Dry storage of liposome-encapsulated hemoglobin: a blood substitute

We have previously demonstrated the stabilization of liposome-encapsulated hemoglobin (LEH) by lyophilization (Cryobiology 25, 277-284, 1988). In the present report, we examine the structural and functional recovery of LEH after 3 months in the dry state. We have investigated the incorporation of the protective carbohydrate trehalose in the production and preservation of lyophilized LEH. Vesicle size, retention of entrapped hemoglobin, oxygen-carrying capacity, and percentage methemoglobin were measured as a function of time stored in the dry state under vacuum at room temperature. The results indicate that 150-300 mM trehalose maintains LEH dry preparations with little change in their size or functional characteristics after 3 months in the dry state. These results are compared to those of LEH that has been stored hydrated at 4 degrees C for the same time period.     

Preparation of giant unilamellar vesicles from damp lipid film for better lipid compositional uniformity

Giant unilamellar vesicles (GUVs) containing cholesterol often have a wide distribution in lipid composition. In this study, GUVs of 1,2-dioleoyl-sn-glycero-3-phosphocholine(DOPC)/1,2-distearoyl-sn-glycero-3-phosphocholine(DSPC)/cholesterol and 1,2-diphytanoyl-sn-glycero-3-phosphocholine(diPhyPC)/1,2-dipalmitoyl-sn-glycero-3-phosphocholine(DPPC)/cholesterol were prepared from dry lipid films using the standard electroformation method as well as a modified method from damp lipid films, which are made from compositional uniform liposomes prepared using the Rapid Solvent Exchange (RSE) method. We quantified the lipid compositional distributions of GUV by measuring the miscibility transition temperature of GUVs using fluorescence microscopy, since a narrower distribution in the transition temperature should correspond to a more uniform distribution in GUV lipid composition. Cholesterol molecules can demix from other lipids in dry state and form cholesterol crystals. Using optical microscopy, micron-sized crystals were observed in some dry lipid films. Thus, a major cause of GUV lipid compositional heterogeneity is the demixing of lipids in the dry film state. By avoiding the dry film state, GUVs prepared from damp lipid films have a better uniformity in lipid composition, and the standard deviations of miscibility transition temperature are about 2.5 times smaller than that of GUVs prepared from dry lipid films. Comparing the two ternary systems, diPhyPC/DPPC/cholesterol GUVs has a larger cholesterol compositional heterogeneity, which directly correlates with the low maximum solubility of cholesterol in diPhyPC lipid bilayers (40.2±0.5mol%) measured by light scattering. Our data indicate that cholesterol interacts far less favorably with diPhyPC than it does with other PCs. The damp lipid film method also has a potential of preparing GUVs from cell membranes containing native proteins without going through a dry state.     

Stability of dry liposomes in sugar glasses.

Sugars, particularly trehalose and sucrose, are used to stabilize liposomes during hydration (freeze-drying and air-drying). As a result, dry liposomes are trapped in a sugar glass, a supersaturated and thermodynamically unstable solid solution. We investigated the effects of the glassy state on liposome fusion and solute retention in the dry state. Solute leakage from dry liposomes was extremely slow at temperatures below the glass transition temperature (Tg); however, it increased exponentially as temperature increased to near or above the Tg, indicating that the glassy state had to be maintained for dry liposomes to retain trapped solutes. The leakage of solutes from dry liposomes followed the law of first-order kinetics and was correlated linearly with liposome fusion. The kinetics of solute leakage showed an excellent fit with the Arrhenius equation at temperatures both above and below the Tg, with a transitional break near the Tg. The activation energy of solute leakage was 1320 kJ/mol at temperatures above the Tg, but increased to 1991 kJ/mol at temperatures below the Tg. The stabilization effect of sugar glass on dry liposomes may be associated with the elevated energy barrier for liposome fusion and the physical separation of dry liposomes in the glassy state. The half-life of solute retention in dry liposomes may be prolonged by storing dry liposomes at temperatures below the Tg and by increasing the Tg of the dry liposome preparation.     

