OBJECTIVE: To find out the optimal conditions of human chromosomal analysis protocol in peripheral blood sample. METHODS: The experiments were made with the variations of phytohaemagglutinin, colcemid, ethidium bromide concentration and the variations of hypotonic solution exposure time. RESULTS: In the experiment on the optimal phytohaemagglutinin concentration, the highest mitotic index in the overall collected cells was obtained in phytohaemagglutinin concentration 15microL/ml. In the experiment on the concentration of mitotic arrestant colcemid, the proper chromosomal state that is meta phase stage and doesn't have many chromosomal crossings or tangles was obtained in colcemid concentration 0.05microg/ml. In the experiment on the optimal exposure time of hypotonic solution(0.075M KCl) treatment, the most suitable intervals between chromosomes were subtained in 20 minutes. In the experiment on the optimal concentration of ethidium bromide to obtain minute chromosomal bands, the best result was when ethidium bromide concentration 5microg/ml or 7.5microg/ml was addition to colcemid concentration 0.02microg/ml. CONCLUSION: The combination of phytohaemagglutinin 15microL/ml, colcemid 0.05microg/ml, hypotonic solution exposure time for 20 minutes is important to the collection of appropriate chromosome state in human chromosomal analysis using peripheral blood. In the case that needs to obtain minute bands, the elongated chromosomes are obtained when ethidium bromide 5microg/ml or 7.5microg/ml in addition to colcemid concentration 0.02microg/ml with the same conditions of phytohaemagglutinin and hypotonic solution.
Demecolcine ; Ethidium ; Humans ; Mitotic Index

The luminous intensity of dark variant (S1) separated from photobacterium phosphoreum (A2) was 1/10,000 less than that of wild-type. Ethidium bromide (EB) (0.6 mg/L), Mytomycin C (MC, 0.05 mg/L), 2-amino fluorene (2-AF, 1.0 mg/L) all could strongly induce reversion mutation for S1 within 24 h and increase reversion ratio significantly. The results of experiments indicated that these revertants had stable genetic characteristic and the mutation may take place at gene levels. The mutagenesis to S1 caused by EB, MC and 2-AF was detected and it may be used as a new rapid, simple and sensitive method for gene toxicant monitoring.
*Chemiluminescent Measurements ; Ethidium/pharmacology ; Ethidium/toxicity ; Luciferases/biosynthesis ; Mitomycins/pharmacology ; Mitomycins/toxicity ; Mutagens ; Mutation/*drug effects ; Photobacterium/*genetics ; Toxicology/methods ; Transcription, Genetic/drug effects ; Variation (Genetics)

Alleles and genotype frequencies and its distribution pattern for the highly polymorphic D1S80 locus were determined in a Korean population sample, especially in Kwangju and Chonnam, by using PCR followed by agarose gel electrophoresis with ethidium bromide staining, a procedure called the amplified-fragment-length polymorphism(Amp-FLP) technique. And the data were compared with the alleles and genotype frequencies of Finnish population, North American Caucasian, and Korean population(Seoul) which had been reported. In 203 unrelated Korean individuals 27 alleles and 84 genotypes were observed. The highest allele frequency was in allele M24(0.128) and tne next orders were inalleles M18(0.126), M29, M30, M31, and M28 and the other alleles showed relatively low frequencies. The highest frequency of genotype was in M18/M24 and the next order frequencies were M18/M30, M19/M27 M29/M29, and M18/M29. The homozyous genotypes were in 9 alleles such as M29, M24, M31, and M18, and most of heterozygous genotypes were composed of alleles of each homozygous genotypes and /or the other alleles, its composition of genotypes was 0.881(74/84), 183(0.901) of the 203 individuals alleles, its composition of genotypes was 0.881(74/84), 183(0.901) of the 203 individuals alleles, its composition of genotypes was 0.881(74/84), 183(0.901) of the 203 individuals were included. The VNTR D1S80 locus demonstrated a heterozygosity of 0.872. From the above results, VNTR D1S80 locus may be a powerful locus to identify individuals, however, the allele frequencies was not closely related to the genotype pattern, and the alleles of homozygous genotypes influenced on the chance of the recombination of the various genotypes. It is necessary to analyze the genotype distribution and the recombination pattern of alleles as well as alleles and genotype frequencies in each populations for statistical test at most highly polymorphic loci.
Alleles* ; Electrophoresis, Agar Gel ; Ethidium ; Gene Frequency ; Genotype* ; Gwangju ; Jeollanam-do ; Polymerase Chain Reaction ; Recombination, Genetic

