Homeoviscous theory under pressure: II. The molecular order of membranes from deep-sea fish

The molecular order of brain and liver membranes isolated from deep sea and continental shelf fish species have been estimated and compared using the fluorescence polarization technique in order to determine whether life in a high pressure habitat is associated with an adjustment of membrane order. Fish were trawled at depths between 200 m and 4000 m, liver and brain membranes were fractionated, and fluorescence polarization was measured at 4°C and ambient pressure. Polarization of the brain myelin fraction provided a statistically significant regression with depth of capture ($\text{P<0.001}$) with a slope of ?0.004 km^?1. This change in polarization with depth was sufficient to offset approximately half of the pressure-induced increase in polarization and thus represents the first structural evidence of homeoviscous adaptation to pressure. Polarization of the brain synaptic and liver mitochondrial fraction was not significantly related to depth. This may be due, at least in part, to a high individual variability of polarization compared to laboratory-acclimated freshwater fish.     

Homeoviscous adaptation under pressure. III. The fatty acid composition of liver mitochondrial phospholipids of deep-sea fish

Homeoviscous adaptation of biological membranes to high hydrostatic pressure has been investigated by determining the differences in lipid composition of membranes from fish obtained from depths between 200 and 4000 m. The fatty acid composition of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine/inositol and cardiolipin from a liver mitochondrial fraction was analysed by capillary gas-liquid chromatography. The ratio of saturated to unsaturated fatty acids significantly and negatively related to depth in PC and PE as predicted by homeoviscous adaptation to pressure. Thus, deep sea species possess greater proportions of unsaturated fatty acids than shallow species. Cardiolipin showed the opposite trend. An unsaturation index was not significantly related to depth in any phospholipid fraction.     

Self-segregation of myelin membrane lipids in model membranes

Rapid conduction of nerve impulses requires coating of axons by myelin sheaths, which are multilamellar, lipid-rich membranes produced by oligodendrocytes in the central nervous system. To act as an insulator, myelin has to form a stable and firm membrane structure. In this study, we have analyzed the biophysical properties of myelin membranes prepared from wild-type mice and from mouse mutants that are unable to form stable myelin. Using C-Laurdan and fluorescence correlation spectroscopy, we find that lipids are tightly organized and highly ordered in myelin isolated from wild-type mice, but not from shiverer and ceramide synthase 2 null mice. Furthermore, only myelin lipids from wild-type mice laterally segregate into physically distinct lipid phases in giant unilamellar vesicles in a process that requires very long chain glycosphingolipids. Taken together, our findings suggest that oligodendrocytes exploit the potential of lipids to self-segregate to generate a highly ordered membrane for electrical insulation of axons.     

Homeoviscous theory under pressure. I. The fatty acid composition of Tetrahymena pyriformis NT-1 grown at high pressure

Tetrahymena was grown at up to 260 atm to see if the bilayer-ordering effect of pressure increased the proportion of unsaturated fatty acids in the membrane lipids. Both whole cells and microsomes showed no such change in their fatty acid composition. The most striking effect was seen in the former which showed a pressure-dependent increase in the proportion of C16:0 in relation to C16:Δ9. Homeoviscous adaptation to pressure does not appear to occur in this cell.     

Mechanism of vestibular adaptation of fish under microgravity.

In a space experiment, the adaptation of goldfish behavior during flight and readaptation after landing were investigated. Six goldfish (1 normal, 1 with otoliths removed on both sides, 4 with otoliths removed on one side) were flown in a fish package (F/P) of Aquatic Animal Experiment Unit (AAEU). The dorsal light responses (DLRs) of fish with otoliths removed were recorded after operation until launch and after landing. The behaviors of the fish were recorded with a video camera on Mission Elapsed Time (MET) Day-00, 02, 05, 08, 12. On MET Day-00, two fish with otoliths removed on one side showed flexion of body toward the operated side. These fish also showed rolling behavior toward the operated side. However, the body flexion disappeared on MET Day-05 or MET Day-08. No rolling behaviors were observed after that time. Five fish showed backward looping behaviors during the mission. Although the frequency of looping episodes decreased after MET Day-08, five fish still showed looping behavior on MET Day-12, that was the last day of video recording on orbit. In microgravity, visual system of fish did not seem to provide sufficient cues to prevent them from looping or rolling. After landing, no looping and rolling behavior was observed. However, the tilt angle of the DLR increased in the fish with otolith removed 5 month before launch but not in normal fish and those with otoliths removed 2 weeks before launch. These results suggest that the behavioral dysfunction and the adaptational process in space are dependent on vestibular inputs.     

