寒武系小碳化石(SCFs)的研究进展及意义

所谓SCFs(Small Carbonaceous Fossils),可译为"小碳化石",其大小跨越了传统的微观与宏观界线(以肉眼可见与否为标准),是一个特定化石群体的统称。它的实质含义,是指通过氢氟酸(HF)等无机酸浸泡泥岩/页岩样品后经过滤、浓缩而获得的一类体积微小的有机碳质薄膜化石,包括后生动物残片、丝状藻/菌类碎片、疑源类等类型。在小碳化石概念被正式提出之前,国外学者曾先后用有机质壁微体化石(Organic-walled microfossils)、碳质微体化石(Carbonaceous microfossils)与布尔吉斯页岩型微体化石(Burgess Shale-type microfossils)来表述此类化石。目前在国际上,与布尔吉斯页岩型(BST)化石库和奥斯坦(Orsten)型化石库一样,小碳化石已经成为寒武纪古生物学及生物埋藏学等领域研究的一个重要的新窗口;同时,通过三类特异埋藏化石的对比研究,实现优势互补,从而能够更好地阐述早期生物的演化与多样性。     

Enzymatic catalysis in cosolvent modified pressurized organic solvents.

An important advantage of carrying out enzymatic catalysis in organic media is the increased solubility of hydrophobic substrates. This study compares a model lipase catalyzed esterification of cholesterol using vinyl acetate (VA) in two such nontraditional media: high-pressure hexane and supercritical (SCF) ethane. The effect of using one of the reactants (VA) as a cosolvent to increase the solubility of the other reactant (cholesterol) in SCF ethane has been investigated. The thermodynamic activity of water (a(w)) in the reaction media was controlled by the direct addition of the salt hydrate pair Na(4)P(2)O(7)/Na(4)P(2)O(7).10H(2)O. The a(w) of the salt hydrate system is shown to be a function of pressure and its variation over the pressure range 104-173 bar has been estimated. The initial reaction rate in pressurized hexane was found to vary linearly with the cholesterol concentration. The reaction rate was also a function of pressure-the effect being more pronounced in ethane than in hexane. This is consistent with the large negative partial molar volumes observed in SCFs, although the sign of the resulting activation volume differs from previous investigations of lipase-catalyzed reactions in SCFs. When corrected for substrate concentration, the initial rate of catalysis in SCF ethane was determined to be greater than in pressurized hexane over the conditions investigated. This study shows that proper solvent choice can be used to regulate reaction rates in pressurized solvents.     

Temperature-dependent effects of high pressure on the bioluminescence of firefly luciferase.

This study measured the effect of high pressure on the enzyme kinetics of firefly luciferase. When firefly luciferase is mixed with luciferin and ATP, a transient flash of light is produced, followed by a weak light, lasting hours. The first stage reaction produces an enzyme-luciferin-AMP complex and pyrophosphate. Addition of pyrophosphate to the reaction mixture decelerated the reaction rate, and the initial flash was prolonged to a plateau, showing a quasi-equilibrium state. The effects of temperature and pressure were analyzed at the plateau. The temperature scan showed that the maximum light intensity was observed at about 22.5 degrees C. When pressurized below the temperature optimum, pressure decreased the light intensity, while increasing it above the temperature optimum. According to the theory of absolute reaction rate, the following values were obtained for the bioluminescent reaction: delta V++ = 823.7 - 2.8 T cm3/mol and delta V = -280.47 + 0.94T cm3/mol, where T is the absolute temperature, delta V++ and delta V are, respectively, activation volume and the volume change due to thermal unfolding. The optimal temperature for the maximum light output occurs because the reaction rate increases with the temperature elevation at low temperature range, but the thermal unfolding of the enzyme decelerates the reaction velocity when the temperature exceeds a critical value. The intensity of luminescence is modified by the influence of pressure on both delta V++ and delta V. So long as the volume of the activated complex (V++) exceeds the average volume of the nonactivated complex (VN), pressure will slow down the reaction. At the point where the volumes become equal, there is no change in the rate under pressure. When the volume of the activated complex is less than that of the reactants, pressure will speed up the rate. This study showed that firefly luciferase is not exceptional to other enzymes in responding to high pressure.     

