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At least 1,153 records · Page 64Linked to original sources
PubMed2001

Thirty-five year mortality following receipt of SV40- contaminated polio vaccine during the neonatal period.

Early poliovirus vaccines, both inactivated and live attenuated, were inadvertently contaminated with simian virus 40 (SV40), a monkey virus known to be oncogenic for newborn hamsters. Although large epidemiologic studies have not identified an elevated cancer risk in persons who received SV40-contaminated vaccines, fragments of SV40 DNA have recently been identified in certain human tumours. We report the follow-up of a cohort of 1073 persons, unique because they received SV40-contaminated poliovirus vaccines as newborns in 1961-63. A previous report of the status of these subjects as of 1977-79 identified 15 deaths, none due to cancer. The present study utilized the National Death Index to identify deaths in the cohort for the years 1979-96. Expected deaths were calculated from Cleveland area sex-, age-, race- and year-specific mortality rates. Increased mortality from all causes was not found. 4 deaths from cancer were found compared to 3.16 expected (P = 0.77). However, 2 deaths from testicular cancer occurred, compared to 0.05 expected (P = 0.002), which may be a chance finding due to multiple comparisons. There were 2 deaths due to leukaemia, a non-significant finding, and no deaths due to tumours of the types putatively associated with SV40. Although these results are, for the most part, consistent with other negative epidemiologic investigations of risks from SV40-contaminated vaccines, further study of testicular cancer may be warranted, and it will be important to continue monitoring this cohort which is now reaching middle-age.

Adult↗
PubMed2006

Biomarkers signal contaminant effects on the organs of English sole (Parophrys vetulus) from Puget Sound.

Fish living in contaminated environments accumulate toxic chemicals in their tissues. Biomarkers are needed to identify the resulting health effects, particularly focusing on early changes at a subcellular level. We used a suite of complementary biomarkers to signal contaminant-induced changes in the DNA structure and cellular physiology of the livers and gills of English sole (Parophrys vetulus) . These sediment-dwelling fish were obtained from the industrialized lower Duwamish River (DR) in Seattle, Washington, and from Quartermaster Harbor (QMH) , a relatively clean reference site in south Puget Sound. Fourier transform-infrared (FT-IR) spectroscopy, liquid chromatography/mass spectrometry (LC/MS) , and gas chromatography/mass spectrometry (GC/MS) identified potentially deleterious alterations in the DNA structure of the DR fish livers and gills, compared with the QMH fish. Expression of CYP1A (a member of the cytochrome P450 multigene family of enzymes) signaled changes in the liver associated with the oxidation of organic xenobiotics, as previously found with the gill. The FT-IR models demonstrated that the liver DNA of the DR fish had a unique structure likely arising from exposure to environmental chemicals. Analysis by LC/MS and GC/MS showed higher concentrations of DNA base lesions in the liver DNA of the DR fish, suggesting that these base modifications contributed to this discrete DNA structure. A comparable analysis by LC/MS and GC/MS of base modifications provided similar results with the gill. The biomarkers described are highly promising for identifying contaminant-induced stresses in fish populations from polluted and reference sites and, in addition, for monitoring the progress of remedial actions.

Animals↗
PubMed1998

Fluorescence-based analysis of DNA ploidy and cell proliferation within fine-needle samplings of breast tumors: a new approach using automated image cytometry.

BACKGROUND: Automated image cytometry can allow concurrent quantification of several parameters in each individual cell within a population, opening new possibilities for diagnosis and prognosis. In this study, the authors investigated the capacity of this method for performing a bivariate analysis of DNA ploidy and synthesis in fine-needle samplings obtained without aspiration from breast tumors. METHODS: Samplings from 25 unselected cases of ductal infiltrative breast adenocarcinoma and 2 cases of fibroadenoma were analyzed. For each case, 3-5 slides (containing approximately 1000 cells each) were quantified to assess experimental precision. Ploidy was determined by fluorescent staining of DNA using 4,6-diamidino-2-phenylindole (DAPI). Contaminating lymphocytes were taken as internal controls to calculate DNA indices. DNA synthesis was analyzed by immunofluorescent detection of 5-bromodeoxyuridine (BrdU) incorporation. Measurements were compared with flow cytometric data obtained from the same patients. RESULTS: Relative error in determination of DNA indices was generally below 5%. Determination of proliferation indices were more variable, with a mean relative error of 25%. Two different populations of BrdU positive cells were detected systematically, one in the diploid and another in the aneuploid fraction. For both cytometric methods, DNA indices were similar in all 27 cases, whereas BrdU labeling indices showed no significant correlation in 13 cases. The remaining cases were not comparable due to lack of flow cytometric data. Labeling indices obtained by image cytometry did not reveal any significant correlation with Scarff-Bloom-Richardson grading or clinical staging. CONCLUSIONS: Automated image cytometry allows concurrent measurement of ploidy and cell proliferation within individual breast carcinoma cells. Statistical reliability can be reached with a relative small number of cells (1000), which is crucial for samples in which the cell number is too low for flow cytometry analysis. Visual control for artifact elimination and better characterization of cell populations makes this a powerful tool for tumor cell investigation. Automated image cytometry allows the obtainment of valuable prognostic parameters of traditional flow cytometry with the relatively small number of cells obtained in aspiration procedures.

