The deconstruction of complex carbohydrates by glycoside hydrolases requires extensive enzyme consortia in which specificity is often conferred by accessory modules and domains that are distinct from the active site. The diverse mechanisms of substrate recognition were explored in this thesis using selected yeast, bacterial and plant polysaccharides as example substrates. Carbohydrate binding modules (CBM) are non-catalytic modules that enhance the catalytic activity of their glycoside hydrolase counterparts through binding to polysaccharide. Normally CBMs are found attached to glycoside hydrolases that target insoluble recalcitrant substrates resulting in a moderate, 2-5 fold, potentiation in enzyme activity. A CBM, defined herein as CBMX40, is found at the C-terminal of a glycoside hydrolase family (GH) 32 enzyme, SacC, which displays exo-levanase activity. CBMX40 binds the non-reducing end of the levan chain targeting the disaccharide fructose--fructose unit. Removal of CBMX40 results in a >100-fold decrease in catalytic activity against levan, compared to the full length native enzyme. The truncated SacC catalytic domain acts as a non-specific exo-β-fructosidase displaying similar activity on β2,1- (inulin) and β2,6-linked fructose polymers, both polysaccharides and oligosaccharides. When CBMX40 was fused to a non-related exo-β-fructosidase, BT 3082, it conferred exo-levanase specificity on the enzyme. Thus CBMX40 is not only able to enhance catalytic activity but is also able to confer catalytic specificity. This led to the hypothesis that the CBM and the active site of the enzyme bind to different terminal residues of branched fructans such as levan. This results in enhanced affinity through avidity effects leading to the potentiation of catalytic activity. The gut bacterium Bacteroides thetaiotaomicron contributes to the maintenance of a healthy human gut. B. thetaiotaomicron is able to acquire and utilise complex carbohydrates that are not attacked by the intestinal enzymes of the host. B. thetaiotaomicron dedicates a large proportion of its genome to glycan degradation with a large expansion of α-mannan degrading enzymes. The B. thetaiotaomicron genome encodes 23 GH92 α-mannanosidases and 10 GH76 α-mannanases. While GH92 has recently been characterised the activities displayed by GH76 relies on the characterization of a single enzyme in this family. B. thetaiotaomicron organises the genes required to sense, degrade, transport and utilise specific complex glycans into genetic clusters defined as Polysaccharide Utilisation Loci (PULs). Transcriptomics revealed that two PULs are up regulated in response to yeast mannan, PUL 36 and PUL 68. These PULs contain both GH76 enzymes along with GH92 enzymes and other CAZy annotated enzymes. Biochemical analysis of the GH76 enzymes found in the two PULs show they are α1, 6 mannanases capable of hydrolysing the α1, 6 mannan backbone of yeast mannan, with the putative periplasmic enzymes generating small oligosaccharides, while the surface mannanases releasing larger products. The three GH92 enzymes encoded by the two PULs have been shown to remove α1, 2 and α1, 3 linked mannose branches from yeast mannan polysaccharide. In addition PUL 68 also encodes a phosphatase that removes the phosphate from mannose-6-phosphate and glucose-6-phosphate but not from intact mannan. Therefore, this study describes the ability of B. thetaiotaomicron to target and degrade yeast α-mannans. The GH5 enzyme CtXyl5A from Clostridium thermocellum is an arabinoxylan specific xylanase that contains a GH5 catalytic module appended to several CBMs. The apo structure of the GH5 catalytic module appended to a family 6 CBM reveals a large pocket abutted to the -1 subsite of the active site. This pocket was thought to bind the arabinose decoration appended to the O3 of the xylan backbone. Here mutational and structural studies showed that the fulfilment of arabinose is this pocket is the key specificity determinant for the novel arabinoxylanase activity. Significantly the bound arabinose displayed a pyranose conformation, rather than a furanose structure which is the typical conformation adopted by arabinose side chains in arabinoxylans. This structural information suggests that CtXyl5A may be able to exploit side chains other than arabinofuranose residues as substrate specificity determinants.

