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)

Phage PBS-2 DNA, which contains uracil in place of thymine, was selectively damaged and then used as substrate for purified Bacillus subtilis uracil-DNA glycosylase. This enzyme releases uracil from DNA in a limited processive manner. Irradiation by ultraviolet light (greater than 305 nm) in the presence of isopropanol and a free radical photoinitiator introduced covalently bound 8-(2-hydroxy-2-propyl)purines into DNA. Methylation by dimethylsulfate yielded 7-methylguanine. Apurinic sites were produced by gentle heating of methylated DNA. Rates of enzymic release of uracil from DNA varied among these three substrates. The Vmax was markedly decreased for DNA containing 8-(2-hydroxy-2-propyl)purines and apurinic sites but was unaffected by the presence of larger quantities of 7-methylguanine. This suggests that certain types of damaged DNA moieties may decrease the capacity for uracil excision. Therefore, interference with enzymic excision of this potentially mutagenic base may constitute a common mechanism of action of the reaction products of several unrelated DNA damaging agents.
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PMID:Perturbations of enzymic uracil excision due to purine damage in DNA. 695 98

We have devised a processing technique to embed calcified tissues, such as bone and tooth enamel, in paraffin, to preserve the delicate antigenic sites of molecules such as growth factors. The same technique, omitting the decalcification step, allows delicate tissues, such as axolotl embryos (Ambystoma mexicanum) containing large yolk masses, to be easily handled during tissue processing and to be serially sectioned. Specimens were all fixed in periodate-lysine-paraformaldehyde (PLP) fixative at 5 degrees C. Bone and teeth were decalcified in an EDTA-G solution at -4 degrees C. Maintaining a temperature of 5 degrees C, the decalcified samples were then washed (with PBS, pH 7.2, under vacuum) to remove glycerol. Both the decalcified tissues and the yolky embryos were dehydrated through an ascending series of isopropanol and embedded in low melting-point paraffin under vacuum. Acidic fibroblast growth factor (aFGF) was located in cells of the expanded cambial layer in the early fracture calluses of male CD-1 mice, demonstrating retention of antigenic sites. The results reported here have not previously been obtained with existing processing and embedding techniques.
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PMID:A histological processing technique that preserves the integrity of calcified tissues (bone, enamel), yolky amphibian embryos, and growth factor antigens in skeletal tissue. 768 84

We have shown that several human malignant glioma cell lines are stimulated by bacterial lipopolysaccharide (E. coli 0111:B4, 1 microgram/ml) to produce a high molecular weight (> 200 kD) growth activity for BALB 3T3, clone A31 cells. This glioma-derived growth factor (GDGF-2) acts like a 'competence' factor. Malignant glioma cell line D-54 MG constitutively produced GDGF-2, which we have partially characterized from serum-free conditioned culture medium. GDGF-2 is resistant to heat (100 degrees C, 5 min), acidic (pH 2, 2 hr) or reducing (0.5 M 2 ME, 30 min) conditions as well as exposure to RNases; however, it is sensitive to > 4 freeze-thaw cycles, alkaline (pH 11, 2 hr) conditions or pre-treatment with proteolytic enzymes. GDGF-2 had a pl of 6.8 determined by preparative isoelectric focusing, bound to DEAE, with elution at 35 and 185 mM NaCl and at 43% acetonitrile from a C4 reversed phase column. GDGF-2 activity was not neutralized by antibodies to TGF alpha, TGF beta, PDGF, VEGF or TNF alpha indicating that it is not immunochemically related to these growth factors. However GDGF-2 co-chromatographed on Superose 12 HPLC (250 x 9 mm; 5% isopropanol, 6 mM CHAPS in PBS) with a substance that suppressed growth of mink lung epithelial cells (Mv1Lu), but not BALB 3T3 cells, and could be neutralized by anti-TGF beta antibodies. GDGF-2 activity eluted from heparin columns in 0.6 M NaCl; thus, it is not a heparin binding growth factor. D-54 MG cell line produced alpha 2-macroglobulin (alpha 2M), which is known to bind TGF beta; however, immunoprecipitation of alpha 2M did not deplete TGF beta or GDGF-2 activity. Further, neither GDGF-2 or TGF beta can be dissociated into lower molecular weight active components by chromatography in high salt (2 M NaCl) or 2-ME (0.5 M). GDGF-2 may be a novel autocrine or paracrine mitogen, stimulating mitotic division or interfering with normal cell growth regulation.
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PMID:Partial characterization of glioma-derived growth factor 2: a novel mitogenic activity from human cell line D-54 MG. 814 64

