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)

Serine proteases, such as alpha-chymotrypsin or elastase, caused an aggregation of rat ascites tumour cell lines, AH-130, AH-109A and YS, in a protein free medium which preserved the cell viability. This aggregation, which was monitored spectrophotometrically, was dependent upon the protease activities and was resistant to treatment with either a calcium chelating reagent (EDTA) or neuraminidase. However, the tumour cell aggregates were redispersed by treatment with deoxyribonuclease I (DNase I). This dispersal effect was dependent upon the DNase activity. A possible relationship between the tumour cell aggregation and development of blood-borne metastasis was studied. An intravenous inoculation in rats of tumour cell aggregates performed by the alpha-chymotrypsin treatment resulted in significantly higher numbers of lung metastatic foci than an injection of single cells. When the re-separated single cells, prepared in vitro by treatment with DNase I following alpha-chymotrypsin treatment, were injected instead of the aggregates, the enhancement of metastasis was reversed. These enhancement and reversal effects were mimicked in vivo by intravenous injections of protease and nuclease following inoculation of a single cell suspension. That is, the number of metastatic foci caused by single cell inoculation followed by an intravenous alpha-chymotrypsin injection, was higher than that in a control group receiving PBS instead of alpha-chymotrypsin. Again, this augmentation was reversed by an injection of DNase I following alpha-chymotrypsin injection. Furthermore, an injection of DNase I alone itself reduced the starting number of metastases resulting from injection of the single tumour cell suspension. These data suggest that the metastatic behaviour of tumour cells may be increased by protease inducible DNA dependent cell aggregation should it occur in the blood stream.
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PMID:Serine protease-induced enhancement of blood-borne metastasis of rat ascites tumour cells and its prevention with deoxyribonuclease. 212 Dec 20

Immunohistochemical and immunochemical analysis using Western blot techniques were carried out with estrogen receptor (ER) monoclonal antibody H-222 to 1) clarify the "nuclear translocation" phenomenon of ER, 2) elucidate the primary nuclear binding site of ER, and 3) to evaluate the binding force between ER and its nuclear binding site in the uterus of ovariectomized adult mice. Exclusive nuclear localization of ER was recognized in the epithelial cells, stroma cells, and smooth muscle cells. Uterine tissues prepared from animals injected with saline, 17 beta-estradiol (E2), estriol (E3), and diethylstilbestrol (DES) exhibited almost the same ER immunostaining when they were fixed prior to sectioning (prefixation method) and frozen sections were used. On the other hand, when fresh-frozen sections were fixed before or after incubation with various solutions (postfixation method) and then treated with various salt solutions, greater differences were seen in immunostaining of ER between saline-injected and hormone-treated animals. Immunostaining of ER in control animals was low after incubation with PBS (0.01 M phosphate buffer containing 0.16 M NaCl, pH 7.2), whereas uterine tissue from hormone-injected mice showed strong nuclear immunostaining after this treatment. After treatment with 0.4 M KCl or 0.5 M NaCl, immunostaining in the uterus of both hormone-injected and control animals was completely abolished. DNase treatment caused an almost complete loss of immunostaining of ER; however, RNase digestion slightly increased immunoreactivity in both E2-injected and control animals. Quantitative analysis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot techniques showed that after incubation of tissue sections for 30 min with PBS, 0.4 M KCl, or DNase, 60%, 10%, and 30% of ER were present, respectively, compared to amount of ER present in unincubated sections. These findings suggest the following for the ER in uterine tissue; nuclear occupancy is a phenomenon that occurs due to a differential affinity between occupied and unoccupied receptors in the nucleus; after hormone treatment, the receptor levels do not fluctuate in the nucleus to the extent demonstrated by binding assays; and the properties of the ER detected in the immunohistochemical analysis are identical to those observed in biochemical studies.
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PMID:Immunological analysis of the biochemical properties of the uterine estrogen receptor. 277 19

INTRODUCTION: Between 20% and 50% of middle ear effusions in otitis media with effusion test positive for bacteria, i.e. H. influenzae, M. catarrhalis and S. pneumoniae. In one study 48% of effusions that tested negative by culture wre positive by polymerase chain reaction (PCR).1 This has been advanced as evidence for the presence of bacterial biofilms, as agents leading to the persistence of glue ear. There is, however, the possibility that the DNA detected is the fossilized remains of bacteria from previously cleared infections. If this is the case, then the effusioin must in some way be protecting the DNA from breakdown by DNases. METHODS: Here we demonstrate, using a viscosity assay, that middle ear effusions taken from children during myringotomy inhibit the breakdown of DNA in a concentration-dependent manner. Middle ear effusion homogenates 0.5-3.0 ml (1 : 10 Vol. vol/effusion: PBS) were incubated with 3 ml of DNA (0.5 mg/ml in PBS) plus 0.2 ml of DNase 1 (500 Kunitz) at 37 degrees C for 24 h. Viscosity measurements were taken at regular intervals and the changes in viscosity expressed as a percentage of time 0. DNase activity was inhibited by 1.5 ml and 3.0 ml of effusion 48% and 91%, respectively, after 30 min incubation. CONCLUSION: This study demonstrated that effusions have the ability to inhibit nuclease activity, and the reported presence of non-culturable bacteria based on DNA detection by PCR could still represent fossilized remains and not viable bacteria. The same could also be true of recent reports of bacterial mRNA.2
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PMID:Does the bacterial DNA found in middle ear effusions come from viable bacteria? 1112 7

