Ca2+ induced phase separations in phospholipid mixtures

We have probed the character of the observed phase separation in mixtures of phosphatidylcholines (PC) and/or phosphatidylethanolamines (PE) in the presence of CaCl2 solutions. Egg yolk phosphatidylethanolamine (EYPE) and a 1:1 molar ratio of dioleoylphosphatidylcholine/dioleoylphosphatidylethanolamine (DOPC/DOPE) were observed to undergo phase separation in CaCl2 solutions, as was previously observed for egg yolk phosphatidylcholine (EYPC) (L.J. Lis et al. Biochemistry, 20 (1981) 1771-1777). However, the mixed chain lipid, palmitoyloleoyl-PC, yielded only a single phase in water or CaCl2 solution. We hypothesize that two lipid species are necessary for the observed phase separation to occur, but that the separation itself is not a function of the individual lipid species, but of the mixture.     

Disentangling population strategies of two cladocerans adapted to different ultraviolet regimes

Zooplankton have evolved several mechanisms to deal with environmental threats, such as ultraviolet radiation (UVR), and in order to identify strategies inherent to organisms exposed to different UVR environments, we here examine life‐history traits of two lineages of Daphnia pulex. The lineages differed in the UVR dose they had received at their place of origin from extremely high UVR stress at high‐altitude Bolivian lakes to low UVR stress near the sea level in temperate Sweden. Nine life‐history variables of each lineage were analyzed in laboratory experiments in the presence and the absence of sub‐lethal doses of UVR (UV‐A band), and we identified trade‐offs among variables through structural equation modeling (SEM). The UVR treatment was detrimental to almost all life‐history variables of both lineages; however, the Daphnia historically exposed to higher doses of UVR (HighUV) showed a higher overall fecundity than those historically exposed to lower doses of UVR (LowUV). The total offspring and ephippia production, as well as the number of clutches and number of offspring at first reproduction, was directly affected by UVR in both lineages. Main differences between lineages involved indirect effects that affected offspring production as the age at first reproduction. We here show that organisms within the same species have developed different strategies as responses to UVR, although no increased physiological tolerance or plasticity was shown by the HighUV lineage. In addition to known tolerance strategies to UVR, including avoidance, prevention, or repairing of damages, we here propose a population strategy that includes early reproduction and high fertility, which we show compensated for the fitness loss imposed by UVR stress.     

Membrane separations in biotechnology

Membranes have always been an integral part of biotechnology processes. The sterile filtration of fermentation media, purification buffers, and protein product pools is standard practice in industry. Microfiltration is also used extensively for medium exchange and harvest. Ultrafiltration can be found in virtually every biotechnology process. A significant number of mammalian cell processes use filtration as an integral part of the overall strategy for viral clearance. Depth filters have also seen widespread use for the clarification of both mammalian and bacterial feed streams. Improvements in membrane technology are now focused on high-resolution applications, including improved protein-virus separation, protein purification by high-performance tangential flow filtration and enhanced membrane chromatography. These developments will allow membranes to play an important role in the evolution of the next generation of biotechnology processes.     

Lipid phase separations induced by the association of cholera toxin to phospholipid membranes containing ganglioside GM1

The interactions of cholera toxin and their isolated binding and active subunits with phospholipid bilayers containing the toxin receptor ganglioside GM1 have been studied by using high-sensitivity differential scanning calorimetry and steady-state and time-resolved fluorescence and phosphorescence spectroscopy. The results of this investigation indicate that cholera toxin associates with phospholipid bilayers containing ganglioside GM1, independent of the physical state of the membrane. In the absence of Ca2+, calorimetric scans of intact cholera toxin bound to dipalmitoylphosphatidylcholine (DPPC) large unilamellar vesicles containing ganglioside GM1 result in a broadening of the lipid phase transition peak and a slight decrease (less than 5%) in the transition enthalpy. In the presence of Ca2+ concentrations sufficient to cause ganglioside phase separation, the association of the intact toxin to the membrane results in a significant decrease of enthalpy change for the lipid transition, indicating that under these conditions the toxin molecule perturbs the hydrophobic core of the bilayer. Calorimetric scans using isolated binding subunits lacking the hydrophobic toxic subunit did not exhibit a decrease in the phospholipid transition enthalpy even in the presence of Ca2+, indicating that the binding subunits per se do not perturb the hydrophobic core of the bilayer. On the other hand, the hydrophobic A1 subunit by itself was able to reduce the phospholipid transition enthalpy when reconstituted into DPPC vesicles. These calorimetric observations were confirmed by fluorescence experiments using pyrene phospholipids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lateral phase separations in binary mixtures of phospholipids having different charges and different crystalline structures

