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PubMed2006

The effects of intermittent hydrostatic pressure on self-assembled articular cartilage constructs.

To date, static culture for the tissue engineering of articular cartilage has shown to be inadequate in conferring functionality to constructs. Various forms of mechanical stimuli accompany articular cartilage development in vivo, and one of these is hydrostatic pressure. This study used histology, biochemistry, and biomechanics to examine the effects of intermittent hydrostatic pressure, applied at 10 MPa and 1 Hz for 4 h per day for 5 days per week for up to 8 weeks on self-assembled chondrocyte constructs. The self-assembling process is a novel approach that allows engineering of articular cartilage constructs without the use of exogenous scaffolds. The self-assembled constructs were found to be capable of enduring this loading regimen. Significant increases in collagen production were only observed in pressurized samples. Intermittent hydrostatic pressure prevented a significant decrease in total GAG, which was significant in controls. Aside from the beneficial effects intermittent hydrostatic pressure may have on ECM synthesis, its effects on mechanical properties may require longer culture periods to manifest. This study demonstrates the successful use of the self-assembling process to produce articular cartilage constructs. It also shows for the first time that long-term culture of tissue-engineered articular cartilage construct benefits from intermittent hydrostatic pressure.

Animals↗

PubMed2002

Inactivation of hepatitis A virus and a calicivirus by high hydrostatic pressure.

Potential application of high hydrostatic pressure processing (HPP) as a method for virus inactivation was evaluated. A 7-log10 PFU/ml hepatitis A virus (HAV) stock, in tissue culture medium, was reduced to nondetectable levels after exposure to more than 450 MPa of pressure for 5 min. Titers of HAV were reduced in a time- and pressure-dependent manner between 300 and 450 MPa. In contrast, poliovirus titer was unaffected by a 5-min treatment at 600 MPa. Dilution of HAV in seawater increased the pressure resistance of HAV, suggesting a protective effect of salts on virus inactivation. RNase protection experiments indicated that viral capsids may remain intact during pressure treatment, suggesting that inactivation was due to subtle alterations of viral capsid proteins. A 7-log10 tissue culture infectious dose for 50% of the cultures per ml of feline calicivirus, a Norwalk virus surrogate, was completely inactivated after 5-min treatments with 275 MPa or more. These data show that HAV and a Norwalk virus surrogate can be inactivated by HPP and suggest that HPP may be capable of rendering potentially contaminated raw shellfish free of infectious viruses.

Caliciviridae↗

PubMed2002

Food processing by high hydrostatic pressure.

The use of high hydrostatic pressures (HHP) for food processing is finding increased application within the food industry. One of the advantages of this technology is that because it does not use heat, sensory, and nutritional attributes of the product remain virtually unaffected, thus yielding products with better quality than those processed traditional methods. HHP have the ability to inactivate microorganisms as well as enzymes responsible for shortening the life of a product. In addition to lengthening the shelf-life of food products, HHP can modify functional properties of components such as proteins, which in turn can lead to the development of new products. Equipment for large-scale production of HHP processed products are commercially available nowadays. Guacamole, sliced ham, oysters, and fruit juices are some of the products currently available on the market. HHP technology is one of the most promising nonthermal processes.

Consumer Behavior↗

PubMed2004

New insights into conformational and functional stability of human alpha-thrombin probed by high hydrostatic pressure.

The effects of high hydrostatic pressure (HHP) and urea on conformational transitions of human alpha-thrombin structure were studied by fluorescence spectroscopy and by measuring the catalytic activity of the enzyme. Treatment of thrombin with urea produced a progressive red shift in the center of mass of the intrinsic fluorescence emission spectrum, with a maximum displacement of 650 cm(-1). HHP (270 MPa) shifted the centre of mass by only 370 cm(-1). HHP combined with a subdenaturing urea concentration (1.5 m) displaced the centre of mass by approximately 750 cm(-1). The binding of the fluorescent probe bis(8-anilinonaphthalene-1-sulfonate) to thrombin was increased by 1.8-, 4.0-, and 2.7-fold after treatment with high urea concentration, HHP or HHP combined with urea, respectively, thus suggesting that all treatments convert the enzyme to partially folded intermediates with exposed hydrophobic regions. On the other hand, treatment of thrombin with urea (but not HHP) combined with dithiothreitol progressively displaced the fluorescent probe, thus suggesting that this condition converts the enzyme to a completely unfolded state. Urea and HHP also led to different conformations when changes in the thrombin catalytic site environment were assessed using the fluorescence emission of fluorescein-d-Phe-Pro-Arg-cloromethylketone-alpha-thrombin: addition of urea up to 2 m gradually decreased the fluorescence emission of the probe to 65% of the initial intensity, whereas HHP caused a progressive increase in fluorescence. Hydrolysis of the synthetic substrate S-2238 was enhanced (35%) in 2 m urea and gradually abolished at higher concentrations, while HHP (270 MPa) inhibited the enzyme's catalytic activity by 45% and abolished it when 1.5 m urea was also present. Altogether, analysis of urea and HHP effects on thrombin structure and activity indicates the formation of dissimilar intermediate states during denaturation by these agents.

