Determination Of DNA Content Research Articles

Cytoplasmic Immunoglobulin Vs. DNA Analysis by Flow Cytometry.

Cytoplasmic immunoglobulin (CIg) vs DNA by flow cytometric (FCM) method allows us to detect DNA content of the neoplastic plasma cells with monotypic cytoplasmic immunoglobulin and also provide us with clear distinction from normal polytypic plasma cells. Abnormalities in cellular DNA content (DNA aneuploidy) and cell cycle determination (proliferative activity) can be measured rapidly by flow cytometry. FCM can measure gross differences in DNA content and distinguish a cell population with normal DNA content, or diploid from a cell population with an abnormal or aneuploid DNA content. With the dual parameter procedure a monoclonal plasma cell population can be identified down to less than 0.1-0.05% of all cells.

Determination of Nuclear DNA Content and Ploidy of Hypericum perforatum L. Accessions Collected From Western Turkey

Abstract Hypericum perforatum L. (St John’s Wort) is a medicinal plant that produces pharmaceutically important compounds with antidepressive, anticancer and antiviral activities. H. perforatum is a facultative apomictic species as it has the ability to reproduce with multiple reproduction mechanisms affecting genetic structure and chemical composition of the plants. The objective of this study was to determine nuclear DNA content and ploidy of H. perforatum L. plants growing naturally in the flora of Turkey. The seeds of 39 Hypericum perforatum L. accessions collected from 23 different locations in Turkey were used in the study. Nuclear DNA content of three different seedlings for each of the 39 H. perforatum accessions was determined using flow cytometry. Based on the results of flow cytometric analysis, nuclear DNA content of the accessions varied between 0.8 - 2.57 pg2C -1 . Nuclear DNA content differences observed among H. perforatum accessions were statistically significant (P< 0.01) and the Duncan test revealed that the accessions formed three clearly distinguishable main groups. Mean nuclear DNA contents of each group were 0.80 pg2C -1 , 1.58 pg2C -1 (1.36-1.73 pg2C -1 ) and 2.38 pg2C -1 (2.15-2.57 pg2C -1 ). Ploidy levels were determined by correlating nuclear DNA content of accessions with the number of their mitotic chromosomes by counting chromosome number of one plant from each group. Findings suggest that H. perforatum accessions used in this study had the following three different ploidy levels: diploid, tetraploid and hexaploid. The percentage of diploid, tetraploid and hexaploid accessions was 2.2, 86.4 and 11.4% respectively.

Determination of DNA content and relative 2C genome sizes of some promising commercial varieties of sugarcane using flow cytometer

&lt;p&gt;In the present study, 2C DNA content and the genome sizes (in picograms-pg and megabase pairs-Mbp respectively) of 19 promising commercial varieties of sugarcane, the derivatives of man-made interspecific hybrids between cultivated and wild species were analyzed using flow cytometry. In this work, 2C nuclear DNA content was determined. Knowing the 2C nuclear DNA content, the unknown chromosome numbers of the varieties could be predicted. Large differences (65 % variation) in DNA content (2C) of 19 varieties were detected, ranging, from 3.80 pg to 10.96 pg, which corresponds to a genome size ranging from 3724.00 Mbp to 10740.80 Mbp due to the variation of ploidy level and are considered the most complex genomes among crop plants. However, the relationship between chromosome number and genome size was highly significant (P &amp;lt; 0.001). In the present study, internode diameter, Sugar juice content and cane yield/ha are also positively correlated with DNA content. The estimated genome sizes would also yield information critical for sugarcane breeding and genome sequencing programs. &lt;/p&gt;&lt;p&gt;&lt;strong&gt;Keywords&lt;/strong&gt;&lt;strong&gt;: &lt;/strong&gt;Genome size, Sugarcane varieties, Flow cytometry, DNA content.&lt;/p&gt;

Comparative analysis of cell cycle parameters in primary and recurrent soft tissue sarcomas.

