ClC-1 is the major skeletal muscle chloride channel and is essential for re-establishing the resting membrane potential of muscle cells after an action potential has occurred. Many mutations throughout the CLCN1 gene, which codes for the CIC-1 protein, have been demonstrated via characterisation in heterologous expression systems, to be causative mutations for either Dominant Myotonia Congenita or Recessive Generalised Myotonia. Recently, increasing numbers of myotonic mutations have been found in the carboxyl tail of CIC-1, which demonstrates its importance as a domain that is essential for the normal function of CIC-1 channels. Previous studies in our laboratory defined a region of 18 amino acids in the immediate post D13 segment of rat CIC-1, essential for the expression of functional channels. / thesis (PhDBiomedicalScience)--University of South Australia, 2002.

Thesis (Ph. D.)--University of Adelaide, Dept. of Agricultural Biochemistry, 1984. / Includes bibliographical references (leaves 125-140).

Thesis (B. Sc. Hons)--University of Adelaide, 1984. / Includes bibliographical references (leaves [86-88]).

Thesis (Ph. D.)--University of Chicago, Dept. of Ecology and Evolution, December 2002. / Includes bibliographical references. Also available on the Internet.

Orientador: Angelo Luiz Cortelazzo / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-07T07:38:47Z (GMT). No. of bitstreams: 1 Ribeiro_ViktoriaKovesdy_M.pdf: 7983680 bytes, checksum: 5d4ac508a6df0a6cc6853aab29ae2d9a (MD5) Previous issue date: 2006 / Resumo: As restingas da região nordeste são pouco estudadas em todos os seus aspectos. Assim, o presente trabalho teve como objetivo analisar sementes de espécies de grande ocorrência na restinga da Reserva Particular do Patrimônio Nacional (RPPN) de Nossa Senhora do Outeiro de Maracaípe em Ipojuca, no litoral pernambucano, dentro de uma perspectiva mais ampla de análise para contribuir com o conhecimento de sua estrutura e das potencialidades de utilização econômica junto à população local. Foram coletadas sementes de 22 espécies, que foram fixadas em solução de FAA 70 ou fixador de Karnovsky para processamento e análise microscópica. Destas, oito espécies foram incluídas em historesina Leica ou em parafina, seguindo-se corte em micrótomo com 5 a 8 cm de espessura, conforme o meio de inclusão: Coccoloba laevis Casar. (Polygonaceae), Licania rigida (Chrysobalanaceae), Myrcia guianensis (Aulb.) DC. (Myrtaceae), Simaba cuneata A.St.-Hil. & Tul. (Simaroubaceae), Manilkara salzmannii (A.DC.) Lam (Sapotaceae), Maytenus impressa Reiss. (Celastraceae), Ocotea gardneri (Meisn.) Meiz (Lauraceae) e Serjania sp (Sapindaceae). Os materiais foram corados por diversos métodos gerais e citoquímicos, visando a determinação de proteínas, polissacarídeos, lipídios, radicais aniônicos totais e compostos fenólicos. Para as quatro primeiras espécies citadas, foram dosadas as principais reservas presentes nas sementes. Os resultados citoquímicos foram confirmados pelas dosagens bioquímicas revelando que C. laevis é uma espécie onde predomina o amido, L. rígida tem maiores teores de proteínas e S. cuneata é oleaginosa. M guianensis mostrou-se moderadamente rica em carboidratos e proteínas. Chamou a tenção a incomum quantidade de açúcares livres presentes nas sementes, que variou de 10% em C. laevis até 25% em L. rígida, podendo representar fator adaptativo interessante à falta de água. Cristais de oxalato, presença de taninos e tegumento lignificado também foram características encontradas na maioria das espécies e também podem estar relacionadas à defesa e estratégias de dispersão das sementes. Os testes citoquímicos revelaram ainda que O. gardneri e Serjania sp são ricas em amido e proteínas, enquanto que M. salzmannii apresenta lipídios em quantidades apreciáveis. Finalmente, pôde ser notado que M. impressa apresenta endosperma bem