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21.
Identification of proteins secreted from axons of embryonic dorsal-root-ganglia neurons 总被引:8,自引:0,他引:8
E T Stoeckli P F Lemkin T B Kuhn M A Ruegg M Heller P Sonderegger 《European journal of biochemistry》1989,180(2):249-258
Secretion of proteins from the growth cone has been implicated in axon growth and synapse formation and might be involved in the transmission of a variety of axon-derived regulatory signals during neurogenesis. In order to identify axonally secreted proteins, dorsal-root-ganglia neurons from chicken embryos were cultured in a compartmentalized cell culture system that allows separate access to neuronal cell somas and axons. The proteins synthesized by the neurons were metabolically labeled by addition of [35S]methionine to the compartment containing the cell somas; the proteins released from the axons were harvested from the culture medium of the axonal compartment. Two-dimensional gel electrophoresis revealed two axonally secreted proteins with apparent molecular mass of 132-140 kDa and 54-60 kDa; they were termed axonin-1 and axonin-2, respectively. Both axonins were found to be secreted from a variety of neuronal cell cultures, but not from any of the nonneuronal cultures investigated, and hence might be neuron-specific. Virtual absence of these proteins from the axonal protein pattern suggests constitutive secretion. The information acquired on coordinates and spot morphology of these proteins in two-dimensional gel electrophoresis provides a useful assay for their purification. 相似文献
22.
Structural comparison of fibroblast growth factor-specific heparan sulfates derived from a growing or differentiating neuroepithelial cell line 总被引:3,自引:1,他引:2
Brickman YG; Nurcombe V; Ford MD; Gallagher JT; Bartlett PF; Turnbull JE 《Glycobiology》1998,8(5):463-471
Heparan sulfate (HS) glycosaminoglycans are essential modulators of
fibroblast growth factor (FGF) activity both in vivo and in vitro, and
appear to act by cross-linking particular forms of FGF to appropriate FGF
receptors. We have recently isolated and characterized two separate HS
pools derived from immortalized embryonic day 10 mouse neuroepithelial 2.3D
cells: one from cells in log growth phase, which greatly potentiates the
activity of FGF-2, and the other from cells undergoing contact-inhibition
and differentiation, which preferentially activates FGF-1. These two pools
of HS have very similar functional activities to those species isolated
from primary neuroepithelial cells at corresponding stages of active
proliferation or differentiation. We present here a structural comparison
between these cell line HS species to establish the nature of the changes
that occur in the biosynthesis of HS. A combination of chemical and
enzymatic cleavage, low pressure chromatography and strong anion-exchange
HPLC were used to generate full chain models of each species. Overall, the
HS pools synthesized in the dividing cell line pools possessed less complex
sulfation than those derived from more differentiated, growth arrested
cells.
相似文献
23.
Heterologous expression of an engineered truncated form of human Lewis fucosyltransferase (Fuc-TIII) by the methylotrophic yeast Pichia pastoris 总被引:1,自引:0,他引:1
Gallet PF; Vaujour H; Petit JM; Maftah A; Oulmouden A; Oriol R; Le Narvor C; Guilloton M; Julien R 《Glycobiology》1998,8(9):919-925
A stable GS115 Pichia pastoris recombinant strain was constructed to
secrete a truncated form of the human alpha(1,3/4) fucosyltransferase
(amino acids 45-361). Enzyme production resulted from a secretory pathway
based on the pre-pro- alpha mating factor signal sequence of the yeast
Saccharomyces cerevisiae . Following its transit through the Golgi
apparatus, the enzyme accumulated in the periplasmic space before its
release in the culture broth (about 30 mg/l). Cell-enclosed enzyme (
approximately 0.16%) proved to be fairly stable for many freezing and
thawing cycles and could be used several times as an immobilized catalyst.
Soluble enzyme (>99.8%) representing the main protein of the culture
broth (10%) has been characterized by Western-blotting, substrate
specificities and kinetic parameters. The two forms (cell- enclosed and
soluble) of recombinant enzyme may be used for in vitro synthesis of
Lewisadeterminants.
相似文献
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With the availability of two-dimensional (2-D) gel electrophoresis databases that have many characterized proteins, it may
be possible to compare a researcher’s gel images with those in relevant databases. This may lead to the putative identification
of unknown protein spots in a researcher’s gel with those characterized in a given database, saving the researcher time and
money by suggesting monoclonal antibodies to try in confirming these identifications. We have developed two tools to help
with this comparison: (1) Flicker, http://www.lecb.ncifcrf.gov/flicker/, a Java applet program running in the researcher’s
Web browser, to visually compare their gels against gels on the Internet; and (2) the 2DWG meta-database, http://www.lecb.ncifcrf.gov/2dwgDB/,
a searchable database of locations of 2-D electrophoretic gel images found on the Internet. Recent additions to Flicker allow
users to click on a protein spot in a gel that is linked to a federated 2D gel database, such as SWISS-2DPAGE, and have it
retrieve a report from that Web database for that protein. 相似文献
27.
