Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Apoptosis, or programmed cell death, is a general mechanism for removal of unwanted cells from the immune system. It is characterized by chromatin condensation, a reduction in cell volume, and endonuclease cleavage of DNA into oligonucleosomal length fragments. Apoptosis is also accompanied by a loss of membrane phospholipid asymmetry, resulting in the exposure of phosphatidylserine at the surface of the cell. Expression of phosphatidylserine at the cell surface plays an important role in the recognition and removal of apoptotic cells by macrophages. Here we describe a new method for the detection of apoptotic cells by flow cytometry, using the binding of fluorescein isothiocyanate-labeled annexin V to phosphatidylserine. When Burkitt lymphoma cell lines and freshly isolated germinal center B cells are cultured under apoptosis inducing conditions, all cells showing chromatin condensation strongly stain with annexin V, whereas normal cells are annexin V negative. Moreover, DNA fragmentation is only found in the annexin V-positive cells. The nonvital dye ethidium bromide was found to stain a subpopulation of the annexin V-positive apoptotic cells, increasing with time. Our results indicate that the phase in apoptosis that is characterized by chromatin condensation coincides with phosphatidylserine exposure. Importantly, it precedes membrane damage that might lead to release from the cells of enzymes that are harmful to the surrounding tissues. Annexin V may prove important in further unravelling the regulation of apoptosis.
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PMID:Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. 806 38

The occurrence of resistance to antiseptics and disinfectants in clinical isolates of coagulase-negative staphylococci (CNS) was examined. Of 164 clinical strains of CNS isolated in the early 1980s, 65 were resistant to cationic antimicrobial compounds such as cetyltrimethylammonium bromide. Further characterisation of 40 resistant isolates by DNA-DNA hybridisation analysis and phenotypic resistance studies revealed that this resistance was mediated by the multidrug export genes qacA and qacC, characterised previously in Staphylococcus aureus. Of the resistant CNS isolates, 50% contained only qacA, 10% contained only qacC, and the remaining 40% contained both qacA and qacC. Both qacA and qacC genes resided on plasmids in all cases, with qacA located on plasmids of > 10 kb, whereas qacC was located primarily on plasmids of 2-3 kb. Representative qacA and qacC plasmids were characterised by restriction endonuclease mapping, and were found to be similar in some cases, but different in others, to those plasmids on which these genes are found in S. aureus.
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PMID:Multidrug resistance to antiseptics and disinfectants in coagulase-negative staphylococci. 811 73

During a 2-week period, Enterobacter cloacae was isolated from throughout the water distribution system in New Haven County, Connecticut. There was no forewarning of this event and no apparent reasons for it. Several epidemiologic and public health questions required rapid answers. Were these E. cloacae isolates the result of treatment failure and breakthrough or was regrowth occurring within the system? Did the E. cloacae isolates represent a health threat and were they causing infection? Pulsed-field gel electrophoresis utilizing whole-cell DNA digestion with restriction endonuclease SpeI permitted the rapid generation of specific information to answer these questions. Gel bands were stained with ethidium bromide and photographed with UV illumination. Homogeneity among isolates was confirmed by repeat digestion with XbaI. From each of the water distribution isolates, a single pattern of restriction endonuclease fragments was generated, indicating that only one clone of E. cloacae was in the distribution system. There was no homogeneity between source and distribution water E. cloacae isolates. Moreover, E. cloacae clinical isolates from patients from New Haven area hospitals showed no identity with E. cloacae isolated from the distribution system. Therefore, pulsed-field gel electrophoresis DNA analysis demonstrated that the E. cloacae from the distribution system was the result of a regrowth bloom within the system and not the result of treatment failure and that this clone was not causing a public health risk.
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PMID:Differentiation of distribution systems, source water, and clinical coliforms by DNA analysis. 812 69

