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21.
Developmental Changes in Polypeptide Composition of, and Precursor Incorporation into, Cellular and Subcellular Fractions of Rat Cerebral Cortex 总被引:4,自引:4,他引:0
Abstract: Neuronal-enriched and glial-enriched fractions from rat cerebral cortex at 2. 5, 9, 14 and 23 days postnatally, and subcellular fractions from 2, 14 and 46 day old rat were prepared. The polypeptide composition of all fractions was analysed by sodium dodecyl sulphate (SDS) polyacrylamide gel electro-phoresis and quantified by densitometry. Fifty-nine polypeptides (mol. wts., 13,200–251,000) were resolved in the cell fractions of which the majority remained unchanged throughout postnatal development. Three polypeptides (mol. wts., 102,000, 56,000, 53,700) were found to increase in amount devel-opmentally in both cellular fractions, the latter two showing a peak in relative amount on day 14 and a subsequent decline. Three polypeptides (mol. wts., 47,000, 28,200, 17,400) were found to be common to the glial-enriched fraction as well as the myelin fraction, and all showed a developmental increase. The neuronal-enriched fraction was found to be enriched in five polypeptides of which one (mol. wt., 51,900) showed a developmental increase after ten days postnatally, the others (mol. wts., 178,700, 142,000, 109,000, 24,000) showing a decrease. In vitro incorporation of [35 S]-methionine into the glial-enriched fraction was carried out, and a developmental decline was observed in the labelling of a polypeptide of 42,000 mol. wt. 相似文献
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Although pharmacological stimulation of a wide variety of transmitter receptors triggers phosphoinositide (PI) turnover, little is known about the type of synaptic activity required to activate this system. To investigate this question, we have used primary cultures of embryonic cortical neurons, which develop functional glutamate and GABA synapses during maturation in vitro. Mature cultures display spontaneous synaptic activity that is totally suppressed by tetrodotoxin (TTX). PI turnover, assayed by the lithium-sensitive accumulation of [3H]CDP-diacylglycerol, was readily detected under basal conditions and was abolished by TTX. Increased excitatory synaptic activity induced by picrotoxin, an antagonist of GABAA receptor-mediated inhibition, further stimulated PI turnover. Similar results were obtained when PI turnover was assayed using [3H]inositol labeling. With either assay, the magnitude of synaptically induced PI turnover was comparable to maximal responses produced by muscarinic receptor stimulation. Although a component of the spontaneous synaptic currents is sensitive to N-methyl-D-aspartate (NMDA)-preferring glutamate receptor antagonists, blockade of NMDA receptors did not affect PI turnover associated with synaptic transmission. To assess the time course of synaptically mediated PI turnover, the amplitude and duration of spontaneous synaptic currents were reduced by lowering the extracellular Ca2+ concentration from 2.25 to 0.5 mM, a maneuver that suppresses basal PI turnover. Increases in PI turnover were detected as early as 5 min following restoration of the extracellular Ca2+ concentration to 2.25 mM. Taken together, these findings indicate that activation of the PI system is associated with physiological levels of glutamatergic synaptic transmission. 相似文献
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The traditional view regarding the pathogenesis of cervical lymphadenitis in guinea pigs is that Lancefield Group C Streptococcus gains access to cervical lymph nodes via an abraded oral mucosa. In this study, it is established that inoculation of intact nasal and conjunctival mucous membranes with Streptococcus zooepidemicus (Lancefield Group C) also can produce the disease. Weanling (SPF) guinea pigs (Cavia porcellus) were divided into two experimental groups of 10 and two control groups of four each. Guinea pigs from each group were individually housed in separate cubicles. Group I was inoculated with 0.05 ml of culture containing 2.8 x 10(7) CFU/ml of S. zooepidemicus into the conjunctiva of the left eye. Group II received a similar inoculum into the left