Diamidino-2-phenylindole Staining Research Articles

Anticancer effects of Mori radicis cortex, Mori ramulus, Angelica tenuissima, and Inonotus obliquus extract mixtures on B16F10 mur ine melanoma cells via up regulation of p53 and down regulation of Akt expression

Purpose: To investigate the anticancer effects of the extract mixture of Mori radicis cortex, Mori ramulus, Angelica tenuissima, and Inonotus obliquus extract (MRME), a new herbal preparation from Korea, on B16F10 cells.
 Methods: The MRME was extracted with water. Cell Counting Kit-8 (CCK-8) assays and 4′,6- diamidino-2-phenylindole (DAPI) staining were used to investigate the effect of MRME on cell proliferation and apoptosis in vitro. The expression of p53, p-p53, and phosphorylated Akt (p-Akt) were analyzed by western blotting. Wound-healing assay was used to analyze cell migration.
 Results: Based on CCK-8 and DAPI staining results, MRME inhibited the growth of B16F10 cells and induced apoptosis in B16F10 cells in a time- and dose-dependent manner (p < 0.05). MRME increased the expression of p53 and p-p53 and decreased the expression of p-Akt in a dose-dependent manner. MRME treatment inhibited cell migration and invasion (p < 0.05).
 Conclusion: Mori Radicis Cortex Mixture Extract exerts anticancer effect by inducing the expression of p53 and p-p53 and decreasing the expression of p-Akt, which may benefit patients with malignant melanoma when used in combination with v-raf murine sarcoma viral oncogene homolog B1 (BRAF) inhibitors.

Kinetochore size scales with chromosome size in bimodal karyotypes of Agavoideae.

In eukaryotes, the total kinetochore size (defined as a chromosomal region containing CENH3-positive nucleosomes) per nucleus strongly correlates with genome size, a relationship that has been hypothesized to stem from general intracellular scaling principles. However, if larger chromosomes within a karyotype required larger kinetochores to move properly, it could also be derived from the mechanics of cell division. We selected seven species of the plant subfamily Agavoideae whose karyotypes are characterized by the presence of small and very large chromosomes. We visualized the kinetochore regions and chromosomes by immunolabelling with an anti-CENH3 antibody and DAPI (6'-diamidino-2-phenylindole) staining. We then employed 2D widefield and 3D super-resolution microscopy to measure chromosome and kinetochore areas and volumes, respectively. To assess the scaling relationship of kinetochore size to chromosome size inside a karyotype, we log-transformed the data and analysed them with linear mixed models which allowed us to control for the inherent hierarchical structure of the dataset (metaphases within slides and species). We found a positive intra-karyotype relationship between kinetochore and chromosome size. The slope of the regression line of the observed relationship (0.277 for areas, 0.247 for volumes) was very close to the theoretical slope of 0.25 for chromosome width based on the expected physics of chromosome passage through the cytoplasm during cell division. We obtained similar results by reanalysing available data from human and maize. Our findings suggest that the total kinetochore size to genome size scaling observed across eukaryotes may also originate from the mechanics of cell division. Moreover, the potential causal link between kinetochore and chromosome size indicates that evolutionary mechanisms capable of leading kinetochore size changes to fixation, such as centromere drive, could promote the size evolution of entire chromosomes and genomes.

PD49-07 ENGINEERING OF CORPORAL TISSUE CONSTRUCTS USING NON-HUMAN PRIMATE CORPUS CAVERNOSAL SMOOTH MUSCLE AND ENDOTHELIAL CELLS FOR CLINICAL APPLICATIONS

