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761.
Immunoblotting is a commonly used technique for the immunodetection of specific proteins which have been fractionated by polyacrylamide gel electrophoresis. We describe here a simple procedure for the double staining of immunoblots, first to detect the immunoreactive component(s) by histochemistry using enzyme-conjugated secondary antibodies, and second to visualize the general protein electrophoretogram using India ink. This procedure permits the direct comparison of electrophoretic mobilities between the immunoreactive protein(s) and the total protein population as well as protein standards of known Mr. The experimental advantage of the procedure is that no additional manipulation of the protein samples or the standards is necessary prior to electrophoretic fractionation. In this report, detection of the vitamin D-dependent calcium-binding protein, calbindin-D28K, is used to illustrate the application of the procedure. 相似文献
762.
E R Hall G L Townsend W M Tuan D L Venton 《Prostaglandins, leukotrienes, and essential fatty acids》1988,32(2):51-56
Thromboxane synthase has been immobilized on phenyl-Sepharose beads by adsorption. The immobilized enzyme is catalytically active and has a slightly lower apparent Km for PGH2 than the detergent-solubilized enzyme. However, both imidazole- and pyridine-based inhibitors are equally effective in inhibiting the immobilized and solubilized enzyme preparations. Although the immobilized enzyme appears to be less stable than the solubilized enzyme it is sufficiently stable to be used as a model for studying the properties of the enzyme. 相似文献
763.
Quantitation of mRNA content in samples of total cellular RNA is required for the analysis of Northern blot hybridization
to estimate the relative level of specific gene expression. Commonly used methods based on UV absorbance and dye staining
measure only total RNA, and mRNA normalization by probing for mRNA levels of housekeeping genes, such as β-actin and glyceraldehyde-3-phosphate
dehydrogenase, assumes a constant level of their expression, which, in fact, may vary as a function of cell proliferation
and differentiation. We describe here a nonradioactive, slot-blotting method for quantifying eukaryotic mRNA levels using
a biotinylated oligo(dT) probe, which hybridizes directly to the 3′-polyadenylated sequence of eukaryotic mRNAs. The method
provides a more accurate estimation of mRNA content in total RNA samples and should be applicable for quantitative Northern
analysis. 相似文献
764.
We have previously shown that cardiovascular anomalies, such as hypertension and tachycardia, develop in Ca(2+)-deficient, shell-less (SL) chick embryos cultured ex ovo, accompanied by elevated circulating catecholamines and higher alpha-adrenergic sensitivity of cardiovascular functions. Results described in the preceding work, using erythrocytes as an experimental system, show that cellular Ca2+ handling properties are also altered as a result of long-term calcium deficiency. To examine the relevance of these findings to cells of the cardiovasculature, we have analyzed and compared the Ca2+ handling characteristics of the heart cells of SL and normal (NL) embryos. For this study, isolated and cultured ventricular myocytes of SL and NL embryos were loaded with Fura-2 via transient membrane damage with glass beads. Compared to Fura-2/AM, bead loading yielded similar values and kinetic profiles of [Ca2+]i-dependent differential fluorescence and, in addition, did not affect cell viability and beating activity. The Fura-2 loaded ventricular myocytes were washed in Ca(2+)-free buffer and then analyzed by ratiometric fluorescence (350 nm/380 nm) microscopy for kinetic changes in [Ca2+]i (R350/380 values) as a function of [Ca2+]o and adrenergic modifiers. At 0.5 and 1.0 mM [Ca2+]o, SL cells showed significantly higher [Ca2+]i, higher beating rates, and faster rate of increase in [Ca2+]i compared to NL cells. At higher [Ca2+]o (3.5 mM), there was no significant difference in [Ca2+]i and beating rate between NL and SL cells. Treatment with norepinephrine (NE; 0.01-1 microM) at 1 mM [Ca2+]o substantially increased [Ca2+]i in both NL and SL cells. In the former, the NE effect was completely inhibited by beta-blockade (1 microM propranolol). In contrast, in SL cells, NE remained effective after beta-blockade, and combined alpha-blockade (1 microM prazosin) and beta-blockade was needed to inhibit completely the NE effect. In both NL and SL cells, treatment with NE substantially increased beating rates in a similar manner. Taken together, these findings suggest that Ca2+ handling and adrenergic regulation of the heart cells are significantly altered in the SL embryos, and that these alterations may be related to the development of impaired cardiovascular functions resulting from systemic Ca2+ deficiency. 相似文献
765.