A new CULLIN 1 mutant has altered responses to hormones and light in Arabidopsis

Regulated protein degradation contributes to plant development by mediating signaling events in many hormone, light, and developmental pathways. Ubiquitin ligases recognize and ubiquitinate target proteins for subsequent degradation by the 26S proteasome. The multisubunit SCF is the best-studied class of ubiquitin ligases in Arabidopsis (Arabidopsis thaliana). However, the extent of SCF participation in signaling networks is unclear. SCFs are composed of four subunits: CULLIN 1 (CUL1), ASK, RBX1, and an F-box protein. Null mutations in CUL1 are embryo lethal, limiting insight into the role of CUL1 and SCFs in later stages of development. Here, we describe a viable and fertile weak allele of CUL1, called cul1-6. cul1-6 plants have defects in seedling and adult morphology. In addition to reduced auxin sensitivity, cul1-6 seedlings are hyposensitive to ethylene, red, and blue light conditions. An analysis of protein interactions with the cul1-6 gene product suggests that both RUB (related to ubiquitin) modification and interaction with the SCF regulatory protein CAND1 (cullin associated and neddylation dissociated) are disrupted. These findings suggest that the morphological defects observed in cul1-6 plants are caused by defective SCF complex formation. Characterization of weak cul1 mutants provides insight into the role of SCFs throughout plant growth and development.     

Dynamics of ANS binding to tuna apomyoglobin measured with fluorescence correlation spectroscopy.

The dynamics of the binding reaction of ANS to native and partly folded (molten globule) tuna and horse apomyoglobins has been investigated by fluorescence correlation spectroscopy and frequency domain fluorometry. The reaction rate has been measured as a function of apomyoglobin and ANS concentrations, pH, and temperature. Examination of the autocorrelation functions shows that the reaction rate is fast enough to be observed in tuna apomyoglobin, whereas the reaction rate in horse apomyoglobin is on the same time scale as diffusion through the volume or longer. Specifically, for tuna apomyoglobin at pH 7 and room temperature the on rate is 2200 microM(-1) s(-1) and the off rate is 5900 s(-1), in comparison with k(on) = 640 microM(-1) s(-1) and k(off) = 560 s(-1) for horse myoglobin as measured previously. The independence of the reaction rate from the ANS concentration indicates that the reaction rate is dominated by the off rate. The temperature dependence of the on-rate shows that this rate is diffusion limited. The temperature dependence of the off rates analyzed by Arrhenius and Ferry models indicates that the off rate depends on the dynamics of the protein. The differences between horse and tuna apomyoglobins in the ANS binding rate can be explained in terms of the three-dimensional apoprotein structures obtained by energy minimization after heme removal starting from crystallographic coordinates. The comparison of the calculated apomyoglobin surfaces shows a 15% smaller cavity for tuna apomyoglobin. Furthermore, a negative charge (D44) is present in the heme cavity of tuna apomyoglobin that could decrease the strength of ANS binding. At pH 5 the fluorescence lifetime distribution of ANS-apomyoglobin is bimodal, suggesting the presence of an additional binding site in the protein. The binding rates determined by FCS under these conditions show that the protein is either in the open configuration or is more flexible, making it much easier to bind. At pH 3, the protein is in a partially denatured state with multiple potential binding sites for ANS molecule, and the interpretation of the autocorrelation function is not possible by simple models. This conclusion is consistent with the broad distribution of ANS fluorescence lifetimes observed in frequency domain measurements.     

Design of bioavailable derivatives of 12-(3-adamantan-1-yl-ureido)dodecanoic acid, a potent inhibitor of the soluble epoxide hydrolase

The soluble epoxide hydrolase (sEH) plays an important role in the metabolism of endogenous chemical mediators involved in blood pressure regulation and vascular inflammation. 12-(3-Adamantan-1-yl-ureido)-dodecanoic acid (AUDA, 1) is a very active inhibitor of sEH both in vitro and in vivo. However, its relatively high melting point and limited solubility in either water or oil-based solvents leads to difficulties in formulating the compound and often results in poor in vivo availability. We investigated the effect of derivatization of the acid functional group of inhibitor 1 on the inhibition potencies, physical properties, and pharmacokinetic properties. For human sEH, similar inhibition potency was obtained when the acid of compound 1 was modified to esters (2-15). The resulting compounds exhibited improved physical properties (23-66 degrees C lower melting point and 5-fold better solubility in oil). Pharmacokinetic studies showed that the esters possess improved oral bioavailability in mice. On the other hand, amide derivatives of AUDA 1 did not show significant improvement in inhibition potencies or physical properties (higher melting points and lower solubility). The esterification of 1 results in compounds that are easier to formulate in animal food and in triglycerides for gavage and other routes of administration, making it easier to study the biological effects of sEH inhibition in vivo.     

