Pressurization facilitates adenovirus-mediated gene transfer into vein graft

We investigated whether application of non-distending hydrostatic pressure facilitates gene transfer into vein grafts. An external jugular vein was placed in a chamber with 100 microl adenovirus solution at a titer of 10(10) pfu/ml and was pressurized to up to 8 atm above ambient pressure for 10 min. Histochemical analysis demonstrated a positive transgene expression in all layers of the vessel wall. Gene transfer with 8 atm pressurization resulted in an approximately 50 times higher transgene expression than that without pressurization. Under 8 atm pressurization, the efficiency of gene transfer reached a plateau at 7.5 min. The application of hydrostatic pressure may improve the effectiveness of intraoperative genetic engineering of vein grafts.     

Pressure-induced depolymerization of spindle microtubules. III. Differential stability in HeLa cells

Evidence from light microscopy (principally polarization microscopy) has demonstrated that hydrostatic pressure can reversibly inhibit mitosis by rapidly depolymerizing the spindle fiber microtubules. We have confirmed this finding in ultrastructural studies of mitotic HeLa cells incubated at 37 degrees C and pressurized at 680 atm (10,000 psi). Althouth there are many spindle microtubules in the cells at atmospheric pressure, electron micographs of cells pressurized for 10 min (and fixed while under pressure in a Landau-Thibodeau chamber) show few microtubules. Pressure has a differential effect on the various types of spindle microtubules. Astral and interpolar MTs appear to be completely depolymerized in pressurized cells, but occasional groups of kinetochore fiber microtubules are seen. Surprisingly, the length and density of microtubules of the stem bodies and midbody of telophase cells appear unchanged by pressurization. In cells fixed 10 min after pressure was released, microtubules were again abundant, the density often appearing to be higher than in control cells. Reorganization seems incomplete, however, since many of the microtubules are randomly oriented. Unexpectedly, kinetochores appeared diffuse and were difficult to identify in sections of pressurized cells. Even after 10 min of recovery at atmospheric pressure, their structure was less distinct than in unpressurized cells.     

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).     

Growth of the extreme thermophile Sulfolobus acidocaldarius in a hyperbaric helium bioreactor

The relationship between pressure and temperature as it affects microbial growth and metabolism has been examined only for a limited number of bacterial species. Because many newly-discovered, extremely thermophilic bacteria have been isolated from pressurized environments, this relationship merits closer scrutiny. In this study, the extremely thermophilic bacterium, Sulfolobus acidocaldarius, was cultured successfully in a hyperbaric chamber containing helium and air enriched with 5% carbon dioxide. Over a pressure range of approximately 1-120 bar and a temperature range of 67-80 degrees C, growth was achieved in a heterotrophic medium with the air mixture at partial pressures up to 3.5 bar. Helium was used to obtain the final, desired incubation pressure. No significant growth was noted above 80 degrees C over the same range of hyperbaric pressures, or at 70 degrees C when pressure was applied hydrostatically. Growth experiments conducted under hyperbaric conditions may provide a means to study these bacteria under simulated in situ conditions and simultaneously avoid the complications associated with hydrostatic experiments. Results indicate that hyperbaric helium bioreactors will be important in the study of extremely thermophilic bacteria that are isolated from pressurized environments.     

The role of microtubules in the regulation of proteoglycan synthesis in chondrocytes under hydrostatic pressure

Chondrocytes of the articular cartilage sense mechanical factors associated with joint loading, such as hydrostatic pressure, and maintain the homeostasis of the extracellular matrix by regulating the metabolism of proteoglycans (PGs) and collagens. Intermittent hydrostatic pressure stimulates, while continuous high hydrostatic pressure inhibits, the biosynthesis of PGs. High continuous hydrostatic pressure also changes the structure of cytoskeleton and Golgi complex in cultured chondrocytes. Using microtubule (MT)-affecting drugs nocodazole and taxol as tools we examined whether MTs are involved in the regulation of PG synthesis in pressurized primary chondrocyte monolayer cultures. Disruption of the microtubular array by nocodazole inhibited [(35)S]sulfate incorporation by 39-48%, while MT stabilization by taxol caused maximally a 17% inhibition. Continuous hydrostatic pressure further decreased the synthesis by 34-42% in nocodazole-treated cultures. This suggests that high pressure exerts its inhibitory effect through mechanisms independent of MTs. On the other hand, nocodazole and taxol both prevented the stimulation of PG synthesis by cyclic 0. 5 Hz, 5 MPa hydrostatic pressure. The drugs did not affect the structural and functional properties of the PGs, and none of the treatments significantly affected cell viability, as indicated by the high level of PG synthesis 24-48 h after the release of drugs and/or high hydrostatic pressure. Our data on two-dimensional chondrocyte cultures indicate that inhibition of PG synthesis by continuous high hydrostatic pressure does not interfere with the MT-dependent vesicle traffic, while the stimulation of synthesis by cyclic pressure does not occur if the dynamic nature of MTs is disturbed by nocodazole. Similar phenomena may operate in cartilage matrix embedded chondrocytes.     

