Characterization of mercury-binding proteins in rat blood plasma

2018 ◽  
Vol 54 (54) ◽  
pp. 7439-7442 ◽  
Author(s):  
Yiling Li ◽  
Bin He ◽  
Qiying Nong ◽  
Guangbo Qu ◽  
Lihong Liu ◽  
...  
Keyword(s):  
Blood Plasma ◽  
Binding Proteins ◽  
Binding Protein ◽  
Protein Profiles ◽  
Rat Blood

Mercury-binding protein profiles in plasma in vitro and in vivo, which were unclear previously, were systematically investigated.

scholarly journals The 36-kilodalton embryonic-type cytoplasmic polyadenylation element-binding protein in Xenopus laevis is ElrA, a member of the ELAV family of RNA-binding proteins.

1997 ◽  
Vol 17 (11) ◽  
pp. 6402-6409 ◽  
Author(s):  
L Wu ◽  
P J Good ◽  
J D Richter
Keyword(s):  
Xenopus Laevis ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Protein ◽  
Dominant Negative ◽  
Cytoplasmic Polyadenylation ◽  
Element Binding Protein

The translational activation of several maternal mRNAs in Xenopus laevis is dependent on cytoplasmic poly(A) elongation. Messages harboring the UUUUUAU-type cytoplasmic polyadenylation element (CPE) in their 3' untranslated regions (UTRs) undergo polyadenylation and translation during oocyte maturation. This CPE is bound by the protein CPEB, which is essential for polyadenylation. mRNAs that have the poly(U)12-27 embryonic-type CPE (eCPE) in their 3' UTRs undergo polyadenylation and translation during the early cleavage and blastula stages. A 36-kDa eCPE-binding protein in oocytes and embryos has been identified by UV cross-linking. We now report that this 36-kDa protein is ElrA, a member of the ELAV family of RNA-binding proteins. The proteins are identical in size, antibody directed against ElrA immunoprecipitates the 36-kDa protein, and the two proteins have the same RNA binding specificity in vitro. C12 and activin receptor mRNAs, both of which contain eCPEs, are detected in immunoprecipitated ElrA-mRNP complexes from eggs and embryos. In addition, this in vivo interaction requires the eCPE. Although a number of experiments failed to define a role for ElrA in cytoplasmic polyadenylation, the expression of a dominant negative ElrA protein in embryos results in an exogastrulation phenotype. The possible functions of ElrA in gastrulation are discussed.

Function of Tubulin Binding Proteins in Vivo

Genetics ◽  
2000 ◽  
Vol 156 (1) ◽  
pp. 69-80 ◽  
Author(s):  
James A Fleming ◽  
Leticia R Vega ◽  
Frank Solomon
Keyword(s):  
Binding Proteins ◽  
Binding Protein ◽  
Yeast Cells ◽  
Gene Products ◽  
Salvage Pathway ◽  
Tubulin Binding ◽  
Mutant Cells ◽  
Β Tubulin

Abstract Overexpression of the β-tubulin binding protein Rbl2p/cofactor A is lethal in yeast cells expressing a mutant α-tubulin, tub1-724, that produces unstable heterodimer. Here we use RBL2 overexpression to identify mutations in other genes that affect formation or stability of heterodimer. This approach identifies four genes—CIN1, CIN2, CIN4, and PAC2—as affecting heterodimer formation in vivo. The vertebrate homologues of two of these gene products—Cin1p/cofactor D and Pac2p/cofactor E—can catalyze exchange of tubulin polypeptides into preexisting heterodimer in vitro. Previous work suggests that both Cin2p or Cin4p act in concert with Cin1p in yeast, but no role for vertebrate homologues of either has been reported in the in vitro reaction. Results presented here demonstrate that these proteins can promote heterodimer formation in vivo. RBL2 overexpression in cin1 and pac2 mutant cells causes microtubule disassembly and enhanced formation of Rbl2p-β-tubulin complex, as it does in the α-tubulin mutant that produces weakened heterodimer. Significantly, excess Cin1p/cofactor D suppresses the conditional phenotypes of that mutant α-tubulin. Although none of the four genes is essential for viability under normal conditions, they become essential under conditions where the levels of dissociated tubulin polypeptides increase. Therefore, these proteins may provide a salvage pathway for dissociated tubulin heterodimers and so rescue cells from the deleterious effects of free β-tubulin.