Adult↗
PubMed1962

Enzyme distribution in membrane, cytoplasm, and nuclear fractions from Pseudomonas fluorescens.

Burrous, S. E. (University of Illinois, Urbana) and W. A. Wood. Enzyme distribution in membrane, cytoplasm, and nuclear fractions from Pseudomonas fluorescens. J. Bacteriol. 84:364-369. 1962.-A procedure is described for fractionation of Pseudomonas fluorescens A3.12 by controlled rupture of spheroplasts and fractional centrifugations to yield cytoplasm, membrane, and deoxyribonucleic acid (DNA) fractions with low cross contamination and little loss of enzymatic activity. Assays for the content of a number of oxidative and nonoxidative enzymes revealed that: (i) little activity is present in the DNA fraction; (ii) only reduced diphosphopyridine nucleotide oxidase is found exclusively in the membranes; (iii) phosphohexosisomerase and probably transketolase and glucose-6-phosphate dehydrogenase are found exclusively in the cytoplasm; and (iv) the remaining five enzymes studied were found in both fractions. In contrast to earlier reports of gluconic dehydrogenase being membrane-bound and 2-keto-3-deoxy-6-phosphogluconate aldolase being soluble in sonic extracts, this procedure produced partly soluble gluconic dehydrogenase and partly membrane-associated 2-keto-3-deoxy-6-phosphogluconate aldolase.

Aldehyde-Lyases↗
PubMed1993

A method for quantitative recovery of DNA from plate lysates of bacteriophage lambda-derived clones.

A method is described for the purification of DNA from recombinant clones in lambda vectors. Plate lysates are used as the starting material, with the phage suspension being either loaded directly onto mini-cesium chloride two-step gradients or first subjected to precipitation with polyethylene glycol-8000 before application to the gradients. A single centrifugation step is used and the DNA is isolated from the harvested bacteriophage by incubation at room temperature with formamide followed by ethanol precipitation. The small-volume gradients were effective in isolating phage particles in the range of 1.0 to 12.5 x 10(11) pfu with quantitative recovery of approximately 7 micrograms DNA/10(11) pfu. The method was tested with a cDNA clone in lambda gt10 (as an example of a lambda-derived vector which gives a plate lysate with a high titer) and rat genomic fragments cloned into EMBL3A (for which the plate lysates are of low titer). For both vectors, the purified DNA appeared free of inhibitory contaminants as assessed by restriction endonuclease digestion and subcloning of fragments.

Bacteriophage lambda↗
PubMed1999

An assessment of the genotoxic impact of the Sea Empress oil spill by the measurement of DNA adduct levels in selected invertebrate and vertebrate species.

The grounding of the Sea Empress oil tanker resulted in the release of 72,000 tonnes of crude oil into Milford Haven, Wales, UK. Our initial studies indicated that this contamination resulted in elevated levels of DNA adducts in one of the area's native marine species Lipophrys pholis [B.P. Lyons, J.S. Harvey, J.M. Parry, An initial assessment of the genotoxic impact of the Sea Empress oil spill by the measurement of DNA adduct levels in the intertidal teleost Lipophrys pholis, Mutat. Res. 390 (1997) 263-268]. These original studies were extended and the genotoxic impact of the oil contamination was investigated in the invertebrates Halichondria panicea and Mytilus edulis, along with the vertebrate fish species L. pholis, Pleuronectes platessa and Limanda limanda. DNA adduct levels were assessed in these species over a period of 2-17 months after the incident. The studies indicate differences in the impact of acute oil contamination upon vertebrate and invertebrate species. The oil contamination did not induce any detectable elevations in adduct levels in the invertebrate species H. panicea and M. edulis. In contrast, the oil contamination did appear to induce adducts in the vertebrate teleost species L. pholis, P. platessa and Lim. limanda. Despite some difficulties in sampling, the data obtained 12-17 months after the spill suggested that the affected species recovered from the oil contamination. While the studies indicate that the genetic impact of the oil contamination was less severe than might have been expected, it remains possible that the DNA adducts detected in the teleosts could lead to genetic changes in these species in the future.