Combined analysis of DNA content and immunofluorescence on single cells by flow cytometry provides information on the proliferative response of cellular sub-populations in mixed cell preparations. However, the presence of considerable numbers of dead (nonviable) cells impairs accurate flow cytometric data analysis, mainly, because dead cells can bind antibodies non-specifically and show alterations in their DNA staining profiles. We developed a rapid method for identification of dead cells by fluorescence in cell preparations that are stained simultaneously for two-color immunofluorescence and DNA content. Cells are stained with 7-aminoactinomycin D (7-AAD) for dead cell discrimination and with fluorescein-isothiocyanate (FITC) and phycoerythrin (PE)-labeled monoclonal antibodies (mAb) for cell surface immunofluorescence. Diffusion of 7-AAD from stained, dead cells into unstained, live cells after cell permeabilization is blocked by the addition of its non-fluorescent analogue actinomycin D (AD). DNA is stained with red-excitable TO-PRO-3 iodide (TP3) which has an emission spectrum that can be effectively separated from the emissions of FITC, PE, and 7-AAD. TP3 staining is performed in the presence of ribonuclease A (RNAse) in phosphate-citrate buffer containing saponin (PCBS) at low pH. FITC fluorescence is sensitive to acid pH; therefore, PCBS is replaced after DNA staining with 1x PBS at pH 7.2 containing saponin to permit accurate detection of FITC immunofluorescence on the flow cytometer. We apply this method to the analysis of differential proliferation of lymphocyte subsets in cultures of human peripheral blood mononuclear cells (PBMC) with low viability.
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PMID:Simultaneous flow cytometric measurement of viability and lymphocyte subset proliferation. 1115 May 48

An increase in oxidizing response above a certain threshold produces, in the absence of a concomitant rise in antioxidant/reducing response, oxidative stress that is associated with complications in diabetes. A simple technique involving reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye has been developed in order to determine quantitatively the antioxidant status of plasma. MTT (50microL; 5.0mg/mL in PBS) was incubated with plasma (100microL) in PBS for 30, 60 or 120min at 37 degrees C, the reaction terminated by addition of 1.0mL of 0.04M hydrochloric acid in isopropanol and the absorbance measured at 570nm. The modulation by plasma of the generation of reactive oxygen species (ROS) in 12,13-phorbol dibutyrate (PDB)-stimulated granulocytes was evaluated using a chemiluminescence luminol-dependent assay. Plasma from healthy subjects (n=15) showed significantly higher antioxidant status (p<0.05) over all time periods studied compared with plasma from diabetic patients (n=27). MTT was directly reduced by plasma although platelets were not involved. Moreover, the reduction of MTT by bovine serum albumin at levels equivalent to the concentration of human serum albumin in plasma was much lower. The antioxidant status of plasma, as evaluated by MTT dye reduction, may reflect an antioxidant response since ROS generation in PDB-stimulated granulocytes was rapidly down-regulated by the presence of plasma (3.3-fold in diabetic patients and 5.8-fold in healthy subjects) confirming the lower antioxidant activity of plasma from diabetic patients. The results demonstrate that extracellular reduction of MTT by plasma may occur via enzymatic and non-enzymatic processes.
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PMID:Determination of the antioxidant status of plasma from type 2 diabetic patients. 1727 Mar 9

The objective of this study was to test the activity of microbicides against herpes simplex virus type 2 (HSV-2) introduced in seminal plasma. We found that seminal plasma interfered with the activity of PRO 2000 and of cellulose sulfate, increasing by 100-fold the concentration of drug required to inhibit 90% of viral plaque formation. Seminal plasma competitively inhibited binding of the microbicides to the HSV-2 envelope. Most of the interference was found in a high molecular-weight fraction; tandem mass spectrometry identified the proteins as fibronectin-1 and lactoferrin. In a murine model, the interference translated in vivo into a loss in protection. We found that 2% PRO 2000 gel protected 100% of mice challenged intravaginally with HSV-2 introduced in PBS, whereas only 55% of mice were protected if virus was introduced in seminal plasma (P=.0007, log rank test). If these findings are reflective of what occurs in humans, modifications to microbicides to ensure that they retain activity in the presence of seminal plasma are indicated.
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PMID:Seminal plasma reduces the effectiveness of topical polyanionic microbicides. 1792 91

