DNA Fluorochrome Research Articles

EVALUATION OF METHODS FOR THE DETECTION OF MYCOPLASMA CONTAMINATION OF CELL CULTURES AND THE EFFECTIVENESS OF ANTIMYCOPLASMA PREPARATIONS

This article presents the results of a study on the detection of mycoplasma infection in various cell lines and the evaluation of the effectiveness of antimycoplasma drugs. To detect mycoplasmas in cell cultures, DNA fluorochrome (DAPI, Apollo Scientific) staining methods, PlasmoTest™ (Invivogen, France), MycoStrip (Invivogen, France) and EZ-PCR™ (Biological Industries, Israel) kits were used, which are widely recommended for detecting mycoplasmas in cell culture. Based on the obtained research results, it was found that the antimycoplasma drugs MycoZap (Lonza Bioscience), Plasmocin (Invivogen, France), MRA (BIO-RAD) and ВМ-Cyclin (Roche) are effective in combating mycoplasma contamination of cell cultures. The method of detecting mycoplasmas with DNA fluorochromes, determining the presence of mycoplasma may be subjective, since its sensitivity to mycoplasmas is low. In turn, the MycoStrip set can give a false negative result due to the presence of mycoplasmas not included in the list of detectable species of this set. The positive and negative results of PlasmoTest were precisely correlated with the results of EZ-PCR. If mycoplasma contamination is suspected, DNA staining and the MycoStrip kit can be used for preliminary analysis, and PlasmoTest™ and EZ-PCR final confirmation.

Reproductive Phases Influence on In Vitro Maturation of Abattoir-Derived Bovine Oocytes

This study investigated the influence of the reproductive phase of oocyte donors originating from abattoir-derived ovaries on in vitro maturation (IVM) of bovine oocytes. The IVM of the oocytes was evaluated by observing the degree of cumulus cell (CC) expansion and extrusion of the first polar body (PB-1) as well as both nuclear progression (NP) and cytoplasmic maturation (CM). For NP, after 24 h of IVM, oocytes were denuded and stained with DNA fluorochrome and observed under an epifluorescence microscope. The male or female pronucleus (PN) forming ability of an oocyte following in vitro fertilization (IVF) was considered as CM. For CM, the fertilized zygotes after 24 h post-insemination were stained with DNA fluorochrome and observed under an epifluorescence microscope. Results revealed that the maturation rate was significantly influenced by the reproductive phase of the oocytes donor, luteal phase ovaries (LPO) versus follicular phase ovaries (FPO) based on the presence or absence of corpus luteum (CL), respectively. The significantly higher CC expansion (2°; 44.9±4.9% versus 35.9±2.7%) and PB-1 extrusion (58.3±1.7% versus 46.7±2.4%) were observed with LPO than FPO oocytes, respectively. A significant (p<0.05) effect of the reproductive phase was also noted with LPO oocytes progressing higher rate of mature metaphase II stage (MII; 58.3±1.7% versus 46.7±2.4%) and PN (54.9±2.3% versus 44.4±3.0%) formation following IVF than FPO oocytes that imply better NP and CM of oocytes, respectively. Therefore, the present study indicates that the reproductive phase of oocyte donors notably from LPO positively influences the IVM of bovine oocytes to produce a higher number of viable embryos in vitro. Ann. Bangladesh Agric. (2022) 26 (1) : 1-19

Reference standards for flow cytometric estimation of absolute nuclear DNA content in plants.

The estimation of nuclear DNA content has been by far the most popular application of flow cytometry in plants. Because flow cytometry measures relative fluorescence intensities of nuclei stained by a DNA fluorochrome, ploidy determination, and estimation of the nuclear DNA content in absolute units both require comparison to a reference standard of known DNA content. This implies that the quality of the results obtained depends on the standard selection and use. Internal standardization, when the nuclei of an unknown sample and the reference standard are isolated, stained, and measured simultaneously, is mandatory for precise measurements. As DNA peaks representing G1/G0 nuclei of the sample and standard appear on the same histogram of fluorescence intensity, the quotient of their position on the fluorescence intensity axis provides the quotient of DNA amounts. For the estimation of DNA amounts in absolute units, a number of well‐established standards are now available to cover the range of known plant genome sizes. Since there are different standards in use, the standard and the genome size assigned to it has always to be reported. When none of the established standards fits, the introduction of a new standard species is needed. For this purpose, the regression line approach or simultaneous analysis of the candidate standard with several established standards should be prioritized. Moreover, the newly selected standard organism has to fulfill a number of requirements: it should be easy to identify and maintain, taxonomically unambiguous, globally available, with known genome size stability, lacking problematic metabolites, suitable for isolation of sufficient amounts of nuclei, and enabling measurements with low coefficients of variation of DNA peaks, hence suitable for the preparation of high quality samples.

