OBJECTIVETo investigate the genomic variation of Epstein-Barr virus (EBV) and its significance in nasopharyngeal carcinogenesis.

METHODSForty nasopharyngeal carcinoma (NPC) biopsy tissues were used for detection of EBV BamHI f variant and LMP1 XhoI-loss by polymerase chain reaction (PCR), nested PCR, and RFLP (restriction fragment length polymorphism). Forty-eight samples of peripheral blood mononuclear cells (PBMC) taken from apparently healthy adult individuals were used for detection of LMP1 XhoI-loss. Three samples of amplified LMP1 exon 1 DNA from B95-8 cell line and 2 NPC tissues (one having XhoI-loss and the other having Wt-XhoI/XhoI-loss) were sequenced.

RESULTSThirty out of the 40 NPC cases (30/40, 75%) harbored EBV BamHI f variant and the remaining 10 (10/40, 25%) harbored BamHI F prototype. Thirty out of the 39 NPCs (30/39, 76.9%) showed single EBV LMP1 XhoI-loss, 7 (7/39, 18.0%) showed single LMP1 Wt-XhoI (presence of a XhoI site in exon 1 of LMP1 gene, as in B95-8 cell line), and 2 (2/39, 5.1%) showed both LMP1 Wt-XhoI and XhoI-loss. Thirty-eight of the 39 NPCs (97.4%) showed EBV LMP1 XhoI-loss or/and BamHI F variant. In the NPC tissue (1 case only) showing the prototype of Wt-XhoI/BamHI "f", there were several base substitutions, including 5 missense mutations and 2 silent mutations present in LMP1 exon 3, on DNA sequencing. On the other hand, 10 out of the 48 samples of PBMC taken from apparently healthy individuals could be amplified successfully by nested PCR for detection of LMP1 XhoI site. All of these 10 samples carried the prototype of EBV LMP1 Wt-XhoI.

CONCLUSIONSThe majority of EBV present in neoplastic cells of NPC is of BamHI "f" variant and/or possesses LMP1 XhoI-loss, as compared with that in healthy individuals. This genomic variation of EBV may bear some roles in the development and progression of NPC.

Adult ; Aged ; Binding Sites ; genetics ; DNA, Viral ; genetics ; metabolism ; Deoxyribonuclease BamHI ; metabolism ; Deoxyribonucleases, Type II Site-Specific ; metabolism ; Female ; Herpesvirus 4, Human ; genetics ; isolation & purification ; Humans ; Male ; Middle Aged ; Mutation ; Nasopharyngeal Neoplasms ; virology ; Sequence Deletion ; Viral Matrix Proteins ; genetics

BACKGROUND: Staphylococcus aureus remains one of the most frequently encountered bacterial pathogens and is responsible for a variety of mild to life- threatening infections. There is a substantial body of evidence that individuals who are asymptomatic nasal carriers of S. aureus are at increased risk of developing serious staphylococcal infections. Approximately 20% to 30% of health care workers at any given time are also nasal carriers of S. aureus. A subset of these may spread the organism to patients by direct contact transmission. CHROMagar Staph aureus (CSA) is a new chromogenic medium for identification of S. aureus on the basis of colony pigmentation. METHODS: The abilities of CSA, thermostable nuclease (DNase), and mannitol salt agar (MSA) to identify S. aureus isolates (n=70) and discriminate between S. aureus and coagluase-negative staphylococci (CoNS; n=8) were compared. RESULTS: CSA proved to be more sensitive and specific than DNase and MSA, allowing a reliable, simple, and rapid method for the identification of S. aureus isolates. All CoNS encountered in this study could be easily differentiated from S. aureus on the medium. The supplementation with 4 microgram/mL of oxacillin allowed simple identification of methicillin resistance in hospital acquired S. aureus strains which show multiple drug resistance profiles. CONCLUSION: CSA proved to be simple and reliable method for the identification of nasal carriers of S. aureus of health care workers.
Agar ; Delivery of Health Care ; Deoxyribonucleases ; Drug Resistance, Multiple ; Humans ; Mannitol ; Methicillin Resistance* ; Methicillin-Resistant Staphylococcus aureus* ; Micrococcal Nuclease ; Oxacillin ; Pigmentation ; Staphylococcal Infections ; Staphylococcus aureus

Multiple DNases were identified from Haemonchus contortus intestine based on previous studies. The DNases detected at 34, 36 and 38.5 kDa had diverse characteristics. Some of them had characteristics similar to those of mammalians and others had unusual characteristics. This study was carried out to fractionate worm intestinal DNases from other proteins using phenyl Sepharose chromatographic methods. All DNases detected from Haemonchus contortus intestine were fractionated in the flowthrough of phenyl Sepharose, indicating the worm DNases are hydrophilic. The DNases were enriched five-fold in the flowthrough fraction while additional steps are required for isolation of the worm DNases. Thus, fractionation with phenyl Sepharose could be used as a good initial step to enrich and separate DNases from other proteins.
Chromatography ; Deoxyribonucleases ; Haemonchus ; Intestines ; Proteins ; Sepharose

