<p>The present investigation is devoted to studies on porcine members of the cytochrome P450 4A (CYP4A) and CYP8B1 subfamilies, which are involved in bile acid biosynthesis and fatty acid metabolism. </p><p>Hyocholic acid is considered to fulfil the requirements for trihydroxy bile acids in the domestic pig (Sus scrofa) in the absence of cholic acid. Hyocholic acid is a 6α-hydroxylated product of chenodeoxycholic acid and the enzyme catalyzing the 6α-hydroxylation was cloned and found to be an atypical member of the CYP4A subfamily. The primary structure of this porcine enzyme, designated CYP4A21, shows about 75% overall sequence identity to members of the CYP4A subfamily expressed in rabbit and man. Divergent amino acids in a “signature sequence” in the active site of all hitherto known CYP4A fatty acid hydroxylases, were found to be important determinants for the 6α-hydroxylase activity of CYP4A21. </p><p>Two homologous CYP4A fatty acid hydroxylases, designated CYP4A24 and CYP4A25, expressed in pig liver and kidney were cloned. These two cDNAs encode proteins of 504 amino acids similar to CYP4A21. The overall identity between CYP4A24 and CYP4A25 is 97% compared to 94% identity to CYP4A21. Whereas CYP4A21 clearly deviates regarding structural features and catalytic activity it is more difficult to establish whether CYP4A24 and CYP4A25 are distinct enzymes or allelic variants of a single enzyme. </p><p>Cloning of the CYP4A21 gene showed a conserved organization compared to CYP4A genes in other species. A segment of the CYP4A24 gene was also cloned and comparison with the CYP4A21 gene revealed an extensive sequence identity also within introns as well as within the proximal promoter regions. This indicates that CYP4A21 and CYP4A fatty acid hydroxylases have a common origin and evolved by gene duplication. The CYP4A21 and CYP4A fatty acid hydroxylases, however, show distinct patterns of expression.</p><p>The key enzyme in cholic acid biosynthesis, CYP8B1, was markedly expressed in fetal pig liver compared to livers from young pigs. The opposite was shown for the expression of CYP4A21. An apparently conserved pig CYP8B1 gene was cloned and was intronless, similar to CYP8B1 genes from other species. The pig gene encoded a protein of 501 amino acids with 81% identity to CYP8B1 expressed in rabbit and man. Unlike other CYP8B1 genes, the pig promoter lacked a TATA-box. This might offer one explanation for the unusual expression pattern, which appears to be restricted to pig fetal life.</p>

<p>Bikunin, a chondroitin sulfate-containing protein of 25 kDa, has protease inhibitory activity and occurs in the plasma in free and complexed form. In inter-α-inhibitor (IαI) and pre-a-inhibitor (PαI) it is covalently linked through its chondroitin sulfate (CS) chain to two or one other polypeptide of about 80 kDa – heavy chains 1 and 2 (H1, H2) and heavy chain 3 (H3) – respectively. Bikunin and the heavy chains are synthesized as precursors, which are proteolytically cleaved and assembled into IαI and PαI in the secretory pathway. The C-terminal extension (CTX) of the heavy chains seems to mediate its own cleavage and theassembly of the complexes. The heavy chains of the IαI family become transferred to hyaluronan during ovulation and inflammation.</p><p>In this thesis, the biosynthesis of PαI, the plasma clearance of bikunin and the binding of IαI to collagen were studied. We found that in H3, a short segment on the N-terminal side of the CTX cleavage site is required for cleavage. Furthermore, the H3 could become linked to free CS chains primed by a xyloside, showing that the bikunin protein core is not needed for coupling. We also identified His649 as a residue essential for coupling, but not for cleavage. </p><p>Bikunin labelled with a residualizing agent, 125I-tyramine cellobiose, was injected into mice to identify tissues involved in its uptake. Half of the radioactivity was recovered in the kidneys, 10% in the liver, and the rest distributed in other tissues. We determined the half-life of bikunin in rat plasma using two independent methods: injection of 125I-bikunin, or hepatectomy followed by assessing the rate of disappearance of endogenous bikunin. Both methods yielded half-time values of 5-7 minutes. Removal of the CS chain did not affect the clearance rate of bikunin.</p><p>IαI and its heavy chains were found to bind to collagen with dissociation constants greater than 2 μM and 0.4-0.6 μM, respectively and this binding was independent of divalent metal ions. We suggest that the interaction of IαI with collagen may play a modulatory role in cell migration or in remodelling of the extracellular matrix.</p>

