Microbial community composition in sediments of the Hydrate Ridge (Cascadian Margin) collected during RV SONNE cruises SO143 (1999) and SO148-1 (2000)

Cold seep environments such as sediments above outcropping hydrate at Hydrate Ridge (Cascadia margin off Oregon) are characterized by methane venting, high sulfide fluxes caused by the anaerobic oxidation of methane, and the presence of chemosynthetic communities. This investigation deals with the diversity and distribution of sulfate-reducing bacteria, some of which are directly involved in the anaerobic oxidation of methane as syntrophic partners of the methanotrophic archaea. The composition and activity of the microbial communities at methane vented and nonvented sediments are compared by quantitative methods including total cell counts, fluorescence in situ hybridization (FISH). Bacteria involved in the degradation of particulate organic carbon (POC) are as active and diverse as at other productive margin sites of similar water depths. The availability of methane supports a two orders of magnitude higher microbial biomass (up to 9.6×10**10cells/cm**3). Sediment samples were obtained during RV SONNE cruises SO143-2 and SO148-1 at the crest of southern Hydrate Ridge at the Cascadia convergent margin off the coast of Oregon. Sediment cores of 20 - 40 cm length were obtained using a video-guided multiple corer from gas hydrate bearing sediments and from reference sites not enriched in methane in the surface sediments. Samples for total cell counts were obtained from 1 cm core slices, fixed with 2% formaldehyde and stored cold (4°C) and the quantification of aggregates was done via epifluorescence microscopy after staining the sediments with Acridine Orange Direct Counts (AODC) according to the method of Meyer- Reil (1983, doi:10.1007/BF00395813). Total cell counts were defined as the sum of single cells plus the aggregated cells in the syntrophic consortia. DAPI staining was used to measure ANME2/DSS aggregate sizes via epifluorescence microscopy of FISH-treated samples. For FISH, subsamples of sediment cores were sliced into 1 cm intervals and fixed for 2-3 h with 3% formaldehyde (final concentration), washed twice with 1×PBS (10 mM sodium phosphate; 130 mM NaCl), and finally stored in 1×PBS/EtOH (1:1) at -20°C.

Geomicrobiology, bulk sediment characteristics and pore water chemistry of sediment cores from Laguna Potrok Aike, southern Patagonia

Authigenic minerals can form in the water column and sediments of lakes, either abiotically or mediated by biological activity. Such minerals have been used as paleosalinity and paleoproductivity indicators and reflect trophic state and early diagenetic conditions. They are also considered potential indicators of past and perhaps ongoing microbial activity within sediments. Authigenic concretions, including vivianite, were described in late glacial sediments of Laguna Potrok Aike, a maar lake in southernmost Argentina. Occurrence of iron phosphate implies specific phosphorus sorption behavior and a reducing environment, with methane present. Because organic matter content in these sediments was generally low during glacial times, there must have been alternative sources of phosphorus and biogenic methane. Identifying these sources can help define past trophic state of the lake and diagenetic processes in the sediments. We used scanning electron microscopy, phosphorus speciation in bulk sediment, pore water analyses, in situ ATP measurements, microbial cell counts, and measurements of methane content and its carbon isotope composition (d13C CH4) to identify components of and processes in the sediment. The multiple approaches indicated that volcanic materials in the catchment are important suppliers of iron, sulfur and phosphorus. These elements influence primary productivity and play a role in microbial metabolism during early diagenesis. Authigenic processes led to the formation of pyrite framboids and revealed sulfate reduction. Anaerobic oxidation of methane and shifts in pore water ion concentration indicated microbial influence with depth. This study documents the presence of active microbes within the sediments and their relationship to changing environmental conditions. It also illustrates the substantial role played by microbes in the formation of Laguna Potrok Aike concretions. Thus, authigenic minerals can be used as biosignatures in these late Pleistocene maar sediments.

