Water Stress and Protein Synthesis: II. Interaction between Water Stress, Hydrostatic Pressure, and Abscisic Acid on the Pattern of Protein Synthesis in Avena Coleoptiles

Water stress causes a reduction in hydrostatic pressure and can cause an increase in abscisic acid in plant tissues. To assess the possible role of abscisic acid and hydrostatic pressure in water stress effects, we have compared the effects of water stress, abscisic acid, and an imposed hydrostatic pressure on the rate and pattern of protein synthesis in Avena coleoptiles. Water stress reduces the rate and changes the pattern of protein synthesis as judged by a double labeling ratio technique, Abscisic acid reduces the rate but does not alter the pattern of protein synthesis. Gibberellic acid reverses the abscisic acid-induced but not the stress-induced inhibition of protein synthesis. The effect of hydrostatic pressure depends on the gas used. With a 19: 1 N₂-air mixture, the rate of protein synthesis is increased in stressed but not in turgid tissues. An imposed hydrostatic pressure alters the pattern of synthesis in stressed tissues, but does not restore the pattern to that found in turgid tissues. Because of the differences in response, we conclude that water stress does not affect protein synthesis via abscisic acid or reduced hydrostatic pressure.     

Estimate of the subepithelial hydrostatic pressure that drives inflammatory transudate into the airway lumen.

Inflammatory diseases of the upper respiratory tract are characterized by flow of plasma filtrate across the epithelium into the airway lumen ("transudation"). Elsewhere, we have proposed that extravasation from microvessels causes edema, and this is associated with elevated subepithelial hydrostatic pressure that drives transudation. To test this hypothesis, we have attempted to block transudation by elevating luminal hydrostatic pressure. We measured the appearance of plasma markers into the lumen of an isolated perfused segment of rat trachea in vivo and found that stimulation of one vagal nerve caused a rapid (half-time approximately 5 min) and nonselective increase in the flow of markers from blood to airway lumen. Leukocyte migration also caused transudation that developed much more slowly (half-time = 2-3 h). In both cases, transudation was blocked by application of luminal hydrostatic pressures. The critical luminal pressure needed to block vagally induced transudation was approximately 4.5 cmH2O, and, to block epithelial transudation induced by leukocyte traffic, it was 3 cmH2O, and we conclude that these are the subepithelial pressures that drive inflammatory transudation into the airway lumen.     

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.     

Effects of transmural pressure on the transport and distribution of low density lipoproteins in the arterial wall

In an attempt to investigate the effects of transmural pressure on LDL transport and distribution across the arterial wall, uptake of labeled LDL has been measured in excised rabbit thoracic aorta, held at in vivo length and pressurized to 70 or 160 mmHg. The transmural distribution of LDL concentration across the wall was determined by examining serial frozen sections cut parallel to the luminal surface at 20 microns intervals from the intima to adventitia. The LDL concentration observed in the first luminal section at 160 mmHg was 20-fold higher than that obtained at 70 mmHg. The LDL concentrations decreased in the subsequent sections of the first half of the media and became similar, in the outer half of the media, to the values observed under normal pressure. These results might provide an account of one of the mechanisms involved in the deleterious effects of hypertension in atherogenesis.     

Fixation of neural tissue for electron microscopy with an electronically controlled perfusion pump

Many attempts to improve the perfusion of mammalian tissues aim at changes of the osmotic pressure. We describe a method for fixation of nervous tissues controlling both the hydrostatic pressure and the flow rate of a perfusion solution. The constancy of these parameters is guaranteed by an electronically controlled perfusion pump. Thus, a more uniform and complete preservation can be achieved. Further advantages of this method include provision for a rapid succession of rinsing and fixation solution and a continuous control of the hydrostatic pressure during perfusion.     

The influence of contact guidance on the orientation of colonies of subcultured vascular smooth muscle cells

Summary Subcultures of smooth muscle cells derived from rat thoracic aorta were grown on plane plastic substrata and on plastic substrata having ridges molded in them by a heated, ruled template. The cells were found to have a very high degree of contact guidance when distributed sparsely on the ridged substrata. When the cell density increased multilayered, elongated colonies formed. On plane substrata these were irregular, curved, and disposed in all directions. On the ridged substrata, however, the colonies were straight, evenly spaced, and positioned at right angles to the ridges. Supported by Grant MT1011 from the Medical Research Council of Canada.     