Exoribonucleases have many important functions in the cell including RNA processing, turnover and quality control. One of the key 3’-5’ exonucleases is the exosome, a multiprotein complex that has been extensively characterised in yeast. Many substrates that undergo maturation and/or degradation involving the yeast exosome have been identified and these include tRNAs, mRNAs, snRNAs, snoRNAs and rRNAs. By comparison, the human exosome is poorly understood and it is not clear whether functions of the yeast exosome are conserved in higher eukaryotes. We show that the human exosome has degradation functions including the turnover, but not the processing, of snoRNAs and the recycling of excised pre-rRNA fragments. We and others have shown that the human exosome also participates in pre-rRNA processing to form the mature 3’ end of 5.8S rRNA. Here we identify a novel role for the exosome in the processing of the pre-rRNA internal transcribed spacer 1 (ITS1). The small (18S) and large (5.8S and 28S) subunit rRNAs are co-transcribed as a single precursor. Processing of ITS1 is a key step in ribosome biogenesis as it separates 18S from the large subunit rRNAs and in higher eukaryotes it involves an additional processing step compared to yeast. We define alternative ITS1 processing pathways in human cells. In the major pathway, following an endonucleolytic cleavage to separate the small and large subunit rRNAs, the exosome, which is not involved in ITS1 processing in yeast, processes to within 25 nucleotides of the 3’ end of 18S. Our data highlight significant differences between the nucleases involved in ITS1 processing in yeast and humans. However, it appears that the roles of several yeast biogenesis factors are conserved in higher eukaryotes. Further, we have investigated mechanisms by which exonucleolytic processing of ITS1 may be regulated and suggest how this could be coordinated with the final maturation steps of the pre-40S complex.

The Sec pathway is the major route for secretion of proteins by bacteria. Its major components are: (i) the translocation channel comprising the SecYEG proteins; (ii) SecA: a chaperone/targeting/molecular motor that drives the movement of proteins across the membrane with participation of SecB or SRP (signal recognition particle) chaperones. Following translocation, proteins are folded to their mature conformation by folding factors such as PrsA. When compared with B. subtilis, the secretory translocase of B. anthracis contains homologues of several Sec pathway components: (i) two homologues of SecA (SecA1 and SecA2), (ii) two homologues of SecY (SecY1 and SecY2), (iii) three homologues of the PrsA foldase (PrsAA, PrsAB and PrsAC). In previous studies, SecA2 was shown to be specific for secretion of S-layer proteins: Sap and EA1, while SecY2 has not been shown to have any substrate specificity. Instead, it seems to ensure high levels of protein secretion in later phases of the growth cycle. A combination of approaches was used to continue the analysis of functioning of the SecA2 secretion pathway. They involved analysis of deletion mutants of B. anthracis, complementation studies, gene expression analysis, protein interaction investigation. We found that a novel protein BA0881 facilitates secretion of Sap and EA1, but is not essential for the processes, and was renamed SecH. Moreover, protein interaction and complementation studies revealed putative interactions between (i) SecA2 and EA1, (ii) SecA2 and SecH, (iii) EA1 and SecH, (iv) SecA1 and SecA2, raising the possibility that SecA2 and SecA1 form a dimer, which might be a functional entity for the secretion of Sap and EA1, with SecH having a role in enhancing interaction between SecA1/SecA2 dimer and its substrates. The role of PrsA-like foldases on secretion was also investigated. PrsAB was found to show substrate specificity for Sap and EA1, while PrsAA showed substrate specificity for penicillin binding proteins. Analysis of cell morphology suggests that PrsAB and SecA2 may also have substrates other than those of Sap and EA1 as their null-mutants show changes in cell length and shape. Lastly, analysis of gene expression showed that the deletion of genes encoding elements of the translocation sytem: SecA2, SecH, PrsAA, PrsAB, PrsAC, SecY2 leads to changes in the level of expression of sap and eag.