You have accessJournal of UrologySexual Function/Dysfunction: Penis/Testis/Urethra: Benign Disease & Malignant Disease I1 Apr 2017PD49-07 ENGINEERING OF CORPORAL TISSUE CONSTRUCTS USING NON-HUMAN PRIMATE CORPUS CAVERNOSAL SMOOTH MUSCLE AND ENDOTHELIAL CELLS FOR CLINICAL APPLICATIONS Joao Zambon, Young-Min Ju, Koudy Williams, Ryan Terlecki, SIta Somara, Alexander Baume, Ashley Dean, John Jackson, Julie Allickson, James Yoo, and Anthony Atala Joao ZambonJoao Zambon More articles by this author , Young-Min JuYoung-Min Ju More articles by this author , Koudy WilliamsKoudy Williams More articles by this author , Ryan TerleckiRyan Terlecki More articles by this author , SIta SomaraSIta Somara More articles by this author , Alexander BaumeAlexander Baume More articles by this author , Ashley DeanAshley Dean More articles by this author , John JacksonJohn Jackson More articles by this author , Julie AllicksonJulie Allickson More articles by this author , James YooJames Yoo More articles by this author , and Anthony AtalaAnthony Atala More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.2231AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Numerous conditions exist, both congenital and acquired, that threaten male sexual health through changes in form and/or function. Efforts in penile reconstruction are often limited by poor availability of functionally intact penile tissue. We have previously demonstrated the ability to reconstruct functional corporal tissue via autologous cell-seeded collagen matrix in a rabbit model. In this study, we investigated the feasibility of engineering corporal constructs using cavernosal smooth muscle (SMCs) and endothelial cells (ECs) from non-human primates (NHPs) seeded onto 3D acellular corporal collagen matrices. METHODS Corpora cavernosa were isolated from NHPs. These specimens were then subjected to an established decellularization process to create acellular corporal collagen matrices. Autologous corporal SMCs and ECs were isolated, expanded in vitro, and seeded onto matrices via a multistep static/dynamic procedure. RESULTS Histologic and immunohistochemical analyses were performed. The corporal construct treated with the TritronX-100 protocol was effectively decellularized based on results of both DAPI (4,6 diamidino 2 phenylindole) staining and DNA assay (< 50 ng dsDNA/mg dry sample weight). Scanning electron microscopy demonstrated highly porous 3D structure and structural integrity. Evenly distributed cellular attachment and phenotype of corporal ECs and SMCs before/after dynamic culture conditioning were evaluated by immunohistochemical staining with anti-von Willebrand factor and anti-alpha smooth muscle actin. CONCLUSIONS We have demonstrated the feasibility of engineering viable and well-organized corpora cavernosa using tissue from NHPs. This represents an important achievement toward clinical utility for humans. Such technology may have application for congenital anomalies, penile cancer, traumatic penile injury, and selected cases of erectile dysfunction and Peyronies disease. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e976 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Joao Zambon More articles by this author Young-Min Ju More articles by this author Koudy Williams More articles by this author Ryan Terlecki More articles by this author SIta Somara More articles by this author Alexander Baume More articles by this author Ashley Dean More articles by this author John Jackson More articles by this author Julie Allickson More articles by this author James Yoo More articles by this author Anthony Atala More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Aggregation and biofilm formation of bacteria isolated from domestic drinking water

The objective of this study was to investigate the autoaggregation, coaggregation and biofilm formation of four bacteria namely Sphingobium, Xenophilus, Methylobacterium and Rhodococcus isolated from drinking water. Auto and coaggregation studies were performed by both qualitative (DAPI staining) and semi-quantitative (visual coaggregation) methods and biofilms produced by either pure or dual-cultures were quantified by crystal violet method. Results from the semi-quantitative visual aggregation method did not show any immediate auto or coaggregation, which was confirmed by the 4′,6 diamidino-2-phenylindole (DAPI) staining method. However, after 2 hours, Methylobacterium showed the highest autoaggregation of all four isolates. The Methylobacterium combinations showed highest coaggregation between dual species at extended period of times (72 hours). Biofilm formation by pure cultures was negligible in comparison to the quantity of biofilm produced by dual-species biofilms. The overall results show that coaggregation of bacteria isolated from drinking water was mediated by species-specific and time-dependent interactions with a synergistic type of biofilm formation. The results of this study are therefore a useful step in assisting the development of potential control strategies by identifying specific bacteria that promote aggregation or biofilm formation in drinking water distribution systems.

