Suppression subtractive hybridization profiles of radial growth phase and metastatic melanoma cell lines reveal novel potential targets

Background: Melanoma progression occurs through three major stages: radial growth phase (RGP), confined to the epidermis; vertical growth phase (VGP), when the tumor has invaded into the dermis; and metastasis. In this work, we used suppression subtractive hybridization (SSH) to investigate the molecular signature of melanoma progression, by comparing a group of metastatic cell lines with an RGP-like cell line showing characteristics of early neoplastic lesions including expression of the metastasis suppressor KISS1, lack of alpha v beta 3-integrin and low levels of RHOC. Methods: Two subtracted cDNA collections were obtained, one (RGP library) by subtracting the RGP cell line (WM1552C) cDNA from a cDNA pool from four metastatic cell lines (WM9, WM852, 1205Lu and WM1617), and the other (Met library) by the reverse subtraction. Clones were sequenced and annotated, and expression validation was done by Northern blot and RT-PCR. Gene Ontology annotation and searches in large-scale melanoma expression studies were done for the genes identified. Results: We identified 367 clones from the RGP library and 386 from the Met library, of which 351 and 368, respectively, match human mRNA sequences, representing 288 and 217 annotated genes. We confirmed the differential expression of all genes selected for validation. In the Met library, we found an enrichment of genes in the growth factors/receptor, adhesion and motility categories whereas in the RGP library, enriched categories were nucleotide biosynthesis, DNA packing/repair, and macromolecular/vesicular trafficking. Interestingly, 19% of the genes from the RGP library map to chromosome 1 against 4% of the ones from Met library. Conclusion: This study identifies two populations of genes differentially expressed between melanoma cell lines from two tumor stages and suggests that these sets of genes represent profiles of less aggressive versus metastatic melanomas. A search for expression profiles of melanoma in available expression study databases allowed us to point to a great potential of involvement in tumor progression for several of the genes identified here. A few sequences obtained here may also contribute to extend annotated mRNAs or to the identification of novel transcripts.

Genes up- and down-regulated by dermcidin in breast cancer: a microarray analysis

Dermcidin (DCD) is a human gene mapped to chromosome 12q13 region, which is co-amplified with multiple oncogenes with a well-established role in the growth, survival and progression of breast cancers. Here, we present a summary of a DNA microarray-based study that identified the genes that are up- and down-regulated in a human MDA-361 pLKO control clone and three clones expressing short hairpin RNA against three different regions of DCD mRNA. A list of 235 genes was differentially expressed among independent clones (> 3-fold change and P < 0.005). The gene expression of 208 was reduced and of 27 was increased in the three DCD-RNAi clones compared to pLKO control clone. The expression of 77 genes (37%) encoding for enzymes involved in amino acid metabolism, glucose metabolism and oxidoreductase activity and several genes required for cell survival and DNA repair were decreased. The expression of EGFR/ErbB-1 gene, an important predictor of outcome in breast cancer, was reduced together with the genes for betacellulin and amphiregulin, two known ligands of EGFR/ErbB receptors. Many of the 27 genes up-regulated by DCD-RNAi expression have not yet been fully characterized; among those with known function, we identified the calcium-calmodulin-dependent protein kinase-II delta and calcineurin A alpha. We compared 132 up-regulated and 12 down-regulated genes in our dataset with those genes up- and down-regulated by inhibitors targeting various signaling pathway components. The analysis showed that the genes in the DCD pathway are aligned with those functionally influenced by the drugs sirolimus, LY-294002 and wortmannin. Therefore, DCD may exert its function by activating the PI3K/AKT/mTOR signaling pathway. Together, these bioinformatic approaches suggest the involvement of DCD in the regulation of genes for breast cancer cell metabolism, proliferation and survival.

Influence of the bacterioplankton community of a tropical eutrophic lagoon on the bacterial community of its neighbouring ocean

