Differential scanning calorimetry study on the inner membrane lipids prepared from barotolerant Pseudomonas sp. BT1.
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We investigated the properties of membrane lipids of barotolerant Pseudomonas sp. BT1 by differential scanning calorimetry and spectrophotometry using a system equipped with a hydrostatic pressure controller. In the case of cells grown under high pressure, an endothermic peak appeared under high-pressure measurement conditions. However, in the case of cells grown at 0.1 MPa, such a peak was not observed. It was also observed on spectrophotometry that the membrane lipids from cells grown at 30 MPa had stable properties in comparison with those grown at 0.1 MPa various hydrostatic pressures and temperatures. (+info)
High hydrostatic pressure can probe the effects of functionally related ligands on the quaternary structures of the chaperonins GroEL and GroES.
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We investigated the effects of high hydrostatic pressure in the range of 1--3 kilobars on tetradecameric GroEL, heptameric GroES, and the GroEL-GroES complex. Unlike GroEL monomers formed by urea dissociation, which can be reassembled back to the tetradecamer, the pressure-dissociated monomers do not reassemble readily. This indicates an alteration of their native structures, an example of conformational drift. Pressure versus time profiles and kinetics of the dissociation of both GroEL and GroES at fixed pressures were monitored by light scattering. Unlike GroEL, GroES monomers do reassociate readily. Reaction conditions were varied by adding ATP, Mg(2+), ADP, AMP-PNP, and KCl. At any individual pressure, the dissociation process is governed by both thermodynamics and kinetics. This leads to the decrease in the yield of monomers at lower pressures. In the presence of Mg(2+) and KCl, GroEL is stable up to 3 kilobars. The presence of either ATP or ADP but not AMP-PNP leads to GroEL dissociation at lower pressures. Interestingly, the GroEL-GroES complex is very stable in the range of 1--2.5 kilobars. However, the addition of ADP destabilizes the complex, which dissociates completely at 1.5 kilobars. The results are rationalized in terms of different degrees of cooperativity between individual monomers and heptameric rings in the GroEL tetradecamer. Such allosteric interactions leading to the alteration of quaternary structure of GroEL in the absence of chemical denaturants are important in understanding the mechanism of chaperonin-assisted protein folding by the GroEL-GroES system. (+info)
Interstitial hydraulic conductivity in a fibrosarcoma.
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Convective transport of therapeutic agents in solid tumors can be improved through intratumoral infusion. To optimize the convection, we investigated the dependence of the hydraulic conductivity on tissue deformation induced by interstitial fluid pressure gradient during the infusion. Two experimental systems were used in the investigation: 1) one-dimensional perfusion through tumor slices and 2) intratumoral infusion using a needle. With these systems, we found that the apparent hydraulic conductivity (K(app)) could be altered by several orders of magnitude in fibrosarcomas through changes in perfusion conditions. When the perfusion pressure was less than a threshold level, fluid flow in tissues could not be detected. When the perfusion pressure was increased above the threshold level, K(app) depended on perfusion system and pressure. The maximum variation in K(app) in fibrosarcomas reached 80,260-fold in our experiments. The large variation in K(app) could be explained by perfusion pressure-induced tissue deformation. These experimental data suggest that the hydraulic conductivity is very sensitive to tissue deformation and imply that it is possible to improve intratumoral infusion of therapeutic agents through optimization of infusion conditions. (+info)
The metastable state of nucleocapsids of enveloped viruses as probed by high hydrostatic pressure.
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Enveloped viruses fuse their membranes with cellular membranes to transfer their genomes into cells at the beginning of infection. What is not clear, however, is the role of the envelope (lipid bilayer and glycoproteins) in the stability of the viral particle. To address this question, we compared the stability between enveloped and nucleocapsid particles of the alphavirus Mayaro using hydrostatic pressure and urea. The effects were monitored by intrinsic fluorescence, light scattering, and binding of fluorescent dyes, including bis(8-anilinonaphthalene-1-sulfonate) and ethidium bromide. Pressure caused a drastic dissociation of the nucleocapsids as determined by tryptophan fluorescence, light scattering, and gel filtration chromatography. Pressure-induced dissociation of the nucleocapsids was poorly reversible. In contrast, when the envelope was present, pressure effects were much less marked and were highly reversible. Binding of ethidium bromide occurred when nucleocapsids were dissociated under pressure, indicating exposure of the nucleic acid, whereas enveloped particles underwent no changes. Overall, our results demonstrate that removal of the envelope with the glycoproteins leads the particle to a metastable state and, during infection, may serve as the trigger for disassembly and delivery of the genome. The envelope acts as a "Trojan horse," gaining entry into the host cell to allow release of a metastable nucleocapsid prone to disassembly. (+info)
Evidence for contribution of neutral trehalase in barotolerance of Saccharomyces cerevisiae.
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In yeast, trehalose accumulation and its hydrolysis, which is catalyzed by neutral trehalase, are believed to be important for thermotolerance. We have shown that trehalose is one of the important factors for barotolerance (resistance to hydrostatic pressure); however, nothing is known about the role of neutral trehalase in barotolerance. To estimate the contribution of neutral trehalase in resisting high hydrostatic pressure, we measured the barotolerance of neutral trehalase I and/or neutral trehalase II deletion strains. Under 180 MPa of pressure for 2 h, the neutral trehalase I deletion strain showed higher barotolerance in logarithmic-phase cells and lower barotolerance in stationary-phase cells than the wild-type strain. Introduction of the neutral trehalase I gene (NTH1) into the deletion mutant restored barotolerance defects in stationary-phase cells. Furthermore, we assessed the contribution of neutral trehalase during pressure and recovery conditions by varying the expression of NTH1 or neutral trehalase activity with a galactose-inducible GAL1 promoter with either glucose or galactose. The low barotolerance observed with glucose repression of neutral trehalase from the GAL1 promoter was restored during recovery with galactose induction. Our results suggest that neutral trehalase contributes to barotolerance, especially during recovery. (+info)
Increased production of tumor necrosis factor-alpha by glial cells exposed to simulated ischemia or elevated hydrostatic pressure induces apoptosis in cocultured retinal ganglion cells.
