IGF2 increases de novo steroidogenesis in prostate cancer cells

Androgen-dependent pathways regulate maintenance and growth of normal and malignant prostate tissues. Androgen deprivation therapy (ADT) exploits this dependence and is used to treat metastatic prostate cancer; however, regression initially seen with ADT gives way to development of incurable castration-resistant prostate cancer (CRPC). Although ADT generates a therapeutic response, it is also associated with a pattern of metabolic alterations consistent with metabolic syndrome including elevated circulating insulin. Because CRPC cells are capable of synthesizing androgens de novo, we hypothesized that insulin may also influence steroidogenesis in CRPC. In this study, we examined this hypothesis by evaluating the effect of insulin on steroid synthesis in prostate cancer cell lines. Treatment with 10 nmol/L insulin increased mRNA and protein expression of steroidogenesis enzymes and upregulated the insulin receptor substrate insulin receptor substrate 2 (IRS-2). Similarly, insulin treatment upregulated intracellular testosterone levels and secreted androgens, with the concentrations of steroids observed similar to the levels reported in prostate cancer patients. With similar potency to dihydrotestosterone, insulin treatment resulted in increased mRNA expression of prostate-specific antigen. CRPC progression also correlated with increased expression of IRS-2 and insulin receptor in vivo. Taken together, our findings support the hypothesis that the elevated insulin levels associated with therapeutic castration may exacerbate progression of prostate cancer to incurable CRPC in part by enhancing steroidogenesis.

The human tissue kallikrein and kallikrein-related peptidase family

The human kallikrein-related peptidases are a subgroup of trypsin and chymotrypsin-like serine peptidases that are characterized by their homology to tissue kallikrein or kallikrein 1 (KLK1) encoded by the KLK1 gene (reviewed in[1-4]). The human KLK locus spans an approximately 320 kb region on chromosome 19q13.3-13.4 and contains fifteen genes encoding KLK1 and fourteen other kallikrein-related peptidases, KLK2-KLK15, which have been named contiguously in the locus in the order of their discovery [5-8] (Figure 606.1). It is the largest contiguous cluster of serine protease encoding genes in the human genome which has evolved from gene duplication of KLK1 and then subsequent reduplication of the newly evolved KLK genes [2]. The high conservation noted for KLK1-KLK3 (62-77%) reflects the proposed duplication of the KLK1 gene that produced the KLK2 gene which further generated the KLK3 gene. In contrast, the newer KLK4-KLK15 proteases share much less similarity, from 24-66%, although strong homology between KLK4 and KLK5, KLK9 and KLK11, and KLK10 and KLK12 suggests these genes are duplications of each other [2]...

Hybrid graphite film–carbon nanotube platform for enzyme immobilization and protection

A novel platform consisting of a multilayered substrate, activated graphite-like carbon film, and dense forest of long, vertically-aligned multiwall carbon nanotubes grown by the chemical vapor deposition is designed, fabricated, and tested for covalent immobilization of enzymatic biocatalysts with the aim of protecting them from shear forces and microbial attacks present in bioreactors. The covalent bonding ensures enzyme retention in a flow, while the dense nanotube forest may serve as a protection of the enzymes from microbial attack without impeding the flow of reactants and products. This platform was demonstrated for the two reference enzymes, horseradish peroxidase and catalase, which were immobilized without degrading their biological activity. This combination of an activated carbon layer for an efficient immobilization of biocatalysts with a protective layer of inert carbon nanotubes could dramatically improve the efficiency and longevity of enzymatic bio-catalysis employed in a large variety of advanced biotechnological processes.

Cytochrome p450 1b1, a new keystone in gene-environment interactions related to human head and neck cancer?

