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71.
经过硫酸铵30%~50%分级沉淀、二步柱层析可获聚丙烯酰胺凝胶电泳均一的粘质赛氏菌胞外蛋白酶制品,收率可达53%,并制备了酶的结晶,该酶以SephadexG100柱层析及SDS-PAGE测得分子量约为81000,该酶的最适pH为7.0,最适温度为45℃,Zn2+、Mn2+、Fe2+、Cu2+、Co2+等重金属离子不同程度地抑制酶活性。 相似文献
72.
Laminin and Neuropeptide Y Are Increased by Synapsin Transfection in Cultured NG108-15 Neuroblastoma/Glioma Hybrid Cells 总被引:2,自引:0,他引:2
† Gabriel Fried Hui Quan Han ‡Björn Meister ‡Tomas Hökfelt Paul Greengard 《Journal of neurochemistry》1995,64(6):2674-2680
Abstract: We have investigated the presence and expression of laminin and neuropeptide Y (NPY) in several NG108-15 cell lines transfected with synapsin Ib, IIa, or IIb. The content of laminin, a basal membrane glycoprotein that promotes adhesion and induces neurite outgrowth and neuronal differentiation, was increased in all transfected cell lines examined. In cells that were chemically differentiated with prostaglandin E1 plus 3-isobutyl-1-methylxanthine, laminin levels were increased even further. The content of NPY, suggested to be a neurotransmitter/neuromodulator in peripheral sympathetic neurons as well as in central neurons, was also increased in all transfected cell lines examined. Immunohistochemical analysis combined with confocal laser microscopy showed that NPY staining was granular and very often enriched in neuritic varicosities. The distribution and the staining pattern of NPY were consistent with storage of NPY in large dense-cored vesicles. The results indicate that, in differentiated neurons, the synapsins increase the levels of a neuropeptide transmitter stored in large dense-cored vesicles and of an extracellular matrix protein associated with neuronal maturation. 相似文献
73.
74.
E. García-Martín F. J. Martín-Romero C. Gutiérrez-Merino 《Journal of neurochemistry》1995,65(6):2757-2764
Abstract: Synaptosomes can be loaded with mag-fura-2 without significant perturbation of their ATP content by incubation for 10 min at 37°C with 10 µM mag-fura-2 acetoxymethyl ester in Hanks'-HEPES buffer (pH 7.45). The intrasynaptosomal free Mg2+ concentration ([Mg2+]i) was found to be dependent on external Mg2+ concentration, increasing from 0.8 to 1.25 mM when the concentration of Mg2+ in the incubation medium increased from 1 to 8 mM. Dissipation of the Na+ gradient across the plasma membrane of synaptosomes by treatment with the Na+ ionophore monensin (0.2 mM) or with veratridine (0.2 mM) and ouabain (0.6 mM) produced a moderate increase of [Mg2+]i, from 1.0 to 1.2–1.3 mM in an incubation medium containing 5 mM Mg2+. Plasma membrane depolarization by incubation of synaptosomes in a medium containing 68 mM KCl and 68 mM NaCl had no effect on [Mg2+]i. Reversal of the Na+ gradient by incubation of synaptosomes in a medium in which external Na+ was replaced by choline increased [Mg2+]i up to 1.6 and 2.2 mM for extrasynaptosomal Mg2+ concentrations of 1 and 8 mM, respectively. We conclude that a Na+/Mg2+ exchange operates in the plasma membrane of synaptosomes. In the presence of Mg2+ in the incubation medium, extrasynaptosomal ATP, but not ADP or adenosine, increased [Mg2+]i from 1.1 ± 0.1 up to 1.6 ± 0.1 mM. The nonhydrolyzable ATP analogue adenosine 5′-(βγ-imido)triphosphate antagonized the effect of ATP, but had no effect by itself on [Mg2+]i. It is concluded that Mg2+ transport across the plasma membrane of synaptosomes is modulated by the activity of an ecto-ATPase or an ecto-protein kinase. 相似文献
75.
