Erythrocyte Lii-Nao countertransport system Inhibition by N-ethylmaleimide probes for a conformational change of the transport system

Human erythrocytes were treated by a series of SH-reagents, including maleimides, iodo compounds, mercurials and oxidizing agents. Rates of Li efflux into Na-rich medium, Li leak and Li_i-Na_o countertransport were then determined. Of the 13 different reagents studied, only N-ethylmaleimide, iodoacetamide and iodoacetate inhibited selectively the countertransport activity. The effect of the various reagents indicates that the sensitive SH-groups of the countertransport system are not externally exposed. N-Ethylmaleimide was used to probe for changes elicited by substrate cations in Li_i-Na_o countertransport. In Na- and Li-free medium, inhibition of Li_i-Na_o countertransport by N-ethylmaleimide of 35% was reached within 2 s. In Na or Li medium, maximal inhibition was twice as great, but was attained much more slowly, within 10 min. Kinetic data and Hill plot analysis indicate the involvement of two classes of SH-groups: one expressed in the various media with and without substrate cations, and an additional one, which becomes specifically available to N-ethylmaleimide in the presence of external Na or Li. The affinity of Na to the site promoting inhibition by N-ethylmaleimide (apparent K_m  12 mM) is higher than the affinity of Na to its external countertransport site (apparent K_m  25 mM), as reported by Sarakadi, B., Alifimoff, J.K., Gunn, R.B. and Tosteson, D.C. (1978) J. Gen. Physiol. 72, 249–265). Reactivity of $\text{N-}\text{ethyl}\text{[}{}^{14}\text{C}\text{]}\text{maleimide}$ was not modified by the media tested. It is concluded that external Na and Li cause a conformational change in the protein(s) of the countertransport system in human erythrocytes.     

Zooplankton food limitation and grazing impact in a eutrophic brackish-water tropical pond (Côte d'Ivoire, West Africa)

Gut fluorescence, feeding and egg production rates of zooplankton assemblages were measured in a shallow, eutrophic brackish-water pond for 24 days. Brachionus plicatilis, Hexarthra intermedia and Apocyclops panamensis successively developed and exhibited differences in food selectivity. Rotifers selected small particles but also had a preference for larger particles (15–21 μm, Equivalent Spherical Diameter, ESD). B. plicatilis appeared less selective than H. intermedia, which fed mostly on particles <6 μm. A. panamensis adults showed a selectivity for 6–21 μm ESD particles. Laboratory experiments suggested that A. panamensis adults were able to shift from seston to carnivorous feeding, depending on the availability of these food resources. Measurements of gut fluorescence and grazing gave comparable ingestion rates. Rotifers displayed the highest ingestion rates (up to 486% body C d-1). Despite high total phytoplankton and seston biomasses, the high ingestion rates and selective grazing of rotifers induced auto food-limitation phenomena and caused major changes in seston abundance and size structure. Grazing impact was less important when A. panamensis dominated. This revised version was published online in July 2006 with corrections to the Cover Date.     

Preparative isolation and purification of dicaffeoylquinic acids from the Ainsliaea fragrans champ by high-speed counter-current chromatography

Two dicaffeoylquinic acids, namely 3,5-dicaffeoylquinic acid and 4,5-dicaffeoylquinic acid, have been successfully separated by high-speed counter-current chromatography (HSCCC) from an extract of Ainsliaea fragrans Champ, followed by an initial clean-up step using AB-8 resin. A two-phase solvent system composed of chloroform:methanol:water (8:8:4) was selected for the isolation with the aqueous-rich phase as the stationary phase and the organic-rich phase as the mobile phase. The developed HSCCC method yielded 34 mg of 3,5-dicaffeoylquinic acid and 17 mg of 4,5-dicaffeoylquinic acid from 150 mg of the crude sample in a one-step separation with purities of 98 and 95%, respectively, as determined by HPLC. The structures of the two compounds were identified from ESI/MS, (1)H- and (13)C-NMR spectroscopic data.     