Cytogenetic markers reveal a reinforcement of variation in the tension zone between chromosome races in the brachypterous grasshopper Podisma sapporensis Shir. on Hokkaido Island

The cytogenetic characteristics of the grasshopper Podisma sapporensis (two races 2n = 23♂ X0/XX and 2n = 22♂ neo-XY/neo-XX) were analysed through fluorescence in situ hybridization with rDNA and telomeric DNA probes, C-banding, fluorochrome and silver staining. For the first time, samples from the neighbourhood of a hybrid population (i.e., Mikuni Pass population) were studied. Our results indicated a significant degree of chromosomal differentiation between P. sapporensis races when comparing the number and position of the rDNA sites, as well as the heterochromatin composition and distribution obtained by C-banding and DAPI/CMA3 staining. Telomeric signals were usually detected at the distal and/or subdistal position of the autosomes; however, some chromosome ends lacked signals, probably due to a low number of telomeric repeats. On the other hand, telomeric DNA sequences were found as interstitial telomeric repeats in some autosomes, which can trigger a variety of genome instability. B chromosomes were found in specimens belonging to both main races from nine out of 22 localities. Four types of X chromosomes in the X0/XX race were identified. It was concluded that the physical mapping of rDNA sequences and heterochromatin are useful as additional markers for understanding the phylogeographic patterns of cytogenetic differentiation in P. sapporensis populations.

Methods to Detect Endoplasmic Reticulum Stress and Apoptosis in Diabetic Nephropathy.

A variety of pathophysiological cellular dysfunctions stress the endoplasmic reticulum (ER), promoting an accumulation of unfolded proteins in the ER lumen. The latter is sensed by intrinsic ER transmembrane proteins: IRE1α (inositol-requiring protein-1α), PERK (protein kinase RNA (PKR)-like ER kinase), and ATF6 (activating transcription factor 6) which when activated trigger the unfolded protein response (UPR), which includes an inhibition of protein translation while inducing specific transcription factors that induce genes aiming to relieve the ER stress response. Collectively, this reduces the burden of unfolded proteins within the ER, eventually restoring ER homeostasis and thus promoting cell survival and adaptation. However, under unresolvable ER stress conditions, the UPR promotes cell death. Diabetic nephropathy (dNP), a leading cause of end-stage renal disease in industrialized countries, is mechanistically closely linked with ER stress and renal cell death. Here, we describe methods (both in vivo and in vitro) for monitoring ER stress, UPR signaling, and cell death in renal cells by analyzing proteins and protein-protein interactions serving as markers of ER stress or cell death. These methods include visualization of interactions of UPR regulators by proximity ligation assay on renal tissue and cells and methods to detect cell death based on DNA fragmentation or fluorochrome substrates for caspases. We include two selected in vivo models to manipulate ER stress regulators and thus the UPR in murine models of dNP. Collectively, these analyses allow assessment of the activation of ER stress-induced signaling pathways and cell death in dNP and manipulation of the UPR in vivo, enabling researchers to probe for causality.

A Sensitive Fluorescence Biosensor for Silver Ions (Ag+) Detection Based on C-Ag+-C Structure and Exonuclease III-Assisted Dual-Recycling Amplification.