Multiple DNases were identified from Haemonchus contortus intestine based on previous studies. The DNases detected at 34, 36 and 38.5 kDa had diverse characteristics. Some of them had characteristics similar to those of mammalians and others had unusual characteristics. This study was carried out to fractionate worm intestinal DNases from other proteins using phenyl Sepharose chromatographic methods. All DNases detected from Haemonchus contortus intestine were fractionated in the flowthrough of phenyl Sepharose, indicating the worm DNases are hydrophilic. The DNases were enriched five-fold in the flowthrough fraction while additional steps are required for isolation of the worm DNases. Thus, fractionation with phenyl Sepharose could be used as a good initial step to enrich and separate DNases from other proteins.
Chromatography ; Deoxyribonucleases ; Haemonchus ; Intestines ; Proteins ; Sepharose

PURPOSE: Aggregatibacter actinomycetemcomitans is associated with localized aggressive periodontitis. It produces cytolethal distending toxin (CDT), which induces cell cycle arrest in the G2/M phase. The CDT holotoxin is composed of CdtA, CdtB, and CdtC. CdtB has structural homology to human DNase I and is an active component of the CDT complex acting as a DNase. In particular, the pattern homology seen in the CdtB subunit has been associated with specific DNase I residues involved in enzyme catalysis, DNA binding, and metal ion binding. So, to study the functions and regulation of recombinant CdtB, we made up a quantity of functional recombinant CdtB and tested it in relation to the metal ion effect. MATERIALS AND METHODS: We constructed the pET28a-cdtB plasmid from A. actinomycetemcomitans Y4 by genomic DNA PCR and expressed it in the BL21 (DE3) Escherichia coli system. We obtained the functional recombinant CdtB by the refolding system using the dialysis method and then analyzed the DNase activity and investigated the metal ion effect from plasmid digestion. RESULTS: The recombinant CdtB subunit was expressed as the inclusion bodies. We were able to obtain functional recombinant CdtB subunit using refolding system. We confirmed that our refolded recombinant CdtB had DNase activity and was influenced by the metal ions Mg2+ and Ca2+. CONCLUSION: We suggest that the factors influencing recombinant CdtB may contribute to CDT associated diseases, such as periodontitis, endocarditic, meningitis, and osteomyelitis.
Aggressive Periodontitis ; Bacterial Toxins ; Catalysis ; Cell Cycle Checkpoints ; Deoxyribonuclease I ; Deoxyribonucleases ; Dialysis ; DNA ; Edetic Acid ; Escherichia coli ; Humans ; Inclusion Bodies ; Ions ; Meningitis ; Osteomyelitis ; Periodontitis ; Plasmids ; Polymerase Chain Reaction

OBJECTIVETo develop a simple method for genotyping of hepatitis E virus (HEV) and to investigate HEV genotype distribution in Nanjing area.

METHODSTwenty-seven full HEV sequences currently-available in GenBank were analyzed with MegAlign and MapDraw programs of DNA STAR software. Degenerate primers were designed and applied to amplify a fragment in HEV ORF1 region. HEV genotypes were determined by the size of the PCR products and by single restriction endonuclease analysis.

RESULTSThe PCR products of HEV genotype 1 and 2 were 275 bp and 269 bp in size. Distinctively, the PCR products of genotype 3 and 4 were 317 bp and 314 bp in size. Moreover, the PCR products of genotype 1 could be digested by Nae 1, but the products of genotype 2 could not. Distinctively, the PCR products of HEV genotype 3 could be digested by Not 1, but the products of genotype 4 could not. Six HEV reference strains standing for different HEV genotypes were clustered into their own types as predicted. Within 43 HEV IgM-positive clinical specimens collected in Nanjing, 19 were HEV PCR-positive and identified as genotype 4.

CONCLUSIONA simple method of PCR combined with single restriction endonuclease analysis is developed for HEV genotyping. This assay allows rapid identification of a large number of HEV isolates directly from clinical specimens. Among patients with hepatitis E in Nanjing, most were infected with HEV genotype 4.

