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At least 1,081 records · Page 60Linked to original sources
PubMed2000

Evaluation of a PCR probe capture assay for the detection of Toxoplasma gondii. Incorporation of uracil N-glycosylase for contamination control.

Toxoplasma gondii is a cyst-forming parasite of clinical relevance in humans primarily because of the neurologic abnormalities it can cause. In some clinical circumstances, it is desirable to detect the pathogen directly. We modified a commercially available Toxoplasma polymerase chain reaction (PCR) probe capture assay by incorporating uracil N-glycosylase (UNG) to prevent carryover amplicon contamination. In addition, UNG inactivation and DNA denaturation were accomplished chemically to simplify the DNA hybridization to the capture probe. The incorporation of UNG effectively eliminated carryover contamination; the probe capture assay showed a log increase in detection sensitivity compared with standard agarose gel electrophoresis. To assess sensitivity and possible inhibition of amplification, different sample types were spiked with Toxoplasma organisms. After DNA extraction and PCR amplification, a sensitivity of 2 tachyzoites for the assay was determined in buffered saline, cerebrospinal fluid (CSF), serum, and amniotic fluid; 20 tachyzoites for whole blood; and 200 tachyzoites for brain tissue. An additional 20 human serum and CSF samples submitted for Toxoplasma serologic testing were run by the PCR method. Of these, only an IgM-positive CSF sample was PCR positive. The Toxoplasma PCR probe capture assay showed good sensitivity and was not substantially inhibited by several different clinically relevant samples.

Amniotic Fluid↗
PubMed1984

Connective tissue activation. XXVIII. A connective tissue activating peptide from human urine.

A protein factor in human urine which has the ability to activate connective tissue cells has been identified and partially purified; it appears to be different from epidermal growth factor and IgG. This urinary connective tissue activating factor (CTAP-U) is nondialyzable, labile to protease, stable to thiols, heat, and acid, and has an acidic isoelectric point. Purified preparations of CTAP-U have biologic activities that cause human connective tissue cells to synthesize incremental amounts of 14C-hyaluronic acid, 35S-proteoglycans, and 3H-DNA in vitro. The cell spectrum responsive to this substance includes human synovial cells, human chondrocytes, and skin fibroblasts. CTAP-U does not react with antisera to connective tissue activating peptide-III or to antibodies against IgG or its Fc and Fab fragments. Furthermore, CTAP-U does not cross-react in a radioreceptor assay for insulin, basic somatomedin, or epidermal growth factor-urogastrone. Utilizing standardized isolation conditions, CTAP-U preparations with these properties have been isolated from the urine of 6 normal individuals.

Cartilage↗
PubMed1984

Metabolism of food toxicants: saccharin and aflatoxin B1, a contrast in metabolism and toxicity.

The artificial sweetener, saccharin, and the secondary mold product, aflatoxin B1, are present in many foods consumed by humans. Both chemicals produce cancer in rats. For this reason, they have aroused concern among scientists and the public as to the carcinogenic risk that they pose to humans. A comparison of these two chemicals reveals striking contrasts in potency, metabolism, mechanism-of-action, and experimental approach to assessing metabolism. These contrasts are examined in detail to illustrate the importance of metabolism in safety evaluation.

Aflatoxin B1↗
PubMed1978

A novel method for the isolation and study of a magnetotactic bacterium.

The magnetococcus, a magnetotactic bacterium, has been grown in a complex simulated natural environment. Sufficiently pure samples of cells were obtained magnetically making axenic cultures unnecessary for many purposes. The magnetococcus is a Gram-negative coccus, 1.6 micron in diameter and readily distinguished by highly refractile inclusions and its magnetotactic behavior. This organism is actively motile by means of two bundles of flagella. Electron dense ferromagnetic inclusions were localized between the flagellar bundles. Collections of magnetococci were morphologically homogeneous and negligibly contaminated by extraneous bacteria. DNA extracted from pooled collections of cells was homogeneous by analytical CsC1 centrifugation. The guanine-cytosine content was 61.7%. Total iron by percent cellular dry weight was 3.8%. Comparisons with a previously described magnetotactic marine coccus were made.

Bacteria↗
PubMedDate not supplied

Organ weights, liver constituents, and serum components in growing chicks fed ochratoxin A.

Chicks were fed diets containing 0, 1, 5 and 10 mg/kg ochratoxin A (OA) over a four-week period to study the effects of OA on growth, weight of internal organs, liver RNA, DNA, protein, and glycogen and serum enzymes. Ochratoxin A depressed the rate of growth and relative weight of the bursa of Fabricius, and increased the relative weights of the liver, kidney, pancreas, and various sections of the gastrointestinal tract, but had no effect on the heart and spleen. Concentrations of liver RNA, DNA and protein were decreased while glycogen was increased. Serum alkaline phosphatase and gamma-glutamyl transferase activities, and uric acid and creatinine concentrations were elevated while serum proteins, albumin, phosphorus, potassium, and cholesterol were depressed. The effects of OA were time- and dose-dependent.