High-pressure inactivation of dried microorganisms

Dried microorganisms are particularly resistant to high hydrostatic pressure effects. In this study, the survival of Saccharomyces cerevisiae was studied under pressure applied in different ways. Original processes and devices were purposely developed in our laboratory for long-term pressurization. Dried and wet yeast powders were submitted to high-pressure treatments (100-150 MPa for 24-144 h at 25 degrees C) through liquid media or inert gas. These powders were also pressurized after being vacuum-packed. In the case of wet yeasts, the pressurization procedure had little influence on the inactivation rate. In this case, inactivations were mainly due to hydrostatic pressure effects. Conversely, in the case of dried yeasts, inactivation was highly dependent on the treatment scheme. No mortality was observed when dried cells were pressurized in a non-aqueous liquid medium, but when nitrogen gas was used as the pressure-transmitting fluid, the inactivation rate was found to be between 1.5 and 2 log for the same pressure level and holding time. Several hypotheses were formulated to explain this phenomenon: the thermal effects induced by the pressure variations, the drying resulting from the gas pressure release and the sorption and desorption of the gas in cells. The highest inactivation rates were obtained with vacuum-packed dried yeasts. In this case, cell death occurred during the pressurization step and was induced by shear forces. Our results show that the mechanisms at the origin of cell death under pressure are strongly dependent on the nature of the pressure-transmitting medium and the hydration of microorganisms.     

Incorporation of l-Leucine 1-13C-Dodecyl Ester to Succinylated αs1-Casein during a Papain-catalyzed Reaction

High hydrostatic pressures of 100 MPa to 300 MPa were applied to beef post-rigor muscle to investigate the efficiency of pressurization as a meat tenderizer.

The fragmentation of myofibrils increased with increasing pressure applied to the muscle, and the degree of fragmentation reached to its maximal level after briefly exposing (5 min) post-rigor muscle to the highest pressure (300 MPa). Electron microscopic studies of the pressurized muscle revealed that marked rupture of I-band and loss of M-line materials had progressed in the myofibrils with increasing applied pressure. However, degradation of the Z-line in myofibrils that can be observed naturally in conditioned muscle was not apparent in the pressurized muscle. There was no significant difference in the electrophoretic pattern of myofibrillar protein among the control and pressurized muscle samples in spite of the marked change of ultrastructure.

From the results, it is suggested that the application of a high hydrostatic pressure to post-rigor muscle causes tenderization of the meat in a different manner from that of conditioning.     

Optical chamber system designed for microscopic observation of living cells under extremely high hydrostatic pressure

A novel pressure chamber system has been developed for the study of living cells under conditions of extremely high hydrostatic pressure up to 100 MPa (1 atm = 0.101325 MPa). The temperature in the chamber is thermostatically controlled in the range from 2 degrees to 80 degrees C. Two high-pressure pumps are employed for continuous perfusion of the chamber with culture medium and a chemical solution under high hydrostatic pressure conditions. The chamber has a 2-mm-thick glass window 2 mm in diameter, with a minimum working distance of 3.8 mm. The chamber system is designed to be adaptable to a variety of microscopic and imaging techniques. Using this chamber system, we successfully carried out real-time observations of elongated Escherichia coli and rounded HeLa cells under pressure.     