Chromatography, High Pressure Liquid ; methods ; Drug Residues ; analysis ; Electrochemistry ; Ethidium ; analysis ; Luminescent Measurements ; methods ; Sensitivity and Specificity

OBJECTIVES: This study was performed to evaluate the critical role of glutathione(GSH) in methyl mercury chloride(MeHgCl)induced cell apoptosis. METHODS: The effect of GSH in MeHgCl induced cell apoptosis was observed in mouse macrophage-derived RAW 264.7 cells in vitro. The cells were cultured in Dulbecco's modified Eagle's medium(DMEM). RESULTS: MeHgCl exerted a dose dependent cytotoxicity,as demonstrated by the MTT assay, which is an assay dependent partially on the mitochondrial function. Moreover, in the presence of NAC, a GSH precursor, the MeHgCl induced cytotoxicity was significantly decreased whereas BSO, a specific GSH synthesis inhibitor,increased the MeHgCl induced cytotoxicity.The MeHgCl induced DNA fragmentation and chromatin condensation was consistent with the morphological alterations. The MeHgCl treated cells exhibited increasing annexin V-FITC binding to the phos-phatidylserine(PS)translocated from the inner to the outer leaflet of the plasma membrane and those cells with NAC pretreatment significantly exhibited decreasing annexin V-FITC binding compared to the cells treated with MeHgCl only. However BSO pretreatment markedly exhibited the increasing annexin V-FITC binding. The MeHgCl treated cells generated ROS, which was evidenced by the oxidation of dihydroethidine and the generation of the fluorescent product, ethidium. In addition, BSO pretreatment further enhanced the extent of ROS generation caused by MeHgCl whereas NAC pretreatment decreased the amount of ROS generation. MeHgCl led to a dose dependent decrease in the GSH content. Although MeHgCl exposure significantly reduced the GSH level, those cells that had a NAC pretreatment contained a higher level of GSH compared to the cells treated with MeHgCl only. In contrast, BSO pretreatment futher enhanced the extent of GSH depletion caused by MeHgCl. CONCLUSIONS: These results indicate that MeHgCl reduced the GSH content and impaired the defense against oxidative damage caused by ROS formation in RAW 264.7 cells. It is possible that these factors leads to the activation of the apoptosis signaling pathway. Ultimately these results suggest that GSH plays a crucial role in protecting the activity against MeHgCl induced apoptosis.
Animals ; Antioxidants ; Apoptosis* ; Cell Membrane ; Chromatin ; DNA Fragmentation ; Ethidium ; Glutathione* ; Mice

Several observation suggest that the antimelanocyte autoantibodies could play a role in melanocyte destruction. Some experiments indicate that melanocyte antibodies from patients with vitiligo can kill melanocyte in vitro. In these experiments, we demonstrated that vitiligo patient's sera containing antimelanocyte antibodies can lyse cultured human melanocytes by complement activation. Melanocyte cytotoxicity was measured using the ethidium bromide/ acridine orange viability assay. Significant melanocyte cytotoxicity was seen in sera from patients with both active and inactive vitiligo(p<0.01). Melanocyte cytotoxicity measured with complement-mediated cytotoxicity decreased after systemic steroid treatment(p<0.05) ; however melanocyte cytotoxicity showed no significant change with systemic PUVA therapy.
Acridine Orange ; Antibodies ; Autoantibodies* ; Complement Activation ; Ethidium ; Humans ; Melanocytes ; PUVA Therapy ; Vitiligo*

OBJECTIVEthe main purpose of this study is to develop an assay for detection of skin viability in situ.