A Luminal Glycoprotein Drives Dose-Dependent Diameter Expansion of the Drosophila melanogaster Hindgut Tube

An important step in epithelial organ development is size maturation of the organ lumen to attain correct dimensions. Here we show that the regulated expression of Tenectin (Tnc) is critical to shape the Drosophila melanogaster hindgut tube. Tnc is a secreted protein that fills the embryonic hindgut lumen during tube diameter expansion. Inside the lumen, Tnc contributes to detectable O-Glycans and forms a dense striated matrix. Loss of tnc causes a narrow hindgut tube, while Tnc over-expression drives tube dilation in a dose-dependent manner. Cellular analyses show that luminal accumulation of Tnc causes an increase in inner and outer tube diameter, and cell flattening within the tube wall, similar to the effects of a hydrostatic pressure in other systems. When Tnc expression is induced only in cells at one side of the tube wall, Tnc fills the lumen and equally affects all cells at the lumen perimeter, arguing that Tnc acts non-cell-autonomously. Moreover, when Tnc expression is directed to a segment of a tube, its luminal accumulation is restricted to this segment and affects the surrounding cells to promote a corresponding local diameter expansion. These findings suggest that deposition of Tnc into the lumen might contribute to expansion of the lumen volume, and thereby to stretching of the tube wall. Consistent with such an idea, ectopic expression of Tnc in different developing epithelial tubes is sufficient to cause dilation, while epidermal Tnc expression has no effect on morphology. Together, the results show that epithelial tube diameter can be modelled by regulating the levels and pattern of expression of a single luminal glycoprotein.     

Effects of hydrostatic pressure and temperature on growth and lipid composition of the inner membrane of barotolerant Pseudomonas sp. BT1 isolated from the deep-sea

A barotolerant member of the genus Pseudomonas was isolated from deep-sea sediment obtained from the Japan Trench, at a depth of 4418 m. The growth temperature was found to affect the hydrostatic pressure range in which the bacterium could grow; the optimum hydrostatic pressure for growth shifted to a higher pressure with increasing temperature. We examined the lipid composition of the inner membrane of cells grown at various hydrostatic pressures and temperatures. The fatty acid components of the inner membrane lipids were C16:0, C16:1, C18:0, and C18:1. The phospholipid components of the inner membrane were phosphatidylethanolamine, cardiolipin, phosphatidylglycerol, and phosphatidylserine. It is evident that the effects of elevated hydrostatic pressure are comparable to the effects of low temperature on both the fatty acid composition of the inner membrane lipids and the phospholipid composition of the inner membrane of this bacterium.     

Analysis of intracellular pH in the yeast Saccharomyces cerevisiae under elevated hydrostatic pressure: a study in baro- (piezo-) physiology

Hydrostatic pressure is a distinctive feature of deep-sea environments, and this thermodynamic parameter has potentially inhibitory effects on organisms adapted to living at atmospheric pressure. In the yeast Saccharomyces cerevisiae, hydrostatic pressure causes a delay in or cessation of growth. The vacuole is a large acidic organelle involved in degradation of cellular proteins or storage of ions and various metabolites. Vacuolar pH, as determined using the pH-sensitive fluorescent dye 6-carboxyfluorescein, was analyzed in a hydrostatic chamber with transparent windows under elevated hydrostatic pressure conditions. A pressure of 40–60 MPa transiently reduced the vacuolar pH by approximately 0.33. A vma3 mutant defective in vacuolar acidification showed no reduction of vacuolar pH after application of hydrostatic pressure, indicating that the transient acidification is mediated through the function of vacuolar H⁺-ATPase. The vacuolar acidification was observed only in the presence of fermentable sugars, and never observed in the presence of ethanol, glycerol, or 3-o-methyl-glucose as the carbon source. Analysis of a glycolysis-defective mutant suggested that glycolysis or CO₂ production is involved in the pressure-induced acidification. Hydration and ionization of CO₂ is facilitated by elevated hydrostatic pressure because a negative volume change (ΔV < 0) accompanies the chemical reaction. Moreover the glucose-induced cytoplasmic alkalization is inhibited by elevated hydrostatic pressure, probably because of inhibition of the plasma membrane H⁺-ATPase. Therefore, the cytoplasm tends to become acidic under elevated hydrostatic pressure conditions, and this could be crucial for cell survival. To maintain a favorable cytoplasmic pH, the yeast vacuoles may serve as proton sequestrants under hydrostatic pressure. We are investigating the physiological effects of hydrostatic pressure in the course of research in a new experimental field, baro- (piezo-) physiology. Received: January 22, 1998 / Accepted: February 16, 1998     