Physiological, morphological, and immunochemical parameters used for the characterization of clinical and environmental isolates ofAcanthamoeba

Parasitology ◽  
2012 ◽  
Vol 140 (3) ◽  
pp. 396-405 ◽  
Author(s):  
A. BECKER-FINCO ◽  
A. O. COSTA ◽  
S. K. SILVA ◽  
J. S. RAMADA ◽  
C. FURST ◽  
...  
Keyword(s):  
Comparative Evaluation ◽  
Polyclonal Antibodies ◽  
Protein Profiles ◽  
Environmental Isolates ◽  
Pathogenic Potential ◽  
Cytopathic Effects ◽  
Specific Proteins

SUMMARYThe factors that characterizeAcanthamoebastrains as harmless or potentially pathogenic have not been elucidated. Analysing thein vitroandin vivoparameters ofAcanthamoebasamples, including heat tolerance at temperatures close to that of the human body, cytopathic effects, and their ability to cause infections in animals, has been proposed to identify their pathogenic potential. Another promising criterion for differentiating strains is the analysis of their biochemical and immunochemical properties. In this study, a comparative evaluation between clinical and environmentalAcanthamoebaisolates was performed on the basis of physiological, morphological, and immunochemical criteria. Crude antigens were used to characterize the protein profiles by electrophoresis and immunize mice to produce polyclonal and monoclonal antibodies. The antibodies were characterized using ELISA, Western blotting, and immunofluorescence techniques. The results obtained with polyclonal antibodies suggest the presence of specific proteins for each studied isolate and co-reactive immunochemical profiles among conserved components. Ten monoclonal antibody clones were obtained; mAb3 recognized 3 out of 4 samples studied. The results of this study may help standardize criteria for identifying and characterizingAcanthamoebastrains. Taken together, our results support the view that a set of features may help differentiateAcanthamoebaspecies and isolates.

scholarly journals Nonequilibrium kinetics of a cyclic GMP-binding protein in dictyostelium discoideum

1982 ◽  
Vol 94 (2) ◽  
pp. 271-278 ◽  
Author(s):  
PJM Van Haastert ◽  
H Van Walsum ◽  
FJ Pasveer
Keyword(s):  
Dictyostelium Discoideum ◽  
Cyclic Gmp ◽  
Binding Proteins ◽  
Binding Protein ◽  
Kinetic Constants ◽  
Nonequilibrium Conditions ◽  
Kinetics Of ◽  
Stimulation Of

Chemoattractants added to cells of the cellular slime mold dictyostelium discoideum induce a transient elevation of cyclic GMP levels, with a maximum at 10 s and a recovery of basal levels at approximately 25 s after stimulation. We analyzed the kinetics of an intracellular cGMP binding protein in vitro and in vivo. The cyclic GMP binding protein in vitro at 0 degrees C can be described by its kinetic constants K(1)=2.5 x 10(6) M(- 1)s(-1), k(-1)=3.5 x 10(-3)s(-1), K(d)=1.4 x 10(-9) M, and 3,000 binding sites/cell. In computer simulation experiments the occupancy of the cGMP binding protein was calculated under nonequilibrium conditions by making use of the kinetic constants of the binding protein and of the shape of the cGMP accumulations. These experiments show that under nonequilibrium conditions by making use of the kinetic constants of the binding protein and the shape of the cGMP accumulations. These experiments show that under nonequilibrium conditions the affinity of the binding protein for cGMP is determined by the rate constant of association (k(1)) and not by the dissociation constant (k(d)). Experiments in vivo were performed by stimulation of aggregative cells with the chemoattractant cAMP, which results in a transient cGMP accumulation. At different times after stimulation with various cAMP concentrations, the cells were homogenized and immediately thereafter the number of binding proteins which were not occupied with native cGMP were determined. The results of these experiments in vivo are in good agreement with the results of the computer experiments. This may indicate that: (a) The cGMP binding protein in vivo at 22 degrees C can be described by its kinetic constants: K(1)=4x10(6)M(-1)s(-1) and K(-1)=6x10(-3)s(-1). (b) Binding the cGMP to its binding protein is transient with a maximum at about 20-30 s after chemotactic stimulation, followed by a decay to basal levels, with a half-life of approximately 2 min. (c) The cGMP to its binding proteins get half maximally occupied at a cGMP accumulation of δ[cGMP](10)=2x10(-8) M, which corresponds to an extracellular stimulation of aggregative cells by 10(-10) M cAMP. (d) Since the mean basal cGMP concentration is approximately 2x10(-7) M, the small increase of cGMP cannot be detected accurately. Therefore the absence of a measurable cGMP accumulation does not argue against a cGMP function. (e) There may exist two compartments of cGMP: one contains almost all the cGMP of unstimulated cells, and the other contains cGMP binding proteins and the cGMP which accumulates after chemotactic stimulation. (f) From the kinetics of binding, the cellular responses to the chemoattractant can be divided into two classes: responses which can be mediated by this binding protein (such as light scattering, proton extrusion, PDE induction, and chemotaxis) and responses which cannot be (solely) mediated by this binding protein such as rlay, refractoriness, phospholipids methylation, and protein methylation.

