DNA content and cytogenetic characteristics of Gymnocalycium quehlianum (Cactaceae) along an altitudinal gradient / Contenido de ADN y características citogenéticas de Gymnocalycium quehlianum (Cactaceae) a lo largo de un gradiente altitudinal

ABSTRACT Important changes in vegetation types occur along elevational gradients. The genus Gymnocalycium is endemic to southern South America, and its species are distributed along elevational gradients. In particular, Gymnocalycium quehlianum is a globular cactus endemic to the Sierras de Córdoba. Studying cytogenetic aspects and DNA content in populations throughout their distribution is key to understanding the species. DNA content and cytogenetic characteristics were analyzed in four populations of G. quehlianum (615, 744, 948 and 1257 masl). The genome size in the four populations varied between 3.55 and 4.30 pg. The populations were diploid (2n = 22). All populations showed the karyotype formula of 10 metacentrics (m) + 1 submetacentric (sm). The species presented symmetrical karyotypes and constitutive heterochromatin CMA+/DAPI- associated with nucleolar organizing regions, always found in the first pair of m chromosomes. The 18-5.8-26S rDNA locus is found in the terminal regions of the first pair of chromosomes m, and the 5S locus is adjacent to the 18-5.8-26S locus. A tendency for DNA content to decrease with increasing altitude was observed.

RESUMEN A lo largo de los gradientes altitudinales se producen cambios importantes en los tipos de vegetación. El género Gymnocalycium es endémico del sur de América del Sur y sus especies se distribuyen en gradientes altitudinales. En particular, Gymnocalycium quehlianum es un cactus globular endémico de las Sierras de Córdoba. Estudiar aspectos citogenéticos y de contenido de ADN en las poblaciones a lo largo de su distribución es clave para comprender a la especie. En cuatro poblaciones de G. quehlianum (615, 744, 948 y 1257 msnm) se analizaron el contenido de ADN y las características citogenéticas. El tamaño del genoma en las cuatro poblaciones varió entre 3,55 y 4,30 pg. Las poblaciones resultaron diploides (2n=22). Todas las poblaciones presentaron la fórmula del cariotipo de 10 metacéntricos (m) + 1 submetacéntrico (sm). La especie presentó cariotipos simétricos y heterocromatina constitutiva CMA+/DAPI- asociados con regiones organizadoras nucleolares, que siempre se encontraban en el primer par de cromosomas m. El locus de ADNr 18-5.8-26S se encuentra en las regiones terminales del primer par de cromosomas m y el locus de 5S está adyacente al locus 18-5.8-26S. Se observó una tendencia del contenido de ADN a disminuir con el aumento de la altitud.

First report on DNA content of three species of Amorphophallus

The Amorphophallus genus is a perennial herb which belongs to the family Araceae. There are more than 170 species in this genus, which is widely distributed in tropical and subtropical areas. As a kind of food and medicine Amorphophallus has been used for more than 2000 years in China. Because of the high content of konjac glucomannan (KGM) and dietary fiber, it has attracted more attention worldwide. In this article, the DNA contents of A. konjac, A. albus and A. bulbifer in China, A. albus, A. paeoniifolius and A. muelleri in Indonesia were estimated by using flow cytometry. In the samples of China, the DNA contents were 12.95 ± 0.73 pg/2C in A. konjac, 10.51 ± 0.05 pg/2C in A. albus and 17.61 pg/2C in A. bulbifer, and for Indonesia, 14.16 ± 0.48 pg/2C in A. albus (flowering), 8.49 ± 0.2 pg/2C in A. paeoniifolius and 17.84 ± 1.46 pg/2C in A. muelleri were used. Interspecific variation was found significantly (P \0.01), suggesting that DNA content might be a parameter that can be used to differentiate the species. Intraspecific variation has also been found significantly (P \ 0.01), whether in the same region or between two regions. As far as we know, this is the first report on genome size estimation of the A. konjac, A. albus and A. muelleri using flow cytometry. Understanding the genome size of Amorphophallus species will help to sequence the genome and analyse the genetic diversity, evolutionary relationship and geographical variation pattern of Amorphophallus species.

