Biomarkers, medical treatment and fetal intrapartum surveillance in intrahepatic cholestasis of pregnancy

Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy-specific disorder characterized by maternal pruritus and elevated liver enzymes. It usually begins in the third trimester of pregnancy and resolves spontaneously after delivery. ICP is considered benign for the pregnant woman, but it is associated with an increased risk for unexplained term stillbirth and preterm delivery. There are no specific laboratory markers to diagnose ICP. The diagnosis is currently based on the presence of maternal pruritus and elevated values of alanine aminotransaminases (ALT) and serum bile acids (BA). Recently, ursodeoxycholic acid (UDCA) has been used for treatment. Mechanisms leading to intrauterine fetal death (IUFD) may be multifactorial and are unknown at present. For this thesis, 415 pregnant women with ICP were studied. The aim was to evaluate the value of the liver enzyme glutathione S-transferase alpha (GSTA) as a specific marker of ICP and to assess the effect of maternal UDCA therapy on maternal laboratory values and fetal outcome. The specific markers predisposing the fetus to heart arrhythmia were studied by comparing waveform analysis of fetal electrocardiograms (FECG) during labor in pregnancies complicated by ICP with controls. The levels of maternal GSTA were high and the values correlated with the value of ALT in patients with ICP. UDCA therapy reduced the values of the liver enzymes and alleviated maternal pruritus, but it did not influence maternal hormonal values. Although the newborns experienced an uneventful perinatal outcome, severe ICP was still associated with preterm birth and admission to the neonatal intensive care unit (NICU). There were no significant differences in intrapartum FECG findings between fetuses born to ICP women and controls.

Structural studies on enzymes of biotechnological and biomedical interest

Structural studies of proteins aim at elucidating the atomic details of molecular interactions in biological processes of living organisms. These studies are particularly important in understanding structure, function and evolution of proteins and in defining their roles in complex biological settings. Furthermore, structural studies can be used for the development of novel properties in biomolecules of environmental, industrial and medical importance. X-ray crystallography is an invaluable tool to obtain accurate and precise information about the structure of proteins at the atomic level. Glutathione transferases (GSTs) are amongst the most versatile enzymes in nature. They are able to catalyze a wide variety of conjugation reactions between glutathione (GSH) and non-polar components containing an electrophilic carbon, nitrogen or sulphur atom. Plant GSTs from the Tau class (a poorly characterized class) play an important role in the detoxification of xenobiotics and stress tolerance. Structural studies were performed on a Tau class fluorodifen-inducible glutathione transferase from Glycine max (GmGSTU4-4) complexed with GSH (2.7 Å) and a product analogue Nb-GSH (1.7 Å). The three-dimensional structure of the GmGSTU4-4-GSH complex revealed that GSH binds in different conformations in the two subunits of the dimer: in an ionized form in one subunit and a non-ionized form in the second subunit. Only the ionized form of the substrate may lead to the formation of a catalytically competent complex. Structural comparison between the GSH and Nb-GSH bound complexes revealed significant differences with respect to the hydrogen-bonding, electrostatic interaction pattern, the upper part of -helix H4 and the C-terminus of the enzyme. These differences indicate an intrasubunit modulation between the G-and Hsites suggesting an induced-fit mechanism of xenobiotic substrate binding. A novel binding site on the surface of the enzyme was also revealed. Bacterial type-II L-asparaginases are used in the treatment of haematopoietic diseases such as acute lymphoblastic leukaemia (ALL) and lymphomas due to their ability to catalyze the conversion of L-asparagine to L-aspartate and ammonia. Escherichia coli and Erwinia chrysanthemi asparaginases are employed for the treatment of ALL for over 30 years. However, serious side-effects affecting the liver and pancreas have been observed due to the intrinsic glutaminase activity of the administered enzymes. Structural studies on Helicobacter pylori L-asparaginase (HpA) were carried out in an effort to discover novel L-asparaginases with potential chemotherapeutic utility in ALL treatment. Detailed analysis of the active site geometry revealed structurally significant differences between HpA and other Lasparaginases that may be important for the biological activities of the enzyme and could be further exploited in protein engineering efforts.

