by more than 6-log colony-forming units (cfu)/g except Bacillus cereus spores. Gram-negative microorganisms were more labile to pressure than Gram-positive ones. Campylobacter jejuni, Pseudomonas aeruginosa, Salmonella typhimurium and Yersinia enterocolitica were inactivated at pressures higher than 3000 atm; Escherichia coli, Saccharomyces cerevisiae and Candida utilis at pressures higher than 4000 atm; Micrococcus luteus, Staphylococcus aureus and Streptococcus faecalis at 6000 atm. Only less than one-log cfu/g of B. cereus spores were inactivated at 6000 atm. Ultraviolet absorption spectra and acridine orange staining suggested that E. coli became permeable and leaked cytoplasmic RNA at lower pressure than S. aureus. From the present findings, the authors propose high hydrostatic pressure treatment as a promising means of preparing wholesome meat and meat products.
F-actin purified from rabbit skeletal muscle undergoes reversible dissociation when subjected to hydrostatic pressures up to 240 MPa. Dissociation and reversibility were detected by the following procedures: fluorescence spectral changes observed under pressure, when either intrinsic tryptophan or pyrenyl emission of N-(1-pyrenyl)iodoacetamide-labeled actin were monitored; electron microscopy of samples fixed under pressure; size-exclusion HPLC of pressurized actin. The effect of pressure upon F-actin that had been polymerized in the presence of either Mg2+, Ca2+ or K+ was studied. The standard volume changes for the association of actin subunits, calculated from pressure/dissociation curves were 74 +/- 14 ml/mol for Mg-F-actin, 79 +/- 12 ml/mol for Ca-F-actin and 328 +/- 63 ml/mol for K-F-actin, indicating that actin subunits are packed differently in the polymer depending on which cation is present. All pressure/dissociation data could be fitted by a model for dissociation of a dimer, which suggests that in the F-actin filament there is a predominant intersubunit interaction interface, most likely the head-to-tail intrastrand interaction between two subunits which repeats itself along the polymer. A tenfold change in total protein concentration from 20 micrograms to 200 micrograms/ml Mg-F-actin did not cause a change in the pressure required for half-maximal dissociation. This indicates a heterogeneity of free energy of association among actin monomers in the Mg-F-actin polymer, suggesting that, in addition to the predominant intersubunit interaction, the disordered interactions in the filament significantly contribute to the heterogeneity of microenvironments in the interface between the subunits.
Previous studies have suggested that the efficiency of oxidative phosphorylation in the freshwater eel (Anguilla anguilla) is increased after acclimation to high hydrostatic pressure. Analysis at atmospheric pressure of the respiratory chain complexes showed that, after 21 days at 10.1 MPa, the activity of complex II was decreased to approximately 50 % (P<0.01) of the control value and that cytochrome c oxidase (complex IV) activity was significantly increased to 149 % of the control value (P<0.05). ADP/O ratios calculated from mitochondrial respiration measurements were significantly increased after acclimation to high hydrostatic pressure (2.87 versus 2.52, P<0.001) when measured in the presence of pyruvate plus malate at atmospheric pressure. These results clearly show an increased oxidative phosphorylation efficiency in response to high-pressure acclimation.
OBJECTIVE: To test the effects of intermittent hydrostatic pressure (IHP) on nitric oxide (NO) release induced by shear stress and matrix macromolecule gene expression in human osteoarthritic chondrocytes in vitro. METHODS: Chondrocytes isolated from cartilage samples from 9 patients with osteoarthritis were cultured and exposed to either shear stress or an NO donor. Nitrite concentration was measured using the Griess reaction. Matrix macromolecule mRNA signal levels were determined using reverse-transcriptase polymerase chain reaction and quantified by imaging analysis software. RESULTS: Exposure to shear stress upregulated NO release in a dose and time-dependent manner. Application of IHP inhibited shear stress induced NO release but did not alter NO release from chondrocytes not exposed to shear stress. Shear stress induced NO or addition of an NO donor (sodium nitroprusside) was associated with decreased mRNA signal levels for the cartilage matrix proteins, aggrecan, and type II collagen. Intermittent hydrostatic pressure blocked the inhibitory effects of sodium nitroprusside but did not alter the inhibitory effects of shear stress on cartilage macromolecule gene expression. CONCLUSION: Our data show that shear stress and IHP differentially alter chondrocyte metabolism and suggest that a balance of effects between different loading forces preserve cartilage extracellular matrix in vivo.