William C. Wetsel 《Cellular and molecular neurobiology》1995,15(1):43-78
Summary 1. Two LHRH neuronal cell lines were developed by targeted tumorigenesis of LHRH neuronsin vivo. These cell lines (GN and GT-1 cells) represent a homogeneous population of neurons. GT-1 cells have been further subcloned to produce the GT1-1, GT1-3, and GT1-7 cell lines. While considerable information is accumulating about GT-1 cells, very little is currently known about the characteristics and responses of GN cells.2. By both morphological and biochemical criteria, GT-1 cells are clearly neurons. All GT-1 cells immunostain for LHRH and the levels of prohormone, peptide intermediates, and LHRH in the cells and medium are relatively high.3. GT-1 cells biosynthesize, process, and secrete LHRH. Processing of pro-LHRH appears to be very similar to that reported for LHRH neuronsin vivo. At least four enzymes may be involved in processing the prohormone to LHRH.4. LHRH neurons are unique among the neurons of the central nervous system because they arise from the olfactory placode and grow back into the preoptic-anterior hypothalamic region of the brain. Once these neurons reach this location, they send their axons to the median eminence. With respect to the immortalized neurons, GN cells were arrested during their transit to the brain. In contrast, GT-1 cells were able to migrate to the preoptic-anterior hypothalamic region but were unable correctly to target their axons to the median eminence. These problems in migration and targeting appear to be due to expression of the simian virus T-antigen.5. While GT-1 cells are a homogeneous population of neurons, they are amenable to coculture with other types of cells. Coculture experiments currently under way should help not only to reveal some of the molecular and cellular cues that are important for neuronal migration and axonal targeting, but they should also highlight the nature of the cellular interactions which normally occurin situ.6. GT-1 cells spontaneously secrete LHRH in a pusatile manner. The interpulse interval for LHRH from these cells is almost identical to that reported for release of LH and LHRHin vivo. GT-1 cells are interconnected by both gap junctions and synapses. The coordination and synchronization of secretion from these cells could occur through these interconnections, by feedback from LHRH itself, and/or by several different compounds that are secreted by these cells. One such compound is nitric oxide.7. GT-1 cells have Na+, K+, Ca2+, and Cl– channels. Polymerase chain reaction experiments coupled with Southern blotting and electrophysiological recordings reveal that GT-1 cells contain at least five types of Ca2+ channels. R-type Ca2+ channels appear to be the most common type of channel and this channel is activated by phorbol esters in the GT-1 cells.8. LHRH is secreted from GT-1 cells in response to norepinephrine, dopamine, histamine, GABA (GABA-A agonists), glutamate, nitric oxide, neuropeptide Y, endothelin, prostaglandin E2, and activin A. Phorbol esters are very potent stimulators of LHRH secretion. Inhibition of LHRH release occurs in response to LHRH, GABA (GABA-B agonists), prolactin, and glucocorticoids.9. Compared to secretion studies, far fewer agents have been tested for their effects on gene expression. All of the agents which have been tested so far have been found either to repress LHRH gene expression or to have no effect. The agents which have been reported to repress LHRH steady-state mRNA levels include LHRH, prolactin, glucocorticoids, nitric oxide, and phorbol esters. While forskolin stimulates LHRH secretion, it does not appear to have any effect on LHRH mRNA levels. 相似文献
76.