desenvolvido, possivelmente com hemiceluloses como sua principal reserva; seus cotilédones apresentaram quantidades apreciáveis de proteínas. As demais espécies coletadas encontram-se processadas para inclusão e corte para futura análise e caracterização citoquímica / Abstract: ¿ Restinga of the northeast have been barely studied in all their aspects. Thus, this work was as objective analyzes seeds of arboreal species of great occurrence in the ¿Reserva Particular do Patrimônio Nacional (RPPN) de Nossa Senhora do Outeiro de Maracaípe¿ in Ipojuca, located at the sea coast of Pernambuco to contribute with the knowledge of structure and these economic potentialities. Twenty-two species of fruits were harvested and the seeds were fixed in FAA70 or Karnovsky solutions and processed for microscopic analysis. Of these, eight species were embedded in Leica Historesin or paraffin and sectioned (5-7cm thick): Coccoloba laevis Casar. (Polygonaceae), Licania rigida (Chrysobalanaceae), Myrcia guianensis (Aulb.) DC. (Myrtaceae), Simaba cuneata A.St.-Hil. & Tul. (Simaroubaceae), Manilkara salzmannii (A.DC.) Lam (Sapotaceae), Maytenus impressa Reiss. (Celastraceae), Ocotea gardneri (Meisn.) Meiz (Lauraceae) and Serjania sp (Sapindaceae). After removing the paraffin and hydration, the materials were stained using several cytochemical methods to detect anionic residues, proteins, polysaccharides, starch, lipids and lignin. The stained sections were examined by light microscopy, using polarized light in some cases. The first four mentioned species of seeds were submitted to extraction to detect their principal reserves. The cytochemical results were confirmed by biochemical dosage revealing that C. laevis is a species where the starch is predominant; L. rigida has got higher contents of proteins and S. cuneata is oleaginous. M guianensis showed moderately rich in carbohydrates and proteins. Free and soluble sugars presented an unusual quantity in the seeds, with variation from 10% in C. laevis up to 25% in L. rigida , showing an possible and interesting adaptative factor to lack of water. Oxalate crystals, presence of tannins and lignified teguments were also characteristics found in most of the species and can be related to the defense and strategies of dispersion of the seeds. The cytochemical analysis revealed that although O. gardneri and Serjania sp are rich in starch and proteins, M. salzmannii presents lipids in appreciable amounts. Finally, it could be found that M. impressa presents endosperm well developed, possibly with hemiceluloses as the principal reserve while their cotyledons presented appreciable amounts of proteins. The other harvested fruits and seeds were processed for embebition and cut for future analysis and cytochemical characterization / Mestrado / Biologia Celular / Mestre em Biologia Celular e Estrutural

Thesis (MSc)-- Stellenbosch University, 2013. / ENGLISH ABSTRACT: Membrane bound cytochrome b5 is a ubiquitous protein with an average molecular weight of 16 kDa. The protein is involved in a number of reactions providing electrons directly to cytochrome P450 enzymes or to other enzymes involved in lipid biosynthesis. It is also known that the protein influences the activities of certain enzymes via an allosteric effect. It has been accepted in the literature that the cytochrome b5 exists primarily in the monomeric form, however, recently it has been shown that it forms homomeric complexes in vivo. In this study, we investigate the cytochrome b5 complex formation using a variety of analytical tools. Cytochrome b5 was isolated from ovine liver microsomes and the purity verified using sodium dodecyl sulphate polyacrylamide gel electrophoresis and electrospray ionisation mass spectrometry. The latter analysis confirmed the presence of a single heme containing protein with Mr=15865 Da, while separation on the polyacrylamide gel