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At least 145 records · Page 8Linked to original sources
PubMed1989

Presence of contaminating mitochondrial DNA from host reticulocytes in experimental infections of Plasmodium berghei.

Superposition of two unrelated processes, namely terminal reticulocyte differentiation and synchronous plasmodial development, takes place in experimental infections of Plasmodium berghei. The first process is shown to be responsible for the appearance of some discrete restriction bands of host origin when DNA is extracted from leucocyte-free blood containing synchronous parasites at early stages of infection. These discrete DNA fragments cross-hybridize with host cell mitochondrial DNA. Purification steps are suggested to reduce this effect, which might be relevant also in the case of other plasmodial species exhibiting preference for reticulocytes as host cell.

Animals↗
PubMed1991

A simple technique for removing plant polysaccharide contaminants from DNA.

A survey of the inhibitory effects of various plant polysaccharides on DNA restrictions (HindIII and EcoRI) revealed that neutral polysaccharides (arabino-galactan, dextran, gum guar, gum locust bean, beta-glucan, inulin, laminaran, mannan and starch) were not very inhibitory. In contrast, acidic polysaccharides (carrageenan, dextran sulfate, gum ghatti, gum karaya, pectin and xylan) were very inhibitory, even at low concentrations. The Elutip-d (RPC-5 type resin) was evaluated for removal of the inhibitory polysaccharides. Used alone or in combination with a phenol/chloroform wash, it proved effective in removing the polysaccharide so that HindIII digestion was possible, except in the cases of carrageenan and dextran sulfate. In addition, the genomic DNA extracts from live oak (Quercus virginiana) and magnolia (Magnolia grandiflora) were sufficiently purified so that the DNAs could be restricted with both EcoRI and HindIII.

DNA↗
PubMed1996

Assays for microbial contamination and DNA analysis based on electrorotation.

A new assay is described for determining the concentration and viability of waterborne micro-organisms. The method exploits the fact that when a latex bead coated with a specific binding agent is complexed to the target analyte, the analyte complex formed assumes new dielectric properties that can be monitored by its electrorotation response. This generic technology is applicable to a wide range of organism and toxicological diagnostic tests, as well as to other areas of biotechnology. An example is given of its possible application to DNA sequence analysis.

Animals↗
PubMed2001

Parvovirus B19 DNA in plasma pools and plasma derivatives.

BACKGROUND AND OBJECTIVES: Human parvovirus B19 (B19) has been transmitted by various plasma-derived medicinal products. The aim of this study was to determine the frequency and the level of B19 DNA contamination in plasma pools destined for fractionation and in a broad range of plasma derivatives. In addition, removal of B19 DNA by the manufacturing process was investigated in cases where corresponding samples from plasma pool and product were available. MATERIALS AND METHODS: Plasma pool samples and blood products were tested for B19 DNA by nested polymerase chain reaction (PCR), and the viral DNA content was determined by TaqMan quantitative PCR. RESULTS: Two-hundred and twenty two of 372 plasma pools for fractionation contained B19 DNA at concentrations of 10(2)-10(8) genome equivalents/ml (geq/ml). While approximately 65% of the DNA-positive plasma pools were only moderately contaminated (< 10(5) geq/ml), 35% contained > 10(6) geq/ml. High frequencies of contamination were detected in Factor VIII (79 of 91), prothrombin complex concentrates (38 of 43) and Factor IX (41 of 62), where the concentration of B19 DNA ranged between 102 and 107 geq/ml. A lower level of B19 DNA contamination was found in antithrombin III (five of 26 samples), in anti-D immunoglobulins (three of 37 samples) and in albumin (four of 51 samples), with levels ranging between 10(2) and 10(3) geq/ml. Furthermore, investigation of plasma pools for solvent/detergent plasma (S/D plasma), from two manufacturers, revealed B19 DNA in 15 of 66 batches at concentrations of 10(2)-10(8) geq/ml. Similar concentrations were detected in the corresponding final S/D plasma products. Anti-B19 immunoglobulin G (IgG) was found in plasma pools and S/D plasma at concentrations of approximately 40 IU/ml. CONCLUSION: Although positive PCR results do not necessarily reflect infectivity, these data show that B19 is a common contaminant in plasma pools and in plasma-derived medicinal products. Considering the resistance of animal parvoviruses to inactivation by heat and chemical agents, and the absence of specific information for B19, the risk of B19 transmission by plasma products should be considered. Physicians should be aware of this problem when treating patients of B19-related risk groups. The plasma fractionation industry should continue their efforts to avoid B19 contamination of plasma derivatives and develop methods which are effective in removing/inactivating parvovirus B19.

Blood↗
PubMed1993

Restriction endonuclease digestion eliminates product contamination in reverse transcribed polymerase chain reaction.

