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

Indução de morte celular em células de adenocarcinoma cervical humano (HeLa) pela lectina da esponja Cinachyrella apion (CaL)

Cancer is a term used to represent a set of more than 100 diseases, including malignant tumors from different locations. The malignancies are the second leading cause of death in the population, representing approximately 17% of deaths of known cause. Strategies that induce differentiation have had limited success in the treatment of established cancers. In this work, a lectin purified from the marine sponge Cinachyrella apion (CaL) was evaluated due to its hemolytic, cytotoxic and antiproliferative properties, besides the ability to induce cell death via apoptosis in tumor cells. The antiproliferative activity of CaL was tested against cell lines, with the highest inhibition of tumor growth for HeLa, reducing cell growth at a dose dependent manner, with a concentration of 10 μg/mL. The hemolytic activity and toxicity against peripheral blood cells were tested using the concentration of IC50 for both trials and twice the IC50 for analysis in flow cytometry, indicating that CaL is not toxic to these cells. To assess the mechanism of cell death caused by CaL in HeLa cells, we performed flow cytometry and western blotting. The results showed the lectin probably induces cell death by apoptosis activation by pro-apoptotic protein Bax, promoting mitochondrial membrane permeabilization, cell cycle arrest in S phase, with accumulation of cells of approximately 57% in this phase, and acting as both dependent and/or independent of caspases pathway. These results suggest that CaL has the potential to be used as drug treatment against cancer.

Lectina da esponja marinha Tedania ignis: purificação, caracterização e interação com leishmanias

Lectin obtained from the marine sponge Tedania ignis was purified and characterized by extraction of soluble proteins (crude extract) in 50mM Borax, pH 7.5. The purification procedure was carried out by crude extract precipitation with ammonium sulfate 30% (FI). The precipitated was resuspended in the same buffer and fractionated with acetone 1.0 volume (F1.0). A lectin was purified from this specific fraction by using an affinity chromatography Sepharose 6B. This lectin preferentially agglutinated human erythrocytes from B type previously treated with papain enzyme. The hemagglutinating activity lectin was dependent of divalent Mn2+ cation and was inhibited by the carbohydrates galactose, xylose and fructose. SDS-PAGE analysis indicated a molecular mass of the lectin around 45 kDa. This protein showed stability until 40°C for 1 h. Further, it showed activity between pH 2.5 and 11.5, with an enhanced activity at pH 7.5. Leishmania chagasi promastigotes stained with Coomassie brilliant blue R-250 were agglutinated by F1,0 and in the presence of galactose this interaction was abolished. These results show that this lectin could be implicated in defense procedures and it will can be used as biological tools in studies with this protozoon

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

Archaea appear to dominate the microbiome of Inflatella pellicula deep sea sponges

Microbes associated with marine sponges play significant roles in host physiology. Remarkable levels of microbial diversity have been observed in sponges worldwide through both culture-dependent and culture-independent studies. Most studies have focused on the structure of the bacterial communities in sponges and have involved sponges sampled from shallow waters. Here, we used pyrosequencing of 16S rRNA genes to compare the bacterial and archaeal communities associated with two individuals of the marine sponge Inflatella pellicula from the deep-sea, sampled from a depth of 2,900 m, a depth which far exceeds any previous sequence-based report of sponge-associated microbial communities. Sponge-microbial communities were also compared to the microbial community in the surrounding seawater. Sponge-associated microbial communities were dominated by archaeal sequencing reads with a single archaeal OTU, comprising similar to ∼60% and similar to ∼72% of sequences, being observed from Inflatella pellicula. Archaeal sequencing reads were less abundant in seawater (similar to ∼11% of sequences). Sponge-associated microbial communities were less diverse and less even than any other sponge-microbial community investigated to date with just 210 and 273 OTUs (97% sequence identity) identified in sponges, with 4 and 6 dominant OTUs comprising similar to ∼88% and similar to ∼89% of sequences, respectively. Members of the candidate phyla, SAR406, NC10 and ZB3 are reported here from sponges for the first time, increasing the number of bacterial phyla or candidate divisions associated with sponges to 43. A minor cohort from both sponge samples (similar to ∼0.2% and similar to ∼0.3% of sequences) were not classified to phylum level. A single OTU, common to both sponge individuals, dominates these unclassified reads and shares sequence homology with a sponge associated clone which itself has no known close relative and may represent a novel taxon.