Joël Chopineau Stéphane Robert Laurence Fenart Roméo Cecchelli Bernard Lagoutte Stéphanie Paitier Marie-Pierre Dehouck Dominique Domurado 《Letters in Peptide Science》1997,4(4-6):313-321
Acylated proteins play a crucial role in cellphysiology because of their increased interaction withmembranes. Their isolation is difficult as aconsequence of their low cellular concentration andtheir chemical preparation is problematic due tosolubility problems. Through the use of reversedmicelles, we produced tens of milligrams of acylatedribonucleases A, chosen as a model, purified them bysemi-preparative high performance liquidchromatography (HPLC) and characterized them by analyticalHPLC, capillary electrophoresis, mass spectrometry, peptide mapping, Edman degradation and enzyme activity. We nextscrutinized the interaction with an in vitro blood–brainbarrier model and demonstrated that palmitoylated andstearoylated ribonucleases A are transported from onecompartment to the other across the cellular monolayer,in contrast to the native enzyme. 相似文献
77.
78.
Comparison of the killer toxin of several yeasts and the purification of a toxin of type K2 总被引:5,自引:0,他引:5
A total of 13 killer toxin producing strains belonging to the genera Saccharomyces, Candida and Pichia were tested against each other and against a sensitive yeast strain. Based on the activity of the toxins 4 different toxins of Saccharomyces cerevisiae, 2 different toxins of Pichia and one toxin of Candida were recognized. The culture filtrate of Pichia and Candida showed a much smaller activity than the strains of Saccharomyces. Extracellular killer toxins of 3 types of Saccharomyces were concentrated and partially purified. The pH optimum and the isoelectric point were determined. The killer toxins of S. cerevisiae strain NCYC 738, strain 399 and strain 28 were glycoproteins and had a molecular weight of Mr=16,000. The amino acid composition of the toxin type K2 of S. cerevisiae strain 399 was determined and compared with the composition of two other toxins. 相似文献
79.
Dr. Stephen R. Shaw 《Cell and tissue research》1978,188(1):35-61
Summary (1) The distribution of the extracellular space (ECS) in the outer part of the locust compound eye has been mapped with lanthanum and ruthenium red, applied to the retina. (2) In the photoreceptor zone, about 2.4% of the volume is ECS, in agreement with radiotracer and electrical estimates. Of this ECS, about 70% lies in lacunae between ommatidia, but only 1–2% adjacent to the photosensitive rhabdom. The lacunae are filled with material which binds applied tracers, and are thought to be structural spaces. (3) It has been suggested several times that such a small cation pool is insufficient to sustain more than a few large photoresponses, but this is shown to be incorrect. Enough Na+ lies within the rhabdomal ECS and within rapid diffusional access to it, to impose no immediate limitation. (4) The palisade vacuoles surrounding the rhabdom are intracellular, and are typical of light as well as dark-adapted eyes. (5) Tracers fail to penetrate more than about 30 m into the axon zone, in agreement with electrical, dye and radiotracer indications of a blood-eye barrier near this point. Septate and gap junctions between glial membranes proliferate at this level, the lacunae disappear, and the axonal clefts narrow, but no tight junctions were seen. Comparison is made with the barrier around the nerve cord. (6) The secondary pigment cells in the retina may function as osmotic/ionic buffers, in conjunction with the blood-eye barrier. 相似文献
80.
A continuous dialysis technique capable of maintaining concentrated suspensions of bloodstream Trypanosoma brucei (up to 4 × 108/ml) at 25 C for 2 hr without loss of viability has been developed in order to measure endocytosis under controlled conditions in vitro. Using this technique, the kinetics and mechanism of uptake of the metabolically inert macromolecule, 125I-labelled polyvinylpyrrolidone (125I-PVP), have been investigated. Binding to the plasma membrane and the rate of uptake of 125I-PVP from the extracellular medium by the trypanosome are both decreased by the addition of unlabelled PVP and human serum albumin. A mechanism for uptake of 125I-PVP by a combination of fluid-phase and adsorptive endocytosis from the flagellar pocket of the trypanosome is proposed. In the presence of serum albumin and unlabelled PVP, endocytosis of 125I-PVP occurs in the fluid phase only, with endocytic indices of 14.5 ± 0.9 and 54.1 ± 11.3 nl/hr/mg protein in vitro at 25 C and in vivo at 37 C, respectively. 相似文献