One of the goals in the field of tissue engineering is the development of artificial cartilage for the treatment of cartilage defects. Therefore autologous chondrocytes are seeded on different artificial matrices to test their possible use as implants (resorption, antigenicity, toxicity and their integration in the tissue). One of the main problems in these experiments is that usually the amount of available chondrocytes is too low for treating large-scale defects or for comparing different matrices. An in-vitro-multiplication of the cells is needed which causes the chondrocytes to dedifferentiate and become fibroblast-like. Therefore parameters which induce a redifferentiation are of great interest. The objective of this study was to determine the influence of intermittent hydrostatic pressure and low oxygen partial pressure on the redifferentiation of dedifferentiated bovine articular chondrocytes in monolayer and three-dimensional alginate bead culture. The redifferentiation process was monitored by immunocytochemical detection of newly synthesized collagen type II. The viability of the cells was determined by the trypanblue exclusion test. The chondrocytes were dedifferentiated by a two week culture in plastic flasks with an oxygen level of 20%. After this they were subcultured in monolayer or three-dimensional alginate culture and subjected to three different stimuli for three weeks in order to redifferentiate: 1.) 20% O2 (= 20.26 kPa PO2) + 5% CO2 + 75% N2; 2.) 5% O2 (= 5.07 kPa PO2) + 5% CO2 + 90% N2; 3.) 5% O2 (= 5.07 kPa PO2) + 5% CO2 + 90% N2 + 8 h/d of intermittent hydrostatic pressure (frequency: 3 bar absolute for 30 min and 1 bar absolute for 2 min). In the monolayer there was no detectable collagen type II found by immunocytochemistry under either of the three culture conditions. Therefore a redifferentiation of dedifferentiated chondrocytes was not possible in monolayer cultures with the tested parameters. In the three-dimensional alginate culture there was no immunocytochemical staining of collagen type II found in the beads cultured with 20% oxygen. With 5% oxygen we found a strong collagen type II-production by chondrocytes throughout the whole bead. The intermittent hydrostatic pressure combined with 5% oxygen lead to a decreased collagen type II-production compared to cells subjected to 5% oxygen only. Also chondrocytes closer to the edge of these beads were more often immunopositive and seemed to produce more immunoreactive collagen type II. The viability of the chondrocytes in the alginate culture was close to 90% after three weeks. Our experiments showed that oxygen partial pressure is an important parameter in the cultivation of articular chondrocytes. Reduced partial oxygen pressure promoted or induced the redifferentiation of dedifferentiated chondrocytes in alginate culture.
We tested the hypothesis that, in asthma, the airway epithelial damage and leakage of blood proteins into the lumen are the result of edema and raised submucosal hydrostatic pressure. Sheets of dog tracheal epithelium were mounted in Ussing chambers, and the effects of transepithelial hydrostatic pressure differences (delta P) on conductance (G), [3H]mannitol flux (Jman), and fluorescein isothiocyanate-albumin flux (Jalb) were determined. delta P values of 20 cmH2O directed from the mucosal to submucosal side of the tissue (m----s) had no significant effects on G, Jman, Jalb, or tissue ultrastructure. delta Ps----m caused increases in conductance (G) with a maximal effect at approximately 20 cmH2O. delta Ps----m of 20 cmH2O significantly (P less than 0.05) increased G (4.3 +/- 0.6 to 10.6 +/- 1.6 mS/cm2), Jman s----m (18 +/- 5 to 411 +/- 54 nmol.cm-2.h-1), J(alb)s----m (0.3 +/- 0.1 to 6.0 +/- 2.0 micrograms.cm-2.h-1), and J(alb)m----s (0.7 +/- 0.3 to 1.8 +/- 0.4 micrograms.cm-2.h-1). Jman m----s was not affected. On removal of delta P, G and Jman s----m returned to preexposure values, though J(alb)s----m remained slightly elevated at 1.1 +/- 0.3 micrograms.cm-2.h-1. Morphologically, delta Ps----m caused dilation of lateral intercellular spaces, disruption of tight junctions, and submucosal edema. The large increases in s----m fluxes of albumin and mannitol are consistent with bulk flow of fluid toward the lumen via the areas of epithelial damage.