Neuroprotective effect of α-lipoic acid on cultured retinal ganglion cells under elevated pressure in vitro

Objective To observe the neuroprotective effect of α-lipoic acid (ALA) on cultured retinal ganglion cells (RGC-5) under elevated pressure in vitro.Methods Cultured RGC-5 cells were divided randomly into 4 groups,including normal control group (group A),negative control group (group B),elevated pressure group (group C) and elevated pressure + ALA group (group D).The cells of group A and C were not intervened with ALA.The cells of group B were treated with 200 μmol/L ALA.The cells of group D were treated with different concentrations of ALA (50,100,200 μmol/L) for one hour.Then cells of group C and D were exerted to 50 mm Hg (1 mm Hg=0.133 kPa) for 24 hours,while the cells of group A and B were exerted to normal pressures for 24 hours.The cell viability was measured using the methyl thiazolyl tetrazolium (MTT) assay and apoptosis was evaluated using 4',6 diamidino-2-phenylindole (DAPI) staining.Expression of MnSOD was determined by real-time polymerase chain reaction (RT-PCR)and Western blot,respectively.Results The cell viability of group B was (65.6 ± 3.4) ％,which lower than that in group D of three concentrations of ALA[ (75.1± 3.3)％,(81.8 ± 2.9 ) ％,(87.9 ± 3.1 ) ％ ],the differences were significantly (t =5.108,10.007,12.513; P＜0.05).DAPI staining revealed that characteristic apoptotic changes,such as chromatin condensation,convoluted nuclei with cavitations,fragmentation of the nucleus,and apoptotic bodies appeared in RGC-5 cells after 24 ours pressure.There was almost no evidence of apoptosis in group D.RT-PCR and Western blot analysis revealed that the expression of MnSOD mRNA and protein were weakly expressed in group C compared with control A (t=22.045,26.979; P＜0.01).Compared group C with group D,the level of MnSOD mRNA and protein in group D increased significantly (t=9.171,12.267,23.567,7.723,12.009,28.198;P＜0.05).In addition,the presence of ALA was found to inhibit hydrostatic pressure induced damage of RGC-5 cells in a dose-dependent manner (F=134.273,194.597;P＜0.01).Conclusion ALA can effectively improve the expression of MnSOD in RGC-5 cells under the condition of elevated pressure,enhance the ability of RGC-5 cells against oxidative damage. Key words: Retinal ganglion cells/ pathology; Thioctic acid/therapeutic use; Cells, cultured; Animal experimentation

Bornavirus Closely Associates and Segregates with Host Chromosomes to Ensure Persistent Intranuclear Infection

Bornaviruses are nonsegmented negative-strand RNA viruses that establish a persistent infection in the nucleus and occasionally integrate a DNA genome copy into the host chromosomal DNA. However, how these viruses achieve intranuclear infection remains unclear. We show that Borna disease virus (BDV), a mammalian bornavirus, closely associates with the cellular chromosome to ensure intranuclear infection. BDV generates viral factories within the nucleus using host chromatin as a scaffold. In addition, the viral ribonucleoprotein (RNP) interacts directly with the host chromosome throughout the cell cycle, using core histones as a docking platform. HMGB1, a host chromatin-remodeling DNA architectural protein, is required to stabilize RNP on chromosomes and for efficient BDV RNA transcription in the nucleus. During metaphase, the association of RNP with mitotic chromosomes allows the viral RNA to segregate into daughter cells and ensure persistent infection. Thus, bornaviruses likely evolved a chromosome-dependent life cycle to achieve stable intranuclear infection.