nares. Control groups received 0.05 ml of TSB broth in the same sites. Five of ten guinea pigs in Group II died four to nine days postinoculation. Surviving guinea pigs were euthanatized at intervals between days 4-13 postinoculation. All guinea pigs were necropsied, cultured and examined for evidence of infection. S. zooepidemicus was recovered from 30/50 and 39/46 sites cultured from Groups I and II, respectively. Lymphadenitis was found in cervical lymph nodes from 8/10 guinea pigs in Group I and 10/10 in Group II. The conjunctival and nasal mucosa, therefore, represent potential sites of entry resulting in cervical lymphadenitis in guinea pigs. 相似文献
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Weiduan Xu Jianmin Chen Glenn Yamasaki John E. Murphy Baisong Mei 《Molecular biotechnology》2010,45(3):248-256
Many therapeutic proteins require appropriate glycosylation for their biological activities and plasma half life. Coagulation
factor VIII (FVIII) is a glycoprotein which has extensive post-translational modification by N-linked glycosylation. The terminal
sialic acid in the N-linked glycans of FVIII is required for maximal circulatory half life. The extent of FVIII sialylation
can be determined by high pH anion-exchange chromatography coupled with a pulse electrochemical detector (HPAEC-PED), but
this requires a large amount of purified protein. Using FVIII as a model, the objective of the present study was to develop
assays that enable detection and prediction of sialylation deficiency at an early stage in the process and thus prevent downstream
product quality excursions. Lectin ECA (Erythrina Cristagalli) binds to unsialylated Galβ1-4 GlcNAc and the ECA-binding level (i.e., terminal Gal(β1-4) exposure) is inversely proportional
to the level of sialylation. By using ECA, a cell-based assay was developed to measure the global sialylation profile in FVIII
producing cells. To examine the Galβ1-4 exposure on the FVIII molecule in bioreactor tissue culture fluid (TCF), an ELISA-based
ECA-FVIII binding assay was developed. The ECA-binding specificity in both assays was assessed by ECA-specific sugar inhibitors
and neuraminidase digestion. The ECA-binding specificity was also independently confirmed by a ST3GAL4 siRNA knockdown experiment.
To establish the correlation between Galβ1-4 exposure and the HPAEC-PED determined FVIII sialylation value, the FVIII containing
bioreactor TCF and the purified FVIII samples were tested with ECA ELISA binding assay. The results indicated an inverse correlation
between ECA binding and the corresponding HPAEC-PED sialylation value. The ECA-binding assays are cost effective and can be
rapidly performed, thereby making them effective for in-process monitoring of protein sialylation. 相似文献
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Sean V. Murphy Rebecca Lim Philip Heraud Marian Cholewa Mark Le Gros Martin D. de Jonge Daryl L. Howard David Paterson Courtney McDonald Anthony Atala Graham Jenkin Euan M. Wallace 《PloS one》2012,7(9)
Cystic fibrosis, an autosomal recessive disorder caused by a mutation in a gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), remains a leading cause of childhood respiratory morbidity and mortality. The respiratory consequences of cystic fibrosis include the generation of thick, tenacious mucus that impairs lung clearance, predisposing the individual to repeated and persistent infections, progressive lung damage and shortened lifespan. Currently there is no cure for cystic fibrosis. With this in mind, we investigated the ability of human amnion epithelial cells (hAECs) to express functional CFTR. We found that hAECs formed 3-dimensional structures and expressed the CFTR gene and protein after culture in Small Airway Growth Medium (SAGM). We also observed a polarized CFTR distribution on the membrane of hAECs cultured in SAGM, similar to that observed in polarized airway cells in vivo. Further, hAECs induced to express CFTR possessed functional iodide/chloride (I−/Cl−) ion channels that were inhibited by the CFTR-inhibitor CFTR-172, indicating the presence of functional CFTR ion channels. These data suggest that hAECs may be a promising source for the development of a cellular therapy for cystic fibrosis. 相似文献