Restriction endonuclease digestion was used to eliminate false-positive signals caused by polymerase chain reaction (PCR) product DNA contamination in a reverse transcribed (RT) PCR for amplifying rubella virus (RV) RNA sequences. A restriction enzyme selected to cut the PCR product DNA between, but not within, the primer binding sites was used to digest reaction mixtures after reverse transcription but before PCR amplification. Because restriction enzymes generally react only with specific double-strand sequences, contaminating DNA was rendered inactive while reverse-transcribed single strand cDNA was amplified. Assays showed that restriction enzyme digestion reduced template activity of product DNA by a factor of 10(7), while leaving sensitivity of the RT-PCR unaffected.

Animals↗
PubMed2002

Analysis of HLA-DRB1*-A* and -B* alleles in prenatal diagnosis for determination of maternal contamination in fetal DNA.

During chorionic villi sampling for prenatal diagnosis with molecular biology techniques, contamination by maternal decidua frequently occurs and can lead to misinterpretation of the test results. To avoid such problems, we present a new method for appraising maternal contamination of fetal DNA, based on genomic typing of the highly variable human leukocyte antigen (HLA) locus-DRB1*, locus A* and locus B* regions by genetic amplification with sequence-specific primers and PCR. Fetal DNA samples obtained for beta-thalassemia diagnosis were analysed after artificial contamination with increasing maternal DNA concentrations ranging from 0.5 to 10% (0.5, 1, 3, 5 and 10%). The approach was found to be rapid, specific, reproducible and highly sensitive and permits recognition of 1-3% contamination by maternal DNA concentrations. The system currently used for detecting maternal DNA contamination in fetal samples is the analysis of polymorphic loci by variable number of tandem repeats and/or short tandem repeats. We propose that the analysis of HLA alleles may provide a valid alternative or complement to this system.

Alleles↗
PubMed2003

Measurement of mitochondrial DNA synthesis in vivo using a stable isotope-mass spectrometric technique.

We describe here a new stable isotope-mass spectrometric technique for measuring mitochondrial DNA (mtDNA) synthesis. Growing (2-4 mo old) and weight-stable (8-10 mo old) Sprague-Dawley rats were primed with (2)H(2)O (deuterated water) to 2.0-2.5% body water enrichment, via intraperitoneal injection, and then given 4% (2)H(2)O in drinking water for 3-11 wk. Mitochondria were isolated from cardiac and hindlimb muscle, and mtDNA was isolated and enzymatically hydrolyzed to deoxyribonucleosides. PCR confirmed the absence of nuclear DNA contamination. The isotopic enrichment of the deoxyribose moiety of deoxyadenosine was determined by GC-MS analysis, and percent new mtDNA was calculated by comparison to genomic DNA enrichments in a tissue with nearly complete turnover (bone marrow). Initial label incorporation into deoxyadenosine of mtDNA was linear, and turnover of mtDNA was observed in nongrowing adult female rats (1.1-1.3% new mtDNA per day in cardiac and skeletal muscle). Die-away curves of mtDNA after discontinuing (2)H(2)O administration gave a similar turnover rate constant. Human subjects were also given (2)H(2)O for up to 6 wk, and mitochondria from platelets were isolated. Incubation with DNase removed any contaminating genomic DNA; platelet mtDNA exhibited linear incorporation from (2)H(2)O and reached plateau values identical to those in genomic DNA from fully turned over cells (circulating monocytes). In conclusion, replication of mtDNA can be directly measured in vivo in rodents and humans without the use of radioactivity. Use of this technique may allow improved understanding of the regulation of mitochondrial biogenesis in health and disease.

Animals↗
PubMed1989

Detection of total DNA with single-stranded DNA binding protein conjugates.

We have developed a rapid and sensitive method for total DNA measurement using single-stranded DNA binding protein from E coli conjugated with horseradish peroxidase or urease. To detect DNA, the sample is heated or alkali treated to denature the DNA and then filtered through nylon or nitrocellulose membranes. After the single-stranded DNA is bound to the membrane, single-stranded DNA binding protein enzyme-conjugate is incubated with the membrane. Next, the unbound conjugate is washed off the membrane and the bound conjugate detected colorimetrically. The assay can detect 10 pg of DNA in less than 3 hr. This method can be applied to the detection of DNA contamination in therapeutic proteins produced by recombinant DNA or hybridoma techniques.

Colorimetry↗
PubMed2006

The application of ultraviolet irradiation to exogenous sources of DNA in plasticware and water for the amplification of low copy number DNA.