Topical gene delivery to the skin shows great potential for painless, non-invasive administration of novel vaccines and therapeutic agents. The challenge is to develop a pharmaceutically acceptable system that can deliver suitable amounts of plasmid DNA to produce the desired level of response. The purpose of this study was to quantitatively assess DNA delivery by a novel lipid-based biphasic delivery system into the viable layers of excised human skin. Biphasic lipid vesicle formulations, incorporating plasmid DNA were evaluated in vitro in flow-through diffusion cells. Fifty mg DNA formulation containing 10 microg DNA was applied to full-thickness human breast skin for 24 hours. Residual formulation was removed and the skin was washed with PBS, then tape-stripped, followed by DNase treatment to remove surface bound DNA. Skin samples were homogenised and digested overnight with Proteinase K. The resulting supernatant was used as a template for quantitative PCR. Three formulations yielded a significant degree of dermal absorption compared to the controls. Formulation 26-3-2-DNA indicated that approximately 1x10(9) copies of plasmid were absorbed per cm2 skin. Other formulations resulted in 5x10(6) copies/cm2 skin (17C3-1-DNA) and 5x10(8) copies/cm2 skin (26-3-1-DNA). Biphasic vesicles delivered significant quantities of plasmid DNA into the 'viable' layers of human skin in vitro. The successful delivery of this large (approximately 4,400 kDa) charged molecule through intact stratum corneum represents a major advance in transdermal macromolecule delivery.
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PMID:Gene delivery into human skin in vitro using biphasic lipid vesicles. 1647 98

A DNase from Bacillus subtilis which specifically hydrolyzes native DNA of phage PBS 1 has been purified and characterized. The mode of action of the enzyme is endonucleolytic, yielding deoxyuridine and oliogonucleotides of various sizes. The primary site of enzymatic attack is deoxyuridylic acid in the DNA. A mild nitrous acid treatment of thymine-containing thymus DNA, which deaminates 30% of the cytosine residues, renders the DNA susceptible to the DNase. Nicked DNA from coli phage T5 and hydroxymethyluracil-containing DNA from phage PBS 15 are not sensitive to this DNase.
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PMID:DNase Specific for Uracil-Containing Bacteriophage DNA. 1678 46

RECIPES: Ammonium chloride lysing solution, 10x. Complete DMEM. Complete RPMI. DTT (DL-dithiothreitol), 0.1 M. EDTA (ethylenediamine tetraacetic acid), 0.5 M, pH 8. Ethidium bromide staining solution. FBS (fetal bovine serum). Formamide, deionized. Gel loading buffer, 6x. L-Glutamine, 0.2 M (100x). HBSS (Hanks' buffered salt solution). PBS (phosphate-buffered saline). RNase A stock solution (DNase-free), 2 mg/ml. SSC, 20x. TAE buffer, 50x. TBE buffer, 10x. TE buffer. TrisCl, 1 M. Trypsin/EDTA solution.
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PMID:Common stock solutions, buffers, and media. 1877 Jun 54

Flow cytometry provides a rapid and high-content multiparameter analysis of individual microorganisms within a population. In the past years, several fluorescent stains were developed in order to monitor DNA content distribution and cell-cycle phases, mainly in eukaryotic cells. Recently, due to its low detection limits, several of these fluorescent stains were also applied to prokaryotic cells. In this study, the ability of a novel far-red fluorescent stain DRAQ5 in assessing intracellular DNA content distribution in Escherichia coli DH5alpha was evaluated. The results showed that a DRAQ5-labelled live E. coli suspension can be obtained by incubation of 1 x 10(6) cells/mL with 5 microM DRAQ5 in PBS buffer supplemented with EDTA (pH = 7.4) during 30 min at 37 degrees C. Flow cytometric analysis of fixed E. coli cells revealed that ethanol should be used in detriment of glutaraldehyde for DRAQ5 labelling. After the analysis of RNase and DNase digested samples, DRAQ5 was proven to be a specific DNA labelling stain. The present study demonstrates that the use of DRAQ5 as a DNA-labelling stain provides an easy assessment of intracellular DNA content and cell-cycle phases in gram-negative bacteria such as E. coli.
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PMID:The use of DRAQ5 to monitor intracellular DNA in Escherichia coli by flow cytometry. 2035 7