Alcohol consumption and tobacco smoking are major causes of head and neck cancers, and regional differences point to the importance of research into gene-environment interactions. Much interest has been focused on polymorphisms of CYP1A1 and of GSTM1 and GSTT1, but a number of studies have not demonstrated significant effects. This has mostly been ascribed to small sample sizes. In general, the impact of polymorphisms of metabolic enzymes appears inconsistent, with some reports of weak-to-moderate associations, and with others of no elevation of risks. The classical cytochrome P450 isoenzyme considered for metabolic activation of polycyclic aromatic hydrocarbons (PAH) is CYP1A1. A new member of the CYP1 family, CYP1B1, was cloned in 1994, currently representing the only member of the CYP1B subfamily. A number of single nucleotide polymorphisms of the CYP1B1 gene have been reported. The amino acid substitutions Val432Leu (CYP1B1*3) and Asn453Ser (CYP1B1*4), located in the heme binding domain of CYP1B1, appear as likely candidates to be linked with biological effects. CYP1B1 activates a wide range of PAH, aromatic and heterocyclic amines. Very recently, the CYP1B1 codon 432 polymorphism (CYP1B1*3) has been identified as a susceptibility factor in smoking-related head-and-neck squamous cell cancer. The impact of this polymorphic variant of CYP1B1 on cancer risk was also reflected by an association with the frequency of somatic mutations of the p53 gene. Combined genotype analysis of CYP1B1 and the glutathione transferases GSTM1 or GSTT1 has pointed to interactive effects. This provides new molecular evidence that tobacco smoke-specific compounds relevant to head and neck carcinogenesis are metabolically activated through CYP1B1 and is consistent with a major pathogenetic relevance of PAH as ingredients of tobacco smoke.

Complement system and alcoholic liver disease

Alcoholic liver disease (ALD) is a well recognized and growing health problem worldwide. ALD advances from fatty liver to inflammation, necrosis, fibrosis and cirrhosis. There is accumulating evidence that the innate immune system is involved in alcoholic liver injury. Within the innate and acquired immune systems, the complement system participates in inflammatory reactions and in the elimination of invading foreign, as well as endogenous apoptotic or injured cells. The present study aimed at evaluating the role of the complement system in the development of alcoholic liver injury. First, in order to study the effects of chronic ethanol intake on the complement system, the deposition of complement components in liver and the expression of liver genes associated with complement in animals with alcohol-induced liver injury were examined. It was demonstrated that chronic alcohol exposure leads to hepatic deposition of the complement components C1, C3, C8 and C9 in the livers of rats. Liver gene expression analysis showed that ethanol up-regulated the expression of transcripts for complement factors B, C1qA, C2, C3 and clusterin. In contrast, ethanol down-regulated the expression of the complement regulators factor H, C4bp and factor D and the terminal complement components C6, C8α and C9. Secondly, the role of the terminal complement pathway in the development of ALD was evaluated by using rats genetically deficient in the complement component C6 (C6-/-). It was found that chronic ethanol feeding induced more liver pathology (steatosis and inflammatory changes) in C6-/- rats than in wild type rats. The hepatic triacylglyceride content and plasma alanine aminotransferase activity increased in C6-/- rats, supporting the histopathological findings and elevation of the plasma pro-/anti-inflammatory TNF-/IL-10 ratio was also more marked in C6-/- rats. Third, the role of the alternative pathway in the development of alcoholic liver steatosis was characterized by using C3-/- mice. In C3-/- mice ethanol feeding tended to reduce steatosis and had no further effect on liver triacylglyceride, liver/body weight ratio nor on liver malondialdehyde level and serum alanine aminotransferase activity. In C3-/- mice alcohol-induced liver steatosis was reduced also after an acute alcohol challenge. In both wild type and C3-/- mice ethanol markedly reduced serum cholesterol and ApoA-I levels, phospholipid transfer protein activity and hepatic mRNA levels of fatty acid binding proteins and fatty acid -oxidation enzymes. In contrast, exclusively in C3-/- mice, ethanol treatment increased serum and liver adiponectin levels but down-regulated the expression of transcripts of lipogenic enzymes, adiponectin receptor 2 and adipose differentiation-related protein and up-regulated phospholipase D1. In conclusion, this study has demonstrated that the complement system is involved in the development of alcohol-induced liver injury. Chronic alcohol exposure causes local complement activation and induction of mRNA expression of classical and alternative pathway components in the liver. In contrast expression of the terminal pathway components and soluble regulators were decreased. A deficient terminal complement pathway predisposes to alcoholic liver damage and promotes a pro-inflammatory cytokine response. Complement component C3 contributes to the development of alcohol-induced fatty liver and its consequences by affecting regulatory and specific transcription factors of lipid homeostasis.