<p>Nitrogen is an essential building block of proteins and nucleic acids and, therefore, crucial for the biosphere. Nearly 79 % of the air consists of nitrogen, but in the form of nitrogen gas (N₂), which cannot be utilized by most organisms. Nitrogen-fixing microorganisms such as cyanobacteria have a central role in supplying biologically useful nitrogen to the biosphere. Therefore, it is important to achieve further understanding of control mechanisms involved in nitrogen fixation and related processes. </p><p>This thesis concerns different molecular aspects of the transcription factor NtcA from the heterocystous cyanobacterium <i>Anabaena</i> PCC 7120. Apart from performing oxygenic photosynthesis, <i>Anabaena</i> PCC 7120 is also capable of fixing nitrogen. NtcA is a protein regulating transcription of a wide range of genes and in particular genes involved in cyanobacterial global nitrogen control. NtcA binds as a dimer to the promoter regions of target genes such as those involved in nitrogen fixation and heterocyst differentiation. </p><p>NtcA from <i>Anabaena</i> PCC 7120 was heterologously expressed in <i>E. coli</i> and a high yield of recombinant protein was achieved through purification by Ni-IMAC chromatography. The purified NtcA was used to examine DNA binding motifs preferred by NtcA <i>in vitro </i>using a semi-random library of DNA sequences. The preferred binding sequence for NtcA is TGTA – N₈ – TACA and at least five of the bases in the palindromic binding site are necessary for binding. Differences in the consensus sequence in vivo may reflect variations in the structural conformation of NtcA under various physiological conditions. </p><p>Since an earlier study suggested redox-regulated NtcA-DNA binding the role of the two cysteine residues of NtcA were investigated. Binding studies using three mutants, Cys157Ala, Cys164Ala, and Cys157Ala / Cys164Ala, demonstrated that all these NtcA variants bind to DNA with a slightly higher affinity in the presence of the reducing agent DTT. The studies indicate that the binding mechanism is not dependent on a conformational change of NtcA caused by breaking of intra-molecular disulfide bonds. </p><p>Crystallization followed by structural studies rendered a partial crystal structure of NtcA. The structure verifies that NtcA is a dimeric protein. Each subunit has three domains: the N-terminal domain, a dimerization helix connecting the N-terminal domain with the C-terminal domain, as well as making up the dimer interface, and a C-terminal domain including the DNA binding helix-turn-helix motif.</p><p>Furthermore, an NtcA binding site was found in the promoter region of the<i> hupSL</i> gene, encoding an uptake hydrogenase in <i>Nostoc punctiforme</i> (ATCC 29133), indicating that yet another gene is transcriptionally controlled by NtcA, thereby further emphasizing the multifaceted role of NtcA in cyanobacteria.</p>

<p>This investigation focused on the mRNA expression of drug metabolising Cytochromes P-450 (CYP) and UDP-glucuronosyltransferases (UGT) and the transport protein P-glycoprotein (Pgp) in the small intestine of humans and rats.</p><p>The mRNA expression of the investigated genes in the human small intestine (duodenum) varies between individuals giving each one of us personal profile. In general, the most dominant forms are Pgp, CYPs 2C9, 2D6, 3A4, and UGTs 1A1, 1A10, 2B7. However, which of these is the highest expressed one varies between individuals.</p><p>The correlation in expression between some CYP forms and UGT forms respectively is relatively high, which indicates that they have some regulatory mechanisms in common. It was also shown that the mRNA expression of both CYPs and UGTs may be affected by endogenous and exogenous factors. Sex and ethnic background, affected the mRNA expression of CYP2A6 and 2E1 respectively. Commonly used drugs such as acetylsalicylicacid (ASA) and omeprazole (omep) affect CYP2A6, CYP2E1 (ASA) and CYP3A4, UGT1A4 (omep). The expression of UGT1A4 is also affected by smoking. All these factors are commonly used and can therefore lead to important drug-drug interactions.</p><p>It was also shown that the human small intestinal CYP mRNA expression pattern differs from that found in the rat. The rat CYP expression is rather constant between the different individuals, and the main rat intestinal forms are CYP1A1, CYP2C, CYP2D6 and CYP3A1. The expression is the same for females and males and no difference can be seen between the different segments of the rat small intestine. As metabolic studies have often been done with rat liver we compared the mRNA expression in the two organs. We found that the mRNA expression of 1A1 was absent in the liver and that the CYP2B1, CYP2Cs, CYP2D1 and Pgp all had a stronger mRNA expression in the small intestine compared to the liver. It is therefore important to realise that results from metabolic studies on liver may not be directly extrapolated to the small intestine.</p><p>Artemisinin is an orally used drug in multidrug treatment of malaria in Southeast Asia. It has been suggested that artemisinin can induce drug metabolism and therefore be involved in drug-drug interactions. This study shows that artemisinin induces mainly the CYP2B via nuclear receptor CAR.</p>