Neutrophils are the host's first line of defense against infections, and their extracellular traps (NET) were recently shown to kill Leishmania parasites. Here we report a NET-destroying molecule (Lundep) from the salivary glands of Lutzomyia longipalpis. Previous analysis of the sialotranscriptome of Lu. longipalpis showed the potential presence of an endonuclease. Indeed, not only was the cloned cDNA (Lundep) shown to encode a highly active ss- and dsDNAse, but also the same activity was demonstrated to be secreted by salivary glands of female Lu. longipalpis. Lundep hydrolyzes both ss- and dsDNA with little sequence specificity with a calculated DNase activity of 300000 Kunitz units per mg of protein. Disruption of PMA (phorbol 12 myristate 13 acetate)- or parasite-induced NETs by treatment with recombinant Lundep or salivary gland homogenates increases parasite survival in neutrophils. Furthermore, co-injection of recombinant Lundep with metacyclic promastigotes significantly exacerbates Leishmania infection in mice when compared with PBS alone or inactive (mutagenized) Lundep. We hypothesize that Lundep helps the parasite to establish an infection by allowing it to escape from the leishmanicidal activity of NETs early after inoculation. Lundep may also assist blood meal intake by lowering the local viscosity caused by the release of host DNA and as an anticoagulant by inhibiting the intrinsic pathway of coagulation.
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PMID:Lundep, a sand fly salivary endonuclease increases Leishmania parasite survival in neutrophils and inhibits XIIa contact activation in human plasma. 2451 88

Listeria monocytogenes is able to form biofilms on various surfaces and this ability is thought to contribute to persistence in the environment and on contact surfaces in the food industry. Extracellular DNA (eDNA) is a component of the biofilm matrix of many bacterial species and was shown to play a role in biofilm establishment of L. monocytogenes. In the present study, the effect of DNaseI treatment on biofilm formation of L. monocytogenes EGD-e was investigated under static and dynamic conditions in normal or diluted complex medium at different temperatures. Biofilm formation was quantified by crystal violet staining or visualized by confocal laser scanning microscopy. Biomass of surface-attached L. monocytogenes varies depending on temperature and dilution of media. Interestingly, L. monocytogenes EGD-e forms DNase-sensitive biofilms in diluted medium whereas in full strength medium DNaseI treatment had no effect. In line with these observations, eDNA is present in the matrix of biofilms grown in diluted but not full strength medium and supernatants of biofilms grown in diluted medium contain chromosomal DNA. The DNase-sensitive phenotype could be clearly linked to reduced ionic strength in the environment since dilution of medium in PBS or saline abolished DNase sensitivity. Several other but not all species of the genus Listeria display DNase-sensitive and -resistant modes of biofilm formation. These results indicate that L. monocytogenes biofilms are DNase-sensitive especially at low ionic strength, which might favor bacterial lysis and release of chromosomal DNA. Since low nutrient concentrations with increased osmotic pressure are conditions frequently found in food processing environments, DNaseI treatment represents an option to prevent or remove Listeria biofilms in industrial settings.
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PMID:DNase-Sensitive and -Resistant Modes of Biofilm Formation by Listeria monocytogenes. 2673 72

Numerous studies have shown the presence of DNA lesions in human spermatozoa affecting sperm quality. However, the nature of this anomaly and its relationship with patient etiology are poorly understood since different mechanisms can be involved in the formation of these novel DNA configurations including the action of a seminal plasma nuclease activity. The objective of this study was to assess the capacity of seminal plasma for producing endogenous DNA cleavage using nuclei of peripheral blood leukocytes as external targets. For this purpose, we used seminal plasma from fertile males with normal semen parameters to produce DNA cleavage in a sample of leukocytes. Three different tests were performed to visualize DNA cleavage: (a) DNase activity detection, (b) DNA Breakage Detection-Fluorescence In Situ Hybridization (DBD-FISH), and (c) Two-dimensional comet assay (Two-tail comet assay). Our results demonstrate that: (i) the seminal plasma is able to cleave DNA compacted with histones in the leukocytes; (ii) this DNA cleavage can be associated with DNase activity and (iii) DNA damage mainly corresponds to single-strand DNA breaks. In conclusion, capacity of seminal plasma for producing DNA cleavage represents a solid contribution to expand the analysis of the standard seminal profile and could constitute a putative diagnostic tool for evaluating male infertility.Abbreviations: ALS: alkali labile sites; ART: Assisted Reproduction Technologies; DBD-FISH: DNA Breakage Detection-Fluorescence In Situ Hybridization; DNA: deoxyribonucleic acid; DSBs-DNA: double-strand DNA; FITC: Fluorescein IsoThioCyanate; GEDA: Gravity Enforced Diffusion Assays; PBS: phosphate-buffered saline; ROS: Reactive Oxigen Species; SSBs-DNA: single-strand DNA; SSC: saline-sodium citrate.
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PMID:Characterization of DNA cleavage produced by seminal plasma using leukocytes as a cell target. 3153 84


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