Thesis (M.Sc.)(Plant Biotechnology))--University of Pretoria, 2002. / Summaries in Afrikaans and English. Includes bibliographical references.

<p> Carrageenan, a mixture of sulphated galactans extractable by hot water from certain marine algae, has been known for its activity in a reversible stimulation of connective tissue growth. One subcutaneous injection of this polysaccharide elicits a granuloma formation which regresses after a few weeks. A single injection after regression results in heightened response; this suggested an immunological reaction. Serial injections of either lambda or kappa carrageenan into rabbits resulted in the formation of precipitins. The present study gives both qualitative and quantitative studies of the reaction involved. Fractions of carrageenans from different species of marine algae were tested for their ability to precipitate either anti-lambda carrageenan antiserum or anti-kappa carrageenan antiserum. Deductions were made from the data as to the structure of the polysaccharides.</p> / Thesis / Master of Science (MSc)

Polygalacturonase-inhibiting proteins (PGIPs) are plant proteins believed to playa role in the defence against pathogenic fungi. In this study. it was hypothesized that apple PGIPI could be used to confer enhanced resistance against Verticillium-wilt. a major disease of potato caused by the fungus Verticillillm dahliae. Transgenic lines containing the apple pgip1 gene under control of the enhanced CaMV 35S (e35S) promoter had been generated previously. Stable integration of the transgene into the potato genome was shown by the polymerase chain reaction (PCR) and Southern blot with a DIG¬labelled apple pgip1 fragment as probe. Polygalacturonase (PG)-inhibiting assays (the agarose diffusion assay and reducing sugar assays) were employed to investigate the inhibiting activity of apple PGIP I extracts, prepared from the transgenic potato lines. on the PGs secreted by V. dahliae grown on pectin medium. Inhibition was successful for all but one of the transgenic lines. Active PGIPI was expressed in the leaves of in vitro- and glasshouse grown plants, as well as in roots of in vitro-grown plants. Due to the success of the in vitro inhibition results. it was anticipated that the apple pgip1 transgene would protect the transgenic lines against Verticillium-wilt in a subsequent glasshouse trial. The transgenic lines and untransformed BP I potato control were planted in soil inoculated with V. dahliae microsclerotia and control soil. Assessments of the visual symptoms of yellowing and wilt were made on a scale of 1-5. Colonisation of stem sections was determined by plating onto potato dextrose agar plates. Disease index values were calculated from the symptom and colonisation data. Analysis of variance indicated six lines to be significantly different from the rest when grown in the inoculated soil, but five of them also showed significantly slower senescence symptoms when grown in the control soil. It is proposed that the physiological effect of an extended juvenile phase resulted in the apparent increased disease resistance. This could be caused by transformation or tissue culture¬-induced somaclonal variation of the potato plants. The hypothesis that transformation of the apple pgip1 gene into potato would confer enhanced resistance against Verticillium-wilt was not supported by the data that was obtained. Expression of antifungal genes by pathogen-inducible promoters is a valuable strategy in the development of disease resistant crops of importance. A construct containing the apple pgipl gene under control of the pathogen-inducible gst1 promoter from Arabidopsis thaliana (L.) Heynh was generated. Agrobacterium tumefaciens GV31OI(pMP90RK) was transfonned with the plant transformation vector pCAMBIA2300 containing the gst1 and e35S promoter-pgip1 inserts. A. thaliana was transformed using the floral-dip method, and putative transgenic progeny were selected by kanamycin selection of the seeds. PCR verified the insertion of the transgene into the genomes of T2 and T3 lines. Gene expression from the two promoters was compared by performing PGIP extractions and the agarose diffusion assay. The gst1 promoter was active even without induction by methyl-salicylate. Both constructs led to the expression of active apple PGIP1 against V. dahliae PG in the heterologous plant A. thaliana. / Dissertation (MSc (Plant Biotechnology))--University of Pretoria, 2006. / Plant Science / unrestricted

