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PubMed1997

Human papillomavirus detection by polymerase chain reaction in benign and malignant prostate tissue is dependent on the primer set utilized.

OBJECTIVES: Prior investigations evaluating the presence of human papillomavirus (HPV) in prostatic tissue by polymerase chain reaction (PCR) technology have yielded detection rates of 0% to 100%. Contamination by viral DNA from prostatic urethral colonization or less than optimal laboratory conditions have been suggested to explain this discrepancy. In addition, the various investigations have differed in the specific oligonucleotide primers utilized for amplification and, therefore, have searched for different segments of the viral genome. The objective of this study is to address these differences. METHODS: Forty-one archival radical prostatectomy specimens were evaluated, identifying areas of normal and abnormal histology. Meticulous technique was used during tissue acquisition, histologic confirmation, DNA isolation, PCR amplification, polyacrylamide gel electrophoresis, and staining. Primers for a 126- and 99-base pair (bp) fragment of the E6 portion of HPV 16 as well as a consensus primer for the L1 portion of the papillomavirus genome were utilized. RESULTS: Of the normal prostatic tissues, 13.5% (5/37) contained the 126-bp E6 viral DNA as did 33.3% (7/21) of benign prostatic hyperplasia (BPH) samples, 25% (5/20) of dysplasia, 18.2% (2/11) of Gleason grades 1 and 2 adenocarcinoma, 25.9% (7/27) of Gleason grade 3 adenocarcinoma, and 6.7% (1/15) of Gleason grade 4 adenocarcinoma. Sections from the urethras of the prostatectomy specimens contained viral DNA in 31.7% (13/41). Viral detection was variable among different specimens in the same patient. With amplification for the 99-bp fragment of HPV 16, 1 of 37 normal (2.7%), 2 of 21 BPH (9.5%), 1 of 20 dysplasia (5.0%), and 2 of 53 cancer (3.8%) specimens revealed HPV DNA. In none of the specimens was DNA amplified using primers for a 450-bp fragment of the L1 portion of HPV. CONCLUSIONS: Previously published discrepancies in HPV detection may be solely due to the differences in primer sets utilized.

Adult↗

PubMed2000

A polymerase chain reaction based method for detecting Mycoplasma/Acholeplasma contaminants in cell culture.

A detection system that utilizes a primer mixture in a nested polymerase chain reaction for detecting Mycoplasma contaminants in cell cultures is described. Primers were designed to amplify the spacer regions between the 16S and 23S ribosomal RNA genes of Mycoplasma and Acholeplasma. This detection system was able to detect 20-180 colony forming units per milliliter of sample. Eight commonly encountered Mycoplasma and Acholeplasma contaminants, which include Mycoplasma (M.) arginini, M. fermentans, M. hominis, M. hyorhinis, M. orale, M. pirum, M. salivarium, and Acholeplasma laidlawii, were consistently amplified. Mycoplasma contaminants generated a single DNA band of 236-365 base pairs (bp), whereas A. laidlawii produced a characteristic two-band pattern of 426 and 219 bp amplicons. Species identification could be achieved by size determination and restriction enzyme digestion. Minor cross-reactions were noted with a few closely related gram positive bacteria and DNA from rat cell lines. A Mycoplasma Detection Kit for detecting Mycoplasma contaminants in cell cultures has been developed based on this approach.

Acholeplasma laidlawii↗

PubMed2003

Novel genes regulated by gonadotropins in granulosa cells: new perspectives on their physiological functions.