The bacterial community architecture associated with four species of sponges, Halichondria panicea, Suberites domuncula, S. carnosus and Pachymatismajohnstonia 'was investigated using culture-dependent and culture-independent strategies. Marine '/ agar was found to be the best of several media used for cultivation of culturable bacteria : associated with sponges. Molecular methods, including denaturing gradient gel ,electrophoresis (DGGE), 16S rDNA cloning and sequence analysis suggested a : bacterial community different from that identified using culture-dependent methods. DGGE can provide a profile of the whole community of the sponge and facilitate screening of large-scale samples. 90% ofthe bacteria associated with these four sponges were sponge species-specific. S. carnosus was also transferred to an aquarium to study kinetic changes of sponge-associ~tedbacterial communities. DGGE analysis showed 'the consistent presence of some particular bands suggesting the continued presence of species of symbiotic bacteria. Four Bacillus species (B. licheniformis SC-43, B. subtilis SD-8, B. pumilus HP-48 and B. cereus HP-22) isolated from the sponges exhibited antagonistic activity against isolates of Gram-positive bacteria obtained from the same .sponges. All strains tested were active against Micrococcus luteus, strain HP-5!6 isolated from H. panicea. This suggests that HP-5!6 can be used in the laboratory as a sensitive indicator of activity. A comparison of several media found Nutrient Agar! Broth containing glycerol and iron (NGF) to be the best medium tested for antimicrobial , compound production. B. licheniformis (SC-43), B. subtilis (SD-8), and Pantoea sp., SC-AF, in the presence of glycerol and ferric ion, could produce antimicrobial '. compounds when grown within ,biofilms; however, the corresponding shaken flask cultures could not. This effect could be related to oxidative stress defence responses. Pantoea sp., SC-AF produced several antimicrobial compounds active against M luteus, HP-5!6 which were different from previously reported Pantocin antimicrobials. In addition, Pantoea sp., SC-AF produced 'jelly-like' extracellular polysaccharide (EPS) ' on NGF and on the nylon membrane in Air-membrane surface bioreactor (AMS) cultures, along with the production of antimicrobial compounds. Only fructose and cellobiose after acid lysis of EPS of Pantoea sp., SC-AF have been identified. In addition, my study confirmed that sponges accommodate large amounts of uncultured bacteria, whose metabolic capability cannot be explored without cultivation. New cultivation strategies should be investigated and biofilm-based culture techniques incorporated in the future search for novel antibiotics.

No description available.

No description available.

Polyamines are small organic molecules which modulate many physiological processes. Here, an inhibitory effect of spermine on rat carotid body chemoreception is reported. Spermine inhibits catecholamine release, from isolated carotid bodies, induced either by high K+ or by hypoxia. This inhibitory effect could be mediated by: the activation of the Ca2+ sensing receptor (CaR) or the inhibition of the voltage-dependent Ca2+ channels. Measurements of intracellular Ca2+ in dissociated type 1 cells, demonstrated that spermine inhibits Ca2+ influx evoked by either high K+ or hypoxia, but did not affect the resting intracellular Ca2+ levels. Then, the expression of the voltage-dependent Ca2+ channels and CaR were assessed by reverse-transcription polymerase chain reaction and immunochemistry in the carotid body. Cav1.2 and Cav2.2 were found to be especially expressed in type 1 cells while Cav1.3, Cav1.4, Cav2.1, Cav2.3, Cav3.1, Cav3.2 and Cav3.3 could not be detected. CaR was detected only in the nerve ending. Having declined a role of the CaR in mediating the spermine inhibition of type 1 cell chemoreception, the effect of spermine on Cav1.2 was investigated using patch-clamp recording of HEK293 cells transiently or stably expressing human Cav1.2. Spermine inhibits Cav1.2 using 2 mM Ba2+ as a charge carrier but not with 20 mM Ba2+. The inhibition of Cav1.2 by spermine in type 1 cells was then confirmed by co-application with nifedipine using Ca2+ imaging. These experiments demonstrate an inhibitory effect of spermine on Cav1.2 and potentially Cav2.2 in rat type 1 cells. In conclusion, spermine inhibits catecholamine release by type 1 cells, via the direct inhibition of Cav1.2 and possibly Cav2.2. This mechanism could act as a negative feedback on the type 1 cells and limit neurotransmitter release.

Haem is the prosthetic moiety of numerous haemoproteins critical for the function of all aerobic cells. Its biosynthesis is a tightly controlled process since high intracellular haem concentrations are cytotoxic, whilst haem deficiency impedes the activity of essential haemoproteins. In the liver and probably all other non- erythroid cells, haem supply is regulated primarily through feedback regulation of the stability of the mRNA encoding aminolevulinic acid synthase 1 (ALAS1), the first and rate-limiting enzyme in the haem biosynthetic pathway. However, the underlying mechanism of this destabilisation is unknown. Consequently, the primary aim of this thesis was to determine how haem regulates ALAS1 mRNA stability to control its own synthesis in non-erythroid cells, using the human hepatoma cell line, HepG2. In humans, the ALAS1 exon lb in the 5'-untranslated region (UTR) is alternatively spliced to produce a minor and major form of the enzyme. This thesis has demonstrated that unlike the major ALAS1 5'-UTR, the minor 5'-UTR causes a downstream heterologous RNA to be poorly translated. In addition, the minor ALAS1 isoform is relatively resistant to haem-mediated decay. Using reporter assays and RT-PCR, we have shown that the human ALAS1 mRNA contains a coding region determinant (CRD) that mediates its haem-sensitivity in HepG2 cells. This CRD can function independently of the ALAS1 5'- and 3'-UTR. Furthermore, this haem-mediated CRD has to be translated to function. RNA- electromobility shift assays (EMSAs) have defined two fragments of the ALAS1 coding region that can bind to HepG2 cytosolic protein. However, this binding does not seem to be affected by the addition or depletion of haem. From the data presented in this thesis, a mechanism into how haem destabilises the ALAS1 mRNA in humans has been proposed.