revealed oligomeric complex formation with the tetrameric form the most prominent oligomer. Using different and particularly harsh denaturing conditions we found that the observed oligomeric aggregates persisted, indicating highly stable complexes. The most prominent tetrameric aggregate was identified to be cytochrome b5 by mass spectrometric sequencing. Further complex formation studies, using a fluorescent dye (1-anilinonaphthalene-8-sulfonic acid) that interact with hydrophobic cavities formed during oligomerisation, provided evidence of protein assembly in oligomeric complexes or aggregation. The formation of the cytochrome b5 complexes was dependent on ionic strength and protein concentration. Previously it was shown that the hydrophobic membrane anchoring domain plays a pivotal role in the cytochrome b5’s homomeric complexes. Using a peptide (IITTIDSNSS), resembling a portion of this domain, together with circular dichroism we showed more organized structure present for the wildtype peptide vs. a mutated control peptide (LLSSLKAVAV). A modified ELISA interaction assay also revealed that the wild-type peptide had a specific interaction with cytochrome b5, providing further evidence that the membrane anchoring domain plays a role in complex formation. These studies also indicated that a hydrogen bond network in this domain may be important for the formation of the homomeric complexes of cytochrome b5. / AFRIKAANSE OPSOMMING: Membraan-gebonde sitochroom b5 is ’n alomteenwoordige proteïen met ’n gemiddelde molekulêre massa van 16 kDa. Die proteïen is betrokke in reaksies waar dit elektrone direk aan sitochroom P450 ensieme verskaf, sowel as ensieme betrokke in lipiedbiosintese. Dit is ook bekend dat die proteïen die aktiwiteite van sekere ensieme via ’n allosteriese effek beïnvloed. Dit is geredelik in die literatuur aanvaar dat sitochroom b5 as ’n monomeer voorkom, maar daar is kort gelede gerapporteer dat homomeriese komplekse in vivo vorm. In hierdie studie is die sitochroom b5-kompleksvorming ondersoek deur gebruik te maak van verskeie analietiese metodes. Sitochroom b5 is vanuit skaaplewer mikrosome geïsoleer en die suiwerheid met behulp van natrium-dodesiel-sulfaat-poliakrielamied-gel-elektroforese en elektrosproei-ionisasie massa-spektrometrie geverifieer. Met die laasgenoemde bevestig dat ’n enkele heem-bevattende proteïen met Mr =15865 teenwoordig was, terwyl poliakrielamied gel-skeiding kompleksvorming getoon het, met tetrameer as die mees prominente oligomeer. Deur verskeie denaturerings kondisies, intsluitend besondere kondisies, is gevind dat hierdie aggregate behoue bly, wat baie stabiele oligomere aandui. Die mees prominente tetrameriese aggregaat is as sitochroom b5 geïdentifiseer met behulp van massa spektrometriese volgordebepaling. Kompleksvorming is verder bewys deur ’n verdere ondersoek met behulp van ’n fluoresserende kleurstof (1-anilinonaftaleen-8-sulfoonsuur) wat met die hirdofobiese holtes, wat vorm tydens oligomermerisasie, interaksie het. Die kompleksvorming was afhanklik van ioniese sterkte, sowel as proteïenkonsentrasie. Voorheen was dit bewys dat die deurslaggewende faktor in die vorming sitochroom b5 se homomeriese komplekse die hidrofobiese membraan-anker-domein is. Deur gebruik te maak van ’n peptied (IITTIDSNSS) wat lyk soos ’n gedeelte van hierdie domein, tesame met sirkulêre dichroisme, is gewys dat meer georganiseerde struktuur teenwoordig was vir die wilde tipe peptied vs. ’n gemuteerde kontrole peptied (LLSSLKAVAV). ’n Gemodifiseerde ELISAinteraksie- essai het ook getoon dat die wilde-tipe peptied spesifieke interaksie met sitochroom b5 het, ’n verdere bewys dat hierdie membraan-anker-domein ’n rol speel in kompleksvorming. Hierdie studies het ook aangedui dat ’n waterstofbinding netwerk in die domein belangrik kan wees vir die vorming van die homomeriese komplekse van sitochroom b5.

