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11.
AA Smith 《Biotechnic & histochemistry》2014,89(3):215-219
One can find the optimal antibody dilution for immunostaining by repeated staining on the same tissue section by using a less dilute antibody for each attempt. Using secondary antibody and horseradish peroxidase conjugated to a dextran polymer, a section stained repeatedly with several dilutions of antibody appears as good as a section stained with only the last dilution. 相似文献
12.
AA Smith 《Biotechnic & histochemistry》2016,91(6):396-400
One can determine the best dilution of a primary antibody for immunohistochemistry that uses horseradish peroxidase conjugated to a secondary antibody by testing increasing concentrations sequentially on the same tissue section. When the same tissue section is incubated repeatedly with increasing concentrations of primary antibodies to epithelial membrane antigen, smooth muscle α-actin, or vimentin using alkaline phosphatase conjugated to a secondary antibody as the reporter, the best staining was obtained with a less concentrated primary antibody than was optimal for a single staining test. The best concentration of primary antibody for single run staining using an alkaline phosphatase reporting system is usually four times the best concentration for staining with multiple runs. The optimal concentration can be determined by denaturing the residual alkaline phosphatase and extracting residual stain by incubating the section in 4:1 diglyme:phosphate buffered saline for 20 min at 80o C between tests of primary antibody concentrations. I tested the method for four chromogens from one supplier and one chromogen from a different supplier. 相似文献
13.
Enhancement of human immunodeficiency virus type 1 envelope-mediated fusion by a CD4-gp120 complex-specific monoclonal antibody. 下载免费PDF全文
S Lee K Peden D S Dimitrov C C Broder J Manischewitz G Denisova J M Gershoni H Golding 《Journal of virology》1997,71(8):6037-6043
The entry of human immunodeficiency virus type 1 (HIV-1) into cells is initiated by binding of the viral glycoprotein gp120-gp41 to its cellular receptor CD4. The gp120-CD4 complex formed at the cell surface undergoes conformational changes that may allow its association with an additional membrane component(s) and the eventual formation of the fusion complex. These conformational rearrangements are accompanied by immunological changes manifested by altered reactivity with monoclonal antibodies specific for the individual components and presentation of new epitopes unique to the postbinding complex. In order to analyze the structure and function of the gp120-CD4 complex, monoclonal antibodies were generated from splenocytes of BALB/c mice immunized with soluble CD4-gp120 (IIIB) molecules (J. M. Gershoni, G. Denisova, D. Raviv, N. I. Smorodinsky, and D. Buyaner, FASEB J. 7:1185-1187 1993). One of those monoclonal antibodies, CG10, was found to be strictly complex specific. Here we demonstrate that this monoclonal antibody can significantly enhance the fusion of CD4+ cells with effector cells expressing multiple HIV-1 envelopes. Both T-cell-line-tropic and macrophage-tropic envelope-mediated cell fusion were enhanced, albeit at different optimal doses. Furthermore, infection of HeLa CD4+ (MAGI) cells by HIV-1 LAI, ELI1, and ELI2 strains was increased two- to fourfold in the presence of CG10 monoclonal antibodies, suggesting an effect on viral entry. These findings indicate the existence of a novel, conserved CD4-gp120 intermediate structure that plays an important role in HIV-1 cell fusion. 相似文献
14.
15.
Photosynthetic enhancement studies performed at 619 nm (excitation of Systems I and II) and at 446 nm (mainly excitation of System I) revealed an 18% photosynthetic enhancement simultaneously with a 31% reduction in glycolate excretion. This observation supports the hypothesis that some glycolate may be consumed in an oxidation process associated with System I when System II is poorly excited and the supply of electrons from the water splitting process of photosynthesis is low. 相似文献
16.
Gloria Salazar Stephanie Zlatic Branch Craige Andrew A. Peden Jan Pohl Victor Faundez 《The Journal of biological chemistry》2009,284(3):1790-1802
The Hermansky-Pudlak syndrome is a disorder affecting endosome sorting.
Disease is triggered by defects in any of 15 mouse gene products, which are
part of five distinct cytosolic molecular complexes: AP-3, homotypic fusion
and vacuole protein sorting, and BLOC-1, -2, and -3. To identify molecular
associations of these complexes, we used in vivo cross-linking
followed by purification of cross-linked AP-3 complexes and mass spectrometric
identification of associated proteins. AP-3 was co-isolated with BLOC-1,
BLOC-2, and homotypic fusion and vacuole protein sorting complex subunits;
clathrin; and phosphatidylinositol-4-kinase type II α (PI4KIIα).