Anilino Naphthalenesulfonates↗

PubMed1988

Prostaglandin blockade blunts the natriuresis of elevated renal interstitial hydrostatic pressure.

Previous studies have shown that renal interstitial volume expansion (RIVE) increases renal interstitial hydrostatic pressure and urinary sodium excretion. In the present study we investigated whether blockade of prostaglandin synthesis inhibits the increase in fractional sodium excretion induced by RIVE. Expansion of the renal interstitial volume was achieved by injecting 50 microliters of 2.5% albumin solution into a polyethylene matrix chronically implanted in the left kidney. Fractional sodium excretion (FENa), renal interstitial hydrostatic pressure (PI), and urinary prostaglandin excretion (UPGE2) were measured before and after RIVE in eight control, seven meclofenamate-treated, and eight indomethacin-treated rats. RIVE in the control animals resulted in significant increases in PI (delta + 4.2 +/- 0.8 mmHg), in FENa (delta + 1.02 +/- 0.27%), and in UPGE2 (% delta + 150 +/- 38%) without significant changes in glomerular filtration rate. Inhibition of prostaglandin synthesis with meclofenamate or indomethacin attenuated the natriuretic response and blocked the increase in UPGE2 associated with RIVE. In summary, direct increases in renal interstitial hydrostatic pressure increase UPGE2 and urinary sodium excretion. This natriuretic response is markedly diminished by inhibition of prostaglandin synthesis. These studies suggest that prostaglandin synthesis may have an important role in mediating the natriuretic effect of increased renal interstitial hydrostatic pressure during renal interstitial volume expansion.

Animals↗

PubMed1993

Intraperitoneal hydrostatic pressure and ultrafiltration volume in CAPD.

The intraperitoneal hydrostatic pressure (IPP) and overall ultrafiltration (UF) volume were measured in 34 patients after a 2-hour exchange with 2 L of dialysate containing 3.86% glucose. These patients had been treated with continuous ambulatory peritoneal dialysis (CAPD) on average for 26 +/- 20 months under stable conditions. The patients were at rest during the 2-hour exchange. The normal IPP measured with the patient lying completely flat was on inspiration (IPPinsp) 14 +/- 4 cm of water (cmH2O) and on expiration (IPPexp) 12 +/- 3 cm H2O, for an intraperitoneal volume of 2820 +/- 319 mL. The mean UF was 744 +/- 323 mL. The mean IPP (IPPmean), defined by (IPPinsp+IPPexp)/2, had a negative linear correlation to the UF volume (r = 0.66; p = 0.0001). The linear regression test showed that an increase of 1 cm H2O in the IPPmean reduced the overall UF volume by 70 mL in 2 hours. In conclusion, even though the UF is produced by the osmotic pressure of the dialysate, it is also influenced to a great extent by the intraperitoneal hydrostatic pressure, which should not be ignored.

Adult↗

PubMed1978

Effects of hydrostatic pressure on fluid transfer by the isolated gallbladder.

The influence of hydrostatic pressure elevation on fluid absorption by the isolated guinea pig gallbladder was determined following addition of various smooth muscle spasmogens to the serosal bathing medium. Acetylcholine, prostaglandin E1 and ouabain increased intraluminal pressure from about 4 cm H2O to 13 cm H2O. The pressure increase was associated with a marked but transient stimulation in fluid transfer. The same phenomenon was observed following an increase in hydrostatic pressure by addition of Ringer's solution to the gallbladder lumen. Evidence is presented to support the hypothesis that this phenomenon represents the extrusion of an edema from the gallbladder wall. Two observations favour this explanation: 1. The wet weights of gallbladder subjected to high intraluminal hydrostatic pressure were only one third of those at low pressure. 2. Microscopic examination of the wall of gallbladders at low pressure showed a marked submucosal edema.

Acetylcholine↗

PubMed2000

Transcriptional activation in chondrocytes submitted to hydrostatic pressure.

