Thiol-induced oligomerization of α-lactalbumin at high pressure

Denaturation and aggregation of-lactalbumin at high pressure (up to 10 kbar, 1000 MPa) were studied by means of circular dichroism, gel-permeation chromatography, sodium dodecyl sulfate and gel electrophoresis. It was found that the unfolding of-lactalbumin at high pressure is reversible even in basic pH and at a protein concentration as large as 10%. In these conditions only a negligible fraction of the protein is denatured irreversibly and aggregates. The rate of aggregation of-lactalbumin at high pressure increases significantly in the presence of low-molecular reducing agents such as cysteine, 2-mercaptoethanol, and dithiothreitol. Maximal yield of-lactalbumin oligomerization (over 90%) was achieved in the presence of cysteine at the molar cysteine/protein ratioq=2 and atpH 8.5. Apparent molecular weight of the obtained oligomers was over 500 kDa. It was shown that the size distribution of oligomers can be modulated by varyingpH and reducing agent. The size distribution shifts in the direction of very large, poorly soluble particles whenpH decreases. Maximal content of the insoluble fraction (about 30%) can be reached at pH 5.5 when cysteine (q=2) is used as reducing agent. The oligomers of-lactalbumin are stabilized mainly by nonnative interchain disulfide bridges. Circular dichroism measurements point to an additional mechanism of cohesion of polypeptide chains in the oligomers, which is formation of intermolecular-sheets.     

Complete mechanical impedance increases the turgor of cells in the apex of pea roots

In this paper we describe an experimental approach which allows turgor (p) in an impeded root to be measured without the need to remove the root from the impeding environment. The maximum axial growth pressure (σ_max) generated by completely impeded pea (Pisum sativum L.) roots was measured using a novel apparatus incorporating a force transducer. The apparatus was designed so that it was possible to gain access to the impeded root with the microcapillary of a pressure probe and so obtain in situ measurements of P. Turgor in cells in the apical region of impeded roots was 0.78 MPa, compared with 0.55 MPa in unimpeded roots. In impeded roots, σ_max was 0.52 MPa, showing that the pressure component resulting from cell wall tension (W, where W=P–σ) decreased from 0.55 to 0.26 MPa as the roots became impeded. When impeded roots were removed from the apparatus, there was no decrease in P over the following 90 min. Impedance did not cause P to change in the non-elongating part of the roots further from the apex.     

Callus formation from protoplasts of Artemisia sphaerocephala Krasch and some factors influencing protoplast division

Round wormwood (Artemisia sphaerocephala Krasch) seeds were germinated on Murashige & Skoog (1962) medium without plant growth regulators. The hypocotyls of seedlings were sliced and cultured on M1 medium with 2,4-dichlorophenoxyacetic acid (9.05 M) to induce callus. The induced calluses were subcultured on the same medium. Ten day old calluses were used to isolate protoplasts in an enzyme solution with 0.65 M mannitol. Protoplast yield strongly depended upon the state of callus cultures. Certain amount of hemicellulase could improve protoplast isolation. Purified protoplasts were cultured in modified Kao & Michayluk (1975) medium with 0.60 M mannitol as osmoticum, suggesting that protoplasts of A. sphaerocephala need a high initial osmolarity. Protoplasts generally divided evenly and the percentage of first division could reach 10%. Kinetin exhibited a positive effect on initial cell division. Furthermore, we studied the effect of protoplast density and vitamin C on sustained growth of protoplasts. After forty days, 1 mm calluses in diameter formed.Abbreviations CH casein hydrolysate - 2,4-D 2,4-dichlorophenoxyacetic acid - KM8P Kao & Michayluk (1975) protoplast medium - MS Murashige & Skoog (1962) medium - MES-2 (N-morpholino)ethanesulfonic acid     

Base water potential of Picea abies as a characteristic of the soil water status

Variation in base water potential (Ψ_b, a daily maximum level of plant water potential, which is presumed to correspond to the condition of equilibrium between the soil and plant water potentials) was examined in shoots of Norway spruce trees growing in well-drained and waterlogged soils. The influence of soil water content, air temperature, and vapour pressure deficit of the atmosphere on Ψ_b was studied using the pressure chamber technique. Maximum daily water potentials were not always observable before dawn; some were registered up to two hours later. This tendency being characteristic of trees growing under stress (shade, waterlogging) conditions, increased with declining soil water availability. In trees growing in well-drained soil, Ψ_b depended asymptotically on the available soil water storage (R²=0.73), while the values were slightly influenced by vapour pressure deficit of the atmosphere as well. In trees growing in waterlogged soil, Ψ_b was independent of the soil water storage, but sensitive to the vapour pressure deficit.     

