<p>Polysaccharides are found in all forms of life and serve diverse purposes. They are enzymatically synthesised from activated monosaccharide precursors, nucleotide sugars. One such nucleotide sugar is UDP-glucuronic acid, which is formed from UDP-glucose by the UDP-glucose dehydrogenase (UGDH) enzyme. UGDH has been proposed to have a regulatory role in the biosynthesis of polysaccharides. The aim of the studies presented in this thesis was to investigate the role of UGDH in the polysaccharide biosynthesis in three different systems: human cell culture, bacterial cultures<i> </i>and growing<i> </i>plants<i>. </i>The effects of UGDH-overexpression on polysaccharide biosyntheses and, when achievable, on UDP-sugar levels, were investigated.</p><p>A mammalian UGDH was cloned from a kidney cDNA library. Transient expression of the cloned enzyme in mammalian cells led to an increased UGDH-activity. Northern blotting analyses revealed a single transcript of 2.6 kb in adult mouse tissues whereas human tissues expressed a predominant transcript of 3.2 kb and a minor transcript of 2.6 kb.</p><p>Overexpression of the bovine UGDH in mammalian cells induced increased synthesis of the glycosaminoglycans; heparan sulphate, chondroitin sulphate and hyaluronan, without changing their relative proportions. The effects on glycosaminoglycan synthesis caused by an increased demand of UDP-glucuronic acid were studied by overexpression of hyaluronan synthase (Has3), which requires UDP-glucuronic acid as substrate. Overexpression of Has3 and coexpression of Has3 and UGDH resulted in highly augmented production of hyaluronan without noticeably affecting heparan sulfate and chondroitin sulfate synthesis.</p><p>Expression of the bacterial UGDH in <i>E. coli</i> resulted in increased formation of UDP-glucuronic acid, but, unexpectedly, also to synthesis of fewer K5 polysaccharide chains. </p><p>Overexpression of UGD1, one of four <i>A. thaliana</i> UGDH genes, in <i>A. thaliana,</i> resulted in dwarfism. Analysis of the cell wall polysaccharides showed alteration in saccharide composition. Paradoxically, the UDP-sugars derived from UDP-glucuronic acid decreased in amount.</p>

Polysaccharides are found in all forms of life and serve diverse purposes. They are enzymatically synthesised from activated monosaccharide precursors, nucleotide sugars. One such nucleotide sugar is UDP-glucuronic acid, which is formed from UDP-glucose by the UDP-glucose dehydrogenase (UGDH) enzyme. UGDH has been proposed to have a regulatory role in the biosynthesis of polysaccharides. The aim of the studies presented in this thesis was to investigate the role of UGDH in the polysaccharide biosynthesis in three different systems: human cell culture, bacterial cultures and growing plants. The effects of UGDH-overexpression on polysaccharide biosyntheses and, when achievable, on UDP-sugar levels, were investigated. A mammalian UGDH was cloned from a kidney cDNA library. Transient expression of the cloned enzyme in mammalian cells led to an increased UGDH-activity. Northern blotting analyses revealed a single transcript of 2.6 kb in adult mouse tissues whereas human tissues expressed a predominant transcript of 3.2 kb and a minor transcript of 2.6 kb. Overexpression of the bovine UGDH in mammalian cells induced increased synthesis of the glycosaminoglycans; heparan sulphate, chondroitin sulphate and hyaluronan, without changing their relative proportions. The effects on glycosaminoglycan synthesis caused by an increased demand of UDP-glucuronic acid were studied by overexpression of hyaluronan synthase (Has3), which requires UDP-glucuronic acid as substrate. Overexpression of Has3 and coexpression of Has3 and UGDH resulted in highly augmented production of hyaluronan without noticeably affecting heparan sulfate and chondroitin sulfate synthesis. Expression of the bacterial UGDH in E. coli resulted in increased formation of UDP-glucuronic acid, but, unexpectedly, also to synthesis of fewer K5 polysaccharide chains. Overexpression of UGD1, one of four A. thaliana UGDH genes, in A. thaliana, resulted in dwarfism. Analysis of the cell wall polysaccharides showed alteration in saccharide composition. Paradoxically, the UDP-sugars derived from UDP-glucuronic acid decreased in amount.