The Rodrigo de Freitas lagoon (RFL) is a tropical eutrophic coastal ecosystem located in the urban area of Rio de Janeiro, Brazil. This environment consists of freshwater but has communication with the ocean through a channel (Jardim de Alah`s Channel). The aim of this study was to evaluate the influence of lagoon water on the nearby ocean using molecular and traditional microbiological methods. We hypothesised that due to the eutrophic low-salinity environment, the bacterioplankton community from the RFL would have a native ""brackish"" composition influenced by both freshwater and marine phylotypes, and that bacterial phylotypes of this community would be detected in oceanic samples closer to the channel between the lagoon and the ocean. The cultivation and microscopy experiments clearly showed this influence. Bacterial cell counts revealed that the greater amounts of bacterial cells present in the lagoon increased the observed values seen at oceanic stations near the channel. The Denaturing gradient gel eletrophoresis community profiles also showed a clear influence of Rodrigo de Freitas lagoon waters on the adjacent beaches. The band patterns found for the stations near the channel showed that these communities were mixtures of the communities of the lagoon and sea, and as the distance from the channel increased, the samples became more similar to ocean bacterial communities. A 16S rRNA gene clone library was constructed using a sample acquired from the connection point between the lagoon and the ocean. Around 52% of the sequences in the library showed similarity to the genus Proteobacteria (1% Alpha, 21% Beta, 19% Gamma and 29% unclassified Proteobacteria), and the second most abundant genus was Bacteroidetes, with 15% of the total clones. The results showed that the structure of the bacterial community had both freshwater and marine characteristics.

Intercellular transport of epidermis-expressed MADS domain transcription factors and their effect on plant morphology and floral transition

P>During the lifetime of an angiosperm plant various important processes such as floral transition, specification of floral organ identity and floral determinacy, are controlled by members of the MADS domain transcription factor family. To investigate the possible non-cell-autonomous function of MADS domain proteins, we expressed GFP-tagged clones of AGAMOUS (AG), APETALA3 (AP3), PISTILLATA (PI) and SEPALLATA3 (SEP3) under the control of the MERISTEMLAYER1 promoter in Arabidopsis thaliana plants. Morphological analyses revealed that epidermal overexpression was sufficient for homeotic changes in floral organs, but that it did not result in early flowering or terminal flower phenotypes that are associated with constitutive overexpression of these proteins. Localisations of the tagged proteins in these plants were analysed with confocal laser scanning microscopy in leaf tissue, inflorescence meristems and floral meristems. We demonstrated that only AG is able to move via secondary plasmodesmata from the epidermal cell layer to the subepidermal cell layer in the floral meristem and to a lesser extent in the inflorescence meristem. To study the homeotic effects in more detail, the capacity of trafficking AG to complement the ag mutant phenotype was compared with the capacity of the non-inwards-moving AP3 protein to complement the ap3 mutant phenotype. While epidermal expression of AG gave full complementation, AP3 appeared not to be able to drive all homeotic functions from the epidermis, perhaps reflecting the difference in mobility of these proteins.

Characterisation of the effect of a simulated hydrocarbon spill on diazotrophs in mangrove sediment mesocosm

An analysis of the effect of an oil spill on mangrove sediments was carried out by contamination of mesocosms derived from two different mangroves, one with a history of contamination and one pristine. The association between N(2) fixers and hydrocarbon degradation was assessed using quantitative PCR (qPCR) for the genes rrs and nifH, nifH clone library sequencing and total petroleum hydrocarbon (TPH) quantification using gas chromatography. TPH showed that the microbial communities of both mangroves were able to degrade the hydrocarbons added; however, whereas the majority of oil added to the mesocosm derived from the polluted mangrove was degraded in the 75 days of the experiment, there was only partially degradation in the mesocosm derived from the pristine mangrove. qPCR showed that the addition of oil led to an increase in rrs gene copy numbers in both mesocosms, having almost no effect on the nifH copy numbers in the pristine mangrove. Sequencing of nifH clones indicated that the changes promoted by the oil in the polluted mangrove were greater than those observed in the pristine mesocosm. The main effect observed in the polluted mesocosm was the selection of a single phylotype which is probably adapted to the presence of petroleum. These results, together with previous reports, give hints about the relationship between N(2) fixation and hydrocarbon degradation in natural ecosystems.

Mechanical properties and cytotoxicity of 3Y-TZP bioceramics reinforced with Al(2)O(3) particles