Mooloolabenes A-E, norsesterterpenes from the Australian sponge Hyattella intestinalis

Five new norsesterterpenes, mooloolabenes A-E (1-5), and the new sesterterpene mooloolaldehyde (6), related to the scalarane family of compounds, were isolated from an acetone extract of the Australian sponge Hyattella intestinalis. Structural elucidation, including relative stereochemical assignment, was based on spectroscopic analysis. All compounds tested showed cytotoxic activity against the P388 cell line.

Climate variability over the last 35,000 years recorded in marine and terrestrial archives in the Australian region : an OZ-INTIMATE compilation

The Australian region spans some 60° of latitude and 50° of longitude and displays considerable regional climate variability both today and during the Late Quaternary. A synthesis of marine and terrestrial climate records, combining findings from the Southern Ocean, temperate, tropical and arid zones, identifies a complex response of climate proxies to a background of changing boundary conditions over the last 35,000 years. Climate drivers include the seasonal timing of insolation, greenhouse gas content of the atmosphere, sea level rise and ocean and atmospheric circulation changes. Our compilation finds few climatic events that could be used to construct a climate event stratigraphy for the entire region, limiting the usefulness of this approach. Instead we have taken a spatial approach, looking to discern the patterns of change across the continent. The data identify the clearest and most synchronous climatic response at the time of the Last Glacial Maximum (LGM) (21 ± 3 ka), with unambiguous cooling recorded in the ocean, and evidence of glaciation in the highlands of tropical New Guinea, southeast Australia and Tasmania. Many terrestrial records suggest drier conditions, but with the timing of inferred snowmelt, and changes to the rainfall/runoff relationships, driving higher river discharge at the LGM. In contrast, the deglaciation is a time of considerable south-east to north-west variation across the region. Warming was underway in all regions by 17 ka. Post-glacial sea level rise and its associated regional impacts have played an important role in determining the magnitude and timing of climate response in the north-west of the continent in contrast to the southern latitudes. No evidence for cooling during the Younger Dryas chronozone is evident in the region, but the Antarctic cold reversal clearly occurs south of Australia. The Holocene period is a time of considerable climate variability associated with an intense monsoon in the tropics early in the Holocene, giving way to a weakened monsoon and an increasingly El Niño-dominated ENSO to the present. The influence of ENSO is evident throughout the southeast of Australia, but not the southwest. This climate history provides a template from which to assess the regionality of climate events across Australia and make comparisons beyond our region. The data identify the clearest and most synchronous climatic response at the time of the Last Glacial Maximum (LGM) (21 ± 3 ka), with unambiguous cooling recorded in the ocean, and evidence of glaciation in the highlands of tropical New Guinea, southeast Australia and Tasmania. Many terrestrial records suggest drier conditions, but with the timing of inferred snowmelt, and changes to the rainfall/runoff relationships, driving higher river discharge at the LGM. In contrast, the deglaciation is a time of considerable south-east to north-west variation across the region. Warming was underway in all regions by 17 ka. Post-glacial sea level rise and its associated regional impacts have played an important role in determining the magnitude and timing of climate response in the north-west of the continent in contrast to the southern latitudes. No evidence for cooling during the Younger Dryas chronozone is evident in the region, but the Antarctic cold reversal clearly occurs south of Australia. The Holocene period is a time of considerable climate variability associated with an intense monsoon in the tropics early in the Holocene, giving way to a weakened monsoon and an increasingly El Niño-dominated ENSO to the present. The influence of ENSO is evident throughout the southeast of Australia, but not the southwest. This climate history provides a template from which to assess the regionality of climate events across Australia and make comparisons beyond our region.