We previously reported that this membrane-anchored enzyme is a regulator of
AP-3 recruitment to membranes and a cargo of AP-3 (Craige, B.,
Salazar, G., and Faundez, V. (2008) Mol. Biol.
Cell
19,1415
-1426). Using cells deficient
in different Hermansky-Pudlak syndrome complexes, we identified that BLOC-1,
but not BLOC-2 or BLOC-3, deficiencies affect PI4KIIα inclusion into
AP-3 complexes. BLOC-1, PI4KIIα, and AP-3 belong to a tripartite
complex, and down-regulation of either PI4KIIα, BLOC-1, or AP-3
complexes led to similar LAMP1 phenotypes. Our analysis indicates that BLOC-1
complex modulates the association of PI4KIIα with AP-3. These results
suggest that AP-3 and BLOC-1 act, either in concert or sequentially, to
specify sorting of PI4KIIα along the endocytic route.Membranous organelles along the exocytic and endocytic pathways are each
defined by unique lipid and protein composition. Vesicle carriers communicate
and maintain the composition of these organelles
(2). Consequently defining the
machineries that specify vesicle formation, composition, and delivery are
central to understanding membrane protein traffic. Generally vesicle
biogenesis uses multiprotein cytosolic machineries to select membrane
components for inclusion in nascent vesicles
(2,
3). Heterotetrameric adaptor
complexes (AP-1 to AP-4) are critical to generate vesicles of specific
composition from the different organelles constituting the exocytic and
endocytic routes
(2-4).The best understood vesicle formation machinery in mammalian cells is the
one organized around the adaptor complex AP-2
(5). This complex generates
vesicles from the plasma membrane using clathrin. Our present detailed
understanding of AP-2 vesicle biogenesis mechanisms and interactions emerged
from a combination of organellar and in vitro binding proteomics
analyses together with the study of binary interactions in cell-free systems
(5-9).
In contrast, the vesicle biogenesis pathways controlled by AP-3 are far less
understood. AP-3 functions to produce vesicles that traffic selected membrane
proteins from endosomes to lysosomes, lysosome-related organelles, or synaptic
vesicles
(10-13).
AP-3 is one of the protein complexes affected in the Hermansky-Pudlak syndrome
(HPS;3 Online
Mendelian Inheritance in Man (OMIM) 203300). So far, mutations in any of 15
mouse or eight human genes trigger a common syndrome. This syndrome
encompasses defects that include pigment dilution, platelet dysfunction,
pulmonary fibrosis, and occasionally neurological phenotypes
(14,
15). All forms of HPS show
defective vesicular biogenesis or trafficking that affects lysosomes,
lysosome-related organelles (for example melanosomes and platelet dense
granules), and, in some of them, synaptic vesicles
(11-13).
Most of the 15 HPS loci encode polypeptides that assemble into five distinct
molecular complexes: the adaptor complex AP-3, HOPS, and the BLOC complexes 1,
2, and 3 (14). Recently binary
interactions between AP-3 and BLOC-1 or BLOC-1 and BLOC-2 suggested that
arrangements of these complexes could regulate membrane protein targeting
(16). Despite the abundance of
genetic deficiencies leading to HPS and genetic evidence that HPS complexes
may act on the same pathway in defined cell types
(17), we have only a partial
picture of protein interactions organizing these complexes and how they might
control membrane protein targeting.In this study, we took advantage of cell-permeant and reversible
cross-linking of HPS complexes followed by their immunoaffinity purification
to identify novel molecular interactions. Cross-linked AP-3 co-purified with
BLOC-1, BLOC-2, HOPS, clathrin, and the membrane protein PI4KIIα. We
previously identified PI4KIIα as a cargo and regulator of AP-3
recruitment to endosomes (1,
18). Using mutant cells
deficient in either individual HPS complexes or a combination of them, we
found that BLOC-1 facilitates the interaction of AP-3 and PI4KIIα. Our
studies demonstrate that subunits of four of the five HPS complexes co-isolate
with AP-3. Moreover BLOC-1, PI4KIIα, and AP-3 form a tripartite complex
as demonstrated by sequential co-immunoprecipitations as well as by similar
LAMP1 distribution phenotypes induced by down-regulation of components of this
tripartite complex. Our findings indicate that BLOC-1 complex modulates the
recognition of PI4KIIα by AP-3. These data suggest that AP-3, either in
concert or sequentially with BLOC-1, participates in the sorting of common
membrane proteins along the endocytic route. 相似文献
17.