Animals↗
PubMed1999

Disinfection of dental diamond burs contaminated with hepatitis B virus.

STATEMENT OF PROBLEM: Hepatitis B infection (HBV) is a significant hazard in the dental environment because the virus may be transmitted through contaminated dental instruments. PURPOSE: This study determined whether cold disinfectants can inactivate HBV DNA and HBV surface antigens on diamond burs contaminated with HBV and whether ultrasonication can increase the antiviral properties of these agents. MATERIAL AND METHODS: Sterile dental diamond burs were contaminated with serum from a patient who tested positive for HBV surface antigen and hepatitis B viral DNA. The burs were air dried and placed in solutions containing either Cidex, Asepsys, TBS, Rotagerm, Virkon disinfectants, or a control phosphate buffered saline. Burs were divided into 2 groups and disinfected for 15 minutes. The first group was ultrasonicated; the second group was not ultrasonicated during disinfection. All the burs were transferred to phosphate buffered saline and ultrasonicated to remove any remaining viral particles. The ultrasonicate was tested for the presence of HBV surface antigen with a microparticle enzyme immunoassay and for hepatitis B viral DNA with a chemiluminescent molecular hybridization assay. RESULTS: TBS did not require ultrasonication to inactivate viral DNA and surface antigen. Rotagerm and Virkon inactivated surface antigen and viral DNA only with ultrasonication. Cidex and Asepsys inactivated viral DNA but not surface antigen with ultrasonication. CONCLUSION: The chlorine containing compound TBS was the most active disinfectant tested and did not require ultrasonication to destroy HBV. The remaining disinfectants should be used with ultrasonication to inactivate HBV.

Analysis of Variance↗
PubMed1989

Dot blot hybridization assay of B19 virus DNA in clinical specimens.

A nonradioactive dot blot hybridization assay for human parvovirus B19 DNA was set up by using a biotin-labeled DNA probe and streptavidin-alkaline phosphatase conjugate. The assay was used to examine 4,895 specimens referred for B19 virus diagnosis during 1987. Of 48 specimens that gave positive reactions for B19 DNA, 41 were confirmed virus positive by electron microscopy (n = 36), radioimmunoassay (n = 26), or counterimmunoelectrophoresis (n = 20). In 7 samples which were not confirmed and in 11 samples giving weak reactions for B19 DNA, there was serological or epidemiological evidence of recent B19 infection. A further 70 specimens gave weak, apparently false-positive reactions. By electron microscopy, 13 of 16 were contaminated by bacteria, and plasmid DNA was demonstrated in one specimen. Of 55 specimens tested, 52 reacted with streptavidin-alkaline phosphatase conjugate alone. These were probable sources of nonspecificity in an otherwise practical and economic screening method for B19 virus.

Adult↗
PubMed1993

Comparison of methods used for detection of mycoplasma contamination in cell cultures, sera, and live-virus vaccines.

Two methods for detection of mycoplasma contamination in cell cultures, sera, and live-virus vaccines were compared: the direct culture test and the DNA staining method employing bisBenzimide (Hoechst No. 33258). Contamination by different species of mycoplasma was found in 39% samples tested. It is recommended to use both techniques for a reliable detection of mycoplasma contamination.

Blood↗
PubMed2001

Detection of Brucella melitensis in semen using the polymerase chain reaction assay.

An evaluation of the polymerase chain reaction (PCR) for detection of Brucella melitensis DNA in bovine and ovine semen was performed. Since semen contains different components that inhibit PCR amplification, a protocol was used to purify Brucella-DNA from bovine and ovine semen samples prior to conducting amplification of the targeted DNA. When separated fractions of naturally Brucella contaminated semen were analyzed by the PCR, most of B. melitensis DNA were present in the seminal fluid and non-sperm fractions. The PCR examination results for detection of B. melitensis DNA in different semen fractions were compared with the results for traditional cultural methods of Brucella from semen. The PCR was more sensitive than the traditional cultural methods since it detected Brucella-DNA in 12 (10%) out of 120 semen samples while direct culture detected only 7 (5.8%) in the same semen samples. The limit of detection by PCR was 100 CFU/ml of semen. In addition, the results of PCR were available in one day, whereas isolation and identification of Brucella organisms required days or even weeks. The PCR may be used as a supplementary test for detection of B. melitensis in semen.

Animals↗
PubMed2004

Laser-capture microdissection: applications in routine molecular dermatopathology.

