Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P30536 (PBS)
 9,886 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The suitability of bromodeoxyuridine (BrdU) immunohistochemistry for the study of myosatellite cell proliferation in three subadult carp (Cyprinus carpio) stages (11, 15, 17 cm) was examined. They were injected intraperitoneally with BrdU and fixed one hour late. After fixation and dehydration, white myotomal muscle and small intestine samples were embedded in Histowax. Cross sections mounted on glass slides, were incubated with monoclonal antiBrdU antibody (1:100) after HCl denaturation. After washing twice in PBS, slides were incubated in goat antimouse IgG FITC secondary antibody (1:20). Single cell suspensions were obtained from gut samples. Cellular DNA partially denatured with 2 N HCl, were immunolabelled with monoclonal antibodies against BrdU. Bivariate distribution of BrdU/total cellular DNA content was measured by flow cytometry. Good visualization of BrdU labelled myosatellite cells (4-6%) and small intestine (8-9%) was obtained. Flow cytometric bivariate BrdU/DNA analysis gave evidence of the same proliferative rate in the gut samples. The applicability of these methods to fish tissues further extend the broad range of biological and biomedical investigation in which BrdU immunohistochemistry has been used.
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PMID:Use of 5'-bromodeoxyuridine immunohistochemistry to examine proliferative activity of fish tissues. 768 4

In order to answer the question of whether there is an optimal buffer system for the preservation and reoxygenation period in liver transplantation, sodium/potassium phosphate, HEPES, TRIS (THAM), MOPS and histidine/His-HCl buffers were investigated. The buffers were added to an "extracellular" electrolyte composition of preservation solution. The solutions were incubated with in vitro cultures of pig hepatocytes in two different models. I: during cold hypoxia (4 degrees C, PO2 < 0.1 mmHg) for 24 h, and II. during the reoxygenation period of 3 h after preservation in UW solution. Cell viability, cell detachment rate, and LDH and GOT liberation were used as parameters of cell alteration. The lowest amount of enzyme release during the preservation period and reoxygenation was obtained using sodium or potassium phosphate buffer. Rising LDH and GOT liberation rates during preservation and reoxygenation were observed with HEPES and TRIS buffer. The enzyme release induced by these three buffer systems correlated with their pKa values. Higher pH of the preservation solution resulted in higher enzyme leakage from the cells. In contrast, the Histidine/HCl buffer system with low pH led to striking cell damage during preservation as well as during reoxygenation. MOPS, a weak acid with the lowest pH in solution, led to the lowest enzyme release during the preservation period, but to high enzyme release after reoxygenation with standard medium. Incubation of the cultures with MOPS after UW preservation resulted in lower enzyme levels in comparison to the controls. In summary, PBS had the best results in our study.
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PMID:[The effect of buffers in liver preservation solutions on hepatocytes in a model of in vitro preservation and reoxygenation]. 799 Jun 20

Artemisinin and its derivatives are a promising new class of antimalarial agents containing an endoperoxide bridge. [14C]Artemisinin alkylated various proteins in vitro. Between 5 and 18% of added drug bound to hemoproteins such as catalase, cytochrome c, and hemoglobin. However, it did not react with heme-free globin. For catalase and hemoglobin, most of the drug reacted with the protein moiety rather than the heme. Artemisinin bound to human serum albumin (HSA) more efficiently at pH 8.6 than 7.4, more efficiently in Dulbecco's PBS than in Tris-HCl buffer, and better when HSA had been made fatty acid-free. Dihydroartemisinin also bound to HSA, whereas deoxyartemisinin, an inactive derivative, did not. There was no binding between DNA and artemisinin. These data provide insight into the mechanism of the reaction between artemisinin and proteins.
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PMID:Alkylation of proteins by artemisinin. Effects of heme, pH, and drug structure. 806 44

Mycoplasma gallisepticum- or M. synoviae-challenged chickens were monitored with serological assays (serum plate agglutination, hemagglutination inhibition, and enzyme-linked immunosorbent assay) and polymerase chain reaction (PCR). The tracheal swabs from M. gallisepticum-challenged chickens received three different treatments (phosphate-buffered saline [PBS], Frey's broth, or 10 mM Tris-HCl/250 mM ethylenediaminetetraacetic acid/ 2.5% sodium dodecyl sulfate [STE]) prior to DNA purification. A nonphenolic method for DNA extraction was utilized. The best PCR results were obtained with PBS swab treatment. The nonphenolic method for DNA extraction was compared with a phenolic method in an experiment with tracheal swabs from M. synoviae-challenged chickens and commercial flocks. Both methods gave comparable results.
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PMID:Polymerase chain reaction optimization for Mycoplasma gallisepticum and M. synoviae diagnosis. 871 37