Dong Tu Nguyen Tuan Cuong Ngo Huy Hoang Tran Thi Phuong Lan Nguyen Binh Minh Nguyen Kouichi Morita & Masahiko Ehara 《FEMS microbiology letters》2009,298(1):37-43
Autoagglutinable strains of Vibrio cholerae O1 (seven nonfimbriate strains and one fimbriate strain) were transformed to obtain resistance to ampicillin. Two distinct mechanisms were found in these strains. One was operating in nonfimbriate strains by reducing OmpU protein production and the other was operating in a fimbriate strain (Bgd17) by newly overproducing cpxP protein. The twitching motility in the fimbriate Bgd17 strain disappeared depending on the production of cpxP protein, suggesting that fimbriation of V. cholerae O1 is controlled by a two-component signal transduction system. 相似文献
766.
Cunningham Sonia A.; Tran Tuan M.; Arrate M. Pia; Bjercke Robert; Brock Tommy A. 《American journal of physiology. Cell physiology》1999,276(1):C176
We have prepared apolyclonal mouse antibody directed against the first threeimmunoglobulin-like domains of the kinase insert domain-containingreceptor (KDR) tyrosine kinase. It possesses the ability to inhibitbinding of the 165-amino acid splice variant of vascular endothelialcell growth factor (VEGF165) torecombinant KDR in vitro as well as to reduceVEGF165 binding to human umbilical vein endothelial cells (HUVEC). These results confirm that the firstthree immunoglobulin-like domains of KDR are involved in VEGF165 interactions. The anti-KDRantibody is able to completely blockVEGF165-mediated intracellularCa2+ mobilization in HUVEC.Therefore, it appears that binding of VEGF165 to the fms-like tyrosinekinase (Flt-1) in these cells does not translate into aCa2+ response. This is furtherexemplified by the lack of response to placental growth factor (PlGF),an Flt-1-specific ligand. Additionally, PlGF is unable to potentiatethe effects of submaximal concentrations ofVEGF165. Surprisingly, theVEGF-PlGF heterodimer was also very inefficient at eliciting aCa2+ signaling event in HUVEC. Weconclude that KDR activation is crucial for mobilization ofintracellular Ca2+ in HUVEC inresponse to VEGF165. 相似文献
767.
The response to light of one ommatidium is reduced or suppressed by simultaneous illumination of neighboring ommatidia. The mechanism of this lateral inhibition may be chemical synaptic transmission, based on the physiological findings of a number of investigators and on the following evidence. The fine structure of the neuropil of the lateral plexus exhibits numerous clear vesicles (ca. 400 A), dense-core vesicles (ca. 700–1400 A), Golgi regions, and other morphological features of neurochemical synapses. The indolealkylamine, serotonin (5-HT), even in nanomolar concentrations, has a potent inhibitory action. An initial, potent inhibitory dose of 5-HT produces a long lasting densensitization to subsequent doses. The desensitization affects lateral inhibition evoked by light stimulation of neighboring receptors, i.e. crossed-desensitization. Eye tissue extracts contain 5-HT and melatonin (MLT) at a level greater than 1 µg/g wet tissue and perhaps as high as 20–30 µg/g, as determined by two-dimensional thin-layer chromatography (TLC) and o-phthaldialdehyde fluorescence assay techniques. Subcellular fractionation on sucrose gradient indicates a peak in 5-HT and MLT content associated with an intermediate density fraction. 5-HT may be an inhibitory transmitter for lateral inhibition. One pathway for metabolism of 5-HT in the lateral eye may be via N-acetylserotonin and melatonin. 相似文献