The influence of Al(2)O(3) addition and sintering parameters on the mechanical properties and cytotoxicity of tetragonal ZrO(2)-3 mol% Y(2)O(3) ceramics was evaluated. Samples containing 0, 10, 20 and 30 wt.% of Al(2)O(3) particles were prepared by cold uniaxial pressing (80 MPa) and sintered in air at 1500, 1550 and 1600 degrees C for 120 min. The effects of the sintering conditions on the microstructure were analyzed by X-ray diffraction analysis and scanning electron microscopy. Hardness and fracture toughness were determined by the Vickers indentation method and the mechanical resistance by four-point bending tests. As a preliminary biological evaluation, ""in vitro"" cytotoxicity tests were realized to determine the cytotoxic level of the ZrO(2)-Al(2)O(3) composites, using the neutral red uptake method with NCTC clones L929 from the American Type Culture Collection (ATCC) bank. Fully dense ceramic materials were obtained with a hardness ranging between 1340 HV and 1585 HV, depending on the amount of Al(2)O(3) in the ZrO(2) matrix. On the other hand, no significant influence of the Al(2)O(3) addition on fracture toughness was observed, exhibiting values near 8 MPa m(1/2) for all compositions and sintering conditions studied. The non-cytotoxic behavior, the elevated fracture toughness, the good bending strength (sigma(f) = 690 MPa) and the elevated Weibull`s modulus (m = 11) exhibited by the material, show that these ceramic composites are highly suitable biomaterials for dental implant applications. (C) 2008 Elsevier Ltd and Techna Group S.r.l. All rights reserved.

Degradation of detergent (linear alkylbenzene sulfonate) in an anaerobic stirred sequencing-batch reactor containing granular biomass

This study aimed to determine the efficiency of an anaerobic stirred sequencing-batch reactor containing granular biomass for the degradation of linear alkylbenzene sulfonate (LAS), a surfactant present in household detergent. The bioreactor was monitored for LAS concentrations in the influent, effluent and sludge, pH, chemical oxygen demand, bicarbonate alkalinity, total solids, and volatile solids. The degradation of LAS was found to be higher in the absence of co-substrates (53%) than in their presence (24-37%). Using the polymerase chain reaction and denaturing gradient gel electrophoresis (PCR/DGGE), we identified populations of microorganisms from the Bacteria and Archaea domains. Among the bacteria, we identified uncultivated populations of Arcanobacterium spp. (94%) and Opitutus spp. (96%). Among the Archaea, we identified Methanospirillum spp. (90%), Methanosaeta spp. (98%), and Methanobacterium spp. (96%). The presence of methanogenic microorganisms shows that LAS did not inhibit anaerobic digestion. Sampling at the last stage of reactor operation recovered 61 clones belonging to the domain bacteria. These represented a variety of phyla: 34% shared significant homology with Bacteroidetes, 18% with Proteobacteria, 11% with Verrucomicrobia, 8% with Fibrobacteres, 2% with Acidobacteria, 3% with Chlorobi and Firmicutes, and 1% with Acidobacteres and Chloroflexi. A small fraction of the clones (13%) were not related to any phylum. Published by Elsevier Ltd.

Assessing the effects of early silvicultural management on long-term site productivity of fast-growing eucalypt plantations: the Brazilian experience

Eucalyptus is the dominant and most productive planted forest in Brazil, covering around 3.4 million ha for the production of charcoal, pulp, sawtimber, timber plates, wood foils, plywood and for building purposes. At the early establishment of the forest plantations, during the second half of the 1960s, the eucalypt yield was 10 m(3) ha(-1) y(-1). Now, as a result of investments in research and technology, the average productivity is 38 m3 ha(-1) y(-1). The productivity restrictions are related to the following environmental factors, in order of importance: water deficits > nutrient deficiency > soil depth and strength. The clonal forests have been fundamental in sites with larger water and nutrient restrictions, where they out-perform those established from traditional seed-based planting stock. When the environmental limitations are small the productivities of plantations based on clones or seeds appear to be similar. In the long term there are risks to sustainability, because of the low fertility and low reserves of primary minerals in the soils, which are, commonly, loamy and clayey oxisols and ultisols. Usually, a decline of soil quality is caused by management that does not conserve soil and site resources, damages soil physical and chemical characteristics, and insufficient or unbalanced fertiliser management. The problem is more serious when fast-growing genotypes are planted, which have a high nutrient demand and uptake capacity, and therefore high nutrient output through harvesting. The need to mobilise less soil by providing more cover and protection, reduce the nutrient and organic matter losses, preserve crucial physical properties as permeability ( root growth, infiltration and aeration), improve weed control and reduce costs has led to a progressive increase in the use of minimum cultivation practices during the last 20 years, which has been accepted as a good alternative to keep or increase site quality in the long term. In this paper we provide a synthesis and critical appraisal of the research results and practical implications of early silvicultural management on long-term site productivity of fast-growing eucalypt plantations arising from the Brazilian context.