A C-Ag+-C structure-based fluorescence biosensor with novel combination design of exonuclease III (Exo III) dual-recycling amplification is proposed for the application of silver ions (Ag+) detection. Since oligo-1 involves C-C mismatches, the presence of Ag+ can be captured to form C-Ag+-C base pairs, which results in a double-helix structure with a blunt terminus. The double-helix structure can be cleaved by EXO III to release short mononucleotide fragments (trigger DNA) and Ag+. Released Ag+ can form new bindings with oligo-1, and other trigger DNA can be produced in the digestion cycles. Hybridization with the signal DNA (oligo-2) transforms a trigger DNA into double-stranded DNA with blunt terminus which can be cleaved by Exo III to reproduce the trigger DNA and form guanine- (G-) quadruplex DNA. The trigger DNA returns free to the solution and hybridizes with another signal DNA, which realizes the dual-recycling amplification. The G-quadruplex DNA can be reported by N-methylmesoporphyrin IX (NMM), a specific G-quadruplex DNA fluorochrome. This method allows Ag+ to be determined in the 5 to 1500 pmol/L concentration range, with a 2 pmol/L detection limit, and it has been successfully applied to the detection of Ag+ in real samples.

Effectiveness of Plasmocure™ in Elimination of Mycoplasma Species from Contaminated Cell Cultures: A Comparative Study versus other Antibiotics.

Objective Mycoplasmas spp. is among major contaminants of eukaryotic cell cultures. They cause a wide range of problems associated with cell culture in biology research centers or biotechnological companies. Mycoplasmas are also resistant to several antibiotics. Plasmocin™ has been used to treat cell lines but Plasmocin™-resistant strains have been reported. InvivoGen has developed a new anti-Mycoplasma agent called Plasmocure™ in order to eliminate resistant Mycoplasma contamination. The aim of this study was the selection of the best antibiotics for treatment of mycoplasma in cell cultures. Materials and Methods In this experimental study, a total of 100 different mammalian cell lines contaminated with different Mycoplasma species were evaluated by microbiological culture (as the gold standard method), indirect DNA fluorochrome staining, enzymatic (MycoAlert™), and universal or species-specific polymerase chain reaction (PCR) detection methods. In this study, animal and human cell lines available in National Cell Bank of Iran, were treated with Plasmocure™. The treatment efficacy and cytotoxicity of Plasmocure™ were compared with those of commonly used antibiotics such as BM- cyclin, Plasmocin™, MycoRAZOR™, sparfloxacin and enrofloxacin. Results Plasmocure™ is comprised of two antibiotics that act through various mechanisms of action than those in Plasmocin™. Two-week treatment with Plasmocure™ was enough to completely eliminate Mycoplasma spp. A moderate toxicity was observed during Mycoplasma treatment with plasmocure™; But, after elimination of Mycoplasma, cells were fully recovered. Mycoplasma infections were eliminated by Plasmocure™, BM-cyclin, Plasmocin™, MycoRAZOR™, sparfloxacin and enrofloxacin. However, the outcome of the treatment process (i.e. the frequency of complete cure, regrowth or cell death) varied among different antibiotics. ConclusionThe highest number of cured cell lines was achieved by using Plasmocure™ which also had the lowest regrowth rate after a period of four months. As a conclusion; Plasmocure™ might be considered an effective antibiotic to treat Mycoplasma infections in mammalian cell cultures especially for precious or vulnerable cells.

Automated quantification of apoptosis in B-cell chronic lymphoproliferative disorders: a prognostic variable obtained with the Cell-Dyn Sapphire (Abbott) automated hematology analyzer.

B-chronic lymphocytic leukemia CLL, a neoplastic clonal disorder with monomorphous small B lymphocytes with scanty cytoplasm and clumped chromatin, can be morphologically differentiated in typical and atypical forms with different prognosis: Smudge cells (Gumprecht's shadows) are one of the well-known features of the typical CLL and are much less inconsistent in other different types CLPD. Abbott Cell-Dyn Sapphire uses the fluorescence after staining with the DNA fluorochrome propidium iodide for the measurement of nucleated red blood cells (NRBCs) and nonviable cells (FL3+ cell fraction): We have studied the possible correlation between presence and number of morphologically identifiable smudge cells on smears and the percentage of nonviable cells produced by Cell-Dyn Sapphire. 305 blood samples from 224 patients with B-cell lymphoproliferative disorders and 40 healthy blood donors were analyzed by CBC performed by Cell-Dyn Sapphire, peripheral blood smear, and immunophenotype characterization. FL3+ fraction in CLPD directly correlated with the percentage of smudge cells and is significantly increased in patients with typical B-CLL. This phenomenon is much less evident in patients with atypical/mixed B-CLL and B-NHL. In small laboratories without FCM and cytogenetic, smudge cells%, can be utilized as a preliminary diagnostic and prognostic tool in differential diagnosis of CLPD.

