Study on separation of conalbumin and lysozyme from high concentration fresh egg white at high flow rates by a novel ion-exchanger

In this report, we show that it is possible to separate valuable proteins from egg-white using a Productiv(TM) CM ion-exchanger column operated at flow rates significantly higher than those than can be achieved using traditional particulate adsorbents. In the approach taken, sample pretreatment is restricted to a simple dilution of the egg-white, which can then be applied to the column at superficial velocities (V(s)) of up to 13.8 m/h. Under a loading of 220 mg total protein per milliliter of ion-exchanger, the resolution (R(s)) between the eluted conalbumin and lysozyme fractions was found to be almost constant during nine consecutive adsorption/desorption cycles. For all nine consecutive batches, the column average adsorption capacity was greater than 30 mg/mL, with 90% recovery of adsorbed protein being achieved in each run. The overall productivity achieved was 12.6 kg/m(3) h for lysozyme and 31.2 kg/m(3) h for conalbumin. (c) 1993 John Wiley & Sons, Inc.     

Solid-phase assay for determination of binding parameters of ligand-protein complexes with high dissociation rates

The binding parameters, the affinity constant (Ka) and binding capacity (Q), of a protein possessing ligand-protein complexes with a high dissociation rate (Sex Steroid Binding protein from Bufo arenarum) were determined using a solid-phase method. The technique is based upon the adsorption of the steroid-protein complex to DEAE-cellulose. This method was compared with a nonequilibrium method (charcoal adsorption of free ligand), and the latter resulted in underestimation of both binding parameters, Ka and Q. The solid-phase method reported here is appropriate to determine the binding parameters of proteins with high dissociation rates because the results are independent of the complex half-time. The method also possesses advantages compared to other equilibrium assays such as dialysis or steady-state electrophoresis. With minor modifications, it may be useful to characterize different proteins, particularly those possessing ligand-protein complexes with very high dissociation rates.     

Efficient inclusion body processing using chemical extraction and high gradient magnetic fishing

In this study we introduce a radical new approach for the recovery of proteins expressed in the form of inclusion bodies, involving (i) chemical extraction from the host cells, (ii) adsorptive capture of the target protein onto small magnetic adsorbents, and (iii) subsequent rapid collection of the product-loaded supports with the aid of high gradient magnetic fields. The manufacture and testing of two types of micron-sized nonporous superparamagnetic metal chelator particles derivatized with iminodiacetic acid is described. In small-scale adsorption studies conducted with a hexahistidine tagged form of the L1 coat protein of human papillomavirus type 16 dissolved in 8 M urea-phosphate buffer, the best binding performance (Q(max) = 58 mg g(-1) and K(d) approximately 0.08 microM) was exhibited by Cu(2+)-charged type II support materials. Equilibrium adsorption of L1 to these nonporous supports was achieved very rapidly (<300 s), and approximately 90% of the tightly bound L1 could be desorbed in just one elution step by including >100 mM imidazole in the equilibration buffer. The influence of feedstock complexity on L1 adsorption to the Cu(2+)-charged type II magnetic chelators was studied using various dilutions of four crude chemical E. coli cell extracts containing denatured L1 protein. Undiminished L1 adsorption to these adsorbents (relative to the 8 M urea-phosphate buffer case) was observed with the least complex of these feed materials, i.e., a partially clarified (12 g dry weight L(-1)) and spermine-treated chemical cell extract (feedstock B). Efficient recovery of L1 from feed B was demonstrated at a 60-fold increased scale using the high gradient magnetic fishing (HGMF) system to collect loaded Cu(2+)-chelator particles following batch adsorption of L1. Over 70% of the initial L1 present was recovered within the HGMF rig in a highly clarified form in two batch elution cycles with an overall purification factor of approximately 10.     

Preparative separation of four individual flavonoids in Scutellaria barbata D. Don based on high selectivity polymeric adsorbents with different polarities

The individual flavonoid component, scutellarin, scutellarein, luteolin and apigenin, in Scutellaria barbata plant was isolated based on the macro porous adsorbent with high adsorption selectivity. These adsorbents were synthesized based on the copolymerization of methyl acrylate and divinylbenzene (MA-co-DVB). So the polarity and the adsorption affinity of these adsorbents can be adjusted through changing MA content in the adsorbents. And then the ability of the adsorbent with different MA contents for isolation of these four individual flavonoid was also investigated. Adsorbents M2 and M4, with MA content of 25% and 45%, respectively, demonstrated the best separation ability. Complete separation of the four flavone compounds was achieved in a continuous process based on combination of adsorbents with different polarities (M2 and M4). Gradient elution using adsorbent M4 separated the four flavonoids into three fractions, which were determined to contain scutellarin, scutellarein and a mixture of luteolin and apigenin. The latter was separated completely by adsorbent M2 subsequently. All four compounds were obtained at high resolution and high recovery yield (96.7%, 94.1%, 95.8% and 93.8%, respectively), suggesting the efficiency of sequentially combined columns with different segregation patterns.     