Molecular adaptation to high pressure in cytochrome P450 1A and aryl hydrocarbon receptor systems of the deep-sea fish Coryphaenoides armatus

Limited knowledge of the molecular evolution of deep-sea fish proteomes so far suggests that a few widespread residue substitutions in cytosolic proteins binding hydrophilic ligands contribute to resistance to the effects of high hydrostatic pressure (HP). Structure-function studies with additional protein systems, including membrane bound proteins, are essential to provide a more general picture of adaptation in these extremophiles. We explored molecular features of HP adaptation in proteins binding hydrophobic ligands, either in lipid bilayers (cytochrome P450 1A - CYP1A) or in the cytosol (the aryl hydrocarbon receptor - AHR), and their partners P450 oxidoreductase (POR) and AHR nuclear translocator (ARNT), respectively. Cloning studies identified the full-length coding sequence of AHR, CYP1A and POR, and a partial sequence of ARNT from Coryphaenoides armatus, an abyssal gadiform fish thriving down to 5000 m depth. Inferred protein sequences were aligned with many non-deep-sea homologs to identify unique amino acid substitutions of possible relevance in HP adaptation. Positionally unique substitutions of various physicochemical properties were found in all four proteins, usually at sites of strong-to-absolute residue conservation. Some were in domains deemed important for protein-protein interaction or ligand binding. In addition, some involved removal or addition of beta-branched residues; local modifications of beta-branched residue patterns could be important to HP adaptation. In silico predictions further suggested that some unique substitutions might substantially modulate the flexibility of the polypeptide segment in which they are found. Repetitive motifs unique to the abyssal fish AHR were predicted to be rich in glycosylation sites, suggesting that post-translational changes could be involved in adaptation as well. Recombinant CYP1A and AHR showed functional properties (spectral characteristics, catalytic activity and ligand binding) that demonstrate proper folding at 1 atm, indicating that they could be used as deep-sea fish protein models to further evaluate protein function under pressure. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone".     

Involvement of brain gangliosides in temperature adaptation of fish

The ganglioside pattern of goldfish brain was investigated after adaptation (acclimatization, acclimation) to different temperatures. Adaptation at lower ambient temperature causes a higher proportion of polysialogangliosides to be formed in fish brain.     

The lateral fluidity of erythrocyte membranes. Temperature and pressure dependence

The pressure and temperature dependence of the lateral and rotational fluidity of erythrocyte membranes was investigated by inserting the excimeric membrane probe 1'-pyrenedodecanoic acid (PDA) into the membranes of intact cells and measuring the probe excimer formation rate and the steady-state polarization of the monomer at pressures up to 2000 atm (2 kbar). At that pressure the lateral diffusivity of PDA was found to decrease by a factor of 10 and its emission anisotropy by a factor of 5 at 22 degrees C. At atmospheric pressure, the local lateral diffusion coefficient of PDA at 2 and 33 degrees C is 1.5 and 4.3 x 10(-8) cm2 s-1, respectively. The activation energy for probe translation was found to decrease from 6 to 3 kcal M-1 in going from atmospheric pressure to 2 kbar, while the entropy decreased by approx. 15 cal M-1 K-1, indicating greater lipid order at the high pressure. The experimental data are consistent with a 'free-area' model for the membrane, analogous to the free-volume model for nonassociated liquids. The lateral diffusivity of PDA was found to be proportional to the free membrane area and linear extrapolation to zero diffusivity indicates that at atmospheric pressure, the fractional free area of the erythrocyte membrane is 6%.     

Bacterial adaptation to high pressure: a respiratory system in the deep-sea bacterium Shewanella violacea DSS12

Shewanella violacea DSS12 is a psychrophilic facultative piezophile isolated from the deep sea. In a previous study, we have shown that the bacterium adapted its respiratory components to alteration in growth pressure. This appears to be one of the bacterial adaptation mechanisms to high pressures. In this study, we measured the respiratory activities of S. violacea grown under various pressures. There was no significant difference between the cells grown under atmospheric pressure and a high pressure of 50 MPa relative to oxygen consumption of the cell-free extracts and inhibition patterns in the presence of KCN and antimycin A. Antimycin A did not inhibit the activity completely regardless of growth pressure, suggesting that there were complex III-containing and -eliminating pathways operating in parallel. On the other hand, there was a difference in the terminal oxidase activities. Our results showed that an inhibitor- and pressure-resistant terminal oxidase was expressed in the cells grown under high pressure. This property should contribute to the high-pressure adaptation mechanisms of S. violacea.     

Prostaglandin dependence of membrane order changes during myogenesis in vitro

Myogenic differentiation in vitro involves at least three events at the cell surface: binding of prostaglandin to cells, cell-cell adhesion, and fusion of the myoblast membranes into syncytia. Previous work has suggested that binding of prostaglandin is causal to the change in cell-cell adhesion and that both are accompanied by a characteristic reorganization of the myoblast membrane detected as a transient increase in membrane order by electron paramagnetic resonance. We show here that this membrane order change, which reaches a maximum at 38 h of development in vitro, was the last membrane order change before bilayer fusion which begins several hours later. This membrane order change, which accompanies the change in cell-cell adhesion, was dependent on the availability of prostaglandin. In myoblasts maintained in indomethacin, where further differentiation is known to be blocked at the prostaglandin binding step, the membrane order change did not occur. However, if myoblasts are provided with exogenous prostaglandin, the membrane order change occurred and differentiation proceeded. The results indicate that the basis of the membrane order change was the reorganization of myoblast membranes to allow increased adhesion and prepare the membrane for bilayer fusion. They also demonstrate that, like the increase in myoblast adhesion, the membrane order change was dependent on prostaglandin being available to bind to its receptor.     