Purification of betulinic acid from Eugenia florida (Myrtaceae) by high-speed counter-current chromatography

A high yield of betulinic acid (up to 17% from the ethanolic extract) was found in the leaves of Eugenia florida collected in south-eastern Brazil, making this species a potential commercial source of the title compound. Extracts of E. florida were subjected to solvent partition, and rapid high-speed counter-current chromatography (HSCCC) was applied to the semi-crude extracts to afford betulinic acid in high purity. The mobile and stationary phases were derived from the two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (10:5:2.5:1). The developing solvent system (stationary and mobile phases) for optimum HSCCC separation was chosen by dissolving the fraction to be chromatographed in the proposed solvent mixture and determining the amount of betulinic acid in each phase by densitometric TLC. Purified betulinic acid was characterized by 13C-NMR, GC-MS and co-injection of its methyl ester with standards in GC-FID. The HSCCC technique is commonly employed to isolate triterpene glycosides, but is applied in this study to an aglycone.     

Oceanic fronts in the Sargasso Sea control the early life and drift of Atlantic eels

Anguillid freshwater eels show remarkable life histories. In the Atlantic, the European eel (Anguilla anguilla) and American eel (Anguilla rostrata) undertake extensive migrations to spawn in the oceanic Sargasso Sea, and subsequently the offspring drift to foraging areas in Europe and North America, first as leaf-like leptocephali larvae that later metamorphose into glass eels. Since recruitment of European and American glass eels has declined drastically during past decades, there is a strong demand for further understanding of the early, oceanic phase of their life cycle. Consequently, during a field expedition to the eel spawning sites in the Sargasso Sea, we carried out a wide range of dedicated bio-physical studies across areas of eel larval distribution. Our findings suggest a key role of oceanic frontal processes, retaining eel larvae within a zone of enhanced feeding conditions and steering their drift. The majority of the more westerly distributed American eel larvae are likely to follow a westerly/northerly drift route entrained in the Antilles/Florida Currents. European eel larvae are generally believed to initially follow the same route, but their more easterly distribution close to the eastward flowing Subtropical Counter Current indicates that these larvae could follow a shorter, eastward route towards the Azores and Europe. The findings emphasize the significance of oceanic physical–biological linkages in the life-cycle completion of Atlantic eels.     

Curve fitting approach for measurement of cellular osmotic properties by the electrical sensing zone method. I. Osmotically inactive volume

We have investigated the confounding effects of dynamic range limitations on measurement of the osmotically inactive volume using electrical sensing zone instruments (e.g., Coulter counters), and propose an improved approach to parameter estimation. The conventional approach for analysis of cell size distributions measured by such particle sizing instruments requires data truncation: the mean cell volume is computed after exclusion of data below a specified lower bound (typically chosen to remove artifacts due to small-volume noise) and above an upper bound (typically governed by instrument limitations). The osmotically inactive volume is then estimated from a Boyle–van’t Hoff plot of the averaged volume data obtained after exposure to various solution osmolalities. We demonstrate that systematic exclusion of data in the conventional approach introduces bias that results in erroneously high estimates of the osmotically inactive volume fraction. To minimize this source of error, we have devised a new algorithm based on fitting a bimodal distribution model to the non-truncated volume data. In experiments with mouse insulinoma (MIN6) cells, the osmotically inactive volume fraction was estimated to be 0.15 ± 0.01 using the new method, which was significantly smaller than the estimate of 0.37 ± 0.02 obtained using the conventional method (p < 0.05). In silico experiments indicated that the parameter estimate obtained by the new method was accurate within 5%, whereas the error associated with the conventional approach was approximately 150%. Parametric analysis was used to elucidate the sensitivity of errors to variations in instrument dynamic range and cell volume distribution width.     

