共查询到20条相似文献,搜索用时 15 毫秒
1.
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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In platelets, PGHS-1-dependant formation of thromboxane A2 is an important modulator of platelet function and a target for pharmacological inhibition of platelet function by aspirin.
Since platelets are anucleated cells, we have used the immortalized human megakaryoblastic cell line MEG-01, which can be
induced to differentiate into platelet-like structures upon addition of TPA as a model system to study PGHS-1 gene expression.
Using a specific antibody to PGHS-1 we have developed a technique using immunofluorescence microscopy and analysis of multiple
digital images to monitor PGHS-1 protein expression as MEG-01 cells were induced to differentiate by a single addition of
TPA (1.6 × 10−8 M) over a period of 8 days. The method represents a rapid and economical alternative to flow cytometry. Using this technique
we observed that TPA induced adherence of MEG-01 cells, and only the non-adherent TPA-stimulated cells demonstrated compromised
viability. The differentiation of MEG-01 cells was evaluated by the expression of the platelet-specific cell surface antigen,
CD-41. The latter was expressed in MEG-01 cells at the later stages of differentiation. We demonstrated a good correlation
between PGHS-1 expression and the overall level of cellular differentiation of MEG-01 cells. Furthermore, PGHS-1 protein expression,
which shows a consistent increase over the entire course of differentiation can be used as an additional and better index
by which to monitor megakaryocyte differentiation.
Published: December 12, 2001 相似文献
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Carine Bécamel Nathalie Galéotti Joël Poncet Patrick Jouin Aline Dumuis Joël Bockaert Philippe Marin 《Biological procedures online》2002,4(1):94-104
There is accumulating evidence that membrane-bound receptors interact with many intracellular proteins. Multiprotein complexes
associated with ionotropic receptors have been extensively characterized, but the identification of proteins interacting with
G protein-coupled receptors (GPCRs) has so far only been achieved in a piecemeal fashion, focusing on one or two protein species.
We describe a method based on peptide affinity chromatography, two-dimensional electrophoresis, mass spectrometry and immunoblotting
to identify the components of multiprotein complexes interacting directly or indirectly with intracellular domains of GPCRs
or, more generally, any other membrane-bound receptor. Using this global approach, we have characterized multiprotein complexes
that bind to the carboxy-terminal tail of the 5-hydroxytryptamine type 2C receptor and are important for its subcellular localization
in CNS cells (Bécamel et al., EMBO J., 21(10): 2332, 2002).
Published: December 9, 2002 相似文献
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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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In synaptic vesicles, the estimated concentration of the excitatory amino acid glutamate is 100–150 mM. It was recently discovered
that VGLUT1, previously characterized as an inorganic phosphate transporter (BNPI) with 9–11 predicted transmembrane spanning
domains, is capable of transporting glutamate. The expression and His-tag based purification of recombinant VGLUT1 from PC12
cells and High Five insect cells is described. Significantly better virus and protein expression was obtained using High Five
rather than Sf9 insect cells. PC12 cell expressed VGLUT1 is functional but not the Baculovirus expressed protein. The lack
of functionality of the Baculovirus expressed VGLUT1 is discussed. The data indicate that VGLUT1 readily oligomerizes/dimerizes.
The data are discussed in the context of developing this system further in order to reconstitute vesicular glutamate uptake
in vitro using lipid-detergent vesicles.
Published: June 7, 2004. 相似文献
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Matthew E. Bechard Sonya Chhatwal Rosemarie E. Garcia Madeline E. Rasche 《Biological procedures online》2003,5(1):69-77
Tetrahydromethanopterin (H4MPT) is a tetrahydrofolate analog originally discovered in methanogenic archaea, but later found in other archaea and bacteria.
The extent to which H4MPT occurs among living organisms is unknown. The key enzyme which distinguishes the biosynthetic pathways
of H4MPT and tetrahydrofolate is ribofuranosylaminobenzene 5′-phosphate synthase (RFAP synthase). Given the importance of
RFAP synthase in H4MPT biosynthesis, the identification of putative RFAP synthase genes and measurement of RFAP synthase activity would provide
an indication of the presence of H4MPT in untested microorganisms. Investigation of putative archaeal RFAP synthase genes
has been hampered by the tendency of the resulting proteins to form inactive inclusion bodies inEscherichia coli. The current work describes a colorimetric assay for measuring RFAP synthase activity, and two modified procedures for expressing
recombinant RFAP synthase genes to produce soluble, active enzyme. By lowering the incubation temperature during expression,
RFAP synthase fromArchaeoglobus fulgidus was produced inE. coli and purified to homogeneity. The production of active RFAP synthase fromMethanothermobacter thermautotrophicus was achieved by coexpression of the geneMTH0830 with a molecular chaperone. This is the first direct biochemical identification of a methanogen gene that codes for an active
RFAP synthase.
