Effect of matrices on affinity purification of protein C

One of the critical problems in scale-up of affinity chromatography is the mechanical strength of the support matrix against pressure. Because the costs of both the gel matrix and the ligand for the affinity chromatography are very high, the reusability of gel matrices is directly related to the total production cost. In certain cases, where the source material is viscous (e.g., blood plasma), irreversible deformation of gel matrices can readily occur, necessitating severe constraints in the flow rate. Consequently, productivity is low.We have characterized the system parameters and investigated the performance of various matrices that are commercially available. The experimental system used for this study was the immunoaffinity purification of protein C (an anticoagulant protein) from human blood plasma. The support matrices studied were cross-linked agarose, polymethyl acrylic, cellulose, and polyvinyl alcohol polymers. The major system parameters studied were pressure tolerance, coupling efficiency, adsorption efficiency, and batch adsorption/desorption kinetics of protein C to/from the monoclonal antibody (MAb)-Matrix complex. In addition, the apparent equilibrium constant and bandwidth of the product concentration profile in the eluate were characterized by performing pulse tests.A methodology was developed for evaluating the immunoaffinity colum performance for the separation of protein C. By utilizing the experimentally measured parameters, the flow rate limitation for each purification step was computed. Then, the purification performance of the matrices were evaluated in terms of productivity per unit time. Among the matrices tested, cellulose was superior in overall performance for the immunoaffinity purification of protein C using a 10 cm x 10 cm column.     

Foliar δ13C within a temperate deciduous forest: spatial,temporal, and species sources of variation

Summary Foliar ¹³C-abundance (¹³C) was analyzed in the dominant trees of a temperate deciduous forest in east Tennessee (Walker Branch Watershed) to investigate the variation in foliar ¹³C as a function of time (within-year and between years), space (canopy height, watershed topography and habitat) and species (deciduous and coniferous taxa). Various hypotheses were tested by analyzing (i) samples collected from the field during the growing season and (ii) foliar tissues maintained in an archived collection. The ¹³C-value for leaves from the tops of trees was 2 to 3%. more positive than for leaves sampled at lower heights in the canopy. Quercus prinus leaves sampled just prior to autumn leaf fall had significantly more negative ¹³C-values than those sampled during midsummer. On the more xeric ridges, needles of Pinus spp. had more positive ¹³C-values than leaves from deciduous species. Foliar ¹³C-values differed significantly as a function of topography. Deciduous leaves from xeric sites (ridges and slopes) had more positive ¹³C-values than those from mesic (riparian and cove) environments. On the more xeric sites, foliar ¹³C was significantly more positive in 1988 (a dry year) relative to that in 1989 (a year with above-normal precipitation). In contrast, leaf ¹³C in trees from mesic valley bottoms did not differ significantly among years with disparate precipitation. Patterns in foliar ¹³C indicated a higher ratio of net CO₂ assimilation to transpiration (A/E) for trees in more xeric versus mesic habitats, and for trees in xeric habitats during years of drought versus years of normal precipitation. However, A/E (units of mmol CO₂ fixed/mol H₂O transpired) calculated on the basis of ¹³C-values for leaves from the more xeric sites was higher in a wet year (6.6±1.2) versus a dry year (3.4±0.4). This difference was attributed to higher transpiration (and therefore lower A/E) in the year with lower relative humidity and higher average daily temperature. The calculated A/E values for the forest in 1988–89, based on ¹³C, were within ±55% of estimates made over a 17 day period at this site in 1984 using micrometeorological methods.     

Carapace movements associated with ventilation and irrigation of the branchial chambers in the semaphore crab,Heloecius cordiformis (Decapoda: Brachyura: Ocypodidae)