DAQ00104 Sponge and dough bread quality of Australian wheat germplasm

The main grade of wheat targeted for the export sponge and dough (S&D) market is Australian prime hard (APH). By association, protein should be a key parameter relating to S&D quality, specifically loaf volume (LV). Surprisingly, the project revealed a low level of correlation between total protein content and LV. It appears that protein composition may be the key to understanding S&D quality, as the glutenin Glu D1 5+10 subunit contributed to the highest LVs. The current varieties KennedyA and SunzellA, together with several breeding lines, provided a consistently high quality over a number of seasons. These varieties performed as well as, if not better than, North American S&D varieties.

Is economic valuation of ecosystem services useful to decision-makers? Lessons learned from Australian coastal and marine management

Economic valuation of ecosystem services is widely advocated as a useful decision-support tool for ecosystem management. However, the extent to which economic valuation of ecosystem services is actually used or considered useful in decision-making is poorly documented. This literature blindspot is explored with an application to coastal and marine ecosystems management in Australia. Based on a nation-wide survey of eighty-eight decision-makers representing a diversity of management organizations, the perceived usefulness and level of use of ecosystem services economic valuation in support of coastal and marine management are examined. A large majority of decision-makers are found to be familiar with economic valuation and consider it useful - even necessary - in decision-making, although this varies across decision-makers groups. However, most decision-makers never or rarely use it. The perceived level of importance and trust in estimated dollar values differ across ecosystem services, and are especially high for values that relate to commercial activities. A number of factors are also found to influence respondent’s use of economic valuation. Such findings concur with conclusions from other existing works, and are instructive to reflect on the issue of the usefulness of ESV in environmental management decision-making. They also confirm that the survey-based approach developed in this application represents a sound strategy to examine this issue at various scales and management levels.

Cruise Report NOAA Ship McARTHUR II Cruise AR-04-04: Leg 2 (June 1-12, 2004): A pilot survey of deepwater coral/sponge assemblages and their susceptibility to fishing/harvest impacts at the Olympic Coast National Marine Sanctuary (OCNMS)

