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Molecular biology study of satellite panicum mosaic virus capsid proteinQi, Dong 15 May 2009 (has links)
Satellite panicum mosaic virus (SPMV) depends on its helper Panicum mosaic virus
(PMV) for replication and movement in host plants. The positive-sense single-stranded
genomic RNA of SPMV encodes a 17-kDa capsid protein (CP) to form 16-nm virions.
Previous studies showed that SPMV CP has multiple functions during infection
including encapsidation, symptom exacerbation, inhibiting the accumulation of SPMV
DIs, and facilitating systemic movement.
This dissertation confirms and extends the results of our previous reports with new
biological and biochemical evidence. For example, the dosage effect of SPMV CP on
symptom severity supports its function as a pathogenicity factor. Biological assays also
demonstrate compensatory effects of SPMV CP on virus mutants defective in systemic
movement. In addition, it is shown for the first time that SPMV CP is involved in cellto-
cell movement of SPMV RNA and associated with the cell wall and membranes, a
signature property of plant virus movement proteins. However, SPMV CP in the cytosol
exists exclusively as virions and is dispensable for symptom exacerbation. SPMV CP contains a distinctive N-terminal arginine-rich motif (N-ARM), which is
required for the in vitro binding of SPMV and PMV genomic RNAs by SPMV CP.
Mutations of this region impair all known functions of SPMV CP. Interestingly,
manipulation of the C-terminus of SPMV CP resulted in the same phenotypes as
alterations in the N-ARM except that this does not affect the RNA binding activity of
SPMV CP. Biological experiments demonstrate that virions are not required for the
properties of SPMV CP to facilitate local and systemic movement and inhibit the
accumulation of SPMV DIs, suggesting that SPMV CP and RNA form alternative
complexes for these purposes. This dissertation study reveals the nucleolar localization
of SPMV CP and its interaction with PMV CP in the form of virions.
The identification of distinct functional domains of SPMV CP and its complex
subcellular localization profile resulted in the proposal of a tentative model on how the
functions of SPMV CP are coordinated for a robust infection. This dissertation provides
a foundation for further understanding of the complex interactions among host plants,
helper viruses, and satellites.
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Investigating cotranslational integration of a multi-spanning membrane protein into the endoplasmic reticulum membraneJongsma, Candice Gene 15 May 2009 (has links)
Most membrane proteins in eukaryotic cells are co-translationally
integrated into the endoplasmic reticulum (ER) membrane at aqueous pores
termed translocons. During multi-spanning membrane protein (MSMP)
integration, the nascent polypeptide is threaded into the translocon pore where
each successive transmembrane segment (TMS) is moved laterally through the
translocon into the bilayer. The hydrophilic polypeptide segments on each side
of the TMS are alternately directed into either the aqueous cytosol or the
aqueous ER lumen. How is the ER membrane permeability barrier maintained
during this process?
For a single-spanning signal-cleaved membrane protein, nascent chain
movement into the lumen occurs while an ion-tight ribosome-translocon junction
prevents ion flow through the translocon pore. Prior to opening this junction to
allow nascent chain movement into the cytosol, BiP (Hsp70 binding protein)
effects closure at the lumenal end of the pore to maintain the membrane
permeability barrier. To determine whether the ribosome and BiP alternately mediate pore closure during the integration of a MSMP, integration
intermediates with nascent chains of different lengths were prepared with a
fluorescent probe positioned in the nascent chain far inside the ribosomal tunnel.
Nascent chain exposure to the cytosol or lumen was then detected by the
collisional quenching of the probe by iodide ions located on either the cytosolic
or lumenal side of the membrane.
While the first TMS through the tunnel caused the ribosome-translocon
junction to open, the second TMS elicited both the closure of this junction and
the opening of the lumenal end of the pore. Movement of a third TMS through
the tunnel caused the ribosome-translocon junction to re-open after closure of
the lumenal end. Pore opening and closing occurred after each TMS was 4-7
residues from the peptidyltransferase center, irrespective of TMS location in the
nascent chain. The ribosome treated all TMSs in the same manner, regardless
of their individual sequence or their native orientation. The ER membrane
permeability barrier is maintained by ribosome-translocon interactions during cotranslational
MSMP integration.