Temperature and leaf osmotic potential as factors in the acclimation of photosynthesis to high temperature in desert plants

Seasonal changes in the high temperature limit for photosynthesis of desert winter annuals growing under natural conditions in Death Valley, California were studied using an assay based upon chlorophyll fluorescence. All species of this group were 6 to 9°C more tolerant of high temperature at the end of the growing season (May) than at its beginning (February). Over this same time period, the mean daily maximum air temperatures increased by 12°C. Laboratory studies have demonstrated that increases in thermal tolerance could be induced by increasing growth temperature alone. For plants growing under field conditions there was also a good correlation between the thermal tolerance of leaves and the osmotic potential of leaf water, indicating that increases in the concentrations of some small molecules might also confer increased thermal tolerance. Isolated chloroplast thylakoids subjected to increasing concentrations of sorbitol could be demonstrated to have increased thermal tolerance.     

Ecophysiology of a lacertid community in the high Moroccan mountains suggests conservation guidelines

Lizard species may differ in their ecophysiology due to adaptation, plasticity and/or phylogeny. In restrictive environments, ecophysiological differences of species living in sympatry are expected to reveal long-term evolutionary responses to the abiotic environment while competitive interactions should be limited. These influences can be disentangled by combining field monitoring with experimental tests. Here, three lacertid lizard species, Atlantolacerta andreanskyi, Scelarcis perspicillata and Podarcis vaucheri sharing high mountain habitats in Oukaimeden (High Atlas, Morocco), were studied. In the field, spatiotemporal variation of the thermal and hydric environment used by the lizards was monitored using data-loggers. In the lab, thermal and hydric ecophysiology was estimated through assessments of preferred temperatures (Tp) and water loss (WL) rates. Species differed in microhabitat use and, hence, in their exposure to variations in temperature and humidity. However, they only differed in their WL (A. andreanskyi > S. perspicillata > P. vaucheri) while their Tp were similar. Such partial differences of species in in the fundamental niche, likely derived from their long-term independent phylogenetic trajectories, can be used to predict their responses to climate and habitat shifts in this and other parts of their respective ranges. Results also confirm previous suggestions that, together with thermal physiology, hydric physiology plays a prominent role in the organisation of lizard communities in the temperate region.     

The cleavage and adsorption of parathyroid hormone at high dilution Implications for receptor binding studies

Like other polypeptide hormones, purified intact parathyroid hormone (1–84)parathyroid hormone is notoriously unstable and is subject to large adsorptive losses in routine laboratory manipulation. The present studies were undertaken with ¹²⁵I-labeled hormone to quantitate the problem and to develop preventative measures, particularly with concentrations of physiological interest, 1 · 10^?10 M.It was found that spontaneous cleavage of the hormone takes place upon its incubation in air or oxygen. This can be prevented by the presence of mercaptoethanol or by plasma levels of cysteine and ascorbate.Under non-cleavage conditions, adsorption was found to be extensive on all materials tested. This adsorption increased with time up to 2 h, was independent of ionic strength, increased with increasing temperature and was presumed to involve hydrophobic interactions. Under given conditions, adsorption was proportional to concentration (constant percentage). However, at very high concentrations, 1 · 10^?6 M, adsorption was markedly reduced.Adsorption was minimized at low pH (2). Bovine serum albumin reduced adsorption under all conditions when present at concentrations of 2 mg/ml or more. Coating laboratory ware with cetyl alcohol also was helpful. Using optimal conditions, cleavage is prevented and losses are less than 5% at neutral pH, and under 2% at pH 2.     

Preparation of high adsorption capacity bio-chars from waste biomass

Bio-chars with high adsorption capacity derived from rice-husks and corncobs were prepared at different retention times (RTs) in a pyrolysis reactor. At a fixed pyrolysis temperature, the pyrolysis RT is a key factor influencing the surface areas and functional group contents of the bio-chars, and further influencing their adsorption capacities. The results indicate that the bio-char prepared at RT of 1.6 s exhibits a higher phenol adsorption capacity (589 mg g⁻¹) than other bio-chars and many activated carbons reported in the literature. An adsorption mechanism based on acid-base interaction and hydrogen binding between phenol and the functional groups was proposed to elucidate the adsorption process. An economic evaluation of the use of bio-chars as adsorbents was made.     