Summary: The offshore shelf and canyon habitats of the OCNMS (Fig. 1) are areas of high primary productivity and biodiversity that support extensive groundfish fisheries. Recent acoustic surveys conducted in these waters have indicated the presence of hard-bottom substrates believed to harbor unique deep-sea coral and sponge assemblages. Such fauna are often associated with shallow tropical waters, however an increasing number of studies around the world have recorded them in deeper, cold-water habitats in both northern and southern latitudes. These habitats are of tremendous value as sites of recruitment for commercially important fishes. Yet, ironically, studies have shown how the gear used in offshore demersal fishing, as well as other commercial operations on the seafloor, can cause severe physical disturbances to resident benthic fauna. Due to their exposed structure, slow growth and recruitment rates, and long life spans, deep-sea corals and sponges may be especially vulnerable to such disturbances, requiring very long periods to recover. Potential effects of fishing and other commercial operations in such critical habitats, and the need to define appropriate strategies for the protection of these resources, have been identified as a high-priority management issue for the sanctuary. To begin addressing this issue, an initial pilot survey was conducted June 1-12, 2004 at six sites in offshore waters of the OCNMS (Fig. 2, average depths of 147-265 m) to explore for the presence of deep-sea coral/sponge assemblages and to look for evidence of potential anthropogenic impacts in these critical habitats. The survey was conducted on the NOAA Ship McARTHUR-II using the Navy’s Phantom DHD2+2 remotely operated vehicle (ROV), which was equipped with a video camera, lasers, and a manipulator arm for the collection of voucher specimens. At each site, a 0.1-m2 grab sampler also was used to collect samples of sediments for the analysis of macroinfauna (> 1.0 mm), total organic carbon (TOC), grain size, and chemical contaminants. Vertical profiles of salinity, dissolved oxygen (DO), temperature, and pressure were recorded at each site with a small SeaCat conductivity-temperature-depth (CTD) profiler. Niskin bottles attached to the CTD also obtained near-bottom water samples in support of a companion study of microbial indicators of coral health and general ecological condition across these sites. All samples except the sediment-contaminant samples are being analyzed with present project funds. Original cruise plans included a total of 12 candidate stations to investigate (Fig. 3). However, inclement weather and equipment failures restricted the sampling to half of these sites. In spite of the limited sampling, the work completed was sufficient to address key project objectives and included several significant scientific observations. Foremost, the cruise was successful in demonstrating the presence of target deepwater coral species in these waters. Patches of the rare stony coral Lophelia pertusa, more characteristic of deepwater coral/sponge assemblages in the North Atlantic, were observed for the first time in OCNMS at a site in 271 meters of water. A large proportion of these corals consisted of dead and broken skeletal remains, and a broken gorgonian (soft coral) also was observed nearby. The source of these disturbances is not known. However, observations from several sites included evidence of bottom trawl marks in the sediment and derelict fishing gear (long lines). Preliminary results also support the view that these areas are important reservoirs of marine biodiversity and of value as habitat for demersal fishes. For example, onboard examination of 18 bottom-sediment grabs revealed benthic infaunal species representative of 14 different invertebrate phyla. Twenty-eight species of fishes from 11 families, including 11 (possibly 12) species of ommercially important rockfishes, also were identified from ROV video footage. These initial discoveries have sparked considerable interests in follow-up studies to learn more about the spatial extent of these assemblages and magnitude of potential impacts from commercial-fishing and other anthropogenic activities in the area. It is essential to expand our knowledge of these deep-sea communities and their vulnerability to potential environmental risks in order to determine the most appropriate management strategies. The survey was conducted under a partnership between NOAA’s National Centers for Coastal Ocean Science (NCCOS) and National Marine Sanctuary Program (NMSP) and included scientists from NCCOS, OCNMS, and several other west-coast State, academic, private, and tribal research institutions (see Section 4 for a complete listing of participating scientists). (PDF contains 20 pages)

Observations of deep coral and sponge assemblages in Olympic Coast National Marine Sanctuary, Washington. Cruise Report: NOAA Ship McArthur II Cruise AR06-07/07

From May 22 to June 4, 2006, NOAA scientists led a research cruise using the ROPOS Remotely Operated Vehicle (ROV) to conduct a series of dives at targeted sites in the Olympic Coast National Marine Sanctuary (OCNMS) with the goal of documenting deep coral and sponge communities. Dive sites were selected from areas for which OCNMS had side scan sonar data indicating the presence of hard or complex substrate. The team completed 11 dives in sanctuary waters ranging from six to 52 hours in length, at depths ranging from 100 to 650 meters. Transect surveys were completed at 15 pre-selected sites, with additional observations made at five other sites. The survey locations included sites both inside and outside the Essential Fish Habitat (EFH) Conservation Area, known as Olympic 2, established by the Pacific Fishery Management Council, enacted on June 12, 2006. Bottom trawling is prohibited in the Olympic 2 Conservation Area for nontribal fishermen. The Conservation Area covers 159.4 square nautical miles or about 15 percent of the sanctuary. Several species of corals and sponges were documented at 14 of the 15 sites surveyed, at sites both inside and outside the Conservation Area, including numerous gorgonians and the stony corals Lophelia pertusa and Desmophyllum dianthus, as well as small patches of the reef building sponge Farrea occa. The team also documented Lophelia sp. and Desmophyllum sp. coral rubble, dead gorgonians, lost fishing gear, and other anthropogenic debris, supporting concerns over potential risks of environmental disturbances to coral health. (PDF contains 60 pages.)

