Efficient somatic gene targeting in the lymphoid human cell line DG75

Among the different approaches used to define the function of a protein of interest, alteration and/or deletion of its encoding gene is the most direct strategy. Homologous recombination between the chromosomal gene locus and an appropriately designed targeting vector results in an alteration or knockout of the gene of interest. Homologous recombination is easily performed in yeast or in murine embryonic stem cells, but is cumbersome in more differentiated and diploid somatic cell lines. Here we describe an efficient method for targeting both alleles of a complex human gene locus in DG75 cells, a cell line of lymphoid origin. The experimental approach included a conditional knockout strategy with three genotypic markers, which greatly facilitated the generation and phenotypic identification of targeted recombinant cells. The vector was designed such that it could be reused for two consecutive rounds of recombination to target both alleles. The human DG75 cell line appears similar to the chicken DT40 pre B-cell line, which supports efficient homologous recombination. Therefore, the DG75 cell line is a favorable addition to the limited number of cell lines amenable to gene targeting and should prove useful for studying gene function through targeted gene alteration or deletion in human somatic cells.     

Influence of diets supplemented with vitamins C and E on pirarucu (Arapaima gigas) blood parameters

This study evaluated the influence of diets supplemented with 500, 800, 1200 mg kg^− 1 of vitamin C (ascorbic acid or AA) and vitamin E (α-tocopherol or α-T) on the physiological responses of pirarucu fed for 2 months. Weight and mortality were not affected by dietary vitamin type or their concentrations. Significant increase (p < 0.05) on the red blood cells count was obtained on treatments with 800 and 1200 mg AA kg^− 1 and on the hemoglobin concentration on treatment with 500 mg α-T kg^− 1 relatively to control. Mean corpuscular volume presented a significant decrease (p < 0.05) on treatment with 800 and 1200 mg AA kg⁻¹ when compared to control. Mean corpuscular hemoglobin concentration was significantly high (p < 0.05) on treatment with 500 mg α-T kg^− 1. Only in vitamin C treatments, we noticed a significant increase (p < 0.05) in the number of leucocytes relative to control. All fish in the vitamin-supplemented treatments, except 500 mg AA kg^− 1, had high total protein values compared to control. Fish treated with 800 or 1200 mg α-T kg^− 1 also showed increases in plasma glucose concentrations. Our results suggest that 800 and 1200 mg AA kg^− 1 are probably the most suitable concentrations for pirarucu diets, although high vitamin E diets are not necessary for quantitative leucocyte increases for this species.     

Carvedilol protects against cisplatin-induced oxidative stress, redox state unbalance and apoptosis in rat kidney mitochondria

Cisplatin is a highly effective chemotherapeutic agent which causes severe nephrotoxicity. Studies have suggested that reactive oxygen species, mainly generated in mitochondria, play a central role in cisplatin-induced renal damage. A wide range of antioxidants have been evaluated as possible protective agents against cisplatin-induced nephrotoxicity; however a safe and efficacious compound has not yet been found. The present study is the first to evaluate the protective potential of carvedilol, a beta-blocker with strong antioxidant properties, against the mitochondrial oxidative stress and apoptosis in kidney of rats treated with cisplatin. The following cisplatin-induced toxic effects were prevented by carvedilol: increased plasmatic levels of creatinine and blood urea nitrogen (BUN); lipid peroxidation, oxidation of cardiolipin; oxidation of protein sulfhydryls; depletion of the non-enzymatic antioxidant defense and increased activity of caspase-3. Carvedilol per se did not present any effect on renal mitochondria. It was concluded that carvedilol prevents mitochondrial dysfunction and renal cell death through the protection against the oxidative stress and redox state unbalance induced by cisplatin. The association of carvedilol to cisplatin chemotherapy was suggested as a possible strategy to minimize the nephrotoxicity induced by this antitumor agent.     

