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101.
Analysis of microRNA expression by in situ hybridization with RNA oligonucleotide probes 总被引:1,自引:0,他引:1
In situ hybridization is an important tool for analyzing gene expression and developing hypotheses about gene functions. The discovery of hundreds of microRNA (miRNA) genes in animals has provided new challenges for analyzing gene expression and functions. The small size of the mature miRNAs ( approximately 20-24 nucleotides in length) presents difficulties for conventional in situ hybridization methods. However, we have described a modified in situ hybridization method for detection of mammalian miRNAs in tissue sections, based upon the use of RNA oligonucleotide probes in combination with highly specific wash conditions. Here, we present detailed procedures for detection of miRNAs in tissue sections or cultured cells. The methods described can utilize either nonradioactive hapten-conjugated probes that are detected by enzyme-coupled antibodies, or radioactively labeled probes that are detected by autoradiography. The ability to visualize miRNA expression patterns in tissue sections provides an additional tool for the analyses of miRNA expression and function. In addition, the use of radioactively labeled probes should facilitate quantitative analyses of changes in miRNA gene expression. 相似文献
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Deo R. Singh Fozia Ahmed Christopher King Nisha Gupta Matt Salotto Elena B. Pasquale Kalina Hristova 《The Journal of biological chemistry》2015,290(45):27271-27279
The EphA2 receptor tyrosine kinase promotes cell migration and cancer malignancy through a ligand- and kinase-independent distinctive mechanism that has been linked to high Ser-897 phosphorylation and low tyrosine phosphorylation. Here, we demonstrate that EphA2 forms dimers in the plasma membrane of HEK293T cells in the absence of ephrin ligand binding, suggesting that the current seeding mechanism model of EphA2 activation is incomplete. We also characterize a dimerization-deficient EphA2 mutant that shows enhanced ability to promote cell migration, concomitant with increased Ser-897 phosphorylation and decreased tyrosine phosphorylation compared with EphA2 wild type. Our data reveal a correlation between unliganded dimerization and tumorigenic signaling and suggest that EphA2 pro-tumorigenic activity is mediated by the EphA2 monomer. Thus, a therapeutic strategy that aims at the stabilization of EphA2 dimers may be beneficial for the treatment of cancers linked to EphA2 overexpression. 相似文献
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106.
Meenakshi Ramaswamy Vinitha Unnikrishnan Suresh Kumar Kizhakkethil Aishwarya Mamiyil Sabu George Thomas 《Acta Botanica Sinica》2014,(8)
We evaluated nine plastid(matK, rbcL, rpoC1, rpoB,rpl36-rps8, ndhJ, trnL-F, trnH-psbA, accD) and two nuclear(ITS and ITS2) barcode loci in family Zingiberaceae by analyzing 60 accessions of 20 species belonging to seven genera from India.Bidirectional sequences were recovered for every plastid locus by direct sequencing of polymerase chain reaction(PCR)amplicons in all the accessions tested. However, only 35(58%)and 40 accessions(66%) yielded ITS and ITS2 sequences,respectively, by direct sequencing. In different bioinformatics analyses, matK and rbcL consistently resolved 15 species(75%)into monophyletic groups and five species into two paraphyletic groups. The 173 ITS sequences, including 138 cloned sequences from 23 accessions, discriminated only 12 species(60%), and the remaining species were entered into three paraphyletic groups. Phylogenetic and genealogic analyses of plastid and ITS sequences imply the possible occurrence of natural hybridizations in the evolutionary past in giving rise to species paraphyly and intragenomic ITS heterogeneity in the species tested. The results support using matK and rbcL loci for barcoding Zingiberaceae members and highlight the poor utility of ITS and the highly regarded ITS2 in barcoding this family, and also caution against proposing ITS loci for barcoding taxa based on limited sampling. 相似文献
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Mj-AMP2, a knottin-type antimicrobial peptide, in vitro inhibits the growth of several plant pathogenic fungi including Magnaporthe oryzae. We demonstrate that transgenic rice (Oryza sativa L.) plants expressing the Mj-AMP2 gene show enhanced resistance to M. grisea, the causal agent of the rice blast disease. Mj-AMP2 was efficiently expressed and the level of Mj-AMP2 ranged from 0.32% to 0.38% of the total protein in the transgenic rice plants. In vitro inhibitory activity assays with the crude protein extract from transgenic rice indicated that the Mj-AMP2 protein produced was biologically active. Constitutive expression of Mj-AMP2 in transgenic rice reduces the growth of M. grisea by 63% with respect to untransformed control plant, and no effect on plant phenotype was observed. Transgene expression of Mj-AMP2 gene was not accompanied by an induction of pathogenesis-related (PR) gene expression indicating that the transgene product itself is directly active against the pathogen. The results presented in this study suggest that the Mj-AMP2 gene could be a useful candidate for protection of rice plants against the rice blast fungus M. grisea. 相似文献
109.
Spector AA Deo N Grosh K Ratnanather JT Raphael RM 《The Journal of membrane biology》2006,209(2-3):135-152
The outer hair cell (OHC) is an extremely specialized cell and its proper functioning is essential for normal mammalian hearing.
This article reviews recent developments in theoretical modeling that have increased our knowledge of the operation of this
fascinating cell. The earliest models aimed at capturing experimental observations on voltage-induced cellular length changes
and capacitance were based on isotropic elasticity and a two-state Boltzmann function. Recent advances in modeling based on
the thermodynamics of orthotropic electroelastic materials better capture the cell’s voltage-dependent stiffness, capacitance,
interaction with its environment and ability to generate force at high frequencies. While complete models are crucial, simpler
continuum models can be derived that retain fidelity over small changes in transmembrane voltage and strains occurring in
vivo. By its function in the cochlea, the OHC behaves like a piezoelectric-like actuator, and the main cellular features can
be described by piezoelectric models. However, a finer characterization of the cell’s composite wall requires understanding
the local mechanical and electrical fields. One of the key questions is the relative contribution of the in-plane and bending
modes of electromechanical strains and forces (moments). The latter mode is associated with the flexoelectric effect in curved
membranes. New data, including a novel experiment with tethers pulled from the cell membrane, can help in estimating the role
of different modes of electromechanical coupling. Despite considerable progress, many problems still confound modelers. Thus,
this article will conclude with a discussion of unanswered questions and highlight directions for future research. 相似文献
110.
Vigmond EJ Weber dos Santos R Prassl AJ Deo M Plank G 《Progress in biophysics and molecular biology》2008,96(1-3):3-18
The bidomain equations are widely used for the simulation of electrical activity in cardiac tissue. They are especially important for accurately modeling extracellular stimulation, as evidenced by their prediction of virtual electrode polarization before experimental verification. However, solution of the equations is computationally expensive due to the fine spatial and temporal discretization needed. This limits the size and duration of the problem which can be modeled. Regardless of the specific form into which they are cast, the computational bottleneck becomes the repeated solution of a large, linear system. The purpose of this review is to give an overview of the equations and the methods by which they have been solved. Of particular note are recent developments in multigrid methods, which have proven to be the most efficient. 相似文献