Genome size in three species of Glandularia and their hybrids / Tamaño del genoma en tres especies de Glandularia y sus híbridos

In this work the relationship between genome size of Glandularia species and the meiotic configurations found in their hybrids are discussed. Glandularia incisa (Hook.) Tronc., growing in two localities of Corrientes and Córdoba provinces, Argentina, with different ecological conditions, showed inter-population variability of the 2C-value. The DNA content found in the Corrientes locality (2.41 pg) was higher than that obtained in the Córdoba locality (2.09 pg) which has more stressful environmental conditions than the former. These values are statistically different from those that were found in Glandularia pulchella (Sweet) Tronc. from Corrientes (1.43 pg) and in Glandularia perakii Cov. et Schn from Córdoba (1.47 pg). The DNA content of the diploid F1 hybrids, G. pulchella × G. incisa and G. perakii × G. incisa, differed statistically from the DNA content of the parental species, being intermediate between them. Differences in the frequency of pairing of homoeologous chromosomes were observed in the hybrids; these differences cannot be explained by differences in genome size since hybrids with similar DNA content differ significantly in their meiotic behavior. On the other hand, the differences in the DNA content between the parental species justify the presence of a high frequency of heteromorphic open and closed bivalents and univalents with different size in the hybrids.

En el presente trabajo se discute la relación entre el tamaño del genoma en especies de Glandularia y las configuraciones meióticas encontradas en sus híbridos. El valor 2C mostró variabilidad interpoblacional en muestras de Glandularia incisa (Hook.) Tronc. coleccionadas en dos localidades con diferentes condiciones ecológicas (provincias de Corrientes y Córdoba, Argentina). El contenido de ADN encontrado en Corrientes (2,41 pg) fue mayor que el obtenido en Córdoba (2,09 pg) donde se registran condiciones ambientales más estresantes. Estos valores son estadísticamente diferentes de los determinados en Glandularia pulchella (Sweet) Tronc. de Corrientes (1.43 pg) y en Glandularia perakii Cov. et Schn de Córdoba (1.47 pg). El contenido de ADN de los híbridos diploides F1, G. pulchella × G. incisa y G. perakii × G. incisa, difirió estadísticamente del contenido de ADN registrado en las especies parentales siendo intermedio entre ellas. Las diferencias observadas en la frecuencia de apareamiento de cromosomas homeólogos no pueden explicarse por diferencias en el tamaño del genoma, ya que híbridos con un contenido de ADN similar difieren significativamente en su comportamiento meiótico. Sin embargo, la diferencia en el contenido de ADN entre las especies parentales explica la presencia de una alta frecuencia de bivalentes heteromórficos tanto abiertos como cerrados y univalentes con diferentes tamaños.

Cytological and genome size data analyzed in a phylogenetic frame: Evolutionary implications concerning Sisyrinchium taxa (Iridaceae: Iridoideae)

Abstract Sisyrinchium is the largest genus of Iridaceae in the Americas and has the greatest amount of cytological data available. This study aimed at investigating how genomes evolved in this genus. Chromosome number, genome size and altitude from species of sect. Viperella were analyzed in a phylogenetic context. Meiotic and pollen analyses were performed to assess reproductive success of natural populations, especially from those polyploid taxa. Character optimizations revealed that the common ancestor of sect. Viperella was probably diploid (2n = 2x =18) with two subsequent polyplodization events. Total DNA content (2C) varied considerably across the phylogeny with larger genomes detected mainly in polyploid species. Altitude also varied across the phylogeny, however no significant relationship was found between DNA content changes and altitude in our data set. All taxa presented regular meiosis and pollen viability (> 87%), except for S. sp. nov. aff. alatum (22.70%), suggesting a recent hybrid origin. Chromosome number is mostly constant within this section and polyploidy is the only source of modification. Although 2C varied considerably among the 20 taxa investigated, the diversity observed cannot be attributed only to polyploidy events because large variations of DNA content were also observed among diploids.