Development of a cellular assay system to study the genome replication of high- and low-risk mucosal and cutaneous human papillomaviruses

We found that recircularized high-risk (type 16 and 18) and low-risk mucosal (type 6b and 11) and cutaneous (type 5 and 8) human papillomavirus (HPV) genomes replicate readily when delivered into U2OS cells by electroporation. The replication efficiency is dependent on the amount of input HPV DNA and can be followed for more than 3 weeks in proliferating cell culture without selection. Cotransfection of recircularized HPV genomes with a linear G418 resistance marker plasmid has allowed subcloning of cell lines, which, in a majority of cases, carry multicopy episomal HPV DNA. Analysis of the HPV DNA status in these established cell lines showed that HPV genomes exist in these cells as stable extrachromosomal oligomers. When the cell lines were cultivated as confluent cultures, a 3- to 10-fold amplification of the HPV genomes per cell was induced. Two-dimensional (2D) agarose gel electrophoresis confirmed amplification of mono- and oligomeric HPV genomes in these confluent cell cultures. Amplification occurred as a result of the initiation of semiconservative two-dimensional replication from one active origin in the HPV oligomer. Our data suggest that the system described here might be a valuable, cost-effective, and efficient tool for use in HPV DNA replication studies, as well as for the design of cell-based assays to identify potential inhibitors of all stages of HPV genome replication.     

Genomic content and new insights on the origin of the B chromosome of the cichlid fish <Emphasis Type="Italic">Astatotilapia latifasciata</Emphasis>

B chromosomes are additional chromosomes widely studied in a diversity of eukaryotic groups, including fungi, plants and animals, but their origin, evolution and possible functions are not clearly understood. To further understand the genomic content and the evolutionary history of B chromosomes, classical and molecular cytogenetic analyses were conducted in the cichlid fish Astatotilapia latifasciata, which harbor 1–2 B chromosomes. Through cytogenetic mapping of several probes, including transposable elements, rRNA genes, a repeated DNA genomic fraction (C ₀ t − 1 DNA), whole genome probes (comparative genomic hybridization), and BAC clones from Oreochromis niloticus, we found similarities between the B chromosome and the 1st chromosome pair and chromosomes harboring rRNA genes. Based on the cytogenetic mapping data, we suggest the B chromosome may have evolved from a small chromosomal fragment followed by the invasion of the proto-B chromosome by several repeated DNA families.     

Diagnosing idiopathic learning disability: a cost-effectiveness analysis of microarray technology in the National Health Service of the United Kingdom

Array based comparative genomic hybridisation (aCGH) is a powerful technique for detecting clinically relevant genome imbalance and can offer 40 to > 1000 times the resolution of karyotyping. Indeed, idiopathic learning disability (ILD) studies suggest that a genome-wide aCGH approach makes 10–15% more diagnoses involving genome imbalance than karyotyping. Despite this, aCGH has yet to be implemented as a routine NHS service. One significant obstacle is the perception that the technology is prohibitively expensive for most standard NHS clinical cytogenetics laboratories. To address this, we investigated the cost-effectiveness of aCGH versus standard cytogenetic analysis for diagnosing idiopathic learning disability (ILD) in the NHS. Cost data from four participating genetics centres were collected and analysed. In a single test comparison, the average cost of aCGH was ￡442 and the average cost of karyotyping was ￡117 with array costs contributing most to the cost difference. This difference was not a key barrier when the context of follow up diagnostic tests was considered. Indeed, in a hypothetical cohort of 100 ILD children, aCGH was found to cost less per diagnosis (￡3,118) than a karyotyping and multi-telomere FISH approach (￡4,957). We conclude that testing for genomic imbalances in ILD using microarray technology is likely to be cost-effective because long-term savings can be made regardless of a positive (diagnosis) or negative result. Earlier diagnoses save costs of additional diagnostic tests. Negative results are cost-effective in minimising follow-up test choice. The use of aCGH in routine clinical practice warrants serious consideration by healthcare providers. Copyright statement The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence (or non exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd, and its Licensees to permit this article (if accepted) to be published in BMJ editions and any other BMJPGL products and to exploit all subsidiary rights, as set out in our licence (bmj.com/advice/copyright.shtml). Authorship The authors included on this paper fulfil the criteria of authorship and no one who fulfils the criteria has been excluded from authorship. The authors made a substantial contribution to the conception, design, analysis and interpretation of data. They were involved in drafting the article or revising it critically for important intellectual content and approving the version to be published. Contributorship Sarah Wordsworth (Guarantor): Planning, conducting and reporting work, interpretation of data, drafting and revising article. James Buchanan: Conducting and reporting work, interpretation of data, revising article. Regina Regan: Completing costing questionnaire, providing protocol details, other costing information, interpretation of data, information about learning disability and genome imbalance and revising article. Val Davison: Completing costing questionnaire, providing protocol details, sharing overall laboratory experience and drafting article. Kim Smith: Completing costing questionnaire, providing protocol details, drafting article. Sara Dyer: Completing costing questionnaire and providing protocol details. Carolyn Campbell: Completing costing questionnaire and providing protocol details. Edward Blair: Critical appraisal of article for clinical content and revising article. Eddy Maher: Completing costing questionnaire, providing protocol details, sharing overall laboratory experience and drafting article. Jenny Taylor: Planning and facilitating work between centres. Drafting and revising article. Samantha JL Knight: Completing costing questionnaire, providing protocol details, other costing information, interpretation of data, providing information about learning disability and genome imbalance, drafting and revising article. Jenny Taylor and Samantha JL Knight contributed equally to the work presented.     