Hartmann B Castelo R Miñana B Peden E Blanchette M Rio DC Singh R Valcárcel J 《RNA (New York, N.Y.)》2011,17(3):453-468
In Drosophila melanogaster, female-specific expression of Sex-lethal (SXL) and Transformer (TRA) proteins controls sex-specific alternative splicing and/or translation of a handful of regulatory genes responsible for sexual differentiation and behavior. Recent findings in 2009 by Telonis-Scott et al. document widespread sex-biased alternative splicing in fruitflies, including instances of tissue-restricted sex-specific splicing. Here we report results arguing that some of these novel sex-specific splicing events are regulated by mechanisms distinct from those established by female-specific expression of SXL and TRA. Bioinformatic analysis of SXL/TRA binding sites, experimental analysis of sex-specific splicing in S2 and Kc cells lines and of the effects of SXL knockdown in Kc cells indicate that SXL-dependent and SXL-independent regulatory mechanisms coexist within the same cell. Additional determinants of sex-specific splicing can be provided by sex-specific differences in the expression of RNA binding proteins, including Hrp40/Squid. We report that sex-specific alternative splicing of the gene hrp40/squid leads to sex-specific differences in the levels of this hnRNP protein. The significant overlap between sex-regulated alternative splicing changes and those induced by knockdown of hrp40/squid and the presence of related sequence motifs enriched near subsets of Hrp40/Squid-regulated and sex-regulated splice sites indicate that this protein contributes to sex-specific splicing regulation. A significant fraction of sex-specific splicing differences are absent in germline-less tudor mutant flies. Intriguingly, these include alternative splicing events that are differentially spliced in tissues distant from the germline. Collectively, our results reveal that distinct genetic programs control widespread sex-specific splicing in Drosophila melanogaster. 相似文献
18.
土壤中棉花黄萎病菌SYBR Green Ⅰ荧光RT-PCR定量检测技术研究 总被引:3,自引:0,他引:3
为实现田间土壤棉花黄萎病菌的早期检测,建立了土壤中棉花黄萎病菌的SYBR Green I荧光定量PCR检测方法。以含342bp PCR扩增产物的阳性质粒为参考,构建了标准曲线,并对该曲线的特异性、敏感性、可重复性进行了评价。结果表明,该方法具有快速、特异性强、敏感度高等特点。检测范围在3.8×103-3.8×108copies/μL之间有良好的线性关系,相关系数R2为0.996,扩增效率为101.5%,灵敏度比常规PCR方法高102倍。 相似文献
19.
Molecular cloning and phylogenetic analysis of the small cytoplasmic RNA from Listeria monocytogenes
A molecular cloning strategy has been designed to isolate the gene that encodes the small cytoplasmic RNA (scRNA) component of bacterial signal recognition particles. Using this strategy a putative Listeria monocytogenes scRNA lambda gt11 recombinant clone was isolated. A previously described complementation assay developed to genetically select functional homologues of 4.5S RNA and scRNA of bacteria confirmed that the lambda gt11 recombinant clone isolated encoded for the scRNA from L. monocytogenes. A secondary structure for this scRNA is proposed and a phylogenetic comparison of the 276 base L. monocytogenes scRNA with previously characterised Gram-positive bacterial scRNAs is also presented. 相似文献
20.
Baillie GS Huston E Scotland G Hodgkin M Gall I Peden AH MacKenzie C Houslay ES Currie R Pettitt TR Walmsley AR Wakelam MJ Warwicker J Houslay MD 《The Journal of biological chemistry》2002,277(31):28298-28309
Here we identify an 11-residue helical module in the unique N-terminal region of the cyclic AMP-specific phosphodiesterase PDE4A1 that determines association with phospholipid bilayers and shows a profound selectivity for interaction with phosphatidic acid (PA). This module contains a core bilayer insertion unit that is formed by two tryptophan residues, Trp(19) and Trp(20), whose orientation is optimized for bilayer insertion by the Leu(16):Val(17) pairing. Ca(2+), at submicromolar levels, interacts with Asp(21) in this module and serves to gate bilayer insertion, which is completed within 10 ms. Selectivity for interaction with PA is suggested to be achieved primarily through the formation of a charge network of the form (Asp(21-):Ca(2+):PA(2-):Lys(24+)) with overall neutrality at the bilayer surface. This novel phospholipid-binding domain, which we call TAPAS-1 (tryptophan anchoring phosphatidic acid selective-binding domain 1), is here identified as being responsible for membrane association of the PDE4A1 cAMP-specific phosphodiesterase. TAPAS-1 may not only serve as a paradigm for other PA-binding domains but also aid in detecting related phospholipid-binding domains and in generating simple chimeras for conferring membrane association and intracellular targeting on defined proteins. 相似文献