Advances in molecular pathology with the introduction of the Southern blot technique and the polymerase chain reaction (PCR) have emerged as important tools, which are frequently used in routine dermato-histopathology. Applications for PCR-based diagnostics are particularly helpful for the determination of clonality in cutaneous lymphocytic infiltrates and for detection of infectious agents, such as herpes simplex virus (HSV), varicella zoster virus (VZV), Borrelia burgdorferi, Mycobacteria, Leishmania, and Treponema pallidum. As biopsies are always composed of different cells, the cells of interest are often only a minor population. As a consequence, their specific DNA is diluted by the majority of contaminating cells. Another problem is the time- and labor-intensive DNA extraction, because usually only formalin-fixed, paraffin-embedded tissue is available, which makes molecular diagnostics a time and labor consuming, and consequently a cost-intensive procedure. To overcome these shortcomings and to eventually shorten the time to generate a result, we introduce a laser-capture microdissection (LCM)-based method for the detection of infectious agents and clonality. Only the cells of interest for the particular indication are microdissected (e.g. epidermal cells for HSV and VZV and lymphocytes for clonality analysis) and subjected to PCR amplification. Due to an accelerated DNA-extraction procedure which generates DNA in 5 h (compared to 3-4 days using conventional DNA extraction), we are able to generate a result within one working day.

Animals↗
PubMed1999

Estimation of bacterial cell numbers in humic acid-rich salt marsh sediments with probes directed to 16S ribosomal DNA

The feasibility of using probes directed towards ribosomal DNAs (rDNAs) as a quantitative approach to estimating cell numbers was examined and applied to study the structure of a bacterial community in humic acid-rich salt marsh sediments. Hybridizations were performed with membrane-bound nucleic acids by using seven group-specific DNA oligonucleotide probes complementary to 16S rRNA coding regions. These included a general eubacterial probe and probes encompassing most members of the gram-negative, mesophilic sulfate-reducing bacteria (SRB). DNA was extracted from sediment samples, and contaminating materials were removed by a series of steps. Efficiency of DNA extraction was 48% based on the recovery of tritiated plasmid DNA added to samples prior to extraction. Reproducibility of the extraction procedure was demonstrated by hybridizations to replicate samples. Numbers of target cells in samples were estimated by comparing the amount of hybridization to extracted DNA obtained with each probe to that obtained with a standard curve of genomic DNA for reference strains included on the same membrane. In June, numbers of SRB detected with an SRB-specific probe ranged from 6.0 x 10(7) to 2.5 x 10(9) (average, 1.1 x 10(9) +/- 5.2 x 10(8)) cells g of sediment-1. In September, numbers of SRB detected ranged from 5.4 x 10(8) to 7.3 x 10(9) (average, 2.5 x 10(9) +/- 1.5 x 10(9)) cells g of sediment-1. The capability of using rDNA probes to estimate cell numbers by hybridization to DNA extracted from complex matrices permits initiation of detailed studies on community composition and changes in communities based on cell numbers in formerly intractable environments.

Journal Article↗
PubMed2001

Excretion and transmission of JCV in human populations.

The potential transmission of JCV through the environment has been analyzed by studying the JC viruses present in raw sewage of urban populations from widely divergent geographical areas. High numbers of JCV were found. JCV was detected in 98% (51/52) of sewage samples from different geographical areas in Europe, Africa, and USA by applying a Nested-PCR procedure. The mean estimated concentration of JCV in sewage was of 10(2)-10(3) viral particles/ml. Sequence analysis shows that JCV found in environmental samples present an archetypal structure in the regulatory region as it has been described in urine samples. Cerebrospinal fluid samples (CSF) of PML (progressive multifocal leucoencephalopathy) patients were also analyzed as control samples in this study presenting tandem repeats and rearrangements at the regulatory region (RR). Sequence analysis of the intergenic region (IGR) allowed the typification and phylogenetic analysis of the JCV sequences detected in sewage. JC viral particles were also found to be stable in sewage samples at 20 degrees C for more than 70 days. This data suggest the idea that the intake of water or food contaminated with JCV could constitute a portal of entry for the virus or the viral DNA to the human organism.

DNA, Viral↗
PubMed1995

Detection of DNA adducts in declining hop plants grown on fields formerly treated with heptachlor, a persistent insecticide.