<p>A 95-kDa protein was purified from normal human granulocytes. The protein reacted with a monoclonal antibody against CEACAM8. MALDI-Tof and MS/MS analyses revealed the protein to be a CGM6 gene product. Thus, the protein was proved to be identical to CEACAM8. </p><p>An ELISA for CEACAM8 was developed with detection range of 1-64μg/L. Data are presented on the levels of CEACAM8 in the blood of healthy individuals and patients undergoing surgery, as well as in patients with acute infection. The highly elevated levels of CEACAM8 in the blood of these patients were significantly correlated with the surface expression of CEACAM8 on neutrophils and the number of circulating neutrophils, which suggests that CEACAM8 could serve as a biological marker for granulocyte activitiy in vivo. </p><p>The cellular content of CEACAM8 in neutrophils was estimated to be 82.4 ± 8.9 ng/10⁶ cells. Subcellular localisation and mobilisation studies showed that the majority of CEACAM8 is present in the secondary granules of human neutrophils, with a small amount on the plasma membranes. Upon stimulation, CEACAM8 translocated to the plasma membranes from the secondary granules and was also released extracellularly (5.5 ± 0.7% of the total content of CEACAM8).</p><p>In eosinophils, the cellular content of CEACAM8 was estimated to be 73.8 ± 6.0 ng/10⁶ cells. In these cells, CEACAM8 is mainly stored in secretory vesicles. Upon activation, eosinophils released 5.1 ± 1.1% of the total content of CEACAM8. </p><p>Administration of granulocyte colony-stimulating factor (G-CSF) to healthy individuals resulted in an increased content of CEACAM8 in neutrophils on day 1, and decreased on day 4. However, the content of CEACAM8 in light membrane fractions was increased on day 4. The translocation of CEACAM8 observed <i>in vivo</i> after G-CSF administration is probably not directly related to this cytokine but to other cytokines such as TNF-a. </p>

<p>Pre-mRNA splicing and alternative pre-mRNA splicing are key regulatory steps controlling geneexpression in higher eukaryotes. The work in this thesis was focused on a characterization of thesignificance of exonic and intronic splicing enhancer elements for pre-mRNA splicing.</p><p>Previous studies have shown that removal of introns with weak and regulated splice sitesrequire a splicing enhancer for activity. Here we extended these studies by demonstrating thattwo "strong" constitutively active introns, the adenovirus 52,55K and the Drosophila Ftzintrons, are absolutely dependent on a downstream splicing enhancer for activity <i>in vitro</i>.</p><p>Two types splicing enhancers were shown to perform redundant functions as activators ofSplicing. Thus, SR protein binding to an exonic splicing enhancer element or U1 snRNP bindingto a downstream 5'splice site independently stimulated upstream intron removal. The datafurther showed that a 5'splice site was more effective and more versatile in activating splicing.Collectively the data suggest that a U1 enhancer is the prototypical enhancer element activatingsplicing of constitutively active introns.</p><p>Adenovirus IIIa pre-mRNA splicing is enhanced more than 200-fold in infected extracts. Themajor enhancer element responsible for this activation was shown to consist of the IIIa branchsite/polypyrimidne tract region. It functions as a Janus element and blocks splicing in extractsfrom uninfected cells while functioning as a splicing enhancer in the context of infected extracts.</p><p>Phosphorylated SR proteins are essential for pre-mRNA splicing. Large amount recombinantSR proteins are needed in splicing studies. A novel expression system was developed to expressphosphorylated, soluble and functionally active ASF/SF2 in <i>E. Coli</i>.</p>

