<p> <i>Candida albicans</i> is a yeast-like fungal pathogen that can cause infections ranging from superficial to life-threatening systemic candidiasis. Current treatments for systemic candidiasis are available but often ineffective and toxic. Consequently, it is necessary to develop new therapeutic approaches. The purpose of this study was to construct antibody-based fusion proteins that can bind to <i>C. albicans</i> cells and eliminate them. Two such fusion proteins were constructed. Each one is composed of M1 Fab as the antibody component that binds to <i> C. albicans</i> mannan and the antifungal peptide HPRP-A1. HPRP-A1 was attached via a 15-amino acid linker to either the C-terminus of the constant light chain of M1 Fab (M1 Fab-HPRP-CL) or the N-terminus of the variable light chain of M1 Fab (M1 Fab-HPRP-VL). Binding of the fusion proteins to purified <i> C. albicans</i> mannan was assessed with enzyme-linked immunosorbent assay and the half maximal effective concentration (EC₅₀) for each fusion protein was estimated. EC₅₀ for M1 Fab-HPRP-CL was 273.6 compared to 74.1 for the original M1 Fab (<i>p</i> &lt; 0.05), whereas M1 Fab-HPRP-VL did not show any binding activity, indicating a negative impact on the antibody binding by the linked peptide. Similarly, M1 Fab-HPRP-CL also showed reduced binding for <i>C. albicans</i> cells when compared to M1 Fab as determined with immunofluorescence microscopy and flow cytometry. The effect of M1 Fab-HPRP-CL on the growth of <i>C. albicans</i> cells was analysed using microdilution and absorbance. At 16 &micro;M, the growth of yeast cells treated with M1 Fab-HPRP-CL was 47.1 % of the growth control, compared to 43.5 % for M1 Fab (<i>p</i> > 0.05) and to 1.9 % for HPRP-A1 by itself (<i>p</i> &lt; 0.001). Moreover, HPRP-A1 killed <i>C. albicans</i> at 32 &micro;M and 64 &micro;M, while M1 Fab and M1 Fab-HPRP-CL did not, indicating a loss of the antifungal activity of HPRP-A1 when attached to the antibody. These data together provide valuable insights into the development of novel antibody-based therapeutics as an alternative treatment for candidiasis.</p><p>

Pronounced opsonic activity has been demonstrated in the coelomic fluid of the Oligochaete Eisenia foetida. This humoral factor enhances the uptake of particles by Eisenia phagocytes by 94% in the case of S. cerevisiae. 31.5% inE.coli and by 34.6% in B.megaterium. In order to determine whether agglutinin molecules are responsible for this activity, the 20,000D haemagglutinin was purified. This agglutinin was found to be heat sensitive, (15 min at 60&deg;C destroying activity completely), partially dependent on calcium ions and with an optimal pH range of 5-8. The agglutinin was found to be active towards a range of vertebrate erythrocytes and various fungal and bacterial cells but is not responsible for opsonic activity. Polyclonal antibodies to this purified agglutinin were raised in rabbits and used in immunocytochemical studies to determine the source of this molecule. It was found that the cell membranes of Eisenia phagocytes bear agglutinin or agglutinin like molecules and their possible roles in internal defence mechanisms is discussed. Eisenia coelomocytes were found to release agglutinins in vitro though the site of synthesis of these molecules is yet to be established.

Two molecular forms I and II (separable by gel filtration) from mature Vicia faba seeds have been studied. The extractability of the enzymes and the in vitro conversion of the low MW form, II, to the larger oligomer, I has been examined over a range of salt concentrations. Specific and total activities of the preparations were high when strong salt solutions were used for extraction. It would appear that α-galactosidase I, in comparison with II, is best extracted from the seeds if solutions of high ionic strength are used. The effects of these salt solutions on the relative levels of the two forms have been investigated by gel filtration. Interpretation of the gel elution profiles obtained is, however, complicated by the in vitro conversion of form II to form I, which is favoured by high salt concentrations and some routine procedures in the purification of α-galactosidases, such as ammonium sulphate fractionation. The relationships between the multiple forms have been studied. α-galactosidase II can be resolved into enzymes II1 and II2 by CM-cellulose chromatography, a-Gal actosidases I, II1 and II2 have been highly purified. Form I (MW 160,000) is a tetramer of enzyme II2 as shown by SDS-PAGE. Immunological studies on the three forms have been carried out and the evidence suggests that they are structurally related, although forms I and II2 are more closely related than forms I and II. On hydrolysis,monosaccharides are released from the three purified enzymes, suggesting they are glycoproteins. The three α-galactosidases also agglutinate red blood cells indicating that they possess lectin activity.