Dissertation (PhD)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: This dissertation describes how the isolation and characterisation of ovine liver cytochrome b5 was accomplished by referring to the following goals achieved in this study: - The optimisation of the isolation and purification procedure for ovine liver microsomal cytochrome b5 in order to obtain sufficient material for aggregation and immunological studies. - The removal of the membrane binding domain of cytochrome b5 by means of tryptic digestion to establish the role of the carboxyl terminal in ovine cytochrome b5 aggregation. - The raising of antibodies against both the trypsin truncated and intact forms of cytochrome b5 to study the aggregation of the protein. - The investigation into the influence of purified cytochrome b5 on steroidogenesis in ovine adrenal microsomes. / AFRIKAANSE OPSOMMING: Die isolering en karakterisering van skaaplewersitochroom b5, soos beskryf in hierdie proefskrif, is uitgevoer deur die volgende doelwitte suksesvol af te handel: - Die optimalisering van die prosedure vir die suksesvolle isolering en suiwering van skaaplewersitochroom b5 ten einde genoegsame hoeveelhede van die suiwer proteïen te hê vir die bestudering van die aggregasie van die proteïen sowel as ‘n immunologiese studie. - Die verwydering van die membraanbindingsdomein van sitochroom b5 om die invloed van die karboksielterminaal op die aggregering van die proteïen te bestudeer. - Die gebruik van sowel die tripties gesnyde as die intakte vorms van sitochroom b5 om ‘n immuunrespons in hase op te wek vir die verkryging van sitochroom b5 spesifieke anti-liggame. - Die gebruik van die gesuiwerde proteïene om die invloed van sitochroom b5 op adrenale steroïdogenese te bestudeer.

Acid phosphatases (apase) are important hydrolytic enzymes that function in the acquisition, production; transport and recycling of inorganic phosphate (Pi), thus making a significant contribution towards nutrients dynamic of many ecological niches. The aim of this study was to characterize the apase enzyme found in the lichen Cladonia portentosa at the molecular level. The initial experiment entailed cloning the apase gene by PCR using degenerate primers designed from close relatives of C. portentosa from the Ascomycete family. The isolation of apase gene from Cladonia portentosa using PCR was not successful. Attempts were then made to purify the secreted apase and to determine its biochemical and molecular properties and to allow comparison with already characterized secreted phosphatases from other fungal sources existing in the NCBI database. It was anticipated that the partial sequence of the purified enzymes would provide a corresponding apase gene. The acid phosphatase enzyme was partial purified to 45 fold by a gel filtration with a yield of 18%. It gave a single, broad glycoprotein band on native PAGE and SDS-PAGE corresponding in size to 250 and 148 kDa, respectively. Under reducing conditions, the purified enzyme migrated as two bands of 116 and 32 kDa, indicating the heterodimer nature of this enzyme. Only one distinct band, (pI 6.4) was observed after electrofocusing. The optimum temperature for the enzyme was 65 °C where an optimal pH was detected at 2.5. The enzyme was inhibited by known acid phosphatase inhibitors (fluoride, molybdate, orthovanadate and tartrate) and the metals (Cu²⁺ and Zn²⁺). The purified enzyme demonstrated broad substrates selectivity and had a KM of 31.2±0.25 μM for phytic acid. Peptide analysis by Mass Spectrometry (MS) MALDI-TOF indicated the presence of two apase proteins. The amino sequences of purified apase/s from Cladonia portentosa were FLAETNPAPFGH, AVGLGYVEELLAR and AQGLGYVQEVLAR. Comparing the amino acids of the sequenced protein with that of already known proteins confirmed the enzyme to be a secreted histidine acid phosphatase, resembling other acid phosphatases and phytase from several filamentous fungi with respect to amino acid composition. To investigate the effect of phosphorus on C. portentosa apase, the mycelium was grown under different concentrations of Pi [0.05, 1.0, 3.0, 10 and 100 mM (KH₂PO₄)]. The aim was to localize the apase enzyme and to screen for the occurrence of the gene coding for the acid phosphatase enzyme. A treatment of 3.0 mM Pi induced high levels of apase compared to all other treatments. In addition, cultures of C. portentosa were grown in axenic cultures to study the effect of pH and Pi versus menadione on the production of acid phosphatase and mycelia growth. A culture media of pH 4.8 and 6.0 resulted in higher apase