Synthetic dipalmitoyl phosphatidylserine exhibits a sharp chain-melting transition temperature at 51 degrees C as judged by partitioning of the spin label 2,2,6,6-tetramethylpiperidine-1-oxyl. Phase diagrams representing lateral phase separations in binary mixtures of dipalmitoyl phosphatidylserine with dipalmitoyl phosphatidylcholine as well as with dimyristoyl phosphatidylcholine are derived from paramagnetic resonance determinations of 2,2,6,6,-tetramethylpiperidine-1-oxyl partitioning, freeze-fracture electron microscopic studies and theoretical arguments that limit the general form of acceptable phase diagrams. The reported phase diagrams are the first to describe binary mixtures in which one lipid is charged and the second lipid uncharged. These phase diagrams also are the first to include the problem of solid phases with different crystalline conformations as it relates to the occurrence of a pretransition in phosphatidylcholines and its absence in phosphatidylserines. In addition to the phase diagrams reported here for these two binary mixtures, a brief theoretical discussion is given of other possible phase diagrams that may be appropriate to other lipid mixtures with particular consideration given to the problem of crystalline phases of different structures and the possible occurrence of second-order phase transitions in these mixtures.     

Thermal behavior of the Pacific sardine (Sardinops sagax) acclimated to different thermal cycles

The study of thermal behavior of fishes provides useful data to enable predictions of the effect of climatic change on populations so as to ensure good management of fisheries. The Pacific sardine has a complex population dynamics; three subpopulation have been proposed (cold, temperate and warm).We exposed Sardinops sagax caeruleus (temperate sub-population) to two different thermal cycles, which were chosen to be consistent with the temperatures reported in two distant places Cedros Island (CIc: 18–23 °C) and San Pedro (SPc: 13–18 °C).The thermal behavior of the SPc and CIc sardines was affected by acclimation treatment: the interval of thermal preference was 17.1–19.9 and 16.0–18.8 °C, while the lethal temperatures interval (LT50) was 7.7–25.6 and 6.9–24.3 °C, and the critical limits CTMax and CTMin were 7.1–32.2 and 5.5–30.4 °C, respectively.The results of thermal behavior showed that sardines of the temperate subpopulation are more tolerant to cold; this might suggest that they would be more able to survive in California and Oregon than in Baja California Sur and the Gulf of California.     

Photoinhibition and repair in Dunaliella salina acclimated to different growth irradiances

The light-dependent rate of photosystem-II (PSII) damage and repair was measured in photoautotrophic cultures of Dunaliella salina Teod. grown at different irradiances in the range 50–3000 mol photons · m^–2· s^–1. Rates of cell growth increased in the range of 50–800 mol photons·m^–2·s^–1, remained constant at a maximum in the range of 800–1,500 mol photons·m^–2 ·s^–1, and declined due to photoinhibition in the range of 1500–3000 mol photons·m^–2·s^–1. Western blot analyses, upon addition of lincomycin to the cultures, revealed first-order kinetics for the loss of the PSII reaction-center protein (D1) from the 32-kDa position, occurring as a result of photodamage. The rate constant of this 32-kDa protein loss was a linear function of cell growth irradiance. In the presence of lincomycin, loss of the other PSII reaction-center protein (D2) from the 34-kDa position was also observed, occurring with kinetics similar to those of the 32-kDa form of D1. Increasing rates of photodamage as a function of irradiance were accompanied by an increase in the steady-state level of a higher-molecular-weight protein complex ( 160-kDa) that cross-reacted with D1 antibodies. The steady-state level of the 160-kDa complex in thylakoids was also a linear function of cell growth irradiance. These observations suggest that photodamage to D1 converts stoichiometric amounts of D1 and D2 (i.e., the D1/D2 heterodimer) into a 160-kDa complex. This complex may help to stabilize the reaction-center proteins until degradation and replacement of D1 can occur. The results indicated an intrinsic half-time of about 60 min for the repair of individual PSII units, supporting the idea that degradation of D1 after photodamage is the rate-limiting step in the PSII repair process.Abbreviations Chl chlorophyll - PSI photosystem I - PSII photosystem II - D1 the 32-kDa reaction-center protein of PSII, encoded by the chloroplast psbA gene - D2 the 34-kDa reactioncenter protein of PSII, encoded by the chloroplast psbD gene - Q_A primary electron-accepting plastoquinone of PSII The work was supported by grant 94-37100-7529 from the US Department of Agriculture, National Research Initiative Competitive Grants Program.     