Hydrochemical and microbiological properties of groundwater from pristine aquifer sampled at the Lower Rhine Embayment

The hydrochemistry and the microbial diversity of a pristine aquifer system near Garzweiler, Germany next to the open-pit lignite mine Garzweiler 1, were characterized. Hydrogeochemical and isotopic data indicate a recent activity of sulfate-reducing bacteria in the Tertiary marine sands. The community structure in the aquifer was studied by fluorescence in situ hybridization (FISH). Up to 7.3 x 10**5 cells/ml were detected by DAPIstaining. Bacteria (identified by the probe EUB338) were dominant, representing 51.9% of the total cell number (DAPI). Another 25.7% of total cell were affiliated with the domain Archaea as identified by the probe ARCH915. Within the domain Bacteria, the beta-Proteobacteria were most abundant (21.0% of total cell counts). Using genusspecific probes for sulfate-reducing bacteria (SRB), 2.5% of the total cells were identified as members of the genus Desulfotomaculum. This reflects the predominant role these microorganisms have been found to play in sulfatereducing zones of aquifers at other sites. Previously, all SRB cultured from this site were from the spore-forming genera Desulfotomaculum and Desulfosporosinus. Samples were taken after pumping for >= 40 min and after parameters such as temperature, pH, redox potential, oxygen and conductivity of the groundwater had remained stable for >= 15 min due to recharge of aquifer water. Hybridization and microscopy counts of hybridized and 4',6'-diamidino-2-phenylindole (DAPI)- stained cells were performed as described in Snaidr et al., (1997, http://aem.asm.org/content/63/7/2884.full.pdf). Means were calculated from 10 to 20 randomly chosen fields on each filter section, corresponding to 800-1000 DAPI stained cells. Counting results were always corrected by subtracting signals observed with the probe NON338. Formamide concentrations and oligonucleotide probes used please see further details.

Microbial community in the sediment of the arctic Smeerenburgfjorden

Fluorescence in situ hybridization (FISH) with 16S rRNA-targeted oligonucleotide probes were used to investigate the phylogenetic composition of a marine Arctic sediment (Svalbard). Hybridization and microscopy counts of hybridized and 4',6'-diamidino-2-phenylindole (DAPI)-stained cells were performed as described previously from Snaidr et al. (1997, http://aem.asm.org/content/63/7/2884.full.pdf). Means were calculated from 10 to 20 randomly chosen fields on each filter section, corresponding to 800 to 1,000 DAPI-stained cells. Counting results were always corrected by subtracting signals observed with the probe NON338. Formamide concentrations are given in further details. FISH resulted in the detection of a large fraction of microbes living in the top 5 cm of the sediment. Up to 65.4% ± 7.5% of total DAPI cell counts hybridized to the bacterial probe EUB338, and up to 4.9% ± 1.5% hybridized to the archaeal probe ARCH915. Besides delta-proteobacterial sulfate-reducing bacteria (up to 16% 52) members of the Cytophaga-Flavobacterium cluster were the most abundant group detected in this sediment, accounting for up to 12.8% of total DAPI cell counts. Furthermore, members of the order Planctomycetales accounted for up to 3.9% of total cell counts. In accordance with previous studies, these findings support the hypothesis that these bacterial groups are not simply settling with organic matter from the pelagic zone but are indigenous to the anoxic zones of marine sediments. Members of the gamma-proteobacteria also constituted a significant fraction in this sediment (6.1% ± 2.5% of total cell counts). A new probe (GAM660) specific for sequences affiliated with free-living or endosymbiotic sulfur-oxidizing bacteria was developed. A significant number of cells was detected by this probe (2.1% ± 0.7% of total DAPI cell counts), showing no clear zonation along the vertical profile. Gram-positive bacteria and the beta-proteobacteria were near the detection limit in all sediments.

Community structure of sulfate reducing bacteria in the sediment of the arctic Smeerenburgfjorden