Relationship of EpCAM expression and DNA copy number alteration in colorectal carcinoma cells / 临床与实验病理学杂志

Purpose To investigate the clinical pathology significance of epithelial cellular adhesion molecule (EpCAM, CD326) expression in colorectal carcinoma cells. Methods Flow cytometry and immunofluorescence assay were used to detect EpCAM expression in 55 cases of fresh colorectal cancer tissues and adjacent normal mucosa. The percentage of positive cells (PPC) and mean fluorescence index (MFI) were calculated. Correlation of EpCAM expression with DNA ploidy change and its value were investigated in the early diagnosis of colorectal cancer. Results The values of PPC and MFI were significantly higher in colorectal carcinoma tissue than that in the normal colorectal tissue [(PPC: (83.48 ± 7.07)％ vs (43.56±5.29)％, t =39.22, P＜0.001. MFI: 28.90(19.60-45.89) vs4.89(3.79-6.28), Z=-6.45, P＜0.001) ]. There were also significant differences (P＜0.01) in the values of PPC and MFI between invasive type and ulcer types, between well-and moderately-differentiated and poorly-differentiated cancers, between Dukes stages A + B and C + D, (between pTNM stages I+ II and III + IV, between pTl + T2 and pT3 + T4, and between pNO and pNl. DNA content analysis showed that DNA polyploid was detected in 39 of 55 colorectal carcinoma (70.90％ ). The DNA index (DI) and ploid were correlated with differentiation degree and Dukes stages, but uncorrelated with lymph node metastasis. At the same time, in the EpCAM positive cases, DI was increased with increased expression of EpCAM (r = 0.668, P =0.000) and the proliferation index of cells in S phase ratio(Sphase fraction, SPF) (r1 =0.664, P1 =0.000, r2 =0.651, P2 = 0.000 ). Conclusion EpCAM expression is obviously upregulated in colorectal carcinoma, and it is closely correlated with the invasion, metastasis and proliferation of tumor cells. The combination of EpCAM expression and DNA content analysis provides references to the early diagnosis and prognosis evaluation in colorectal carcinoma.

Endoreduplication in floral structure, vegetative and fruits of red pitaya with white pulp / Endorreduplicação em estrutura floral, vegetativa e frutos de pitaya vermelha de polpa branca

Endoreduplication is the change of cellular cycle that result DNA duplication without cell division and could result endopolyploid cells. This phenomenon is common in plants and animals and considered as evaluative strategy. Although endoreduplication reported in various plant species, the information about these phenomena in red pitaya is rare. Therefore, this work was done with the objective of studying the endoreduplication in Hylocereus undatus Haw. using flow cytometry analysis. In this study were used the tissue from the flower structure, fruits, roots, cladode, and thorns of the pitaya plant.To determine the DNA content approximately 50 mg the sample of each treatment with Pisum sativum (the internal standard reference) were grind in plate of petri dishes contained 1 mL of cold Marie buffer to release the nucleus.The nuclear suspension was filtered through 50 µm mesh. The nucleuses were colored with 25 µL of 1 mg/L mL of propidium iodide. For each treatment, three samples of 10 thousand nucleuses were analyzed in cytometry Facscalibur (Becton Dickinson). The content of nuclear DNA (pg) was estimated as the ratio between fluorescence intensity of the G1 nucleus of the standard and the G1nucleus of the sample multiplied by the quantity of DNA of the internal reference. In conclusion, the endoreduplication occurred in all part of the plants analyzed except in the aculeus and the roots. The analysis evidenced different index of DNA content in the tissues analyzed being observed up to four different ploidy levels. The phenomena of endoreduplication occurs in all parts of the plant analyzed except in aculeus and roots.