Effects of flooding under hydrostatic pressure on the respiratory metabolism of germinated wheat seeds

Germinated wheat seeds ( Triticum aestivum L. cv. Barqai) that had been subjected to short hydrostatic pressure treatments (0.3–1.2 MPa) changed their normal metabolism into one which is characterized by a high ethanol production, a low O₂ consumption and a low CO₂ evolution. Alcoholic fermentation could account for ca half of the CO₂ evolved from the pressurized seeds. The level of acetaldehyde was low, though significantly higher in the pressurized seeds than in the controls. Subjection of wheat seeds to osmotic stress under aerobic conditions lowered their O₂ uptake and CO₂ evolution but did not induce ethanol production. Exposure of pressurized seeds to NaCl stress did not alter their ethanol production beyond that which had been induced by pressure. Ethanol production by pressurized seeds increased following either the addition of sucrose or by excision of the embryos from the endosperms. More electrolytes leaked into the embedding solution from pressurized seeds than from control seeds. Exogenous ethanol was toxic to wheat seeds at concentrations as low as 343 m M . The effects of hydrostatic pressure and of the consequently induced ethanol production on the mortality of flooded seeds is discussed.     

Effects of pressure on germination of seeds of wheat (Triticum aestivum cv. Barqai) in saline and in non-saline media

The effects of an increase in the absolute environmental pressure (air, N₂, O₂ or hydrostatic), up to 1 MPa, on the germination of wheat seeds and the survival of wheat seedlings were studied. Seeds were exposed to saline and non-saline media, in Petri dishes, on a double layer of filter paper. They were then introduced for different time periods into a pressure chamber and pressurized by the addition of N₂ to the chamber in the range of ambient to 1 MPa. Subsequently the seeds were left to germinate under normal atmospheric conditions. Seed germination and subsequent growth decreased during the first 6 h and then regained the control levels. Nevertheless, application of similar pressures to seeds which had been submerged under water was highly inhibitory. Such effects of pressure seem to be the result of flooding with water of some crucial intercellular spaces and a consequent disturbance of O₂ supply to the germinating embryo. The additional flood-water comprised only 1–3% of the total water content of 24-h-old seedlings. Sensitivity of the submerged seeds and the germinating seedlings to pressure varied with age and developmental stage. Highest sensitivity to pressure was obtained with 12 to 72-h-old submerged seedlings. Removal of the excess water after the pressure treatment restored the germinability of the seeds.     

高免疫原性的高压力失活病毒

以鸡传染性法氏囊病病毒（ＩＢＤＶ）强毒株为对象研究了高压力对ＩＢＤＶ活性的影响。以鸡胚成纤维细胞和ＳＰＦ雏鸡为模型的研究表明,ＩＢＤＶ的感染活性随压力和作用时间的增加而降低,直至完全丧失,但高压力失活的ＩＢＤＶ可有效诱导雏鸡血清抗体大量产生,对雏鸡的保护作用达１００％,且无发病现象,说明ＩＢＤＶ经一定高压力条件处理后仍具有高免疫原性,即具备了疫苗的特性,内源荧光光谱、内源荧光偏振,散射光及ｂｉｓ－     

Water flow dynamics in the respiratory tract of the carp (Cyprinus carpio L.).

Simultaneous measurements of water velocity in the buccal chamber, and buccal and opercular hydrostatic pressure of carp have revealed surprisingly high water velocities. The high flow velocities mean that, at times, the kinetic energy of flow makes a substantial contribution to the total fluid energy. This suggests that there may be unequal distribution of hydrostatic pressures within the buccal chamber. Anatomical examinations showed that fluid channels in the buccal chamber and gill raker sieve are complex and can be expected to vary spatially and temporally throughout the respiratory cycle. It appears that there is a potential for error in many of the previous analyses of 'gill resistance and energetics of fish breathing based solely on hydrostatic pressure measurements and the simplifying assumption of steady-state conditions.     

Proboscis extrusion in Bullia (Nassariidae): A study of fluid skeletons in Gastropoda

The pleurembolic proboscis of Bullia functions in the manner of a classical hydrostatic skeleton. Its protrusion occurs both with high pressure pulses (2 KPa) in the cephalic sinus and at a steady standing pressure (0-2 KPa). The proboscis probably functions as a haemocoelic chamber separate from the cephalic sinus and pressure pulses from within the everted proboscis, but not the sinus, suggest that muscle antagonism occurs in a manner common to hydrostatic skeletons. The advantages of the proboscis having a hydrostatic skeleton are discussed.     

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

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

Bilirubin Oxidase from Trachyderma tsunodae K-2593, a Multi-copper Enzyme

The gel-setting and gel-melting temperatures of aqueous gelatin solutions (0.5 and 1.0 wt%) were measured by a steady state hot-wire method during treatment under high hydrostatic pressure up to 200 MPa. The high-pressure treatment caused both the gel-setting and gel-melting temperatures to increase with increasing pressure. The hot-wire method was proved to be effective for assessing the coagulation or gelation of food materials under pressurized conditions.     