The community structure of sulfate-reducing bacteria (SRB) of a marine Arctic sediment (Smeerenburgfjorden, Svalbard) was characterized by both fluorescence in situ hybridization (FISH) by using group- and genus-specific 16S rRNA-targeted oligonucleotide probes. Samples stored in PBS-ethanol were diluted and treated by mild sonication. A 10-ml aliquot of a 1:40 dilution was filtered onto a 0.2-mm-pore-size type GTTP polycarbonate filter (Millipore, Eschborn, Germany). Hybridization and microscopic counting of hybridized and 49,69-diamidino-2-phenylindole (DAPI)-stained cells were performed as described previously from Snaidr et al. (1997, http://aem.asm.org/content/63/7/2884.full.pdf). Details of probes and formamide concentrations which were used are listed in futher details.. Means were calculated by using 10 to 20 randomly chosen fields for each filter section, which corresponded to 800 to 1,000 DAPI-stained cells. Counting results were always corrected by subtracting signals observed with probe NON338. The SRB community was dominated by members of the Desulfosarcina-Desulfococcus group. This group accounted for up to 73% of the SRB detected. The predominance was shown to be a common feature for different stations along the coast of Svalbard. In a top-to-bottom approach we aimed to further resolve the composition of this large group of SRB by using probes for cultivated genera. While this approach failed, directed cloning of probe-targeted genes encoding 16S rRNA was successful and resulted in sequences which were all affiliated with the Desulfosarcina-Desulfococcus group. A group of clone sequences (group SVAL1) most closely related to Desulfosarcina variabilis (91.2% sequence similarity) was dominant and was shown to be most abundant in situ, accounting for up to 54.8% of the total SRB detected.

Microbial community composition of sandy intertidal sediments of Sylt-Rømø Basin, Wadden Sea

Molecular biological methods were used to investigate the microbial diversity and community structure in intertidal sandy sediments near the island of Sylt (Wadden Sea) at a site which was characterized for transport and mineralization rates in de Beer et al., (2005, hdl:10013/epic.21375). The sampling was performed during low tide in the middle of the flat, approximately 40 m in the offshore direction from the high water line on October 6, 1999, March 7, 2000, and July 5, 2000. Two parallel cores were collected from each season for molecular analyses. Within 2 h after sampling the sediment cores were sub-sampled and fixed in formaldehyde for FISH analysis. The cells were hybridized, stained with 4',6'-diamidino-2-phenylindole (DAPI) and microscopically counted as described previously [55]. Details of probes and formamide concentrations which were used are shown in further details. Counts are reported as means calculated from 10-15 randomly chosen microscopic fields corresponding to 700-1000 DAPI-stained cells. Values were corrected for the signals counted with the probe NON338. Fluorescence in situ hybridization (FISH)with group-specific rRNA-targeted oligonucleotide probes were used to characterize the microbial community structure over depth (0-12 cm) and seasons (March, July, October). We found high abundances of bacteria with total cell numbers up to 3×109 cells ml-1 and a clear seasonal variation, with higher values in July and October versus March. The microbial community was dominated by members of the Planctomycetes, the Cytophaga/Flavobacterium group, Gammaproteobacteria, and bacteria of the Desulfosarcina/Desulfococcus group. The high abundance (1.5×10**7 - 1.8×10**8 cells/ml accounting for 3-19% of all cells) of presumably aerobic heterotrophic polymer-degrading planctomycetes is in line with the high permeability, deep oxygen penetration, and the high rates of aerobic mineralization of algal biomass measured in the sandy sediments by de Beer et al., (2005, hdl:10013/epic.21375). The high and stable abundance of members of the Desulfosarcina/Desulfococcus group, both over depth and season, suggests that these bacteria may play a more important role than previously assumed based on low sulfate reduction rates in parallel cores de Beer et al., (2005).

Haemulidae, modelo cariotípico de estase evolutiva

Conselho Nacional de Desenvolvimento Científico e Tecnológico

Avaliação do efeito citotóxico da lectina da esponja marinha Cliona varians contra células de leucemia mielóide crônica

In this study, a BCR-ABL expressing human chronic myelogenous leukaemia cell line (K562) was used to investigate the antitumoral potential of a novel lectin (CvL) purified from the marine sponge Cliona varians. CvL inhibited the growth of K562 cells with an IC50 value of 70 g/ml, but was ineffective to normal human peripheral blood lymphocytes in the same range of concentrations tested (180 g/ml). Cell death occurred after 72 h of exposure to the lectin and with sign of apoptosis as analysed by DAPI staining. Investigation of the possible effectors of this process showed that cell death occurred in the presence of Bcl-2 and Bax expression, and involved a caspase-independent pathway. Confocal fluorescence microscopy indicated a major role for the lysosomal protease cathepsin B in mediating cell death. Accordingly, pre-incubation of K562 cells with the cathepsin inhibitor L-trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64) abolished the cytotoxic effect of CvL. Furthermore, we found upregulation of tumor necrosis factor receptor 1 (TNFR1) and down-modulation of p65 subunit of nuclear factor kappa B (NFB) expression in CvL-treated cells. These effects were accompanied by increased levels of p21 and downmodulation of pRb, suggesting that CvL is capable of cell cycle arrest. Collectively, these findings suggest that cathepsin B acts as death mediator in CvL-induced cytotoxicity possibly in a still uncharacterized connection with the membrane death receptor pathway

