A study of the proteolytic enzymes of the sea anemone Metridium senile was undertaken in search of the proposed archetype 'serine protease' from which the family of homologous serine proteases of higher animals is believed to have arisen. Activity towards the chymotrypsin specific substrate N-acetyl-L-tyrosine ethyl ester (ATEE) was found to be initially very low in homogenates of gastric filaments of Metridium but during incubation at 0°C an autocatalytic increase in the activity was observed. This 'activation' was inhibited by a competitive inhibitor of trypsin, benzamidine and by di-isopropylphosphorofluoridate (DFP) but not by the chymotrypsin competitive inhibitor indole. These observations suggested a process somewhat similar to the activation of serine proteases of higher animals. General steps in the purification of the anemone proteases were as follows: (i) 2 M LiCl extraction of acetone powder of gastric filaments (ii) acetone fractionation of the 2 M LiCl extract at -15°C, (iii) DEAE-Sephadex chromatography of the 40-80% acetone fraction, (iv) cation exchange chromatography of the DEAE-breakthrough peak on Bio-Rex 70 at pH 6.0 in the presence of .005 M indole and (v) re-chromatography on Bio-Rex 70 at pH 6.0 in the presence of indole. ATEE-ase activity was resolved into 3 components, designated A, BI and B2 in step (iv). Peaks A and BI migrated as single major Amido Black staining bands at pH 4.0 on polyacrylamide disc electrophoresis but there were several minor components present in both cases. Protease B2 appeared homogeneous on electrophoresis. 8 M urea-starch gel electrophoresis 32 of Dl³² P-labelled proteases followed by autoradiography; and Amido Black staining showed that the major Amido Black staining band was also the major radioactive band in each case. Gel filtration of protease A on G-100 Sephadex gave an approximate molecular weight of 40-50, 000. Sedimentation velocity of the enzymes gave values of S₂₀ of 2.0, 2.5. and 2.2 for DIP-proteases A, BI and B2, respectively, indicative of a lower molecular weight. Sedimentation equilibrium studies with protease A indicated considerable polydispersity but gave a weight-average molecular weight (Mw) of around 28, 000. The behavior on Sephadex G-100 and evidence of polydispersity upon sedimentation equilibrium may be a result of dimerization. Features of the amino acid composition showing similarity to α-chymotrypsin were a high half-cystine content and a similar number of aromatic and basic amino acids. Major differences from mammalian α-chymotrypsin included a higher proline and histidine content and a lower content of hydroxylic amino acids. Enzymic properties of the anemone enzymes were very similar to those of mammalian α-chymotrypsin. Protease A showed 3-5 fold higher k[subscript: cat] values in the hydrolysis of several chymotrypsin ester substrates but a markedly lower k[subscript: cat] in the hydrolysis of N-acetyl-L- tryptophan ethyl ester. The anemone enzyme was shown to be rapidly inhibited by DFP, phenyl methane sulphonyl fluoride (PMSF) and N-tosyl-L-phenylalanyl chloromethylketone (TPCK) but was not inhibited by the trypsin specific inhibitor N-[symbol omitted]tosyl-L-lysine chloromethylketone (TLCK). Competitive inhibition of the hydrolysis of ATEE was observed with indole and 3-phenyl propionate. Protease A hydrolysed the polypeptide chain of glucagon on the carboxyl side of tyrosine, phenylalanine and leucine, provided the latter was not adjacent to tyrosine or phenylalanine. A lower but significant cleavage at both sides of the two arginine residues was also observed. The latter cleavages were not affected by the chymotrypsin inhibitor TPCK but were eliminated by prior reaction of the enzyme with soybean trypsin inhibitor. Thus it was concluded that they represent the activity of a contaminating trypsin-like enzyme in the preparation of protease A. In studies on the short active centre sequence of proteased A, BI and B2 using the technique of co-electrophoresis of partial acid hydrolysates of Dl³² P-labelled enzymes and Dl³² P-chymotrypsin followed by high voltage electrophoresis and autoradiography, the active centre sequence of the three anemone proteases was found to be -Asp. Ser. Gly-, the sequence common to all of the mammalian serine proteases. In conclusion, the anemone enzymes show remarkable structural and enzymic similarity with mammalian chymotrypsin and they probably represent present day relics of early forms of chymotrypsin, homologous with the familiar enzyme of higher animals. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

No description available.