Certain aspects of the flagella and flagellar proteins of selected members of the Proteus and Providence groups have been compared. Proteus flagella, negatively stained and examined in the electron microscope, presented both "beaded" and "lined" surface appearance. Structures which possibly represent cross sections of flagella were observed; these show two groups of three sub-units with a slight gap between the triplets. Reaggregation of flagellin occurred in the presence of high concentrations of salt. Purified flagellins of P. vulgaris, P. mirabilis, P. morganii and P. rettgerii and the Providence group gave single bands when electro-phoresed in starch or polyacrylamide gels. Comparison of the mobility of purified flagellins revealed great differences, even between those flagellins isolated from strains of a single accepted species. In addition, tryptic peptide maps of purified flagellins each had a distinctive pattern. Molecular weights of flagellins of the four Proteus species were assessed by electrophoresis jui polyacrylamide gels containing SDS; in all cases values of about 40,000 were obtained. Anri no acid analysis revealed the presence of N-methyl lysine and histidine; neither have been reported conclusively in this group before. N-methyl lysine was restricted to flagellins from certain P. morganii strains while histidine was found in approximately half of the flagellins examined, irrespective of species. The C terminal amino acid of all the flagellins examined is arginine; the C terminal sequence is ser - leu - leu - arg COOH. In every case alanine is the common N terminal amino acid. Seven out of thirty-three of the peptides found on tryptic peptide maps of P. vulgaris NCTG 100 20 flagellin have been completely or partially sequenced. The possibilities of using cyanogen bromide to cleave Proteus flagellin were investigated; citraconylation and maleylation were both extremely effective in keeping the resultant cyanogen bromide fragments in solution. The results are discussed with regard to inter-relationships between the species and the possible uses of these results in the taxonomy of the genus Proteus and the Providence group.

Caleosins were originally described as one of the two major protein components of storage lipid bodies in the seeds of higher plants, the other being oleosins. In contrast with oleosins, caleosins have a single calcium-binding EF-hand domain plus several potential phosphorylation sites and have been hypothesised as playing a role in lipid-body formation and possibly mobilisation in seeds. In Arabidopsis, there are six functional caleosin genes, two of which encode seed-specific proteins while the other isoforms are expressed in a variety of vegetative and reproductive tissues. More recently, seed caleosins have been shown to have a peroxygenase activity but the function of this was uncertain. This study describes the characterisation of a specific membrane-bound caleosin isoform, termed Clo-3 in Arabidopsis and Brassica spp, that appears to be present in all plant tissues and is responsive to a variety of biotic and abiotic stresses. Bioinformatic analysis reveals that similar caleosin-like genes/proteins are present in all vascular plants, as well as in nonvascular plants such as mosses, and even in single-celled algae. Intriguingly, caleosin-like genes are also present in the genomes of most fungi described to date, with the surprising exception of the yeasts. In order to understand the function of caleosins in plants, a detailed structural and functional analysis of this novel class of protein is reported here. Biochemical studies demonstrate that the Clo-3 isoform binds calcium (one atom per molecule), can be phosphorylated most likely, by a casein kinase 2 (CK2) protein kinase, and has putative peroxygenase activity. In addition to biochemical data, microscopy analysis shows that Clo-3 may be located both on the endoplasmic reticulum and chloroplastid envelope membranes. specifically the chloroplast envelope. Biochemical evidence of cell membrane localisation is also presented. Protease digestion experiments show that the membrane bound Clo-3 has a Type I transmembrane orientation, where its N-terminal domain faces the lumen of microsomes while the C-terminal is on the cytosolic face. Such an orientation is common for receptors or proteins that may be activated by signalling molecules. The Clo-3 gene and its encoded protein are each upregulated by salt and drought stresses and by abscisic acid (ABA) treatment. Reverse genetics using RNAi knockdown mutants demonstrate specific transcription factors involved in regulating Clo-3 during different stresses. Peroxygenase activities of Clo-3 enriched microsomes were higher following salt stress. Although the data is representative of potentially many peroxygenases, it does provide indirect evidence that Clo-3 abundance increases and/or catalytic activity is induced during stress. The study also presents evidence of the response of Clo-3 to biotic stress and related signalling molecules. Arabidopsis Clo-3 is highly responsive to the phytohormone salicylic acid, to the salicylic acid synthetic analogue DCINA, the biotic signalling molecule hydrogen peroxide, and to infection by the common fungal pathogen of Brassicas, Leptosphaeria maculans (Phoma), while experiments utilising the non-expressor of pathogenesis related protein 1 (npr1) knockout mutant plant demonstrates Clo-3 response to salicylic acid (SA) is chiefly via npr1 translocation to the nucleus. The type of peroxygenase epitomised by Clo-3 is similar to those involved in the formation of epoxy alcohols from fatty acid hydroperoxides. The latter are a class of oxylipins that are seen in fungal infection, and also play a role in various aspects of fungal spore development including sporulation and a role in cuticle synthesis. As such, Clo-3 in Arabidopsis and possibly similar caleosins in other species might play roles in oxylipin signalling pathways that are involved in a protective role during both biotic and abiotic stress responses.