e22538 Background: The purpose of the study was to determine DNA content and distribution of cells in cell cycle phases by flow cytometry in patients with primary and recurrent soft tissue sarcomas. Methods: 60 patients with soft tissue sarcomas (STS) were recruited: 30 with primary tumors and 30 with recurrent ones. Mean age of patients with primary STS was 56±5.4 years, with recurrent STS – 55±6.7 years. DNA content was determined using the BD Facs Cantoo II flow cytometer with CycleTEST PLUS DNA Reagent Kit (Becton Dickinson). The data were processed using ModFit LT program. Results: Comparative analysis of the cell cycle kinetics showed an increase in the percentage of cells in G2+M phase by 2 times in diploid and by 2.1 times in aneuploid recurrent tumors in comparison with primary ones (1.8±0.5% vs. 0.9±0.1% for diploid tumors; 5.4±2.2% vs. 2.6±0.7% for aneuploid tumors). An increase in the percentage of aneuploid tumors was found in recurrent G2 and G3 tumors (from 50% in primary to 66.7% in recurrent G2 tumors and from 63.25% in primary to 85% in recurrent G3 tumors). Mean content of aneuploid cells in recurrent G2 tumors was 2.2 times higher (p≤0.05), while the differences in primary and recurrent G3 tumors were not significant. The percentage of aneuploid tumors depended on the disease stage and increased in stages IIb and III in recurrent tumors, compared to primary ones (from 37.5% to 71.4% in recurrent st. IIb; and from 65% in primary st. III to 72.7% in recurrences) (p≤0.05). Conclusions: DNA analysis by flow cytometry demonstrated a high biologic potential of both primary and recurrent tumors. Some values in the mitotic cycle in recurrent tumors were probably associated with adjuvant therapy, as well as influenced by the coefficient of two parameters – the percentage of cells in G2+M phase and the cell loss factor determining a high malignant potential of these tumors.

Blue intensity matters for cell cycle profiling in fluorescence DAPI-stained images

In the past decades, there has been an amazing progress in the understanding of the molecular mechanisms of the cell cycle. This has been possible largely due to a better conceptualization of the cycle itself, but also as a consequence of technological advances. Herein, we propose a new fluorescence image-based framework targeted at the identification and segmentation of stained nuclei with the purpose to determine DNA content in distinct cell cycle stages. The method is based on discriminative features, such as total intensity and area, retrieved from in situ stained nuclei by fluorescence microscopy, allowing the determination of the cell cycle phase of both single and sub-population of cells. The analysis framework was built on a modified k-means clustering strategy and refined with a Gaussian mixture model classifier, which enabled the definition of highly accurate classification clusters corresponding to G1, S and G2 phases. Using the information retrieved from area and fluorescence total intensity, the modified k-means (k=3) cluster imaging framework classified 64.7% of the imaged nuclei, as being at G1 phase, 12.0% at G2 phase and 23.2% at S phase. Performance of the imaging framework was ascertained with normal murine mammary gland cells constitutively expressing the Fucci2 technology, exhibiting an overall sensitivity of 94.0%. Further, the results indicate that the imaging framework has a robust capacity to both identify a given DAPI-stained nucleus to its correct cell cycle phase, as well as to determine, with very high probability, true negatives. Importantly, this novel imaging approach is a non-disruptive method that allows an integrative and simultaneous quantitative analysis of molecular and morphological parameters, thus awarding the possibility of cell cycle profiling in cytological and histological samples.

In vitro Antiproliferative and Apoptosis Inducing Effect of Allium atroviolaceum Bulb Extract on Breast, Cervical, and Liver Cancer Cells.