Physical map of repetitive DNA and karyotype evolution in three species of the genus Omophoita (Coleoptera: Alticinae)

The order Coleoptera comprises species that are used as models in evolutionary studies, such as Alticinae, which features giant sex chromosomes. Thus, the aim of the present study was to investigate the differentiation and evolution of the sex chromosome in species of the Omophoita genus (Alticinae) using repetitive DNA (total C0t-1), C-banding and base-specific fluorochromes. Analyses using probes generated through C0t-1 reassociation kinetics showed markers distributed across all the chromosomes, especially the sex chromosomes. In conclusion, repetitive sequences are distributed across the sex chromosomes and autosomes, demonstrating that the heterochromatin of the species is largely composed of repetitive DNA. Cross-hybridisation among the species produced a very similar staining pattern for the probes. Thus, we conclude that the majority of the genome of the species of Omophoita is shared, showing the heterochromatin to be largely composed of repetitive DNA distributed along the sex chromosomes and autosomes.

Larger Daphnia at lower temperature: a role for cell size and genome configuration?

Experiments with Daphnia magna and Daphnia pulex raised at 10 and 20 °C yielded larger adult size at the lower temperature. This must reflect increased cell size, increased cell numbers, or a combination of both. As it is difficult to achieve good estimates on cell size in crustaceans, we, therefore, measured nucleus and genome size using flow cytometry at 10 and 20 °C. DNA was stained with propidium iodide, ethidium bromide, and DAPI. Both nucleus and genome size estimates were elevated at 10 °C compared with 20 °C, suggesting that larger body size at low temperature could partly be accredited to an enlarged nucleus and thus cell size. Confocal microscopy observations confirmed the staining properties of fluorochromes. As differences in nucleotide numbers in response of growth temperature within a life span is unlikely, these results seem accredited to changed DNA-fluorochrome binding properties, presumably reflecting increased DNA condensation at low temperature. This implies that genome size comparisons may be impacted by ambient temperature in ectotherms. It also suggests that temperature-induced structural changes in the genome could affect cell size and for some species even body size.

Roscovitine is a novel agent that can be used for the activation of porcine oocytes reconstructed with adult cutaneous or fetal fibroblast cell nuclei

The present study was undertaken to investigate the preimplantation developmental competence of cloned pig embryos that were derived from fibroblast cell nuclei by different methods for the activation of reconstructed oocytes. In subgroups IA and IB, nuclear-transferred (NT) oocytes derived from either adult cutaneous or fetal fibroblast cells that had been classified as nonapoptotic by intra vitam analysis for programmed cell death using the YO-PRO-1 DNA fluorochrome underwent sequential physical (i.e., electrical) and chemical activation (SE-CA). This novel method of SE-CA, which was developed and optimized in our laboratory, involves treatment of reconstituted oocytes with direct current pulses and subsequent exposure to 7.5 μM calcium ionomycin, followed by incubation with 30 μM R-roscovitine (R-RSCV), 0.7 mM 6-dimethylaminopurine and 3.5 μg/mL cycloheximide. In subgroups IIA and IIB, NT oocytes were subjected to the standard method of simultaneous fusion and activation mediated by direct current pulses. The proportion of cloned embryos in subgroup IA that reached the morula and blastocyst stages was 145/248 (58.5%) and 78/248 (31.5%), respectively. The proportions of cloned embryos in subgroup IB that reached the morula and blastocyst stages were 186/264 (70.5%) and 112/264 (42.4%), respectively. In turn, subgroup IIA yielded proportions at the morula and blastocyst stages of 110/234 (47.0%) and 49/234 (20.9%), respectively. Subgroup IIB yielded proportions at the morula and blastocyst stages of 144/243 (59.3%) and 74/243 (30.5%), respectively. In summary, the SE-CA of NT oocytes reconstructed from either type of nonapoptotic/nonnecrotic (i.e., YO-PRO-1-negative) fibroblast cell resulted in porcine cloned embryos with considerably better in vitro developmental outcomes than those of cloned embryos generated using the simultaneous fusion and activation approach. To our knowledge, this is the first report of the successful stimulation of porcine NT oocytes using electric pulses followed by an additional activation with a higher dose (1.5 times) of calcium ionomycin and subsequent exposure to a combination of 30 μM R-RSCV and lower concentrations (by 3 times) of 6-dimethylaminopurine and cycloheximide. Moreover, we report here the first use of R-RSCV, a novel meiosis-promoting factor-related p34cdc2 kinase inhibitor, in the oocyte activation protocol for the somatic cell cloning of pigs.

