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1.
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
2.
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
3.
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
4.
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
5.
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
6.
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
7.
8.
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
9.
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
10.
11.
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
12.
Kienberger Ferry Zhu Rong Moser Rosita Rankl Christian Blaas Dieter Hinterdorfer Peter 《Biological procedures online》2004,6(1):120-128
Dynamic force microscopy (DFM) allows imaging of the structure and the assessment of the function of biological specimens
in their physiological environment. In DFM, the cantilever is oscillated at a given frequency and touches the sample only
at the end of its downward movement. Accordingly, the problem of lateral forces displacing or even destroying bio-molecules
is virtually inexistent as the contact time and friction forces are reduced. Here, we describe the use of DFM in studies of
human rhinovirus serotype 2 (HRV2) weakly adhering to mica surfaces. The capsid of HRV2 was reproducibly imaged without any
displacement of the virus. Release of the genomic RNA from the virions was initiated by exposure to low pH buffer and snapshots
of the extrusion process were obtained. In the following, the technical details of previous DFM investigations of HRV2 are
summarized.
Published: June 29, 2004. 相似文献
13.
Suppression of gene expression by small interfering RNA (siRNA) has proved to be a gene-specific and cost effective alternative
to other gene suppression technologies. Short hairpin RNAs (shRNAs) generated from the vector-based expression are believed
to be processed into functional siRNAs in vivo, leading to gene silencing. Since an shRNA library carries a large pool of potential siRNAs, such a library makes it possible
to knock down gene expression at the genome wide scale. Although much of research has been focused on generating shRNA libraries
from either individually made gene specific sequences or cDNA libraries, there is no report on constructing randomized shRNA
libraries, which could provide a good alternative to these existing libraries. We have developed a method of constructing
shRNAs from randomized oligonucleotides. Through this method, one can generate a partially or fully randomized shRNA library
for various functional analyses. We validated this procedure by constructing a p53-specific shRNA. Western blot revealed that
the p53-shRNA successfully suppressed expression of the endogenous p53 in MCF-7 cells. We then made a partially randomized
shRNA library. Sequencing of 15 randomly picked cloned confirmed the randomness of the library. Therefore, the library can
be used for various functional assays, such as target validation when a suitable screening or selection method is available. 相似文献
14.
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
15.
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
16.
Seok Hwee Koo Tan Ching Ong Kok Ting Chong Caroline Guat Lay Lee Fook Tim Chew Edmund Jon Deoon Lee 《Biological procedures online》2007,9(1):27-42
We have developed and validated a consolidated bead-based genotyping platform, the Bioplex suspension array for simultaneous
detection of multiple single nucleotide polymorphisms (SNPs) of the ATP-binding cassette transporters. Genetic polymorphisms
have been known to influence therapeutic response and risk of disease pathologies. Genetic screening for therapeutic and diagnostic
applications thus holds great promise in clinical management. The allele-specific primer extension (ASPE) reaction was used
to assay 22 multiplexed SNPs for eight subjects. Comparison of the microsphere-based ASPE assay results to sequencing results
showed complete concordance in genotype assignments. The Bioplex suspension array thus proves to be a reliable, cost-effective
and high-throughput technological platform for genotyping. It can be easily adapted to customized SNP panels for specific
applications involving large-scale mutation screening of clinically relevant markers. 相似文献
17.
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
18.
We have developed a system to visualize functionally activated neurons and their projections in the brain. This system utilizes
a transgenic mouse, fos-tau-lacZ (FTL), which expresses the marker gene, lacZ, in neurons and their processes after activation by many different stimuli. This system allows the imaging of activation
from the level of the entire brain surface, through to individual neurons and their projections. The use of this system involves
detection of neuronal activation by histochemical or immunohistochemical detection of β-galactosidase (βgal), the product
of the lacZ gene. Furthermore, the underlying brain state of the FTL mice determines the basal levels of expression of βgal. Here we describe in detail our protocols for detection of FTL expression in these mice and discuss the main variables which need to be considered in the use of these mice for the detection
and mapping of functionally activated neurons, circuits and regions in the brain. 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 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