Follicular stimulating hormone (FSH) is a key hormone secreted from the pituitary, which controls the development of the follicle-enclosed oocytes in the mammalian ovary by interacting with specific receptors located exclusively on granulosa cells. Its biological activity involves stimulation of intercellular communication and upregulation of steroidogenesis, yet the entire spectrum of genes which are regulated by FSH are not fully characterized. We have established rat and human FSH responsive granulosa cell lines, which express FSH receptors at 20-times higher rates compared to primary cells. Since the lines are monoclonal, they are expected to have a homogeneous composition of RNA among the entire cell population, which increases the probability of yielding a distinct view of genes modulated by FSH eliminating the possibility of other cell types contamination. Using Affymetrix DNA microarrays to uncover novel FSH-regulated genes, we discovered genes not reported earlier to be regulated by FSH. These include genes coding for (1) proteases; (2) growth factors and cytokines; (3) proteins involved in intercellular communication and connection with the nervous system; (4) protein phosphatases and kinases; (5) anti oxidants and anti-toxicants; (6) G-coupled proteins. These findings can deepen our understanding in the mechanism of FSH action in stimulation of the development of the ovarian follicular cells, in the modulation of ovarian intracellular and intercellular communication and in the process of selection of the dominant follicle. When human granulosa cells, obtained from in vitro fertilization patients were exposed to either hLH- or hFSH stimulation and mRNAs of these cells were analyzed by DNA microarrays, novel genes, similar to those found modulated by FSH in FSH responsive cell lines, were discovered in the human primary cells. This suggests that the immortalized cell systems established in our laboratory could serve as a useful system expanding the spectrum of authentic genes modulated by gonadotropin stimulation in normal ovarian function and in ovarian malfunction.

Animals↗

PubMedDate not supplied

Properties of RNase A immobilized on magnetic Poly(2-hydroxyethyl methacrylate) microspheres.

Magnetic hydrogel microspheres 1.5 microm in size were prepared by dispersion copolymerization of 2-hydroxyethyl methacrylate and ethylene dimethacrylate in the presence of magnetite, which formed the core of the particles. RNase A was coupled to the particles by the cyanuric chloride method. Gel electrophoresis of plasmid DNA pUC 19 (contaminated by bacterial RNA) confirmed RNA degradation with the immobilized enzyme. The effect of temperature and pH on the relative activity of immobilized RNase A was estimated after incubation of the samples at different temperatures (30-80 degrees C) and pH (4.0-8.0). Maximum relative activity was observed at 70 degrees C and pH 6.5. The matrices based on magnetic poly(HEMA) had a low tendency to adsorb RNA.

Biotechnology↗

PubMed2005

Metal-free catalysts for the hydrolysis of RNA derived from guanidines, 2-aminopyridines, and 2-aminobenzimidazoles.

2-aminopyridine and 2-aminobenzimidazole were chosen as structural analogues to substitute guanidinium groups in receptor molecules designed as phosphoryl transfer catalysts. Shifting the pKa of the guanidinium analogues toward 7 was expected to raise catalytic activities in aqueous buffer. Although the pKa's of both heterocycles are similar (6.2 and 7.0), only 2-aminobenzimidazole led to active RNA cleavers. All cleavage assays were run with fluorescently labeled substrates and a DNA sequencer. RNase contaminations would degrade RNA enantioselectively. In contrast, achiral catalysts such as 9b and 10b necessarily induce identical cleavage patterns in RNA and its mirror image. This principle allowed us to safely rule out contamination effects in this study. The most active catalysts, tris(2-aminobenzimidazoles) 9b and 10b, were shown by fluorescence correlation spectroscopy (FCS) to aggregate with oligonucleotides. However, at very low concentrations the compounds are still active in the nonaggregated state. Conjugates of 10b with antisense oligonucleotides or RNA binding peptides, therefore, will be promising candidates as site specific artificial ribonucleases.

Aminopyridines↗

PubMed2003

Increased expression and no mutation of the Flap endonuclease (FEN1) gene in human lung cancer.

