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1.
Integrin adhesion molecules have important adhesion and signaling functions. They also play a central role in the pathogenesis
of many autoimmune diseases. Over the past few years we have described a T cell adoptive transfer model to investigate the
role of T cell integrin adhesion molecules in the development of autoimmunity. This report summarizes the methods we used
in establishing this murine model. By treating murine CD4+ T cells with DNA hypomethylating agents and by transfection we
were able to test thein vitro effects of integrin overexpression on T cell autoreactive proliferation, cytotoxicity, adhesion and trafficking. Furthermore,
we showed that the ability to inducein vivo autoimmunity may be unique to the integrin lymphocyte function associated antigen-1 (LFA-1).
Published: October 24, 2003 相似文献
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The cell cycle machinery consists of regulatory proteins that control the progression through the cell cycle ensuring that
DNA replication alternates with DNA segregation in mitosis to maintain cell integrity. Some of these key regulators have to
be degraded at each cell cycle to prevent cellular dysfunction. Mitotic exit requires the inactivation of cyclin dependent
kinase1 (cdk1) and it is the degradation of the cyclin subunit that inactivates the kinase. Cyclin degradation has been well
characterized and it was shown that it is ubiquitin proteasome pathway that leads to the elimination of cyclins. By now, many
other regulatory proteins were shown to be degraded by the same pathway, among them members of the aurora kinase family, degraded
many other regulatory proteins. Aurora kinases are involved in mitotic spindle formation as well as in cytokinesis. The abundance
and activity of the kinase is precisely regulated during the cell cycle. To understand how proteolysis regulates transitions
through the cell cycle we describe two assays for ubiquitination and degradation of xenopus aurora kinase A using extracts
from xenopus eggs or somatic cell lines.
Published: November 11, 2002 相似文献
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Optimization of naked DNA delivery for interferon subtype immunotherapy in cytomegalovirus infection
Type I interferon (IFN) gene therapy modulates the immune response leading to inflammatory heart disease following cytomegalovirus
(CMV) infection in a murine model of post-viral myocarditis. Efficacy of different immunisation protocols for the IFN constructs
was influenced by the dose of DNA, subtype choice, combination use, pre-medication, and timing of DNA administration. Optimal
efficacy was found with bupivacaine treatment prior to DNA inoculation of 200μgIFN DNA 14 days prior to virus challenge. Maximal antiviral and antimyocarditic effects were achieved with this vaccination schedule.
Furthermore, inoculation of synergistic IFN subtypes demonstrated enhanced efficacy when delivered either alone or with CMVgB DNA vaccination in the CMV model. Thus naked DNA delivery of IFN provides an avenue of immunotherapy for regulating herpesvirus-induced
diseases.
Published: February 17, 2003 相似文献
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Joe J. Harrison Howard Ceri Jerome Yerly Carol A. Stremick Yaoping Hu Robert Martinuzzi Raymond J. Turner 《Biological procedures online》2006,8(1):194-215
Microbes frequently live within multicellular, solid surface-attached assemblages termed biofilms. These microbial communities
have architectural features that contribute to population heterogeneity and consequently to emergent cell functions. Therefore,
three-dimensional (3D) features of biofilm structure are important for understanding the physiology and ecology of these microbial
systems. This paper details several protocols for scanning electron microscopy and confocal laser scanning microscopy (CLSM)
of biofilms grown on polystyrene pegs in the Calgary Biofilm Device (CBD). Furthermore, a procedure is described for image
processing of CLSM data stacks using amira™, a virtual reality tool, to create surface and/or volume rendered 3D visualizations
of biofilm microorganisms. The combination of microscopy with microbial cultivation in the CBD — an apparatus that was designed
for highthroughput susceptibility testing — allows for structure-function analysis of biofilms under multivariate growth and
exposure conditions. 相似文献
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Most behavioral experiments within circadian research are based on the analysis of locomotor activity. This paper introduces
scientists to chronobiology by explaining the basic terminology used within the field. Furthermore, it aims to assist in designing,
carrying out, and evaluating wheel-running experiments with rodents, particularly mice. Since light is an easily applicable
stimulus that provokes strong effects on clock phase, the paper focuses on the application of different lighting conditions. 相似文献
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Assembly of DNA into chromatin allows for the formation of a barrier that protects naked DNA from protein and chemical agents
geared to degrade or metabolize DNA. Chromatin assembly occurs whenever a length of DNA becomes exposed to the cellular elements,
whether during DNA synthesis or repair. This report describes tools to study chromatin assembly in the model systemSaccharomyces cerevisiae. Modifications to anin vitro chromatin assembly assay are described that allowed a brute force screen of temperature sensitive (ts) yeast strains in order to identify chromatin assembly defective extracts. This screen yielded mutations in genes encoding
two ubiquitin protein ligases (E3s):RSP5, and a subunit of the Anaphase Promoting Complex (APC),APC5. Additional modifications are described that allow for a rapid analysis and anin vivo characterization of yeast chromatin assembly mutants, as well as any other mutant of interest. Our analysis suggests that
thein vitro andin vivo chromatin assembly assays are responsive to different cellular signals, including cell cycle cues that involve different
molecular networks.