Various aspects of microtubule nucleation and organization have been investigated m Drosophila and MDCK cells. Drosophila indirect fibrillar flight muscles (IFMs) do not possess centriole-containing centrosomes. The ends of both microtubules and developing myofibrils closely associate with specialized cell junctions called myotendon junctions which are located at the ends of developing muscle cells. Antisera raised against pericentrin, a protein involved in microtubule nucleation, stain myotendon junctions throughout myofibrillogenesis. The antisera also stain the centrosome/spindle poles of Drosophila embryos and cultured Drosophila epithelial cells. Hence, microtubules may be nucleated at the myotendon junctions of IFMs rather than by sites associated with nuclear envelopes as in vertebrate muscle cells. Incipient MDCK daughter cells form an extremely short intercellular bridge. This contains the mid-body and ends of two mid-body associated microtubule bundles. Eventually the two microtubule bundles completely lose contact with the mid-body. These microtubules may have been severed. Consequently, the furrow base can constrict around the mid-body to complete daughter cell separation. A non-affinity purified y-tubulin antiserum stains an antigen closely associated with both sides of the mid-body. This antigen is not y-tubulin. The antigen appears to be associated with a recently discovered mitotic organelle (the telophase disc). Taxol induced microtubule arrays contain 'chains' of microtubules in interphase neuroblast cells of Drosophila third instar larvae. Adjacent microtubules in these 'chains' appear to share several protofilaments. Preliminary results indicate that microtubules of taxol induced asters are composed of 12 rather than the normal 13 protofilaments in MDCK cells. The centrosome may nucleate and release microtubules in interphase MDCK cells. y-Tubulin is only concentrated at the centrosome of control and taxol-incubated interphase MDCK cells. Microtubule ends do not focus at the centrosome in either case. The centrosomally-associated microtubule array increases substantially in size from anaphase to late telophase. At late telophase the array spreads throughout each incipient daughter cell. Subsequently, the array ceases to focus at the centrosome. y-Tubulin and pericentrin initially concentrate at the centre of two of the several taxol-induced microtubule asters in each mitotically arrested MDCK cell. Subsequently, y-tubulin and pericentrin are concentrated at two closely associated discrete spherical sites. These sites are not associated with microtubule asters. This may represent a very clear example of microtubule release from the centrosomes in these cells.

The effect of photolysis of nitroprusside was investigated in both frog ventricular trabeculae and rabbit ear arterial strips. Unphotolysed nitroprusside failed to elicit any effect on frog ventricular twitch tension. However, upon photolysis it had a potent negative inotropic action. The extent of twitch depression was shown to depend on the degree of photolysis. It was postulated that these effects are due to a labile physiologically active photolytic product. This was positively identified as nitric oxide. Preliminary results of the negative inotropic action of thiols and synthesised nitrosothiols are also presented. In contrast to frog ventricle, intact nitroprusside does exert a relaxing effect on precontracted mammalian smooth muscle. This effect is markedly potentiated by photolysis. It is concluded that the mechanism of action of nitroprusside on both tissues involves the release of nitric oxide which is postulated to activate guanylate cyclase. This suggests that mammalian vascular smooth muscle has a mechanism for degrading nitroprusside which is absent in frog ventricle.

abstract: The Multiple Antibiotic Resistance Regulator Family (MarR) are transcriptional regulators, many of which forms a dimer. Transcriptional regulation provides bacteria a stabilized responding system to ensure the bacteria is able to efficiently adapt to different environmental conditions. The main function of the MarR family is to create multiple antibiotic resistance from a mutated protein; this process occurs when the MarR regulates an operon. We hypothesized that different transcriptional regulator genes have interactions with each other. It is known that Salmonella pagC transcription is activated by three regulators, i.e., SlyA, MprA, and PhoP. Bacterial Adenylate Cyclase-based Two-Hybrid (BACTH) system was used to research the protein-protein interactions in SlyA, MprA, and PhoP as heterodimers and homodimers in vivo. Two fragments, T25 and T18, that lack endogenous adenylate cyclase activity, were used for construction of chimeric proteins and reconstruction of adenylate cyclase activity was tested. The significant adenylate cyclase activities has proved that SlyA is able to form homodimers. However, weak adenylate cyclase activities in this study has proved that MprA and PhoP are not likely to form homodimers, and no protein-protein interactions were detected in between SlyA, MprA and PhoP, which no heterodimers have formed in between three transcriptional regulators. / Dissertation/Thesis / Masters Thesis Biology 2018

