Human neutrophils can activate the food-derived carcinogen MeIQx.
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Biotransformation↗
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Results for “DNA Contamination”
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Lack of association of herpesviruses with brain tumors.
Gliomas are the most frequent primary brain tumors in humans. Many studies have been carried out on their etiology; however, the only confirmed risk factors are hereditary predisposing conditions and high dose of ionizing radiation. Recently, human cytomegalovirus (HCMV) gene products and nucleic acids were reported to be present in all of 27 glioma samples investigated in contrast to other brain tissues, and it was hypothesized that HCMV might play a role in glioma pathogenesis. To evaluate these findings, samples of 40 gliomas, 31 meningiomas, and 6 acoustic neurinomas (ACNs) were analyzed for the presence of HCMV macromolecules using polymerase chain reaction (PCR) and immunohistochemistry. Additionally, corresponding blood samples from 72 patients were analyzed for the presence of HCMV DNA to check for a possible contamination of tumor tissues with HCMV-infected blood cells. No HCMV DNA sequences were found, neither in brain tumor tissues nor in corresponding blood samples. Immunohistochemistry did not detect HCMV-specific proteins. Addressing a possible role of other herpesviruses as has been suggested in seroepidemiological studies, seroprevalence of antibodies to HCMV, herpes simplex virus (HSV), Epstein-Barr virus (EBV), and varicella-zoster virus (VZV) were determined by enzyme-linked immunosorbent assay (ELISA). Serological analyses of brain tumor patients showed no significant differences in the prevalences of antibodies to HCMV, HSV, EBV, or VZV compared to the general population. Thus, the data of the present study do not support the hypothesis of an association of herpesviruses with the development of primary brain tumors.
Adolescent↗
The role of the human acetylation polymorphism in the metabolic activation of the food carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ).
The metabolic activation of the heterocyclic food carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) by two human cytochrome P450 monoxygenases (P4501A1 and P4501A2) and two human N-acetyltransferases (NAT1 and NAT2) was investigated. Various combinations of these enzymes were functionally expressed in COS-1 cells. DNA adducts resulting from the activation of IQ were assayed quantitatively by the 32P-postlabeling procedure. The highest adduct frequency was observed in cells expressing both CYP1A2 and NAT2. CYP1A2 in combination with NAT1 was 3-6 times less active. When expressed alone these enzymes gave rise to low adduct frequencies. Experiments with N-acetyl-IQ as substrate suggest that NAT1 and NAT2 in addition to their known role in N-acetylation display arylhydroxamic acid N, O-acetyltransferase (AHAT) activity. Quantitative differences in adduct formation between IQ and N-acetyl-IQ indicated that metabolic activation of these arylamines preferentially occurs by P4501A2-catalyzed N-hydroxylation followed by O-acetylation mediated through NAT1 and/or NAT2. These data, in combination with the known genetic polymorphism of NAT2, may explain the clinical observation that the acetylation polymorphism constitutes a risk factor in the carcinogenic activation of environmental mutagens.
Acetylation↗
Clinical experience with preimplantation genetic diagnosis and intracytoplasmic sperm injection.
Preimplantation genetic diagnosis (PGD) is a novel procedure whose use may be considered to obtain a very early prenatal diagnosis for couples at risk for transmitting genetic diseases. Using the polymerase chain reaction (PCR) or fluorescence in-situ hybridization (FISH) the genotype or the sex of biopsied cleavage-stage embryos obtained after in-vitro fertilization can be determined and selected embryos can then be transferred. In-vitro fertilization with intracytoplasmic sperm injection is the method of choice to obtain embryos to be analysed through PCR to reduce contamination by residual sperm DNA. In our series of 61 PGD cycles for 29 couples at risk over a period of 4 years the ongoing pregnancy rate per cycle was 15%, per transfer 19% and per patient 31%. One of the six morphologically normal children born, who is still alive and doing well, weighed 850 g after birth at 25 weeks following a complicated triplet pregnancy. More experience is needed to correctly evaluate the efficiency and safety of this novel technique as well as to determine its place in the prevention of genetic disease.
Adult↗
Dealing with contamination: enzymatic control of carryover contamination in PCR.
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Base Sequence↗
AIDS origins. Disputed AIDS theory dies its final death.
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Acquired Immunodeficiency Syndrome↗
Enumeration by DNA Colony Hybridization of Virulent Yersinia enterocolitica Colonies in Artificially Contaminated Food.
[This corrects the article on p. 441 in vol. 51.].