Thesis (Ph. D.)--Oregon State University, 2007. / Printout. Includes bibliographical references. Also available on the World Wide Web.

No description available.

Thesis (M.S.)--Western Washington University, 2010. / Includes bibliographical references (leaves 70-73). Also issued online.

xv, 151 p. : ill. A print copy of this thesis is available through the UO Libraries. Search the library catalog for the location and call number. / The sea anemone Metridium senile occurs along a salinity gradient in the South Slough Estuary, Oregon, where it is subjected to frequent and sometimes large fluctuations in salinity. This study determined how hypo-osmotic stress contributes to the survival and distribution of this population. In the laboratory, chronic exposure ofM. senile to 50% and 75% seawater for twenty-eight days resulted in partial regulation of volume and magnesium ions. Anemones transplanted to the field exhibited increased mortality and partial regulation of volume, osmolality, and magnesium ions with decreased salinity during the wet season (December-February) and less regulation during the dry season (June-August). This pattern of physiological tolerance coincides with observed trends of seasonal abundance and distribution. Previous studies describe M senile as a marine osmoconformer, however this estuarine population is able to withstand moderate hypo-osmotic stress through partial regulation of tissue osmolality and magnesium ions. / Committee in Charge: Dr. Craig M. Young, Chair; Dr. Nora B. Terwilliger; Dr. Steven S. Rumrill; Dr. Caren E. Braby

Resumo: O presente estudo tem como objetivos (1) realizar avaliação metodológica para determinação da medida mais adequada para estimar o tamanho das anêmonas, (2) obter dados sobre a variação temporal na riqueza, abundância e zonação das espécies de actiniários no entremarés rochoso e (3) verificar e quantificar o deslocamento de Bunodosma caissarum. Para o objetivo (1) foram tomadas variadas medidas in situ e ex situ de B. caissarum. O parâmetro morfométrico que apresentou menor variação entre campo e laboratório foi o diâmetro do disco pedal considerado, portanto, o mais adequado para estimativas de tamanho. O objetivo (2) foi estudado sazonalmente, na Praia da Enseada, Ubatuba (SP), por meio da demarcação de estratos com diferentes distâncias do limite inferior do entremarés. As espécies presentes foram Actinia bermudensis, Bunodosoma caissarum, Anthopleura cascaia, B. cangicum, Phyllactis flosculifera e um táxon não identificado. A distribuição das três primeiras espécies, mais abundantes, foi inversamente proporcional ao aumento da distância da água. As demais espécies não foram registradas em todas as estações e, com exceção do táxon não identificado, parecem ser mais relacionadas às regiões mais distantes da água. O objetivo (3) foi realizado em campo, na Ilha Urubuqueçaba, Santos (SP), e em laboratório, ambos por meio de mapeamento fotográfico. Em campo, as anêmonas em fenda apresentaram maior velocidade do que as anêmonas expostas. Em adição, demonstrou-se que sob ventos mais fortes e maiores ondas, as anêmonas deslocaram-se menos, provavelmente pela necessidade de maior adesão ao substrato. Em laboratório verificou-se a influência de organismos em contato físico, salinidade e profundidade sobre o deslocamento... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The objectives of this study are to (1) perform a methodological evaluation in order to determine the most suitable measure to estime anemones' size, (2) obtain data regarding to seasonal variation of richness, abundance and zonation of Anthozoa species, and (3) verify and quantify the locomotion of Bunodosoma caissarum. In objective (1) several morphometric parameters of B. caissarum were measured in situ and ex situ. Pedal disk diameter showed the smallest variation among field and laboratory, this way being considered the most suitable to size estimates. The objective (2) was seasonally realized at Enseada Beach, Ubatuba (SP), through the demarcation of horizontal transects with distinct distances from the lower limit of intertidal zone. Six species were found: Actinia bermudensis, Bunodosoma caissarum, Anthopleura cascaia, B. cangicum, Phyllactis flosculifera and a nonidentified taxa. The distribution of the first three species was inversely proportional to the increasing of distance from water. The other species were not recorded in all seasons and, except for the non-identified taxa, seem to be more adapted to areas farther from the water. The objective (3) was performed in field at Urubuqueçaba Island, Santos (SP) and in laboratory both through photographic mapping. In field anemones located in fences moved faster than those in exposed sites, probably because they were in search of more favorable locations. Such conclusion matches the literature which indicates that sea anemones locomotion is associated with unfavorable conditions. In addition, it was demonstrated that anemones have lower mobility under stronger winds and larger waves probably due to the need of adhesion to substrate. It was verified in laboratory the influence of organisms in contact with anemones... (Complete abstract click electronic access below) / Orientador: Fernando José Zara / Coorientador: Alexander Turra / Banca: Ronaldo Adriano Christofoletti / Banca: Alvaro Esteves Migotto / Mestre