Some yeast strains secrete extracellular polypeptide toxins known to have potential growth inhibitory activity on sensitive yeast cells. These yeast strains are known as killer yeasts and their toxins are named as killer toxins or killer proteins. Yeast killer proteins are found inhibitory to Gram-positive bacteria in several studies which were based on microbial interactions of the producer strains tested with sensitive strains. K5 type yeast killer protein produced by Pichia anomala NCYC 434 was previously purified and characterized in our laboratory. The protein is glycosilated and has a pI value of 3,7 and molecular mass of 49 kDa, with exo &amp / #946 / -1,3-glucanase activity. Antibacterial activity of the pure K5 type yeast killer protein was tested against 19 clinical isolates of gram-positive bacteria causing skin infections and 2 quality control strains and found to have inhibitory activity on the isolates of Methicillin-sensitive Staphylococcus aureus (MSSA) and Enterococcus faecium. Toxin MIC and MBC ranges were 32 - 256 &micro / g/ml and 64 - &gt / 512 &micro / g/ml respectively. Cell killing analysis revealed that toxin has a bacteriostatic activity and the inhibitory effect starts between 8. and 12. hours. Regrowth of the bacteria is retarded with the increased dose of the toxin. K5 type yeast killer protein might be used as a topical antibacterial agent with its bacteriostatic activity for skin and wound infections caused by MSSA and Enterococcus faecium with appropriate formulation studies upon the antibacterial spectrum determination of the toxin in this study.

No description available.

No description available.

Encapsulated Escherichia coli are responsible for a number of life threatening infections of man. These range from urinary tract infections to septicemia and neonatal meningitis. A common property of these E. coli strains is the expression of a polysaccharide capsule or K antigen. The expression of a capsule is an essential virulence factor protecting the bacterium from host defenses. Like many virulence factors capsule gene expression is regulated by temperature, such that at 37 0C inside the host the capsule is expressed whereas at 20 0C it is not. The project used the K5 capsule gene cluster as a model system to study in detail the regulation of capsule gene expression. Expression of E. coli K5 gene cluster is regulated at the transcriptional level by two convergent promoters PR1 and PR3. The temperature regulation-dependent expression is in part controlled at the level of transcription by complex regulatory network involving the regulators SlyA, H-NS and IHF acting at PR1 and PR3. A large 5’ untranslated region (5’ UTR) is involved in transcriptional regulation by interacting with global regulator proteins. In this study, a combination of lacZ reporter gene fusions, 5’ RACE analysis and site-direct mutagenesis at promoter functional elements were used to investigate the promoter. These studies identified that the PR1 promoter was more complex than initially thought and contains, in addition to previously characterized PR1-1 promoter at +1, three additional tandem promoters PR1-2, PR1-3 and PR1-4 transcribing in the same direction from the site +133, +142 and +182, respectively. In order to analyse the contribution for the transcription from PR1 among these multiple promoters, these multiple tandem promoters’ activities were measured by β-galactosidase assay and Real-time quantitative reverse PCR assay. We determined that PR1-2 and PR1-3 are two cryptic promoters with very low transcription activity while PR1-1 and PR1-4 are the major promoters that contributed evenly to the total transcripts into kps operon in the mid-exponential phase. Furthermore, we demonstrated that the promoter PR1-1 and PR1-4 are tightly coupled and the activity of PR1-4 can be co-ordinately reduced by disrupted PR1-1.Different minimal PR1-lacZ promoter fusions were also transformed into strains with mutations in the genes that encode these regulatory proteins (IHF, SlyA and H-NS) and the transcription activity was examined by β-galactosidase assay at both 37 0C and 20 0C. IHF is required indirectly for maximum transcription at PR1-1 promoter but directly represses transcription from PR1-4 due to binding at +160 region at 37 0C. Global regulator H-NS represses the transcription at both 37 0C and 20 0C at PR1 and plays an important role for transcriptional temperature regulation at PR1 region. The anti-repressor SlyA activates transcription at PR1-1 at 37 0C. This study identified for the first time growth phase dependent expression from the PR1 promoter. Also, this study discovered different temporal patterns of promoter PR1-1 and PR1-4 transcription was coordinated with bacterial growth cycle. Overall this study will be helpful to decipher the complex regulation of capsule gene expression in E. coli.

Some yeasts secrete polypeptide toxins, which are lethal to other sensitive yeast cells, gram-positive pathogenic bacteria and pathogenic fungi. Therefore these are designated as killer toxins. Killer toxins are suggested as potent antimicrobial agents especially for the protection of fermentation process against contaminating yeasts, biological control of undesirable yeasts in the preservation of foods. Moreover they are promising antimicrobial agents in the medical field / due to immune system suppressing diseases like AIDS, there is an increase in the incidence of fungal diseases and current antimycotics have low selectivity and severe side effects. In this study our aim was to explain the cytocidal effect and enzymatic properties of K5 type yeast killer protein, which is secreted by Pichia anomala NCYC 434 cells, and known to have a broad range of killing spectrum. Competitive inhibition of the toxin with cell wall polysaccharides showed that primary binding site of toxin is &amp / #946 / -1,3-glucans of sensitive cells. Toxin showed exo-&amp / #946 / -1,3-glucanase activity which causes loss of cell wall rigidity leading cell death. Km and Vmax were found to be 0,3 mg/ml and 372,3 &micro / mol/min/mg for laminarin hydrolysis. The toxin exerted its cytocidal effect after 2 h contact with the target cells. Toxin production was found to be dependent on &amp / #946 / -1,3-glucan content of the media. Toxin activity was completely inhibited by Hg+2 ,while several metal ions and DTT increased the activity to different extends. Our findings revealed the characteristics of K5 type killer toxin which will help for its possible uses in near future.