A endorreduplicação é uma alteração do ciclo celular, ocorrendo a replicação do DNA sem a divisão celular, podendo resultar em células endopoliploides. É um fenômeno comum em plantas e animais, sendo considerada uma estratégia evolutiva. Embora a endorreduplicação seja relatada em diversas espécies de plantas, as informações sobre este fenomeno em pitaia vermelha são escassas. Assim, objetivou-se com este trabalho estudar a endorreduplicação em Hylocereus undatus Haw. pela técnica de citometria de fluxo. Foram analisados tecidos de estruturas florais, de frutos, raiz, cladódio e acúleos de plantas de pitaia. Para a determinação do conteúdo de DNA, aproximadamente 50 mg de amostra de cada tratamento, juntamente com Pisum sativum (padrão de referência interno) foram triturados em placa de Petri contendo 1 mL de tampão Marie gelado para a liberação dos núcleos. A suspensão de núcleos foi filtrada através de malha de 50 µm. Os núcleos foram corados com 25 µL de solução de 1 mg/1 mL de iodeto de propídeo para cada amostra. Para cada tratamento, três amostras de 10 mil núcleos foram analisadas em citômetro Facscalibur (Becton Dickinson). O conteúdo de DNA nuclear (pg) foi estimado utilizando-se a razão entre as intensidades de fluorescência dos núcleos G1 do padrão de referência e dos núcleos G1 da amostra, multiplicando-se esta razão pela quantidade de DNA do padrão de referência. Conclui-se que a endorreduplicação ocorre em todas as partes da planta analisada, exceto no acúleo e na raiz. As análises evidenciam diferentes índices de conteúdo de DNA nos tecidos, sendo observados até quatro diferentes níveis de ploidia. O fenômeno da endorreduplicação ocorreu em todas as partes da planta analisadas, exceto nos acúleos e raízes.

Estimation of Nuclear DNA Content in Tannin-rich Medicinal Plant Cornus officinalis by Flow Cytometry / 中草药·英文版

Objective: The amount of nuclear DNA (C-value) is a key biodiversity character that provides strong unifying elements in revealing the phylogenetic regularity and relationship between genome size and functional traits for plant resource. The estimation of C-values could primarily extend our knowledge on the genetic background and genome diversity for medicinal plants, and thereby the variation of pharmacological constituents and phylogenetic mechanism of medicinal plant taxa will be revealed. However, a large number of medicinal plants (e.g. Cornus officinalis) typically contain a series of secondary metabolites, especially tannic acid, which would significantly affect the estimation of DNA content by flow cytometry (FCM). Methodological discussions and improvement need to be made to solve this problem. Methods: Two isolation buffers LB01 and Otto 1 were selected to prepare nuclear suspension with additional treatments of pre-soaking and centrifugation combination of gradient centrifugal force and duration. The best isolation and estimation methods were determined by FCM measurement in C. officinalis. Results: The dry leaves were pre-soaked in Otto I buffer for 15 min and the Otto I nuclear suspension was centrifugated at 1.0103 g for 2 min. The results showed that debris and nuclei were better separated and the scatterplots of good quality were obtained with low coefficient of variation (CV). Contrarily, the nuclear DNA content of C. officinalis could not be accurately estimated for nuclei extracted by LB01 buffer. Finally, 2C-value and genome size of C. officinalis were first estimated as 5.92 pg and 2893 Mbp, respectively. Conclusion: The new methods proposed here are able to accurately estimate DNA content of C. officinalis, which provides valuable references for the estimation of genome size in other tannin-rich medicinal plants. © 2014 Tianjin Press of Chinese Herbal Medicines.

Establishment of an in vitro plantlet regeneration protocol for unique varieties of brinjal (Solanum melongena L.) var. Mattu Gulla and Perampalli Gulla.

Brinjal (Solanum melongena L.) var. Mattu Gulla (MG) and var. Perampalli Gulla (PG) are unique varieties with distinct flavour cultivated in Udupi, Karnataka State, and are exposed to several biotic and abiotic stresses. An efficient and reproducible in vitro regeneration method is required to expedite the manipulation of these brinjal varieties to cope up with stress by tissue culture and gene transfer methods. The present study, reports a rapid and efficient in vitro regeneration protocol for these two varieties. The in vitro growth response was studied on Murashige and Skoog (MS) medium supplemented with 2, 4-D, BAP and IAA, and the plantlets were regenerated efficiently from callus cultures of leaf, cotyledon and hypocotyl explants. Among the three explants, the hypocotyl explants were found to have better callus induction and multiple shoot regeneration. High frequency of shoot initiation was achieved from hypocotyl derived calluses in MS media with 2.0 mg/L BAP and 0.5 mg/L IAA in MG and PG. Efficient and rapid shoot proliferation, and elongation were noted in MS medium with 1.0 mg/L BAP and 0.3 mg/L GA3. The in vitro regenerated shoots produced healthy roots when they were cultured on MS medium supplemented with 0.5 mg/L IBA. A significant difference was observed in percentage of callus induction, number of shoots per callus, shoot elongation and number of hardened plantlets of MG and PG. MG showed maximum response in all stages of culture than PG. Hardening of plantlets in tissue culture was achieved in three weeks. The hardened plantlets were grown in pots for further acclimatization in green house and finally transplanted to experimental garden where they developed into flowering plants and produced mature fruits with viable seeds.