The underlying molecular mechanisms leading to microsatellite alteration and mutations in human lung cancer remain unknown. Since Flap endonuclease1 (Fen1), which functions in the base excision repair system, has been shown to be involved in tumor progression of mouse models with microsatellite instability in a haplo-insufficient manner, we performed expression and mutation analyses for FEN1 in human lung cancer cell lines. Reverse transcriptase PCR analysis revealed that all 49 lung cancer cell lines (20 small cell lung cancers (SCLCs) and 29 non-small cell lung cancers (NSCLCs)) expressed FEN1. In addition, microarray analysis showed that FEN1 expression was elevated significantly by 1.65-fold (P=0.001) in SCLC cell lines compared to normal lung controls (normal human lung cultures and immortalized normal human bronchial epithelial cell lines). FEN1 protein was abundantly expressed in all 23 lung cancer cell lines (10 SCLCs and 13 NSCLCs) and was expressed at lower levels in three of four normal lung epithelial culture controls. Direct sequencing of genomic DNAs revealed no FEN1 mutation in seven SCLCs and nine NSCLCs. As part of this analysis we discovered and sequenced a FEN1 pseudogene (GenBank accession #AY249897) located at 1p22.2. This pseudogene is amplified from cDNA preparations contaminated with genomic DNA and must be taken into account in any FEN1 mutation analysis studies. Our results suggest that alterations of FEN1 are not likely to contribute to development of lung cancer.

Carcinoma, Non-Small-Cell Lung↗

PubMed2001

Quantitative polymerase chain reaction for the rapid prenatal diagnosis of homozygous alpha-thalassaemia (Hb Barts hydrops fetalis).

A quantitative polymerase chain reaction (Q-PCR) method based on the TaqMan technology has been devised for the prenatal diagnosis of homozygous alpha*-thalassaemia (south-east Asian type deletion). Primers and TaqMan probes were designed to specifically amplify an alpha*-thal chromosomal fragment or a normal alpha-chromosomal fragment. Variations in input target DNA in individual sample wells were normalized by the simultaneous amplification of a beta-actin gene fragment and results expressed as a ratio to that of beta-actin. There was no overlap of the data between the homozygous alpha*-thal, alpha*-thal and normal subjects. Up to 5% maternal DNA (alpha*-thal) contamination did not affect the specificity of the result. In 31 prenatal diagnoses, the result using Q-PCR compared favourably with the gold standard of Southern hybridization of alpha-genes.

Actins↗

PubMed1997

Monoclonal origin of multicentric Kaposi's sarcoma lesions.

BACKGROUND: Kaposi's sarcoma has features of both hyperplastic proliferation and neoplastic growth. Multiple lesions, in which spindle cells are prominent, often arise synchronously over widely dispersed areas. We tested the hypothesis that the spindle cells in these multicentric lesions originate from a single clone of precursor cells. METHODS: To determine whether Kaposi's sarcoma is a monoclonal disorder, we assessed the methylation patterns of the androgen-receptor gene (HUMARA) in multiple lesions from women with the acquired immunodeficiency syndrome. In polyclonal tissues, about half the copies of each HUMARA allele are methylated, whereas in cells derived from a single clone all the copies of only one allele are methylated. To minimize contamination by normal DNA, we used microdissection to isolate areas composed primarily of spindle cells, the putative tumor cells. RESULTS: Eight patients with a total of 32 tumors were studied. Of these tumors, 28 had highly unbalanced methylation patterns (i.e., predominant methylation of one HUMARA allele). In all the tumors that had unbalanced methylation from a given patient, the same allele predominated. CONCLUSIONS: These data indicate that Kaposi's sarcoma is a disseminated monoclonal cancer and that the changes that permit the clonal outgrowth of spindle cells occur before the disease spreads.

Acquired Immunodeficiency Syndrome↗

PubMed1994

Differential expression of mRNAs for JC virus large and small tumor antigens in brain tissues from progressive multifocal leukoencephalopathy patients with and without AIDS.