UV-Induced Apoptosis in Lens: Prevention by Caffeine

Purpose: UV irradiation is a well-known etiological factor in the genesis of human cataracts. Irradiation with such wavelengths is known to initiate formation of reactive oxygen species implicated in the onset and progression of cataracts. As a corollary to our previous reports showing that caffeine can protect against UV-induced physiological damage to the lens, apparent by the effect against the decrease in the levels of adenosine triphosphate (ATP) and glutathione (GSH) as well as the associated inhibition of the active transport activity, further experiments were undertaken to find out whether caffeine would also be effective in inhibiting the eventual UV-triggered apoptotic cell death. Method: Freshly isolated mice lenses were incubated exposed to UV in the absence and presence of caffeine (2.5 mM) for a period of 5 hours. The blank controls were run in the dark. The medium of incubation was Tyrode. Subsequently, they were fixed in 10% buffered formalin and processed for sectioning and staining. Nuclear localization was done by staining with diamidino-2-phenylindole (DAPI). Apoptosis was histochemically detected with terminal deoxynucleotidyl transferase–catalyzed labeling of the 3′-OH termini in the DNA breaks using fluorescein-labeled dUTP as substrate, that is, the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) reaction. Results: As expected, incubation of the lenses under UV led to extensive apoptosis of the epithelium. The majority of the epithelial cells became TUNEL positive. Such transformation was thwarted if incubation was conducted in the medium fortified with caffeine. Another notable cellular abnormality, also apparent by DAPI staining, was the partial denudation and loss of epithelial cells from the anterior surface of the lens. A significant number of apoptotic cells also appeared to have migrated to the posterior capsule. Caffeine prevented these effects. Conclusion: The results demonstrate that caffeine has a significant inhibitory effect against the onset of apoptotic cell death of the lens epithelium induced by UV irradiation. The caffeine effect is apparently exerted by its property of scavenging reactive oxygen species produced photochemically. We therefore hypothesize that the compound could be useful in attenuating the cataractogenic effect of UV.

Lack of Tyrosylprotein Sulfotransferase-2 Activity Results in Altered Sperm-Egg Interactions and Loss of ADAM3 and ADAM6 in Epididymal Sperm

Tyrosine O-sulfation is a post-translational modification catalyzed by two tyrosylprotein sulfotransferases (TPST-1 and TPST-2) in the trans-Golgi network. Tpst2-deficient mice have male infertility, sperm motility defects, and possible abnormalities in sperm-egg membrane interactions. Studies here show that compared with wild-type sperm, fewer Tpst2-null sperm bind to the egg membrane, but more of these bound sperm progress to membrane fusion. Similar outcomes were observed with wild-type sperm treated with the anti-sulfotyrosine antibody PSG2. The increased extent of sperm-egg fusion is not due to a failure of Tpst2-null sperm to trigger establishment of the egg membrane block to polyspermy. Anti-sulfotyrosine staining of sperm showed localization similar to that of IZUMO1, a sperm protein that is essential for gamete fusion, but we detected little to no tyrosine sulfation of IZUMO1 and found that IZUMO1 expression and localization were normal in Tpst2-null sperm. Turning to a discovery-driven approach, we used mass spectrometry to characterize sperm proteins that associated with PSG2. This identified ADAM6, a member of the A disintegrin and A metalloprotease (ADAM) family; members of this protein family are associated with multiple sperm functions. Subsequent studies revealed that Tpst2-null sperm lack ADAM6 and ADAM3. Loss of ADAM3 is strongly associated with male infertility and is observed in knockouts of male germ line-specific endoplasmic reticulum-resident chaperones, raising the possibility that TPST-2 may function in quality control in the secretory pathway. These data suggest that TPST-2-mediated tyrosine O-sulfation participates in regulating the sperm surface proteome or membrane order, ultimately affecting male fertility.

Calcium- and polyphosphate-containing acidic granules of sea urchin eggs are similar to acidocalcisomes, but are not the targets for NAADP