Haight, Roger D. (University of Nebraska, Lincoln) and Richard Y. Morita. Interaction between the parameters of hydrostatic pressure and temperature on aspartase of Escherichia coli. J. Bacteriol. 83:112-120. 1962.-The data obtained from studies of an aspartase preparation and aspartase in cells of Escherichia coli indicate that there is an interaction between the parameters of hydrostatic pressure and temperature. Pressure was found to decrease aspartase activity at 45 C and lower in vitro and below 53 C in vivo, thereby indicating that when the enzyme-substrate complex is formed there is an increase in molecular volume which is counteracted by pressure. Above 53 C in vivo and above 45 C in vitro, temperature probably starts the unfolding process of the enzyme to expose more reactive sites, while pressure then pushes the enzyme and substrate into closer proximity with each other. Thus, pressure stimulated activity and also prevents further unfolding of the enzyme. Since the enzyme preparation retains about the same level of activity after being subjected first to 1000 atm at 56 C, the aspartase probably refolds into its original configuration or one similar to it, when subjected to 1 atm at 37 C. In all cases, the presence of the substrate was found necessary to protect aspartase from thermal inactivation or denaturation.
Plasminogen undergoes a large conformational change when it binds 6-aminohexanoate. Using ultraviolet absorption spectroscopy and native PAGE, we show that hydrostatic pressure brings about the same conformational change. The volume change for this conformational change is -33 mL.mol-1. Binding of ligand and hydrostatic pressure both cause the protein to open up to expose surfaces that had previously been buried in the interior.
Investigations have been made into the effects of temperature (between 10 and 19 degrees C) on the one hand, and hydrostatic pressure per se (101 ATA) on the other, on recovery time from anesthesia (pentobarbital) in a fish (Salmo gairdneri R). At each of the two pressures studied (1 and 101 ATA) the rise in temperature shortens the recovery period, after a treatment by a constant dose of pentobarbital. Under hyperbaric pressure conditions (101 ATA) the duration of anesthesia is lower than the control value obtained at barometric pressure, whatever the temperature. The analysis of the results shows a similarity between the effects of temperature and pressure on the recovery from pentobarbital anesthesia on the one hand and on chemical kinetics on the other. The results support the hypothesis of the direct action of hydrostatic pressure per se at molecular level.
OBJECTIVE: This study tested the hypothesis that intermittent hydrostatic pressure applied to human osteoarthritic chondrocytes modulates matrix metalloproteinase and pro-inflammatory mediator release in vitro. DESIGN: Human osteoarthritic articular chondrocytes were isolated and cultured as primary high-density monolayers. For testing, chondrocyte cultures were transferred to serum-free medium and maintained without loading or with exposure to intermittent hydrostatic pressure (IHP) at 10 MPa at a frequency of 1 Hz for periods of 6, 12 and 24 h. Levels of matrix metalloproteinase-2, -9 (MMP-2, -9), tissue inhibitor of metalloproteinase-1 (TIMP-1), and the pro-inflammatory mediators, interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1), released into the culture medium were assessed by ELISA. Matrix metalloproteinase activity was confirmed by zymographic analysis. RESULTS: In the absence of IHP, levels of MMP-2, TIMP-1, IL-6, and MCP-1 in the chondrocyte culture medium increased in a time-dependent manner. Application of IHP decreased MMP-2 levels at all time periods tested, relative to unloaded control cultures maintained for the same time periods. Although 84/82 kDa bands were faintly detectable by zymography, MMP-9 levels were not quantifiable in medium from loaded or unloaded cultures by ELISA. TIMP-1 levels were not altered in response to IHP at any time period tested. IL-6 and MCP-1 levels decreased in cultures exposed to IHP at 12 and 24 h, relative to unloaded control cultures maintained for the same time periods. CONCLUSION: IHP decreased release of MMP-2, IL-6 and MCP-1 by osteoarthritic chondrocytes in vitro suggesting that pressure influences cartilage stability by modulating chondrocyte expression of these degradative and pro-inflammatory proteins in vivo.