Chitinase and peroxidase activity in different stages of eucalypt leaves after inoculation with Puccinia psidii and acibenzolar-S-metil

Chitinase and peroxidase activity in different stages of eucalypt leaves after inoculation with Puccinia psidii and acibenzolar-S-metil To elucidate some biochemical processes during infection in the pathosystem Puccinia psidii x eucalyptus, the defense metabolism in different-stage leaves was compared between rust-resistant and susceptible clones, respectively. In addition, chitinase and peroxidase activities were assayed. Each treatment consisted of 4 replicates, in a completely randomized design: 2 clones, inoculated and not inoculated with P. psidii; sprayed with acibenzolar-S-methyl (ASM) and distilled water; and represented by the 1(st) leaf pair (size equivalent to 1/5 total leaf development), 2(nd) pair (2/5 total development), and 4(th) pair (4/5 total leaf length). Leaves were harvested in 4 periods: 0, 24, 72 and 96 hours after inoculation. Results indicated that ASM treatment or P. psidii action led to higher chitinase and peroxidase activity level but did not alter the expression of these activities in developed leaves (4(th) pair) during the experiment. Alterations in enzyme levels after inoculation were only observed in developing leaves (1(st) and 2(nd) pairs), which suggests that the response to infection was concomitant to chitinase and peroxidase synthesis. The highest increases in enzymatic activities were observed in resistant clones at 72 hours after inoculation and in susceptible ones previously treated with ASM and later inoculated with the pathogen.

Inheritance of resistance to Puccinia psidii G. Winter in a eucalyptus interspecific hybrid progeny evaluated under conditions of natural infection

Inheritance of resistance to Puccinia psidii G. Winter in a eucalyptus interspecific hybrid progeny evaluated under conditions of natural infection Rust caused by the fungus Puccinia psidii is currently the most important disease of eucalyptus. It is widely disseminated in Brazil, and causes serious damage in nurseries and plantation areas. The identification of resistant germplasm along with knowledge of the genetic basis of resistance heredity are the first requirements for the success of breeding programs aiming to develop resistant varieties. Earlier studies carried out under controlled conditions suggested a monogenic control as well as the participation of at least two genes promoting resistance to rust. The goal of this study was to evaluate the resistance to P. psidii under field conditions in fourteen progenies from controlled crosses and self-crosses among four hybrid clones of Eucalyptus grandis Hill ex Maiden x Eucalyptus urophylla ST Blake that contrast for resistance to the fungus. Results indicated that resistance could be explained by one locus with main effects and at least three different alleles. However, loci with minor effects may influence the resistance, since variation on severity classes was observed. Differences in segregation of resistance between reciprocal crosses were not observed, indicating absence of cytoplasmic effects.

Effect of acibenzolar-S-methyl and Saccharomyces cerevisiae on the activation of Eucalyptus defences against rust

Tree defence mechanisms against the fungus Puccinia psidii were examined by comparing the activities of defence-related enzymes (chitinase, peroxidase and phenylalanine ammonia-lyase) of two Eucalyptus grandis x E. urophylla (urograndis) hybrids, previously classified as either susceptible to rust (VR hybrid) or moderately resistant to rust (C0 hybrid). Furthermore, the potential of disease control by artificial activation of host defences using either acibenzolar-S-methyl (ASM) or Saccharomyces cerevisiae extract was also investigated. Greenhouse inoculation trials revealed that the C0 hybrid had lower disease severity than the VR hybrid but following foliar applications of either ASM or S. cerevisiae extract treatment, disease severity (evaluated at 15 days after inoculation) was reduced in both hybrids. This enhanced resistance was associated with the induction of a hypersensitive reaction which appeared to be effective in controlling rust in both clones. The activity of all enzymes differed between clones and inducer treatment. The role of the defence-related enzymes in imparting resistance to eucalypt hybrids against rust is discussed.

Evaluation of GFP Tag as a Screening Reporter in Directed Evolution of a Hyperthermophilic beta-Glucosidase

By applying a directed evolution methodology specific enzymatic characteristics can be enhanced, but to select mutants of interest from a large mutant bank, this approach requires high throughput screening and facile selection. To facilitate such primary screening of enhanced clones, an expression system was tested that uses a green fluorescent protein (GFP) tag from Aequorea victoria linked to the enzyme of interest. As GFP`s fluorescence is readily measured, and as there is a 1:1 molar correlation between the target protein and GFP, the concept proposed was to determine whether GFP could facilitate primary screening of error-prone PCR (EPP) clones. For this purpose a thermostable beta-glucosidase (BglA) from Fervidobacterium sp. was used as a model enzyme. A vector expressing the chimeric protein BglA-GFP-6XHis was constructed and the fusion protein purified and characterized. When compared to the native proteins, the components of the fusion displayed modified characteristics, such as enhanced GFP thermostability and a higher BglA optimum temperature. Clones carrying mutant BglA proteins obtained by EPP, were screened based on the BglA/GFP activity ratio. Purified tagged enzymes from selected clones resulted in modified substrate specificity.