secretion than when compared with pH 2.5 medium. The presence of 2.0 μM menadione marginally increased levels of the apase compared to the control treatment. Apase was further localized cytochemically using fluorescent substrate-enzyme-labelled fluorescence (ELF-97) which forms a fluorescent crystalline precipitate at the site of phosphate activity. Fluorescent microscope revealed that the enzyme was present in all treatments, irrespective of Pi concentration, however, the fluorescence signals were intense in low Pi concentrations (0.05 and 1.0 and 3.0 mM Pi). Ultrastructure localization using live mycelium under confocal microscopy using Vector blue III substrate revealed that the enzyme was localized in the cytoplasm, cell membrane, vacuole and small organelles, presumed to be endosomes. Co-staining with FM4-64, confirmed the punctuate structure to be secretory vesicles or a vacuolar network. To investigate the effect of P starvation on C. portentosa at a molecular level, the effect of Pi on the gene expression profile was examined. The generation of a cDNA library from axenic grown mycelium treated with P provided a foundation for the identification and characterization of genes expressed in the P treated mycelium through expressed sequence tags (ESTs). Several genes were identified whose transcriptional profiles have been significantly changed by phosphorus treatment and menadione. They include genes required for signal transduction and vesicular transport, cell biosynthesis and protein metabolism and stress response. In conclusion, this study constitutes the first step towards understanding the molecular mechanism governing acid phosphatase in C. portentosa. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.

Cytochromes c are electron transfer proteins in which haem is covalently attached to the polypeptide chain via thioether bonds formed from thiol groups of the two cysteines and the two vinyl groups of haem. This attachment is a post translational process and in many species of bacteria as many as approximately twelve gene products, the functions of which are largely unknown, are required. In Gram-negative bacteria the assembly of the c-type cytochromes occurs in the periplasm. Cytochrome c₅₅₂ from the thermophilic organism Hydrogenobacter thermophilus is known to be expressed in the cytoplasm of Escherichia coli. This unique example of cytoplasmic assembly of a c-type cytochrome has previously been postulated to result from insertion of haem into the folded apoform of the cytochrome followed by non-catalysed attachment of the haem. This postulate is supported by the present work which has shown that the cytoplasmic assembly of this cytochrome c₅₅₂ continues in the absence of the E. coli ccm genes which are needed for 'normal' c-type cytochrome assembly in that organism. Attempts to test the postulate of spontaneous assembly of the cytochrome c₅₅₂ with in vitro experiments require large amounts of cytochrome c₅₅₂ and its apo protein. A number of procedures for preparing these proteins were investigated. Although a T7-based expression produced lower amounts than was expected, its use led to detection of the apo form of cytochrome c₅₅₂ in E. coli. It was shown that this apoform has some secondary structure, whereas mitochondrial apocytochrome c has a random coil conformation. This observation is consistent with, but does not prove, the postulate for cytochrome c₅₅₂ assembly. It was unexpectedly found that a strain of E. coli that produces abnormally large amounts of its endogenous c-type cytochromes also made large amounts of cytoplasmic cytochrome c₅₅₂. NMR studies on this material are consistent with a single and 'normal' attachment of the haem to the polypeptide. Thus the unusual cytoplasmic assembly was not different from the usual periplasmic assembly that occurs in the H. thermophilus itself. In E. coli there is a periplasmic cytochrome b₅₆₂ that is presumed to acquire its haem in the periplasm. Some of the ccm genes, required for c-type cytochrome assembly, are postulated to code for a system that transports haem to the periplasm. Cytochrome b- ₅₆₂ synthesis was not blocked by the absence of these genes. This implies that haem provision for cytochrome b₅₆₂ synthesis occurs independently of the ccm system. Apocytochrome b<sub>562 could be detected in E. coli with the ratio apo:holo being higher in a strain that produces c-type cytochromes to relatively low levels. It is suggested that the synthesis of both cytochrome b₅₆₂ and c-type cytochromes is at least partly a reflection of the rate of production of haem by the cells.