At present, only a little is known about the transcriptional regulation in chondrocytes submitted to various physicomechanical factors known to exist in articular cartilage. Recently, we have investigated the effects of hydrostatic pressure on transcriptional control in chondrocytes using human chondrosarcoma and immortalized chondrocyte cell lines for the experiments. Hydrostatic pressure was applied on the cells in a special computer-controlled, water-filled pressure chamber, where cyclic and static pressures up to 32 MPa can be created. Differential display RT-PCR and probing of cDNA arrays are the methods we have used to study differential gene expression due to hydrostatic pressure. By differential display RT-PCR experiments, we have observed several differentially expressed cDNA bands under continuous 30 MPa hydrostatic pressure, while 30 MPa cyclic pressure at 1 Hz produced much fewer changes. In the first phase of our studies, we have focused on the effects of 30 MPa hydrostatic pressure because it causes a unique hsp70-mediated stress response in immortalized chondrocytes. Differential display RT-PCR screening provided us with several clones that derive from low-abundance mRNAs, such as death-associated protein 3 (DAP3), a nucleotide-binding protein which increases due to interferon-gamma induced cell death; PTZ-17 (or p311), a seizure-related protein; H-NUC, a nuclear DNA binding protein; and one new gene of unknown function. In Northern blots, an induction was confirmed for the new gene, DAP3 and PTZ-17 were down-regulated in some but not in all parallel experiments; however, basal level of H-NUC mRNA was too low to be detected in Northern blots. We then chose to widen our screening to a number of known genes arrayed as cDNA blots. Under 30 MPa continuous hydrostatic pressure, four different time points were chosen (0, 3, 6 and 24 h) for the experiments. The screening of 588 cDNAs showed 15 up-regulated and 6 down-regulated genes. Consistently with our previous results hsp70 was highly induced, as well as hsp40, a chaperone protein functioning together with hsp70. Gadd45 and to a lesser extent Gadd153 (stress genes induced by, e.g., ionizing radiation and ischaemia) were up-regulated, as well as p21waf1,cip1, a protein participating in cell cycle regulation that can interact with Gadd45. Northern blots confirmed Gadd45 induction. Down-regulated transcripts included, e.g., DAD-1, glutathione S-transferase pI, DNA-binding inhibitor ID-1H, and cytoplasmic dynein light chain.

Blotting, Northern↗

PubMedDate not supplied

Physiological levels of hydrostatic pressure alter morphology and organization of cytoskeletal and adhesion proteins in MG-63 osteosarcoma cells.

The response of human MG-63 osteosarcoma cells to physiological levels of hydrostatic pressure was studied. Cell cultures were subjected to a 20-min, 4-MPa hydrostatic pressure pulse. Adhesion was measured at 20 min and 2 h post-hydrostatic pressure. Morphometric measurements of cell shape and immunofluorescent assays of cytoskeletal and adhesion proteins were done pre- and post-hydrostatic pressure. Pressure-treated cells showed increased adhesion (resistance to deadhesion by trypsinization)-with increased recovery time. Indirect immunofluorescence demonstrated increased heterotypic adhesion receptor at cell-cell interfaces and increased alpha 3, beta 1-integrin at cell-substrate interfaces. Indirect immunofluorescence demonstrated depolymerization of alpha-tubulin, vimentin, and actin during the pressure pulse. Actin reorganization was slower than that of alpha-tubulin and vimentin, with stress filaments not well organized even after 1 h postpressure. The depolymerization of alpha-tubulin, vimentin, and actin observed at relatively low levels of hydrostatic pressure suggests disintegration of the integrin-cytoskeletal attachment complex. The increased resistance of the cells to trypsinization and the increase in both heterotypic adhesion receptor and the alpha 3, beta 1-integrin at cell interfaces suggest that cells compensate for loss of cytoskeletal integrity by increasing attachment to both adjacent cells and the extracellular matrix.

Bone and Bones↗

PubMed2002

Dogfish hair cells sense hydrostatic pressure.

Many marine invertebrates and fish respond to hydrostatic pressure in order to regulate their depth and synchronize their behaviour to tidal cycles. Here we investigate the effect of hydrostatic pressure on the vestibular hair cells located in the labyrinth of the dogfish Scyliorhinus canicula, and find that it modulates their spontaneous activity and response to angular acceleration. This may explain not only the low resting activity of vertebrate hair cells but also how fish that do not have swim bladders can sense hydrostatic cues.

Animals↗

PubMed2003

Hydrostatic pressure modulates proteoglycan metabolism in chondrocytes seeded in agarose.