METHODSTwo nucleic acid dyes, SYTO 13 and ethidium bromide (EB), are used to assess the viability of skin tissue. The viability of cells can be determined by different colors under fluorescence microscope. In this study, the recovery of skin restored at 4 degrees C for different periods is measured by SYTO/EB dyes and a type of indirect assessment, WST-1. Then both results are compared.

RESULTSThe results measured by two methods showed that the viability of skin tissue was nearly 30% after two weeks storage. At any point of experiment, the data from two methods have not obvious variance.

CONCLUSIONThe assessment of skin viability by SYTO/EB is also quick, sensitive and convenient. At the same time the viability of any type of cells can be observed in situ. So it can be considered as an available method for detection of skin viability.

Animals ; Cell Survival ; physiology ; Cryopreservation ; Ethidium ; Male ; Models, Animal ; Rats ; Skin ; cytology ; metabolism ; Skin Physiological Phenomena ; Tissue Preservation ; methods

BACKGROUND: Chlamydia trachomatis is currently classified into 15 serovars (A, B, Ba, C, D, E, F, G, H, I, J, K, L1, L2, L3) and a large number of serovariants. Typing of C. trachomatis serovars has generally been performed by the MOMP (major outer membrane protein) serotyping method, which requires a large panel of polyclonal and monoclonal antibodies. Recently the PCR was successfully applied to the genotyping of different C. trachomatis serovars by means of restriction fragment length polymorphism (RFLP) analysis and direct sequencing of amplified omp1 DNA. The objective of this study was to evaluate the serotyping of C. trachomatis by PCR-FLP and to determine the serovars of C. trachomatis urogenital isolates. METHODS: The 15 reference strains of C. trachomatis and 27 clinical isolates were analyzed by PCR-FLP. The C. trachomatis omp1 gene was amplified by PCR. For serotyping by RFLP analysis, the omp1 PCR products were digested with AluI and were electrophoresed through a 7% polyacrylamide gel with ethidium bromide staining. If necessary, the PCR products were analyzed with HinfI, a combination of EcoRI and DdeI, or CfoI. RESULTS: In serotyping of 15 reference strains of C. trachomatis, serovars A, B, Ba, E, F, G, K, and L2 were clearly identified after AluI digestion. However serovars C, H, I, J, L3 and serovars D, L1 were respectively identical patterns after AluI digestion. Serovars C and J and serovars D and L1 were further discriminated by a second step of enzyme digestion with HinfI. Serovar H, I, and L3 were distinguished by enzyme digestion with EcoRI and DdeI. In serotyping of C. trachomatis from 27 urogenital isolates, 25 isolates were clearly typed. Nine were typed as serovar E, 8 were typed as serovar D, 6 were typed as F, 2 were typed as serovar H. Two isloates showed unidentifiable RFLP pattern which was not in accordance with any of the existing C. trachomatis prototypes. CONCLUSIONS: PCR-FLP analysis is a rapid, simple, and powerful tool for differentiating serovars of C. trachomatis. Therefore, this approach is recommended for future epidemiological studies of C. trachomatis. And the serovar E, D, and F are the most prevalent types found in urogenital specimens, representing 92% of those investigated.
Antibodies, Monoclonal ; Chlamydia trachomatis* ; Chlamydia* ; Digestion ; DNA ; Ethidium ; Membranes ; Polymerase Chain Reaction* ; Polymorphism, Restriction Fragment Length* ; Serotyping*