Survivin overexpression promotes proliferation of laryngeal carcinoma / 实用肿瘤学杂志

Objective The aim of this study is to investigate the expression of Survivin in laryngeal car-cinoma and to elucidate the relationship between cell proliferation and the expression of Survivin .Methods The expression and DNA quantification of Survivin in 63 cases of laryngeal carcinoma and 15 cases of normal laryngeal tissues were detected by immunohistochemical staining ( SP) and flow cytometry .Results Forty-five sections of laryngeal carcinoma were positive for survivin as determined by immunohistochemical staining ,but Survivin was undetected in normal laryngeal tissues .The intensity of Survivin expression was significantly increased with tumor differentiation and lymph node metastasis (P0.05).High DNA index(DI)and proliferation index(PI)were detected in laryngeal carcino-ma(P<0.05),and PI was positively correlated with expression of Survivin (P<0.05).Conclusion Survivin overexpression triggers laryngeal carcinoma cell hyperproliferation ,especially in development of laryngeal carcino-ma.These data identify Survivin as an important target in laryngeal carcinoma and provide a translational pathway for developing new therapies against this target .

Karyotype analysis, DNA content and molecular screening in Lippia alba (Verbenaceae)

Cytogenetic analyses, of pollen viability, nuclear DNA content and RAPD markers were employed to study three chemotypes of Lippia alba (Mill.) (Verbenaceae) in order to understand the genetic variation among them. Different ploidy levels and mixoploid individuals were observed. This work comprises the first report of different chromosome numbers (cytotypes) in L. alba. The chromosome numbers of La2-carvone and La3-linalool chemotypes suggested that they are polyploids. Flow cytometric analysis showed an increase of nuclear DNA content that was not directly proportional to ploidy level variation. A cluster analysis based on RAPD markers revealed that La3-linalool shares genetic markers with La1-citral and La2-carvone. The analysis showed that the majority of genetic variation of La3-linalool could be a consequence of ixoploidy. ur data indicates that sexual reproduction aong those three chemotypes is unlikely and suggests the beginning of reproductive isolation. The results demonstrated that chromosome analysis, nuclear DNA content estimation and RAPD markers constitute excellent tools for detecting genetic variation among L. alba chemotypes.

Análises citogenéticas, de viabilidade do pólen, do conteúdo de DNA nuclear e marcadores RAPD foram empregadas no estudo de três quimiotipos de Lippia alba (Mill.) (Verbenaceae) visando contribuir para o entendimento da variação genética entre os mesmos. Diferentes níveis de ploidia e indivíduos mixoploides foram observados. Este trabalho compreende o primeiro relato de diferentes números cromossômicos (citótipos) em L. alba. Os números cromossômicos dos quimiotipos La2-carvona e La3-linalol sugere que eles seja poliploides. A análise da citometria de fluxo mostrou um aumento do conteúdo de DNA nuclear que não foi diretamente proporcional à variação no nível de ploidia. A análise de agrupamento baseada nos marcadores RAPD demonstrou que La3-linalol compartilha marcadores genéticos com La1-citral e La2-carvona. A análise mostrou que a maior parte da variação genética de La3-linalol pode ser consequência da mixoploidia. Nossos dados indicam que a reprodução sexual entre os três quimiotipos parece improvável, sugerindo o início de isolamento reprodutivo. Os resultados demonstraram que a análise cromossômica, a quantificação do DNA nuclear estimado e os marcadores RAPD constituem excelentes ferramentas para detecção de variação genética entre quimiotipos de L. alba.