Structural transitions in the intrinsically disordered plant dehydration stress protein LEA7 upon drying are modulated by the presence of membranes

Dehydration stress-related late embryogenesis abundant (LEA) proteins have been found in plants, invertebrates and bacteria. Most LEA proteins are unstructured in solution, but some fold into amphipathic α-helices during drying. The Pfam LEA_4 (Group 3) protein LEA7 from the higher plant Arabidopsis thaliana was predicted to be 87% α-helical, while CD spectroscopy showed it to be largely unstructured in solution and only 35% α-helical in the dry state. However, the dry protein contained 15% β-sheets. FTIR spectroscopy revealed the β-sheets to be largely due to aggregation. β-Sheet content was reduced and α-helix content increased when LEA7 was dried in the presence of liposomes with secondary structure apparently influenced by lipid composition. Secondary structure was also affected by the presence of membranes in the fully hydrated state. A temperature-induced increase in the flexibility of the dry protein was also only observed in the presence of membranes. Functional interactions of LEA7 with membranes in the dry state were indicated by its influence on the thermotropic phase transitions of the lipids and interactions with the lipid headgroup phosphates.     

Oxygen isotope composition of CAM and C3 Clusia species: non-steady-state dynamics control leaf water 18O enrichment in succulent leaves

Leaf gas exchange and leaf water (18)O enrichment (Delta(18)O(L)) were measured in three Clusia species under field conditions during dry and wet seasons and in Miconia argentea during the dry season in the Republic of Panama. During the dry season, all three Clusia species used crassulacean acid metabolism (CAM); during the wet season Clusia pratensis operated in the C(3) mode, while Clusia uvitana and Clusia rosea used CAM. Large departures from isotopic steady state were observed in daytime Delta(18)O(L) of the Clusia species, especially during the dry season. In contrast, daytime Delta(18)O(L) was near isotopic steady state in the C(3) tree M. argentea. Across the full data set, non-steady-state predictions explained 49% of variation in observed Delta(18)O(L), whereas steady-state predictions explained only 14%. During the wet season, when Delta(18)O(L) could be compared with Clusia individuals operating in both C(3) and CAM modes, steady-state and non-steady-state models gave contrasting predictions with respect to interspecific variation in daytime Delta(18)O(L). The observed Delta(18)O(L) pattern matched that predicted for the non-steady state. The results provided a clear example of how non-steady-state control of leaf water (18)O dynamics can shift the slope of the relationship between transpiration rate and daytime Delta(18)O(L) from negative to positive.     

Thermal denaturation and structural changes of α-helical proteins in keratins

To gain insight into the thermal stability of intermediate filaments and matrix in the biological composite structure of α-keratins, the thermal denaturation performance of human hair fibers was investigated by Modulated Differential Scanning Calorimetry (MDSC) in the dry and the wet state. Denaturation enthalpy ΔH(D) in water was found to be independent of heating rate (11.5J/g) and to be approximately double as high as in the dry state (5.2J/g). The lower enthalpy (dry) and its dependency on heating rate are attributed to effects of pyrolysis. The stepwise change of reversing heat capacity ΔC(p) marks the denaturation process as a classic two-stage transition. The increase of ΔC(p) with heating rate reflects a continuous shift of the nature of the denaturation of the α-helical material, first, into random coil and then towards random β-materials for lower heating rates. Denaturation temperatures follow Arrhenius relationships with heating rate, yielding activation energies of 416kJ/mol (dry) and 263kJ/mol (wet), respectively. A decrease of activation energy (wet) for high heating rates supports the hypothesis of systematic changes of the pathway of denaturation.     

Assessment of the physiological state of the common carp Cyprinus carpio L. by the water content in the organism

A simple method is proposed for distinguishing between two levels of the physiological state of the common carp by the water content in the organism. It is shown that low water content (2.61 ± 0.04 g H₂O per 1 g of dry weight, 72.3 ± 0.27%) in the body of the fish is associated with a state providing for the growth of the organism. Higher water content (3.63 ± 0.06 g H₂O per 1 g of dry weight, 78.4 ± 0.26%) in the body of the fish is associated with a physiological state that helps it survive through environmental conditions unfavorable for the growth of the organism.     