Morphological characterization of two new species of Dicontophrya (Ciliophora: Astomatia: Contophryidae) commensal of earthworms (Oligochaeta: Annelida) of ebebda and nkolbikogo (Cameroon)

Article

Mapeamento cromossômico de genes ribossomais em espécies estuarinas da família Gerreidae Identificação de uniformidade cariotípica e sua empregabilidade com fins biotecnológicos - REPROGEN

Perciformes are dominant in the marine environment, characterized as the largest and most diverse fish group. Some families, as Gerreidae, popularly known as silver jennies, carapebas, or mojarras have a high economic potential to marine fish farming, natural explotation and game fishing. Genetic information of these species are of fundamental importance for their management and production. Despite exist over 13,000 marine fish species described, only 2% were cytogenetically analyzed and less than 1% have some reproductive characteristics known. Induced breeding, cytogenetic characterization and cryopreservation of gametes, represent important areas in applied fish studies. In this project cytogenetic analyzes were performed to acess genetic aspects of Gerreidae species, distributed in coastal and estuarine regions of Northeast Brazil. Different methods for identifying chromosomal regions were employed using conventional techniques (Ag-NORs, C-banding), staining with base-specific fluorochromes (DAPI-CMA3), and physical mapping of ribosomal genes 18S and 5S rDNA, through hybridization in situ with fluorescent probes (FISH). The six species analyzed showed remarkable chromosome conservatism. The 18S and 5S ribosomal genes when analyzed in phylogenetic perspective demonstrate varied evolutionary dynamics, suggesting ocurrence of stasis process in some groups and greater dynamism in others. Double FISH with 18S and 5S probes showed both how efficient cytotaxonomic markers in the homogeneous karyotypes of this group of species. The karyotypic pattern identified in addition to the evolutionary aspects of karyotype, are suggestive of existence of low potential of post-zygotic barrier, prompting further research to prospect for artificial interspecific hybridization of these species of commercial importance

Antitumor activity of physcion 8-o-β-glucopyranoside against cervical cancer by induction of apoptosis

Purpose: To investigate the antitumor activity of physcion8-O-β-glucopyranoside (PSG) against cervical cancer, as well as its mechanisms. Methods: The anti-proliferative effects of PSG on HeLa cells were determined by CCK-8 assay and the half maximal inhibitory concentration (IC50) values were calculated. Subsequently, a mouse xenograft model of HeLa cell line was established to investigate the antitumor effect of PSG in vivo. Furthermore, cell apoptosis was investigated by fluorescence microscopy via DAPI staining, and other mechanisms were determined by Western blot assay. Results: In vitro, PSG exhibited significant anti-proliferative effect on HeLa cells (p <0.05) in concentration-and time-dependent manners, with an IC50 value of 41.34 μg/mL. In vivo, PSG also had significant anti-tumor activity in nude mouse xenograft model (p < 0.05), inhibiting tumor growth. Furthermore, the results showed that treatment with PSG (20, 40 and 60 μg/mL) for 24 h resulted in significantly increased apoptosis in HeLa cells (p < 0.05). Additionally, Western blot analysis revealed that after exposure to 20, 40 and 60 μg/mL of PSG for 24 h, protein expressions of C-caspase-3, Ccaspase-9 and Bax were markedly up-regulated (p < 0.05) while Bcl-2 was significantly down-regulated (p < 0.05). These results confirmed that PSG inhibited HeLa cell growth by inducing mitochondriamediated apoptosis via up-regulation of caspase-3 and caspase-9 and Bax, and down-regulation of Bcl2. Conclusion: The results demonstrate that PSG possesses notable anti-tumor activity against cervical cancer and that the mechanisms involve induction of apoptosis by mitochondria-mediated signaling pathway.