Radioimmunoassay of cyclic AMP can provide a highly sensitive assay for adenylate cyclase, even at very high ATP concentrations

Modifications of the cyclic AMP radioimmunoassay of Cailla et al. [in Hormones and Cell Regulation (J. Dumont and J. Nunez, eds.), Vol. 4, pp. 1-24, Elsevier/North-Holland, Amsterdam/New York (1980)] allowed its use in the determination of adenylate cyclase activity, which was otherwise precluded by high blank values. These high values originate mainly from chemically formed cyclic AMP and from ATP cross-reactivity. The simultaneous presence of ATP and magnesium ions generates cyclic AMP under the alkaline conditions used to succinylate the sample; this interference can be dealt with either by chelation of Mg2+ ions with EDTA during succinylation or by periodic acid oxidation of samples prior to succinylation. In addition, ATP itself contributes to blank values by its cross-reactivity, especially when working with high concentrations of this substrate. This interference can be decreased by a batch adsorption of ATP or oxidized ATP on alumina. Detailed procedures were discussed, with the choice of the additional steps to the standard method of Cailla et al. having to be made on the basis of the sensitivity requirements. When preventing ATP cyclization, the radioimmunoassay was as sensitive as methods using [alpha-32P]ATP as substrate. Elimination of ATP can improve the sensitivity by one order of magnitude. This method is especially interesting with high ATP concentrations and/or with low cyclic AMP production.     

A molecular dynamics study on the thermal rectification effect at the solid–liquid interfaces between the face-centred cubic (FCC) of gold (Au) with the surfaces of (100), (110) and (111) crystal planes facing the liquid methane (CH4)

A molecular dynamics study on the solid–liquid (S-L) interfaces for solid wall of gold having the face-centred cubic of (100), (110) and (111) crystal planes contacting liquid methane was examined using non-equilibrium molecular dynamics simulations. An investigation on the thermal rectification effect was performed by measuring the thermal boundary conductance (TBC) at the S-L interface. Thermal rectification can be defined as the differences in the TBC at the interface between the two opposite heat flow directions; one is from the liquid to solid and vice versa. The thermal rectifications are up to 13% for (110) crystal plane, followed by 6% and 0.3% for (111) and (100) crystal planes, respectively. It was found that the TBC at the S-L interface was influenced by the magnitude of the adsorption of liquid molecules at the vicinity of the interface. The results show that due to the different temperature distribution, different magnitude of the adsorption of liquid molecules is generated for the two opposite heat flow directions. On the surface of the solid walls for (110) crystal plane, where lattice-scale corrugation exists, it was found that there exists difference in distance between the surface layers of the solid and liquid across the interface between the cases of the two opposite heat flow directions, which affects the TBC at the interface. The present results suggest that the factors that influence the thermal rectification at the S-L interface are the magnitude of the adsorption of liquid molecules and the surface structure of the solid walls that differ significantly among the three types of crystal planes.     

Design and selection of nitrogen-rich bridged di-1,3,5-triazine derivatives with high energy and reduced sensitivity

The heats of formation (HOFs), electronic structures, energetic properties, and thermal stabilities of a series of energetic bridged di-1,3,5-triazine derivatives with different substituents and linkages were studied using density functional theory. It was found that the groups -N(3) and -N=N- are effective structural units for improving the HOF values of the di-1,3,5-triazine derivatives. The effects of the substituents on the HOMO-LUMO gap combine with those of the bridge groups. The calculated detonation velocities and detonation pressures indicate that substituting the -ONO(2), -NF(2), or -N=N- group is very useful for enhancing the detonation performance of these derivatives. Analysis of the bond dissociation energies for several relatively weak bonds suggests that most of the derivatives have good thermal stability. On the whole, the -NH(2), -N(3), -NH-, and -CH=CH- groups are effective structural units for increasing the thermal stabilities of the derivatives. Based on detonation performance and thermal stability, nine of the compounds can be considered potential candidates for high energy density materials with reduced sensitivity.     

Process development for purifying tacrolimus from <Emphasis Type="Italic">Streptomyces</Emphasis> sp. using adsorption

The immunosuppressant tacrolimus produced by Streptomyces sp. TST10 was purified in a process that included extraction, pre-adsorption using HP20 resin, adsorption using CG161M resin, and crystallization. In this study, the purification process using the adsorption resin CG161M was optimized by correlating tacrolimus yield with analogue load. One-step adsorption and two-step adsorption using CG161M can be applied selectively to the purification process, according to the analogue load of the input solution. We determined a correlation between the analogue load and the first adsorption yield in the two-step adsorption. We also observed yields according to the analogue loads in the one-step adsorption and the second adsorption of the two-step adsorption. As a result, the purification yields can be predicted by input conditions (analogue load). The purification strategy can be modified to achieve specific goals of purity, yield, and cost.     