Dissolving carbon dioxide in high viscous substrates to accelerate biocatalytic reactions

Solvent free biotransformation of polyglycerol-3 and lauric acid yields polyglycerol-3-laurate and water. This conversion can be catalyzed by Novozym 435. However, the performance is limited by the viscosity of polyglycerol as well as of polyglycerol-3-laurate. A decrease of viscosity by increasing reaction temperature is only possible in a certain temperature range because of the limited stability of the applied enzyme. By dissolving high dense carbon dioxide into the reaction system the viscosity could be reduced, keeping the temperature at an acceptable level at the same time. Thus the reaction rate was increased by a factor of 4 while working at a pressure of 280 bar and 60°C.     

Thermoelasticity of large lecithin bilayer vesicles.

Micromechanical experiments on large lecithin bilayer vesicles as a function of temperature have demonstrated an essential feature of bilayer vesicles as closed systems: the bilayer can exist in a tension-free state (within the limits of experimental resolution, i.e., less than 10(-2) dyn/cm). Furthermore, because of the fixed internal volume, there is a critical temperature at which the vesicle becomes a tension-free sphere. Below this temperature, thermoelastic tension builds up in the membrane and the vesicle's internal pressure increases while the surface area remains constant. Above this temperature, the vesicle's surface area increases while the tension and internal pressure are negligible. Without mechanical support, the vesicles fragment into small vesicles because they have insufficient surface rigidity. In the upper temperature range we have measured the increase of surface area with temperature. These data established the thermal area expansivity to be 2.4 X 10(-3)/degrees C. At constant temperature, we used either pipet aspiration with suction pressures up to 10(4) dyn/cm2 or compression against a flat surface with forces up to 10(-2) dyn to produce area dilation of the vesicle surface on the order of 1%. The rate of increase of membrane tension with area dilation was calculated, which established the elastic area compressibility modulus to be 140 dyn/cm. The tension limit that produced lysis was observed to be 3-4 dyn/cm (equivalent to 2-3% area increase). The product of the elastic area compressibility modulus, the thermal area expansivity, and the temperature gives the reversible heat of expansion at constant temperature for the bilayer. This value is 100 ergs/cm2 at 25 degrees C, or approximately 5 kcal/mol of lecithin. Similarly, the product of the thermal area expansivity multiplied by the area compressibility modulus determines the rate of increase of thermoelastic tension with decrease in temperature when the area is held constant, i.e., -0.34 dyn/cm/degrees C.     

Mechanisms of inhibition of (Na,K)-ATPase by hydrostatic pressure studied with fluorescent probes

To investigate the mechanisms by which hydrostatic pressure inhibits (Na,K)-ATPase, we measured enzyme activity, as a function of pressure and temperature, of purified (Na,K)-ATPase from dog kidney and eel electroplax, and we monitored protein conformation, possible subunit interactions, and the fluidity of the membrane with fluorescent probes. The (Na,K)-ATPase and p-nitrophenylphosphatase activities were inhibited reversibly by pressures below 1.5 kilobars (eel enzyme) and 2.5 kilobars (dog kidney enzyme). Above these pressures, the enzymes were inactivated irreversibly. The plots of 1n(activity) versus pressure were curvilinear; this indicates that the reversible inhibition by pressure involves two or more rate-limiting steps. The calculated activation volumes varied with temperature and pressure and were larger for the (Na,K)-ATPase activity compared to the p-nitrophenylphosphatase activity. The fluorescence polarization of three hydrophobic probes decreased with increasing temperature (10-36 degrees C) and increased with increasing pressure (10(-3)-1.5 kilobars), reversibly, without any evidence of a lipid phase transition. Plots of enzyme activity versus fluorescence polarization of the lipid probes showed an inverse relationship; this indicates that enzyme activity was directly related to the fluidity of the membrane as measured by the lipid probes. Pressure had no effect on the fluorescence polarization of two cardiac glycoside probes nor on the efficiency of resonance energy transfer between either donor and acceptor cardiac glycosides specifically bound to the ouabain sites of different alpha-subunits, or tryptophan and the bound cardiac glycoside probe. These results suggest that dissociation of dimeric alpha-subunits is not related to the inhibition by pressure, and that the cardiac glycoside-complexed enzyme conformation is stabilized by pressure. It is concluded that increased pressure decreases the membrane fluidity which hinders conformational transitions associated with rate-limiting steps of the (Na,K)-ATPase reaction. It is proposed that ion-bound or -occluded forms of (Na,K)-ATPase are stabilized by pressure because they occupy a smaller volume.     