Mechanisms of antioxidant action of zinc dialkvldithio­ phosphate (ZnDRP) and several structurally related compounds (basic zinc dialkyldithiophosphate (b-ZnDRP), dialkylthio-phosphoryl disulphide (DRDS), dialkyldithiophosphoric acid (DRDPA), dialkylthiophosphoric acid (DRTPA)) were studied in both the presence and absence of a soluble iron catalyst (iron (III) stearate (FeST) ). Oxygen absorption studies in two hydrocarbon substrates, white mineral oil and decalin, show that each of the above thiophosphoryl compounds is an effective inhibitor of oxidation at 130°C in the absence of FeST. In the presence of FeST, however, their effectiveness is severely reduced and a higher concentration of antioxidant is required to provide stabilisation against oxidation. The initial presence of hydroperoxide is shohl1 to have a marked effect on the antioxidant activity of each of the above thiophosphoryl compounds in both the presence and absence of FeST. All the above compounds are shown to be capable of decomposing cumene hydroperoxide (CHP) in both the presence and absence of FeST at 110°C. The long term stabilisation against hydrocarbon oxidation provided by the above thio-phosphoryl compounds in the presence of added CHP may be attributed to ionic hydroperoxide decomposition, promoted by sulphur containing acids formed as a result of the oxidation of the parent additive molecule by CHP. Although reaction with FeST may lead to the removal of some of the sulphur acids, hydroperoxide decomposition is shown to still be occurring in the presence of soluble iron. Of the above thiophosphoryl compounds, only DRDPA gives iron (III) dialkyldithiophosphate (FeDRP) on reaction with FeST. In the absence of CHP, FeDRP was shown to be an inhibitor of hydrocarbon oxidation. In the presence of CHP, however, FeDRP becomes inactive due to free radical decomposition of the hydroperoxide by the iron complex. FeDRP is shown to be an effective hydroperoxide decomposer even when present in small amounts, the mechanism of decomposition depending on the initial CHP:FeDRP ratio. The initial homolytic decomposition of hydroperoxide is caused by the FeDRP itself, but the subsequent ionic reactions are associated with the oxidation product(s) of the iron complex.

<p> Rod and cone photoreceptors in the retina are polarized sensory neurons that possess uniquely modified primary cilium, called the outer segment, to capture photons. Circadian-mediated shedding and renewal of outer segment membrane discs requires extensive vesicular transport of protein cargo from the endoplasmic reticulum and Golgi to the base of the cilium. Endocytosis is vesicle transport process of capturing and/or recycling extrinsic components and is shown to occur in retina of early vertebrates, such as <i>Xenopus</i> laevis. In this thesis, I have explored the hypothesis that a critical endocytosis-associated protein Rabgef1 is critical for the genesis of photoreceptor outer segments in the mammalian retina. After demonstrating high expression of Rabgef1 concordant with photoreceptor maturation, I characterized morphology and function of retina from <i>Rabgef1</i>-loss of function (<i>Rabgef1</i>^{&ndash;/&ndash;}) mice. Though no gross defect was observed by histology and immunohistochemistry before eye opening (postnatal day 14), transmission electron microscopy demonstrated ultrastructural defects in photoreceptor outer segments by P8. Progressive, yet rapid, photoreceptor degeneration and near-complete ablation of the visual response were evident at and after P15. I show that the outer segment defect noted in <i>Rabgef1</i>^{&ndash;/&ndash;} mice was not due to defective ciliogenesis or trafficking of cargo proteins to the cilium. In concordance with other systems, Rabgef1 was enriched in purified endocytic vesicles from the retina and interacted with Rabaptin5, confirming its role in Rab5-mediated endocytosis. Curiously, <i>Rabgef1</i>^{&ndash;/&ndash;} photoreceptors accumulated enlarged vesicular/endosomal structures within the inner segment, similar to loss of function mutations in the yeast orthologue of Rabgef1, Vps9p. My studies provide the first evidence of an essential role of Rabgef1-mediated fusion and recycling of endocytic vesicles in the formation and/or renewal of outer segment membrane discs in the mammalian retina. Rabgef1 and other components of the endocytic pathway should therefore be considered as candidates for human retinopathies. </p><p>