Hemoglobin recognition by imprinting in semi-interpenetrating polymer network hydrogel based on polyacrylamide and chitosan

Semi-interpenetrating polymer network (semi-IPN) hydrogel was prepared to recognize hemoglobin, by molecularly imprinted method, in the mild aqueous media of chitosan and acrylamide in the presence of N,N'-methylenebisacrylamide as the cross-linking agent. The hydrogel obtained has been investigated by using thermal analysis, X-ray diffraction, differential scanning calorimetry (DSC), and environmental scanning electron microscope (ESEM). Langmuir analysis showed that an equal class of adsorption was formed in the hydrogel, and the adsorption equilibrium constant and the maximum adsorption capacity were evaluated to be 4.27 g/mL and 36.53 mg/g wet hydrogel, respectively. The imprinted semi-IPN hydrogel has a much higher adsorption capacity for hemoglobin than the nonimprinted hydrogel with the same chemical composition and also has a higher selectivity for the imprinted molecule.     

A sensitive diagnostic assay of rheumatoid arthritis using three-dimensional ZnO nanorod structure

We synthesized a three-dimensional nanorod structure of zinc oxide (ZnO) using a simple sol-gel process and systematically investigated properties of the ZnO nanorods regarding protein adsorption and effect on fluorescence emission. As compared to conventional polystyrene plate that has been widely used for strong protein adsorption, the ZnO nanorods had a superior protein adsorption capacity and significantly amplified fluorescence emission, suggesting the ZnO nanorods are attractive for fluorescence-based biomolecular detection assays. When applied to diagnostic assay of rheumatoid arthritis (RA) using cyclic citrullinated peptide (CCP) probe with a RCGRS motif that reportedly has a strong affinity for ZnO, the ZnO nanorods gave apparently high positive signals for all the RA-positive standards and patient sera, whereas upon the detection using conventional polystyrene plate, all the detection signals were relatively negligible. Moreover, the streptavidin-mediated immobilization of well oriented CCP further enhanced sensitivity, even for a 5000-times diluted patient serum. A highly sensitive detection of a very small amount of RA autoantibodies is important because individuals at high risk of developing RA can be identified several years before the clinical onset. Consequently, the fluorescence-based sensitive assay of RA was successfully performed using the three-dimensional ZnO nanorods, owing to the fluorescence amplification and protein/peptide adsorption properties and dimensionality of ZnO nanorods that in turn increases probe accessibility to anti-CCP RA autoantibodies. Although RA was assayed here for proof-of-concept, the ZnO nanorods-based assay can be applied in general to sensitive detection of a wide variety of antibody or protein targets.     

Diurnal variation around an optimum and near‐critically high temperature does not alter the performance of an ectothermic aquatic grazer

The growing threat of global climate change has led to a profusion of studies examining the effects of warming on biota. Despite the potential importance of natural variability such as diurnal temperature fluctuations, most experimental studies on warming are conducted under stable temperatures. Here, we investigated whether the responses of an aquatic invertebrate grazer (Lymnaea stagnalis) to an increased average temperature differ when the thermal regime is either constant or fluctuates diurnally. Using thermal response curves for several life‐history and immune defense traits, we first identified the optimum and near‐critically high temperatures that Lymnaea potentially experience during summer heat waves. We then exposed individuals that originated from three different populations to these two temperatures under constant or fluctuating thermal conditions. After 7 days, we assessed growth, reproduction, and two immune parameters (phenoloxidase‐like activity and antibacterial activity of hemolymph) from each individual. Exposure to the near‐critically high temperature led to increased growth rates and decreased antibacterial activity of hemolymph compared to the optimum temperature, whilst temperature fluctuations had no effect on these traits. The results indicate that the temperature level per se, rather than the variability in temperature was the main driver altering trait responses in our study species. Forecasting responses in temperature‐related responses remains challenging, due to system‐specific properties that can include intraspecific variation. However, our study indicates that experiments examining the effects of warming using constant temperatures can give similar predictions as studies with fluctuating thermal dynamics, and may thus be useful indicators of responses in nature.     