Evidence for a super-reduced cobamide as the major corrinoid fraction in vivo and a histidine residue as a cobalt ligand of the p-cresolyl cobamide in the acetogenic bacterium Sporomusa ovata

The redox state of cobalt in p-cresolyl cobamide and one of its axial ligands were determined by EPR spectroscopy of Sporomusa ovata as harvested. The analyses revealed that less than 2% (less than 30 nmol/g dry cells) of the total corrinoids (greater than 2400 nmol/g dry cells) were in a low-spin Co(II) complex. The amount increased to about 15% (190-450 nmol/g dry cells) upon partial oxidation by air, indicating that the original valence state of cobalt was a Co(I) prior to this treatment. The cob(I)amide was quantified as Co(III)-CH3 after methylation by iodomethane. More than 45% (1100 nmol/g dry cells) of the extractable corrinoids were in the methylated form, whereas non-treated cells revealed less than 1% (less than 15 nmol g dry cells) of light-sensitive corrinoids. EPR spectra of the Co(II) complex exhibited a threefold N-hyperfine splitting in the gz region, which was similar to vitamin B12. Cells grown with [1.3-15N2]histidine showed a twofold N-hyperfine splitting, demonstrating that the axial N ligand of the corrinoid was derived from the imidazole group of histidine. It is concluded that the super-nucleophilic p-cresolyl cob(I)amide is the major corrinoid complex in vivo and that it is stabilized by its protein(s). The Co(II) ion of the prosthetic group was coordinated by one histidine residue of the apoprotein(s).     

Determination of the interparticular effective diffusion coefficient for CO(2) and O(2) in solid state fermentation

A simple experimental diffusion controlled fermentor (DCF), coupled with the use of a mathematical model based on mass balance, is proposed to measure the variation of the gas (CO(2) and O(2)) diffusion coefficients in solid state fermentation. The DCF was packed with an ion-exchange resin impregnated with a nutritive medium and inoculated with Aspergillus niger. The growth conditions in the DCF were very similar to those found in equipment operated with convective oxygen supply. The diffusion coefficient was shown to be very dependent on the biomass concentration within the solid state fermentor, and attained values of less than 5% of the molecular diffusion in air when the biomass in the fermentor reached 27 mg dry/g dry support.     

The effect of fructan on membrane lipid organization and dynamics in the dry state

Fructans are a group of fructose-based oligo- and polysaccharides, which appear to be involved in membrane preservation during dehydration by interacting with the membrane lipids. To get further understanding of the protective mechanism, the consequences of the fructan-membrane lipid interaction for the molecular organization and dynamics in the dry state were studied. POPC and DMPC were investigated in the dry state by (2)H, (31)P NMR, and Fourier transform infrared spectroscopy using two types of fructan and dextran. The order-disorder transition temperature of dry POPC was reduced by 70 degrees C in the presence of fructan. Fructan increased the mobility of the acyl chains, but immobilized the lipid headgroup region. Most likely, fructans insert between the headgroups of lipids, thereby spacing the acyl chains. This results in a much lower phase transition temperature. The headgroup is immobilized by the interaction with fructan. The location of the interaction with the lipid headgroup is different for the inulin-type fructan compared to the levan-type fructan, since inulin shows interaction with the lipid phosphate group, whereas levan does not. Dextran did not influence the phase transition temperature of dry POPC showing that reduction of this temperature is not a general property of polysaccharides.     

Chemical States of Bacterial Spores: Dry-Heat Resistance

Mature bacterial spores can be manipulated by chemical pretreatments between states sensitive and resistant to dry heat. The two chemical forms of the spore differ in dry-heat resistance by about an order of magnitude. Log survivor curves for each chemical state were approximately straight lines. The temperature dependence of dry-heat resistance for each chemical state was similar to that usually found for dry-heat resistance. A method of testing spore resistance to dry heat has been designed to minimize artifacts resulting from (i) change of chemical state during the test, (ii) effects of water vapor activity, (iii) incomplete recovery of spores from the test container and clumping of spores. Implications of the existence of different chemical resistance states for experimental strategy and testing of dry-heat resistance are discussed.     