Development and application of high-throughput amplified fragment length polymorphism technique in Calluna vulgaris (Ericaceae)

Calluna vulgaris is an important ornamental crop of the horticultural industry in Europe. In order to improve breeding of this species, especially of the most important trait of ‘bud-flowering', the implementation of molecular techniques that allow rapid, reproducible and efficient screening of whole segregating populations e.g. for molecular marker and mapping approaches is a requirement. We therefore aimed to introduce the powerful tool of amplified fragment length polymorphisms (AFLP®), a widely and successfully applied method, into our methodological assortment. As an essential prerequisite, the isolated DNA should be of adequate quality which is a common obstacle when dealing with woody species and their interfering secondary components/metabolites. The results of screening different and modified DNA isolation protocols are described. As the outcome of our evaluations of reaction conditions during the AFLP® procedure, we circumstantiate a functional protocol ranging from DNA extraction to visualization of AFLP® banding patterns for the woody crop C. vulgaris. This method is suitable for high throughput genetic applications and may even be transferable to other species. In addition, costs are reduced by reasonable reagents and multiplexing assays.

A postmortem semi-quantitative study on time-dependent changes of DNA content in chondrocytes in rat's costal cartilage / 中国法医学杂志

Objective To study the relationship between the DNA content of chondrocytes in the costal cartilage and postmortem interval in putrefactive rat cadavers.Methods Nuclear DNA wag visualized by modified Feulgen's staining method.DNA content of ehondrocytes in the costal cartilage was semi-quantita tively determined by a computerized image analysis system in rats within 35d postmortem.Results Staining intensity of the nuclei was gradually reduced within from 1d to 28d postmortem.The nuclej could not be detected at 35d.The DNA content of chondrocytes decreased time-dependently within 28 days after death as determined semi-quantitatively,which revealed a linear relationship between DNA content and postmortem interval.Conclusion DNA content of chondrocytes in the costal cartilage reduces time-dependently with the extension of postmortem interval.

Cytogenetic studies in some species of Passiflora L. (Passifloraceae): a review emphasizing Brazilian species

The Passifloraceae is represented by species of tropical and subtropical origin. The Passiflora is the richest genus with approximately 450 species, 200 of them being native to Brazil. Recent karyological studies have reported the basic chromosome number for the Passiflora genus as x = 6, whereas x = 9, x = 10 and x = 12 were established as secondary basic numbers. High rates of fertility occur in most Passiflora species, since both meiotic index and pollen viability are above 90 percent. Unusual meiotic behavior has been described in some taxa. Unviable pollen were observed in some diploids species. The genome size varies from 1.83 to 5.36 pg, and significant interspecific variance has been observed. Studies using the FISH methodology have shown that there are two to three rDNA 45S sites and one 5S site in the species analyzed. In this review, information about the above-mentioned studies is presented and discussed in detail.

A família Passifloraceae é representada por espécies de origem tropical e subtropical. Passiflora é o gênero mais rico, com aproximadamente 450 espécies, cerca de 200 delas nativas do Brasil. Recentes estudos cariológicos têm relatado o número básico de cromossomos para o gênero Passiflora como sendo x = 6, enquanto x = 9, x = 10 e x = 12 foram considerados números básicos secundários. Altas taxas de fertilidade são observadas na maioria das espécies de Passiflora, uma vez que o índice meiótico e a viabilidade polínica apresentam-se acima de 90 por cento.Comportamento meiótico irregular tem sido descrito para alguns taxas. Grãos de pólen inviáveis foram observados em espécies diplóides. O tamanho do genoma varia de 1,83 a 5,36 pg, e variação interespecífica significativa tem sido observada. Estudos usando a metodologia de hibridização in situ (FISH) tem demonstrado haver de dois a três sites de DNAr 45S e um site de DNAr 5S nas espécies analisadas. Nesta revisão, informações sobre os estudos acima mencionados são apresentados e discutidos em detalhes.