Journal Article↗
Interaction of Simian Virus 40 chromatin with Simian Virus 40 T-antigen.
We have studied the binding of the tumor antigen (T-antigen) of simian virus 40 to simian virus 40 chromatin (minichromosomes). The minichromosomes isolated from infected cells by a modification of standard techniques were relatively free of contaminating RNA and cellular DNA and had a ratio (by weight) of protein to DNA of approximately 1; their DNA was 50 to 60% digestible to an acid-soluble form by staphylococcal nuclease. Cleavage of this chromatin with restriction endonucleases indicated that the nuclease-resistant regions were randomly distributed in the population of minichromosomes, but were not randomly distributed within minichromosomes. Only 20 to 35% of these minichromosomes adsorbed nonspecifically to nitrocellulose filters, permitting binding studies between simian virus 40 T-antigen and chromatin to be performed. Approximately two to three times as much T-antigen was required to bind chromatin as to bind an equivalent amount of free DNA. When T-antigen was present in excess, both chromatin and free DNA were quantitatively retained on the filters. On the other hand, when DNA or chromatin was present in excess, only one-third as much chromatin as DNA was retained. We suggest that T-antigen-chromatin complexes may be formed by the cooperative binding of T-antigen to chromatin, whereas T-antigen-DNA complexes may be formed by simple bimolecular interactions.
Antigens, Viral↗
Human chorionic gonadotropin stimulates iodide uptake, adenylate cyclase, and deoxyribonucleic acid synthesis in cultured rat thyroid cells.
hCG stimulates thyroid function, but it has been suggested that it is impurities in commercial hCG preparations or a variant of hCG that are responsible for the thyrotropic activity. In this study, we tested the thyrotropic activity of purified and commercial hCG and compared its action with that of bovine TSH (bTSH) in cultured rat FRTL-5 cells in regard to stimulation of iodide uptake, activation of adenylate cyclase, and synthesis of DNA. Iodide uptake was measured after incubation of the cells for 48-72 h with the test hormones, followed by a 40-min incubation with 0.1 microCi Na125I and 10 mumol/L carrier NaI; the 125I in the washed cells was counted. Adenylate cyclase was measured after incubation of the cells with the test stimulators for 3 h in hypotonic medium by RIA of cAMP in the medium. DNA synthesis was measured after incubation of the cells with the test substances for 24 h, followed by addition of [3H]thymidine for 3 h and then measuring the incorporation of [3H]thymidine into the cells. Both purified and commercial hCG produced a dose-related increase in iodide uptake. The relative potency of commercial hCG was 0.024 microU bTSH/U hCG and that of purified hCG was 0.042 microU bTSH/U hCG; compared with human TSH, the potency of purified hCG was 0.72 microU/U hCG. hCG caused a dose-related increment of adenylate cyclase and [3H]thymidine incorporation. The effect of hCG on iodide uptake and [3H]thymidine incorporation was additive with that of bTSH; hCG was not an antagonist of TSH in these cultured rat thyroid cells. We conclude that hCG has intrinsic thyrotropic activity in FRTL-5 cells in regard to stimulation of iodide uptake, activation of adenylate cyclase, and stimulation of DNA synthesis.
Adenylyl Cyclases↗
Advantages and disadvantages of current diagnostic tests for the detection of Helicobacter pylori.
Current tests used to detect Helicobacter pylori are either invasive (histological detection, culture, the polymerase chain reaction (PCR), smear examination) or non-invasive (serology, 13C-urea breath test). These tests vary in their sensitivity and specificity, and the choice of test will depend on the situation, for example, whether the test is to detect infection or the success of eradication treatment. The accuracy of histological tests depends, to a large degree, on the expertise of the pathologist, while the accuracy of culture can depend on the conditions in which the specimen is transported and processed. When performed under optimal conditions, both techniques give very good results. The PCR test has similar sensitivity and specificity to histological and culture tests but a strict protocol must be followed to avoid contamination with H. pylori DNA. The rapid urease test (with a reading taken 1 hour later) is suitable for diagnosis before treatment but its sensitivity decreases after treatment. Smear examination has limited sensitivity. The urea breath test and serology (specific IgG detected by enzyme-linked immunosorbent assay with purified antigens) have sensitivities close to those using the best of the biopsy methods. Other points to consider when selecting a test are its availability, the rapidity of the results (which can range from a few minutes to 2 weeks), possibilities for retrospective analysis, quantification and the detection of pathogenic properties, the globality of certain tests that present an overall picture of the stomach, thus avoiding errors in sampling, and the cost of the test. Important added value can be gained from certain tests: histology allows evaluation of the status of the mucosa, culture allows strain typing and tests for antibiotic susceptibility, and the breath test can confirm successful eradication without endoscopy. When the diagnostic tests are performed correctly, most of them are highly accurate.