Animal Feed↗
PubMed1996

Multiparameter flow cytometric characterization of epidermal cell suspensions prepared from normal and hyperproliferative human skin using an optimized thermolysin-trypsin protocol.

Reliable flow cytometric analysis of normal and diseased skin requires pure epidermal single-cell suspensions. Several methods to separate the dermis from the epidermis are available. The proteolytic enzyme thermolysin separates the epidermis from the dermis at the lamina lucida and therefore permits reliable dermoepidermal separation. In the present study an optimized cell isolation procedure using thermolysin and trypsin is described, which is particularly suitable for punch biopsies. A 16-20-h (overnight) incubation of biopsies taken from normal and hyperproliferative skin with thermolysin (0.5 mg/ml) at 4 degrees C produced a selective separation of the dermis and epidermis. After a 30-min trypsin incubation (0.25 mg/ml) at 37 degrees C a cell suspension was produced which was characterized by minimal cell damage (cellular debris and clumps), a high recovery of basal cells and high quality DNA histograms. Furthermore, dermal contamination was very low. The thermolysin-trypsin separation methodology followed by triple-labelling flow cytometry provided a precise quantification of the percentage of keratin 10-positive cells, vimentin-positive cells and cells in S and G2M phases. Proliferative activity was selectively measured in the basal, the suprabasal and the non-keratinocyte compartment at various time intervals during epidermal regeneration after adhesive tape stripping. In contrast to the non-keratinocytes, the percentage of cells in S and G2M phases in the basal keratinocytes and in the suprabasal compartment increased 44-48 h after stripping. The increased proliferation following tape stripping was paralleled by an increased invasion of vimentin-positive cells into the epidermis and preceded by a decreased number of keratin 10-positive cells. Thermolysin-trypsin separation followed by three-colour flow cytometry permits a highly selective characterization of normal and hyperproliferative epidermis.

Adolescent↗
PubMed1994

Purification and analytical characterization of an anti-CD4 monoclonal antibody for human therapy.

A purification process for the monoclonal anti-CD4 antibody MAX.16H5 was developed on an analytical scale using (NH4)2SO4 precipitation, anion-exchange chromatography on MonoQ or Q-Sepharose, hydrophobic interaction chromatography on phenyl-Sepharose and gel filtration chromatography on Superdex 200. The purification schedule was scaled up and gram amounts of MAX.16H5 were produced on corresponding BioPilot columns. Studies of the identity, purity and possible contamination by a broad range of methods showed that the product was highly purified and free from contaminants such as mouse DNA, viruses, pyrogens and irritants. Overall, the analytical data confirm that the monoclonal antibody MAX.16H5 prepared by this protocol is suitable for human therapy.

Animals↗
PubMed1979

Mutagenic, cancerogenic and teratogenic effects of alcohol.

Alcohol is mutagenic, cancerogenic and teratogenic in man. Ethanol is mutagenic via its first metabolite, acetaldehyde. This is substantiated by the findings that acetaldehyde induces chromosomal aberrations, sister-chromatid exchanges and cross-links between DNA strands. Methanol, a contaminant of many alcoholic beverages, is also mutagenic via its metabolite, formaldehyde. In addition, different indirect pathways may lead to mutations by alcohol. The cancerogenic activity of alcohol remains unverified by modern standard carcinogenicity tests. Ethanol and other alcohols, as well as aldehydes, inhibit RNA synthesis in cells and in cell-free transcriptional systems. A reduction of cellular RNA synthesis may play an important role in the mutagenic, carcinogenic and teratogenic activity of alcohol.

Acetaldehyde↗
PubMed1996

A cellular mechanism for imidocarb retention in edible bovine tissues.

Imidocarb dipropionate, formulated as Imizol, is used for the treatment and prophylaxis of bovine babesiosis. Several studies have shown that imidocarb remains detectable in edible ovine and bovine tissues for several months after dosing but the mechanism of retention remains unknown. In this study, the mechanism of imidocarb retention was investigated by measuring the binding of [14C]imidocarb to bovine hepatocytes, erythrocytes, sub-cellular fractions and isolated bovine macromolecules. The proportion of [14C]imidocarb (10 microM) bound to cells in suspension culture (1 x 10(7) cells.ml-1) was found to be substantially greater to hepatocytes (56.5%) than to erythrocytes (4.6%). Studies with washed erythrocytes reconstituted in plasma indicated that approximately 70% of the [14C]imidocarb was bound to plasma proteins, 10% to erythrocytes, and 20% remained free. Measurement of [14C]imidocarb binding to sub-cellular fractions prepared from bovine liver revealed preferential accumulation in the nuclear, rather than in the mitochondrial, microsomal or cytosolic fractions. Binding capacities of selected bovine macromolecules for [14C]imidocarb were in the order deoxy-ribonucleic acid (DNA) = ribonucleic acid (RNA) > > alpha 1-acid glycoprotein (AGP) > serum albumin (BSA) > haemoglobin (Hb). DNA binding sites for imidocarb remained unsaturated over the concentration range 0-100 microM [14C]imidocarb. Competitive binding studies between imidocarb and pentamidine or spermidine provided evidence for common DNA binding sites. These studies indicated that preferential binding of [14C]imidocarb to hepatocytes compared with erythrocytes observed in vitro was a result of substantial reversible binding to nucleic acids and that the same cellular mechanism may be implicated in the slow elimination of imidocarb from edible tissues in vivo.