Acidocalcisomes are acidic calcium-storage compartments described from bacteria to humans and characterized by their high content in poly P (polyphosphate), a linear polymer of many tens to hundreds of Pi residues linked by high-energy phosphoanhydride bonds. In the present paper we report that millimolar levels of short-chain poly P (in terms of Pi residues) and inorganic PPi are present in sea urchin extracts as detected using 31P-NMR, enzymatic determinations and agarose gel electrophoresis. Poly P was localized to granules randomly distributed in the sea urchin eggs, as shown by labelling with the poly-P-binding domain of Escherichia coli exopolyphosphatase. These granules were enriched using iodixanol centrifugation and shown to be acidic and to contain poly P, as determined by Acridine Orange and DAPI (4′,6′-diamidino-2-phenylindole) staining respectively. These granules also contained large amounts of calcium, sodium, magnesium, potassium and zinc, as detected by X-ray microanalysis, and bafilomycin A1-sensitive ATPase, pyrophosphatase and exopolyphosphatase activities, as well as Ca2+/H+ and Na+/H+ exchange activities, being therefore similar to acidocalcisomes described in other organisms. Calcium release from these granules induced by nigericin was associated with poly P hydrolysis. Although NAADP (nicotinic acid–adenine dinucleotide phosphate) released calcium from the granule fraction, this activity was not significantly enriched as compared with the NAADP-stimulated calcium release from homogenates and was not accompanied by poly P hydrolysis. GPN (glycyl-L-phenylalanine-naphthylamide) released calcium when added to sea urchin homogenates, but was unable to release calcium from acidocalcisome-enriched fractions, suggesting that these acidic stores are not the targets for NAADP.

New image analysis tool to study biomass and morphotypes of three major bacterioplankton groups in an alpine lake

We present an image analysis routine to de- termine the contribution of distinct morphotypes to the total abundance and biomass (carbon) of freshwater bac- terioplankton and to the fraction of cells detected by fluorescence in situ hybridization via catalyzed reporter deposition (CARD-FISH). The method was tested on bacterial assemblages from an alpine lake (Piburger See, Austria) at characteristic time points during the lim- nological year. Although on average 51% of 4',6'-di- amidino-2-phenylindole (DAPI)-stained objects were hy- bridized with the oligonucleotide probe EUB I-III, we detected on average 80% of total biomass determined from DAPI staining. The assemblage was numerically dominated by cocci and rods <0.6 µm (mean cell volume = 0.024 µm 3 ). Only a minor part of these morphotypes could be hybridized (18 and 50%, respectively). In con- trast, larger rods (0.087 µm 3 ), cocci (0.155 µm 3 ) and vibrio-shaped cells (0.073 µm 3 ) showed much higher probabilities to be detected by CARD-FISH. These mor- photypes per se formed the highest contribution to total biomass, which explained the high detection efficiency of biomass with CARD-FISH. In addition, we determined the seasonal dynamics of morphotype distributions within 3 distinct phylogenetic lineages. Actinobacteria were predominately small rods and cocci, whereas bac- teria from the Cytophaga -Flavobacterium-Bacteroides group formed mainly large rods, cocci and filaments. Be- taproteobacteria showed the highest morphological vari- ability. Within all lineages, distinct spatio-temporal dy- namics of dominant morphotypes were observed. Thus, the approach presented here will allow for more detailed studies of the amount of carbon bound by different bac- terial taxa. This is of relevance as distinct lineages can contribute much more to total bacterial biomass than to total bacterioplankton abundance.

Bacterial biomass and activity in the marginal ice zone of the northern Barents Sea

Abstract Bacteria in the Arctic Waters are well adapted to low temperatures and play a key role in the transformation of organic matter. However, the activity of planktonic bacteria at cellular level remains poorly understood. In this study, we use fluorescent markers (4′,6′-diamidino-2-phenylindole (DAPI), 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), Live/Dead BacLight viability kit) to discriminate between bacterial cells with a variety of physiological activities in the 0–200 m water column and sinking particles. During two field studies (July 2003 and 2004), we covered nine stations in the northern Barents Sea. The median bacterial abundance (DAPI staining) in the upper 50 m layer was 0.9×106 cells ml−1 (range 0.2–3.2×106 cells ml−1) in 2003 and 0.5×106 cells ml−1 (range 0.2–1.0×106 cells ml−1) in 2004. Bacteria with sufficient electron transport activity to be stained with CTC were on average 10% of the total count and ca. 20% of the total cells had intact cell membranes. In the water column, proxies of substrate availability (POC, PON, chlorophyll a, primary production) and bacterial production (thymidine and leucine uptake) correlated strongly with total bacterial count, CTC-stained cells and cells with ‘leaky’ membrane (stained with propidium iodine), but not with the concentration of cells with intact cell membrane. Contrary to expectations, the proportion of CTC-stained bacteria was not higher in the sinking particles (captured with sediment traps) compared to the ambient water. However, out of the bacteria with intact cell membranes, a higher proportion scored as CTC positive in the aggregates compared to the ambient water. Bacterial cells with ‘leaky’ cell membranes formed the largest part of total cell count in all samples, and accumulated in sites with high microbial activity (sinking aggregates, chlorophyll maxima, layers of high primary and bacterial production). We hypothesize that the source of the bacterial cells with ‘leaky’ cell membranes was metabolically the most active fraction of the bacterial assemblage (stained with CTC).