Synchronous effects of temperature, hydrostatic pressure, and salinity on growth, phospholipid profiles, and protein patterns of four Halomonas species isolated from deep-sea hydrothermal-vent and sea surface environments

Four strains of euryhaline bacteria belonging to the genus Halomonas were tested for their response to a range of temperatures (2, 13, and 30 degrees C), hydrostatic pressures (0.1, 7.5, 15, 25, 35, 45, and 55 MPa), and salinities (4, 11, and 17% total salts). The isolates were psychrotolerant, halophilic to moderately halophilic, and piezotolerant, growing fastest at 30 degrees C, 0.1 MPa, and 4% total salts. Little or no growth occurred at the highest hydrostatic pressures tested, an effect that was more pronounced with decreasing temperatures. Growth curves suggested that the Halomonas strains tested would grow well in cool to warm hydrothermal-vent and associated subseafloor habitats, but poorly or not at all under cold deep-sea conditions. The intermediate salinity tested enhanced growth under certain high-hydrostatic-pressure and low-temperature conditions, highlighting a synergistic effect on growth for these combined stresses. Phospholipid profiles obtained at 30 degrees C indicated that hydrostatic pressure exerted the dominant control on the degree of lipid saturation, although elevated salinity slightly mitigated the increased degree of lipid unsaturation caused by increased hydrostatic pressure. Profiles of cytosolic and membrane proteins of Halomonas axialensis and H. hydrothermalis performed at 30 degrees C under various salinities and hydrostatic pressure conditions indicated several hydrostatic pressure and salinity effects, including proteins whose expression was induced by either an elevated salinity or hydrostatic pressure, but not by a combination of the two. The interplay between salinity and hydrostatic pressure on microbial growth and physiology suggests that adaptations to hydrostatic pressure and possibly other stresses may partially explain the euryhaline phenotype of members of the genus Halomonas living in deep-sea environments.     

Luminal localization of blood-brain barrier sodium, potassium adenosine triphosphatase is dependent on fixation.

Cytochemical data in the literature reporting localization of sodium, potassium adenosine triphosphatase (Na(+), K(+)-ATPase) in the blood-brain barrier (BBB) have been contradictory. Whereas some studies showed the enzyme to be located exclusively on the abluminal endothelial plasma membrane, others demonstrated it on both the luminal and abluminal membranes. The influence of fixation on localization of the enzyme was not considered a critical factor, but our preliminary studies showed data to the contrary. We therefore quantitatively investigated the effect of commonly used fixatives on the localization pattern of the enzyme in adult rat cerebral microvessels. Fixation with 1%, 2%, and 4% formaldehyde allowed deposition of reaction product on both the luminal and abluminal plasma membranes. The luminal reaction was reduced with increasing concentration of formaldehyde. Glutaraldehyde at 0.1%, 0.25%, 0.5%, in combination with 2% formaldehyde, drastically inhibited the luminal reaction. The abluminal reaction was not significantly altered in all groups. These results show that luminal localization of BBB Na(+), K(+)-ATPase is strongly dependent on fixation. The lack of luminal localization, as reported in the literature, may have been the result of fixation. The currently accepted abluminal polarity of the enzyme should be viewed with caution.     

Influence of hydrostatic pressure on the effects of the heavy metal cations of manganese, copper, cobalt, and nickel on the growth of three deep-sea bacterial isolates.

Increases hydrostatic pressure varied the 72-h growth yield of three bacterial isolates from the deep sea in the presence of heavy metal cations of Mn, Cu, Co, and Ni, depending on the bacterial isolate, the metal cation and its concentration, and the level of hydrostatic pressure. Above atmospheric, hydrostatic pressure was found to have one of the following four effects on the response of culture growth to a heavy metal cation. (i) It could be without effect; (ii) it could enhance inhibition by a metal cation; (iii) it could increase the 72-h growth yield by a metal cation; or (iv) it could protect against a growth inhibitory effect noted at a lower pressure. Possible reasons for these varied responses are discussed.     

