A fluorometric method for direct detection of inorganic polyphosphate in enterohemorrhagic O157:H7 / 南方医科大学学报

OBJECTIVE@#To establish a quantitative fluorescent detection method using DAPI for detecting inorganic polyphosphate (polyP) in enterohemorrhagic Escherichia coli (EHEC) O157:H7.@*METHODS@#The DNA of wild-type strain of EHEC O157:H7 was extracted and purified. DAPI was combined with the extracted DNA and polyP45 standards for measurement of the emission spectra at 360 nm and 415 nm fluorescence spectrophotometry. The fluorescence of DAPI-DNA and DAPI-polyP complexes was detected by fluorescence confocal microscopy to verify the feasibility of DAPI for detecting polyP. To determine the optimal pretreatment protocol for improving the cell membrane permeability, the effects of 6 pretreatments of the cells (namely snap-freezing in liquid nitrogen, freezing at -80 ℃, and freezing at -20 ℃, all followed by thawing at room temperature; heating at 60 ℃ for 10 min; treatment with Triton x-100; and placement at room temperature) were tested on the survival of EHEC O157:H7. The fluorescence values of the treated bacteria were then measured after DAPI staining. A standard calibration curve of polyP standard was established for calculation of the content of polyP in the live cells of wildtype EHEC strain and two mutant strains.@*RESULTS@#At the excitation wavelength of 360 nm, the maximum emission wavelength of DAPI-DNA was 460 nm, and the maximum emission wavelength of DAPI-polyP was 550 nm at the excitation wavelength of 415 nm. The results of confocal microscopy showed that 405 nm excitation elicited blue fluorescence from DAPIDNA complex with the emission wavelength of 425-475 nm; excitation at 488 nm elicited green fluorescence from the DAPIpolyP complex with the emission wavelength of 500-560 nm of. Snap-freezing of cells at -80 ℃ followed by thawing at room temperature was the optimal pretreatment to promote DAPI penetration into the live cells. The standard calibration curve was =1849+127.5 (R=0.991) was used for determining polyP content in the EHEC strains. The experimental results showed that wild-type strain had significantly higher polyP content than the mutant strains with deletion.@*CONCLUSIONS@#We established a convenient quantitative method for direct and reliable detection polyP content to facilitate further study of polyP and its catalytic enzymes in EHEC O157:H7.

Screening of antiproliferative activity mediated through apoptosis pathway in human non-small lung cancer A-549 cells by active compounds present in medicinal plants

Objective:To explore the antiproliferative activity and apoptosis in cells caused by active compounds present in plants using different techniques.Methods:We investigated the antiproliferative effects of methanolic extracts from different parts of seven plants on A-549 (lung cancer) cells and primary cell culture (chick embryo fibroblast cells, as normal cells) using MTT assay and the potent plant was fractioned further. All these fractions were screened again for anti-proliferative activity. DNA fragmentation and DAPI staining were used to study apoptosis. Quantitative real-time was used to investigate the expression of apoptotic-related genes. LC-MS andResults:Methanolic extract of Vitex negundo (V. negundo) was selected as a potent fraction. Among all fractions screened, ethylacetate fraction of V. negundo was selected as the most potent antiproliferative fraction and phytochemical analysis of the extract revealed the presence of secondary metabolites. Ethaylacetate fraction of V. negundo was found to cause characteristic apoptotic morphological changes and generation of ROS in A-549 cells. Ethaylacetate fraction of V. negundo also induced apoptosis in A-549 which was supported by DNA fragmentation and DAPI staining. To investigate the molecular mechanism behind the cytotoxic effect of ethaylacetate fraction of V. negundo, quantitative real-time PCR was used to measure expression levels of p53, bax, bcl2, casp-3 and casp-9. Using LC-MS andConclusions:It is concluded from the present study that V. negundo is capable of triggering growth-inhibitive and apoptosis effects in A-549 cells, signifying that V. negundo may possesses anti-lung cancer activity.

Screening of antiproliferative activity mediated through apoptosis pathway in human non-small lung cancer A-549 cells by active compounds present in medicinal plants

Objective: To explore the antiproliferative activity and apoptosis in cells caused by active compounds present in plants using different techniques. Methods: We investigated the antiproliferative effects of methanolic extracts from different parts of seven plants on A-549 (lung cancer) cells and primary cell culture (chick embryo fibroblast cells, as normal cells) using MTT assay and the potent plant was fractioned further. All these fractions were screened again for anti-proliferative activity. DNA fragmentation and DAPI staining were used to study apoptosis. Quantitative real-time was used to investigate the expression of apoptotic-related genes. LC-MS and

Anticancer Activity of Oscillatoria Terebriformis Cyanobacteria in Human Lung Cancer Cell Line A549.