Using high sensitivity forensic STR polymerase chain reaction (PCR) typing procedures, we have found low concentrations of DNA contamination in plasticware and water assumed to be sterile, which is not detected by standard DNA procedures. One technique commonly used to eliminate the presence of DNA is ultraviolet (UV) irradiation; we optimized such a protocol used in the treatment of water, tubes, plates, and tips for low copy number DNA (LCN) amplification. UV light from a Stratalinker((R)) 2400 was administered to 0.2, 1.5 mL tubes, and PCR plates contaminated with up to 500 pg of DNA. They were subsequently quantified with an ALU-based real-time PCR method using the Rotorgene 3000. Overall, there was a decrease in concentration of DNA recovered as the duration of treatment increased. Nonetheless, following 45 min of irradiating a PCR plate with 500 pg of DNA, nearly 6 pg were still detected. However, when the plate was raised within an inch of the UV source, less than 0.2 pg of DNA was detected. Additionally, lining the area around the samples with aluminum foil further reduced the amount of time necessary for irradiation, as only 30 min eliminated the presence DNA in the raised PCR plate. Similar experiments were conducted using tubes filled with a solution of DNA and water in equivalent concentrations for 50, 15, and 1.5 mL tubes with comparative results. It is plausible that the aluminum foil increased the amount of reflection in the area thereby enhancing penetration of UV rays through the walls of the plasticware. This protocol was tested for the possibility of inhibitors produced from irradiation of plastic tubes. As our protocols require less irradiation time than previous studies, PCR sensitivity was not affected. Moreover, the lifespan of the UV lamps was extended. Our findings demonstrate that this method is useful as an additional precautionary measure to prevent amplification of extraneous DNA from plasticware and water without compromising the sensitivity of LCN DNA amplifications.

DNA↗
PubMed1994

Lipopolysaccharide is a frequent contaminant of plasmid DNA preparations and can be toxic to primary human cells in the presence of adenovirus.

Endotoxin (lipopolysaccharide, LPS) is commonly found as a contaminant in plasmid DNA preparations. We demonstrate here that the quantities of LPS typically contaminating DNA preparations can generate a toxicity to primary cells (primary human skin fibroblasts, primary human melanoma cells) in the presence of entry-competent adenovirus particles. Toxicity can be observed with as little as 100 ng/ml free LPS or 100 pg/ml LPS when the LPS is assembled into polylysine/adenovirus complexes. Simple and effective methods of removing the contaminating LPS using either a polymyxin B resin or Triton X-114 extraction are described. Treatment of DNA samples to remove LPS eliminates the toxicity to primary cells.

Adenoviridae↗
PubMedDate not supplied

Adenosine diphosphate ribosyltransferase and protein acceptors associated with cytoplasmic free messenger ribonucleoprotein particles.

ADP-ribosyltransferase activity has been characterized in free messenger ribonucleoprotein particles (mRNP) from mouse plasmacytoma cells. This enzymatic activity appears to be associated with the free mRNP and not due to nuclear contamination. The enzyme activity is not stimulated by added DNA or histone H1 and represents 34 per cent of the total cellular ADP-ribosyltransferase activity while the DNA contamination in free mRNP is less than 4 per cent of the total cellular DNA. Moreover, the ADP-ribosyltransferase specific activity per mg of DNA is about 75-fold higher in free mRNP than in the nuclei. During CsCl gradient centrifugation of the cytoplasmic fraction, the ADP-ribosylated material separates out at a buoyant density similar to that of free mRNP. This ADP-ribosyltransferase activity is inhibited by thymidine, nicotinamide and 3-aminobenzamide, while it is highly stimulated by exogenous pancreatic RNase. The in vitro synthesized acid insoluble material is rendered partly soluble by treatment by a proteolytic enzyme or by snake venom phosphodiesterase resulting in phosphoribosyl-AMP formation: the pancreatic RNase does not solubilize this material. Several ADP-ribosylated proteins are detected by lithium dodecylsulfate gel electrophoresis. Such an ADP-ribosyltransferase activity has also been detected in free mRNP from rat liver. It is suggested that this ADP-ribosylation of specific free mRNP proteins may be associated with free mRNP structure and/or with some chemical covalent type of modification rendering mRNA available for translation.

Adenosine Diphosphate Ribose↗
PubMed2006

Characterization of differential gene expression profiles in diabetic embryopathy using DNA microarray analysis.