Published: July 3, 2003 相似文献
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Sergei V. Saveliev 《Biological procedures online》2002,4(1):70-80
The described method allows for detection of rare linear DNA fragments generated during genomic deletions. The predicted limit
of the detection is one DNA molecule per 107 or more cells. The method is based on anchor PCR and involves gel separation of the linear DNA fragment and chromosomal DNA
before amplification. The detailed chemical structure of the ends of the linear DNA can be defined with the use of additional
PCR-based protocols. The method was applied to study the short-lived linear DNA generated during programmed genomic deletions
in a ciliate. It can be useful in studies of spontaneous DNA deletions in cell culture or for tracking intracellular modifications
at the ends of transfected DNA during gene therapy trials.
Published: November 11, 2002 相似文献
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Krishnan Neeraja M. Raina Sameer Z. Pollock David D. 《Biological procedures online》2004,6(1):180-188
Substitution patterns among nucleotides are often assumed to be constant in phylogenetic analyses. Although variation in the
average rate of substitution among sites is commonly accounted for, variation in the relative rates of specific types of substitution
is not. Here, we review details of methodologies used for detecting and analyzing differences in substitution processes among
predefined groups of sites. We describe how such analyses can be performed using existing phylogenetic tools, and discuss
how new phylogenetic analysis tools we have recently developed can be used to provide more detailed and sensitive analyses,
including study of the evolution of mutation and substitution processes. As an example we consider the mitochondrial genome,
for which two types of transition deaminations (C⇒T and A⇒G) are strongly affected by single-strandedness during replication,
resulting in a strand asymmetric mutation process. Since time spent single-stranded varies along the mitochondrial genome,
their differential mutational response results in very different substitution patterns in different regions of the genome.
Published: September 2, 2004. 相似文献
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Satellite DNA sequences are known to be highly variable and to have been subjected to concerted evolution that homogenizes
member sequences within species. We have analyzed the mode of evolution of satellite DNA sequences in four fishes from the
genusDiplodus by calculating the nucleotide frequency of the sequence array and the phylogenetic distances between member sequences. Calculation
of nucleotide frequency and pairwise sequence comparison enabled us to characterize the divergence among member sequences
in this satellite DNA family. The results suggest that the evolutionary rate of satellite DNA inD. bellottii is about two-fold greater than the average of the other three fishes, and that the sequence homogenization event occurred
inD. puntazzo more recently than in the others. The procedures described here are effective to characterize mode of evolution of satellite
DNA.