Summary Carapace movements in crabs are briefly reviewed. While on land and recirculating branchial water, the Australian semaphore crab Heloecius cordiformis (Decapoda: Ocypodidae), a semi-terrestrial air-breathing mangrove crab, sequentially depresses and elevates its carapace relative to its thorax (0.5–1 mm excursion) in a regular pump-like manner. In quiescent crabs each carapace-pumping cycle lasts about 4 s; carapace depression takes 3 s and elevation 1 s. Carapace movements are brought about by pressures generated within the branchial chambers by the scaphognathites, probably in combination with carapace muscles. Carapace movements are associated with bilaterally synchronised scaphognathite activity. Unilateral scaphognathite activity was not observed. During normal forward recirculation of branchial water the scaphognathites beat at about 1.5 Hz (slow-forward pumping) and the lungs (epibranchial chambers) are not ventilated. In Heloecius, the lungs are not physically separated from the gills below by an anatomical barrier. Lung ventilation is accomplished during the following sequence of events: the carapace is lowered and the scaphognathites pump in a fast-forward mode at about 2.8 Hz. This activity preferentially pumps air out of the lungs and generates suction within the branchial chambers (4–10 cm H₂O below ambient) which draws water from external body surfaces into the hypobranchial space below and around the gills. At the end of the carapace's downward travel the scaphognathites switch from fast-forward to fastreverse beating at about 4 Hz. This pumps air into the lungs and the carapace elevates. As a result, during carapace elevation the water which had previously been drawn into the branchial chambers by fast-forward pumping activity is released and flows out between the legs and into the abdominosternal cavity. When the carapace reaches its original resting or up position the scaphognathites switch from fast-reverse to slowforward beating to re-establish water recirculation through the branchial chambers. This cycle is subsequently repeated. In stationary crabs, there are 2 carapace-pumping cycles per minute, increasing to 14 per minute in active crabs (walking). When water is absent, the lungs are preferentially ventilated by slow-reverse scaphognathite pumping activity. Carapace movements do not occur in the absence of branchial water. Carapace pumping is thought to provide a mechanism which permits the scaphognathites to ventilate the lungs in the presence of recirculating branchial water, without this water interfering with lung ventilation or being lost to the environment.Abbreviations FF, FR, SF, SR fast-forward, fast-reverse, slowforward, slow-reverse scaphognathite pumping - MEA Milne Edwards aperture     

大豆光合速率和气孔导度对水分胁迫的响应

土壤水分胁迫使两个供试大豆品种(系)光合速率和气孔导度降低,“鲁豆四号”降低的幅度大于小粒大豆品系“7605”。在相同的叶水势下,“7605”的光合速率和气孔导度均高于“鲁豆四号”,但“7605”气孔随水势下降而关闭的速率大于“鲁豆四号”。水分胁迫使叶片温度升高,“7605”比“鲁豆四号”升温较快,但在同一水分处理中,“鲁豆四号”的叶温高于“7605”。水分胁迫降低了大豆的水分利用效率,且“鲁豆四号”降低的速率大于“7605”。结果表明,“7605”对水分胁迫具有较好的适应能力。     

Interrelationships between water and cell metabolism in Artemia cysts X. Microwave dielectric studies

Artemia cysts are composed of an inner mass of about 4000 cells surrounded by an acellular shell. This system can undergo cycles of hydration-dehydration without viability loss, and is a useful model for the study of intracellular water. We have measured the relative permittivity (ε′) of these cysts as a function of water content over the frequency range 0.8–70 GHz. Detailed analysis of the data for cysts containing close to 1 g H₂O/g dry weight indicates that a significant fraction of the total water in this system exhibits dielectric behavior different from that of pure water: the distribution parameter (α) for the dispersion analyzed by the Cole-Cole equation deviates from zero, and the permittivity of cyst water appears to be significantly lower than that of pure liquid.     

Plasmodium falciparum: isolation and purification of spontaneously released merozoites by nylon membrane sieves

A new procedure for isolating spontaneously released merozoites from in vitro cultures of Plasmodium falciparum (FVO and FCB strains) is described. The mature forms of relatively synchronous cultures containing predominantly trophozoites and few schizonts were concentrated with Plasmagel and then incubated at 37 C, without adding fresh red blood cells, until trophozoites matured into schizonts. Merozoites which were subsequently released were harvested and freed from host red blood cell material by low-speed centrifugations and nylon membrane sieves (3- and 1.2-μm pore size). From a culture containing about 5.2 × 10⁹ mature-form parasites, a total of about 10.7 × 10⁹ merozoites were released during three consecutive harvests and about 69% of these merozoites were recovered after the isolation and purification procedures. As demonstrated by both light and electron microscopy, most merozoites were morphologically intact and the merozoite preparations were free of host cell constituents. SDS-acrylamide gel electrophoresis confirmed the absence of host cell material and also showed that merozoites had a complex protein pattern of apparent molecular weights between 225 and 15 kdaltons. Such purified merozoite preparations will be invaluable for malaria immunization studies, for identification of protective antigens of P. falciparum, and for other immunological and biochemical studies.     