Survival of Mycobacterium avium in Drinking Water Biofilms as Affected by Water Flow Velocity, Availability of Phosphorus, and Temperature

Mycobacterium avium is a potential pathogen occurring in drinking water systems. It is a slowly growing bacterium producing a thick cell wall containing mycolic acids, and it is known to resist chlorine better than many other microbes. Several studies have shown that pathogenic bacteria survive better in biofilms than in water. By using Propella biofilm reactors, we studied how factors generally influencing the growth of biofilms (flow rate, phosphorus concentration, and temperature) influence the survival of M. avium in drinking water biofilms. The growth of biofilms was followed by culture and DAPI (4',6'-diamidino-2-phenylindole) staining, and concentrations of M. avium were determined by culture and fluorescence in situ hybridization methods. The spiked M. avium survived in biofilms for the 4-week study period without a dramatic decline in concentration. The addition of phosphorus (10 microg/liter) increased the number of heterotrophic bacteria in biofilms but decreased the culturability of M. avium. The reason for this result is probably that phosphorus increased competition with other microbes. An increase in flow velocity had no effect on the survival of M. avium, although it increased the growth of biofilms. A higher temperature (20 degrees C versus 7 degrees C) increased both the number of heterotrophic bacteria and the survival of M. avium in biofilms. In conclusion, the results show that in terms of affecting the survival of slowly growing M. avium in biofilms, temperature is a more important factor than the availability of nutrients like phosphorus.

Sleeping Beauty Transposase Has an Affinity for Heterochromatin Conformation

The Sleeping Beauty (SB) transposase reconstructed from salmonid fish has high transposition activity in mammals and has been a useful tool for insertional mutagenesis and gene delivery. However, the transposition efficiency has varied significantly among studies. Our previous study demonstrated that the introduction of methylation into the SB transposon enhanced transposition, suggesting that transposition efficiency is influenced by the epigenetic status of the transposon region. Here, we examined the influence of the chromatin status on SB transposition in mouse embryonic stem cells. Heterochromatin conformation was introduced into the SB transposon by using a tetracycline-controlled transrepressor (tTR) protein, consisting of a tetracycline repressor (TetR) fused to the Kruppel-associated box (KRAB) domain of human KOX1 through tetracycline operator (tetO) sequences. The excision frequency of the SB transposon, which is the first step of the transposition event, was enhanced by approximately 100-fold. SB transposase was found to be colocalized with intense DAPI (4',6'-diamidino-2-phenylindole) staining and with the HP1 family by biochemical fractionation analyses. Furthermore, chromatin immunoprecipitation analysis revealed that SB transposase was recruited to tTR-induced heterochromatic regions. These data suggest that the high affinity of SB transposase for heterochromatin conformation leads to enhancement of SB transposition efficiency.