Natural products are considered potent sources for novel drug discovery and development. The multiple therapeutic effects of natural compounds in traditional medicine motivate us to evaluate the cytotoxic activity of bulb of Allium atroviolaceum in MCF7 and MDA-MB-231, HeLa and HepG2 cell lines. The bulb methanol extract of A. atroviolaceum was found to be an active cell proliferation inhibitor at the time and dose dependent manner. Determination of DNA content by flow cytometry demonstrated S and G2/M phase arrest of MCF-7 cell, correlated to Cdk1 downregulation, S phase arrest in MDA-MB-231 which is p53 and Cdk1-dependent, sub-G0 cell cycle arrest in HeLa aligned with Cdk1 downregulation, G0/G1, S, G2/M phase arrest in HepG2 which is p53-dependent. Apoptosis as the mechanism of cell death was confirmed by morphology study, caspases activity assay, as well as apoptosis related gene expression, Bcl-2. Caspase-8, -9, and -3 activity with downregulation of Bcl-2 illustrated occurrence of both intrinsic and extrinsic pathways in MCF7, while caspase-3 and -8 activity revealed extrinsic pathway of apoptosis, although Bcl-2 downregulated. In HeLa cells, the activity of caspase-9 and -3 and downregulation of Bcl-2 shows intrinsic pathway or mitochondrial pathway, whereas HepG2 shows caspase independent apoptosis. Further, the combination of the extract with tamoxifen against MCF7 and MDA-MB-231 and combination with doxorubicin against HeLa and HeG2 demonstrated synergistic effect in most concentrations, suggests that the bulb of A. atroviolaceum may be useful for the treatment of cancer lonely or in combination with other drugs.

Determination of Ploidy Level and Nuclear DNA Content in the Droseraceae by Flow Cytometry

Determination of Ploidy Level and Nuclear DNA Content in the Droseraceae by Flow Cytometry

Chromosome numbers and DNA content in some species of Mecardonia (Gratiolae, Plantaginaceae).

Cytogenetic characterization and determination of DNA content by flow cytometry of five species of Mecardonia Ruiz et Pavon, 1798 (Gratiolae, Plantaginaceae) was performed. This is the first study of nuclear DNA content carried out in the genus. Mitotic analysis revealed a base chromosome number x = 11 for all entities and different ploidy levels, ranging from diploid (2n = 2x = 22) to hexaploid (2n = 6x = 66). The results include the first report of the chromosome numbers for Mecardonia flagellaris (Chamisso & Schlechtendal, 1827) (2n = 22), Mecardonia grandiflora (Bentham) Pennell, 1946 (2n = 22), Mecardonia kamogawae Greppi & Hagiwara, 2011 (2n = 66), and Mecardonia sp. (2n = 44). The three ploidy levels here reported suggest that polyploidy is common in Mecardonia and appear to be an important factor in the evolution of this genus. The 2C- and 1Cx-values were also estimated in all the species. The 2C-values ranged from 1.91 to 5.29 pg. The 1Cx-values ranged from 0.88 to 1.03 pg. The general tendency indicated a decrease in the 1Cx-value with increasing ploidy level. The significance of the results is discussed in relation to taxonomy of the genus.

RDNA mapping, heterochromatin characterization and AT/GC content of Agapanthus africanus (L.) Hoffmanns (Agapanthaceae).

Agapanthus (Agapanthaceae) has 10 species described. However, most taxonomists differ respect to this number because the great phenotypic plasticity of the species. The cytogenetic has been an important tool to aid the plant taxon identification, and to date, all taxa of Agapanthus L'Héritier studied cytologically, presented 2n = 30. Although the species possess large chromosomes, the group is karyologically little explored. This work aimed to increase the cytogenetic knowledge of Agapanthus africanus (L.) Hoffmanns by utilization of chromosome banding techniques with DAPI / CMA3 and Fluorescent in situ Hybridization (FISH). In addition, flow cytometry was used for determination of DNA content and the percentage of AT / GC nitrogenous bases. Plants studied showed 2n = 30 chromosomes, ranging from 4.34 - 8.55 µm, with the karyotype formulae (KF) = 10m + 5sm. Through FISH, one 45S rDNA signal was observed proximally to centromere of the chromosome 7, while for 5S rDNA sites we observed one signal proximally to centromere of chromosome 9. The 2C DNA content estimated for the species was 2C = 24.4 with 59% of AT and 41% of GC. Our data allowed important upgrade for biology and cytotaxonomy of Agapanthus africanus (L.) Hoffmanns.