An unusual combination in papaya ( Carica papaya): the good (glucosinolates) and the bad (cyanogenic glycosides)

Glucosinolates are a group of sulphur-containing glycosides found in the plant order Brassicales which includes the Brassica vegetables such as broccoli, cabbage and cauliflower. When brought into contact with the plant enzymes, myrosinases, the glucosinolates break down releasing glucose and other products which serve principally in plant defence against herbivores. The most important of the products from a human nutritional viewpoint, are the isothiocyanates. These potent inducers of detoxifying enzymes bestow the distinct anti-cancer properties on these plants. Unique among tropical fruits, papaya is known to contain an abundance of one particular glucosinolate, glucotropaeolin. Other compounds that play a pivotal role in the chemical defence system of many plants are the cyanogenic glycosides. Cyanogenic glycosides are activated by plant enzymes in the event of pest attack, releasing the deterrent: toxic hydrogen cyanide. Papaya, in addition to glucosinolates, also contains low levels of cyanogenic glycosides, an unusual occurrence because it was assumed that the two classes of metabolites were mutually exclusive. Studies measuring the levels of both in the edible parts of the papaya fruit and other utilised tissues are discussed and considered in the context of potential human health ramifications. All rights reserved, Elsevier.

GDP-L-fucose: synthesis and role in inflammation

GDP-L-fucose: synthesis and role in inflammation The migration of leukocytes from intravascular locations to extravascular sites is essential to the immune responses. The initial attachment of leukocytes to the endothelium and the rolling step of the leukocyte extravasation cascade are mediated by selectins, a family of cell adhesion molecules on cell surfaces. Selectins are able to recognize glycoproteins and glycolipids containing the tetrasaccharide sialyl Lewis x (sLex, Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAc). Several glycosyltransferases are involved in the biosynthesis of sLex, fucosyltransferase VII (Fuc-TVII) being the last enzyme to modify the sLex structure. Fuc-TVII transfers L-fucose from GDP-L-fucose to sialylated N-acetyllactosamine. GDP-L-fucose is synthesized in the cytosol via two different metabolic pathways. The major, constitutively active de novo pathway involves conversion of GDP-α-D-mannose to GDP-β-L-fucose. In the alternative salvage pathway, L-fucokinase synthesizes from free fucose L-fucose-1-phosphate, which is further converted to GDP-L-fucose by GDP-L-fucose pyrophosphorylase. GDP-L-fucose is translocated from the cytosol to Golgi for fucosylation via the GDP-fucose transporter. This thesis involved the study of the synthesis of GDP-L-fucose via the salvage pathway: cloning and expression of murine L-fucokinase and GDP-L-fucose pyrophosphorylase. The gene expression levels of these enzymes were found to be relatively high in various tissues; the mRNA levels were highest in brain, ovary and testis. This study also describes molecular cloning of rat fucosyltransferase VII (FUT7) and its expression as a functional enzyme. Gene expression levels of GDP-L-fucose synthesizing enzymes, GDP-fucose transporter and FUT7 were determined in inflamed tissues as well as cancer cells. Our results revealed a clear upregulation of the enzymes involved in the synthesis of GDP-L-fucose via de novo pathway, GDP-fucose transporter and FUT7 in inflamed tissues and in cancer cells. On the contrary, the GDP-L-fucose salvage pathway was found to be irrelevant in inflammation and in tumorigenesis. Furthermore, our results indicated the transcriptional coregulation of Golgi transporters involved in the synthesis of sulfo sLex, i.e. CMP-sialic acid, GDP-fucose and 3 phosphoadenosine 5 -phosphosulfate transporters, in inflammation.

Proton translocation coupled to electron transfer reactions in terminal oxidases

Terminal oxidases are the final proteins of the respiratory chain in eukaryotes and some bacteria. They catalyze most of the biological oxygen consumption on Earth done by aerobic organisms. During the catalytic reaction terminal oxidases reduce dioxygen to water and use the energy released in this process to maintain the electrochemical proton gradient by functioning as a redox-driven proton pump. This membrane gradient of protons is extremely important for cells as it is used for many cellular processes, such as transportation of substrates and ATP synthesis. Even though the structures of several terminal oxidases are known, they are not sufficient in themselves to explain the molecular mechanism of proton pumping. In this work we have applied a complex approach using a variety of different techniques to address the properties and the mechanism of proton translocation by the terminal oxidases. The combination of direct measurements of pH changes during catalytic turnover, time-resolved potentiometric electrometry and optical spectroscopy, made it possible to obtain valuable information about various aspects of oxidase functioning. We compared oxygen binding properties of terminal oxidases from the distinct heme-copper (CcO) and cytochrome bd families and found that cytochrome bd has a high affinity for oxygen, which is 3 orders of magnitude higher than that of CcO. Interestingly, the difference between CcO and cytochrome bd is not only in higher affinity of the latter to oxygen, but also in the way that each of these enzymes traps oxygen during catalysis. CcO traps oxygen kinetically - the molecule of bound dioxygen is rapidly reduced before it can dissociate. Alternatively, cytochrome bd employs an alternative mechanism of oxygen trapping - part of the redox energy is invested into tight oxygen binding, and the price paid for this is the lack of proton pumping. A single cycle of oxygen reduction to water is characterized by translocation of four protons across the membrane. Our results make it possible to assign the pumping steps to discrete transitions of the catalytic cycle and indicate that during in vivo turnover of the oxidase these four protons are transferred, one at a time, during the P→F, F→OH, Oh→Eh, and Eh→R transitions. At the same time, each individual proton translocation step in the catalytic cycle is not just a single reaction catalyzed by CcO, but rather a complicated sequence of interdependent electron and proton transfers. We assume that each single proton translocation cycle of CcO is assured by internal proton transfer from the conserved Glu-278 to an as yet unidentified pump site above the hemes. Delivery of a proton to the pump site serves as a driving reaction that forces the proton translocation cycle to continue.