球形红假单胞菌反应中心中蛋白的量子化学研究

采用重叠二体近似方法和建立在从算水平上的扩展负本征值因数计算方法（extended negative factor counter method）研究了球形红假单胞菌（Rhodobacter sphaeroides(Van Niel)Imhoff,Truper et Pfennin）中兴合反应中心中蛋白链L及M的电子结构。结果表明：（1）对组成蛋白链L（M）的前线轨道有重要贡献的氨基酸残基分布在L链的自由螺旋区（M链的α螺旋区）。由于自由螺旋是有柔曲性的,它易于在电子转移的过程中改变其构象并降低体系的能量,而α螺旋结构却相对稳定,这种差别有可能是光合反庆中心中电子转移只沿L支进行的原因之一。（2）与特殊对分子及辅助叶绿素分子形成轴向配位的组氨酸残基对于特殊对P和辅助叶绿素分子的ELUMO有重要影响,但此组氨酸的相应分子轨道的贡献并没有出现在蛋白链的前线轨道组成中。这意味着色素分子与蛋白链之间的相互作用对蛋白链前线轨道的贡献没有影响,但却能影响相应色素分子的ELUMO能级。     

Comparison of discrete-point vs. dimensionality-reduction techniques for describing performance-related aspects of maximal vertical jumping

The aim of this study was to assess and compare the ability of discrete point analysis (DPA), functional principal component analysis (fPCA) and analysis of characterizing phases (ACP) to describe a dependent variable (jump height) using vertical ground reaction force curves captured during the propulsion phase of a countermovement jump. FPCA and ACP are continuous data analysis techniques that reduce the dimensionality of a data set by identifying phases of variation (key phases), which are used to generate subject scores that describe a subject?s behavior. A stepwise multiple regression analysis was used to measure the ability to describe jump height of each data analysis technique. Findings indicated that the order of effectiveness (high to low) across the examined techniques was: ACP (99%), fPCA (78%) and DPA (21%). DPA was outperformed by fPCA and ACP because it can inadvertently compare unrelated features, does not analyze the whole data set and cannot examine important features that occur solely as a phase. ACP outperformed fPCA because it utilizes information within the combined magnitude-time domain, and identifies and examines key phases separately without the deleterious interaction of other key phases.     

UNDERSTANDING AND INTERPRETING HEMATOCRIT MEASUREMENTS IN PINNIPEDS

Hematocrit data are relatively easily obtained from blood samples of pinnipeds but differences in methodology and variable physiological conditions of the subjects can significantly alter their values. This two-fold problem makes comparative data and modeling efforts difficult. To quantify the difficulty of obtaining accurate and representative hematocrit values in pinnipeds, hematocrit was measured by both microcentrifugation and Coulter counter methods in a range of pinnipeds under a variety of physiological and handling conditions. The data show that the Coulter counter hematocrit values were 4%-15% higher than those measured by microcentrifugation. In addition, blood samples from restrained animals showed consistently elevated hematocrit values relative to resting subjects. A significant difference was also found between hematocrit values from pups and adults. Finally, hematocrit was shown to decline over the course of isofluorane anesthesia. Taken together, these results suggest that laboratory methodology, developmental state, and animal handling techniques can significantly alter hematocrit values in pinnipeds. Thus, modeling efforts that require representative hematocrit values, such as calculations of total blood oxygen stores, can be markedly impacted by variations in hematocrit measurement techniques and sampling regimes.     

Comparison of coulter volumes with radiometrically determined intracellular water volumes for cultured cells

Summary During methotrexate-induced differentiation of cultured human choriocarcinoma (BeWo) cells, proliferation is inhibited, morphologic and biochemical changes occur, and giant, often multinucleated, cells form. We have used the increase in cell volume as a marker of the mature syncytiotrophoblastlike phenotype. Uninduced and differentiated BeWo cells are not spherical, and theoretical considerations suggested that deviations in shape could result in significant errors in Coulter volume. To determine if the values obtained by electrical pulse sizing reflected the actual mass of BeWo cells, we have evaluated the relationship between Coulter volumes and intracellular water volumes obtained using a shape-independent estimate for eight cell types. A close correlation (r ²=0.97) was found, indicating that cell volume changes in populations of irregularly shaped cells can be accurately measured using a Coulter instrument. Supported by an operating grant from the National Cancer Institute of Canada. N.S.B. was a recipient of a studentship award from the Alberta Heritage Foundation for Medical Research. C.E.C. is a Senior Research Scientist of the National Cancer Institute of Canada. The McEachern Laboratory is a research facility of the Faculty of Medicine, University of Alberta, and the Cross Cancer Institute, Edmonton, Alberta.