Hierarchical status and colour preference in Nile tilapia (<Emphasis Type="Italic">Oreochromis niloticus</Emphasis>)

We studied the colour preference of isolated Nile tilapia (Oreochromis niloticus) and whether previous residence or body size can affect environmental colour choice. In the first phase, a cylindrical tank was divided into five differently coloured compartments (yellow, blue, green, white and red), a single fish was introduced into the tank and the frequency at which this fish visited each compartment was recorded over a 2-day study period. An increasingly larger fish (approx +2 cm in length each time) was then added into the tank on each of days 3, 5 and 7 (=four fish in the tank by day 7), and the frequency at which each fish visited the different compartments of the tank was observed twice a day to obtain visit frequency data on the differently sized fishes. This experiment was replicated six times. In the first phase, the solitary fish established residence inside the yellow compartment on the first and second days. Following the introduction of a larger fish, the smaller fish was displaced from the occupied compartment. Nile tilapia possibly shows this preference for yellow as a function of its visual spectral sensitivity and/or the spectral characteristics of its natural environment. Moreover, body size is an important factor in determining hierarchical dominance and territorial defence, and dominant fish chose the preferred environmental colour compartment as their territory.     

Structural and Biological Characterization of Two Crotamine Isoforms IV-2 and IV-3 Isolated from the <Emphasis Type="Italic">Crotalus durissus cumanensis</Emphasis> Venom

In this work, we isolated the two new crotamine isoforms from the Crotalus durissus cumanensis rattlesnake venom and its “in vitro” neurotoxic, myotoxic and lethality (DL₅₀) intracerebroventricular (i.c.v.) effects were characterized. These proteins were named IV-2 and IV-3 and were purified by combination of two chromatographic steps on molecular exclusion chromatography on Superdex 75 and reverse phase HPLC (μ-Bondapack C18). The molecular mass of the crotamine isoforms was 4905.96 Da for isoform IV-2 and 4956.97 Da for IV-3 and, as determined by mass spectrometry, and both contained six Cys residues. Enzymatic hydrolysis followed by de novo sequencing by tandem mass spectrometry was used to determine the primary structure of both isoforms. The positions of five sequenced tryptic peptides, including the N-terminal of the isoform IV-2 and four from isoform IV-3 were deduced by comparison with a homologous protein from the crotamine family. The isoforms IV-2 and IV-3 had a sequence of amino acids of 42 amino acid residues IV-2: YKRCHIKGGH CFPKEKLICI PPSSDIGKMD CPWKRKCCKK RS and pI value 9.54 and IV-3: YKQCHKKGGH CFPKEVLICI PPSSDFGKMD CRWKRKCCKK RS with a pI value of 9.54. This protein showed high molecular amino acid sequence identity with other crotamine-like proteins from Crotalus durissus terrificus. These new crotamine isoforms induced potent blockade of neuromuscular transmission in young chicken biventer cervicis preparation and potent myotoxic effect. In mice, both isoforms induced myonecrosis, upon intramuscular or subcutaneous injections. These activities were modulated by the presence of positively charged amino acid residues. The LD₅₀ of isoform IV-2 was 0.07 mg/kg and isoform IV-3 was 0.06 mg/kg the animal weight, by i.c.v. route.     