Made available in DSpace on 2014-12-17T14:03:26Z (GMT). No. of bitstreams: 1 EduardoHCF.pdf: 473372 bytes, checksum: 2fca55fdea3773bfb6942b7901966772 (MD5) Previous issue date: 2006-07-07 / Since the first description of sulfated polysaccharides from seaweeds, the biological activities of these compounds have been evaluated under different aspects and experimental procedures. Among the broad biological activities presented by seaweed polysaccharides, anticoagulant action appears as a promising function. In this present study we have obtained sulfated polysaccharides from the green seaweed Codium isthmocladium by proteolytic digestion, followed by separation into five fractions (0.3, 0.5, 0.7, 0.9 and 1.2) by sequential acetone precipitation. The chemical analyses have demonstrated that all fractions are composed mainly by sulfated polysaccharides. The anticoagulant activity of these fractions was determined by activated partial thromboplastin time (aPTT) and prothrombin time test (PT) using citrate normal human plasma. None fraction has shown anticoagulant activity by PT test. Furthermore, all of them have shown anticoagulant activity by aPTT test. These results indicated that the molecular targets of these sulfated polysaccharides are mainly in the intrinsic via of the coagulation cascade. Agarose gel electrophoresis in 1,3-diaminopropane acetate buffer, pH 9.0, stained with 0.1% toluidine blue showed the presence of two or three bands in several fractions while the fraction 0.9 showed a single spot. By anion exchange chromatography, the acid polysaccharides from the 0.9 acetone fraction were separated into two new fractions eluted respectively with 2.0 and 3.0 M NaCl. These compounds showed a molecular weight of 6.4 and 7.4 kDa respectively. Chemical analyses and infrared spectroscopy showed that Gal 1 and Gal 2 are sulfated homogalactans and differ one from the other in degree and localization of sulfate groups. aPPT test demonstrated that fractions 2,0 and 3,0M (Gal1 and Gal 2, respectively) have anticoagulant activity. This is the first time that anticoagulant sulfated homogalatans have been isolated from green algae. To prolong the coagulation time to double the baseline value in the aPTT, the required amount of sulfated galactan 1 (6,3mg) was similar to low molecular heparin Clexane?, whereas only 0,7mg of sulfated galactan 2 was needed to obtain the same effect. Sulfated galactan 2 in high doses (250mg) induces platelet aggregation. These results suggest that these galactans from C. isthmocladum have a potential application as an anticoagulant drug / Desde a primeira descri??o de polissacar?deos sulfatados em algas marinhas, as atividades biol?gicas destes compostos foram avaliadas sob diferentes aspectos e procedimentos experimentais. Dentre as diversas atividades biol?gicas apresentadas pelos polissacar?deos de algas marinhas, a a??o do anticoagulante aparece como sendo uma fun??o promissora. Neste presente estudo foram obtidas cinco fra??es de polissacar?deos sulfatados da alga Codium isthmocladium (F0.3, F0.5, F0.7, F0.9 e F1.2) , atrav?s de prote?lise seguida de fracionamento com volumes crescentes de acetona. An?lises qu?micas demonstraram que todas as fra??es s?o compostas por polissacar?deos sulfatados. A atividade anticoagulante destas fra??es cet?nicas foram determinadas por testes de tempo de tromboplastina parcial ativada (aPTT) e tempo de protrombina (PT), utilizando plasma humano normal citratado. Nenhuma fra??o apresentou atividade via teste de PT. No entanto todas as fra??es demonstraram atividade anticoagulante para teste de aPPT. Estes resultados indicam que o(s) seus alvo(s) molecular(es) esta(?o) localizado(s) principalmente na via intr?nseca da cascata de coagula??o. Eletroforese em gel de agarose demonstrou a presen?a de 2 ou 3 bandas em varias fra??es cet?nicas, enquanto a F0.9, somente uma banda. Os polissacar?deos da F 0.9, quando submetidos a cromatografia da troca i?nica, foram separados em duas novas fra??es elu?das com 2,0 e 3,0 M de NaCl. Estes compostos apresentaram um peso molecular de 6.4 e 7.4 kDa respectivamente. Analises qu?micas e espectroscopia de infravermelho das Gal 1 e Gal 2 demonstraram que essas s?o homogalactanas sulfatadas e que diferenciavam uma da outra apenas no grau e localiza??o dos grupamentos sulfato. Teste de aPTT mostrou que as fra??es 2,0 e 3,0 M (Gal 1 e Gal 2, respectivamente) possuem atividade anticoagulante. Esta ? a primeira vez que homogalactanas sulfatadas anticoagulantes s?o extra?das de macroalgas verdes. A massa necess?ria de Gal 1 (6,3mg) para prolongar o tempo normal de coagula??o em duas vezes, via teste de aPTT, foi semelhante a Clexane?, no entanto somente 0,7mg da Gal 2 s?o necess?rios para se obter o mesmo efeito. A Gal 2 em altas doses (250mg) induziu a agrega??o plaquet?ria. Estes resultados sugerem que as galactanas da alga C. isthmocladum tem potencial aplica??o como f?rmacos anticoagulantes