Occurrence,properties and biological implications of the active uptake of water vapour from the atmosphere in Psocoptera

Water vapour absorption is shown to occur in 22 species of Psocoptera inhabiting diverse environments and representing all major groups of this insect order. Evidently the faculty is a common feature of the whole order and it seems not to be related to specific environmental conditions. For the first time water vapour uptake could be demonstrated in fully winged and flying insects. The critical equilibrium humidities vary considerably among different species ranging from 58 to 85% r.h. Marked interspecific differences are also observed in water loss and uptake rates but no clear correlation with habitat or systematic group is recognizable. The uptake rates of Psocoptera are among the highest of all arthropods investigated so far. From weight recordings with a sensitive microbalance it could be seen that continuous operation of the uptake mechanism is restricted to limited periods of time of less than 1 hr regardless of the water status of the animals. Initiation and termination of the uptake process are abrupt and continuous uptake proceeds at a constant rate at a given relative humidity. Uptake rates are humidity-dependent decreasing with falling relative humidity whereas the adjustment of the equilibrium level of body water is independent of ambient humidity. Equilibrium is maintained by intermittent operation of the uptake mechanism within ca. 3% of body water mass. The uptake mechanism exhibits marked sensitivity to starvation in most members of the Psocomorpha. Some features of the uptake process of Psocoptera are in close agreement with those of Mallophaga reflecting the close relationship between the two groups.     

The purification and characterization of homologous high molecular weight storage proteins from grain of wheat,rye and barley

Summary Homologous high molecular weight storage prolamins were purified from grain of wheat, rye and barley using combinations of gel filtration, ion-exchange chromatography and preparative isoelectric focusing. Sodium dodecylsulphate polyacrylamide gel electrophoresis showed that the components were single bands with apparent mol.wts. of above 100,000. Molecular weights determined by sedimentation equilibrium ultracentrifugation were considerably lower; 54,700, 67,600 and 69,600 for the components from barley, rye and wheat respectively. Amino acid analysis showed the presence of 13.6 to 16.5 mol% glycine, 29.6 to 34.0 mol% glutamate + glutamine, 11.4 to 13.7 mol% proline and a total of 4.0 to 5.7 mol% basic amino acids. Automated N-terminal amino acid sequencing of the component from wheat showed the presence of cysteine residues at positions 5 and 10, and this is discussed in relation to the possible role of these proteins in the visco-elastic gluten network.     

The isolation of abscisic acid (ABA) deficient mutants by selection of induced revertants in non-germinating gibberellin sensitive lines of Arabidopsis thaliana (L.) heynh.

Summary By selecting for germinating seeds in the progeny of mutagen-treated non-germinating gibberellin responsive dwarf mutants of the ga–1 locus in Arabidopsis thaliana, germinating lines (revertants) could be isolated. About half of the revertants were homozygous recessive for a gene (aba), which probably regulates the presence of abscisic acid (ABA). Arguments for the function of this gene were obtained from lines homozygous recessive for this locus only, obtained by selection from the F₂ progeny of revertant X wild-type crosses. These lines are characterized by a reduced seed dormancy, symptoms of withering, increased transpiration and a lowered ABA content in developing and ripe seeds and leaves.Abbreviations ABA Abscisic acid - GA₄₊₇ Mixture of gibberellin A₄ and A₇ - EMS Ethylmethanesulfonate - NG Non-germinating - G Germinating     

Water balance in Collembola and its relation to habitat selection: Water content,haemolymph osmotic pressure and transpiration during an instar

In the two collembolan species Orchesella cincta and Tomocerus minor the water content, haemolymph osmotic pressure and transpiration rate fluctuate with the feeding rhythm during each instar. The changes in water content, however, are due to changes in dry weight, because the absolute water weight stays constant during the instar.The intake of food is probably the cause of the increase in haemolymph osmotic pressure. Increase of osmotically active substances in the blood and/or blood volume reduction may be responsible for the rise in osmotic pressure. This change in osmotic pressure in turn may affect the responsiveness of the animals to water as well as their feeding behaviour.Changes in the epicuticle and in epidermal cell membranes may cause changes in the rate of transpiration. The high rate observed during ecdysis and during the mid-instar may explain the behaviour of the animals in varied water conditions.Dehydration during the instar causes an equivalent rise in osmotic pressure for both Tomocerus minor and Orchesella cincta. The water loss appears to involve the haemolymph. The physiological state of the animal does not influence the rise in osmotic pressure. There are no signs of any osmoregulation in the two species.