北京鸭红细胞铜锌超氧化物歧化酶电荷异构体的分离和某些性质

等电聚焦表明,北京鸭红细胞铜锌超氧化物歧化酶由等电点分别为５．０,５．３,５．９,６．１和６．５的五个主要的活性组分（电荷异构体）构成,利用分析型聚丙烯酰胺凝胶等电聚焦电泳进行电荷异构体的制备级分离,采用三氯乙酸沉淀法快速确定蛋白条带的位置,电渗洗脱法回收蛋白,获得其中两个电荷异构体,对比研究结果表明电荷异构体的活性,氨基酸组成,二级结构等性质无明显差异。     

Rapid regulation of electrolyte absorption in sweat duct

Even though the same Cl channel (CFTR) is common to certain fluid transport functions that are oppositely directed, i.e., secretion and absorption, only fluid secretion has clearly been shown to be acutely regulated. It is now clear that fluid secretion activated by -adrenergic stimulation is controlled by cAMP-mediated opening and closing of CFTR-Cl channels. Since the conductance of the human sweat duct is almost wholly due to CFTR-Cl conductance (CFTR-G_Cl), we sought to determine whether salt absorption via CFTR-Cl channels could also be subject to acute regulation in this purely absorptive epithelium. After -toxin permeabilization, we found that addition of cAMP resulted in a large increase in Cl diffusion potentials across the apical membrane and a more than twofold increase in the average membrane conductance. Since the cAMP effects were dependent on Cl alone, not on Na, and since apical Cl conductance appears to be almost exclusively comprised of CFTR-G_Cl, we surmise that this form of electrolyte absorption like secretion is also subject to acute control through CFTR-G_Cl. Acute regulation of absorption involves both activation by phosphorylation (PKA) and inactivation by dephosphorylation (unknown endogenous phosphatase) of CFTR. Phosphorylation of CFTR was shown by the facts that CFTR-G_Cl could be activated by cAMP and inhibited by the kinase antagonist staurosporine, or by removal of either substrate ATP or Mg²⁺ cofactor. Inactivation of CFTR-G_Cl by endogenous phosphatase(s) was indicated by a spontaneous but reversible loss of CFTR-G_Cl upon removal of cAMP. Such loss of CFTR-G_Cl activity could be prevented either by application of phosphatase inhibitors or by using phosphatase-resistant ATP--S as substrate to phosphorylate CFTR. We surmise that absorptive function is subject to rapid regulation which can be switched on and off acutely by a control system that is common to both absorptive and secretory processes and that this control is crucial to switching between conductive and nonconductive transport mechanisms during salt absorption.The authors are grateful to Mr. Kirk Taylor for expert technical assistance, and to numerous volunteer human subjects for participating in the experiments with informed consent. Supported by grants from the National Institutes of Health, DK-41329-04 and the National Cystic Fibrosis Foundation, Z 439.     

L-Alanine absorption in human intestinal Caco-2 cells driven by the proton electrochemical gradient

In human Caco-2 intestinal epithelial layers, xxxl-alanine absorption can be energized by a proton gradient across the brush-border membrane. Acidification of the apical medium, even in Na⁺-free media, is associated with a saturable net transepithelial absorption of xxxl-alanine. xxxl-Alanine transport causes cytosolic acidification consistent with proton/amino acid symport. xxxl-Alanine transport in Na⁺-free media is rheogenic, stimulating an inward short-circuit current in voltageclamped epithelial monolayers. By measurement of rapid xxxl-alanine influx across the apical membrane, xxxl-alanine-stimulated inward short-circuit current and intracellular acidification in the same cell batch, we estimate xxxl-alanine/proton stoichiometry to be 10.62 ±0.25 (xxxsd) (short-circuit current) or 10.73 ±0.19 (intracellular acidification). From competition studies, it is likely that xxxl-proline, -aminoisobutyric acid, and -alanine, but not xxxl-valine and xxxl-serine, are substrates for protonlinked, substrate transport in the brush border of Caco-2 cells.This study was supported by the Wellcome Trust (to D.T.T. and N.L.S.) and the LINK Programme in Selective Drug Delivery and Targeting (funded by the SERC/MRC/DTI and Industry). Charlotte Ward gave excellent technical assistance.     

Resource-mediated effects of stream pollution on food absorption of Asellus aquaticus (L.) populations

The role of interactions between chemical perturbations and biological constraints on detritivores occurring in polluted streams were investigated by analysing food absorption variation with stress. Absorption rate and efficiency of four Asellus aquaticus (L.) populations from differently polluted habitats were quantified with respect to the microbial guilds colonizing detritus. A twin tracer method was used. Detritus was microbially colonized in standard conditions and on each stream bottom to control for potential resource-independent variations among individuals. The relationship between length and weight was also determined on a random sample of individuals of each population. Differences of 14.6% in potential absorption efficiency and 11.3% in potential absorption rate were observed between populations from the least and the most polluted habitat. Actual (realized) variations were much stronger: from a minimum of a 60.1% reduction in absorption efficiency to a maximum of 93.8% for the rate. The realized food absorption and the individual weight per length showed the same pattern of variation among populations. This suggested that the availability of energy to isopods in nature was related to stream pollution and resource quality. Bottomup interactions appear to be the most relevant pathway through which chemical water pollution affects the Asellus populations studied. The potential resource-independent variations among individuals are also likely to be explained by temporal cascading of resource-mediated effects.     