Engineering challenges in high density cell culture systems

High density cell culture systems offer the advantage of production of bio-pharmaceuticals in compact bioreactors with high volumetric production rates; however, these systems are difficult to design and operate. First of all, the cells have to be retained in the bioreactor by physical means during perfusion. The design of the cell retention is the key to performance of high density cell culture systems. Oxygenation and media design are also important for maximizing the cell number. In high density perfusion reactors, variable cell density, and hence the metabolic demand, require constant adjustment of perfusion rates. The use of cell specific perfusion rate (CSPR) control provides a constant environment to the cells resulting in consistent production. On-line measurement of cell density and metabolic activities can be used for the estimation of cell densities and the control of CSPR. Issues related to mass transfer and mixing become more important at high cell densities. Due to the difference in mass transfer coefficients for oxygen and CO₂, a significant accumulation of dissolved CO₂ is experienced with silicone tubing aeration. Also, mixing is observed to decrease at high densities. Base addition, if not properly done, could result in localized cell lysis and poor culture performance. Non-uniform mixing in reactors promotes the heterogeneity of the culture. Cell aggregation results in segregation of the cells within different mixing zones. This paper discusses these issues and makes recommendations for further development of high density cell culture bioreactors.     

Detection of mining impacts on aquatic macroinvertebrate communities: Results of a disturbance experiment and the design of a multivariate BACIP monitoring programme at Coronation Hill,Northern Territory

Abstract Multivariate methods for the detection of impacts on communities are: (i) explored in the context of a disturbance experiment at a site of previous uranium mining in Kakadu National Park, Northern Territory; and (ii) applied to the design of a monitoring programme for a proposed mining development at nearby Coronation Hill. The disturbance experiment in Rockhole Mine Creek used a gravity-fed redirection of polluted water from the mine adit to create a point-source pollutant. Weekly samples of the macroinvertebrate communities were taken both before and after disturbance at the control and‘impact’ site, as part of a multivariate BACIP (Before-After-Control-Impact-Pairs) design. The results of the experiment show that the multivariate approach provided high sensitivity in detecting impacts on the community, with little difference in sensitivity between species and family level analyses. A simple procedure for deleting those taxa that show poor discrimination between pristine and presently disturbed areas further increased the sensitivity of the tests. In addition to the BACIP analyses, robust ordination of the community data suggested a time/treatment interaction effect. Application of the multivariate BACIP approach also was explored using baseline data for benthic communities from the South Alligator River near Coronation Hill. The method provides high statistical power for monitoring; a relatively small change in community dissimilarity would be detectable in a programme with 5 years of observations before and after commencement of mining. A robust ordination of the yearly samples for the control and potential-impact sites showed that, although samples varied along identifiable gradients over time, the two areas changed in parallel so that the difference between the areas was approximately constant over years. This result demonstrates that the multivariate BACIP approach can provide relative constancy of pre-impact difference values, a key requirement of BACIP designs that is difficult to satisfy using abundance data for a single species.     

水稻伸长生长的数学模型

水稻地上部诸器官的伸长生长,可以分为3个阶段和两个过渡期．器官在前期的凹型曲线生长为加速生长（定为第1阶段）,中期的直线生长为等速生长（第2阶段）,后期的凸型曲线生长为减速生长（第3阶段）;每两个阶段临界处均存在特殊的生长过程,前面的定为第1过渡期,做变加速生长,后面的定为第2过渡期,做变减速生长.组成器官的各个细胞的伸长生长,也可以分为前期凹型曲线阶段、中期的直线阶段以及这两个阶段临界处的过渡期.运用类比原理推断;细胞在凹型曲线阶段,其原生质的膨压大于壁压,而且这两个压差始终维持稳定,这就使细胞做等加速生长;细胞经过过渡期的变加速生长过渡到直线生长阶段,在这个阶段中,膨压大小与壁压相等,这就使细胞以过渡期最末的生长速度做等速生长;最后壁压大于膨压,而且这两个相差压会阻止细胞的“惯性”生长直至停止生长,其结果产生减速生长．     

Physiology of cold hardiness in cocoons of five earthworm taxa (Lumbricidae: Oligochaeta)