scholarly journals Heterogeneity and properties of hepatic dexamethasone-binding proteins

1979 ◽  
Vol 180 (1) ◽  
pp. 187-193 ◽  
Author(s):  
S L H Liu ◽  
T E Webb
Keyword(s):  
Rat Liver ◽  
Binding Proteins ◽  
Binding Protein ◽  
Novikoff Hepatoma ◽  
Liver Cytosol ◽  
Activated Complex ◽  
Simple Dissociation ◽  
Rat Liver Cytosol

Evidence from experiments in vivo and in vitro is presented for the presence of three species of dexamethasone-binding proteins in rat liver, which are identified by chromatography on Sepharose 6B or by isoelectric focusing. Although two of these species (DI and DII) possess properties characteristic of a true receptor, the third binding protein (i.e. DIII), which migrates most slowly on Sepharose 6B, but has stability properties similar to protein DII, exhibits a 3-fold lower affinity for dexamethasone and the activated complex neither binds to DNA-cellulose nor translocates to the nucleus. Only the predominant liver receptor (DI), which is eluted first from Sepharose 6B, is present in Novikoff-hepatoma cytosol, suggesting that the major and minor species are not interconverted through simple dissociation during their isolation. The binding activities of all three species in the liver cytosol increase approx. 2-fold in vivo after adrenalectomy and show a transient 2-fold fall in vivo after the administration of cortisol. These changes in vivo in protein DIII shows a marked lag compared with those in proteins DI and DII, which change in parallel. It is therefore proposed that rat liver cytosol contains two dexamethasone receptors and a dexamethasone-binding protein that may be derived from these receptors.

Lactoferrin-binding proteins in Bifidobacterium bifidum

2002 ◽  
Vol 80 (1) ◽  
pp. 91-94 ◽  
Author(s):  
Woan-Sub Kim ◽  
Tetsuya Tanaka ◽  
Haruto Kumura ◽  
Kei-ichi Shimazaki
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Binding Proteins ◽  
Binding Protein ◽  
Bifidobacterium Bifidum ◽  
Molecular Weights ◽  
In Vivo Experiments ◽  
Bactericidal Effects

Lactoferrin is an iron-binding glycoprotein and its bacteriostatic and bactericidal effects on gram-positive and gram-negative bacteria are well known. On the other hand, it is known that certain kinds of lactic acid bacteria are resistant to its antibacterial effects. Moreover, it is reported that lactoferrin promotes the growth of bifidobacteria in in vitro and in vivo experiments. In our experiments, lactoferrin-binding protein was found both in the membrane and cytosolic fractions of Bifidobacterium bifidum Bb-11. The bifidobacteria were grown in anaerobic conditions with lactobacilli MRS broth containing cysteine, harvested by centrifugation, and processed by sonication. The lactoferrin-binding proteins on the PVDF-membrane transferred after SDS-PAGE were detected by far-Western (western-Western) method using biotinylated lactoferrin and streptavidin-labelled horse radish peroxidase. The molecular weights of the lactoferrin binding protein detected in the membrane fraction were estimated to be 69 kDa and those in cytosolic fractions were 20, 35, 50, and 66 kDa.Key words: lactoferrin, Bifidobacterium, lactic acid bacteria, binding protein.

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

2019 ◽  
Author(s):  
Priya Prakash ◽  
Travis Lantz ◽  
Krupal P. Jethava ◽  
Gaurav Chopra
Keyword(s):  
Amyloid Beta ◽  
Western Blots ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Set Up ◽  
Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

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