JC virus (JCV) causes progressive multifocal leukoencephalopathy (PML), the fatal demyelinating infection of oligodendrocytes, in up to 5% of AIDS patients. An intron-differential RNA PCR was developed to study the expression of alternately spliced JCV early mRNAs in brain tissues from PML patients with and without AIDS and in JCV-induced hamster brain tumors. The method utilizes primers that span the large tumor (T) and small tumor (t) antigen introns allowing amplification of specific cDNAs in the presence of contaminating viral genomic DNA. Hybridization with specific junctional probes and DNA sequence analysis confirmed the identity of the PCR products. Sequencing showed that JCV early mRNA is alternatively spliced as previously predicted by analogy to simian virus 40. Large T antigen mRNA was detected in all the brain tissues from PML patients with and without AIDS. The expression of small t antigen mRNA varied depending upon the association of PML with AIDS and upon other unknown factors. Of the 12 PML/AIDS brain tissue samples, 11 (92%) expressed small t antigen mRNA, whereas only 8 of 13 (62%) brain samples from patients with PML alone showed detectable levels of small t antigen mRNA. Human immunodeficiency virus 1 proviral DNA was detected in 10 of 12 PML/AIDS brain samples. The results indicate that alternative splicing of JCV early mRNA is regulated in the human brain and that the production of small t antigen may not be essential for the pathogenesis of PML.

Acquired Immunodeficiency Syndrome↗

PubMed1996

Putative effects of mitogenic lectin therapy corroborated by alloactivation data.

A firm theoretical case for PHA and other plant mitogens as superior immunomodulators has previously been presented, but direct confirmatory evidences have been compromised by experimental studies involving excessive dosages of erythroagglutinating PHA that often compromised the circulation in smaller animals, while inadequate amounts were applied in humans because this mitogen's availability in nonagglutinating form was restricted. The resulting underestimation of efficacy has failed to inspire the production of industrial quantities of mitogens required for reliable clinical trials. As a means of circumventing this dilemma, past favorable results from the immuno-stimulating activities of allocativation have been extrapolated to forecast the effects to be anticipated from mitogenic modulation. Such an extrapolation would underrate the latter's impact, which would neither be confined to stimulation nor dependent on the uncertainties of engraftment. This review cites clear examples showing that all recognized immune system pathways have been stimulated by alloactivation except perhaps the ADCC, and an example of this pathway's activation has been shown to have occurred with PHA therapy itself. Of the mitogenic lectins currently available, PHA, Con A, and PWM have each shown rare instances of hypersensitization that hopefully might be eliminated by exclusion of contaminants with recombinant DNA methods of production.

Graft vs Host Disease↗

PubMed2000

Infrequency of detection of particle-associated MSRV/HERV-W RNA in the synovial fluid of patients with rheumatoid arthritis.

OBJECTIVES: To determine whether the recently identified multiple sclerosis-associated retrovirus, MSRV, is detectable in the serum and synovial fluid of patients with rheumatoid arthritis (RA). METHODS: A reverse transcription-polymerase chain reaction (RT-PCR) assay was used to seek evidence of particle-associated MSRV/HERV-W RNA in the plasma and synovial fluid of patients with RA and controls. Stringent precautions were taken to avoid detection of contaminating human genomic DNA and cellular RNA sequences. RESULTS: Thirty-seven plasma samples were tested (20 from RA patients and 17 from controls) but none had detectable MSRV/HERV-W RNA. Synovial fluid samples were available from nine patients with RA and 10 controls. Particle-associated MSRV/HERV-W RNA was reproducibly detected in two of nine synovial fluid samples from RA patients and in one control sample. The identity of RT-PCR products was confirmed by sequencing. CONCLUSION: MSRV/HERV-W RNA sequences are detectable in the synovial fluid of a small proportion of RA patients, but this phenomenon may not be specific to RA.

Arthritis, Rheumatoid↗

PubMed2004

Investigation of the effect of the coronal restoration quality on the composition of the root canal microflora in teeth with apical periodontitis by means of T-RFLP analysis.