OBJECTIVE: To investigate the effect of isolated hydrostatic pressure on proteoglycan metabolism in chondrocytes. METHODS: Bovine articular chondrocytes cultured in agarose gels were subjected to 5 MPa hydrostatic pressure for 4 hours in either a static or a pulsatile (1 Hz) mode, and changes in glycosaminoglycan (GAG) synthesis, hydrodynamic size, and aggregation properties of proteoglycans and aggrecan messenger RNA (mRNA) levels were determined. RESULTS: The application of 5 MPa static pressure caused a significant increase in GAG synthesis of 11% (P < 0.05). Column chromatography showed that this increase in GAG synthesis was associated with large proteoglycans. In addition, semiquantitative reverse transcriptase-polymerase chain reaction showed a 4-fold increase in levels of aggrecan mRNA (P < 0.01). CONCLUSION: Hydrostatic pressure in isolation, which does not cause cell deformation, can affect proteoglycan metabolism in chondrocytes cultured in agarose gels, indicating an important role of hydrostatic pressure in the regulation of extracellular matrix turnover in articular cartilage.

Aggrecans↗

PubMed2004

Use of hydrostatic pressure to produce 'native' monomers of yeast enolase.

The effects of hydrostatic pressure on yeast enolase have been studied in the presence of 1 mm Mn(2+). When compared with apo-enolase, and Mg-enolase, the Mn-enzyme differs from the others in three ways. Exposure to hydrostatic pressure does not inactivate the enzyme. If the experiments are performed in the presence of 1 mm Mg(2+), or with apo-enzyme, the enzyme is inactivated [Kornblatt, M.J., Lange R., Balny C. (1998) Eur. J. Biochem 251, 775-780]. The UV spectra of the high pressure forms of the Mg(2+)- and apo-forms of enolase are identical and distinct from the spectrum of the form obtained in the presence of 1 mm Mn(2+); this suggests that Mn(2+) remains bound to the high pressure form of enolase. With Mn-enolase, the various spectral changes do not occur in the same pressure range, indicating that multiple processes are occurring. Pressure experiments were performed as a function of [Mn(2+)] and [protein]. One of the changes in the UV spectra shows a dependence on protein concentration, indicating that enolase is dissociating into monomers. The small changes in the UV spectrum and the retention of activity lead to a model in which enolase, in the presence of high concentrations of Mn(2+), dissociates into native monomers; upon release of pressure, the enzyme is fully active. Although further spectral changes occur at higher pressures, there is no inactivation as long as Mn(2+) remains bound. We propose that the relatively small and polar nature of the subunit interface of yeast enolase, including the presence of several salt bridges, is responsible for the ability of hydrostatic pressure to dissociate this enzyme into monomers with a native-like structure.

Enzyme Stability↗

PubMed1992

Response of plasma membrane to applied hydrostatic pressure in chondrocytes and fibroblasts.

Effects of applied hydrostatic pressure on transmembrane potentials were investigated in sheep articular chondrocytes and human skin fibroblasts in non-confluent monolayer cultures. Resting potentials in chondrocytes (about -12 mv) and in fibroblasts (about -15 mV) were increased and decreased respectively by over 40% after pressure was applied cyclically (0.33 Hz, 120 mm Hg, 20 minutes). Continuous pressure (120 mm Hg, 20 minutes) caused deplorization in both cell types. Low frequency pressure application (less than 0.08 Hz) caused depolarization in chondrocytes and hyperpolarization in fibroblasts. Quinidine (2 x 10(-5) M) blocked and verapamil (10(-5) M) reduced hyperpolarization responses, suggesting involvement of Ca(2+)-dependent K+ channels. A23187 (1.9 x 10(-6) M) caused hyperpolarization in chondrocytes, augmented further by subsequent pressure application (0.33 Hz). Tetrodotoxin (10(-6) M) blocked depolarization responses indicating that these were due to Na+ influx. Blockade of histamine H1 receptors by chlorpheniramine maleate (5.1 x 10(-6) M), H2 receptors by cimetidine (7.9 x 10(-6) M) and beta-adrenoreceptors by sotolol (1.3 x 10(-4) M) had no effect on hydrostatic pressure-induced hyperpolarization in chondrocytes. Cytochalasin B (2 x 10(-5) M and at 4 x 10(-6) M) abolished pressure-induced hyperpolarization in chondrocytes; in contrast, applied cyclical hydrostatic pressure to cytochalasin-treated fibroblasts caused hyper-polarization, suggesting that cytoskeletal changes were involved.

Animals↗

PubMed2002

The effect of hydrostatic pressure on S-layer-supported lipid membranes.