Development of hydroxyapatite-based nanomaterials to enhance biological response of osteoblast cells for clinical application

Purpose: To develop a novel chitosan/gelatin-hydroxyapatite (CGHaP) microspheres for evaluating the biological response of pre-osteoblast cells. Methods: The microsphere was prepared by water-in-oil emulsion method. Cell proliferation was studied using AlamarBlue colorimetric assay and DAPI staining while alkaline phosphatase assay was carried out by colorimetric assay method. Chitosan microspheres as well as chitosan-hydroxyapatite microspheres was prepared and tested for biological response from MC3T3-E1 cell line. Results: The results showed that CGHaP promotes MC3T3-E1 cell proliferation and spread on the surface of microspheres. The cells were clustered with more actin filaments and well-linked with neighbouring cells or adjacent cells when cultured in CGHaP microspheres whereas fewer cells were spread on chitosan (CH) microspheres. CGHaP microspheres significantly (p < 0.05) promoted cell attachment, proliferation and extracellular matrix mineralization. CGHaP microspheres presented significantly (p < 0.02) higher calcium deposition (0.5 ng) than CH microspheres (0.28 ng). Specifically, CGHaP microspheres exhibited high ALP activity (8 units; 2-fold) compared to CH with 3 units, after 7 days of incubation. The results suggest that CGHaP possesses a great ability to facilitate bone ingrowth formation and possibility of good osteointegration in vivo. Conclusion: The nanomaterial enhances the proliferation of pre-osteoblast cells in tissue engineering microspheres. The outcome of this study may have a major impact on the development of novel nanomaterials for bone tissue engineering.

Haemulidae, modelo cariotípico de estase evolutiva

Conselho Nacional de Desenvolvimento Científico e Tecnológico

Avaliação do efeito citotóxico da lectina da esponja marinha Cliona varians contra células de leucemia mielóide crônica

In this study, a BCR-ABL expressing human chronic myelogenous leukaemia cell line (K562) was used to investigate the antitumoral potential of a novel lectin (CvL) purified from the marine sponge Cliona varians. CvL inhibited the growth of K562 cells with an IC50 value of 70 g/ml, but was ineffective to normal human peripheral blood lymphocytes in the same range of concentrations tested (180 g/ml). Cell death occurred after 72 h of exposure to the lectin and with sign of apoptosis as analysed by DAPI staining. Investigation of the possible effectors of this process showed that cell death occurred in the presence of Bcl-2 and Bax expression, and involved a caspase-independent pathway. Confocal fluorescence microscopy indicated a major role for the lysosomal protease cathepsin B in mediating cell death. Accordingly, pre-incubation of K562 cells with the cathepsin inhibitor L-trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64) abolished the cytotoxic effect of CvL. Furthermore, we found upregulation of tumor necrosis factor receptor 1 (TNFR1) and down-modulation of p65 subunit of nuclear factor kappa B (NFB) expression in CvL-treated cells. These effects were accompanied by increased levels of p21 and downmodulation of pRb, suggesting that CvL is capable of cell cycle arrest. Collectively, these findings suggest that cathepsin B acts as death mediator in CvL-induced cytotoxicity possibly in a still uncharacterized connection with the membrane death receptor pathway

Cytogenetic characterization of the Passiflora edulis Sims x Passiflora cincinnata Mast. interspecific hybrid and its parents.

The genus Passiflora L. consists of approximately 530 widely distributed species, including Passiflora edulis, which has drawn interest because of its commercial and agronomic value. Passiflora cincinnata is another important species owing to its long flowering period and resistance or tolerance to diseases and pests. In the present study, the meiotic segregation and pollen viability of an interspecific hybrid (P. edulis x P. cincinnata) and its parents were analyzed. The genomic contents were characterized using chromomycin A3 (CMA3)/40-60-diamidino-2-phenylindole (DAPI) staining, fluorescent in situ hybridization with 5S/45S ribosomal DNA (rDNA), genomic in situ hybridization (GISH), and inter-simple sequence repeat (ISSR) markers. The results indicated the diploid chromosome number for the parents and interspecific hybrid was 2n = 18. We also observed regular meiosis, one pair of S rDNA sites, and two pairs of 45S rDNA sites that colocalized with two pairs of CMA3 /DAPI- bands. The GISH data revealed three distinct chromosomal groups in the hybrid. The genetic origins of the interspecific hybrid, and its relationship with its parents were also confirmed using ISSR markers.