AIM: To investigate the effect of the radiographic and clinical quality of coronal restorations on the composition of the root canal flora of teeth with necrotic pulps and teeth with root fillings associated with apical periodontitis. METHODOLOGY: Twenty-eight necrotic pulps and 35 root filled canals with signs of apical periodontitis were studied. Both the coronal filling (presence of radiographically or clinically deficient margins and/or secondary caries) and the root filling (homogeneity and length) were scored. Bacterial root canal samples were taken with sterile paper points under rubber dam and using measures to prevent contamination. A DNA-based nonculture bacterial identification technique was used, namely terminal restriction fragment length polymorphism (T-RFLP) analysis. RESULTS: Twelve samples were negative for bacterial DNA. A total of 33 different terminal restriction fragments (TRFs) were detected. The Fusobacterium nucleatum/Streptococcus mitis group was the most frequently encountered TRF. The mean number of TRFs per necrotic pulp was 6.2 and 5.8 for the groups with acceptable and unacceptable coronal restorations, respectively. This difference was not significant. In the root filled group, these values (respectively, 5.2 and 8.6) were statistically significantly different (P < 0.05). The following parameters in root filled teeth had no significant influence on the mean numbers of TRFs detected: the length and homogeneity of the root filling and the type of tooth (anterior-premolar-molar). CONCLUSION: T-RFLP allowed the rapid assessment of bacterial biodiversity in root canal samples. The technique revealed the presence of bacteria that have rarely been described in the root canals of teeth with apical periodontitis. Biodiversity in the root filled group was high, as compared with culture-dependent studies where monoinfections were more frequently reported. Only in root filled teeth did defective coronal restorations have a statistically significant influence on the mean numbers of detected TRFs per sample.

Adult↗

PubMed2006

A 'dirty' business: testing the limitations of terminal restriction fragment length polymorphism (TRFLP) analysis of soil fungi.

Terminal restriction fragment length polymorphism (TRFLP) is an increasingly popular method in molecular ecology. However, several key limitations of this method have not been fully examined especially when used to study fungi. We investigated the impact of spore contamination, intracollection ribosomal DNA internal transcribed spacer (ITS) region variation, and conserved restriction enzyme recognition loci on the results produced by TRFLP to characterize soil fungal communities. We find that (i) the potential for nontarget structures such as spores to contribute DNA to target sample extractions is high; (ii) multiple fragments (i.e. 'extra peaks') per PCR primer-restriction enzyme combination can be detected that are caused by restriction enzyme inefficiency and intracollection ribosomal DNA ITS variation; and (iii) restriction enzyme digestion in conserved vs. variable gene regions leads to different characterizations of community diversity. Based on these results, we suggest that studies employing TRFLP need to include information from known, identified fungi from sites within which studies take place and not to rely only on TRFLP profiles as a short cut to fungal community description.

Base Sequence↗

PubMed1978

Rapid purification of plasmid DNAs by hydroxyapatite chromatography.

A method is described for the rapid preparation of plasmid DNAs of molecular weight up to 14 X 10(6). This method involves the chromatography, at room temperature, of bacterial cleared lysates on hydroxyapatite in the presence of high concentrations of phosphate and urea. All detectable protein and RNA contamination of plasmid DNA is removed by this procedure and the conformation of the plasmid DNA is unaffected. Less than 0.5% chromosomal DNA is present in the purified preparation and even this can be removed if necessary by a simple extention of the procedure to include a heat-denaturation step. The method is extremely rapid and amenable to large-scale plasmid preparation; 5 mg ColE1 DNA have been purified within 40 min. The yield of plasmid DNA is similar to that obtained with the conventional dye-centrifugation technique, however the purity is greater.

Chromatography↗

PubMed1994

Simple method for extracting plasmid DNA from lactic acid bacteria.

Rapid screening and large-scale plasmid DNA isolation procedures are described for lactic acid bacteria, using glass beads to break cells. The rapid screening procedure allows one to obtain plasmid DNA pellets in less than 1 h. This method has been successfully tested on various bacteria from the genera Lactococcus, Leuconostoc, Lactobacillus, Pediococcus, Streptococcus, Enterococcus and Propionibacterium. This procedure yields plasmid DNA with minor chromosomal and plasmid DNA-degraded form contaminations.

Bacteriological Techniques↗

PubMed1993

Transmission of human papillomaviruses from mother to child.

