Molecular phylogenetics of Black Cobra (Naja naja) in Pakistan

Background: Snakes are found on every continent in the world except Antarctica, and on smaller land masses. Being ecologically important, they also cause a large number of bites, leading to millions of deaths. Mitochondrial and nuclear gene sequences are being used to identify, characterize, and infer genetic biodiversity among different snake species. Furthermore, phylogenetics helps in inferring the relationships and evolutionary histories among these species. Black cobra is one of the four most venomous snakes in Pakistan. Four mitochondrial (ND4, Cytochrome b, 12S rRNA, and 16S rRNA) and four nuclear (C-mos, RAG-1, BDNF, and NT3) genes were used to trace diversity and infer the phylogenetic relationship of black cobra in Pakistan. Results: Almost similar phylogenies were obtained through maximum likelihood and Bayesian inference, showing two species of cobra in Pakistan, namely, black cobra (Naja naja) and brown cobra (Naja oxiana). All Naja species were divided into three clades: black cobra (N. naja) and brown cobra (N. oxiana) cladding with different species of Naja; N. naja (Pakistan) cladding with N. naja from Nepal; and N. oxiana showed close relationship with Naja kaouthia from Thailand and Naja siamensis from Thailand. Conclusion: It was confirmed genetically that there are two cobra species in Pakistan, i.e., black and brown cobras. This study will help in not only genetic conservation but also developing anti-venom against snake species.

Research progress on the relationship between mitochondrial DNA variation and tu-morigenesis / 中国肿瘤临床

The occurrence and development of tumors is a biological process that is extremely complex.In depth research gradually revealed that mitochondria play an important role in the metabolism of tumor cells.The types of mitochondrial DNA(mtDNA)muta-tions identified contain point mutations,deletions,insertions,and copy-number variations.These mutations may be involved in the proliferation,growth,invasion,and metastasis of tumor cells.In this study,mtDNA mutations,copy-number changes,correlation of mtDNA with nuclear DNA,and the progress gained in the development of the relationship between mtDNA mutations and tumorigene-sis are detailed.This review aims to provide additional references to improve the understanding of the relationship between mtDNA mutations and tumor progression.

Prospecting molecular markers to distinguish Cichla kelberi, C. monoculus and C. piquiti / Prospecção de marcadores moleculares para distinção de Cichla kelberi, Cichla monoculus e Cichla piquiti

Peacock bass, a fish of the genus Cichla, is an exotic species from the upper river Paraná floodplain in which the species Cichla kelberi and C. piquiti have been confirmed, coupled to the specie C. monoculus upstream in the Capivara and Taquaruçu dams. The introduction of this genus has caused negative impacts on the diversity of native species. Current research prospects DNA sequences capable of distinguishing the three species and provide molecular data for the taxonomic characterization of the species in the upper Paraná River basin. Sequencing of nuclear (tmo4c4, dlx2 and bmp4) and mitochondrial (cox1, cytb) loci were done from fish of the three species of the genus Cichla reported in the literature of the upper Paraná River basin. Sequence analysis provided molecular differentiation for the species through the usage of loci cytb, dlx2 and cox1. Since the latter only distinguished C. piquiti from the other Cichla species, the loci bmp4 and tmo4c4 were not adequate to accomplish our aim.

O tucunaré é um peixe pertencente ao gênero Cichla, sendo exótico na planície de inundação do alto rio Paraná, onde foi confirmada a presença das espécies Cichla kelberi e C. piquiti e, logo a montante, C. monoculus, nas represas de Capivara e Taquaruçu. A introdução deste gênero tem ocasionado impacto negativo à diversidade de espécies nativas. Visando providenciar dados moleculares para auxiliar na caracterização taxonômica das espécies deste gênero na bacia do alto rio Paraná, os objetivos deste trabalho foram prospectar sequências de DNA capazes de distinguir as três espécies. Procedeu-se o sequenciamento de loci nucleares (tmo4c4, dlx2 e bmp4) e mitocondriais (cox1, cytb) de peixes das três espécies do gênero Cichla relatadas na literatura da bacia do alto rio Paraná. As análises das sequências possibilitaram a diferenciação molecular para estas espécies pela utilização dos loci cytb, dlx2 e cox1; este último, somente para distinguir C. piquiti das demais espécies de Cichla. A utilização dos loci bmp4 e tmo4c4 não foi adequada para este propósito.

Effect of surgical stress on nuclear and mitochondrial DNA from healthy sections of colon and rectum of patients with colorectal cancer.

Surgical resection at any location in the body leads to stress response with cellular and subcellular change, leading to tissue damage. The intestine is extremely sensitive to surgical stress with consequent postoperative complications. It has been suggested that the increase of reactive oxygen species as subcellular changes plays an important role in this process. This article focuses on the effect of surgical stress on nuclear and mitochondrial DNA from healthy sections of colon and rectum of patients with colorectal cancer. Mitochondrial DNA copy number, mitochondrial common deletion and nuclear and mitochondrial 8-oxo-2′-deoxyguanosine content were measured. Both the colon and rectal tissue were significantly damaged either at the nuclear or mitochondrial level. In particular, mitochondrial DNA was more damaged in rectum than in colon. The present investigation found an association between surgical stress and nuclear and mitochondrial DNA damage, suggesting that surgery may generate an increase in free radicals, which trigger a cascade of molecular changes, including alterations in DNA.