Thermal maxima for juvenile shortnose sturgeon acclimated to different temperatures

Many populations of shortnose sturgeon, Acipenser brevirostrum, in the southeastern United States continue to suffer from poor juvenile recruitment. High summer water temperatures, which may be exacerbated by anthropogenic activities, are thought to affect recruitment by limiting available summer habitat. However, information regarding temperature thresholds of shortnose sturgeon is limited. In this study, the thermal maximum method and a heating rate of 0.1°C min⁻¹ was used to determine critical and lethal thermal maxima for young-of-the-year (YOY) shortnose sturgeon acclimated to temperatures of 19.5 and 24.1°C. Fish used in the experiment were 0.6 to 35.0 g in weight and 64 to 140 days post hatch (dph) in age. Critical thermal maxima were 33.7°C (±0.3) and 35.1°C (±0.2) for fish acclimated to 19.5 and 24.1°C, respectively. Critical thermal maxima significantly increased with an increase in acclimation temperature (p < 0.0001). Lethal thermal maxima were 34.8°C (±0.1) and 36.1°C (±0.1) for fish acclimated to 19.5 and 24.1°C, respectively. Lethal thermal maxima were significantly affected by acclimation temperature, the log₁₀ (fish weight), and the interaction between log₁₀(fish weight) and acclimation temperature (p < 0.0001). Thermal maxima were used to estimate upper limits of safe temperature, thermal preferences, and optimal growth temperatures of YOY shortnose sturgeon. Upper limits of safe temperature were similar to previous temperature tolerance information and indicate that summer temperatures in southeastern rivers may be lethal to YOY shortnose sturgeon if suitable thermal refuge cannot be found.     

Membrane leakage of solutes after thermal shock or freezing

Washed human erythrocytes were cooled at different rates from +37 °C to 0 °C in hypertonic solutions of either NaCl (1.2 m) or of a mixture of sucrose (40% $\text{w}\text{v}$) with NaCl (2.53% $\text{w}\text{v}$). Thermal shock hemolysis was measured and the surviving cells were examined for their mass and cell water content and also for net movements of sodium, potassium, and ¹⁴C-sucrose. The results were compared with those obtained from cells in sucrose (40% $\text{w}\text{v}$) initially, cooled at different rates to ?196 °C and rapidly thawed.The cells cooled to 0 °C in NaCl (1.2 m) showed maximal hemolysis at the fastest cooling rate studied (39 °C/min). In addition in the surviving cells this cooling rate induced the greatest uptake of ¹⁴C-sucrose and increase in cell water and cell mass and also entry of sodium and loss of cell potassium. A different dependence on cooling rate was seen with the cells cooled from +37 °C to 0 °C in sucrose (40% $\text{w}\text{v}$) with NaCl (2.53% $\text{w}\text{v}$). In this solution, survival decreased both at slow and fast cooling rates correlating with the greatest uptake of cell sucrose and increase in cell water. There was extensive loss of cell potassium and uptake of sodium at all cooling rates, the cation concentrations across the cell membrane approaching unity.The cells frozen to ?196 °C at different cooling rates in sucrose (40% $\text{w}\text{v}$) initially, also showed sucrose and water entry on thawing together with a loss of cell potassium and an uptake of cell sodium. More sucrose entered the cells cooled slowly (1.8 ° C/min) than those cooled rapidly (318 ° C/min).These results show that cooling to 0 °C in hypertonic solutions (thermal shock) and freezing to ?196 °C both induce membrane leaks to sucrose as well as to sodium and potassium. These leaks are not induced by the hypertonic solutions themselves but are due to the effects of the added stress of the temperature reduction on the membranes modified by the hypertonic solutions. The effects of cooling rate are explicable in terms of the different times of exposure to the hypertonic solutions. These results indicate that the damage observed after thermal shock or slow freezing is of a similar nature.     

Light induction of nonphotochemical quenching,CO2 fixation,and photoinhibition in woody and fern species adapted to different light regimes