Comparison of 4', 6'-diamidino-2-phenylindole and Giemsa Stainings in Preimplantation Mouse Embryos Micronucleus Assay Including a Triple Dose Study

Analysis of micronuclei (MN) in preimplantation embryos is a good method for the evaluation of cytogenetic damage induced by occupational and environmental mutagen during early pregnancy. To examine whether conventional Giemsa staining produced the same accuracy of micronuclei as the DNA-specific 4', 6'-diamidino-2-phenylindole (DAPI) staining in preimplantation embryo induced by maternal exposure to chlorpyrifos, we conducted assays on 469 mouse (3 groups) preimplantation embryos micronucleus. Slides were stained with DAPI. After DAPI staining, the slides were de-stained and restained with Giemsa. Giemsa staining showed similar frequencies in MN to DNA-specific DAPI staining in all three groups. Both staining techniques revealed significant increases in frequency of MN in the treated group in comparison to the control group. Both methods showed a statistically significant correlation between MN frequency and the dose of chlorpyrifos. Compared with DAPI staining, the sensitivity of Giemsa staining was 85.0%, 86.0% and 90.9% for control, 40 mg/kg, and 80 mg/kg chlorpyrifos treated group, respectively. The specificity was 97.9%, 91.4% and 96.5% for control, 40 mg/kg, and 80 mg/kg chlorpyrifos treated group, respectively. Thus, we recommend that Giemsa staining technique be a standard staining method in detecting MN of preimplantation embryos induced by occupational or environmental hazards.

Diversity of bacteria growing in natural mineral water after bottling.

Bacterial growth occurs in noncarbonated natural mineral waters a few days after filling and storage at room temperature, a phenomenon known for more than 40 years. Using the full-cycle rRNA approach, we monitored the development of the planktonic bacterial community in a noncarbonated natural mineral water after bottling. Seven 16S rRNA gene libraries, comprising 108 clones in total, were constructed from water samples taken at various days after bottling and from two different bottle sizes. Sequence analyses identified 11 operational taxonomic units (OTUs), all but one affiliated with the betaproteobacterial order Burkholderiales (6 OTUs) or the class Alphaproteobacteria (4 OTUs). Fluorescence in situ hybridization (FISH) was applied in combination with DAPI (4',6'-diamidino-2-phenylindole) staining, viability staining, and microscopic counting to quantitatively monitor changes in bacterial community composition. A growth curve similar to that of a bacterium grown in a batch culture was recorded. In contrast to the current perception that Gammaproteobacteria are the most important bacterial components of natural mineral water in bottles, Betaproteobacteria dominated the growing bacterial community and accounted for 80 to 98% of all bacteria detected by FISH in the late-exponential and stationary-growth phases. Using previously published and newly designed genus-specific probes, members of the betaproteobacterial genera Hydrogenophaga, Aquabacterium, and Polaromonas were found to constitute a significant proportion of the bacterial flora (21 to 86% of all bacteria detected by FISH). For the first time, key genera responsible for bacterial growth in a natural mineral water were identified by applying molecular cultivation-independent techniques.

Multifunctional centromere binding factor 1 is essential for chromosome segregation in the human pathogenic yeast Candida glabrata.

The CBF1 (centromere binding factor 1) gene of Candida glabrata was cloned by functional complementation of the methionine biosynthesis defect of a Saccharomyces cerevisiae cbf1 deletion mutant. The C. glabrata-coded protein, CgCbf1, contains a basic-helix-loop-helix leucine zipper domain and has features similar to those of other budding yeast Cbf1 proteins. CgCbf1p binds in vitro to the centromere DNA element I (CDEI) sequence GTCACATG with high affinity (0.9 x 10(9) M(-1)). Bandshift experiments revealed a pattern of protein-DNA complexes on CgCEN DNA different from that known for S. cerevisiae. We examined the effect of altering the CDEI binding site on CEN plasmid segregation, using a newly developed colony-sectoring assay. Internal deletion of the CDEI binding site led only to a fivefold increase in rates of plasmid loss, indicating that direct binding of Cbf1p to the centromere DNA is not required for full function. Additional deletion of sequences to the left of CDEI, however, led to a 70-fold increase in plasmid loss rates. Deletion of the CBF1 gene proved to be lethal in C. glabrata. C. glabrata cells containing the CBF1 gene under the influence of a shutdown promoter (tetO-ScHOP) arrested their growth after 5 h of cultivation in the presence of the reactive drug doxycycline. DAPI (4',6'-diamidino-2-phenylindole) staining of the arrested cells revealed a significant increase in the number of large-budded cells with single nuclei, 2C DNA content, and short spindles, indicating a defect in the G(2)/M transition of the cell cycle. Thus, we conclude that Cbf1p is required for chromosome segregation in C. glabrata.