Published: March 4, 2003 相似文献
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In proteins, some processes require conformational changes involving structural domain diffusion. Among these processes are
protein folding, unfolding and enzyme catalysis. During catalysis some enzymes undergo large conformational changes as they
progress through the catalytic cycle. According to Kramers theory, solvent viscosity results in friction against proteins
in solution, and this should result in decreased motion, inhibiting catalysis in motile enzymes. Solution viscosity was increased
by adding increasing concentrations of glycerol, sucrose and trehalose, resulting in a decrease in the reaction rate of the
H+-ATPase from the plasma membrane ofKluyveromyces lactis. A direct correlation was found between viscosity (η) and the inhibition of the maximum rate of catalysis (V
max). The protocol used to measure viscosity by means of a falling ball type viscometer is described, together with the determination
of enzyme kinetics and the application of Kramers’ equation to evaluate the effect of viscosity on the rate of ATP hydrolysis
by the H+-ATPase.
Published: May 1, 2003 相似文献
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An improved method for constructing and selectively silanizing double-barreled,neutral liquid-carrier,ion-selective microelectrodes 总被引:1,自引:0,他引:1
We describe an improved, efficient and reliable method for the vapour-phase silanization of multi-barreled, ion-selective
microelectrodes of which the silanized barrel(s) are to be filled with neutral liquid ion-exchanger (LIX). The technique employs
a metal manifold to exclusively and simultaneously deliver dimethyldichlorosilane to only the ion-selective barrels of several
multi-barreled microelectrodes. Compared to previously published methods the technique requires fewer procedural steps, less
handling of individual microelectrodes, improved reproducibility of silanization of the selected microelectrode barrels and
employs standard borosilicate tubing rather than the less-conventional theta-type glass. The electrodes remain stable for
up to 3 weeks after the silanization procedure. The efficacy of a double-barreled electrode containing a proton ionophore
in the ion-selective barrel is demonstrated in situ in the leaf apoplasm of pea (Pisum) and sunflower (Helianthus). Individual leaves were penetrated to depth of ∼150 μm through the abaxial surface. Microelectrode readings remained stable
after multiple impalements without the need for a stabilizing PVC matrix. 相似文献
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Activation of lymphocytes in mammals is often quantified by measuring the amount of proliferation during the expansion phase
of an immune response. Bromodeoxyuridine (BrdU) incorporation and carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution
assays are some of the techniques widely used in mammalian studies of pathogeninduced proliferation and provide a convenient
way of quantifying the cellular response. We have extended the use of these proliferation assays to the amphibian Xenopus laevis. We have developed this species as a valuable comparative model to study immunity against a wellknown amphibian pathogen,
Frog Virus 3 (FV3). Fluorescence activated cell sorting was used to assess the level of BrdU incorporation of lymphocytes
in vivo and CFSE dilution in an in vitro activation assay. Both techniques have shown that splenic lymphocytes proliferate specifically upon FV3 challenge. This indicates
that common methods for detection of proliferation upon immunologic challenge are easily applied to other vertebrate species,
as it highlights the evolutionary conservation of the proliferative nature of immune responses throughout vertebrate phyla. 相似文献
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The Histone Association Assay provides an easy approach for detecting proteins that bind chromatin in vivo. This technique
is based on a chromatin immunoprecipitation protocol using histone H3-specific antibodies to precipitate bulk chromatin from
crosslinked whole cell extracts. Proteins that co-precipitate with chromatin are subsequently detected by conventional SDS-PAGE
and Western blot analysis. Unlike techniques that separate chromatin and nonchromatin interacting proteins by centrifugation,
this method can be used to delineate whether a protein is chromatin associated regardless of its innate solubility. Moreover,
the relative amount of protein bound to DNA can be ascertained under quantitative conditions. Therefore, this technique may
be utilized for analyzing the chromatin association of proteins involved in diverse cellular processes. 相似文献
20.
Isolated epithelial cells from intestinal mucosae are a suitable object for the study of the regulation of ion transport in
the gut. This regulation possesses a great importance for human and veterinary medicine, as diarrheal diseases, which often
are caused by an inadequate activation of intestinal anion secretion, are one of the major lethal diseases of children or
young animals. The aim of this paper is to describe a method for the isolation of intact colonic crypts, e.g. for the subsequent
investigation of the regulation of anion secretion by the intracellular second messenger, Ca2+ using electrophysiological and imaging techniques.
Published: April 8, 2002 相似文献