In an appropriate ionic environment, the resting potential of canine cardiac purkinje fibers may have either of two value. By changing the external K concentration, [K](0), in small steps, it was shown that, in the low (1 mM) Cl, Na-containing solutions used in this study, the two levels of resting potential could be obtained only within a narrow range of [K](0) values; that range was usually found between 1 and 4 mM. Within the critical [K](0) range the resting potential could be shifted from either level to the other by the application of small current pulses. It was shown that under these conditions the steady-state current- voltage relationship was “N-shaped,” and that a region of both negative slope, and negative chord conductance lay between the two stable zero-current potentials. The negative chord conductance was largely due to inward sodium current, only part of which was sensitive to tetrodotoxin (TTX). Under appropriate conditions, the negative chord conductance could be abolished by several experimental interventions and the membrane potential thereby shifted from the lower to the higher resting level: those interventions which were effective by presumably diminishing the steady-state inward current included reducing the external sodium concentration, adding TTX, or adding lidocaine; those which presumably increased the steady-state outward current included small increases in [K](0), brief depolarizations to around -20 mV, or the addition of acetylcholine chloride. 相似文献
28.
Luís H Franco Pryscilla F Wowk Célio L Silva Ana PF Trombone Arlete AM Coelho-Castelo Constance Oliver Maria C Jamur Edson L Moretto Vânia LD Bonato 《Genetic vaccines and therapy》2008,6(1):1-11
Background
Application of plasmid DNA for immunization of food-producing animals established new standards of food safety. The addition of foreign products e.g. pDNA into the food chain should be carefully examined to ensure that neither livestock animals nor consumers develop unpredicted or undesirable side-effects.Methods
A quantitative real-time PCR (QRTPCR) methodology was developed to study the biodistribution and persistence of plasmid DNA vaccine pDNAX (pVAX-Hsp60 TM814) in mice and beef cattle. The linear quantification range and the sensitivity of the method was found to be 10 – 109 copies per reaction (500 ng/gDNA) and 3 copies per reaction, respectively.Results
Persistence of pDNAX in mice muscle tissue was restricted to injection site and the amount of pDNAX showed delivery formulation dependent (naked pDNA, electroporation, cationic liposome complexes) and mouse age-dependent clearance form injection site but pDNAX was still detectable even after 365 days. The QRTPCR analysis of various muscle tissue samples of vaccinated beef bulls performed 242–292 days after the last revaccination proved that residual pDNAX was found only in the injection site. The highest plasmid levels (up to 290 copies per reaction) were detected in the pDNAX:CDAN/DOPE group similarly to mice model. No pDNA was detected in the samples from distant muscles and draining lymph nodes.Conclusion
Quantitative real-time PCR (QRTPCR) assay was developed to assess the residual pDNA vaccine pVAX-Hsp60 TM814 in mice and beef cattle. In beef cattle, ultra low residual level of pDNA vaccine was only found at the injection site. According to rough estimation, consumption of muscles from the injection site represents almost an undetectable intake of pDNA (400 fg/g muscle tissue) for consumers. Residual plasmid in native state will hardly be found at measurable level following further meat processing. This study brings supportive data for animal and food safety and hence for further approval of pDNA vaccine field trials. 相似文献29.
The microarray explorer tool for data mining of cDNA microarrays: application for the mammary gland 总被引:6,自引:0,他引:6 下载免费PDF全文
The Microarray Explorer (MAExplorer) is a versatile Java-based data mining bioinformatic tool for analyzing quantitative cDNA expression profiles across multiple microarray platforms and DNA labeling systems. It may be run as either a stand-alone application or as a Web browser applet over the Internet. With this program it is possible to (i) analyze the expression of individual genes, (ii) analyze the expression of gene families and clusters, (iii) compare expression patterns and (iv) directly access other genomic databases for clones of interest. Data may be downloaded as required from a Web server or in the case of the stand-alone version, reside on the user’s computer. Analyses are performed in real-time and may be viewed and directly manipulated in images, reports, scatter plots, histograms, expression profile plots and cluster analyses plots. A key feature is the clone data filter for constraining a working set of clones to those passing a variety of user-specified logical and statistical tests. Reports may be generated with hypertext Web access to UniGene, GenBank and other Internet databases for sets of clones found to be of interest. Users may save their explorations on the Web server or local computer and later recall or share them with other scientists in this groupware Web environment. The emphasis on direct manipulation of clones and sets of clones in graphics and tables provides a high level of interaction with the data, making it easier for investigators to test ideas when looking for patterns. We have used the MAExplorer to profile gene expression patterns of 1500 duplicated genes isolated from mouse mammary tissue. We have identified genes that are preferentially expressed during pregnancy and during lactation. One gene we identified, carbonic anhydrase III, is highly expressed in mammary tissue from virgin and pregnant mice and in gene knock-out mice with underdeveloped mammary epithelium. Other genes, which include those encoding milk proteins, are preferentially expressed during lactation. MAExplorer may be accessed at http://www.lecb.ncifcrf.gov.MAExplorer. 相似文献
30.
The use of an algorithm with a broad range of utility in digital image processing (the positive difference transform) is illustrated. Its effectiveness as a procedure for selective elimination of hemoglobin-containing images is demonstrated. A set of heuristics, employing information concerning nuclear hemoglobin content, is shown to discriminate nucleated erythrocytic cells from those of the leukocyte series. 相似文献