Bacillus subtilis SMC is required for proper arrangement of the chromosome and for efficient segregation of replication termini but not for bipolar movement of newly duplicated origin regions.

SMC protein is required for chromosome condensation and for the faithful segregation of daughter chromosomes in Bacillus subtilis. The visualization of specific sites on the chromosome showed that newly duplicated origin regions in growing cells of an smc mutant were able to segregate from each other but that the location of origin regions was frequently aberrant. In contrast, the segregation of replication termini was impaired in smc mutant cells. This analysis was extended to germinating spores of an smc mutant. The results showed that during germination, newly duplicated origins, but not termini, were able to separate from each other in the absence of SMC. Also, DAPI (4',6'-diamidino-2-phenylindole) staining revealed that chromosomes in germinating spores were able to undergo partial or complete replication but that the daughter chromosomes were blocked at a late stage in the segregation process. These findings were confirmed by time-lapse microscopy, which showed that after duplication in growing cells the origin regions underwent rapid movement toward opposite poles of the cell in the absence of SMC. This indicates that SMC is not a required component of the mitotic motor that initially drives origins apart after their duplication. It is also concluded that SMC is needed to maintain the proper layout of the chromosome in the cell and that it functions in the cell cycle after origin separation but prior to complete segregation or replication of daughter chromosomes. It is proposed here that chromosome segregation takes place in at least two steps: an SMC-independent step in which origins move apart and a subsequent SMC-dependent step in which newly duplicated chromosomes condense and are thereby drawn apart.

Non-surgical transfer of 4’,6’-diamidino-2-phenylindole-stained equine embryos

4’,6’-diamidino-2-phenylindole (DAPI) is a fluorescent dye that binds only to the DNA of dead blastomeres of an embryo. The use of DAPI for assessing viabilities of equine Day 6 to 7 embryos was studied. The pregnancy and normal development of the embryo until 20 days after transfer (day of pregnancy termination) was used as an indicator ofembryonic viability. Eleven embryos were stained with DAPI at room temperature for 15 min. They were then exposed to UV light to visualize staining, and cultured for 2 h before non-surgical transfer to recipient mares. Eight control embryos were cultured for 2 h before transfer to recipient mares. The recipient mares were scanned for pregnancies every other day starting 6 days after transfer. Twenty days after the transfer the pregnant recipient mares received luprostiol to induce abortion of the foetus. The pregnancy rate 6 days after transfer was 82% in the treatment group, and 75% in the control group (P&gt;0.05). One DAPI-stained embryo resorbed and was no longer visible 12 days after transfer. However, this particular recipient mare had a uterine inflammation as shown by examination in the oestrus following embryo transfer. Although the number of embryos is insufficient to demonstrate lack of treatment effect, the high pregnancy rates following DAPI staining should encourage further studies on this subject.

Resolution of mitochondrial DNA structures in the large yeast Wickerhamia fluorescens

DAPI (4′6 Diamidino-2-phenylindole) staining of the large yeast Wickerhamia fluorescens revealed extensive cytoplasmic staining material attributable to mitochondrial DNA (mtDNA), often present in large network-like structures. The maintenance of this mtDNA appears to be insensitive to a variety of mitochondrial specific mutagens, suggesting that W. fluorescens may be classified as a petite negative yeast. Restriction enzyme analysis generated a unit genome size of 42(10 6) D for this mtDNA which, together with determinations of the average mtDNA per cell, allowed an estimate of the cellular copy number of mitochondrial genomes. A physical map of this mtDNA was also derived. These experiments suggested models which might reflect the cytological structures resolved by DAPI staining in W. fluorescens relative to other yeasts.