Physiological factors affecting O2 transport by hemoglobin in an in vitro capillary system

O2 transport was examined by measuring the fractional saturation of concentrated hemoglobin solutions flowing through an artificial capillary that was approximately 27 micron in diameter and embedded in a silicone rubber film approximately 170 micron thick. The effects of pH, hemoglobin concentration, O2 tension, temperature, and organic phosphate were measured and analyzed quantitatively by a rigorous mathematical model that included the geometry of the capillary in the silicone film, parabolic flow velocity distributions inside the lumen, and cooperative O2 binding by hemoglobin. The rates of both oxygenation and deoxygenation were limited by diffusion and governed by the magnitude of the O2 gradient between the intracapillary fluid phase and the external gas space. In uptake experiments, O2 flux is determined primarily by the external O2 tension (16-160 mmHg in our experiments) because the internal O2 pressure is kept small due to chemical combination with hemoglobin. In release experiments, the external O2 tension is maintained at zero, and the transport rate is determined by the intracapillary partial pressure of O2 that is proportional to the O2 half-saturation pressure of hemoglobin value of the hemoglobin sample. As a result, factors that change the affinity of hemoglobin for O2, such as pH, temperature, and organic phosphate concentration, influence strongly the rate of O2 release but have little effect on the rate of O2 uptake. These properties are physiologically advantageous, since a decrease in pH or an increase in temperature during exercise increases both the rate and extent of deoxygenation while not altering the kinetics of oxygenation.     

On the temperature and pressure dependence of a range of properties of a type of water model commonly used in high-temperature protein unfolding simulations

Molecular dynamics simulations of protein folding and unfolding are often carried out at temperatures (400-600 K) that are much higher than physiological or room temperature to speed up the (un)folding process. Use of such high temperatures changes both the protein and solvent properties considerably, compared to physiological or room temperature. Water models designed for use in conjunction with biomolecules, such as the simple point charge (SPC) model, have generally been calibrated at room temperature and pressure. To determine the distortive effect of high simulation temperatures on the behavior of such "room temperature" water models, the structural, dynamic, and thermodynamic properties of the much-used SPC water model are investigated in the temperature range from 300 to 500 K. Both constant pressure and constant volume conditions, as used in protein simulations, were analyzed. We found that all properties analyzed change markedly with increasing temperature, but no phase transition in this temperature range was observed.     

Chill injury in the eggs of the migratory locust, Locusta migratoria （Orthoptera： Acrididae）： the time-temperature relationship with high-temperature interruption

Abstract Mortality of the overwintering egg of the migratory locust, Locusta migratoria L. , was attributed to chill injury because of its occurrence well above the egg's super cooling point. In this study, two parameters, upper limit of chill injury zone (ULCIZ) and sum of the injurious temperature (SIT), were used to examine the locust egg's cold hardiness. The value of ULCIZ for the locust egg is 1.06 ± 0.54°C, and the SIT is -329.7 (hour · degree). The superoxide dismutase (SOD) and catalase (CAT) activities changed dramatically after cold stress, indicating that oxygen and hydroxide free radicals are probably efficiently detoxified at low temperatures. It was suggested that the nature of chill injury in locust egg might be a complex of metabolic disorder and a non-proportional decrease in enzymatic reaction and transports, because the LDH activity at low temperature increased significantly and the ATPase activity decreased with prolonged duration of exposure to low temperatures. The results from high temperature interruption revealed that the high temperature intervals significantly increased the survival of locust eggs.     