Some yeast strains under certain conditions secrete into the medium polypeptide toxins which are inhibitory to sensitive cells. These yeast strains are termed as killer yeasts and their toxins are designated as killer proteins or killer toxins. Killer proteins are classified into 11 typical types (K1-K11). These toxins have different killing mechanisms on sensitive cells. Some of them hydrolyze major cell wall component &amp / #946 / -1,3- glucans. As mammalian cells lack cell walls research and development of novel highly selective antifungals are mostly focused on the agents which target the components of the fungal cell wall. We have previously characterized the K5 type killer protein. This protein is an exo &amp / #946 / -1,3-glucanase which is stable at pH&rsquo / s and temperatures appropriate for its medical usage. &amp / #946 / -1,3- glucan hydrolyzing activity of the K5 type killer protein highlighted the potential use of this protein as a selective antimycotic agent. Antifungal activity of the K5 type yeast killer protein was tested against 26 human pathogenic yeast and 9 dermathophyte strains and found to be affective on all of the tested strains. Toxin MIC50, MIC100 and MFC values were found to be between 0.25-4, 0.5-8, 1-8 &micro / g/ml respectively except Candida krusei isolates. Cell killing analysis revealed that toxin activity starts within first 2 hours and complete cell death time differs due to the susceptibility of strains to the K5 type yeast killer protein. K5 type yeast killer protein would be used as a novel and selective agents with the results obtained from this study.

No description available.

The optically stimulated luminescence (OSL) characteristics of a suite of quartz and feldspar samples from a range of modern glaciofluvial sediments have been explored to determine the use of OSL as a depositional pathway tracer. Paraglacial and subglacial source material and various glaciofluvial deposits have been analysed from the glacial catchments of Bergsetbreen, Fåbergstølsbreen, and Nigardsbreen as well as the Fåbergstølsgrandane sandur, Jostedalen, Norway. The OSL distribution signatures have been characterised through exploration of sample skewness, kurtosis and overdispersion, and dose distributions of the different depositional settings and source materials are distinct for both quartz and feldspar. Residual ages are greatest for feldspar, indicating significant potential age overestimation where feldspar is used to date glaciofluvial deposits. Sample dose distributions and overdispersion characteristics are driven by source sediment properties, whereas residual ages are controlled by transport and depositional processes. Those transport and depositional processes which result in significant light exposure, also influence dose distributions, and processes that sort sediments least effectively have the highest residual doses. Sample OSL characteristics, transport distance and grain size distributions have been investigated using factor analysis, as a means of predicting sediment source, facies, depositional process and deposit type. Although the depositional processes of the quartz samples can be clearly differentiated based upon OSL characteristics, factor analyses of feldspar and grain size characteristics are inconclusive. The application of quartz OSL to the Norwegian samples was limited by its very poor luminescence sensitivity. Quartz is the preferred mineral for OSL, however, despite the plethora of successful quartz OSL applications, the precise origin of the UV/blue luminescence emission, measured during OSL, remains unclear. The origins of this emission and controls on its intensity were explored using a variety of spectroscopic techniques including photoluminescence, cathodoluminescence, radioluminescence (RL), ionoluminescence (IL) and x-ray excited optical luminescence (XEOL). Exciting sample luminescence at a range of energies enables exploration of the different donor centres responsible for the luminescence emission. Cathodoluminescence and RL emission spectra are similar, comprising broad emissions at 1.5, 2.0 and 2.7 eV (detection in the UV part of the spectrum was not possible for these experiments). Ionoluminescence emission spectra were dominated by the ~ 3.3 eV emission, which is a component of the signal conventionally monitored during OSL. This emission depleted as a function of dose, to the benefit of the red emission (1.8-2.0 eV) for all samples throughout IL, and similar observations were made for the 3.4 eV emission observed from the XEOL emission spectra. The XEOL spectra are dominated by an emission at ~ 3.8 eV, not widely reported for quartz, which has tentatively been attributed to peroxy linkages. Differences between the IL and XEOL emission spectra are interpreted as evidence for the presence of multiple excited states.