Phylogenetic and cytogenetic relationships among species of Oxalis section Articulatae (Oxalidaceae)

Oxalis section Articulatae Knuth (Oxalidaceae) comprises a group of five species with a restricted distribution in the southern part of South America. To assess the current circumscription of section Articulatae and the evolutionary mechanisms involved in its diversification, we used three plastid DNA sequences and the nuclear ribosomal transcribed spacer (ITS) to build a molecular phylogeny. A cytogenetic analysis of the group including chromosome number, heterochromatin distribution and determination of nuclear DNA content was also performed. Cytologically, all species presented symmetrical karyotypes except for O. linarantha with a bimodal karyotype although it maintained the same haploid number as the other species (n = 7). Nuclear DNA contents in the Articulatae species are among the lowest for the genus. The O. linarantha 2C value represents the minimum value known for Oxalis, extending the genome size range from 0.58 to 41.10 pg, representing approximately a 70-fold difference, and one of the highest ranges known for an angiosperm genus. Molecular, chromosomal and morphological data suggest that section Articulatae should be redefined to include only those species with rhizomatous stems while the tunicate-bulbous species O. linarantha should be placed elsewhere. Different ploidy levels were found, ranging from 2x to 7x. Diploids and polyploids showed a geographic structure; diploids are widely distributed, while polyploids are found in the uplands of eastern Uruguay and southern Brazil. Haplotype sharing and chromosome data suggest that at least some of section Articulatae polyploids may be the result of interspecific hybridization and appear to have originated very recently.

Flow cytometric analyses of somatic and pollen nuclei in midday flowers (Aizoaceae)

Abstract Endoreduplication (continuous DNA replication without chromosomal or cellular division) is known to be extraordinarily pronounced in a number of succulent plants, including midday flower species. Cytological investigations in different genotypes of the Aizoaceae genera Lampranthus and Delosperma were performed with three objectives: characterization of endoreduplication in several plant organs; determination of absolute DNA contents (pg/2C); and screening for unreduced male gametes. Flow cytometric determinations of absolute DNA contents using internal standards were carried out on nuclei extracted from pollen tubes. Further analyses of pollen nuclei were intended to verify the occurrence of unreduced gametes in the two genera. In contrast to the majority of angiosperms, in which endoreduplication is often restricted to certain tissues or cell types, the investigated succulents primarily consisted of cells with elevated DNA contents. Absolute DNA contents ranged from 1.6 to 2.36 pg/2C in Lamprant...

Influence of saHnity and food deprivation on growth and RNA-DNA ratio in red drum Sciaenops ocellatus (Pisces: Sciaenidae)

Juvenile Red drum Sciaenops ocellatus held in 30 ppl natural seawater or fresh water were starved or fed ad libitum for su weeks, and sampled each week for growth and for determination of RNA, DNA, and protein contents of white and red muscles and liver. Specific growth Tate and condition factor were used lo compare changes in nucleic acid and protein concentrations in fish tissues. Salinity had liule effect on growth, while starvalIon caused a significant reducuon in all growth parameters and in the RNA-DNA ratio after three weeks. The RNA-DNA ralIos of red and white muscles of the fed fish were greater than those of the starved fish and also were related to specific growth Tate and fish condition factor. Nucleic acid concentralIons in liver were highly variable. Since wrute muscle predorninates and is more easily sampled in red drum than red muscle, and because it has less variable RNA-DNA ratios than liver, it is the tissue recornmended for nucleic acid analysis of physiological condition and growth.

Near-infrared Spectroscopy Applications in the Quantitative Determination of Bovine Genomic DNA Content from Milk

This study was to provide a convenient method for the determination of DNA concentration from milk. Near infrared (NIR) spectroscopy technology with orthogonal experiment method was conducted to determine bovine genomic DNA content in milk for the first time. DNA was extracted from milk samples with a novel method that was constructed based on our pre-experiments results. All samples were scanned (570-1850 nm), and 74 samples were used to establish the model of DNA content in NIR analysis. The results showed that the optimal model's performance for DNA content was excellent, with SEC = 0.26%, SECV = 0. 69%, SEP = 0. 28%, RSQ = 0.913 and R 2 = 0.9846, RPD = 3.57 respectively. Thus NIR was feasible to nondestructively determine DNA content in milk. Therefore, NIR technology would be valuable in meeting with the increasing demand for monitoring the dairy food chain and ensuring labeling authenticity to the consumers.