Effects of elevated atmospheric CO(2) and temperature on leaf optical properties in Acer saccharum

Elevated partial pressures of atmospheric carbon dioxide, similar to numerous causes of plant stress, may alter leaf pigmentation and structure and thus would be expected to alter leaf optical properties. Hypotheses that elevated CO(2) pressure and air temperature would alter leaf optical properties were tested for sugar maple (Acer saccharum) in the middle of its fourth growing season under treatment. The saplings had been growing since 1994 in open-top chambers and partial shade at Oak Ridge, Tennessee under the following treatments: (1) ambient CO(2) pressure and air temperature (control); (2) CO(2) pressure approximately 30 Pa above ambient; (3) air temperatures 3 degrees C above ambient; and (4) elevated CO(2) and air temperature. Under elevated CO(2) or temperature, spectral reflectance, transmittance and absorptance in the visible spectrum (400-720 nm) tended to change in patterns that generally are associated with chlorosis, with maximum differences from the control near 700 nm. However, these changes were not significant at P=0.05. Although reflectance, transmittance and absorptance at 700 nm correlated strongly with leaf chlorophyll concentration, variability in chlorophyll concentration was greater within than among treatments. The lack of treatment effects on pigmentation explained the non-significant change in optical properties in the visible spectrum. Optical properties in the near-infrared (721-850 nm) were similarly unresponsive to treatment with the exception of an increased absorptance throughout the 739-850 nm range in leaves that developed under elevated air temperature alone. This response might have resulted from effects of air temperature on leaf internal structure.     

Heat-induced changes of chlorophyll fluorescence in isolated chloroplasts and related heat-damage at the pigment level

The heat-induced changes of chlorophyll fluorescence excitation and emission properties were studied in isolated chloroplasts of Larrea divaricata Cav. An analysis of the temperature dependency of fluorescence, under Fo and Fmax conditions, of temperature-jump fluorescence induction kinetics, and of 77 degrees K emission spectra of preheated chloroplasts revealed two major components in the heat-induced fluorescence changes: (1) a fluorescence rise, reflecting the block of Photosystem II reaction centers; and (2) a fluorescence decrease, caused by the functional separation of light-harvesting pigment protein complex from the rest of the pigment system. Preferential excitation of chlorophyll a around 420 nm, produced a predominant fluorescence rise. Preferential excitation of chlorophyll b, at 480 nm, gives a predominant fluorescence decrease. It is proposed that the overlapping of the fluorescence decrease on the somewhat faster fluorescence rise, results in the biphasic fluorescence rise kinetics observed in isolated chloroplasts. Both the rise component and the decay component are affected by the thermal stability of the chloroplasts, acquired during growth of the plants in different thermal environments. Mg2+ enhances the stability against heat-damage expressed in the decrease component, but has no effect on the rise component. Heat pretreatment leads to a decrease of the variable fluorescence in the light-induced 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) rise curve, but no change in half-rise time is observed. It is concluded that the block of Photosystem II reaction centers precedes the loss of the light-harvesting pigment protein complex. However, the approximately antiparallel heat-induced Fmax decrease and Fo increase suggest a common cause for the two events. A heat-induced perturbation of the thylakoid membrane is discussed.     

Mechanism-based irreversible inactivation of horseradish peroxidase at 500 MPa

The effects of high-pressure treatment on the reaction rates of horseradish peroxidase (HRP) with guaethol or guaiacol as a hydrogen donor were evaluated from direct transmission measurements in a high-pressure optical cell at 435 nm. Peroxidases are known to be very barostable and insensitive to heat. With guaethol the reaction velocity was independent of pressure up to 500 MPa, but with guaiacol the cytochrome c oxidase underwent a mechanism-based irreversible inhibition of catalytic activity when subjected to pressure; in the resting states (fully oxidized or reduced), it was insensitive to pressure. The enzyme inactivation took place with an inactivation rate constant of 5.15 x 10(-1) min(-1) at 500 MPa, 25 degrees C and pH 7. The degree of inactivation was correlated to the concentration of guaiacol. This is the first report on a mechanism-based pressure inactivation of HRP triggered at moderate pressure and temperature and mediated by the hydrogen donor.     