Expression of PTEN and DNA content and invasion and metastasis of lung cancer / 中国基层医药

Objective This study was designed to determine the expression ofgeneproducts of phosphatase and tensin homoligy deleted onehromoseten(PTEN)and DNA content in non small cell lung cancer(NSCLC)tissue and investigate their association with the invasion and metastasis of NSCLC.Methods The expression of PTEN were detected in 78 cases of lung cancer tissues by immunohistochemistry SP methods,DNA content in 30 cases of lung cancer tissues was examined by flow cytometry.Results The rate of total expression depletion of PTEN was 42.3%.The rates of PTEN expression depletion in lymph node metastasis group and no lymph node metastasis group were 52.1%and 26.7%respectively,with significantant difference(P＜0.05).The patients who had high expression of PTEN had a longer survival time.The range of the DNA index(DI)distribution ranged from 1.04 to 1.93,the DNA index and aneuploid tumor had a positive correlation with the lymph node metastasis,with the increased of TNM stage,the DI and the rate of DNA aneuploid increased(P＜0.05).Conclusions Expression of PTEN is associated strongly with lymph node metastasis of lung cancer,correlations exist between DNA content and TNM stage,lymph node metastasis.They are indications of metastasis potential and prognosis of lung cancer.

Molecular phylogenetic relationships among four species of the mangrove tree genus Bruguiera (Rhizophoraceae), as revealed by chromosome and RAPD markers

Analysis of karyotype, nuclear DNA content and RAPD markers were performed in four species of Bruguiera (Rhizophoraceae) of Bhitarkanika mangrove forests, Orissa, India. Detailed karyotype analysis revealing 2n=34 in B. cylindrica and 2n=36 in B. gymnorrhiza was reported for the first time and 2n=34 in B. parviflora and B. sexangula was confirmed. On the basis of the common types of chromosomes present among Bruguiera, two distinct groups were found; one consists of B. cylindrica and B. parviflora and the other of B. gymnorrhiza and B. sexangula. The symmetrical karyotype with same chromosome types grouped B. cylindrica and B. parviflora together and presence of Type E chromosomes placed B. gymnorrhiza and B. sexangula in a separate group, suggesting their closer affinity in their respective group. Analysis of chromosome length, volume, INV and 4C DNA content confirmed this division. Nuclear DNA content was two-fold higher (~17.0 pg) in the second group than in the first (~8.0 pg). The amplification products generated through RAPD revealed 1-9 amplicons with size variations from 600 bp to 2 500 bp with 49.31% genetic similarity between B. gymnorrhiza and B. sexangula and 47.10% in between B. cylindrica and B. parviflora. The high copy number marker band (~ 1 100 bp) yielded in OPN-15 primer in B. parviflora the characteristic DNA marker, which was cloned and used as probes for assessment of genetic diversity, and demonstrated its close genetic affinity to B. cylindrica. B. gymnorrhiza and B. sexangula also produced similar marker bands of ~600 bp and ~2 200 bp in the same primer. All of the cytological, 4C DNA content and RAPD data confirmed the existence of two taxonomically distinct groups of Bruguiera: one consisting of B. cylindrica and B. parviflora and the other of B. gymnorrhiza and B. sexangula as placed earlier (1862) in the tribe Rhizophoreae by Bentham and Hooker, on the basis of the flowering habits of Bruguiera. Genetically, the B. sexangula and B. gymnorrhiza group was found to be very closely, rather than distantly, related to B. parviflora and B. cylindrica. Our results demonstrate that molecular markers together with cytological evidence provide an effective tool to access the existing interspecific genetic polymorphism in mangrove species, to solve the taxonomic problems and to design their conservation strategy. Rev. Biol. Trop. 55 (2): 437-448. Epub 2007 June, 29.

Estudiamos cuatro especies del mangle Bruguiera (Rhizophoraceae) en Orissa, India. Los cromosomas indican queB. cylindrica y B. parviflora son un grupo taxonómico, y que B. gymnorrhiza y B. sexangula son otro. Genéticamente, el par B. sexangula y B. gymnorrhiza está cercanamente emparentado con B. parviflora and B. cylindrica. Nuestros datos indican que el uso combinado de marcadores genéticos y evidencia citológica permiten discernir el polimorfismo genético interespecífico en los mangles, tanto para resolver problemas taxonómicos como para diseña estrategias eficaces de conservación.