Floristic composition and similarity of 15 hectares in Central Amazon, Brazil

The Amazon region is one of the most diverse areas in the world. Research on high tropical forest diversity brings up relevant contributions to understand the mechanisms that result and support such diversity. In the present study we describe the species composition and diversity of 15 one-ha plots in the Amazonian terra firme dense forest in Brazil, and compare the floristic similarity of these plots with other nine one-ha plots. The 15 plots studied were randomly selected from permanent plots at the Embrapa Experimental site, Amazonas State in 2005. The diversity was analysed by using species richness and Shannon's index, and by applying the Sorensen's index for similarity and unweighted pair-group average (UPGMA) as clustering method. Mantel test was performed to study whether the differences in species composition between sites could be explained by the geographic distance between them. Overall, we identified 8 771 individuals, 264 species and 51 plant families. Most of the species were concentrated in few families and few had large number of individuals. Families presenting the highest species richness were Fabaceae (Faboideae: 22spp., Mimosoideae: 22spp.), Sapotaceae: 22spp., Lecythidaceae: 15 and Lauraceae: 13. Burseraceae had the largest number of individuals with 11.8% of the total. The ten most abundant species were: Protium hebetatum (1 037 individuals), Eschweilera coriacea (471), Licania oblongifolia (310), Pouteria minima (293), Ocotea cernua (258), Scleronema micranthum (197), Eschweilera collina (176), Licania apelata (172), Naucleopsis caloneura (170) and Psidium araca (152), which represented 36.5% of all individuals. Approximately 49% of species had up to ten individuals and 13% appeared only once in all sampled plots, showing a large occurrence of rare species. Our study area is on a forest presenting a high tree species diversity with Shannon's diversity index of 4.49. The dendrogram showed two groups of plots with low similarity between them (less than 0.25), and the closer the plots were one to another, more similar in species composition (Mantel R = 0.3627, p < 0.01). The 15 plots in our study area share more than 50% of their species composition and represent the group of plots that have the shortest distance between each other. Overall, our results highlight the high local and regional heterogeneity of environments in terra firme forests, and the high occurrence of rare species, which should be considered in management and conservation programs in the Amazon rainforest, in order to maintain its structure on the long run.     

Purification, and biochemical and biophysical characterization of cellobiohydrolase I from Trichoderma harzianum IOC 3844

Because of its elevated cellulolytic activity, the filamentous fungus Trichoderma harzianum has a considerable potential in biomass hydrolysis applications. Trichoderma harzianum cellobiohydrolase I (ThCBHI), an exoglucanase, is an important enzyme in the process of cellulose degradation. Here, we report an easy single-step ion-exchange chromatographic method for purification of ThCBHI and its initial biophysical and biochemical characterization. The ThCBHI produced by induction with microcrystalline cellulose under submerged fermentation was purified on DEAE-Sephadex A-50 media and its identity was confirmed by mass spectrometry. The ThCBHI biochemical characterization showed that the protein has a molecular mass of 66 kDa and pI of 5.23. As confirmed by smallangle X-ray scattering (SAXS), both full-length ThCBHI and its catalytic core domain (CCD) obtained by digestion with papain are monomeric in solution. Secondary structure analysis of ThCBHI by circular dichroism revealed alpha- helices and beta-strands contents in the 28% and 38% range, respectively. The intrinsic fluorescence emission maximum of 337 nm was accounted for as different degrees of exposure of ThCBHI tryptophan residues to water. Moreover, ThCBHI displayed maximum activity at pH 5.0 and temperature of 50 degrees C with specific activities against Avicel and p-nitrophenyl-β-D-cellobioside of 1.25 U/mg and 1.53 U/mg, respectively.