Failure strength of the bovine caudal disc under internal hydrostatic pressure

The structure of the disc is both complex and inhomogeneous, and it functions as a successful load-bearing organ by virtue of the integration of its various structural regions. These same features also render it impossible to assess the failure strength of the disc from isolated tissue samples, which at best can only yield material properties. This study investigated the intrinsic failure strength of the intact bovine caudal disc under a simple mode of internal hydrostatic pressure. Using a hydraulic actuator, coloured hydrogel was injected under monitored pressure into the nucleus through a hollow screw insert which passed longitudinally through one of the attached vertebrae. Failure did not involve vertebra/endplate structures. Rather, failure of the disc annulus was indicated by the simultaneous manifestation of a sudden loss of gel pressure, a flood of gel colouration appearing in the outer annulus and audible fibrous tearing. A mean hydrostatic failure pressure of 18+/-3 MPa was observed which was approximated as a thick-wall hoop stress of 45+/-7 MPa. The experiment provides a measurement of the intrinsic strength of the disc using a method of internal hydrostatic loading which avoids any disruption of the complex architecture of the annular wall. Although the disc in vivo is subjected to a much more complex pattern of loading than is achieved using simple hydrostatic pressurization, this latter mode provides a useful tool for investigating alterations in intrinsic disc strength associated with prior loading history or degeneration.     

Pressure effects on the lateral distribution of cholesterol in lipid bilayers: a time-resolved spectroscopy study.

The effects of hydrostatic pressure and temperature on the phase behavior and physical properties of the binary mixture palmitoyloleoylphosphatidylcholine/cholesterol, over the 0-40 molar % range of cholesterol compositions, were determined from the changes in the fluorescence lifetime distribution and anisotropy decay parameters of the natural lipid trans-parinaric acid (t-PnA). Pressurized samples were excited with a Ti-sapphire subpicosecond laser, and fluorescence decays were analyzed by the quantified maximum entropy method. Above the transition temperature (T(T) = -5 degrees C), at atmospheric pressure, two liquid-crystalline phases, alpha and beta, are formed in this system. At each temperature and cholesterol concentration below the transition pressure, the fluorescence lifetime distribution pattern of t-PnA was clearly modulated by the pressure changes. Pressure increased the fraction of the liquid-ordered beta-phase and its order parameter, but it decreased the amount of cholesterol in this phase. Palmitoyloleoylphosphatidylcholine/cholesterol phase diagrams were also determined as a function of temperature and hydrostatic pressure.     

Movement of horseradish peroxidase in rabbit submandibular glands after ductal injection

Synopsis Horseradish peroxidase (HRP) has been used as a tracer to study movements of solutions injected retrogradely via the duct of submandibular glands in rabbits. 0.1 ml of solution was injected either manually or by a constant hydrostatic pressure, and the subsequent distribution of HRP in the gland and duct at different times after injection has been examined histochemically at light and electron microscopical levels.Shortly after the injections, strong interstitial staining for peroxidase resulted from passage between acinar cells. Some sites of cellular uptake were observed and staining occurred in some ductal cells even when the duct had been cut at the hilum to minimize pressure effects. It is not known whether this diffuse uptake represents a physiological or pathological phenomenon. Some interstitial activity still remained 24 hr after injection but had disappeared by 48 hr. Inflammatory cells first appeared in the gland about 4 hr after the injection and slowly increased up to about 24 hr after injection.The results indicate that the HRP reaches the interstices of the gland principally by penetration between acinar cells, and that the junctional complexes between striated duct cells appear to be more resistant to disruption by luminal pressures.     

Synchronous Effects of Temperature, Hydrostatic Pressure, and Salinity on Growth, Phospholipid Profiles, and Protein Patterns of Four Halomonas Species Isolated from Deep-Sea Hydrothermal- Vent and Sea Surface Environments