>Magister Scientiae - MSc / Sea anemones (Order Actinaria) are a diverse order from the Class Anthozoa. They are found in all marine habitats at all depths and their symbiotic relationships play an important role in energy transfers especially in the benthic-pelagic community. The evolutionary background and phylogenetics of the class is poorly understood due to a lack of correspondence between taxonomic and molecular data (Daly et al. 2008). Therefore, a deeper exploration into Cnidarian molecular biology is needed to establish these as an evolutionary model organism. Gene discovery from various marine invertebrates has facilitated the recovery of anti-cancer drugs, antibiotics and reporter genes (Faulkner, 2000; Allen and Jaspars, 2009). The most commercially lucrative products from sea anemones are fluorescent and chromoproteins (FP/CP), which are used as non-invasive real-time reporter genes. The applications for these proteins are extensive and range from monitoring cellular processes such as protein localisation and interactions to imaging (Alieva et al. 2008). Therefore, novel FP and CPs have potential for commercialization. The aims of the project were to analyze basic molecular diversity of the sea anemones Pseudactinia varia, Pseudactina flagellifera and Bunodosoma capensis and evaluate a new screening method to isolate novel FP and CPs. To assess the basic molecular diversity, of the sea anemones and their associated symbionts 16S rRNA and 18S rRNA clone libraries were generated. The sea anemones used in this study clustered together with those of the Family Actiniidae. The bacterial associations observed based on the closest relative BLAST analysis were dominated by Proteobacteria (gamma, alpha and epsilon) as well as Bacteroides. The associate bacterial symbionts possibly produce compounds that range from polyunsaturated fatty acids, polyhydroxyalkanoates to anti-microbial compounds that aid the host in various processes. In order to screen for FPs and CPs from sea anemones three types of cDNA libraries were generated to be screened either by sequence based or activity based approaches. Novel primers were designed which could be applied for the screening of a variety of Anthozoans. A positive control was also designed and synthesised in order to test the capability of the designed primers and optimise the amplification. Although amplicons were generated from gDNA and cDNA libraries from each of the sea anemones they were found to be non-specific products. The detection limit is likely to be the limiting factor. The construction of an activity based library was not achieved due to technical constraints, which highlights the need for new molecular tools in this field or improvements to the existing ones. / National Research Foundation (NRF)

The sea anemone Aiptasia pallida is a biological model for anthozoan research. Like all cnidarians, A. pallida possesses nematocysts for food capture and defense. Studies have shown that anthozoans, such as corals, can rapidly increase nematocyst concentration when faced with competition or predation, suggesting that nematocyst production may be an induced trait. The potential effects of two types of tissue damage, predator induced (Lysmata wurdemanni) and artificial (forceps), on nematocyst concentration was assessed. Nematocysts were identified by type and size to examine the potential plasticity associated with nematocyst production. While no significant differences were found in defensive nematocyst concentration between shrimp predation treatments versus controls, there was a significant difference in small-sized nematocyst in anemones damaged with forceps. The proportions of the different types of nematocysts between treatment types were also found to be different suggesting that nematocyst production in A. pallida is a plastic trait. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2014. / FAU Electronic Theses and Dissertations Collection

Thesis (M.Sc.)--Victoria University of Wellington, 2008. / Includes bibliographical references.