Biopsy↗
A decaepitope polypeptide primes for multiple CD8+ IFN-gamma and Th lymphocyte responses: evaluation of multiepitope polypeptides as a mode for vaccine delivery.
Proteins are generally regarded as ineffective immunogens for CTL responses. We synthesized a 100-mer decaepitope polypeptide and tested its capacity to induce multiple CD8(+) IFN-gamma and Th lymphocyte (HTL) responses in HLA transgenic mice. Following a single immunization in the absence of adjuvant, significant IFN-gamma in vitro recall responses were detected for all epitopes included in the construct (six A2.1-, three A11-restricted CTL epitopes, and one universal HTL epitope). Immunization with truncated forms of the decaepitope polypeptide was used to demonstrate that optimal immunogenicity was associated with a size of at least 30-40 residues (3-4 epitopes). Solubility analyses of the truncated constructs were used to identify a correlation between immunogenicity for IFN-gamma responses and the propensity of these constructs to form particulate aggregates. Although the decaepitope polypeptide and a pool of epitopes emulsified in IFA elicited similar levels of CD8(+) responses using fresh splenocytes, we found that the decaepitope polypeptide more effectively primed for in vitro recall CD8(+) T cell responses. Finally, immunogenicity comparisons were also made between the decaepitope polypeptide and a corresponding gene encoding the same polypeptide delivered by naked DNA immunization. Although naked DNA immunization induced somewhat greater direct ex vivo and in vitro recall responses 2 wk after a single immunization, only the polypeptide induced significant in vitro recall responses 6 wk following the priming immunization. These studies support further evaluation of multiepitope polypeptide vaccines for induction of CD8(+) IFN-gamma and HTL responses.
Animals↗
Sequence mutations and amplification of PIK3CA and AKT2 genes in purified ovarian serous neoplasms.
Sequence mutations and gene amplifications lead to activation of the PIK3CA-AKT2 signaling pathway and have been reported in several types of neoplasms including ovarian cancer. Analysis of such genetic alterations, however, is usually complicated by contamination of normal cell DNA, artifacts associated with formalin-fixed tissues and the sensitivity of the techniques employed. In this study, we analyzed the sequence mutations in PIK3CA and AKT2 genes using purified tumor cells that were isolated from high-grade ovarian serous carcinomas and serous borderline tumors (SBTs) and assessed gene amplification using a dual-color FISH on tissue microarrays. Somatic sequence mutations in the kinase domain of AKT2 were not detected in any of the 65 ovarian tumors analyzed. Mutations of PIK3CA were rare, occurring only in one (2.3%) of 44 high-grade serous carcinomas and in only one (4.8%) of 21 SBTs. Dual-color FISH demonstrated that PIK3CA and AKT2 were not amplified in SBTs but amplified in 13.3% and 18.2% high-grade carcinomas, respectively. High-level amplification (>3 fold) was more frequently observed in AKT2 than in PIK3CA. Unlike mutations in ERBB2, KRAS and BRAF which are mutually exclusive in SBTs, coamplification of PIK3CA and AKT2 was present in five high-grade carcinomas including the OVCAR3 cells. Amplification in either of the genes occurred in 27% high-grade serous carcinomas. In conclusion, the methods we employed provide unambiguous evidence that somatic sequence mutations of PIK3CA and ATK2 are rare in ovarian serous tumors but amplification of both genes may play an important role in the development of high-grade ovarian serous carcinoma.
Carcinoma↗
Species origin of milk in Italian mozzarella and Greek feta cheese.
Several animal species such as cattle, goats, sheep, and water buffalo provide milk for dairy products. We describe a simple procedure for detecting the species origin of milk used for cheese production. DNA was isolated from Italian mozzarella or Greek feta by sequential organic extractions and resin purification. This DNA was analyzed by polymerase chain reaction-restriction fragment length polymorphism as described previously for meat samples. This procedure differentiated mozzarella made from water buffalo milk and from less expensive bovine milk and also feta cheeses made from bovine, ovine, and caprine milk.
Animals↗
Genetic identification of nine hake species for detection of commercial fraud.