Survey report of NOAA Ship McArthur II cruises AR-04-04, AR-05-05 and AR-06-03: Habitat classification of side scan sonar imagery in support of deep-sea coral/sponge explorations at the Olympic Coast National Marine Sanctuary

Habitat mapping and characterization has been defined as a high-priority management issue for the Olympic Coast National Marine Sanctuary (OCNMS), especially for poorly known deep-sea habitats that may be sensitive to anthropogenic disturbance. As a result, a team of scientists from OCNMS, National Centers for Coastal Ocean Science (NCCOS), and other partnering institutions initiated a series of surveys to assess the distribution of deep-sea coral/sponge assemblages within the sanctuary and to look for evidence of potential anthropogenic impacts in these critical habitats. Initial results indicated that remotely delineating areas of hard bottom substrate through acoustic sensing could be a useful tool to increase the efficiency and success of subsequent ROV-based surveys of the associated deep-sea fauna. Accordingly, side scan sonar surveys were conducted in May 2004, June 2005, and April 2006 aboard the NOAA Ship McArthur II to: (1) obtain additional imagery of the seafloor for broader habitat-mapping coverage of sanctuary waters, and (2) help delineate suitable deep-sea coral/sponge habitat, in areas of both high and low commercial-fishing activities, to serve as sites for surveying-in more detail using an ROV on subsequent cruises. Several regions of the sea floor throughout the OCNMS were surveyed and mosaicked at 1-meter pixel resolution. Imagery from the side scan sonar mapping efforts was integrated with other complementary data from a towed camera sled, ROVs, sedimentary samples, and bathymetry records to describe geological and biological (where possible) aspects of habitat. Using a hierarchical deep-water marine benthic classification scheme (Greene et al. 1999), we created a preliminary map of various habitat polygon features for use in a geographical information system (GIS). This report provides a description of the mapping and groundtruthing efforts as well as results of the image classification procedure for each of the areas surveyed. (PDF contains 60 pages.)

A pilot survey of deepwater coral/sponge assemblages and their susceptibility to fishing/harvest impacts at the Olympic Coast National Marine Sanctuary (OCNMS). Cruise report for NOAA Ship McARTHUR II Cruise AR-04-04: Leg 2 (June 1-12, 2004)

The offshore shelf and canyon habitats of the OCNMS are areas of high primary productivity and biodiversity that support extensive groundfish fisheries. Recent acoustic surveys conducted in these waters have indicated the presence of hard-bottom substrates believed to harbor unique deep-sea coral and sponge assemblages. Such fauna are often associated with shallow tropical waters, however an increasing number of studies around the world have recorded them in deeper, cold-water habitats in both northern and southern latitudes. These habitats are of tremendous value as sites of recruitment for commercially important fishes. Yet, ironically, studies have shown how the gear used in offshore demersal fishing, as well as other commercial operations on the seafloor, can cause severe physical disturbances to resident benthic fauna. Due to their exposed structure, slow growth and recruitment rates, and long life spans, deep-sea corals and sponges may be especially vulnerable to such disturbances, requiring very long periods to recover. Potential effects of fishing and other commercial operations in such critical habitats, and the need to define appropriate strategies for the protection of these resources, have been identified as a high-priority management issue for the sanctuary.

Antimicrobial screening and active compound isolation from marine bacterium NJ6-3-1 associated with the sponge Hymeniacidon perleve

Twenty-nine marine bacterial strains were isolated from the sponge Hymeniacidon perleve at Nanji island, and antimicrobial screening showed that eight strains inhibited the growth of terrestrial microorganisms. The strain NJ6-3-1 with wide antimicrobial spectrum was identified as Pseudoalteromonas piscicida based on its 16S rRNA sequence analysis. The major antimicrobial metabolite, isolated through bioassay-guide fractionation of TLC bioautography overlay assay, was identified as norharman (a beta-carboline alkaloid) by EI-MS and NMR.