Animals↗
PubMed2006

Improved detection of episomal Banana streak viruses by multiplex immunocapture PCR.

Banana streak viruses (BSV) are currently the main viral constraint to Musa germplasm movement, genetic improvement and mass propagation. Therefore, it is necessary to develop and implement BSV detection strategies that are both reliable and sensitive, such as PCR-based techniques. Unfortunately, BSV endogenous pararetrovirus sequences (BSV EPRVs) are present in the genome of Musa balbisiana. They interfere with PCR-based detection of episomal BSV in infected banana and plantain, such as immunocapture PCR. Therefore, a multiplex, immunocapture PCR (M-IC-PCR) was developed for the detection of BSV. Musa sequence tagged microsatellite site (STMS) primers were selected and used in combination with BSV species-specific primers in order to monitor possible contamination by Musa genomic DNA, using multiplex PCR. Furthermore, immunocapture conditions were optimized in order to prevent Musa DNA from interfering with episomal BSV DNA during the PCR step. This improved detection method successfully allowed the accurate, specific and sensitive detection of episomal DNA only from distinct BSV species. Its implementation should benefit PCR-based detection of viruses for which homologous sequences are present in the genome of their hosts, including transgenic plants expressing viral sequences.

Badnavirus↗
PubMed2000

alpha(1)-antitrypsin can increase insulin-induced mitogenesis in various fibroblast and epithelial cell lines.

alpha(1)-Antitrypsin (AT), the archetypal member of the superfamily of serine proteinase inhibitors, inhibits leukocyte elastase activity and thereby can prevent lung damage. Here we show that in fibroblasts from human fetal lung and mouse embryo as well as in certain epithelial cells AT can also enhance the stimulatory effects of insulin on DNA synthesis and cell proliferation. Warming of AT at a moderate (41 degrees C) temperature for a longer time (21 h) or at a higher (65 degrees C) temperature for 30 min before treatment increased its stimulatory effects on both DNA synthesis and activating phosphorylation of p42/p44 mitogen-activated protein kinases. The results suggest that AT may promote regeneration of damaged tissues under pathophysiological conditions which are associated with fever.

Alkaline Phosphatase↗
PubMed2001

Progress on methods of gene detection in preimplantation embryos.

The advent of the polymerase chain reaction (PCR) and the development of fluorescence in situ hybridization (FISH) have had a tremendous impact on preimplantation genetic diagnosis (PGD). While PCR is a powerful tool in detecting genetic diseases or molecular markers affecting quantitative trait loci, the main use of FISH is screening for chromosomal aberrations. This presentation reviews the recent progress in preimplantation genetic diagnosis with an emphasis on bovine embryos. In particular the importance of biopsy size and strategies to avoid PCR contamination are discussed. Alternative DNA amplification and detection methods as well as methods to meet the challenge of multiple locus detection for marker assisted selection are presented.

Animals↗
PubMed2000

Use of crypt isolation to determine loss of heterozygosity of multiple tumor suppressor genes in colorectal carcinoma.

Analysis of loss of heterozygosity (LOH) is very important in the study of tumor suppressor genes. However, accurate LOH analysis of tumor suppressor genes is difficult because of dilution by contaminating non-tumor DNA. Thus, enrichment of tumor DNA is required to accurately determine LOH of the tumor. We developed a new application of the fluorescent polymerase chain reaction by coupling it with crypt isolation to accurately assess the incidence of LOH of tumor suppressor genes in 45 colorectal carcinomas. LOH was observed at p53 in 26 of 37 tumors (70.3%), at APC in 13 of 35 (37.1%), at DCC in 16 of 25 (64.0%), at NF-2 in 5 of 23 (21.7%), and at nm23 H-1 in 7 of 30 (23.3%). We could clearly determine LOH of these genes because the crypt isolation technique was used. Although the incidence of LOH at each of these loci, as determined by using this technique, was similar to that obtained in previous studies using conventional methods, this method provides a simpler, more accurate way to assess LOH. In addition, the morphology of the samples can be analyzed before genetic analysis.

Adenocarcinoma, Mucinous↗