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Although glial cells in the optic nerve head undergo a reactivation process in glaucoma, the role of glial cells during glaucomatous neurodegeneration of retinal ganglion cells is unknown. Using a coculture system in which retinal ganglion cells and glial cells are grown on different layers but share the same culture medium, we studied the influences of glial cells on survival of retinal ganglion cells after exposure to different stress conditions typified by simulated ischemia and elevated hydrostatic pressure. After the exposure to these stressors, we observed that glial cells secreted tumor necrosis factor-alpha (TNF-alpha) as well as other noxious agents such as nitric oxide into the coculture media and facilitated the apoptotic death of retinal ganglion cells as assessed by morphology, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, and caspase activity. The glial origin of these noxious effects was confirmed by passive transfer experiments. Furthermore, retinal ganglion cell apoptosis was attenuated approximately 66% by a neutralizing antibody against TNF-alpha and 50% by a selective inhibitor of inducible nitric oxide synthase (1400W). Because elevated intraocular pressure and ischemia are two prominent stress factors identified in the eyes of patients with glaucoma, these findings reveal a novel glia-initiated pathogenic mechanism for retinal ganglion cell death in glaucoma. In addition, these findings suggest that the inhibition of TNF-alpha that is released by reactivated glial cells may provide a novel therapeutic target for neuroprotection in the treatment of glaucomatous optic neuropathy. (+info)
Displacement of the contents of dentinal tubules and sensory transduction in intradental nerves of the cat.
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Experiments were performed on anaesthetized cats to test the hypothesis that fluid flow through dentinal tubules is part of the mechanism involved in the transduction of pain-producing stimuli in teeth. In 11 animals, fluid flow through dentine and single- and multi-unit activity in intradental nerves were recorded simultaneously during the application of changes in hydrostatic pressure (-500 to +500 mm Hg) to exposed dentine. Seventeen A-fibres (conduction velocity (CV), 10.6-55.1 m s(-1)) were isolated that were pressure sensitive. The thresholds of these units in terms of dentinal fluid flow were in the range 0.3-2.1 nl s(-1) mm(-2) during outward flow from the pulp and 2.0-3.5 nl s(-1) mm(-2) during inward flow. All the units were more sensitive to outward than inward flow. Twenty-eight units (CV, 0.6-48.8 m s-1) were not pressure sensitive, and 12 of these had conduction velocities in the C-fibre range (< 2.5 m s(-1)). The velocities of the tubular contents were calculated by estimating the number and diameters of dentinal tubules exposed. At the threshold of single-fibre responses these velocities were in the range 31.7-222.9 microm s(-1) during outward flow 211.4-369.6 microm s-1 during inward flow. Repetitive pressure stimulation of dentine resulted in a progressive reduction in the evoked discharge, which was probably due to pulp damage. In seven animals, 10 single intradental nerve fibres were selected that responded to hydrostatic pressure stimuli and their responses to the application of hot, cold, osmotic, mechanical and drying stimuli to exposed dentine were investigated. With these stimuli dentinal fluid flow could not be recorded in vivo for technical reasons and was therefore recorded in vitro after completion of the electrophysiological recordings. With each form of stimulus, the discharge evoked in vivo was closely related to the flow predicted from the in vitro measurements. The results were therefore consistent with the hypothesis that the stimuli act through a common transduction mechanism that involves fluid flow through dentine. (+info)
Inactivation of gram-negative bacteria by lysozyme, denatured lysozyme, and lysozyme-derived peptides under high hydrostatic pressure.
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We have studied the inactivation of six gram-negative bacteria (Escherichia coli, Pseudomonas fluorescens, Salmonella enterica serovar Typhimurium, Salmonella enteritidis, Shigella sonnei, and Shigella flexneri) by high hydrostatic pressure treatment in the presence of hen egg-white lysozyme, partially or completely denatured lysozyme, or a synthetic cationic peptide derived from either hen egg white or coliphage T4 lysozyme. None of these compounds had a bactericidal or bacteriostatic effect on any of the tested bacteria at atmospheric pressure. Under high pressure, all bacteria except both Salmonella species showed higher inactivation in the presence of 100 microg of lysozyme/ml than without this additive, indicating that pressure sensitized the bacteria to lysozyme. This extra inactivation by lysozyme was accompanied by the formation of spheroplasts. Complete knockout of the muramidase enzymatic activity of lysozyme by heat treatment fully eliminated its bactericidal effect under pressure, but partially denatured lysozyme was still active against some bacteria. Contrary to some recent reports, these results indicate that enzymatic activity is indispensable for the antimicrobial activity of lysozyme. However, partial heat denaturation extended the activity spectrum of lysozyme under pressure to serovar Typhimurium, suggesting enhanced uptake of partially denatured lysozyme through the serovar Typhimurium outer membrane. All test bacteria were sensitized by high pressure to a peptide corresponding to amino acid residues 96 to 116 of hen egg white, and all except E. coli and P. fluorescens were sensitized by high pressure to a peptide corresponding to amino acid residues 143 to 155 of T4 lysozyme. Since they are not enzymatically active, these peptides probably have a different mechanism of action than all lysozyme polypeptides. (+info)