Earthworm cocoons are mostly found in the uppermost soil layers and are therefore often exposed to low temperatures during winter. In the present study, cocoons of five taxa of earthworms were investigated for their tolerance to freezing, melting points of cocoon fluids and dehydration of cocoons when exposed to a frozen environment. Embryos of the taxa investigated were freeze intolerant. The melting points of fully hydrated cocoon fluids were high (above –0.3°C) and thermal hysteresis factors were absent. Exposure to a frozen environment caused the cocoons to dehydrate drastically and dehydrated cocoons showed significantly lower super-cooling points than fully hydrated cocoons, reducing the risk of freezing for dehydrated cocoons. It is proposed therefore that the cold-hardiness strategy of the earthworm cocoons is based on dehydration upon exposure to subzero temperatures in the frozen environment. Cocoons of three surface-dwelling taxa, Dendrobaena octaedra, Dendrodrilus rubidus tenuis and Dendrodrilus rubidus norvegicus had lower supercooling points and survived frost exposure better than cocoons of two deeper-dwelling taxa, Aporrectodea caliginosa and Allolobophora chlorotica. One of the investigated taxa, D. r. norvegicus, was collected from a cold alpine habitat. However, it was not more cold hardy than the closely related D. r. tenuis collected from a lowland temperate habitat. D. octaedra was the most cold hardy taxon, its cocoons being able to withstand –8°C for 3 months and –13.5°C for 2 weeks in frozen soil.Abbreviations dw dry weight - fw fresh weight - SCP supercooling point     

The influence of the cytosolic oncotic pressure on the permeability of the mitochondrial outer membrane for ADP: implications for the kinetic properties of mitochondrial creatine kinase and for ADP channelling into the intermembrane space

Summary Cytosolic proteins as components of the physiological mitochondrial environment were substituted by dextrans added to media normally used for incubation of isolated mitochondria. Under these conditions the volume of the intermembrane space decreases and the contact sites between the both mitochondrial membranes increase drastically. These morphological changes are accompanied by a reduced permeability of the mitochondrial outer compartment for adenine nucleotides as it was shown by extensive kinetic studies of mitochondrial enzymes (oxidative phosphorylation, mi-creatine kinase, mi-adenylate kinase). The decreased permeability of the mitochondrial outer membrane causes increased rate dependent concentration gradients in the micromolar range for adenine nucleotides between the intermembrane space and the extramitochondrial space. Although all metabolites crossing the outer membrane exhibit the same concentration gradients, considerable compartmentations are detectable for ADP only due to its low extramitochondrial concentration. The consequences of ADP-compartmentation in the mitochondrial intermembrane space for ADP-channelling into the mitochondria are discussed.     

Consumption of benthic invertebrates by waterbirds in the Oosterschelde estuary,SW Netherlands

The number of waders in the Oosterschelde, S.W. Netherlands, declined after a reduction in intertidal area due to the construction of a storm surge barrier and secondary dams, suggesting that the carrying capacity had been reached (Schekkerman et al., 1993). In this paper we present data on consumption and predation pressure by birds to explore whether the reduction in their numbers is due to prey depletion or to other factors.The total annual consumption of benthic invertebrates by birds in the Oosterschelde amounted to 1573 × 10³ g ADW y^–1 in the period before the coastal engineering works (pre-barrier) and 1500 × 10³ kg ADW y^–1 in the post-barrier period. More than half of the total amount of biomass is eaten by the Oystercatcher, and only seven (pre-barrier) or even six (post-barrier) bird species together take 90% of the total.Although the consumption by individual species may vary considerably among years, the total consumption was remarkably stable, with a CV of only 3–4% of the mean, especially compared to the variability of the prey populations. In the pre-barrier period, consumption was lowest in mid summer, increased sharply from August onwards until a peak was reached in January. A sharp decrease took place in March. In the post-barrier period, consumption peaked in October.The total consumption per unit area per year does not differ much between different sectors of the Oosterschelde, apart from a distinctly lower value in the eastern part. Of the total amount of food taken by birds, only 0.1–0.4% is taken in the subtidal compartment. In several study plots on an individual tidal flat, there was a clear relation between consumption and benthic biomass.The predation pressure was 13 and 23% of the standing stock, in the post- and pre-barrier period respectively. When cockles, mussels and their main predator, the Oystercatcher, are excluded from the calculations, the predation pressure of the other species was 30 and 37% of the biomass, respectively.Predation pressure of Oystercatchers in individual study plots varied from less than 10% to more than 70% of the standing stock. On cockle beds the predation pressure was positively related to the average length of the cockles present.Based on these results and a comparison with the literature we conclude that, at least for several species that feed intertidally, carrying capacity could be limited by the stocks of food. This does not mean that birds face food shortage each season. As the variability of the benthos populations is much higher than that of the bird densities it is likely that at some times food is not limiting, at other times it is. On the other hand, consumption is very low in the subtidal compartment and species feeding here could potentially increase substantially in numbers in the Oosterschelde.