We have developed a polymerase chain reaction (PCR)-based method to measure glutathione peroxidase (GSH-Px) mRNA levels. Expression was measured by multiplex competitive PCR amplification of (a) cDNA from GSH-Px and the "housekeeping" gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and (b) two internal standards consisting of single-base mutants of GSH-Px and GAPDH cDNA that cause either a loss (GSH-Px) or a gain (GAPDH) of an EcoRI restriction endonuclease recognition site. RNA extracted from a human papillomavirus-immortalized human bronchial epithelial cell line (BEP2D) was reverse transcribed. Serial dilutions of cDNA were PCR amplified in the presence of GSH-Px and GAPDH primers and quantified amounts of mutated internal standards. The amplified DNA was restriction digested with EcoRI and electrophoresed on an agarose gel stained with ethidium bromide, separating native from mutated products. Densitometry was performed to quantitate the bands. Our studies demonstrate that this technique measures the relative expression of GSH-Px to GAPDH precisely and reproducibly for studies done with the same master mixture and dilution of internal standards. Ratios of relative gene expression varied less than 25% from the mean. This technique will be useful to measure changes in gene expression, particularly when the amount of study sample is limited or the level of gene expression is low.
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PMID:Measurement of gene expression by multiplex competitive polymerase chain reaction. 823 2

Molecular biology is changing the face of diagnostic medicine, and infectious diseases of the oral soft tissues are among the targets of these advances in biotechnology. As an illustration of these concepts, a PCR-based detection and typing system for human papillomaviruses (HPVs) will be discussed. A single "consensus" set of oligomeric nucleotide primers can be used to amplify a 571- to 594-base-pair region of the E1 open reading frame of HPV 6, 11, 16, and 18. These HPV types are commonly associated with preneoplastic and cancerous lesions of the genital, respiratory, and digestive tracts. PCR amplification yields single bands of similar size for these viruses by agarose gel electrophoresis. Digestion of the resultant products by the restriction endonuclease AccI yields distinctive and reproducible banding patterns by polyacrylamide gel electrophoresis (with ethidium bromide) due to their internal sequence diversity. The system is sensitive; without radioisotopes, it can detect and type HPV 18 in as little as 100 pg of DNA from HeLa cells. We have used it to confirm HPV in fresh-frozen tumors. Computer sequence analysis can be used to modify the system for the detection of new HPV types as they are characterized. Other applications of molecular-biology-based detection systems for infectious diseases of the head and neck region will be discussed.
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PMID:New approaches to the diagnosis of oral soft-tissue disease of viral origin. 826 10

An assay has been developed (restriction endonuclease digestion assay--RED100) based on inhibition of the restriction endonuclease BamHI that is capable of quantitative evaluation of the relative DNA-binding affinity of pyrrolo[2,1-c] [1,4]benzodiazepine (PBD) antitumour antibiotics. This method provides comparable results to those obtained from thermal denaturation and ethidium bromide displacement assays but is much more sensitive, discriminating between molecules of similar structure such as DC-81, iso-DC-81 and neothramycin. The results reveal a trend between relative DNA-binding affinity and in vitro cytotoxicity for the PBDs in two tumour cell lines studied.
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PMID:A quantitative assay to measure the relative DNA-binding affinity of pyrrolo[2,1-c] [1,4]benzodiazepine (PBD) antitumour antibiotics based on the inhibition of restriction endonuclease BamHI. 836 84

A sensitive assay based on the polymerase chain reaction for the detection of Ockelbo virus RNA was developed. Two primer pairs from the gene coding for the E2 glycoprotein were chosen. By use of a nested strategy for the primers, as few as 1 to 10 PFU could be detected. The amplified products were visualized as bands of appropriate size on ethidium bromide-stained agarose gels. The primer pairs allowed amplification of several Ockelbo and Sindbis virus isolates but discriminated between these and other alphaviruses. Ockelbo virus RNA was detected in 4 of 10 skin biopsy specimens collected during the acute stage of the disease. The identities of the amplified products were confirmed by restriction endonuclease cleavage. Acute- and convalescent-phase sera as well as lymphocytes collected during the convalescent phase were negative by the polymerase chain reaction. No infectious virus could be recovered from any of the specimens.
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PMID:Detection of Ockelbo virus RNA in skin biopsies by polymerase chain reaction. 839 82