Glutathione Transferases as Detoxification Agents: Structural and Functional Studies

Glutathione transferases (GSTs) are a diverse family of enzymes that catalyze the glutathione-dependent detoxification of toxic compounds. GSTs are responsible for the conjugation of the tripeptide glutathione (GSH) to a wide range of electrophilic substrates. These include industrial pollutants, drugs, genotoxic carcinogen metabolites, antibiotics, insecticides and herbicides. In light of applications in biomedicine and biotechnology as cellular detoxification agents, detailed structural and functional studies of GSTs are required. Plant tau class GSTs play crucial catalytic and non-catalytic roles in cellular xenobiotic detoxification process in agronomically important crops. The abundant existence of GSTs in Glycine max and their ability to provide resistance to abiotic and biotic stresses such as herbicide tolerance is of great interest in agriculture because they provide effective and suitable tools for selective weed control. Structural and catalytic studies on tau class GST isoenzymes from Glycine max (GmGSTU10-10, GmGSTU chimeric clone 14 (Sh14), and GmGSTU2-2) were performed. Crystal structures of GmGSTU10-10 in complex with glutathione sulfenic acid (GSOH) and Sh14 in complex with S-(p-nitrobenzyl)-glutathione (Nb-GSH) were determined by molecular replacement at 1.6 Å and 1.75 Å, respectively. Major structural variations that affect substrate recognition and catalytic mechanism were revealed in the upper part of helix H4 and helix H9 of GmGSTU10-10. Structural analysis of Sh14 showed that the Trp114Cys point mutation is responsible for the enhanced catalytic activity of the enzyme. Furthermore, two salt bridges that trigger an allosteric effect between the H-sites were identified at the dimer interface between Glu66 and Lys104. The 3D structure of GmGSTU2-2 was predicted using homology modeling. Structural and phylogenetic analysis suggested GmGSTU2-2 shares residues that are crucial for the catalytic activity of other tau class GSTs–Phe10, Trp11, Ser13, Arg20, Tyr30, Leu37, Lys40, Lys53, Ile54, Glu66 and Ser67. This indicates that the catalytic and ligand binding site in GmGSTU2-2 are well-conserved. Nevertheless, at the ligandin binding site a significant variation was observed. Tyr32 is replaced by Ser32 in GmGSTU2-2 and thismay affect the ligand recognition and binding properties of GmGSTU2-2. Moreover, docking studies revealed important amino acid residues in the hydrophobic binding site that can affect the substrate specificity of the enzyme. Phe10, Pro12, Phe15, Leu37, Phe107, Trp114, Trp163, Phe208, Ile212, and Phe216 could form the hydrophobic ligand binding site and bind fluorodifen. Additionally, side chains of Arg111 and Lys215 could stabilize the binding through hydrogen bonds with the –NO2 groups of fluorodifen. GST gene family from the pathogenic soil bacterium Agrobacterium tumefaciens C58 was characterized and eight GST-like proteins in A. tumefaciens (AtuGSTs) were identified. Phylogenetic analysis revealed that four members of AtuGSTs belong to a previously recognized bacterial beta GST class and one member to theta class. Nevertheless, three AtuGSTs do not belong to any previously known GST classes. The 3D structures of AtuGSTs were predicted using homology modeling. Comparative structural and sequence analysis of the AtuGSTs showed local sequence and structural characteristics between different GST isoenzymes and classes. Interactions at the G-site are conserved, however, significant variations were seen at the active site and the H5b helix at the C-terminal domain. H5b contributes to the formation of the hydrophobic ligand binding site and is responsible for recognition of the electrophilic moiety of the xenobiotic. It is noted that the position of H5b varies among models, thus providing different specificities. Moreover, AtuGSTs appear to form functional dimers through diverse modes. AtuGST1, AtuGST3, AtuGST4 and AtuGST8 use hydrophobic ‘lock–and–key’-like motifs whereas the dimer interface of AtuGST2, AtuGST5, AtuGST6 and AtuGST7 is dominated by polar interactions. These results suggested that AtuGSTs could be involved in a broad range of biological functions including stress tolerance and detoxification of toxic compounds.