Size structure of the metazoan community in a Piedmont stream

We characterized the size structure of virtually the entire metazoan community in a fourth order, sandybottomed Piedmont stream during late summer. Our study, the first to sample across all habitat types and sizes of metazoans in an aquatic ecosystem, indicates that at the community level, stream size spectra may be bimodal for the benthos or trimodal when fish are included. Animals spanning 10 orders of magnitude in dry mass (from gastrotrichs to fish) were quantitatively collected from nine habitat types. The bimodal benthic size spectrum was characterized by a meiofaunal component (mostly oligochaetes and micro-crustacea) and a macrobenthic component (mostly the introduced asiatic clam, Corbicula fluminea). Insects contributed little to overall standing crop. Size-specific contribution to whole-community metabolism was assessed using allometric equations for respiration, and we found a distinctly bimodal distribution across the entire metazoan size range, with peaks in the meiofaunal and benthic macrofaunal size ranges. Our bimodal benthic size spectrum is similar to that observed for marine benthos but not to other freshwater benthic systems, possibly because the entire range of habitat types and/or animal sizes were not sampled in the latter. Numerous factors may influence size spectra in stream ecosystems, including local geomorphic (habitat) conditions, water level fluctuations, species introductions, and predation processes.     

蜂毒素与紫膜的相互作用机理研究──与三种表面活性剂的比较

用吸收光谱和圆二色谱的方法研究了蜂毒素与嗜血菌紫膜的相互作用机理．通过与三种在结构和电荷上不同的表面活性剂与紫膜的作用相比较,可以年出蜂毒作为带正电荷的分子与同样带正电的表面活性剂ＤＴＡＢ在引起紫膜凝聚方面表现相同;但在对紫膜可见光区的吸收光谱和圆二色谱的影响上却与具有刚性结构的ＣＨＡＰＳ相似,表明蜂毒可在紫膜表面以一种刚性较大的构象（如α螺旋）存在,不能进入膜蛋白流水区的深层．另外,从紫膜－Ｔｒｉｔｏｎ－蜂毒混合作用体系的研究中得到如下推测：蜂毒与Ｔｒｉｔｏｎ竞争菌紫质分子周围的结合位点,可排斥位于菌紫质周围的Ｔｒｉｔｏｎ分子．表明蜂毒具有比Ｔｒｉｔｏｎ更强的与菌紫质的亲和力,从而提供了支持蜂毒分子存在与膜蛋白－菌紫质的直接相互作用的有力证据．     

The influence of the type of contraction on the masseter muscle EMG power spectrum

Different behaviours of the EMG power spectrum across increasing force levels have been reported for the masseter muscle. A factor that could explain these different behaviours may be the type of contraction used, as was recently shown for certain upper limb muscles⁵. The purpose of this study was to compare, between two types of isometric contractions, the behaviour of EMG power spectrum statistics (median frequency (MF) and mean power frequency (MPF)) obtained across increasing force levels. Ten women exerted, while biting in the intercuspal position, three 5 s ramp contractions that increased linearly from 0 to 100% of the maximal voluntary contraction (MVC). They also completed three step contractions (constant EMG amplitude) at each of the following levels: 20, 40, 60 and 80% MVC. EMG signals from the masseter muscle were recorded with miniature surface electrodes. The RMS, as well as the MPF and MF of the power spectrum were calculated at 20, 40, 60 and 80% MVC for each type of contraction. As expected, the RMS values showed similar increases with increasing levels of effort for both types of contractions. Different behaviours for both MPF (contraction^*force interaction, ANOVA, P<0.05) and MF (contraction^*force interaction, ANOVA, P>0.05) across increasing levels of effort were found between the two types of contraction. The use of step contractions gave rise to a decrease of both MPF and MF with increasing force, while the use of ramp contractions gave rise to an increase in both statistics up to at least 40% MVC followed by a decrease at higher force levels. These findings suggest that the type of contraction used does influence the behaviour of the spectral statistics across increasing force levels and that this could explain the differences obtained in previous studies for the masseter muscle.     

Preservation and computer-aided microscopic analysis of planktonic Protozoa and algae

Samples of microplankton and larger nanoplankton (5 to 200 m) are preserved with a combination of Lugol's solution and DaFano's fixative. Organisms are then settled on a gelatin-coated slide, dried and embedded in 40 percent glycerin. Counting and sizing is performed under a microscope using a drawing tube, which facilitates measuring the organisms with a microcomputer-interfaced caliper. An interactive computer program, written in BASIC, allows for estimating the volumes of cells in up to 40 shape/species categories. The program then saves data on a disk, retrieves them, and calculates the results either for individual species (abundance, biomass, and mean cell volume) or as a pooled size spectrum of all organisms measured.     

Influence of phosphate ion on the fluorescence of 3-fluorotyrosine

Summary 3-Fluorotyrosine fluorescence is quenched effectively by phosphate ions not only by a dynamic but also by a static mechanism owing to H-bond complex formation in ground state. 3-Fluorotyrosine pK_a values both in the ground and first excited state (8.3 and 4, respectively) are appreciably lower than those of tyrosine, thus promoting 3-fluorotyrosinate ion formation in the excited state. Additional emission owing to 3-fluorotyrosinate ion (near 350 nm) may be taken erroneously for tryptophan fluorescence.