Improving post-thaw survival of cryopreserved mouse blastocysts by hydrostatic pressure challenge

The purpose of the experiments was to study the effect of high hydrostatic pressure treatment prior to vitrification to the survival of expanded mouse blastocysts. High hydrostatic pressure has been reported to induce the production of "shock proteins" in bacteria, which can provide a possibility of cross-protection to other environmental stresses. The possible beneficial effects of this alleged principle was examined on embryo vitrification. First, the behaviour of blastocysts was studied at altered pressure conditions. In the second part of the study, pressure treatment was combined with a cryopreservation protocol. Our results indicate that the survival of pressurized mouse embryos depends on the magnitude and the duration of pressure applied. We demonstrated that a preceding pressure treatment strikingly increases the survival of the frozen blastocysts as well as the speed of resumption of the development, and hatching rate.     

Effects of hydrostatic pressure on a membrane-enveloped virus: high immunogenicity of the pressure-inactivated virus.

A new approach to the preparation of antiviral vaccines relying on the inactivation of the virus particle by hydrostatic pressure is described. The enveloped virus vesicular stomatitis virus was utilized as a model; a pressure of 260 MPa applied for 12 h reduced infectivity by a factor of 10(4), and the antibodies against pressurized material were as effective as those against the intact virus when measured by their neutralization titer. Fluorescence measurements indicate that application of pressure results in perturbations of the particle interactions that permit binding of specific molecular probes. Electron microscopy showed that the membrane of the pressurized virus was partially preserved, presenting the spike pattern of the membrane G protein. Unlike the icosahedral viruses, dissociation into smaller particles was not observed, but a constant change in the morphology was the presence of a bulge in the surface of the pressurized virus, indicating a displacement of the capsid subunits, retained under the lipid and protein membrane.     

In planta mobilization of mPing and its putative autonomous element Pong in rice by hydrostatic pressurization

The miniature Ping (mPing) is a recently discovered endogenous miniature inverted repeat transposable element (MITE) in rice, which can be mobilized by tissue culture or irradiation. It is reported here that mPing, together with one of its putative transposase-encoding partners, Pong, was efficiently mobilized in somatic cells of intact rice plants of two distinct cultivars derived from germinating seeds subjected to high hydrostatic pressure, whereas the other autonomous element of mPing, Ping, remained static in the plants studied. mPing excision was detected in several plants of both cultivars in the treated generation (P0), which were selected based on their novel phenotypes. Southern blot analysis and transposon-display assay on selfed progenies (P1 generation) of two selected P0 plants, one from each of the cultivars, revealed polymorphic banding patterns consistent with mobilization of mPing and Pong. Various mPing excisions and de novo insertions, as detected by element-bracketing, locus-specific PCR assays, occurred in the different P1 plants of both cultivars. Pong excision at one locus for each cultivar was also detected by using a Pong internal primer together with locus-specific flanking primers in the P1 plants. In contrast to the pressurized plants, immobility of both mPing and Pong in control plants, and the absence of within-cultivar heterozygosity at the analysed loci were verified by Southern blotting and/or locus-assay. Sequencing at 18 mPing empty donor sites isolated from the pressurized plants indicated properties characteristic of the element excision. Sequence-based mapping of 10 identified mPing de novo insertions from P1 progenies of pressurized plants indicated that all were in unique or low-copy regions, conforming with the targeting propensity of mPing. No evidence for further mPing activity was detected in the P2 plants tested. In spite of the high activity of mPing and Pong in the pressurized plants, amplified fragment length polymorphism (AFLP) analysis denoted their general genomic stability, and several potentially active retrotransposons also remained largely immobile. Further investigation showed that the same hydrostatic pressure treatments also caused mobilization of mPing in the standard laboratory cultivar for japonica rice, Nipponbare. Thus, a simple and robust approach for in planta MITE-mobilization in rice has been established by using high hydrostatic pressure treatment, which may be useful as an alternative for gene-tagging in this important crop plant.     

Method for Correcting Laboratory Model Deep-Well Disposal System Data for Hydrostatic Pressure Effects

A pressure chamber for determining the effect of increased hydrostatic pressure on growth and metabolic activities of groundwater bacteria is described. The chamber was used to show that moderate increases in pressure (to about 100 atmospheres) result in increased growth of mixed cultures of industrial-injection-well bacteria and in the more complete degradation of formate and nitrate by these bacteria, as compared with identical cultures at atmospheric pressure.