Inferring the Relative Resilience of Alternative States

Ecological systems may occur in alternative states that differ in ecological structures, functions and processes. Resilience is the measure of disturbance an ecological system can absorb before changing states. However, how the intrinsic structures and processes of systems that characterize their states affects their resilience remains unclear. We analyzed time series of phytoplankton communities at three sites in a floodplain in central Spain to assess the dominant frequencies or “temporal scales” in community dynamics and compared the patterns between a wet and a dry alternative state. The identified frequencies and cross-scale structures are expected to arise from positive feedbacks that are thought to reinforce processes in alternative states of ecological systems and regulate emergent phenomena such as resilience. Our analyses show a higher species richness and diversity but lower evenness in the dry state. Time series modeling revealed a decrease in the importance of short-term variability in the communities, suggesting that community dynamics slowed down in the dry relative to the wet state. The number of temporal scales at which community dynamics manifested, and the explanatory power of time series models, was lower in the dry state. The higher diversity, reduced number of temporal scales and the lower explanatory power of time series models suggest that species dynamics tended to be more stochastic in the dry state. From a resilience perspective our results highlight a paradox: increasing species richness may not necessarily enhance resilience. The loss of cross-scale structure (i.e. the lower number of temporal scales) in community dynamics across sites suggests that resilience erodes during drought. Phytoplankton communities in the dry state are therefore likely less resilient than in the wet state. Our case study demonstrates the potential of time series modeling to assess attributes that mediate resilience. The approach is useful for assessing resilience of alternative states across ecological and other complex systems.     

飞来峡水库蓄水初期营养状态及浮游生物分布特征

于2000年丰水期和枯水期调查分析了新建河流型水库-飞来峡水库在蓄水初期的营养状态及浮游生物特征。结果表明:水库处于营养累积高峰期,已开始回落,但流域上游大量含磷废水使水库中磷含量继续上升,目前属于中营养型。共记录到浮游植物29种(属),以蓝藻门、绿藻门、硅藻门种类较多,其中丰水期以蓝藻和绿藻为主,枯水期是硅藻占优势,除大坝附近外,浮游植物两个季节密度变化不大,为0.39×10⁶cells·L^-1.浮游动物53种(属),其中包括僧帽溞和透明薄皮溞两种嗜寒性种类,丰度为52ind.·L^-1,枯水期高于丰水期,数量以轮虫和无节幼体为多。     

Characteristics and specificity of the interaction of a fluorochrome from aniline blue (sirofluor) with polysaccharides

The effects of polysaccharide structure and environment on the formation of fluorescent complexes between the polysaccharide and a fluorochrome (4,4′ - [carbonylbis (benzene-4,1-diyl) bis(imino)] bisbenzenesulfonic acid (Sirofluor) isolated from the triarylmethane dye, aniline blue, have been studied. Amongst the wide range of water-soluble polysaccharides tested, fluorescent complexes are formed only with glucans, the strongest fluorescence being obtained with linear (1 → 3)-β-d-glucans and with linear (1 → 3)-β-d-glucans bearing single glucose residues attached at the 6-position. The fluorescence of complexes formed with water-insoluble polysaccharides depends on the ionic environment as well as the polysaccharide structure. (1 → 3)-β-d-Glucans form strongly fluorescent complexes in the dry state and in the presence of water or phosphate buffer. Various cellulose ((1 → 4)-β-d-glucan) samples form strongly fluorescent complexes in the dry state and in the presence of phosphate buffer, but are significantly reduced in the presence of water alone.     