Leukocytes play a central role in protecting the body against infectious organisms and their research is essential for understanding the mechanisms of immunity. By studying leukocytes across a range of species, insights are provided into differing strategies employed to ensure resistance to disease. Surprisingly, the structure and function of marsupial leukocytes has received very limited study. Marsupials represent a major evolutionary pathway with distinct differences in reproduction and development from placental mammals. These differences in the life history of marsupials place unique challenges on the immune system, and differences in leukocyte structure and function could be reasonably expected. In this thesis, studies were undertaken to examine the cytochemical, ultrastructural and functional features of leukocytes from species of marsupials, belonging to the family Macropodidae (kangaroos and wallabies). The aim of these studies was to elucidate the characteristics of macropodid leukocytes and to compare and contrast these features with the known characteristics of other mammalian leukocytes. Leukocytes from two species of macropodid, the tammar wallaby (Macropus eugenii) and the western grey kangaroo (Macropus fuliginosis), formed the basis of this study with additional material provided from quokka (Setonix brachyurus), woylie (Bettongia pencillata) and red kangaroo (Macropus rufus). Staining characteristics of cells were examined following reaction with Sudan black B, peroxidase, chloroacetate esterase, naphthyl butyrate esterase, alkaline phosphatase and periodic acid-Schiff. Peroxidase and Sudan Black B reactions were similar to domestic animal species but chloroacetate esterase and naphthyl butyrate esterase were unreliable as markers for macropodid neutrophils and monocytes, respectively. Significant variation in staining for alkaline phosphatase was seen between species of macropodid. Tammar wallabies and quokka demonstrated strong neutrophil alkaline phosphatase activity whereas western grey kangaroos, red kangaroos and woylies contained no activity within their leukocytes. Peroxidase and alkaline phosphatase cytochemistry were also assessed at the ultrastructural level with transmission electron microscopy. This allowed the identification of distinct granule populations within macropodid neutrophils. Two subcellular compartments containing alkaline phosphatase activity were identified within tammar wallaby neutrophils. These were considered equivalent to secretory vesicles and a subpopulation of specific granules. Tubular vesicles containing alkaline phosphatase were also identified within the eosinophils of tammar wallabies. These structures were a novel finding having not been reported previously in the eosinophils of other animal species. In addition to cytochemistry, the general ultrastructure of leukocytes from tammar wallabies and western grey kangaroos were reported. Results were similar to previous reports for other marsupial species. The current body of knowledge was extended by the first detailed description of the ultrastructure of basophils in a marsupial. To assess functional aspects of macropdid neutrophils, flow cytometric assays were performed examining oxidative burst responses and phagocytosis. Reactive oxygen species were generated by neutrophils from tammar wallabies and western grey kangaroos in response to phorbol 12-myristate 13-acetate but not N-formyl-Met-Leu-Phe or opsonised bacteria. Phagocytosis of opsonised bacteria was also measured in neutrophils from tammar wallabies, which was poor in contrast to ovine neutrophils. However, flow cytometric studies were limited by sample preparation. Further optimisation of isolation methods for tammar wallaby leukocytes should be undertaken before dogmatic conclusions are drawn. Overall, the results of this thesis demonstrate that, in the areas examined, the general characteristics of leukocyte structure and function of mammals are present in macropodids. However differences were identified both within and outside of the macropodid group. These differences have important ramifications for the use of ‘model’ species in the study of leukocyte biology in marsupials. The results also provide potentially useful tools for the clinical diagnosis of haematological disease in macropodids and may be of interest to those studying comparative and evolutionary aspects of leukocyte structure and function.