Florida Agricultural Experiment Station Journal Series no. R-09353
Published: March 4, 2003 相似文献
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Due to the involvement of calcium as a main second messenger in the plant signaling pathway, increasing interest has been
focused on the calcium signatures supposed to be involved in the patterning of the specific response associated to a given
stimulus. In order to follow these signatures we described here the practical approach to use the non-invasive method based
on the aequorin technology. Besides reviewing the advantages and disadvantages of this method we report on results showing
the usefulness of aequorin to study the calcium response to biotic (elicitors) and abiotic stimuli (osmotic shocks) in various
compartments of plant cells such as cytosol and nucleus.
Published: December 9, 2002 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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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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Microtubule inhibitors such as Vinblastine and Paclitaxel are chemotherapy agents that activate the mitotic spindle checkpoint,
arresting cells in mitosis and leading to cell death. The pathways that connect mitotic arrest to cell death are not well
characterized. We developed a mammalian cell-based cDNA cloning method to isolate proteins and protein fragments whose expression
inhibits colony formation in the presence of microtubule inhibitors. Understanding how these proteins impact cellular responses
to microtubule drugs will lead to better understanding of the biochemical pathways connecting mitotic arrest and cell death
in mammalian cells and may provide novel targets that can enhance microtubule inhibitor-mediated chemotherapy. 相似文献
13.
To assess the role of sortilin in the sorting and trafficking of sphingolipid activator proteins (SAPs) the function of sortilin
was abolished by a dominant-negative mutant and by the use of RNAi. Mutant sortilin lacking the carboxyl-terminal region that
contains the sorting signal abolished the trafficking of SAPs to the lysosomes. Both sortilin and SAPs were retained in the
Golgi apparatus. The use of chemically synthesized siRNA effectively blocked the trafficking of SAPs to the lysosomes as well.
Additionally, we created a stable COS-7 cell line transfected with the pSilencer 3.1 H1 neo vector containing a selected siRNA
template oligonucleotide (small hairpin interference RNA) where the levels of sortilin were greatly suppressed. The elimination
of sortilin by this method will permit to determine whether or not sortilin is involved in a general mechanism of lysosomal
sorting that involves the trafficking of various soluble lysosomal proteins other than SAPs. 相似文献
14.
Recombinant production and biochemical analysis of actin mutants has been hampered by the fact that actin has an absolute
requirement for the eukaryotic chaperone CCT to reach its native state. We therefore have developed a method to rapidly screen
the folding capacity and functionality of actin variants, by combining in vitro expression of labelled actin with analysis on native gels, band shift assays or copolymerization tests. Additionally, we
monitor, using immuno-fluorescence, incorporation of actin variants in cytoskeletal structures in transfected cells. We illustrate
the method by two examples. In one we show that tagged versions of actin do not always behave native-like and in the other
we study some of the molecular defects of three β-actin mutants that have been associated with diseases.
Published: October 25, 2004. 相似文献
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Véronique Masson Laetitia Devy Christine Grignet-Debrus Sarah Bernt Khalid Bajou Silvia Blacher Guy Roland Yawen Chang Timothy Fong Peter Carmeliet Jean-Michel Foidart Agnès Noël 《Biological procedures online》2002,4(1):24-31
Angiogenesis, a key step in many physiological and pathological processes, involves proteolysis of the extracellular matrix.
To study the role of two enzymatic families, serine-proteases and matrix metalloproteases in angiogenesis, we have adapted
to the mouse, the aortic ring assay initially developed in the rat. The use of deficient mice allowed us to demonstrate that
PAI-1 is essential for angiogenesis while the absence of an MMP, MMP-11, did not affect vessel sprouting. We report here that
this model is attractive to elucidate the cellular and molecular mechanisms of angiogenesis, to identify, characterise or
screen “pro- or anti-angiogenic agents that could be used for the treatment of angiogenesis-dependent diseases. Approaches
include using recombinant proteins, synthetic molecules and adenovirus-mediated gene transfer.
Published: October 28, 2002 相似文献
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To assess the role of genes required for skin organogenesis, tissue regeneration and homeostasis, we have established in vitro
skin equivalents composed of primary cells or cell lines, respectively. In these organotypic cocultures keratinocytes generate
a normal epidermis irrespective of the species and tissue origin of fibroblasts. The combination of cells derived from mouse
and human tissues facilitates the identification of the origin of compounds involved in epidermal tissue reconstitution and
thus the precise analysis of growth regulatory mechanisms.
Published: April 12, 2004 相似文献