固氮酶铁钼辅因子FeMo-co在水与非水体系内重组活力与催化活力的比较研究

 本文提出一种测定FeMo-co催化活力的反应体系,用此反应体系,在测定FeMo-co催化活力的过程中,FeMo-co与变种UW45抽提液的重组活性始终保持不变。讨论了水含量、还原剂对FeMo-co催化活力和重组活力的影响。     

Size Effects on the Uptake of Particles by Populations of Tetrahymena pyriformis Cells

Flow cytometry has been used to make direct measurements of rates of uptake of latex microspheres from dilute, monodisperse suspensions by Tetrahymena pyriformis. Measurements were made for five different sizes of microspheres, ranging from 1.09 to 6.17 μm diameter. Fractions of cells in the population that did not ingest the microspheres offered were also determined. In addition, the size distributions, as indicated by the forward angle light scattering intensity which is measured by the instrument, were determined for the whole population and for the subpopulations of cells that did and did not ingest the particles, for each particle size used. It was found that the fraction of cells that did not ingest the particles was small and independent of particle size when this was less than about 2.7 μm, but increased with particle size when particle size was increased above this value. The so-called maximum clearance rate, which can be calculated from the data, was found to increase monotonically with particle size if it were based only on those cells which actually ingested the particles offered. However, a plot of maximum clearance rate vs. particle size exhibited a maximum if the clearance rate were based on all cells present in the population.     

A simple and reliable turbidimetric and kinetic assay for alpha-amylase that is readily applied to culture supernatants and cell extracts

Summary A simple, reliable and sensitive assay for alpha-amylase activity is reported, together with its theoretical derivation, that overcomes many of the problems encountered with other assays, especially when attempting to assay alpha-amylase activity in crude cell extracts or culture supernatants. The method relies on the reduction in turbidity that occurs upon digestion of a starch suspension with alpha-amylase. The initial rate of decrease in turbidity is shown to be proportional to a wide range of enzyme concentrations, permitting a rapid spectrophotometric and kinetic determination of alpha-amylase activity.     

Reductions in genetic variation inDrosophila andE. coli caused by selection at linked sites

Selection at linked sites has important consequences for the properties of neutral variation and for tests of the predictions of the neutral theory of molecular evolution. We review the theory of the effect of adaptive gene substitutions on neutral variability at linked sites (hitchhiking or selective sweeps) and discuss theoretical results on the effect of selection against deleterious alleles on variation at linked sites (background selection). InDrosophila melanogaster there is a clear relation between the frequency of recombination in a given region of the chromosome and the amount of natural variability in that region. Attempts to predict this relation have given rise to models of selective sweeps and background selection. We describe possible methods of discriminating between these models, and also discuss the probable strong influence of selective sweeps on variation in largely nonrecombining genomes, with particular reference toEscherichia coll. Finally we present some unresolved questions and possible directions for future research.     

Specificities of Red Cell Membrane Sites Transporting Three Long Chain Fatty Acids

We studied the specificities of human red cell membrane bindings of three long chain fatty acids, palmitic- arachidonic- and oleic acid, using resealed membranes, ghosts. Previously estimated binding capacities, affinities and inside/outside distributions [6, 10, 11, 12], suggest separated binding sites. This possibility is explored by estimating the binding properties of one fatty acid in the presence of one or two of the others. Binding capacities, nmol g⁻¹ ghosts, of palmitic and arachidonic acid estimated simultaneously vs. separately are 27.4 ± 2.7 vs. 29.0 ± 2.1 (P < 0.6) and 6.5 ± 0.6 vs. 5.5 ± 0.5 (P < 0.2) respectively. The corresponding estimates for oleic- and palmitic acid are 36.5 ± 2.0 vs. 34.0 ± 2.2 (P < 0.4) and 28.4 ± 1.8 versus 29.1 ± 2.1 (P < 0.8). The binding sites are therefore independent. For each of the three fatty acids in the absence or in the presence of one or two of the others, the inside/outside distributions of the binding sites and the membrane transfer rate constants are elucidated by exchange efflux kinetics at 0°C from ghosts with and without enclosed albumin. Packed ghosts loaded with radioactive acids are injected rapidly into a large volume of vigorously stirred buffer with albumin. With a resolution time of about 1-sec serial filtered ghost-free aliquots are collected and counted. The analyses show that palmitic- and oleic acid sites of transport are entirely independent but do not exclude that palmitic- and/or oleic acid binding may diminish the arachidonic acid affinity a little. The diversity combined with specificity suggests that the transport sites for long chain fatty acids are protein-determined microdomains of phospholipids. Received: 26 June 1995/Revised: 11 October 1995     