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The Investigation of the pH Effect on Slow Exchange Dynamics in Amino Acids and Proteins with NMR Relaxation Dispersion ExperimentsChen, Yan-wen 09 July 2007 (has links)
None
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Applications of Water-Soluble Fullerene Derivatives in Biological-Field for Affinity Mass SpectrometryTeng, Chia-Fang 04 July 2000 (has links)
none
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Applications of polyaniline-protein-A modified electrode in immunoassayLiao, Kuo-Tang 19 July 2001 (has links)
none
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Molecular Interaction of Tau and MicrotubuleYen, Yi-Chen 21 August 2002 (has links)
Tau protein is one of the microtubule-associated proteins (MAPs) and mainly expressed in neuronal cells. It hasbeen demonstrated that Tau may play an important role in regulating microtubule dynamic in neurons. Structurally and functionally, Tau protein composed of regulatory projection domain in N-terninus and microtubule-binding domain in C-terminus. It has been shown that the biological function of Tau protein was regulated by phosphorylation and dephosphorylation. In Alzheimer¡¦s disease (AD) brain, hyperphosphorylated Tau caused by over active kinases may contribute to the disassociation of Tau from microtubule and form the pathologically hallmarker, paired helical filaments (PHFs). The reason to study cdc2 and GSK3£] is two folds. First, both cdc2 and GSK3£] activities are raised abbrently in AD brain. Second, the phosphorylation sites of cdc2 and GSK3£] have been identified as those in PHFs.These prompted us to regard cdc2 and GSK3£] as candidates that hyperphosphorylated Tau in AD.
In the following study, we used immunofluorescence analysis, co-immunoprecipitation and GST-fusion protein pull down assay to clarify the subcellular localization of Tau. We also shown that the interaction between tubulin withfull length Tau (Tau WT) and some Tau mutants that we found that not only Tau WT, but also N-terminus of Tau (Tau-N) and C-terminus of Tau (Tau-C) can bind to tubulin. Surprisingly, we observed that a fragment of N-terminus, Tau 122-244, localized in nucleus. Furthermore, we used tubulin assembly assay to test if tau or its mutants can promote tubulin assembly in vitro. Results showed that only Tau WT can promote tubulin assembly in vitro but not Tau-N or Tau-C. Although Tau-N or Tau-C can bind to tubulin in vivo and in vitro, these mutants did not remain the ability to promote tubulin assembly that suggested both functional domains, N-terminus and C-terminus of Tau, are necessary and essential for the biological function of Tau. On the other hand, we used of phosphorylation assay and site directed mutagenesis to demonstrate that T231 of Tau is one of important phosphorylation sites of cdc2 and GSK3£]. Finally, we used tubulin assembly assay to show that phosphorylated Tau by GSK3£] can negatively regulate the ability of Tau to promote tubulin assembly that indicated that the phosphorylation at T231 may play a role in regulating Tau.
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Prediction of Protein Structures Based on Curve AlignmentChen, Yi-Ying 27 August 2002 (has links)
Various proteins with specific properties and functions exist in organisms, they
perform all important biochemical activities. The biochemical functions of proteins
are determined by their structures. One of the most important issues in the life
science is to predict the three-dimensional structures with protein sequences, and
then to deduce their biochemical functions. To predict protein structure precisely
will accelerate biochemical research. However, it is a challenge task to obtain the real
structure of a protein. The objective of this study is to develop a protein structure
prediction methodology based on a structure-known protein (such as the proteins
in the PDB database), where the two protein sequences are extremely similar.
Some folding algorithms, such as U-fold and S-fold, have been developed to
predict protein structures. However, the folding algorithms work on a grid lattice,
which is very different from the real structure of a protein. Here we use the curve
fitting technique, such as B-splines, to convert the lattice model and a real structure
to the same domain, that is, the curve. We therefore perform curve (structure)
alignment on them. The curve alignment can also be used to evaluate the similarity
between two structures. By the experimental results, our protein structure prediction
method performs well when we get two protein sequences with similarity that
is not too high.