<p>Heparan sulfate (HS), a linear negatively charged polysaccharide located at the cell surface and in the extracellular matrix, interacts with, and thereby regulates the functions of numerous proteins. HS-protein interactions depend on the fine structure of HS, especially its sulfation pattern. This thesis aimed to understand how differently sulfated domains in HS are generated. Specifically, the substrate specificities of HS hexuronic acid 2-<i>O</i>-sulfotransferase (2OST) and HS glucosaminyl 6-<i>O</i>-sulfotransferases (6OSTs) were investigated. </p><p>Three different 6OSTs (6OST1-3) have been cloned and characterized. To study the mechanisms controlling 6-<i>O</i>-sulfation we incubated the recombinant purified 6-OST isoforms with different 6-<i>O</i>-desulfated poly- and oligosaccharide substrates and the active sulfate donor 3'-phosphoadenosine 5'-phospho[³⁵S]sulfate (³⁵S-labeled PAPS). All three enzymes catalyzed 6-<i>O</i>-sulfation of both <i>N</i>-acetylated (GlcNAc) as well as <i>N</i>-sulfated (GlcNS) glucosamines next to a nonreducing iduronic acid (IdoA) or glucuronic acid (GlcA). Similar specificities were demonstrated, although some differences in substrate preferences were noted.</p><p>To understand how pre-existing 2-<i>O</i>-sulfates affects 6-<i>O</i>-sulfation, 6OST2 and 6OST3 were incubated with pair-wise mixed octasaccharide substrates with different contents of 2-<i>O</i>-sulfates. The specificities for substrates with two or three 2-<i>O</i>-sulfates were higher compared to octasaccharides with no or one 2-<i>O</i>-sulfate indicating that 2-<i>O</i>-sulfate groups substantially promote the subsequent 6-<i>O</i>-sulfation. </p><p>Overexpression of the 6OSTs in a mammalian cell line resulted in increased 6-<i>O</i>-sulfation of -GlcA-GlcNS- and -GlcA-GlcNAc- sequences. The results were not isoform specific, but affected by the overexpression level. </p><p>The 2OST catalyzes 2-<i>O</i>-sulfation of both IdoA and GlcA residues, with high preference for IdoA units. To study how 2-<i>O</i>-sulfation of GlcA and IdoA is regulated, we incubated the enzyme with different substrates and ³⁵S-labeled PAPS. Our findings revealed that the 2OST almost exclusively sulfated IdoA also with a ratio of GlcA to IdoA of 99:1, suggesting that 2-<i>O</i>-sulfation of GlcA occurs before IdoA is formed.</p>

<p>This thesis is focused on enzymatic studies of the secreted aspartic proteases (Saps) from <i>Candida albicans</i> as a tool for discovery of anti-<i>candida</i> drugs. <i>C. albicans</i> causes infections in a number of different locations, which differ widely in the protein substrates available and pH. Since <i>C. albicans</i> needs Saps during virulent growth, these enzymes are good targets for drug development.</p><p>In order to investigate the catalytic characteristics of Saps and their inhibitor affinities, substrate-based kinetic assays were developed. Due to the low sensitivity of these assays, especially at the sub-optimal pH required to mimic the different locations of infections, these assays were not satisfactory. Therefore, a biosensor assay was developed whereby, it was possible to study interaction between Saps and inhibitors without the need to optimise catalytic efficacy. Furthermore, the biosensor assay allowed determination of affinity, as well as the individual association and dissociation rates for inhibitor interactions.</p><p>Knowledge about substrate specificity, Sap subsite adaptivity, and the pH dependencies of catalytic efficacy has been accumulated. Also, screening of transition-state analogue inhibitors designed for HIV-1 protease has revealed inhibitors with affinity for Saps. Furthermore, the kinetics and pH dependencies of their interaction with Saps have been investigated. One of these inhibitors, BEA-440, displayed a complex interaction with Saps, indicating a conformational change upon binding and a very slow dissociation rate. A time dependent interaction was further supported by inhibition measurements. The structural information obtained affords possibilities for design of new more potent inhibitors that might ultimately become drugs against candidiasis. The strategy to combine substrate specificity studies with inhibitor screening has led to complementary results that generate a framework for further development of potent inhibitors.</p>