DNA Restriction Enzymes ; metabolism ; DNA, Complementary ; genetics ; metabolism ; Deoxyribonucleases, Type II Site-Specific ; metabolism ; Genotype ; Hepatitis E ; blood ; genetics ; immunology ; Hepatitis E virus ; genetics ; Humans ; Polymerase Chain Reaction ; methods ; RNA, Viral ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

BACKGROUND: Thyroid Transcription Factor-1(TTF-1) acts as a tissue specific transcription factor in the regulation of lung specific gene expression and as morphogenic protein during lung organogenesis. Currently, there is very little information on the cis-acting sequences and transcription and transcription factors that direct the TTF-1 gene expression. DNAse 1 hypersensitive (DH) sites represent a marker for active or potentially active chromatin and are likely to be especially important in gene regulation, being associated with many DNA sequences that regulate gene expression. It is clear that DH regions correlate with genetic regulatory loci and binding for sequence-specific DNA-binding proteins. METHODS: We have used DH site assays to identify putative distal regulatory elements in H441 lung adenocarcinoma cells, which express the TTF-1 gene and HeLa cells. RESULTS: There are four DH sites 5' of the TTF-1 gene. These sites are located at base pair approximately +150, -450, -800, and -1500 from the start of transcription. CONCLUSION: These data suggest that there may be at least one intragenic site and regulatory region 5' prime to the promotor region.
Adenocarcinoma ; Base Pairing ; Base Sequence ; Chromatin ; Deoxyribonuclease I* ; Deoxyribonucleases* ; DNA-Binding Proteins ; Gene Expression ; HeLa Cells ; Humans ; Lung* ; Organogenesis ; Promoter Regions, Genetic ; Regulatory Sequences, Nucleic Acid ; Thyroid Gland ; Transcription Factors*

Trypsin and DNase digestion technique has become a retinal digestion technique for studying diabetic retinopathy. We tried osmotic digestion method with DNase and compared the quality of preparation of retina and microvascular change with trypsin digestion in normal and diabetic rats. Streptozotocin-induced diabetic rats were sacrificed at 9, 18, 27weeks in 6 rats. Right retinas were digested with 3% trypsin while left wer digested with 0.1% DNase. For control, 18 normal rats were sacrificed at the same time. DNase was superior to trypsin for retinal preparation on the stainability, the degree of separating nonvascular fissue from vascular net, the degree of preservation of vascular net in normal & diabetic rats. In diabetic rats, the number of pericyte decreased significantly with age, which not in normal rats(p=0.0023) We suggest DNase digestion technique as an new alternative for trypsin digestion technique in the study of microvascular change of diabetic retinopathy.
Animals ; Deoxyribonucleases* ; Diabetes Mellitus ; Diabetic Retinopathy ; Digestion* ; Endothelial Cells ; Microvessels* ; Pericytes ; Rats* ; Retina ; Retinaldehyde* ; Trypsin*

BACKGROUND: Staphylococcus aureus is most frequently isolated Gram-positive cocci from clinical specimens. The accurate identification of S. aureus is required. Lipovitellin-salt-mannitol (LSM) agar is medium for differentiating S. aureus and coagulase-negative staphylococci (CNS) by mannitol acidification and lipovitellin lipase reaction. In this study, we compare LSM agar with the other conventional methods for differentiating S. aureus and CNS in clinical laboratories, coagulase test, mannitol-salt agar (MSA), and DNase agar. METHODS: One hundred and forty-five isolates of staphylococci from clinical specimens are used. S. aureus and CNS were identified by coagulase test, MSA, DNase agar, and LSM agar. RESULTS: Eighty-nine isolates were identified as S. aureus and 59 isolates were revealed CNS. Compared ability of methods to differntiate S. aureus from CNS, sensitivity and specificity were 98.7% and 96.6% with LSM agar, 92.1% and 96.6% with coagulase test, 96.6% and 93.2% with MSA, 93.3% and 98.3% with DNase agar, respectively. CONCLUSIONS: LSM agar show good discrimination between S. aureus and CNS. LSM agar may be used for identification of S. aureus in clinical laboratories.
Agar* ; Coagulase ; Deoxyribonucleases ; Discrimination (Psychology) ; Gram-Positive Cocci ; Lipase ; Mannitol ; Sensitivity and Specificity ; Staphylococcus aureus* ; Staphylococcus*

Zinc finger nuclease (ZFN), which is a chimeric fusion structure between a Cys2-His2 zinc-finger protein (ZFP) and the cleavage domain of Fok I endonuclease, can be used to introduce targeted double-stranded breaks (DSBs). ZFN-mediated cleavage leads to mutations when double-stranded breaks are repaired by homologous recombination (HR) or nonhomologous end joining (NHEJ). In recent years, ZFNs are widely used in the fields of genetic research. In this review, the methodology and technical advantages of ZFNs were briefly discussed.
Animals ; Deoxyribonucleases, Type II Site-Specific ; chemistry ; genetics ; metabolism ; Humans ; Zinc Fingers