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Expression of alpha-1-microglobulin mRNA change after oxalate challenge in renal epithelium cellTsai, Jeng-Yu 25 August 2002 (has links)
Alpha-1-microglobulin is co-synthesized with bikunin on the AMBP gene (
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pH-biased isoelectric trapping separationsShave, Evan Eric 30 October 2006 (has links)
The classical isoelectric trapping (IET) technique, using the multicompartment
electrolyzer (MCE), has been one of the most successful electrophoretic techniques in
preparative-scale protein separations. IET is capable of achieving high resolution
discrimination of proteins, by isolating proteins in between buffering membranes, in
their isoelectric state. However, due to the inherent nature of the IET process, IET has
suffered several shortcomings which have limited its applicability. During a classical
IET separation, a protein gets closer and closer to its pI value, thus the charge of the
protein gets closer and closer to zero. This increases the likelihood of protein
precipitation and decreases the electrophoretic velocity of the protein, thus making the
separation very long. Furthermore, the problems are aggravated by the fact that the
instrumentation currently used for IET is not designed to maximize the efficiency of
electrophoretic separations.
To address these problems, a new approach to IET has been developed, pH-biased IET.
By controlling the solution pH throughout the separation, such that it is not the same as
the proteinâÂÂs pI values, the problems of reduced solubility and low electrophoretic
migration velocity are alleviated. The pH control comes from a novel use of isoelectric buffers (also called auxiliary isoelectric agents or pH-biasers). The isoelectric buffers are
added to the sample solution during IET and are chosen so that they maintain the pH at a
value that is different from the pI value of the proteins of interest. Two new pieces of
IET instrumentation have been developed, resulting in major improvements in protein
separation rates and energy efficiency. A variety of separations, of both small molecules
and proteins, have been successfully performed using the pH-biased IET principle
together with the new instrumentation.
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Influence of dietary composition on coccidiosis vaccination efficacy in broilersLee, Jason Thomas 25 April 2007 (has links)
This research program included a series of experiments to investigate the effect
of starter diet protein level on the performance of broilers vaccinated with Coccivac®-B
and subsequently challenged with a mixed species Eimeria challenge compared to nonvaccinated
broilers. Pre-challenge performance data indicates that vaccination may
decrease body weights and increase feed conversion ratio (FCR) with vaccination. The
time period associated with the observed effects is between 13 to 17 d of age. This
reduction in performance of vaccinated broilers versus non-vaccinated broilers was
eliminated by the conclusion of the experiments (27 d) in the higher protein diets.
Vaccination was effective at generating protective immunity against the Eimeria
challenge evidenced by significantly increased body weight gains, improved feed
conversions, reduced post-challenge mortality, and reduced lesion development in
vaccinated broilers compared to non-vaccinated.
The final experiment included the comparison of Coccivac®-B to Bio-Cox®
(salinomycin) for controlling field strain Eimeria in broilers reared on two different dietary rations varying in protein concentration. Diet A had a lower protein
concentration than Diet B. On day 14, Eimeria collected from commercial broiler farms
in Texas were spray applied to the litter in all pens. Broilers reared on Diet B were
heavier at Day 40 while body weights at day 50 were similar for all groups. Broilers fed
Diet B had lower FCR during the starter and finisher diets. Broilers fed salinomycin had
lower FCR for the starter and grower diets while vaccinated broilers had lower FCR
during the withdrawal period. Cumulative FCR for the entire grow out period were
similar for all groups.
These data indicate that vaccination can be utilized as an anticoccidial preventive
and are suggestive that reduced protein concentration of starter diets can lead to
significant losses in broiler performance when utilizing a vaccination program to prevent
coccidiosis. Feeding an appropriately formulated diet while vaccinating broilers with
Coccivac®-B as an alternative to the use of salinomycin yields at least equivalent if not
elevated performance in the presence of field-strain Eimeria during grow-out with no
effect on the cost of production.
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