We aimed to find out relations among nonphotochemical quenching (NPQ), gross photosynthetic rate (P _G), and photoinhibition during photosynthetic light induction in three woody species (one pioneer tree and two understory shrubs) and four ferns adapted to different light regimes. Pot-grown plants received 100% and/or 10% sunlight according to their light-adaptation capabilities. After at least four months of light acclimation, CO₂ exchange and chlorophyll fluorescence were measured simultaneously in the laboratory. We found that during light induction the formation and relaxation of the transient NPQ was closely related to light intensity, light-adaption capability of species, and P _G. NPQ with all treatments increased rapidly within the first 1–2 min of the light induction. Thereafter, only species with high P _G and electron transport rate (ETR), i.e., one pioneer tree and one mild shade-adapted fern, showed NPQ relaxing rapidly to a low steady-state level within 6–8 min under PPFD of 100 μmol(photon) m^?2 s^?1 and ambient CO₂ concentration. Leaves with low P _Gand ETR, regardless of species characteristics or inhibition by low CO₂ concentration, showed slow or none NPQ relaxation up to 20 min after the start of low light induction. In contrast, NPQ increased slowly to a steady state (one pioneer tree) or it did not reach the steady state (the others) from 2 to 30 min under PPFD of 2,000 μmol m^?2 s^?1. Under high excess of light energy, species adapted to or plants acclimated to high light exhibited high NPQ at the initial 1 or 2 min, and showed low photoinhibition after 30 min of light induction. The value of fastest-developing NPQ can be quickly and easily obtained and might be useful for physiological studies.     

Multi-dimensional liquid phase based separations in proteomics

This review covers recent developments towards the implementation of multi-dimensional (MuD) liquid phase based systems for proteome investigations. Although two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) has been used as a standard approach in proteomics, its drawbacks including the limited dynamic range and molecular mass range, together with lack of on-line integration with biological mass spectrometery (Bio-MS) have limited its widespread use and applications in proteomics. In the meantime, various liquid-phase based multi-dimensional separation techniques have been explored. Especially, with the emergence of the combination of nanoflow capillary high-performance liquid chromatography (cHPLC) and Bio-MS, attention is again refocused on utilizing multi-dimensional liquid-phase based separation of proteins. Some remarkable applications of on-line analysis of intact proteins and on-column digested proteins, and the emergence of approaches such as multiple HPLC-electrospray ionization tandem MS and capillary array electrophoresis-matrix assisted laser desorption ionization MS, have stimulated thinking towards developing a automated multi-dimensional system (MuDSy) that integrates liquid phase based separation, digestion and identification of proteins in complex biological mixtures.     

Low light acclimated submerged freshwater plants show a pronounced sensitivity to increasing irradiances

The high light sensitivity of three submerged aquatic freshwater plant species, Egeria densa, Elodea nuttallii and Myriophyllum heterophyllum, which have been cultivated at a photosynthetically active radiation (PAR, 400-700 nm) of 70 μmol photons m⁻² s⁻¹, was studied by means of chlorophyll fluorescence and pigment analyses. Exposure of plants to 100, 300, 600 and 1000 μmol photons m⁻² s⁻¹ PAR for up to 360 min induced a strong reduction of the F_v/F_m ratio, indicating a pronounced inactivation of PSII even at the lowest PAR applied. These changes were accompanied by a reduction of the chlorophyll content to about 60-70% of control values at the highest PAR. Rapidly inducible photoprotective mechanisms were not affected, as derived from the rapid generation of pH-dependent energy dissipation under these conditions. At PAR higher than 100 μmol photons m⁻² s⁻¹, however, the primary quinone acceptor of photosystem II, Q_A, was reduced to about 80% and the effective quantum yield of photosystem II, Φ_PSII, dropped to values of about 10%, indicating a high reduction state of the photosynthetic electron transport chain. These data support the notion that the three aquatic macrophytes have a very low capacity for the acclimation to higher light intensities.     

Ribulose-1,5-bisphosphate carboxylase from plants adapted to extreme environments

The K_m and V_max of ribulose-1,5-bisphosphate carboxylase (RuBPCase)in selenium absorbing plants (Astragalus flavus Barn., Astragalusrafaelensis Barn. and Stanleya pinnata Bril.) were similar toRuBPCase from tomato (Lycopersicon esculentum L. var. tropic).The pH optima for RuBPCase activity was 8.0 for L. esculentumand A. flavus and 7.0 for A. rafaelensis and S. pinnata. TheActivation Energy (E) values for the enzymes were as follows:A.flavus (21.37), S.pinnata (19.85), A. rafaelensis (19.12)and L. escudentum (18.58). The energy of activation was higherfor the desert plants as compared to the tomato. The Arrheniusplot curves were linear to 50?C far the desert plants as comparedto 45?C for tomato. Enzyme kinetics of RuBPCase from halophytic plants (Salicorniapacifica Stand., var. utahensis (Tidestrom) Munz. and Salicorniarubra Nels.) indicated the enzyme was at least as sensitiveto NaCl concentrations as the enzyme from tomato. (Received November 9, 1976; )     

Using combined measurements of gas exchange and chlorophyll fluorescence to investigate the photosynthetic light responses of plant species adapted to different light regimes