Made available in DSpace on 2014-12-17T14:03:38Z (GMT). No. of bitstreams: 1 MoniqueGCFA_DISSERT_PARCIAL.pdf: 1765775 bytes, checksum: 3815c2c3560cb6ce59df27ea29b474ca (MD5) Previous issue date: 2011-07-18 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Marine algae are one of the major sources of biologic compounds. In extracellular matrix of these organisms there are sulfated polysaccharides that functions as structural components and provides protection against dehydration. The fraction 1.0 (F1.0) rich in sulfated galactans obtained from red seaweed Hypnea musciformis was physicochemical characterized and evaluated for pharmacologic activity through antioxidant activity, cytotoxic action on erythrocytes, anticoagulant, stimulatory action under antithrombotic heparan sulfate synthesis and their effects on cell proliferation and cycle cell progression. The main components of F1.0 were carbohydrates (49.70 ? 0.10%) and sulfate (44.59 ? 0.015%), presenting phenolic compounds (4.79 ? 0.016%) and low protein contamination (0.92 ? 0.001%). Fraction 1.0 showed polidisperse profile and signs in infrared analysis in 1262, 1074 and 930, 900 and 850 attributed to sulfate esters S=O bond, presence of a 3,6- anidrogalactose C-O bond, non-sulfated ?-D-galactose and a C-O-SO4 bond in galactose C4, respectively. The fraction rich in sulfated galactans exhibited strong antioxidant action under lipid peroxidation assay with IC50 of 0.003 mg/mL. Besides the inhibition of hemolysis induced by H2O2 in erythrocytes treated with F1.0, this fraction did not promote significant cytotoxity under erythrocytes membranes. F1.0 exhibited low anticoagulant activity causing moderate direct inhibition of enzimatic activity of thrombin. This fraction promoted stimulation around of 4.6 times on this synthesis of heparan sulfate (HS) by rabbit aortic endothelial cells (RAEC) in culture when was compared with non treated cells. The fraction of this algae displayed antiproliferative action under RAEC cells causing incresing on cell number on S fase, blocking the cycle cell progression. Thus F1.0 presented cytostatic and no cytotoxic action under this cell lineage. These results suggest that F1.0 from H. musciformis have antioxidant potential which is a great effect for a compound used as food and in food industry which could be an alternative to food industry to prevent quality decay of lipid containing food due to lipid peroxidation. These polysaccharides prevent the lipid peroxidation once the fraction in study exhibited strong inhibitory action of this process. Furthermore that F1.0 present strong antithrombotic action promoting the stimulation of antithrombotic HS synthesis by endothelial cells, being important for thrombosis preventing, by its inhibitory action under reactive oxygen species (ROS) in some in vitro methods, being involved in promotion of hypercoagulability state. / Algas marinhas s?o uma das principais fontes de compostos biologicamente ativos. Na matriz extracelular desses organismos existem os polissacar?deos sulfatados que funcionam como componente estrutural prevenindo-a contra desidrata??o. A fra??o 1,0 (F1,0) rica em galactanas sulfatadas obtida da alga vermelha Hypnea musciformis foi caracterizada fisicoquimicamente e avaliada quanto a atividade farmacol?gica por meio de ensaios de atividade antioxidante, a??o citot?xica sobre hem?cias, atividade anticoagulante, a??o estimulat?ria sobre a s?ntese de heparam sulfato antitromb?tico e seus efeitos na prolifera??o e progress?o do ciclo celular. Os principais componentes da F1,0 foram carboidratos (49,70 ? 0,10%) e sulfato (44,59 ? 0,015%), apresentando compostos fen?licos (4,79 ? 0,016%) e baixa contamina??o prot?ica (0,92 ? 0,001%). F1,0 mostrou perfil polidisperso e sinais na an?lise de infravermelho em 1262, 1074 e 930, 900 e 850 cm-1 atribu?dos a liga??es S=O de ?steres de sulfato, presen?a de liga??o C-O de 3,6-anidrogalactose, ?-D-galactose n?o sulfatada e liga??o C-O-SO4 no C4 da galactose, respectivamente. A fra??o rica em galactanas sulfatadas exibiu forte a??o antioxidante sobre o ensaio de peroxida??o lip?dica com IC50 de 0,003 mg/mL. Al?m da alta inibi??o da hem?lise induzida por H2O2 em hem?cias humanas tratadas com F1,0, esta fra??o n?o promoveu citotoxicidade significativa sobre a membrana de hem?cias. F1,0 exibiu baixa atividade anticoagulante, causando moderada inibi??o direta da atividade enzim?tica da trombina. Esta fra??o promoveu estimula??o de cerca de 4,6 vezes na s?ntese de heparam sulfato (HS) pelas c?lulas endoteliais da aorta de coelho (RAEC), em cultura, quando comparadas com as c?lulas n?o tratadas com F1,0. A fra??o dessa alga mostrou atividade antiproliferativa sobre as c?lulas RAEC, causando aumento no n?mero de c?lulas na fase S, impedindo a progress?o do ciclo celular. Assim, F1,0 apresentou a??o citost?tica e n?o citot?xica sobre esta linhagem celular. Esses resultados sugerem que F1,0 de H. musciformis tem potencial antioxidante, efeito importante para um composto utilizado como alimento e na ind?stria aliment?cia, podendo ser uma alternativa na ind?stria aliment?cia para a preven??o do decaimento da qualidade dos alimentos contendo lip?dio devido a peroxida??o lip?dica, uma vez que a fra??o em estudo exibiu forte a??o inibit?ria sobre a peroxida??o lip?dica. Al?m disso F1,0 apresenta forte a??o antitromb?tica promovendo a estimula??o da s?ntese de HS antitromb?tico pelas c?lulas endoteliais, sendo ?til na preven??o da trombose, devido tamb?m a sua a??o inibit?ria sobre as esp?cies reativas do oxig?nio (ROS) em alguns sistemas in vitro, estando envolvidos na promo??o de estado de hipercoagulabilidade.