A rapid in situ procedure for determination of bacterial susceptibility or resistance to antibiotics that inhibit peptidoglycan biosynthesis

BackgroundAntibiotics which inhibit bacterial peptidoglycan biosynthesis are the most widely used in current clinical practice. Nevertheless, resistant strains increase dramatically, with serious economic impact and effects on public health, and are responsible for thousands of deaths each year. Critical clinical situations should benefit from a rapid procedure to evaluate the sensitivity or resistance to antibiotics that act at the cell wall. We have adapted a kit for rapid determination of bacterial DNA fragmentation, to assess cell wall integrity.ResultsCells incubated with the antibiotic were embedded in an agarose microgel on a slide, incubated in an adapted lysis buffer, stained with a DNA fluorochrome, SYBR Gold and observed under fluorescence microscopy. The lysis affects the cells differentially, depending on the integrity of the wall. If the bacterium is susceptible to the antibiotic, the weakened cell wall is affected by the lysing solution so the nucleoid of DNA contained inside the bacterium is released and spread. Alternatively, if the bacterium is resistant to the antibiotic, it is practically unaffected by the lysis solution and does not liberate the nucleoid, retaining its normal morphological appearance. In an initial approach, the procedure accurately discriminates susceptible, intermediate and resistant strains of Escherichia coli to amoxicillin/clavulanic acid. When the bacteria came from an exponentially growing liquid culture, the effect on the cell wall of the β-lactam was evident much earlier that when they came from an agar plate. A dose-response experiment with an E. coli strain susceptible to ampicillin demonstrated a weak effect before the MIC dose. The cell wall damage was not homogenous among the different cells, but the level of damage increased as dose increased with a predominant degree of effect for each dose. A microgranular-fibrilar extracellular background was evident in gram-negative susceptible strains after β-lactam treatment. This material was digested by DNase I, hybridised with a specific whole genome probe, and so recognized as DNA fragments released by the bacteria. Finally, 46 clinical strains from eight gram-negative and four gram-positive species were evaluated blind for susceptibility or resistance to one of four different β-lactams and vancomycin, confirming the applicability of the methodology.ConclusionThe technique to assess cell wall integrity appears to be a rapid and simple procedure to identify resistant and susceptible strains to antibiotics that interfere with peptidoglycan biosynthesis.

How endo- is endo-? Surface sterilization of delicate samples: a Bryopsis (Bryopsidales, Chlorophyta) case study

In the search for endosymbiotic bacteria, elimination of ectosymbionts is a key point of attention. Commonly, the surface of the host itself or the symbiotic structures are sterilized with aggressive substances such as chlorine or mercury derivatives. Although these disinfectants are adequate to treat many species, they are not suitable for surface sterilization of delicate samples. In order to study the bacterial endosymbionts in the marine green alga Bryopsis, the host plant’s cell wall was mechanically, chemically and enzymatically cleaned. Merely a chemical and enzymatic approach proved to be highly effective. Bryopsis thalli treated with cetyltrimethylammonium bromide (CTAB) lysis buffer, proteinase K and bactericidal cleanser Umonium Master showed no bacterial growth on agar plates or bacterial fluorescence when stained with a DNA fluorochrome. Moreover, the algal cells were intact after sterilization, suggesting endophytic DNA is still present within these algae. This new surface sterilization procedure opens the way to explore endosymbiotic microbial communities of other, even difficult to handle, samples.