OBJECTIVE: The molecular mechanisms by which maternal diabetes impairs embryogenesis are not established. This study aimed to determine the developmental genes and molecular pathways that are involved in diabetic embryopathy, by comparing gene expression profiles in the yolk sacs between the embryos of diabetic and control rats by using DNA microarray analysis. STUDY DESIGN: Diabetes was induced in female rats by injecting streptozotocin (65 mg/kg) intravenously. Glucose levels were controlled by subcutaneously implanting insulin pellets. The female rats were mated with normal male rats. At gestation day 4, the insulin pellets were removed from a group of animals, making them hyperglycemic. The animals with insulin pellets served as controls. At gestational day 12, embryos were explanted, and yolk sacs were collected. Total RNA, free of DNA contamination, was extracted from the yolk sacs. Complementary DNA probes were synthesized, labeled with Cy3 and Cy5 fluorescent dyes, and used to hybridize rat oligo-array containing 10,000 genes. Data were analyzed by using 1-sample t test on log2 ratios, with P < .05 representing a significant difference. The changes in expression levels of important genes were verified with the use of a real-time polymerase chain reaction (PCR). RESULTS: Five microarray experiments produced consistent results. A total of 101 genes were found to be differentially expressed between the embryos of diabetic and control rats. Analyses that used PathwayAssist (Ariadne Genomics, Rockville, MD) revealed a number of potential signaling pathways and genes involved in insulin signaling and stress response (insulin 2, insulin-binding protein 1, GST pi1), cell growth (GAP43, CSF1R, HGF), calcium signaling (calbindin 3, CBP A6), and PKC signaling (PKCBP beta15, FABP5), in concert with prior biochemical and molecular findings. CONCLUSION: These observations show significant alterations in expression of developmental and stress response genes in diabetic embryopathy, and demonstrate, for the first time, that the yolk sac cells express insulin during early development. In addition, these data also demonstrate that hyperglycemia induces altered gene expression, resulting in aberrant cell signaling, morphogenesis, and embryopathy.

Animals↗
PubMed2005

Detection of bovine papillomavirus DNA on the normal skin and in the habitual surroundings of horses with and without equine sarcoids.

The purpose of the present study was to examine whether bovine papillomavirus (BPV) DNA can be detected on the normal skin and in the habitual surroundings of horses with and without equine sarcoids by means of superficially taken swabs. In affected horses, no significant difference in presence of BPV-DNA could be observed between samples obtained from the equine sarcoid surface, from normal skin close to the tumour and from a normal skin site in direct contact with the tumour. From the group of healthy horses living in contact with affected horses, 44% were BPV-DNA positive. The surroundings of affected and non-affected horses are probably not a major source of BPV-DNA contamination. It can be concluded that BPV-DNA is present on the normal skin of horses affected by equine sarcoid and to a lesser degree, on the normal skin of horses living in contact with affected horses.

Animals↗
PubMed1989

Autoantibodies to DNA topoisomerase II in cryptogenic fibrosing alveolitis and connective tissue disease.

Sera from patients with autoimmune lung and connective tissue diseases were investigated for antibodies to topoisomerase II. Anti-topoisomerase II antibodies were detected by ELISA in 37% of sera from patients with cryptogenic fibrosing alveolitis (CFA), in one (8%) case of sarcoidosis and in 31% of sera from systemic lupus erythematosus (SLE) patients. Sera from rheumatoid arthritis, juvenile rheumatoid arthritis, progressive systemic sclerosis, Sjögren's syndrome and undifferentiated connective tissue disease were negative. CFA and sarcoidosis sera strongly reacted in immunoblotting with a 170 kD protein, also recognized by rabbit antiserum to recombinant topoisomerase II, while SLE sera presented a weak reaction. Pre-adsorption with dsDNA dramatically decreased the topoisomerase II binding in ELISA by the most positive SLE serum, but did not affect the binding by the most positive CFA serum, thus indicating that anti-topoisomerase II reactivity of SLE sera is probably due either to cross-reacting antibodies or, in part, to minor DNA contamination of our enzyme preparation. The determination of DNA topoisomerase II relaxation activity, performed after incubation with antibody-positive sera, showed that only CFA sera precipitate enzymatic activity. The finding that CFA presents antibodies to an enzyme essential to cell survival stresses the role of autoimmunity in the pathogenesis of idiopathic pulmonary fibrosis. This may offer further insight into the cause of autoimmune disease and prove a valuable tool in the study of enzyme molecular biology.

Adolescent↗
PubMed1998

Host cells in the spiral canal of Taenia solium (Cestoda) cysticerci.

The presence of host inflammatory cells inside the spiral canal of all viable Taenia solium cysts obtained from naturally infected pigs is described. Cells can penetrate into the vicinity of suckers and rostellum, although most appear damaged, suggesting that conditions in the canal are deleterious for them. These observations extend the localization of host inflammatory infiltrate to this intricate microniche, which may offer new approaches for the treatment of cysticercosis, based on a scolex-targeted action. The presence of host cells in the canal of cysts also poses the problem of the resulting contamination with host materials in studies using cysts extracts. As an example, host DNA contamination is readily detectable in genomic DNA isolated from T. solium and Taenia taeniaeformis cysts, as demonstrated by polymerase chain reaction amplification and subsequent sequencing of a segment of the 18S ribosomal gene.

Animals↗