This study describes the 5S rRNA gene as a simple and reliable one-step PCR-based genetic marker that allows unambiguous identification of Merluccius paradoxus, Merluccius senegalensis, Merluccius australis, Merluccius gayi, Merluccius bilinearis, Merluccius hubbsi, and Macruronus magellanicus. The marker is based on differences in length at the nontranscribed spacer within the 5S rRNA genes and is robust enough for identification of processed products, such as fish fingers and other precooked commercial hake. Further restriction fragment length polymorphism analysis at the mitochondrial cytochrome b gene can distinguish Merluccius merluccius and Merluccius capensis. The application of these markers for routine works such as surveys of quality and labeling of commercial hakes is suggested due to their accuracy, low price, and possible automation for large-scale surveys.
Animals↗
Detection of crustacean DNA and species identification using a PCR-restriction fragment length polymorphism method.
The detection of potentially allergenic proteins, such as those derived from crustaceans, in food products is a major concern for the food processing industry. A PCR-restriction fragment length polymorphism (PCR-RFLP) method was designed to detect the presence of crustacean DNA in food products and to determine the species source of the DNA. This PCR assay amplifies an approximately 205-bp fragment of the 16S rRNA gene in crustacean species, including shrimp, crab, lobster, and crawfish. This reaction will not amplify DNA derived from mammals, such as cow and sheep. After amplification, the PCR product is digested with differential restriction endonucleases to determine the species source of the crustacean DNA. The specificity of this assay was demonstrated using four species of shrimp, three species of crab, and two species of lobster and crawfish. This assay is sensitive enough to detect crustacean DNA in a raw meat mixture containing <0.1% shrimp.
Animals↗
High-throughput method for isolating plasmid DNA with reduced lipopolysaccharide content.
Isolating plasmid DNA from bacteria is a fundamental step in molecular biology. It is often accomplished by an alkaline lysis of bacteria and the subsequent adsorption of nucleic acids to silica oxide in the presence of chaotropic substances. Here we show that the addition of such chaotropic reagents is not required for the efficient DNA isolation with silica oxide. This surprising finding allowed us to purify plasmid DNA with significantly less lipopolysaccharides (LPS), which is otherwise a common bacterial contaminant of silica oxide-isolated DNA and inhibits subsequent applications. In addition, we have implemented a precipitation step that altogether leads to a reduction of the LPS content by a factor of 900 relative to published methods. Our novel protocol facilitates an inexpensive high-throughput analysis of pure plasmids in a 96-well format without the addition of hazardous reagents.
Cell Line↗
[Molecular genetic marker identification of traditional Chinese drug hippocampus].
Ancient DNA technique was used to extract DNA from 5 species of Chinese traditional drugs Hippocampus. The 12S rRNA gene fragment and cytochrome b gene fragment were amplified from DNA extract using Polymerase Chain Reaction (PCR) method. Restriction Fragment Length Polymorphism (RFLP) analysis and DNA sequence analysis were performed. The RFLP analysis method can identify 2 species of Hippocampus. The molecular genetic markers produced by DNA sequence method can identify all 5 species of Hippocampus. This method will be valuable for the identification of other animal drugs.
Cytochrome b Group↗
An improved purification procedure of alkaline phosphatase from calf intestine by applying partition in aqueous two-phase systems and dye-ligand chromatography.
Aqueous two-phase partitioning has been elaborated in order to improve the purification of alkaline phosphatase from calf intestine in larger scale. The laborious precipitation and centrifugation steps for the removal of the enzyme from the cell debris and from the bulk protein were replaced by this technique yielding a high recovery (88%) and a significant lower time requirement. For the preparation of 100.000 units (46 mg) of a homogeneous enzyme 2.0 kg of a system containing 200 g PEG 4000 and only 10 g dextran M 70 is necessary. Affinity partitioning in aqueous two-phase systems was used to screen 41 dyes for selecting a suitable ligand for the dye-ligand chromatography of the enzyme. In the case of alkaline phosphatase the results obtained by affinity partitioning coincide with the experimental requirements for the affinity chromatography of the enzyme. Procion Navy HE-R (Blue 171) exhibits a high affinity, selectivity and binding capacity for the enzyme compared with other dyes investigated. The purification procedure provided the same degree in purity (2200 U/mg) and yield (59%) if mucosa or chyme was applied as starting material. In the range of practical use the purified enzyme contains no detectable activities of DNAses (endonucleases) and DNA-nicking activities. The contamination with phosphodiesterase I (EC. 3.1.4.1.) is less than 0.01%.
Alkaline Phosphatase↗