Hop decline was observed in Alsace, eastern France, in reparcelled sugar beet fields formerly abundantly treated with an insecticide, heptachlor. Leaves were collected from 'declining hops' grown in an heptachlor-contaminated area and from 'healthy hops' grown in a soil not contaminated by heptachlor. These two samples came from hop vines treated with other usual pesticides. 'Control' hop leaves came from soil neither treated with pesticide nor contaminated with heptachlor. Hypermodified nucleotides (DNA adducts) were detected using the (32)P-postlabelling method. No detectable DNA adducts were found in the 'control' specimen, whereas eight adducts were detected in the 'healthy hops' specimen, probably due to the usual pesticide treatment. However, 16 adducts, nine of which were new adducts, could be detected in the 'declining hops' specimen. It may therefore be supposed that the presence of these hypermodified nucleotides perturbs gene expression and so contributes to the hop decline. In addition, to confirm the genotoxicity of heptachlor, it is shown that it induces DNA adducts in bean-cell suspension culture as well. Finally, it is proposed, in the case of alternate cultures scheduled in fields which were formerly treated with pesticides, adapted to other cultures, that particular attention should be given to the history of the soils.

Journal Article↗
PubMed2001

Rapid detection of Listeria monocytogenes in foods, by a combination of PCR and DNA probe.

Listeria monocytogenes is a frequent contaminant of water and foods. Its rapid detection is needed before some foods can be prepared for marketing. In this work L. monocytogenes has been searched for in foods, by a combination of polymerase chain reaction (PCR) and a DNA probe. Both PCR and the probe were prepared for recognizing a specific region of the internalin gene, which is responsible for the production of one of the most important pathogenic factors of Listeria. The combined use of PCR and the DNA probe was used for the detection of L. monocytogenes in over 180 environmental and food samples. Several detection methods were compared in this study, namely conventional culture methods; direct PCR; PCR after an enrichment step; a DNA probe alone; a DNA probe after enrichment and another commercially available gene-probe. Finally PCR and the DNA probe were used in series on all the samples collected. When the DNA probe was associated with the PCR, specific and accurate detection of listeria in the samples could be obtained in about a working-day. The present molecular method showed some advantages in terms of rapidity and specificity in comparison to the other aforementioned tests. In addition, it resulted as being easy to handle, even for non-specialized personnel in small diagnostic microbiology laboratories.

Aged↗
PubMed1998

DNA-cleaving activity of superoxide dismutase specific for circular supercoiled double-stranded DNA in vitro.

Several superoxide dismutases (SODs), N. tabacum Mn-SOD, porcine erythrocyte Cu/Zn-SOD and bovine erythrocyte Cu/Zn-SOD, have been found to exhibit the activity to cleave the circular supercoiled double-stranded DNA into nicked and further linear form in vitro. The fact that porcine erythrocyte Cu/Zn-SOD did not cleave the linear double-stranded DNA excluded the possibility that nuclease contaminated the SOD preparations and showing the cleaving activity was dependent on the supercoiled form of DNA. Porcine erythrocyte Cu/Zn-SOD inactivated by H2O2 or guanidine still remained its supercoiled DNA-cleaving activity. However, when the SOD was digested with proteases, its activity to cleave supercoiled DNA was completely abolished. These results suggested that the supercoiled DNA-cleaving activity was relative to the apoenzyme moiety, less relevant to the O2- dismutation site of SOD. The enzymatic mechanism of cleaving activity of SOD to supercoiled DNA was discussed.

Animals↗
PubMed2005

Random amplified polymorphic DNA-PCR based cloning of markers to identify the beer-spoilage strains of Lactobacillus brevis, Pediococcus damnosus, Lactobacillus collinoides and Lactobacillus coryniformis.

AIMS: Beer-spoilage ability of lactic acid bacteria such as Lactobacillus brevis is a strain-dependent phenomenon in which the mechanism has not yet been completely clarified. In order to systematically identify genes that contribute to beer-spoilage, large-scale random amplified polymorphic DNA (RAPD)-based cloning methods was carried out. METHODS AND RESULTS: A systematic RAPD polymerase chain reaction (PCR) analysis using 600 primers was performed on beer-spoilage and on nonspoilage strains of L. brevis. Among 600 primers, three were found to amplify a single locus highly specific to beer-spoilage strains. DNA sequencing of this locus revealed a three-part operon encoding a putative glycosyl transferase, membrane protein and teichoic acid glycosylation protein. PCR analysis of typical beer-spoilage lactic acid bacteria suggested that this locus is highly specific to beer-spoilage strains. CONCLUSION: The cloned markers are highly specific to identify the beer-spoilage strains not only in L. brevis but also in Pediococcus damnosus, Lactobacillus collinoides and Lactobacillus coryniformis. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper proves that RAPD-PCR is an efficient method for cloning the strain-specific genes from bacteria. The markers described here is one of the most useful tools to identify the beer-spoilage strains of lactic acid bacteria.

Beer↗