LSU rDNA-BASED RFLP ASSAYS FOR DISCRIMINATING SPECIES AND STRAINS OF ALEXANDRIUM (DINOPHYCEAE)1

In a previous study large-subunit ribosomal RNA gene (LSU rDNA) sequences from the marine dinoflagellates Alexandrium tamarense (Lebour) Balech, A. catenella (Whedon et Kofoid) Balech, A. fundyense Balech, A. affine (Fukuyo et Inoue) Balech, A. minutum Halim, A. lusitanicum Balech, and A. andersoni Balech were compared to assess inter- and intraspecific relationships. Many cultures compared in that study contained more than one class of LSU rDNA. Sequencing pooled clones of rDNA from single cultures revealed length heterogeneities and sequence ambiguities. This complicated sequence comparisons because multiple rDNA clones from a single culture had to be sequenced individually to document the different classes of molecules present in that culture. A further complication remained as to whether or not the observed intraculture sequence variations were reliable genetic markers or were instead artifacts of the polymerase chain reaction (PCR) amplification, cloning, and/or sequencing methods employed. The goals of the present study were to test the accuracy of Alexandrium LSU rDNA sequences using restriction fragment-length polymorphism (RFLP) analysis and to devise RFLP-based assays for discriminating among representatives of that group. Computer-assisted examination of the sequences allowed us to identify a set of restriction enzymes that were predicted to reveal species, strain, and intraculture LSU rDNA heterogeneities. All groups identified by sequencing were revealed independently and repeatedly by RFLP analysis of PCR-amplified material. Five ambiguities and one length heterogeneity, each of which ascribes a unique group of Alexandrium species or strains, were confirmed by restriction digests. Observed intraculture LSU rDNA heterogeneities were not artifacts of cloning and sequencing but were instead a good representation of the spectrum of molecules amplified during PCR reactions. Intraculture LSU rDNA heterogeneities thus serve as unique genetic markers for particular strains of Alexandrium, particularly those of A. tamarense, A. catenella, and A. fundyense. However, some of these “signature heterogeneities” represented a smaller portion of PCR product than was expected given acquired sequences. Other deviations from predicted RFLP patterns included incomplete digestions and appearance of spurious products. These observations indicate that the diversity of sequences in PCR product pools were greater than that observed by cloning and sequencing. The RFLP tests described here are useful tools for characterizing Alexandrium LSU rDNA to define the evolutionary lineage of cultures and are applicable at a fraction of the time, cost, and labor required for sequencing.     

五种野生稻叶绿体DNA多态性研究

对野生稻 5个种的18个材料的叶绿体DNA(cpDNA)进行了EcoRI的RFLP分析。 结果显示,共有10种酶切模式,不同种野生稻的cpDNA的RFLP类型都不同,而且在其中一些 种内也有变化,尤以O.rufipogon的种内多态性最为显著,并主要与地理来源有关。本研究还在O.punctata的材料中发现一种以往的分析都不曾描述过的多态性模式。通过对结果的分析,探讨了不同种类野生稻的叶绿体基因组之间以及它们与核基因组之间的进化关系。 Abstract:The polymorphisms of chloroplast DNA from 18 materials of 5 wild rice species were investigated using RFLP analysis.10 restriction patterns were obtained from the analysis of these materials.Different species had different of its RFLP patterns chloroplast DNA,and the polymorphisms existed even with species,especially in O.rufipogon varieties of different geographical origins.In O.punctata a new type of rice chloroplast DNA restriction pattern was discovered which had not been reported before.According to the results obtained,the evolutionary relationships among chlorplast genomes,and between chloroplast and nuclear genomes in different wild rice are discussed.     