Regulation of 5'AMP-activated protein kinase activity and substrate utilization in exercising human skeletal muscle

The metabolic role of 5'AMP-activated protein kinase (AMPK) in regulation of skeletal muscle metabolism in humans is unresolved. We measured isoform-specific AMPK activity and beta-acetyl-CoA carboxylase (ACCbeta) Ser(221) phosphorylation and substrate balance in skeletal muscle of eight athletes at rest, during cycling exercise for 1 h at 70% peak oxygen consumption, and 1 h into recovery. The experiment was performed twice, once in a glycogen-loaded (glycogen concentration approximately 900 mmol/kg dry wt) and once in a glycogen-depleted (glycogen concentration approximately 160 mmol/kg dry wt) state. At rest, plasma long-chain fatty acids (FA) were twofold higher in the glycogen-depleted than in the loaded state, and muscle alpha1 AMPK (160%) and alpha2 AMPK (145%) activities and ACCbeta Ser(221) phosphorylation (137%) were also significantly higher in the glycogen-depleted state. During exercise, alpha2 AMPK activity, ACCbeta Ser(221) phosphorylation, plasma catecholamines, and leg glucose and net FA uptake were significantly higher in the glycogen-depleted than in the glycogen-loaded state without apparent differences in muscle high-energy phosphates. Thus exercise in the glycogen-depleted state elicits an enhanced uptake of circulating fuels that might be associated with elevated muscle AMPK activation. It is concluded that muscle AMPK activity and ACCbeta Ser(221) phosphorylation at rest and during exercise are sensitive to the fuel status of the muscle. During exercise, this dependence may in part be mediated by humoral factors.     

Photo-oxidation modulates green fern spore longevity during dry storage

Desiccation tolerance and longevity of plant propagules in the dry state have significant implications for biotechnological applications. In this study fern spores were used as a unicellular model to characterize some of the mechanisms of ageing during dry storage of plant propagules (at relative humidity ca. 15%). More specifically, we compared the potential relationships among indicators of photo-oxidative stress and spore viability during dry storage between green (chlorophyllous) spores of Todea barbara and non-green spores of Christella dentata. Green spores stored under the light aged faster than those stored in the dark, and faster than light- and dark-stored non-green spores of C. dentata. This rapid ageing in light-stored green spores was associated with significantly lower antioxidant activity (relative to time zero and dark-stored spores) during storage, and a burst of hydrogen peroxide during the latter stages of storage, which was not a feature of dark-stored spores. We attribute these signs of enhanced oxidative-stress mediated ageing in light-stored spores to photo-oxidative processes, similar to those described in other homoiochlorophyllous organisms. Additionally, high antioxidant activity and low levels of reactive oxygen species in green spores compared with non-green spores suggests differing mechanisms of coping with life in the dry state.     

Enhanced production of pectinase by Bacillus sp. DT7 using solid state fermentation

Bacillus sp. DT7 produced very high levels of alkaline and thermotolerant pectinase by solid state fermentation. Production of this enzyme was affected by nature of solid substrate, level of moisture content, presence or absence of carbon, nitrogen, mineral and vitamin supplements. Maximum enzyme production of 8050 U/g dry substrate was obtained in wheat bran supplemented with polygalacturonic acid (PGA; 1%, w/v) and neurobion (a multivitamin additive; 27 micro l/g dry substrate) with distilled water at 75% moisture level, after 36 h of incubation at 37 degrees C.     

Photoinduced kinetics of bacteriorhodopsin in a dried xerogel glass

Time-resolved absorption measurements of the formation and decay kinetics of the M (deprotonated) photocycle intermediate of bR purple membranes entrapped within a dried xerogel glass have been investigated. The dramatic change observed for the M state decay time is in contrast to the relatively insensitive half life reported for the M intermediate of the D96N mutant entrapped within a dried sol-gel glass. The decay kinetics of the M intermediate was observed to slow by a factor of almost 100 when the solvent was removed from the wet-gel to form the dry xerogel glass. Very long aging times for wet-gels resulted in highly biexponential M state decay kinetics. Upon drying, the M state formation rate initially decreased relative to that in solution before increasing in the dry xerogel to a formation rate nearly three times faster than in solution.     

The action of ionizing radiation on DNA in the presence of quinacrine

Strand breakage of DNA irradiated in solution and in the dry state in the presence of quinacrine was investigated by sedimentation analysis. Determination of single strand breaks in solution combined with binding studies permits to conclude that bound quinacrine protects DNA more effectively than the free compound. In the dry state quinacrine is without detectable effect on both single and double strand break formation, neither under aerobic nor anaerobic conditions.