Bone turnover is regulated by a cocktail of hormones and signalling factors controlling key cell processes such as proliferation, differentiation, mineralisation and apoptosis. Disruption to the overall mineralisation-resorption balance leads to bone disorders, such as osteoporosis - a 'silent' disease affecting around 7 million people in England and Wales. Ion channels that are presumed targets for bone signalling factors include voltage-gated K channels, ATP-dependent K channels and transient receptor potential TRP channels, and several of these channel-types reportedly have roles in cell proliferation, apoptosis, and differentiation in various tissues. This Thesis shows that human osteoblasts express a number of channels in these families, including maxi-K, ATP-dependent K channels, TRPV1 and TRPM7. The maxi-K channel, displaying characteristic electrophysiological hallmarks, is abundant in patch-clamp recordings of primary human osteoblasts implying a functional role, and the Katp agonist pinacidil is shown to promote osteoblast proliferation. Electrophysiological evidence for the TRPVI channel is not found, although the mRNA signal for a TRPVI splice variant TRPVlb may provide an answer, as it renders the channel less sensitive to capsaicin and protons. However, Ca imaging indicates that osteoblastic TRPV1 channels allow Ca2 influx, and are sensitive to 1 µM capsaicin and protons. In functional studies the TRPVI ligands capsaicin and capsazepine do not influence mineralisation, but interestingly the TRPVI agonists capsaicin, resiniferatoxin and anandamide appear to prevent differentiation of osteoblastic pre-cursor cells to adipocytes, and instead encourage maturation along the osteoblast pathway, whilst TRPV1 antagonists do not affect adipocyte differentiation. In conclusion, a number of K channels and the TRPV1 channel are expressed in osteoblasts and may have important putative roles in osteoblast cell function. Further steps are required to confirm this before the channels can be considered targets for drug development to treat bone disorders.

In the adult, changes in free ionized plasma calcium concentration (Ca2+ G) are monitored by the G-protein-coupled, extracellular calcium-sensing receptor, CaR, but whether CaR plays a role in lung development is unknown. CaR has the potential to be a key regulator of Ca2+ dependent cell fate during development. It is hypothesized that extracellular calcium is an important extrinsic factor that modulates the intrinsic lung developmental programme, through activation of the CaR. CaR is expressed in the developing mouse lung in the pseudoglandular phase, from embryonic day 10.5 (El0.5), with a peak of expression at El2.5 and a subsequent decrease by El8, after which the receptor is absent. Lung branching morphogenesis in vitro is sensitive to Ca2+ G, being negatively modulated by the higher, fetal (i.e., 1.7 mM) Ca2+ 0 yet optimal at physiological adult Ca2+ Q (i.e., 1.05-1.2 mM). Administration of the specific CaR positive allosteric modulator, the calcimimetic R-568, mimics the suppressive effects of high Ca2+G on branching morphogenesis while both phospholipase C and PI3 kinase inhibition reverse these effects. CaR activation suppresses cell proliferation while it enhances lung distension, fluid secretion and intracellular calcium signalling. Conditions which are restrictive to branching and fluid secretion can be rescued by manipulating Ca2+ 0 in the culture medium. Lung explant cultures from the current mouse model of CaR inactivation respond in a similar manner to Ca2+ Q and the calcimimetic R-568. These results indicate the presence of expression of a functional CaR splice variant, which is detected at El 1.5, 12.5 and 15.5 in CaR knockout lungs. The observations presented here support a novel role for the CaR in preventing hyperplastic lung disease in utero and present two potential models for its mode of action within this system.