One broad-leaved pioneer tree, Alnus formosana, two broad-leaved understory shrubs, Ardisia crenata and Ardisia cornudentata, and four ferns with different light adaptation capabilities (ranked from high to low, Pyrrosia lingus, Asplenium antiquum, Diplazium donianum, Archangiopteris somai) were used to elucidate the light responses of photosynthetic rate and electron transport rate (ETR). Pot-grown materials received up to 3 levels of light intensity, i.e., 100%, 50% and 10% sunlight. Both gas exchange and chlorophyll (Chl) fluorescence were measured simultaneously by an equipment under constant temperature and 7 levels (0?C2,000 ??mol m^?2 s^?1) of photosynthetic photon flux density (PPFD). Plants adapted to-or acclimated to high light always had higher light-saturation point and maximal photosynthetic rate. Even materials had a broad range of photosynthetic capacity [maximal photosynthetic rate ranging from 2 to 23 ??mol(CO₂) m^?2 s^?1], the ratio of ETR to gross photosynthetic rate (P _G) was close for A. formosana and the 4 fern species when measured under constant temperature, but the PPFD varied. In addition, P. lingus and A. formosana grown under 100% sunlight and measured at different seasonal temperatures (15, 20, 25, and 30°C) showed increased ETR/P _G ratio with increasing temperature and could be fitted by first- and second-order equations, respectively. With this equation, estimated and measured P _G were closely correlated (r ² = 0.916 and r ² = 0.964 for P. lingus and A. formosana, respectively, p<0.001). These equations contain only the 2 easily obtained dynamic indicators, ETR and leaf temperature. Therefore, for some species with near ETR/P _G ratio in differential levels of PPFD, these equations could be used to simulate dynamic variation of leaf scale photosynthetic rate under different temperature and PPFD conditions.     

Photosynthetic efficiency of Betula pendula acclimated to different quantum flux densities

Abstract. Seedlings of Betula pendula were grown in a controlled environment chamber at quantum flux densities of 50, 250 and 600 μmol m⁻² s⁻¹. The relationship between the flux densities of absorbed CC₂ and quanta was determined for shoots of whole seedlings. Rates of both light-saturated and in situ (measured under the growing conditions) net photosynthesis were determined and the pholosynthetic quantum yields under light-limiting conditions were calculated. Anatomical leaf characteristics, chlorophyll contents and sizes and densities of the photosynthetic units (chlorophyll/P700) were determined. Chloroplasts were isolated and their rates of 2,6-dichlorophenol indophenol photoreduction were measured together with their pool sizes of the electron transport carriers plastoquinone and cylochrome ƒ.
Although acclimated to different quantum flux densities, the three birch populations showed the same quantum yield of net photosynthesis. This was approximately 0.028 in normal air (21.2 kPa oxygen) and about 0.040 when photorespiration was largely inhibited in 2.0 kPa oxygen. In addition, the in situ net photosynthesis rates were limited by the absorbed quantum flux density for low, intermediate and high light grown seedlings. It was concluded that birch acclimated to the three light regimes at different levels of organization (metabolic and anatomical). Thus, the quanta which were absorbed in situ could be transferred into chemical equivalents at an optimal and constant efficiency. The use of different reference bases for expressing rates of net photosynthesis are also discussed.     

Membrane phospholipid organization in calcium-loaded human erythrocytes

Intracellular Ca2+ levels in human erythrocytes were increased by incubating them with variable concentrations of Ca2+ in the presence of ionophore A23187. Experiments were done to confirm that the Ca2+ loading did induce changes in the cell shape and membrane protein composition. The effect of the increased cytoplasmic Ca2+ levels on the membrane phospholipid organization was analysed using bee venom and pancreatic phospholipases A2, Merocyanine 540 and fluorescamine as the external membrane probes. About 20% phosphatidylethanolamine (PE) and 0% phosphatidylserine (PS) were hydrolysed by the phospholipases in intact control cells, whereas in identical conditions these enzymes readily degraded, 20-30% PE and 7-30% PS, in Ca2+-loaded erythrocytes, depending on the cytoplasmic Ca2+ concentration. Also, Merocyanine 540 failed to stain the fresh or control erythrocytes, but it labeled the cells loaded with Ca2+. Furthermore, fluorescamine labeled approx. 20% PE in fresh or control erythrocytes while in identical conditions, significantly higher amounts of PE were modified in intact Ca2+-loaded cells. These results demonstrate that Ca2+ loading in human erythrocytes leads to loss of the transbilayer phospholipid asymmetry, and suggest that, together with spectrin, polypeptides 2.1 and 4.1 may also play an important role in maintaining the asymmetric distribution of various phospholipids across the erythrocyte membrane bilayer.