Imaging techniques applied for detection of secretion, transgene delivery and G proteincoupled receptors in reproductive and endocrine cells in vivo and in vitro

Post-testicular sperm maturation occurs in the epididymis. The ion concentration and proteins secreted into the epididymal lumen, together with testicular factors, are believed to be responsible for the maturation of spermatozoa. Disruption of the maturation of spermatozoa in the epididymis provides a promising strategy for generating a male contraceptive. However, little is known about the proteins involved. For drug development, it is also essential to have tools to study the function of these proteins in vitro. One approach for screening novel targets is to study the secretory products of the epididymis or the G protein-coupled receptors (GPCRs) that are involved in the maturation process of the spermatozoa. The modified Ca2+ imaging technique to monitor release from PC12 pheochromocytoma cells can also be applied to monitor secretory products involved in the maturational processes of spermatozoa. PC12 pheochromocytoma cells were chosen for evaluation of this technique as they release catecholamines from their cell body, thus behaving like endocrine secretory cells. The results of the study demonstrate that depolarisation of nerve growth factor -differentiated PC12 cells releases factors which activate nearby randomly distributed HEL erythroleukemia cells. Thus, during the release process, the ligands reach concentrations high enough to activate receptors even in cells some distance from the release site. This suggests that communication between randomly dispersed cells is possible even if the actual quantities of transmitter released are extremely small. The development of a novel method to analyse GPCR-dependent Ca2+ signalling in living slices of mouse caput epididymis is an additional tool for screening for drug targets. By this technique it was possible to analyse functional GPCRs in the epithelial cells of the ductus epididymis. The results revealed that, both P2X- and P2Y-type purinergic receptors are responsible for the rapid and transient Ca2+ signal detected in the epithelial cells of caput epididymides. Immunohistochemical and reverse transcriptase-polymerase chain reaction (RTPCR) analyses showed the expression of at least P2X1, P2X2, P2X4 and P2X7, and P2Y1 and P2Y2 receptors in the epididymis. Searching for epididymis-specific promoters for transgene delivery into the epididymis is of key importance for the development of specific models for drug development. We used EGFP as the reporter gene to identify proper promoters to deliver transgenes into the epithelial cells of the mouse epididymis in vivo. Our results revealed that the 5.0 kb murine Glutathione peroxidase 5 (GPX5) promoter can be used to target transgene expression into the epididymis while the 3.8 kb Cysteine-rich secretory protein-1 (CRISP-1) promoter can be used to target transgene expression into the testis. Although the visualisation of EGFP in living cells in culture usually poses few problems, the detection of EGFP in tissue sections can be more difficult because soluble EGFP molecules can be lost if the cell membrane is damaged by freezing, sectioning, or permeabilisation. Furthermore, the fluorescence of EGFP is dependent on its conformation. Therefore, fixation protocols that immobilise EGFP may also destroy its usefulness as a fluorescent reporter. We therefore developed a novel tissue preparation and preservation techniques for EGFP. In addition, fluorescence spectrophotometry with epididymal epithelial cells in suspension revealed the expression of functional purinergic, adrenergic, cholinergic and bradykinin receptors in these cell lines (mE-Cap27 and mE-Cap28). In conclusion, we developed new tools for studying the role of the epididymis in sperm maturation. We developed a new technique to analyse GPCR dependent Ca2+ signalling in living slices of mouse caput epididymis. In addition, we improved the method of detecting reporter gene expression. Furthermore, we characterised two epididymis-specific gene promoters, analysed the expression of GPCRs in epididymal epithelial cells and developed a novel technique for measurement of secretion from cells.