共查询到20条相似文献,搜索用时 15 毫秒
1.
Teleost gut associated lymphoid tissue (GALT) consists of leucocyte populations located both intraepithelially and in the
lamina propria with no structural organization. The present study aims to assess different protocols for the isolation of
GALT cells from an important fish species in the Mediterranean aquaculture, the gilthead seabream. Mechanical, chemical and
enzymatic treatments were assayed. Nylon wool columns and continuous density gradients were used for further separation of
cell subpopulations. Light microscopy and flow cytometry showed that the highest density band (HD) consisted of a homogeneous
lymphocytic population, whereas the intermediate density band (ID) corresponded to epithelial and secretory cells and some
lymphocytes. Respiratory burst activity of total cell suspensions revealed very low numbers of potential phagocytic cells,
reflecting results from light microscopy and reports in other teleost species. The present data set up the basis for future
functional characterization of GALT in seabream. 相似文献
2.
The gilthead seabream is a protandrous seasonal breeding teleost that is an excellent model for studying the testicular regression
process which occurs in both seasonal testicular involution and sex reversion. Little is known about the cell types and the
molecular mechanisms involved in such processes, mainly because of the lack of appropriate methods for testis dissociation,
and testicular cell isolation, culture and functional characterization. We have previously reported that gilthead seabream
acidophilic granulocytes infiltrate the testis at post-spawning stage, settle close to the spermatogonia and accumulate intracellular
interleukin-1β. In this paper, we report several flow cytometry based assays which allow to establish the role played by gilthead
seabream testicular acidophilic granulocytes and permits their quantification.
Published: June 29, 2004. 相似文献
3.
4.
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
5.
Pierre Antony Kristen Hoek Bhaskarjyoti Sarmah Wasif N. Khan 《Biological procedures online》2007,9(1):73-83
B cell subpopulations in the spleen have been extensively characterized phenotypically; however, biochemical properties of
these cell populations following B cell antigen receptor engagement have not been fully determined due to technical difficulties
and limiting cell numbers. We therefore employed mini-scale protocols to assess lipid signaling, particularly that of diacylglycerol
and inositol trisphosphate, with as few as 0.5×106 purified early (T1) and late (T2) transitional B cells. Additionally, utilizing flow cytometric techniques, we determined
levels of phosphatidylinositol bisphosphate and calcium mobilization in T1 and T2 cells, as well as mature follicular and
marginal zone B cells using less than 1×106 primary B cells. Thus, these biochemical and flow cytometric methodologies can be used to analyse signal-induced changes
in phosphatidylinositol bisphosphate levels, diacylglycerol and inositol triphosphate production and calcium in each B cell
population.
These authors contributed equally. 相似文献
6.
7.
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
8.
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
9.
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
10.
Pancreatic β-cell apoptosis is known to participate in the β-cell destruction process that occurs in diabetes. It has been
described that high glucose level induces a hyperfunctional status which could provoke apoptosis. This phenomenon is known
as glucotoxicity and has been proposed that it can play a role in type 1 diabetes mellitus pathogenesis. In this study we
develop an experimental design to sensitize pancreatic islet cells by high glucose to streptozotocin (STZ) and proinflammatory
cytokines [interleukin (IL)-1β, tumor necrosis factor (TNF)-α and interferon (IFN)-γ]-induced apoptosis. This method is appropriate
for subsequent quantification of apoptotic islet cells stained with Tdt-mediated dUTP Nick-End Labeling (TUNEL) and protein
expression assays by Western Blotting (WB). 相似文献
11.
12.
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
13.
Jianrun Xia Song Hon H. Kim Susan Macmillan Ray Truant 《Biological procedures online》2006,8(1):63-68
Live cell fluorescence microscopy using fluorescent protein tags derived from jellyfish and coral species has been a successful
tool to image proteins and dynamics in many species. Multi-colored aequorea fluorescent protein (AFP) derivatives allow investigators to observe multiple proteins simultaneously, but overlapping spectral
properties sometimes require the use of sophisticated and expensive microscopes. Here, we show that the aequorea coerulescens fluorescent protein derivative, PS-CFP2 has excellent practical properties as a blue fluorophore that are distinct from green
or red fluorescent proteins and can be imaged with standard filter sets on a widefield microscope. We also find that by widefield
illumination in live cells, that PS-CFP2 is very photostable. When fused to proteins that form concentrated puncta in either
the cytoplasm or nucleus, PSCFP2 fusions do not artifactually interact with other AFP fusion proteins, even at very high levels
of over-expression. PSCFP2 is therefore a good blue fluorophore for distinct three color imaging along with eGFP and mRFP
using a relatively simple and inexpensive microscope. 相似文献
14.
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
15.
The UBA domain is a conserved sequence motif among polyubiquitin binding proteins. For the first time, we demonstrate a systematic,
high throughput approach to identification of UBA domain-interacting proteins from a proteome-wide perspective. Using the
rabbit reticulocyte lysatein vitro expression cloning system, we have successfully identified eleven proteins that interact with p62’s UBA domain, and the majority
of the eleven proteins are associated with neurodegenerative disorders, such as Alzheimer’s disease. Therefore, p62 may play
a novel regulatory role through its UBA domain. Our approach provides an easy route to the characterization of UBA domain
interacting proteins and its application will unfold the important roles that the UBA domain plays.
Published: December 12, 2003. 相似文献
16.
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
17.
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
18.
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
19.
Human herpesvirus (HHV)-6B is a pathogen causing latent infection in virtually all humans. Nevertheless, the interaction of
HHV-6B with its host cells is poorly understood. Although HHV-6B is approximately 90% homologous to HHV-6A, it expresses certain
B-specific genes. In order to quantify the amount of expressed viral mRNA we have developed a method using real-time PCR on
a LightCycler instrument. Here we describe an assay for the detection of the HHV-6B B6 mRNA, but our approach can easily be
extended to involve other mRNAs. This method is useful during the study of HHV-6B biology and offers reliable and reproducible,
quantitative detection of viral mRNA below the attomol range.
Published: December 9, 2002 相似文献
20.
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