Simultaneous quantitative assessment of circulating cell-free mitochondrial and nuclear DNA by multiplex real-time PCR

Quantification of circulating nucleic acids in plasma and serum could be used as a non-invasive diagnostic tool for monitoring a wide variety of diseases and conditions. We describe here a rapid, simple and accurate multiplex real-time PCR method for direct synchronized analysis of circulating cell-free (ccf) mitochondrial (mtDNA) and nuclear (nDNA) DNA in plasma and serum samples. The method is based on one-step multiplex real-time PCR using a FAM-labeled MGB probe and primers to amplify the mtDNA sequence of the ATP 8 gene, and a VIC-labeled MGB probe and primers to amplify the nDNA sequence of the glycerinaldehyde-3-phosphate-dehydrogenase (GAPDH) gene, in plasma and serum samples simultaneously. The efficiencies of the multiplex assays were measured in serial dilutions. Based on the simulation of the PCR reaction kinetics, the relative quantities of ccf mtDNA were calculated using a very simple equation. Using our optimised real-time PCR conditions, close to 100 percent efficiency was obtained from the two assays. The two assays performed in the dilution series showed very good and reproducible correlation to each other. This optimised multiplex real-time PCR protocol can be widely used for synchronized quantification of mtDNA and nDNA in different samples, with a very high rate of efficiency.

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.

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.

The Changes of Histopathology, Proliferation Activity, and Nuclear DNA Content N-butyl-N-(4-hydroxybuty1) Nitrosamine(BBN) Induced Bladder Cancer in Rats / 대한비뇨기과학회지

To evaluate the process of tumor progression in chemical carcinogenesis of the bladder cancer. 0.05% BBN was administered to female Wistar rats for 12 weeks. The rats were divided into six groups and sacrificed every or every two weeks from the 12th to the 20th weeks. Cellular touch imprints of urinary bladder for DNA content analysis by image analyzer and mean AgNORs count per nucleus were performed immediately after sacrifice. Thereafter, the urinary bladder was embedded in paraffin for histopathological examination.On histopathological findings, simple hyperplasia was found in all cases after 12 weeks therapy of BBN. Atypical hyperplasia of the bladder, indicative of a precancerous state, was found in 88.9% of the 12 weeks group, 83.3% of the 13 weeks and in all cases after 14 weeks therapy of the BBN. Bladder cancer was found in 33.3% of the 13 weeks, 55.6% of the 14 weeks, and 100% of the above 16 weeks therapy group. The nuclear DNA content of 21 cases of atypical hyperplasia was diploid in 19 cases(91.5%) and aneuploid in 2 cases(9.5%). DNA aneuploidy was found in 18 cases(66.7%) among the 27 cases of the cancer group. Mean AgNORs count per nucleus and proliferation index by flowcytometry were higher in atypical hyperplasia and cancer group than these of simple hyperplasia and control group, but those differences according to histologic type were not statistically significant. And there was statistically significant correlation between proliferation index and mean AgNORs count per nucleus(r=0.57, p<0.05). These data suggest that the change of nuclear DNA content might occur during the early phase of the carcinogenesis in BBN-induced bladder cancer of rats.

The relationship between nuclear DNA content and the expressions of cyclin D1 and P53 proteins in carcinogenesis of breast carcinoma induced by DMBA in rats / 第三军医大学学报

Objective To investigate the interrelation between nuclear DNA content and the expressions of cyclin D1 and P53 in carcinogenesis of breast carcinoma induced by 7,12 dimethylbenz(a)anthracence(DMBA) in rats. Methods Animal model of breast carcinoma was replicated by gastric perfusion of DMBA at 20 mg/100 g. Nuclear DNA content was detected with image analysis instrument and the expressions of cyclin D1 and P53 at every stage of carcinogenesis of breast carcinoma induced by DMBA in rats observed by Sp immunohistochemical staining. Results In mammary gland tissue at grade Ⅰ, grade Ⅱ and grade Ⅲ atypical hyperplasia of breast carcinoma, there was overepression of cyclin D1(3/20, 6/20 and 11/20, respectively). The overexpression of cyclin D1 was detected to be present in grade Ⅰ, grade Ⅱ and grade Ⅲ breast carcinoma (7/9, 1/4 and 0/7, respectively). The overexpression of P53 was detected to be present in grade Ⅰ, grade Ⅱ and grade Ⅲ breast carcinoma (1/9, 1/4 and 6/7, respectively). Conclusion Nuclear DNA content reflects the state of cell proliferation. Cyclin D1 overexpression participates in the carcinogenesis of breast carcinoma, which may be the early event in breast carcinoma. P53 expression is associated with the development of breast carcinoma, which may be the late event in breast carcinoma. The expression of cyclin D1 is negatively correlated with P53 overexpression.

Molecular organization of great millet (Sorghum vulgare) DNA.

Approximately 52% of the nuclear genome of great millet (Sorghum vulgare) consists of repetitive DNA which can be grouped into very fast, fast and slow components. The reiteration frequencies of the fast and slow reassociating components are 7000 and 92 respectively. Approximately 90% of the genome consists of repeated sequences interspersed amongst themselves and with single copy sequences. The interspersed repeat sequences are of three sizes viz. > 1·5 kilobase pairs, 0·5–1·0 kilobase pairs and 0·15–0·30 kilobase pairs while the size of the single copy sequences is 3·0 kilobase pairs. Hence the genome organization of great millet is essentially of a mixed type.

VALUE OF NUCLEAR DNA MEASUREMENT IN MITOTIC CELLS FROM GASTRIC CARCINOMA OF INTESTINAL TYPE AND ITS PRECANCEROUS LESIONS / 解放军医学杂志

Quantitative DNA measurements were done in mitotic figures in cells from early gastric carcinoma of intestinal type(n = 8), slight (n=10), moderate (n=10) and severe dysplasia (n = 8), and simple intestinal metaplasia (n=10). The mean DNA values were found to increase gradually from intestinal metaplasia to early gastric carcinoma, with the sequence of from slight, moderate to severe dysplasia, and the differences between each two adjacent groups being statistically signi ficant (P