Expression and localization of Lewisx glycolipids and GD1a ganglioside in human glioma cells

We analysed the glycolipid composition of glioma cells (N-370 FG cells), which are derived from a culture of transformed human fetal glial cells. The neutral and acidic glycolipid fractions were isolated by column chromatography on DEAE-Sephadex and analysed by high-performance thin-layer chromatography (HPTLC). The neutral glycolipid fraction contained 1.6 µg of lipid-bound glucose/galactose per mg protein and consisted of GlcCer (11.4% of total neutral glycolipids), GalCer (21.5%), LacCer (21.4%), Gb4 (21.1%), and three unknown neutral glycolipids (23%). These unknown glycolipids were characterized as Lewis^x (fucosylneolactonorpentaosyl ceramide; Le^x), difucosylneolactonorhexaosyl ceramide (dimeric Le^x), and neolactonorhexaosyl ceramide (nLc6) by an HPTLC-overlay method for glycolipids using specific mouse anti-glycolipid antibodies against glycolipid and/or liquid-secondary ion (LSI) mass spectrometry. The ganglioside fraction contained 0.6 µg of lipid-bound sialic acid per mg protein with GD1a as the predominant ganglioside species (83% of the total gangliosides) and GM3, GM2, and GM1 as minor components. Trace amounts of sialyl-Le^x and the complex type of sialyl-Le^x derivatives were also present. Immunocytochemical studies revealed that GD1a and GalCer were primarily localized on the surface of cell bodies. Interestingly, Le^x glycolipids and sialyl-Le^x were localized not only on the cell bodies but also on short cell processes. Especially, sialyl-Le^x glycolipid was located on the tip of fine cellular processes. The unique localization of the Le^x glycolipids suggests that they may be involved in cellular differentiation and initiation of cellular growth in this cell line.     

Membrane receptors for estrogen,progesterone, and testosterone in the rat brain: Fantasy or reality

Summary 1. There are numerous circumstantial evidence supporting the concept that steroid hormones control cellular function by means other than the nuclear receptor steroid binding mechanism. It is the intent of this report to present evidence indicating that steroids bind to specific sites in neuronal membranes.2. Some of the criteria to define steroid membrane receptors using steroid-BSA conjugates that can be radioiodinated to desired specific activity have been fulfilled for each of the three sex steroids using crude synaptosomal membrane preparations (P2 fractions) from the CNS of female and male rats. Ligand binding for each of the three steroids indicate high-affinity and high-capacity sites with distinct brain selectivity and stereospecificity. For example, 17-E-6-[¹²⁵I]BSA binds hypothalamic P2 fractions (HYP-P2) with an estimatedK _d of about 3±0.7 nM (X ± SE;n=3), whereas the cerebellum P2 (CB-P2) fractions bind the ligand with aK _d of 34±7 nM and, aB _max of 3 and 42 pmol/mg protein, respectively. Estrogen and testosterone binding fit best a one-single site, while progesterone binding sites can be best represented by a two-binding site, one high-affinity (K _d=1–2 nM) and one low affinity (K _d=62 nM), in CB-P2 fractions from intact adult female rat brain. Kinetics studies for T-3-[¹²⁵I]BSA indicate that the estimatedK _d of 30±2 nM for the olfactory bulb P2 fractions (OB-P2) from male rats is in good agreement withK _d values computed from Scatchard-derived data using the LIGAND algorithm.3. 17-E-6-[¹²⁵I]BSA binding sites are stereospecific and appears to be present as early as 5 days of age in both the OB- and the CB-P2 fractions without changes during development. In contrast, P-6-[¹²⁵I]BSA binding sites are practically absent during days 5 and 12 and appear by day 22.4. Finally, membrane receptor molecules for estrogen and progesterone have been isolated and purified by affinity chromatography and characterized by PAGE and Western blot. Microsequencing of one of the membrane estrogen binding proteins indicates that the high-affinity site corresponds to the OSCP subunit of the proton ATP synthase.5. It remains to be determined if P and T also bind to this complex enzyme or if they bind to other subunits of the family of proton ATPases. Overall the data indicate that steroid hormones conjugated to BSA are important tools to study the reality of membrane steroid receptors.