Cultivation of hitherto-uncultured bacteria belonging to the Verrucomicrobia subdivision 1 from the potato (Solanum tuberosum L.) rhizosphere

The role of dominant bacterial groups in the plant rhizosphere, e.g., those belonging to the phyla Acidobacteria and Verrucomicrobia, has, so far, not been elucidated, and this is mainly due to the lack of culturable representatives. This study aimed to isolate hitherto-uncultured bacteria from the potato rhizosphere by a combination of cultivation approaches. An agar medium low in carbon availability (oligotrophic agar medium) and either amended with potato root exudates or catalase or left unamended was used with the aim to improve the culturability of bacteria from the potato rhizosphere. The colony forming unit numbers based on colonies and microcolonies were compared with microscopically determined fluorescence-stained cell numbers. Taxonomical diversity of the colonies was compared with that of library clones made from rhizosphere DNA, on the basis of 16S rRNA gene comparisons. The oligotrophic media amended or not with catalase or rhizosphere extract recovered up to 33.6% of the total bacterial numbers, at least seven times more than the recovery observed on R2A. Four hitherto-uncultured Verrucomicrobia subdivision 1 representatives were recovered on agar, but representatives of this group were not found in the clone library. The use of oligotrophic medium and its modifications enabled the growth of colony numbers, exceeding those on classical agar media. Also, it led to the isolation of hitherto-uncultured bacteria from the potato rhizosphere. Further improvement in cultivation will certainly result in the recovery of other as-yet-unexplored bacteria from the rhizosphere, making these groups accessible for further investigation, e.g., with respect to their possible interactions with plants.

The bacterial diversity in a Brazilian non-disturbed mangrove sediment

The bacterial diversity present in sediments of a well-preserved mangrove in Ilha do Cardoso, located in the extreme south of So Paulo State coastline, Brazil, was assessed using culture-independent molecular approaches (denaturing gradient gel electrophoresis (DGGE) and analysis of 166 sequences from a clone library). The data revealed a bacterial community dominated by Alphaproteobacteria (40.36% of clones), Gammaproteobacteria (19.28% of clones) and Acidobacteria (27.71% of clones), while minor components of the assemblage were affiliated to Betaproteobacteria, Deltaproteobacteria, Firmicutes, Actinobacteria and Bacteroidetes. The clustering and redundancy analysis (RDA) based on DGGE were used to determine factors that modulate the diversity of bacterial communities in mangroves, such as depth, seasonal fluctuations, and locations over a transect area from the sea to the land. Profiles of specific DGGE gels showed that both dominant (`universal` Bacteria and Alphaproteobacteria) and low-density bacterial communities (Betaproteobacteria and Actinobacteria) are responsive to shifts in environmental factors. The location within the mangrove was determinant for all fractions of the community studied, whereas season was significant for Bacteria, Alphaproteobacteria, and Betaproteobacteria and sample depth determined the diversity of Alphaproteobacteria and Actinobacteria.

IBA application for rooting of Eucalyptus benthamii Maiden and Cambage x Eucalyptus dunnii Maiden minicuttings

IBA application for rooting of Eucalyptus benthamii Maiden and Cambage x Eucalyptus dunnii Maiden minicuttings. Eucalyptus has great importance in the forestry sector and many advances in the area of improvement have been achieved with the advent of biotechnological techniques. However, some promising genotypes still do not have multiplication protocols with cloning techniques, such as minicutting. The study aimed to evaluate IBA concentrations for survival, rooting and vegetative vigor of E. benthamii x E. dunnii minicuttings, and determine the maximum technical efficiency dose. Ministumps H12, H19 and H20 clones were cultivated in a clonal minigarden under a semi-hydroponic system. For rooting, the rninicutting basal portion was plunged in hydro-alcoholic solutions, whose concentrations were: 0; 2,000; 4,000; 6,000 and 8,000 mg L(-1) of IBA. The experiment was conducted in a completely randomized design, with the factors consisting of three clones and five IBA concentrations, with five replications, containing 10 minicuttings per replication. IBA positively influenced the minicutting rooting processes, with differentiated behavior between the clones, with 30.32 to 55.45% rooting variation. The positives increments occurred until the highest IBA concentration for H12 and H19 clones. However, the range between 4,000 and 6,000 mg L(-1) of IBA treatments promoted the best rooting results for the H20 clone.