We report on the behavior of unsupported and surface layer (S-layer)-supported lipid membranes at the application of a uniform hydrostatic pressure. At a hydrostatic pressure gradient higher than 6 N/m(2), unsupported lipid membranes, independent from which side pressurized and S-layer-supported lipid membranes pressurized from the lipid-faced side revealed a pronounced increase in capacitance. A maximal hydrostatic pressure gradient of 11.0 N/m(2) resulted in an almost doubling of the capacitance of the (composite) membranes. S-layer-supported lipid membranes showed a hysteresis in the capacitance versus pressure plot, indicating that this composite structure required a certain time to reorient when the pressure gradient acting from the lipid-faced side was balanced. By contrast, the S-layer-supported lipid membrane pressurized from the protein-faced side revealed only a minute increase in capacitance (C/C(0,max)=1.17+/-0.05), reflecting only minor pressure-induced area expansion. In addition, no hysteresis could be observed, indicating that no rearrangement of the composite membrane occurred. The maximal induced tension was with 4.3+/-0.2 mN/m, significantly higher than that of unsupported (2.5+/-0.3 mN/m) and S-layer-supported lipid membranes pressurized from the lipid-faced side (2.6+/-0.1 mN/m).

Bacterial Proteins↗

PubMed1962

Malic dehydrogenase activity at 101 C under hydrostatic pressure.

Morita, Richard Y. (University of Nebraska, Lincoln) and Roger D. Haight. Malic dehydrogenase activity at 101 C under hydrostatic pressure. J. Bacteriol. 83:1341-1346. 1962.-No malic dehydrogenase activity was found to occur at 101 C at various hydrostatic pressures from 1 to 700 atm. However, activity was demonstrated with hydrostatic pressures above 700 atm, with optimal activity at 1,300 atm at the same temperature. Explanation of the data is based upon thermal denaturation of the enzyme, which involves a molecular volume increase of the enzyme. The molecular volume increase is counteracted by hydrostatic pressure. Pressures above 700 atm to 1,500 atm (highest employed) were sufficient to offset the denaturation by 101 C which probably resulted in an incomplete denaturation of malic dehydrogenase.

Atmospheric Pressure↗

PubMed1995

Antagonistic effects of hydrostatic pressure and osmotic pressure on cytochrome P-450cam spin transition.

The combined effects of hydrostatic pressure and osmotic pressure, generated by polyols, on the spin equilibrium of fenchone-bound cytochrome P-450cam were investigated. Hydrostatic pressure indices a high spin to low spin transition, whereas polyols induce the reversed reaction. Of the four solutes used, glycerol, glucose, stachyose, and sucrose, only the last two would act on the spin transition by osmotic stress. The spin volume changes measured by both techniques are different, 29 and -350 ml/mol for hydrostatic pressure and osmotic pressure, respectively. It suggests that even if the two are perturbing water molecules, different properties are probed. From the volume change induced by osmotic stress, 19 water molecules are deduced that would be implicated in the spin transition of the fenchone-bound protein. This result suggests that water molecules other than the well defined ones located in the active site play a key role in modulating the spin equilibrium of cytochrome P-450cam.

Camphor 5-Monooxygenase↗

PubMed1976

Hydrostatic pressure effects on deswelling of de-epithelialized and de-endothelialized corneas.

The effects of varying hydrostatic pressure on the thinning rate of preswollen de-epithelialized or de-endothelialized corneas has been determined in the specular microscope. The appropriate membrane was removed, the cornea given access to Ringer to swell, and then fluid exchange at that surface blocked with oil. De-epithelialized corneas thin more slowly as hydrostatic pressure on the posterior surface is increased, until fluid movement ceases at 60 to 70 mm. Hg. Fluid movement can occur, therefore, against a considerable hydrostatic pressure. De-endothelialized corneas thin at a higher rate as hydrostatic pressure is increased; this effect is probably a mechanical one with increasing pressure forcing fluid out across the epithelium.

Animals↗

PubMed2004

Injection by hydrostatic pressure in conjunction with electrokinetic force on a microfluidic chip.

A simple method was developed for injecting a sample on a cross-form microfluidic chip by means of hydrostatic pressure combined with electrokinetic forces. The hydrostatic pressure was generated simply by adjusting the liquid level in different reservoirs without any additional driven equipment such as a pump. Two dispensing strategies using a floating injection and a gated injection, coupled with hydrostatic pressure loading, were tested. The fluorescence observation verified the feasibility of hydrostatic pressure loading in the separation of a mixture of fluorescein sodium salt and fluorescein isothiocyanate. This method was proved to be effective in leading cells to a separation channel for single cell analysis.

Animals↗