Sunny side up: lethally high, not low, nest temperatures may prevent oviparous reptiles from reproducing at high elevations

Oviparous (egg-laying) lizards and snakes generally inhabit warmer climates than do related viviparous (live-bearing) taxa. This pattern is widely attributed to the failure of oviparous reproduction in cold climates, but the thermal regimes of potential nest-sites above and below the elevational cut-off for oviparous reproduction have never been quantified. We studied oviparous ( Bassiana duperreyi ) and viviparous ( Eulamprus heatwolei ) scincid lizards at such a site in the Brindabella Range of south-eastern Australia. Miniature data-loggers monitored temperatures of nest-sites and lizards in midsummer, partway through the incubation period of eggs in natural nests. Our results contradict the simplistic notion that mean nest temperatures determine this elevational limit for oviparity. Instead, potential nest-sites with average temperatures suitable for embryogenesis in Bassiana are available well above the threshold elevation. However, thermal minima decrease consistently with elevation and thus the maximum temperature needed for any given mean incubation temperature increases rapidly with elevation. Potential nest-sites above the elevational threshold can only attain mean temperatures high enough to sustain embryogenesis by having lethally high thermal maxima. Such nest-sites are available close to the soil surface, but cannot support development. In contrast, behavioural thermoregulation allows viviparous lizards to maintain high mean body temperatures concurrently with relatively low maximum temperatures, regardless of elevation. Paradoxically, oviparous reptiles may be restricted to low elevations not because nests that provide appropriate mean incubation temperatures are unavailable further up the mountain, but because eggs laid in such shallow nests would overheat.  © 2003 The Linnean Society of London, Biological Journal of the Linnean Society , 2003, 78, 325–334.     

Immobilization of α-amylase and amyloglucosidase onto ion-exchange resin beads and hydrolysis of natural starch at high concentration

α-Amylase was immobilized on Dowex MAC-3 with 88 % yield and amyloglucosidase on Amberlite IRA-400 ion-exchange resin beads with 54 % yield by adsorption process. Immobilized enzymes were characterized to measure the kinetic parameters and optimal operational parameters. Optimum substrate concentration and temperature were higher for immobilized enzymes. The thermal stability of the enzymes enhanced after the immobilization. Immobilized enzymes were used in the hydrolysis of the natural starch at high concentration (35 % w/v). The time required for liquefaction of starch to 10 dextrose equivalent (DE) and saccharification of liquefied starch to 96 DE increased. Immobilized enzymes showed the potential for use in starch hydrolysis as done in industry.     

The cleavage and adsorption of parathyroid hormone at high dilution: implications for receptor binding studies.

Like other polypeptide hormones, purified intact parathyroid hormone (1-84)parathyroid hormone is notoriously unstable and is subject to large adsorptive losses in routine laboratory manipulation. The present studies were undertaken with 125I-labeled hormone to quantitate the problem and to develop preventative measures, particularly with concentrations of physiological interest, 1 . 10(-10) M. It was found that spontaneous cleavage of the hormone takes place upon its incubation in air or oxygen. This can be prevented by the presence of mercaptoethanol or by plasma levels of cysteine and ascorbate. Under non-cleavage conditions, adsorption was found to be extensive on all materials tested. This adsorption increased with time up to 2 h, was independent of ionic strength, increased with increasing temperature and was presumed to involve hydrophobic interactions. Under given conditions, adsorption was proportional to concentration (constant percentage). However, at very high concentrations, 1 . 10(-6) M, adsorption was markedly reduced. Adsorption was minimized at low pH (2). Bovine serum albumin reduced adsorption under all conditions when present at concentrations of 2 mg/ml or more. Coating laboratory ware with cetyl alcohol also was helpful. Using optimal conditions, cleavage is prevented and losses are less than 5% at neutral pH, and under 2% at pH 2.     

Mating success at high temperature in highland‐ and lowland‐derived populations as well as in heat knock‐down selected Drosophila buzzatii

Thermal‐stress selection can affect multiple fitness components including mating success. Reproductive success is one of the most inclusive measures of overall fitness, and mating success is a major component of reproduction. However, almost no attention has been spent to test how mating success can be affected by thermal‐stress selection. In this study, we examine the mating success in the cactophilic Drosophila buzzatii Patterson & Wheeler (Diptera: Drosophilidae) derived from two natural populations that nearly represent the ends of an altitudinal cline for heat knock‐down resistance. Furthermore, we extended the analysis using laboratory lines artificially selected for high and low heat knock‐down resistance. Mating success at high temperature was found to be higher in the lowland than the highland population after a heat pre‐treatment. Moreover, individuals selected for heat knock‐down resistance showed higher mating success at high temperature than did individuals selected for low knock‐down resistance. These results indicate that adaptation to thermal stress can confer an advantage on fitness‐related traits including mating success and highlight the benefits of earlier heat exposure as an adaptive plastic response affecting mating success under stress of higher temperature.