Previously we have recrystallized estradiol with various organic solvents and investigated solvate molecules within estradiol crystals by using CP/MAS solid-state NMR. To investigate the effect of recrystallization solvents on the physicochemical properties of recrystallized estradiol, four different crystal habits of estradiol were recrystallized and their physicochemical properties were characterized by optical microscopy, solubility, and FT-IR measurements. Various crystal habits in size and shape were produced by the interaction between the estradiol and different solvents. Although the estradiol crystal habits prepared from ethanol and methanol had larger particle size, they were more soluble in PBS than those recrystallized from isopropanol and acetone. In spite of the low solubilities, the estradiols prepared from isopropanol and acetone were released in PBS and permeated through the hairless mouse skin similar to the others. Thus, although microscopic observation of recrystallized estradiols revealed that the estradiol had different crystal habits, the release and permeation properties of different estradiol crystals might be independent on the solvate molecules associated with the solvent used for recrystallization.
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PMID:Solvent effects on physicochemical behavior of estradiols recrystallized for transdermal delivery. 1827 16

Prior understanding on the in vitro release profile of the drug from drug eluting devices such as stent (DES) is crucial in designing and optimizing the drug embedded coating or matrices. In fact, assessing in vitro release profile is a mandatory requirement prior to the clinical evaluation of DES. The in vitro release is also employed to estimate parameters such as T1/2. The release profile largely depends on the release medium selected for the studies. Normally PBS with a pH of 7.4 is used for assessing the release kinetics of the drug. Often drug undergoes irreversible changes such as hydrolysis in PBS leading to erroneous assessment of the release profile. This is particularly true in the case of sirolimus, one of the widely used drugs in various applications. We studied the influence of various media on the release profile of sirolimus from DES. The data generated suggested that a release medium consisting of 9:1 (v/v) of normal saline and isopropanol is a most suitable one for assessing in vitro the release kinetics of sirolimus from DES.
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PMID:The release kinetics of drug eluting stents containing sirolimus as coated drug: role of release media. 2013 93

An effective and fast RNA isolation method of activated sludge was established and five different methods were compared based on RNA yield, purity, integrity, RT-PCR amplification of 16S rRNA genes and subsequent terminal restriction fragment length polymorphism (T-RFLP) analysis. That is, the precipitated activated sludge was washed with TENP and PBS buffer, followed by using lysozyme and TRIzol to direct lysis of microbial cells, chloroform to remove protein and most of the DNA from bacterial lysate, isopropanol to precipitate nucleic acid and DNase I to hydrolyze residual DNA. To further purify RNA, RNA purifying column was utilized. The results demonstrated that the extraction method, with the aid of TRIzol and RNA purification kit, can effectively extract high-quality RNA. It not only means low degradability and high quantity, purity and diversity, but also the genes of 16S rRNA and amoA can be amplified by RT-PCR. Compared with other methods, it showed great advantage of low cost and high efficiency and can be applied to RNA extraction of activated sludge in a large number. Furthermore, T-RFLP results indicated that the community composition as well as the abundance of individual members was affected by the kind of RNA extraction methods. This work established a rapid and effective method to extract high-quality RNA from activated sludge and would show great potential for monitoring microbial changes and studying metabolism and community array of activated sludge.
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PMID:[Rapid method to extract high-quality RNA from activated sludge]. 2032 49

New rat monoclonal antibodies (mAbs) for DDT [1,1,1 -trichloro-2,2-bis (4-chlorophenyl) ethane], namely DDT 7C12, DDT 1C1, and DDT 1B2, were developed, characterized, and applied in ELISA both in coating antigen and in enzyme-tracer format. The latter used horseradish peroxidase (HRP) or glucose oxidase as enzymes. The lowest concentration of p,p'-DDT was determined with mAb DDT 7C12 and DDT-hapten HRP, with a test midpoint (IC50) of 0.5 +/- 0.2 microg/L (n=10) in 40 mM PBS (phosphate-buffered saline). The mouse anti-rat immunoglobulin lambda-light chain mAb LA1B12 was used as capture mAb. The best IC50 for o,p'-DDT in 40 mM PBS was 1.0 +/- 0.3 microg/L (n=12) and was obtained with mAb DDT 1C1 and DDT-hapten HRP, whereas mAb DDT 1B2 was very selective for p,p'-DDT with an IC50 of 4.2 + 1.6 microg/L (in 40 mM PBS, n=9). An optical immunosensor was optimized and applied for the analysis of DDT (or DDT equivalents). This immunosensor consists of a bench-top optical readout device and disposable sensor chips, which include the fluidic system. Evanescent field excitation and emission of the fluorophore Oyster-645 was used. An IC50 for p,p'-DDT [in 5% (v/v) isopropanol in 40 mM PBS] of 4 microg/L was obtained using DDT 7C12-Oyster-645. ELISA and immunosensor were used for the analysis of p,p'-DDT in unspiked and spiked surface water samples. Within the working ranges of these immunotechniques, recoveries ranged from 80 to 120%.
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PMID:Analysis of DDT isomers with enzyme-linked immunosorbent assay and optical immunosensor based on rat monoclonal antibodies as biological recognition elements. 2033 65


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