Telomere length and transrenal DNA isolated from transplanted kidney recipients' urine

Transplantation is the preferred method of end stage renal insufficiency treatment due to better quality of life and extended life of transplanted patients. Currently a non-invasive test, which evaluates the risk of acute or chronic rejection or deterioration of the transplanted organ's function, is being sought. An increase of the transrenal DNA concentration in the urine of urinary tract infection patients and in renal graft recipients during an episode of acute rejection was observed. There were also reports on shortening of telomeres in transplanted organ chromosomes, as the result of accelerated aging of cells, and its connection with the onset of chronic allograft nephropathy and the degree of its completion, and thus the deterioration of kidney function. The aim of this paper is to describe the urine genetic analysis through determining the length of the telomeres and the content of transrenal DNA to monitor kidney function and to evaluate the prevalence of acute and chronic rejection in patients after kidney transplantation. The genetic analysis of the biological material collected from patients relies on the determination of transrenal DNA content and length of DNA telomeres isolated from the urine of kidney recipients. The presented methods assume that the genetic profile of the transplanted organ recipient as well as kidney donor can be determined, so the source of the genetic material in the urine of the patient can be identified. A measurable effect of these methods' use would be to complement the evaluation of the prevalence of acute and chronic rejection and transplanted kidney function with a modern, non-invasive method, which is the analysis of telomere length from sediment of urine and the content of transrenal DNA in the urine.

Determination of the ruminant origin of bone particles using fluorescence in situ hybridization (FISH).

Molecular biology techniques such as PCR constitute powerful tools for the determination of the taxonomic origin of bones. DNA degradation and contamination by exogenous DNA, however, jeopardise bone identification. Despite the vast array of techniques used to decontaminate bone fragments, the isolation and determination of bone DNA content are still problematic. Within the framework of the eradication of transmissible spongiform encephalopathies (including BSE, commonly known as “mad cow disease”), a fluorescence in situ hybridization (FISH) protocol was developed. Results from the described study showed that this method can be applied directly to bones without a demineralisation step and that it allows the identification of bovine and ruminant bones even after severe processing. The results also showed that the method is independent of exogenous contamination and that it is therefore entirely appropriate for this application.

Cultured Hepatocytes Adopt Progenitor Characteristics and Display Bipotent Capacity to Repopulate the Liver

Clinical studies have proved the therapeutic potential of hepatocyte transplantation as a promising alternative to whole organ liver transplantation in the treatment of hereditary or end-stage liver disease. However, donor shortage seriously restricts cell availability, and the lack of appropriate cell culture protocols for the storage and maintenance of donor cells constitutes a significant obstacle. The aim of this study was to stimulate mature hepatocytes in culture to multiply in vitro and track their fate on transplantation. Rat hepatocytes isolated nonenzymatically were cultured serum free for up to 10 days. They were stimulated into proliferation in the presence of growth factors and conditioned media from nonparenchymal and hepatocyte culture supernatants, as well as 10 mM lithium chloride (LiCl). Cell proliferation was assessed by determining DNA content. Additionally, the extent of cell differentiation was estimated using immunofluorescence staining of hepatic, biliary, progenitor, and mesenchymal markers and gene expression analyses. Transplantation studies were performed on the Fischer CD26-mutant rat following pretreatment with retrorsine and partial hepatectomy. Proliferating hepatocytes increasingly adopted precursor characteristics, expressing progenitor (OV6, CD133), hepatic lineage (CK18), biliary (CD49f, CK7, CK19), and mesenchymal (vimentin) markers. The supplement of LiCl further enhanced the proliferative capacity by 30%. Transplantation studies revealed extensive repopulation by large donor hepatocyte clusters. Furthermore, bile duct-like structures deriving from donor cells proved to be immunoreactive to ductular markers and formed in close proximity to endogenous bile ducts. Mature hepatocytes reveal their potential to "switch" between phenotypes, adopting progenitor characteristics during proliferation in vitro. Following transplantation, these "retrodifferentiated" cells further expanded in vivo, thereby generating bipotentially differentiated progenies (hepatocytes and bile duct-like structures). This apparent plasticity of mature hepatocytes may open new approaches for cell-based strategies to treat liver disease.