Bovine herpesvirus 1 DNA has been isolated by SDS lysis of the virus purified from potassium tartrate (10-50%) density gradient centrifugation. The quality and quantity of viral DNA was checked by UV spectrophotometry and ethidium bromide stained agarose gel electrophoresis. The 0.4 kb Hin dIII'O' fragment of BHV-1 DNA was selectively cloned into Hin dIII cut pUC9 plasmid DNA (2.665 kb). Recombinants were screened by white/blue colonies as well as Hin dIII restriction enzyme analysis. On restriction endonuclease analysis of recombinant plasmid DNA (p-BH-0) with several restriction enzymes, viz., Sau 3A, Hin fI, Rsa I, Sal I, Dra I, Bgl I, Bgl II, Sma I, Hpa I, Stu I, Mlu I, Xho I, Kpn I, Hae III, Eco RI, Bam HI, Pst I, Pal I, revealed insert viral DNA having sites for Hin fI, Hae III, Rsa I, Sma I, only. Further, the partial restriction map of the recombinant plasmid DNA was constructed using above enzymes.
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PMID:Molecular cloning and restriction endonuclease analysis of 0.4 kb Hin dIII'O' fragment of bovine herpesvirus 1 DNA. 840 44

The amounts of nuclear DNA and the AT and GC content of four Eulemur (Prosimii, Lemuridae) species and of an E. coronatus x E. macaco hybrid were measured by flow cytometry in peripheral blood leukocytes, following propidium iodide, Hoechst 33258, and mithramycin staining. Hoechst 33258 and mithramycin were also used to evaluate the base composition of genomic DNA in the chromosomes. The amount of DNA resisting C-banding pretreatment (C-heterochromatic DNA) was measured in metaphase chromosomes by static fluorometry. The genome of E. coronatus was significantly larger than the genomes of all other species examined, due to a higher content of pericentromeric, mainly GC-rich, heterochromatic DNA. The restriction banding patterns produced by BamHI digestion and ethidium bromide staining on extracted DNA were studied in the hybrid and its parental species (E. coronatus and E. macaco). The restriction banding pattern of the sole E. coronatus individual showed two bands which were repeated in the restriction banding pattern of the hybrid. The qualitative and quantitative differences of C-heterochromatic DNA in E. coronatus confirm the "splitting" processes and the phylogenetic relationships in the genus Eulemur suggested by Jung et al. (1992) on the basis of the restriction banding patterns produced by endonuclease digestion.
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PMID:Genome size and qualitative and quantitative characteristics of C-heterochromatic DNA in Eulemur species and in a viable hybrid. 844 31

DNA fragmentation was evaluated in three instances of programmed cell death, interdigital cell death in embryonic mouse limbs, and metamorphic death of both the labial glands and intersegmental muscle in the tobacco hornworm Manduca sexta. In the mouse, we evaluated both developmental cell death and expanded-range cell death induced by retinoic acid. The status of DNA was examined in several ways. Nuclei were examined by electron microscopy and Feulgen staining. Quantitative assessment of total DNA content in Feulgen-stained degenerating nuclei was made for the gland. In the labial gland, DNA content does not drop during the early phases of cell death; nor is an endonucleolytic ladder seen when DNA was examined by ethidium bromide staining or prelabeling with [3H]thymidine. Only by using end labeling of DNA could we detect DNA fragmentation at a very late stage in cell death, day 4 of the collapse of the gland. In contrast, WEHI 7.1 lymphoma cells display an early and extensive ladder after treatment with glucocorticoids. In mouse limb, for which cell death follows a more classic apoptotic morphology, a ladder is likewise not seen. We conclude that activation of an endonuclease is neither a trigger nor a necessary or defining component of the early phases of developmental programmed cell death, and that reported failure by others to find such a ladder may depend on limitations in the system that is under investigation.
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PMID:Delayed internucleosomal DNA fragmentation in programmed cell death. 846 89


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