<p>Macrolides are a large group of clinically relevant antibiotics that inhibit protein synthesis by binding to the large ribosomal subunit in the peptide exit tunnel, close to the peptidyl transferase center (PTC). We have shown that the peptide length of the resulting peptidyl-tRNA drop-off products is proportional to the distance between the PTC and the respective macrolide in the tunnel. This indicates that macrolides act by sterically blocking the nascent peptide exit path.</p><p>A substantial amount of read-through into full-length product was observed for some macrolides and depends on the relation between the dissociation rate constants for peptidyl-tRNA and the macrolide, respectively. The dissociation rate constant for josamycin is 60 times lower than the dissociation rate constant for erythromycin, which explains why no read-through is seen for josamycin in contrast to erythromycin.</p><p>Macrolides do not compete with binding of ternary complexes, hence they are non-competitive inhibitors. However, the text-book description is not valid for macrolide antibiotics, and we show that this is due to the equilibrium assumption generally used to describe non-competitive inhibitors. Our results suggest that a more thorough mechanistic investigation is required to classify inhibitors than what has been proposed previously.</p><p>Further, we have examined the phenomenon of peptide mediated resistance to macrolides. Our results show that expression of a resistance peptide increases the dissociation rate constant for erythromycin.</p><p>In addition, we have examined the accuracy of protein synthesis on three different levels: (<i>i</i>) How do the three initiation factors accomplish fast and accurate initiation of protein synthesis, (<i>ii</i>) how does proof-reading work on the isoleucyl-tRNA synthetase, and (<i>iii</i>) what is the accuracy in the tRNA selection and how is it accomplished? Our data propose a change of the view on all these mechanisms.</p><p>In conclusion this thesis presents new results on protein synthesis, macrolide antibiotics and macrolide resistance.</p>

<p>Megalin is an endocytotic receptor belonging to the low-density lipoprotein family. It has often been viewed only as merely a scavenger receptor of absorptive and secretory epithelia. Recent work has revealed that the megalin intracellular domain contains several motifs potentially binding proteins involved in signal transduction. </p><p>To find potential intracellular proteins binding to megalin, a yeast two-hybrid screening was initiated with the intracellular tail of megalin as the bait. A partial clone encoding the scaffolding protein postsynaptic protein 95 (PSD-95) was found to bind to megalin with its second PDZ-domain. Co-localization experiments in HEK-293 cells and kidney, placenta and parathyroid tissue confirmed this interaction. The PSD-95 related proteins PSD-93 and SAP102 were also confirmed to bind megalin with their PDZ2-domains, but the corresponding domain from SAP97 did not bind. Mutation analysis revealed that an amino acid residue change Ala to Thr was the cause of this.</p><p>Megalin has within the central nervous system (CNS) been shown to be expressed only in the ependymal cells and choroid plexus. Nothing has been known about megalin expression in the spinal cord. To study spatio-temporal expression of megalin in the spinal cord, extensive staining of prenatal and postnatal mouse spinal cord was undertaken. Megalin expression was found in the dorsal part of the embryonic spinal cord. Most of these cells also expressed vimentin, suggesting that megalin has a role in the normal development of astrocytes. In the postnatal mouse, megalin seems to be expressed in oligodendrocytes only in the spinal cord white matter, and co-incident with myelination. This suggests that megalin is involved in the formation and maintenance of myelin along long spinal pathways. Megalin staining was clearly seen in the nucleus of these cells, indicating that megalin works in a notch-like signalling pathway.</p><p>Uptake of retinol to the retina pigment epithelium (RPE) has long been thought to be a diffusion process. Staining for megalin in RPE revealed strong expression, and uptake experiments with 3H-retinol bound to retinol-binding protein and blocking with the LDL-receptor family specific antagonist receptor-associated protein (RAP) showed that megalin is a receptor for uptake of retinol to the RPE.</p>