Irises: A practical tool for image-based analysis of cellular DNA content.

The DNA content of nuclei is a valuable measure of cell cycle status. Irises is a software tool to facilitate systematic in situ determination of DNA content for cell cycle analysis at single-nucleus resolution within complex tissues. We demonstrate the utility of the tool with analysis of DNA content in germline nuclei of C. elegans. Compared with results obtained by manual analysis, we find the tool greatly facilitates analysis by improving speed at least 5-fold while maintaining accuracy. The source code and instruction manual (including installation for both Mac and PC) are provided.

Noninvasive Quantification of In Vitro Osteoblastic Differentiation in 3D Engineered Tissue Constructs Using Spectral Ultrasound Imaging

Non-destructive monitoring of engineered tissues is needed for translation of these products from the lab to the clinic. In this study, non-invasive, high resolution spectral ultrasound imaging (SUSI) was used to monitor the differentiation of MC3T3 pre-osteoblasts seeded within collagen hydrogels. SUSI was used to measure the diameter, concentration and acoustic attenuation of scatterers within such constructs cultured in either control or osteogenic medium over 21 days. Conventional biochemical assays were used on parallel samples to determine DNA content and calcium deposition. Construct volume and morphology were accurately imaged using ultrasound. Cell diameter was estimated to be approximately 12.5–15.5 µm using SUSI, which corresponded well to measurements of fluorescently stained cells. The total number of cells per construct assessed by quantitation of DNA content decreased from 5.6±2.4×104 at day 1 to 0.9±0.2×104 at day 21. SUSI estimation of the equivalent number of acoustic scatters showed a similar decreasing trend, except at day 21 in the osteogenic samples, which showed a marked increase in both scatterer number and acoustic impedance, suggestive of mineral deposition by the differentiating MC3T3 cells. Estimation of calcium content by SUSI was 41.7±11.4 µg/ml, which agreed well with the biochemical measurement of 38.7±16.7 µg/ml. Color coded maps of parameter values were overlaid on B-mode images to show spatiotemporal changes in cell diameter and calcium deposition. This study demonstrates the use of non-destructive ultrasound imaging to provide quantitative information on the number and differentiated state of cells embedded within 3D engineered constructs, and therefore presents a valuable tool for longitudinal monitoring of engineered tissue development.

Caracterização anatômica e citométrica em biribazeiro (Rollinia mucosa [Jacq.

O biribazeiro (Rollinia mucosa [Jacq.]) é uma frutífera nativa da América Central e América do Sul que se destaca das demais espécies do gênero por apresentar frutos grandes e comestíveis. Tendo em vista que pouco se conhece a respeito da anatomia da planta, tipo de reserva da semente, como também sobre o conteúdo de DNA de espécies da família anonáceas, este trabalho teve por objetivos caracterizar histoquimicamente as sementes e anatomicamente as plântulas, e determinar o conteúdo de DNA de biribazeiro. As plântulas foram obtidas de sementes coletadas na região Amazônica pela Embrapa Roraima e enviadas à Universidade Federal de Lavras. Os frutos foram despolpados, e suas sementes foram previamente lavadas e semeadas em bandejas de 48 células, contendo como substrato pó de serragem, permanecendo em câmara de germinação a 30ºC por 90 dias. Nas plantas obtidas, procedeu-se à análise histoquímica, à caracterização anatômica e à determinação do conteúdo de DNA. As principais conclusões deste trabalho foram que: (1) as sementes da Rollinia mucosa apresentam reserva principal lipídica; (2) as secções transversais da lâmina foliar indicam organização dorsiventral, do tipo hipoestomática com estômatos paracíticos e tricomas em ambas as faces da folha; (3) as folhas de biribazeiro apresentam, em média, 4,77 pg de DNA.

Efficient analysis of ploidy levels in plant evolutionary ecology

Flow cytometry (FCM) has provided an efficient method to determine DNA content in cells, thus providing a feasible alternative to microscopy for the estimation of ploidy for population level studie...