Exfoliated cervical epithelial cells from women 6 weeks postpartum were analyzed for human papillomavirus (HPV) DNA using the polymerase chain reaction, and results were compared with those from buccal mucosal smears from their babies. Eleven mothers had genital genotypes of HPV in their cervical smears, and the children of 8 of these had HPV of the same genotype in buccal mucosal cell samples. Nineteen mothers had no HPV DNA detected in their cervical smears, and 1 of the buccal mucosal cell samples from their children was positive for HPV DNA (p < 0.0001). Contamination of a child's mouth with 'genital' HPV from a mother's cervix appears to occur commonly at birth or in the perinatal period, and to persist for at least 6 weeks. This observation has implications for the epidemiology and management of HPV associated cancer and precancerous conditions in the cervix and the mouth.

Cervix Uteri↗

PubMed1994

Development of PCR assays to detect ampicillin resistance genes in cerebrospinal fluid samples containing Haemophilus influenzae.

We developed PCR primers specific for the blaTEM and blaROB ampicillin resistance genes. The specificity of the primers was confirmed by testing a series of Escherichia coli isolates containing a variety of ampicillin resistance genes and a series of ampicillin-resistant and ampicillin-susceptible Haemophilus influenzae isolates. There was a perfect correlation between ampicillin MICs, the presence of beta-lactamase (as determined by the nitrocefin test), and the results with the blaTEM and blaROB primers. Isolates of H. influenzae and Streptococcus pneumoniae obtained from 25 frozen cerebrospinal fluid (CSF) specimens were also tested. Four of 14 H. influenzae isolates were positive with the blaTEM primers; none were positive with the blaROB primers. Ampicillin MICs were determined for the H. influenzae isolates, and penicillin MICs were determined for the S. pneumoniae isolates. Only the four PCR-positive H. influenzae isolates had elevated MICs of ampicillin and were beta-lactamase positive. None of the H. influenzae isolates contained the blaROB gene, and none of the S. pneumoniae isolates produced positive reactions with either primer set. We then used universal primers directed to conserved regions of rRNA and a Haemophilus detection probe to identify which of the 25 frozen samples of CSF contained H. influenzae. Fourteen of the 25 CSF specimens were positive for H. influenzae, which correlated with the number of organisms obtained by culture of the CSF samples. Four of the CSF samples were positive with the blaTEM primer set, and these correlated with the four H. influenzae isolates that were positive when tested directly by PCR. The blaTEM assay required the use of native Taq polymerase because Amplitaq preparations were contaminated with vector DNA that contained the blaTEM-1 gene.

Ampicillin Resistance↗

PubMed2000

Free radical-mediated transgene inactivation of macrophages by endotoxin.

Endotoxin, the lipopolysaccharide component of gram-negative bacteria, is a common contaminant of plasmid DNA preparations. The present study investigated the effect of endotoxin on gene transfection efficiency and the role of reactive oxygen species (ROS) in this process. Gene transfection studies were performed in various cell types with cytomegalovirus-luciferase as a reporter plasmid and cationic liposome as a transfecting agent. The presence of endotoxin in plasmid DNA preparations severely limited transgene expression in macrophages but had little or no effect in other cell types tested. This decreased transfection was dependent on ROS-mediated cellular toxicity induced by endotoxin. Neutralizing the endotoxin by the addition of polymyxin B effectively increased transfection efficiency and reduced toxicity. Electron spin resonance studies confirmed the formation of ROS in endotoxin-treated cells and their inhibition by free radical scavengers. The ROS scavenger N-t-butyl-alpha-phenylnitrone, the H(2)O(2) scavenger catalase, and the.OH scavenger sodium formate effectively inhibited endotoxin-induced effects, whereas the O(2)(-) scavenger superoxide dismutase had lesser effects. These results indicate that multiple oxidative species are involved in the transfection inactivation process and that.OH formed by H(2)O(2)-dependent, metal-catalyzed Fenton reaction play a major role in this process.

Animals↗