Semi-automated relative quantification of cell culture contamination with mycoplasma by Photoshop-based image analysis on immunofluorescence preparations

Mycoplasma contamination in cell culture is a serious setback for the cell-culturist. The experiments undertaken using contaminated cell cultures are known to yield unreliable or false results due to various morphological, biochemical and genetic effects. Earlier surveys revealed incidences of mycoplasma contamination in cell cultures to range from 15 to 80%. Out of a vast array of methods for detecting mycoplasma in cell culture, the cytological methods directly demonstrate the contaminating organism present in association with the cultured cells. In this investigation, we report the adoption of a cytological immunofluorescence assay (IFA), in an attempt to obtain a semi-automated relative quantification of contamination by employing the user-friendly Photoshop-based image analysis. The study performed on 77 cell cultures randomly collected from various laboratories revealed mycoplasma contamination in 18 cell cultures simultaneously by IFA and Hoechst DNA fluorochrome staining methods. It was observed that the Photoshop-based image analysis on IFA stained slides was very valuable as a sensitive tool in providing quantitative assessment on the extent of contamination both per se and in comparison to cellularity of cell cultures. The technique could be useful in estimating the efficacy of anti-mycoplasma agents during decontaminating measures.

Observations of chromosomal behaviour in living meiotic zygotes of Chlamydomonas reinhardtii (Chlorophyceae)

Clarification of chromosome number and behaviour during meiosis is important for genetic investigations of the unicellular alga Chlamydomonas reinhardtii. However, the chromosome number and their morphology remain uncertain in this alga. We stained living zygotes of C. reinhardtii with the highly sensitive DNA fluorochrome SYBR Green I and observed chromosomal behaviour during meiosis. Zygotes with two and four cells, resulting from the first and second meiotic divisions, appeared approximately 16 and 20 h, respectively, after mature zygotes were exposed to light under experimental conditions. In prophase I, chromosomes in the nucleus changed from long, thread-like structures to bivalents. At metaphase I and anaphase I, chromosomes assumed raft-like configurations but chromosomal configurations were less distinct at metaphase II and anaphase II. The nuclei were always localized near the zygotic surface. By observing bivalents at diakinesis we determined the total number of chromosomes to be 18. Chromosome lengths ranged from 0.5 to 2 µm. Using approximate fluorescence intensities, we estimated DNA content of the largest chromosome to be 3–6 times greater than that of the smallest.

Microbial community analysis of a full-scale membrane bioreactor treating industrial wastewater

A Kubotatrade mark submerged membrane bio-reactor was applied to treat wastewater from a sugar manufacturing industry. To achieve optimal results, fundamental and extended understanding of the microbiology is important. Fluorescence in situ hybridization was used to evaluate the microbial community present. The majority of cells visualized in the sludge flocs by staining with the DNA fluorochrome DAPI, hybridized strongly with a bacterial probe. Probes specific for the alpha-, beta-, and gamma-subclasses of proteobacteria and high G + C Gram positive bacteria were used to characterize the community structures by in situ hybridization. Sampling was carried out over 12 weeks and samples were fixed with 4% paraformaldehyde for gram positive organisms and ice cold ethanol for gram negative organisms. The activated sludge population usually constitutes about 80 to 90% of proteobacteria. However, in this study it was found that a relatively small amount of proteobacteria was present within the system. No positive hybridization signal was observed with any of the applied eubacterial family- level probes.

DNA Fragmentation in Microorganisms Assessed In Situ

Chromosomal DNA fragmentation may be a direct or indirect outcome of cell death. Unlike DNA fragmentation in higher eukaryotic cells, DNA fragmentation in microorganisms is rarely studied. We report an adaptation of a diffusion-based assay, developed as a kit, which allows for simple and rapid discrimination of bacteria with fragmented DNA. Intact cells were embedded in an agarose microgel on a slide, incubated in a lysis buffer to partially remove the cell walls, membranes, and proteins, and then stained with a DNA fluorochrome, SYBR Gold. Identifying cells with fragmented DNA uses peripheral diffusion of DNA fragments. Cells without DNA fragmentation show only limited spreading of DNA fiber loops. These results have been seen in several gram-negative and gram-positive bacteria, as well as in yeasts. Detection of DNA fragmentation was confirmed by fluoroquinolone treatment and by DNA breakage detection-fluorescence in situ hybridization. Proteus mirabilis with spontaneously fragmented DNA during exponential and stationary growth or Escherichia coli with DNA damaged after exposure to hydrogen peroxide or antibiotics, such as ciprofloxacin or ampicillin, was clearly detected. Similarly, fragmented DNA was detected in Saccharomyces cerevisiae after amphotericin B treatment. Our assay may be useful for the simple and rapid evaluation of DNA damage and repair as well as cell death, either spontaneous or induced by exogenous stimuli, including antimicrobial agents or environmental conditions.