BACKGROUND: Chlamydia trachomatis is currently classified into 15 serovars (A, B, Ba, C, D, E, F, G, H, I, J, K, L1, L2, L3) and a large number of serovariants. Typing of C. trachomatis serovars has generally been performed by the MOMP (major outer membrane protein) serotyping method, which requires a large panel of polyclonal and monoclonal antibodies. Recently the PCR was successfully applied to the genotyping of different C. trachomatis serovars by means of restriction fragment length polymorphism (RFLP) analysis and direct sequencing of amplified omp1 DNA. The objective of this study was to evaluate the serotyping of C. trachomatis by PCR-FLP and to determine the serovars of C. trachomatis urogenital isolates. METHODS: The 15 reference strains of C. trachomatis and 27 clinical isolates were analyzed by PCR-FLP. The C. trachomatis omp1 gene was amplified by PCR. For serotyping by RFLP analysis, the omp1 PCR products were digested with AluI and were electrophoresed through a 7% polyacrylamide gel with ethidium bromide staining. If necessary, the PCR products were analyzed with HinfI, a combination of EcoRI and DdeI, or CfoI. RESULTS: In serotyping of 15 reference strains of C. trachomatis, serovars A, B, Ba, E, F, G, K, and L2 were clearly identified after AluI digestion. However serovars C, H, I, J, L3 and serovars D, L1 were respectively identical patterns after AluI digestion. Serovars C and J and serovars D and L1 were further discriminated by a second step of enzyme digestion with HinfI. Serovar H, I, and L3 were distinguished by enzyme digestion with EcoRI and DdeI. In serotyping of C. trachomatis from 27 urogenital isolates, 25 isolates were clearly typed. Nine were typed as serovar E, 8 were typed as serovar D, 6 were typed as F, 2 were typed as serovar H. Two isloates showed unidentifiable RFLP pattern which was not in accordance with any of the existing C. trachomatis prototypes. CONCLUSIONS: PCR-FLP analysis is a rapid, simple, and powerful tool for differentiating serovars of C. trachomatis. Therefore, this approach is recommended for future epidemiological studies of C. trachomatis. And the serovar E, D, and F are the most prevalent types found in urogenital specimens, representing 92% of those investigated.
Antibodies, Monoclonal ; Chlamydia trachomatis* ; Chlamydia* ; Digestion ; DNA ; Ethidium ; Membranes ; Polymerase Chain Reaction* ; Polymorphism, Restriction Fragment Length* ; Serotyping*

Ror fetal sex determination by the PCR method, oilgoprimers to Y- chromosome gene, DYZI, SRY, and AMGL were synthesized genomic DNA was extracted from male and female placenta for the control use. DYZI represented 154 bp single band to 0.001 pg/ml male genomic DNA but did not represent 154 bp band in female genomic DNA, SRY represented 341 bp bandto 1 pg/ml male genomic DNA in 2% agarose gel eleftrophoresis stained with ethidium bromide. DYZI was 1,000 fold sensitive than Sry and AMGL. DYZI and SRY could not identify the PDR failure from female but AMGL identified to 1,000-fold. During the dyal ampiification of female genomic DNA mixed with male genomic DNA, 0.00125 pg/nl, 1:400 part, male genomic DNA contamination represented male band but SRY amplification did not represent male band. It was suggested that SRY gene was deleted in two 46,XY felmle cases. For fetal sex determination, PCR with DYZL, SRY, and AMGL was performed in 10 cases. For fetal sex determination, PCR with DYZL, SRY, and AMGL with karyotyping in 10 cases of chorionic villi sex dietermination, PCR with DYZI, SRY, and AMGL was performed in 10 cases. For feral sex determination, PCR with DYZI, SRY, and AMGL with karyothping result, fetal sex determination, PCR with DYZI, SRY, and AMGL was performed in 10 Cases of choricinic villi and 15 cases of amnionic cells. By the comparison with karyotyping result, fetal sex determination was achieved successfully in all 23 samlies using PCR of SRY and AMGL but false result was detected in 3 cases(13%) using DYZI. Acording to our results, it was concluded that DYZL was 1,000-fold sensitive than SRY and AMGL but could not be used because of its false results, and AMGL and SRY must be used concomitantly for precise sex determination.
Amnion ; Chorionic Villi ; DNA ; DNA Contamination ; Ethidium ; Female ; Genes, sry ; Humans ; Karyotyping ; Male ; Placenta ; Polymerase Chain Reaction* ; Sepharose