Four strains of euryhaline bacteria belonging to the genus Halomonas were tested for their response to a range of temperatures (2, 13, and 30°C), hydrostatic pressures (0.1, 7.5, 15, 25, 35, 45, and 55 MPa), and salinities (4, 11, and 17% total salts). The isolates were psychrotolerant, halophilic to moderately halophilic, and piezotolerant, growing fastest at 30°C, 0.1 MPa, and 4% total salts. Little or no growth occurred at the highest hydrostatic pressures tested, an effect that was more pronounced with decreasing temperatures. Growth curves suggested that the Halomonas strains tested would grow well in cool to warm hydrothermal-vent and associated subseafloor habitats, but poorly or not at all under cold deep-sea conditions. The intermediate salinity tested enhanced growth under certain high-hydrostatic-pressure and low-temperature conditions, highlighting a synergistic effect on growth for these combined stresses. Phospholipid profiles obtained at 30°C indicated that hydrostatic pressure exerted the dominant control on the degree of lipid saturation, although elevated salinity slightly mitigated the increased degree of lipid unsaturation caused by increased hydrostatic pressure. Profiles of cytosolic and membrane proteins of Halomonas axialensis and H. hydrothermalis performed at 30°C under various salinities and hydrostatic pressure conditions indicated several hydrostatic pressure and salinity effects, including proteins whose expression was induced by either an elevated salinity or hydrostatic pressure, but not by a combination of the two. The interplay between salinity and hydrostatic pressure on microbial growth and physiology suggests that adaptations to hydrostatic pressure and possibly other stresses may partially explain the euryhaline phenotype of members of the genus Halomonas living in deep-sea environments.     

Effects of cyclic and constant hydrostatic pressure on norepinephrine and epinephrine levels in the brain of flounder

The effects of cyclic and constant hydrostatic pressure on the norepinephrine and epinephrine content in discrete regions of the brain of European flounder Platichthys flesus were studied. After a 14 day exposure to cyclic hydrostatic pressure with a tidal period of 12·4 h and with a maximum peak of 800 kPa (range 200–800 kPa of absolute hydrostatic pressure), fish showed a highly significant decrease in norepinephrine content when compared to control animals held at constant atmospheric pressure. No changes were detected in fish submitted to a constant hydrostatic pressure of 800 kPa.     

Pathology of black bass hepatic tissue infected with larvae of the tapeworm Proteocephalm ambloplitis

Intra- and interspecific differences in pigmentation between finfold larvae of the three most abundant cyprinids in Dutch eutrophic waters, bream, white bream and roach, were studied, using laboratory-raised larvae in the length range 8–11 mm. Because the internal pigmentation of the larvae has been used for identification, some attention is paid to the effects of different ways of fixation and preservation on transparency. The size and the shape of the melanophores, as described in the literature, could not be used as identification characters because of too much intraspecific variation and the effects of light conditions at the moment of fixation. Three characters proved to be significant for the identification of the larvae of the three species. Roach can be distinguished from bream and white bream by the pattern of melanophores on the belly. A second character is the pigmentation of the ventral aorta, which is only found in white bream. Lastly bream shows an irregular pattern of melanophores on the dorsal side, in contrast to roach which has a regular pattern.     

Hydrostatic pressure induced changes in the cytoarchitecture of pheochromocytoma (PC-12) cells.

Confocal microscopy, in association with three-dimensional reconstruction, revealed that microtubules and microfilaments in differentiating PC-12 cells were disrupted in a dose-dependent manner following pressure treatment. Hydrostatic pressure caused cell rounding, microtubule and microfilament disorganization, neurite retraction and the formation of a microtubule ring adjacent to the cell surface. Volume analysis from computer-generated reconstructed cells, at atmospheric pressure, showed that the apparent volume of microtubules and microfilaments, normalized to 100 units, was 22 and 11 respectively. At 4000 and 8000 psi, the apparent microtubule volume was reduced to 16 and 12 units, respectively, and the apparent microfilament volume was reduced to 8 and 5 units, respectively. Thus, the apparent microtubule and microfilament volumes in PC-12 cells decreased as pressure increased. In the presence of taxol and phalloidin which stabilize the cytoarchitecture, cells resist the effects of hydrostatic pressure. In the presence of colchicine and cytochalasin D compounds which destabilize the cytoarchitecture, cells are more susceptible to the disrupting effects of hydrostatic pressure. The effects of hydrostatic pressure on cell morphology were reversible.     

The effect of helium and of hydrogen at high pressure on the cell division of Tetrahymena pyriformis W.

The rate of cell division of Tetrahymena growing in an observational high pressure vessel was measured at selected pressures of helium, hydrogen and at high hydrostatic pressure. Pressures greater than 100 atm reduced the rate of division, but the gases inhibited division to a lesser degree than pure hydrostatic pressure. Hydrogen's effect was distinguishable from that of hydrostatic pressure at 130 atm or more, while helium's effect appeared at 175 atm. These inert gases probably counteract the action of pressure by stabilising apolar pressure-labile targets.