The aim of this work was to re-examine, on a quantitative basis, the relationship between banding pattern after Giemsa staining and the amount (and distribution) of DNA along the length of the chromatid arms. To do this, we investigated by cytofluorometric methods the occurrence of possibly different extraction of chromosome DNA after some alternative G-banding procedure, i.e. treatment of chromosomes with saline solutions, or DNasi I digestion in situ. The G-banding procedure entailing trypsin pretreatment is known to be difficult to standardize; in the present investigation, it was also found that trypsin induced a massive, although quantitatively variable, extraction of DNA from fixed metaphase chromosomes. G-banding-like patterns may be obtained, by treating chromosomes preparations with saline solutions. Both PBS and Tris-HCl treatment for the times considered induced a G-banding-like pattern after Giemsa staining, regardless of the age of chromosome preparations; no banding was observed after staining DNA with PI, nor extraction of DNA was found to occur. DNase I digestion initially induced a G-banding in both human and mouse chromosomes after Giemsa staining, with concomitant extraction of DNA (but without apparent G-banding-like pattern after PI staining); after 30 min digestion, a C-banding-like pattern was observed after both Giemsa and PI staining. Exposure to PBS or Tris-HCl buffer at room temperature may therefore be recommended as a G-banding inducing treatment, since it allows the classification of single chromosomes after Giemsa staining, without determining significant displacement of genomic DNA, which can be submitted to further analysis in situ.
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PMID:Treatments with saline solutions and DNase I have different effects on DNA content and distribution in human and in mouse chromosomes. 883 3

The subunit structure of soluble goat hepatic lectin was studied by determining molecular weight under nondenaturing conditions by gel filtration and denaturing conditions by SDS PAGE. Affinity purified lectin was subjected to HPLC on asahipack column equilibrated with 10 mM Tris-HCl pH 7.5, containing 1 mM CaCl2, 1mM 2-mercaptoethanol and 0.1M NaCl. The lectin was eluted under single peak at retention time of 12 min. corresponding to molecular weight of 38Kd. On SDS-PAGE in the presence and absence of 2-mercaptoethanol protein moved as single band with Rm 0.45, which corresponds to molecular weight of 20 Kd. The results suggest that soluble goat hepatic lectin is a dimmer of identical subunits which are linked together by noncovalent interactions. The interaction of monoclonal antibodies raised against soluble goat hepatic lectin (MGHL/20) with hemagglutinin from different species as sheep, human, rat, bovine and chicken was studied in PBS by solid phase binding assay. MGHL/20 showed 29.89% binding with these lectin. However no binding was found with Ca++ dependent membrane bound lectin. These results indicate that soluble goat hepatic lectin possesses antigenic structural relationship with soluble 14 K lectin family.
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PMID:Subunit structure of Ca++ dependent soluble goat hepatic lectin: evidence that it has antigenic structural relationship with soluble 14K lectin family. 886 13

The functional properties of 125I-labeled antibodies and antigens adsorbed on polystyrene and silicone were compared to their counterparts immobilized by non-adsorptive methods. Less than 20% of polyclonal (pAb) and 1-2% of monoclonal (mAb) capture antibody equivalents remained functional after adsorption as a monolayer. Survivability circa doubled or was totally rescued, when the same antibodies were immobilized via a streptavidin bridge or by using a first stage polyclonal antiglobulin capture antibody, respectively. Similarly, the antigenicity of bovine IgGs for pAb and mAb anti-IgGs was highest when the IgGs were immobilized via a streptavidin bridge or when secondarily adsorbed to an albumin monolayer. IgGs in these configurations were significantly more antigenic than when directly adsorbed on polystyrene or a silicone elastomer. Similar activity was seen after adsorption on polystyrene or silicone. Interestingly, these IgGs were equally antigenic when denatured and subsequently adsorbed in 6M guanidine-HCl versus adsorption in PBS without prior denaturation. Although many of the above finding on antibodies and antigens could be explained by the greater accessibility of antigenic epitopes or antibody binding sites when molecules are immobilized by some type of underlying molecular layer, we also show that certain mAb and pAbs preferentially recognized allotopes on IgG2a when IgG2a was adsorbed. Furthermore, such antigenicity was highest when IgG2a was adsorbed at low, sub-monolayer concentrations. Finally, we show that differences in antigenicity need not be related to the method of immobilization, but can also result from differences in the microenvironment of the epitope. This was demonstrated using a filamentous phage clone specific for fluorescein (FLU). This clone recognizes the fluorescein hapten differently depending on the carrier protein used and the method of conjugation. Data presented in this report indicate that antibodies and antigens adsorbed on hydrophobic polymers undergo changes in their functional properties. Data suggest that both changes in conformation and the accessibility of antigen epitopes or antibody binding sites, most likely occur. Especially in the case of the latter, the functional concentration may be 1-2 orders of magnitude lower than the antibody protein concentration. These observations have implications for immunodiagnostics and emphasize the need to determine the specificity of an antibody in the assay in which it is employed and to make no assumptions about the behavior of solid-phase antigens and antibodies from their behavior in solution. Our studies are also relevant to the use of silicone medical prostheses. The antigenicity of IgGs adsorbed on silicone as a multilayer (secondary layer) is much higher than when directly adsorbed. Since such surfaces would be exposed to very high protein concentrations in vivo, multilayers not a monolayer, would be expected. Thus it would seem from these studies that host protein adsorbed on silicone would be expressed to the immune system at the surface of multilayers. This being the case, it seems unlikely that the adsorption of host protein in vivo would generate new epitopes against which the host's immune system could respond and subsequently initiate an autoimmune syndrome.
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PMID:Adsorption-induced antigenic changes and their significance in ELISA and immunological disorders. 903 11