High-pressure stopped-flow spectrometry at low temperatures

A stopped-flow instrument operating over temperature and pressure ranges of +30 to -20 degrees C and 10(-3) to 2 kbar , respectively, is described. The system has been designed so that it can be easily interfaced with many commercially available spectrophotometers of fast response time, with the aid of quartz fiber optics. The materials used for the construction are inert, metal free and the apparatus has proven to be leak free at temperatures as low as -20 degrees C under a pressure of 2 kbar . The performance of the instrument was tested by measuring the rate of reduction of cytochrome c with sodium dithionite and the 2,6-dichloroindophenol/ascorbate reaction. The dead time of the system has been evaluated to be 20, 50, and congruent to 100 ms in water at 20 degrees C, in 40% ethylene glycol/water, and at 20 degrees C and -15 degrees C, respectively. These values are rather pressure independent up to 2 kbar . Application of the bomb was demonstrated using the cytochrome c peroxidase/ethyl peroxide reaction. This process occurred in two phases and an increase in pressure decreased the rates of reactions indicating two positive volumes of activation (delta V not equal to app (fast) = 9.2 +/- 1.5 ml X mol-1; delta V not equal to app (slow) = 14 +/- 1.5 ml X mol-1, temperature 2 degrees C). The data suggest that the fast reaction could involve a hydrophobic bond, whereas the slow process could be associated with a stereochemical change of the protein. The problem of temperature equilibrium for high-pressure experiments is also discussed.     

High hydrostatic pressure treatment for the inactivation of Staphylococcus aureus in human blood plasma

For the past 30years, pressure inactivation of microorganisms has been developed in biosciences, in particular for foods and more recently for biological products, including pharmaceutical ones. In many past studies, the effect of high hydrostatic pressure (HHP) processes on pathogens focused mainly on the effect of an increase of the pressure value. To assure the safety of pharmaceutical products containing fragile therapeutic components, development of new decontamination processes at the lowest pressure value is needed to maintain their therapeutic properties. The aim of this study was therefore to evaluate the impact of the process parameters characterizing high-pressure treatments [such as the pressurization rate (PR) and the application mode (AM)] on the inactivation of pathogens, in particular to determine how these parameters values could help decrease the pressure value necessary to reach the same inactivation level. The effect of these physical parameters was evaluated on the inactivation of Staphylococcus aureus ATCC 6538 which is an opportunistic pathogen of important relevance in the medical, pharmaceutical and food domains. Human blood plasma was chosen as the suspension medium because of its physiological importance in the transfusion field. It was shown that the optimization of all the selected parameters could lead to a high inactivation level (≈5log(10) decrease of the initial bacterial load) at a pressure level as low as 200MPa, underlining some synergistic effects among these parameters. Complete inactivation of the initial bacterial population was achieved for the following conditions: PR=50MPas(-1), AM=5×2min, T≈-5°C and P=300MPa.     

Physicochemical properties of blue fluorescent protein determined via molecular dynamics simulation

Blue fluorescent protein (BFP) is a mutant of green fluorescent protein (GFP), where the chromophore has been modified to shift the emitted fluorescence into the blue spectral region. In this study, MD calculations were performed with the GROMACS simulation package and AMBER force field to investigate the dependence of BFPs physicochemical properties on temperature and applied pressure. The MD approach enabled us to calculate the compressibility of protein itself, separately from the nontrivial contribution of the hydration shell, which is difficult to achieve experimentally. The computed compressibility of BFP (3.94 x10(-5) MPa(-1)) is in agreement with experimental values of globular proteins. The center-of-mass diffusion coefficient of BFP and its dependence on temperature and pressure, which plays an important role in its application as a probe for intracellular liquid viscosity measurement, was calculated and found to be in good agreement with photobleaching recovery experimental data. We have shown that decreased temperature as well as applied pressure increases the water viscosity, but the concomitant decrease of the BFP diffusion coefficient behaves differently from Stokes-Einstein formula. It is shown that the number of hydrogen bonds around the protein grows with pressure, which explains the aforementioned deviation. Pressure also reduces root mean square (RMS) fluctuations, especially those of the most flexible residues situated in the loops. The analysis of the RMS fluctuations of the backbone Calpha atoms also reveals that the most rigid part of BFP is the center of the beta-barrel, in accord with temperature B factors obtained from the Protein Data Bank.