Purpose: To evaluate the anti-cancer properties of the cyanobacterial extract of Oscillatoria terebriformis Methods: The extract was tested in Human lung cancer cell lines and examined for its effect on cell viability, nuclear morphology and sub-G1 formation. Cell viability was determined by micro culture tetrazolium technique (MTT), nuclear morphology investigated using 4’-6-diamidino-2-phenylindole (DAPI) staining technique, and apoptosis assay using DNA fragmentation. Results: The results showed decreasing cell viability in a concentration-dependent manner. Altered cell morphology after treatment with the extract demonstrated that cells experienced apoptosis. Conclusion: The data demonstrate that Oscillatoria Terebriformis extract induced apoptosis in Human lung cancer A549 cells, and therefore, has a potential as an anti-cancer agent.

Quantitative and Qualitative Estimation of Bacteria Contaminating Human Hairs

Human hairs have been known to be easily contaminated with microorganisms. This study was performed in order to measure what bacterial species and how much microorganisms contaminate human hairs in specific place. Virgin human hairs were left at 6 positions in inside corner and beside window in a laboratory for 7 days. The number of viable bacterial cells, which were determined by most probable number method, contaminating the human hairs was measured at a maximum of 10(6)/g hair and a minimum of 10(3)/g hair in inside corner and maximum of 10(6)/g hair and a minimum of 10(3)/g hair beside window. The bacterial cells-contaminating human hairs were observed via fluorescence light microscopy after 4',6-diamino-2-phenylindole (DAPI) staining. The bacterial community contaminating human hairs was analyzed via the thermal gradient gel electrophoresis (TGGE) technique, based on the diversity of the 16S-rDNA variable region. In total, approximately 20 bacterial species were detected from 12 groups of hair samples. In this study, general experimental methods-fluorescence staining, TGGE and MPN-were combined to develop new method for observation and estimation of bacteria contaminating human hairs.

Quantitative and Qualitative Estimation of Bacteria Contaminating Human Hairs

Human hairs have been known to be easily contaminated with microorganisms. This study was performed in order to measure what bacterial species and how much microorganisms contaminate human hairs in specific place. Virgin human hairs were left at 6 positions in inside corner and beside window in a laboratory for 7 days. The number of viable bacterial cells, which were determined by most probable number method, contaminating the human hairs was measured at a maximum of 10(6)/g hair and a minimum of 10(3)/g hair in inside corner and maximum of 10(6)/g hair and a minimum of 10(3)/g hair beside window. The bacterial cells-contaminating human hairs were observed via fluorescence light microscopy after 4',6-diamino-2-phenylindole (DAPI) staining. The bacterial community contaminating human hairs was analyzed via the thermal gradient gel electrophoresis (TGGE) technique, based on the diversity of the 16S-rDNA variable region. In total, approximately 20 bacterial species were detected from 12 groups of hair samples. In this study, general experimental methods-fluorescence staining, TGGE and MPN-were combined to develop new method for observation and estimation of bacteria contaminating human hairs.

The karyotype of Nothoscordum arenarium Herter (Gilliesioideae, Alliaceae): a populational and cytomolecular analysis

The genus Nothoscordum Kunth comprises approximately 20 species native to South America. Karyologically, the genus is remarkable for its large chromosomes and Robertsonian translocations. Variation in chromosome number has been recorded in a few polyploid species and it is unknown among diploids. This study presents the chromosome number and morphology of 53 individuals of seven populations of N. arenarium Herter (2n = 10). In addition, karyotype analyses after C-banding, staining with CMA and DAPI, and in situ hybridization with 5S and 45S rDNA probes were performed in six individuals from one population. All individuals exhibited 2n = 10 (6M + 4A), except for one tetraploid (2n = 20, 12M + 8A) and one triploid (2n = 15, 9M + 6A) plant. C-banding revealed the presence of CMA+/DAPI- heterochromatin in the short arm and in the proximal region of the long arm of all acrocentric chromosomes. The 45S rDNA sites co-localized with the CMA+ regions of the acrocentrics short arms, while the 5S rDNA probe only hybridized with the subterminal region of a pair of metacentric chromosomes. A change in the pattern of CMA bands and rDNA sites was observed in only one individual bearing a reciprocal translocation involving the long arm of a metacentric and the long arm of an acrocentric chromosome. These data suggest that, despite isolated cases of polyploidy and translocation, the karyotype of N. arenarium is very stable and the karyotypic instability described for other species may be associated with their polyploid condition.