Please do not disturb: Destruction of chromatin structure by supravital nucleic acid probes revealed by a novel assay of DNA‐histone interaction

The biomarkers designed to be used supravitally are expected to have minimal effect on structure and function of the cell. Unfortunately nearly all fluorochromes developed to probe live cells interact in undesired way with cellular constituents and affect functional pathways. Herein we comment on potential applications of diverse DNA binding probes in view of the recent article by Wojcik & Dobrucki on DRAQ 5 and SYTO 17. The approach used by these authors to assess DNA-histone interactions using the cells having histones tagged with fluorescent proteins offers a valuable tool to study mechanism of action of antitumor drugs targeting DNA. While the effect of many intercalating drugs may be similar to that of DRAQ5, it may be of particular interest to observe the effects induced by intra-strand and inter-strand DNA crosslinking drugs, alkylating agents, histone deacetylase inhibitors or even anti-metabolites. The cells having histones tagged with fluorescent proteins thus may serve as biomarkers to probe mechanism of action of drugs targeting DNA or affecting chromatin structure. In fact, because such gross chromatin changes as revealed by dissociation and segregation of histones from DNA are most likely incompatible with long-term cell survival, the methodology may be applied for rapid screening of investigational antitumor agents.

Cytological investigation of Haplopappus gracilis (Nutt.) Gray: 5-methylcytosine-rich regions, fluorochrome banding and chromatin sensitivity to DNase I digestion

Haplopappus gracilis (Nutt.) Gray, one of the five known higher plants with a chromosome number of 2n = 4, was studied from a cytological point of view. The chromosome complement of this species was characterized by means of automated karyotype analysis. Moreover, the DNA methylation pattern and fluorochrome banding were determined and compared with cytological data present in the literature. DNA methylation distribution along metaphase chromosomes involved all chromosome territories evidenced by C-banding. Other methylated bands correlated positively with aceto-orcein-positive heterochromatic portions and/or with late replicating bands and/or fluorochrome bands. Some methylated bands showed differences between homologous chromosomes. These bands belonged partly to certain heterochromatic domains and partly to intercalary sites not defined by other standard banding techniques. Differences between the homologues were also indicated by our DNA content data obtained after DNase I digestion.

The Effect of Doxorubicin on MEK-ERK Signaling Predicts Its Efficacy in HCC

Hepatocellular cancer (HCC) is a leading cause of cancer-related death worldwide. Historically, doxorubicin (DOX) has been widely used against unresectable HCC with variable response rates. We hypothesized that DOX combined with mitogen-activated protein kinase kinase-extracellular signal-regulated kinase (MEK-ERK) targeted therapy may provide enhanced anti-cancer effects. Human HCC cell lines (HepG2, Hep3B) were treated with DOX and MEK enzyme inhibitors, U0126 or PD184161, alone or in combination. Growth, apoptosis, and ERK expression/MEK activity were respectively determined by proliferation assay, DNA fragmentation enzyme-linked immunoassay or fluorochrome inhibitor of caspases, and Western blot. DOX (0.01-1 microM) decreased cell proliferation in Hep3B cells (IC(50) approximately 0.12 microM) at 48 to 72 h; DOX was less effective in HepG2 cells (IC(50) approximately 0.25 microM). At early time points (30 min) after DOX treatment of Hep3B cells, MEK activity was unchanged at low doses and decreased at higher doses; after 24 h, phospho-ERK levels increased at higher doses. Contrarily, in HepG2 cells, DOX caused a sustained, dose-dependent increase in phospho-ERK levels at early and late time points. The MEK inhibitor U0126 decreased phospho-ERK in both HCC lines. In contrast to DOX, HepG2 cells were more sensitive than Hep3B cells to U0126. The combination of DOX with U0126 (or PD184161) resulted in greater inhibition of proliferation in HepG2 but not in Hep3B cells. This effect may be mediated in part by enhanced apoptosis. The effect of DOX on early and late induction of MEK activity predicts its chemotherapeutic response in HCC. Furthermore, this effect may also determine the utility of MEK inhibitor combination treatment.