A novel metalloproteinase originally isolated from brain myelin membranes is present in many tissues.

A monoclonal antibody, CG4, was raised to a novel 60 kDa metalloproteinase purified from a bovine brain myelin glycoprotein fraction. Glycoproteins extracted from both myelin and nine different bovine tissues showed the 60 kDa CG4-immunoreactive band by immunoblotting in amounts that broadly paralleled enzymic activity of this metalloproteinase and varied relatively little among the tissues.     

Temperature dependence of the acetylcholinesterase activity in synaptic membranes of the rat brain

The temperature dependence (5-40 degrees C) of the acetylcholinesterase activity in synaptic membranes of the rat brain at different substrate concentrations was studied. At low substrate concentrations, the Arrhenius plot has two linear sections. At high concentration, there is one linear section throughout the temperature range. The addition of glycerol to incubation medium to final concentrations of 1 and 2% (w/v) increases the Michaelis constant, without affecting the maximal rate and the inhibition constant. The role of diffusion in the temperature dependence of the acetylcholinesterase activity is discussed.     

Immunochemical measurement of myelin basic protein in developing rat brain: an index of myelin synthesis.

The initial time and rate of myelin basic protein synthesis in neural tissues of the rat have been measured from birth to 120 days. The protein was quantitated by a radioimmunoassay directly applied to unfractionated cerebrum, cerebellum, olfactory bulb, midbrain, brain stem, optic and trigeminal nerve, and areas of the spinal cord. Because the protein is a specific myelin constituent and its appearance correlates precisely with the synthesis of myelin lipids, the data in this report can be interpreted in terms of myelin synthesis and oligodendrocyte activity. The results show striking heterogeneity in the initial time and rate of myelin synthesis in neural tissue.     

Plasma membrane dehydrogenases in rat brain synaptic membranes. Multiplicity and subunit composition

Plasma membrane redox enzymes have been investigated in synaptic membranes from rat brain nerve terminals. UV-Vis spectra of intact synaptic plasma membranes are presented and the presence of ab-type cytochrome, detectable at 77°K and sensitive to NADH or NADPH, is shown. The molecular characterization of rat synaptic NADH-dehydrogenases was further performed on solubilized enzymes using a recently developed nondissociating polyacrylamide gel electrophoresis technique. Synaptic plasma membranes were solubilized with 1% sodium cholate or Triton X-114 and centrifuged. The supernatant retained over 60% of the NADH-dehydrogenase activity, tested with either DCIP or ferricyanide as substrates, together with NADH. Both enzyme activities were insensitive toward rotenone. This extraction procedure also solubilized about 50% of the proteins. When submitted to polyacrylamide gel electrophoresis under nondenaturing conditions and stained for NADH-dehydrogenase activity, five bands of different mobilities were detected. The multiple NADH-dehydrogenases of synaptic plasma membranes were investigated by means of band excision and the five excised bands each submitted to amino acid analysis and to 2-D electrophoresis. The subunit composition of each band was then deduced, together with the molecular weight and pI of each respective subunit. NADH-dehydrogenases have also been purified by means of FPLC on Mono-P (chromatofocusing) followed by gel filtration on Superose 12. NADH-Dehydrogenase IV and V could be purified in their active forms by this approach.Abbreviations DCIP dichlorophenol-indophenol - FPLC fast protein liquid chromatography.     

Effect of pressure on circumferential order of adventitial collagen in human brain arteries.

A key factor in the contribution of collagen fibres to tissue mechanics is the alignment of the fibres, which we studied in brain arteries, focussing on alignment changes with distending pressure. Arteries from autopsy were cannulated and fixed at different distending pressures from 0 to 200 mmHg (1 mmHg = 133.32 Pa), alcohol dehydrated, paraffin embedded, sectioned, and stained for birefringent enhancement. The polarized light microscope was set for extinction and fibre orientations were precisely determined at the rotational position of extinction for 200 positions around the artery wall. Results from 22 arterial cross sections revealed, with fixation pressure, a significant but incomplete straightening of collagen (even at 200 mmHg). The mean angular deviation of alignment of fibres was +/- 30 degrees for arteries fixed at zero transmural pressure, which in contrast was +/- 7 degrees for the inner and +/- 13 degrees for the mid-adventitia for arteries fixed at 200 mmHg transmural pressure. We verified on vessels fixed at low pressure, by using a full wave plate in conjunction with the specificity of the interference colours, that the measurements were correct and not confused with angles at 90 degrees to the morphological axis. Alternative tissue processing was done with two arteries fixed at 120 mmHg and processed for frozen sections; the results showed diminished variability in alignment but within the range of measurements for wax embedded tissue. We concluded that the collagen fabric could contribute to the mechanics of brain arteries but that it would be with sinusoidal rather than straightened fibres of collagen.