In the previous paper we described the effect of several different solvents on the structure of antibodies and demonstrated that 0.1 M glycine, pH 2.9, 7 M urea, pH 4.0, and 6 M guanidine-HCl, pH 4.0, unfold the antibodies to different degrees. Antibodies can be refolded from all of these solvents by dialysis. Polyclonal antibodies (pAbs) are a mixture of antibodies which recognize and bind different epitopes on the same antigen, with the strength of the antigen-antibody binding varying with each subpopulation. When rabbit antisera to the extracellular domain of Her2 receptor (sHer2), derived from Chinese hamster ovary cells, was applied to an antigen column, bound pAbs were recovered with a step-wise elution of 0.1 M glycine, pH 2.9 (44% of the total recovered pAb), 7 M urea, pH 4.0 (29%), and 6 M guanidine-HCl, pH 4.0 (27%), with baseline resolution between them. Fluorescence spectra of the pAbs confirmed that the 0. 1 M glycine pH 2.9 sample had near-native structure, the pAbs in 7 M urea, pH 4.0, were partially unfolded, and the pAbs in the 6 M guanidine-HCl, pH 4.0, were totally unfolded. The glycine- or urea-eluted sample was refolded by dialysis into PBS, while the guanidine-HCl-eluted sample was first dialyzed into the 7 M urea pH 4.0 buffer and then into PBS. The refolded material from glycine or urea had native-like spectra, while the spectrum of the protein refolded from 6 M guanidine-HCl was slightly perturbed. All three of these subpopulations of pAbs formed antigen-antibody complexes which could be isolated by gel-filtration chromatography, precipitated sHer2 during immunoprecipitation, and recognized sHer2 in Western blots. The guanidine-HCl-eluted material was most sensitive for Western blotting. Identical results were obtained with pAbs applied either in the batch mode or to the top of the column, indicating that antibody aggregation which may occur when applied from the top of the column is not responsible for the distribution of pAbs into different subpopulations. These results indicate that the sequential use of these three increasingly chaotropic solvents to elute antibodies results in both increased recovery of antibodies and fractionation of pAbs into subpopulations with potentially different antigen binding characteristics.
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PMID:Fractionation and characterization of polyclonal antibodies using three progressively more chaotropic solvents. 936 10

The effect of ionic strength and pH of carrier solutions on the separation of liposomes by flow field-flow fractionation (flow FFF) has been studied for the determination of accurate vesicle size distribution of liposomes. Retention behaviors of liposomes (PC/PG/cholesterol) are observed in typical buffer solutions (PBS and Tris-HCl) of various ionic strengths as carrier liquids in flow FFF. The average diameters of collected fractions at each flow FFF run are measured by photon correlation spectroscopy (PCS) for the comparison with FFF calculations at corresponding time interval of collected fractions. A reasonable separation of liposomes is observed at I = 0.016 M for both buffer solutions. Retention of liposomes is found to be elongated at ionic strengths higher than an optimum condition found experimentally, but it is shortened at a lower ionic strength due to the electrostatic interaction between the channel wall and the liposomes. Finally, size distributions of liposomes are provided comparing the liposome preparations by flow FFF.
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PMID:Size characterization of liposomes by flow field-flow fractionation and photon correlation spectroscopy. Effect of ionic strength and pH of carrier solutions. 969 17

In this study, the stability of poly(butyl cyanoacrylate) (PBCA) nanoparticle suspensions was examined for up to 1 year by measuring the nanoparticle sizes. The nanoparticles were prepared with different stabilizers (dextran 70.000, poloxamer 188, or polysorbate 85), and the particle size was determined before and after purification by centrifugation and after dilution with different solutions (0.1 N HCl, 0.01 N HCl, H2O, and PBS). The most constant sizes were with the untreated acidic nanoparticle suspensions. In all other cases, agglomeration of the particles occurred: the extent of this agglomeration and the time at which the agglomeration occurred depended on the experimental conditions. Nanoparticle polymer degradation, as indicated by size decrease, was not observed. Thus, PBCA nanoparticles can be stored as suspensions, making the lyophilization and the sometimes problematic resuspension by ultrasonication, unnecessary, which is advantageous for clinical applications.
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PMID:Long-term stability of PBCA nanoparticle suspensions. 1067 Sep 41


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