Genetic diversity and DNA content of three South American and three Eurasiatic Trifolium species

Six species of Trifolium (T. polymorphum Poir., T. riograndense Burkart, T. argentinense Speg., T. medium L., T. pratense L. and T. repens L.) were analyzed using inter-simple sequence repeats (ISSR) markers. Six selected primers generated 186 polymerase chain reaction (PCR) products exploring 112 loci in 34 genotypes analyzed with molecular sizes ranging from 200 to 1300 bp. These primers were able to discriminate among and within species, with the PCR products being on average 41.6 percent species-specific and 59.9 percent polymorphic at the within species level. Nuclear DNA content was determined by flow cytometry and revealed variation among species. The 1Cx genome size values were calculated and were found to range from 0.46 pg (T. pratense) to 0.96 pg (T. polymorphum). Genome size values of South American species were higher than those of Eurasiatic origin. The analyses of the molecular data grouped the six species in agreement with their geographical origin and clearly differentiate T. polymorphum from T. argentinense. The Eurasiatic group showed the highest average of species-specific bands (45.3 percent) and the South American group exhibited the highest amount of total bands (59.7). The highest level of intra-species polymorphisms was detected in T. argentinense (92.9 percent), followed by T. medium (89.5 percent).

Relationship between DAPI-fluorescence fading and nuclear DNA content: An alternative method to DNA quantification?

In observations by confocal or conventional fluorescence microscopy, important factors should be considered in order to obtain accurate images. One of them, such as the fluorescence bleaching from highest intensity to lowest signal of fluorescence is a common problem with several DNA fluorochromes and especially for DAPI stain. The fluorescence of DAPI fades rapidly when it is exposed to UV light, under optimal conditions of observation. Although the fading process can be retarded using a mounting medium with antifading reagents, the photochemical process underlying the fluorescence decay has not yet been fully explained. In addition, no relationship between fluorescence fading and nuclear DNA content has been tested. In order to test this relationship, we measured by means of image analysis the DAPI-fluorescence intensity in several cellular types (spermatozoa, erythrocytes and haemocytes) during their fluorescence bleaching. An algorithm specifically built in MATLAB software was used for this approach. The correlation coefficient between nuclear DNA content and DAPI-fluorescence fading was found equal to 99 percent. This study demonstrates the feasibility to measure nuclear DNA content by fluorescence fading quantification, as an alternative method concurrently with image analysis procedures.

Investigation on the Expression of Heparanase and DNA Content in Gastric Carcinoma by Flow Cytometry / 医学研究杂志

Objective To investigate the relation of heparanase expression and DNA content to the clinicopathological characteristic in gastric carcinoma. Methods FCM was used to detect the expression of heparanase and DNA content in 50 gastric carcinoma,25 adjacent carcinoma and 10 normal mucosa.Results FI value of Heparanase in carcinoma group, adjacent carcinoma group and normal mucosa group was 1.86?0.44,1.23?0.45,1.00?0.21,There was a significant difference between them(P

Tripterygium Hypoglaucum (Levl) Hutch alkaloids induce DNA strand breaks and DNA fragmentation in Jurkat T lymphoma cells / 第三军医大学学报

Objective To investigate the effects of Tripterygium Hypoglaucum (Levl) Hutch alkaloids on the nuclear DNA strand breaks and DNA fragmantation in Jurkat T lymphoma cells to understand the mechanisms of the apoptosis. Methods After Jurkat cells were induced by the alkaloids, DNA stand breaks were labeled by TUNEL assay and DNA fragmentation was analyzed by DNA content analysis. Flow cytometry was performed to determine these phenomena. Results THH alkaloids could effectively induce DNA stand breaks and DNA fragmentation. Conclusion There are great changes in nuclear DNA in the apoptosis of Jurkat T lymphoma cells induced by THH alkaloids.

Influence of matrine on the cell cycle and DNA content of fibroblasts derived from hypertrophic scar / 中华医学美学美容杂志

Objective To study the effect of matrine on the cell cycle and DNA content of fibroblasts derived from hypertrophic scarring tissues. Methods The fibroblasts were derived from 3～5 generations of the cultured hypertrophic scarring tissues. After the cells were treated with different concentration of matrine, the population doubling time was calculated and the cell cycle and DNA content were measured with flow cytometry. The fibroblasts treated with normal saline were as control. Results Compared with the control group, the distributions in cell cycle showed that the percentage of G 2 M and S phase cells decreased, and the percentage of G 0 G 1 phase cells increased significantly ( P