This review of available literature attempts to interpret net effects of metabolically inert light gases (He, H2, and Ne) as the resultant of hydrostatic pressure and intrinsic pharmacological effects associated with exposure to these gases, and to assess the relative importance of each component with respect to a number of biological responses. A common pattern is recognizable for pressure reversal of anesthesia, high pressure convulsions, high pressure bradycardia, and certain characteristics of liposome model systems. Using the method of analysis proposed, these lightest gases can be shown to conform to the pattern of relation of potency to physical properties characteristic of more potent gaseous anesthetics, including N2, N2O, and Xe. The relations between effect produced and partial pressure of the acting gas are approximately linear to total pressures of 100 ATA for anesthesia or pressure reversal of anesthesia and (or to a much smaller extent) for the liposome model systems, but not for high pressure convulsions. As a result of these general factors no single gas can be expected to neutralize the effects of hydrostatic pressure with regard to all of the biological responses tested over any significant pressure range. A series of experiments with single cells and tissue cultures have revealed interactions between high pressure and inert gas that do not conform to the pattern set by the responses mentioned so far. These responses cannot yet be shown to constitute a homogeneous group and may represent at least two subgroups. Responses falling into this second heterogeneous category include cell motility, development of cell abnormalities and lysis, and cell and perhaps virus replication or multiplication. The implication of these results for the formulation of biophysical hypotheses to explain interactions between inert gas and high pressure, for considerations of high pressure effects as a safety hazard, and for the problem of experimental approaches to the study of pressure acclimation are discussed briefly.
The pressure conditions at the distal end of the interlobular arteries and in the interlobular veins were investigated from the pressures obtained in superficial small arteries and veins, accidentally found on the kidney surface, during the subsequent blockade of the blood stream in the down-stream and up-stream direction, respectively. The results suggested a hydrostatic pressure in the distal end of the interlobular arteries of about 85 mm Hg under normotensive conditions-a pressure which remained fairly constant when the perfusion pressure in the renal artery was decreased within the autoregulation range. The results indicate a considerable pressure drop of about 40 mm Hg along the interlobular arteries. During hypotension this pressure drop decreased, implying a decreased resistance in the interlobular arteries, i.e. a typical autoregulative response. The pressure in the interlobular veins amounted to about 5 mm Hg, which is a few mm Hg higher than that in the renal vein and about 7 mm lower than that in the peritubular capillary network. The results suggest a flow resistance located somewhere between the peritubular capillaries and the intrarenal veins. This resistance is not influenced by vasoactive substances but it is decreased when the systemic venous pressure is raised above 10 mm Hg. The resistance seems to act in the direction of protecting the peritubular capillaries from minor changes in the central venous pressure.
Barophilic bacteria inhabit the deep oceans, and the specific functional modifications and regulatory mechanisms which govern adaptation to hydrostatic pressure are beginning to be understood. For example, the rate of production of several proteins by some hydrothermal vent archaebacteria and the degree of saturation of membrane lipids in other deep-sea bacteria have been found to change as a result of cultivation at high pressure. We report here the cloning of gene, ompH, which encodes a major pressure-inducible protein of strain SS9, a gram-negative eubacterium isolated from a depth of 2.5 kilometres in the Sulu Sea. Messenger RNA encoded by ompH is expressed when cells are grown at 280 atm but not at 1 atm, indicating that transcription of the ompH gene is controlled by hydrostatic pressure. The function of the OmpH protein in adaptation to high pressure and the use of the ompH gene in studying how bacteria sense and respond to pressure is discussed.