Cytochrome P450-mediated drug interactions affecting lidocaine

Lidocaine is a widely used local anaesthetic agent that also has anti-arrhythmic effects. It is classified as a type Ib anti-arrhythmic agent and is used to treat ventricular tachycardia or ventricular fibrillation. Lidocaine is eliminated mainly by metabolism, and less than 5% is excreted unchanged in urine. Lidocaine is a drug with a medium to high extraction ratio, and its bioavailability is about 30%. Based on in vitro studies, the earlier understanding was that CYP3A4 is the major cytochrome P450 (CYP) enzyme involved in the metabolism of lidocaine. When this work was initiated, there was little human data on the effect of inhibitors of CYP enzymes on the pharmacokinetics of lidocaine. Because lidocaine has a low therapeutic index, medications that significantly inhibit lidocaine clearance (CL) could increase the risk of toxicity. These studies investigated the effects of some clinically important CYP1A2 and CYP3A4 inhibitors on the pharmacokinetics of lidocaine administered by different routes. All of the studies were randomized, double-blind, placebo-controlled cross-over studies in two or three phases in healthy volunteers. Pretreatment with clinically relevant doses of CYP3A4 inhibitors erythromycin and itraconazole or CYP1A2 inhibitors fluvoxamine and ciprofloxacin was followed by a single dose of lidocaine. Blood samples were collected to determine the pharmacokinetic parameters of lidocaine and its main metabolites monoethylglycinexylidide (MEGX) and 3-hydroxylidocaine (3-OH-lidocaine). Itraconazole and erythromycin had virtually no effect on the pharmacokinetics of intravenous lidocaine, but erythromycin slightly prolonged the elimination half-life (t½) of lidocaine (Study I). When lidocaine was taken orally, both erythromycin and itraconazole increased the peak concentration (Cmax) and the area under the concentration-time curve (AUC) of lidocaine by 40-70% (Study II). Compared with placebo and itraconazole, erythromycin increased the Cmax and the AUC of MEGX by 40-70% when lidocaine was given intravenously or orally (Studies I and II). The pharmacokinetics of inhaled lidocaine was unaffected by concomitant administration of itraconazole (Study III). Fluvoxamine reduced the CL of intravenous lidocaine by 41% and prolonged the t½ of lidocaine by 35%. The mean AUC of lidocaine increased 1.7-fold (Study IV). After oral administration of lidocaine, the mean AUC of lidocaine in-creased 3-fold and the Cmax 2.2-fold by fluvoxamine (Study V). During the pretreatment with fluvoxamine combined with erythromycin, the CL of intravenous lidocaine was 53% smaller than during placebo and 21% smaller than during fluvoxamine alone. The t½ of lidocaine was significantly longer during the combination phase than during the placebo or fluvoxamine phase. The mean AUC of intravenous lidocaine increased 2.3-fold and the Cmax 1.4-fold (Study IV). After oral administration of lidocaine, the mean AUC of lidocaine increased 3.6-fold and the Cmax 2.5-fold by concomitant fluvoxamine and erythromycin. The t½ of oral lidocaine was significantly longer during the combination phase than during the placebo (Study V). When lidocaine was given intravenously, the combination of fluvoxamine and erythromycin prolonged the t½ of MEGX by 59% (Study IV). Compared with placebo, ciprofloxacin increased the mean Cmax and AUC of intravenous lidocaine by 12% and 26%, respectively. The mean plasma CL of lidocaine was reduced by 22% and its t½ prolonged by 7% (Study VI). These studies clarify the principal role of CYP1A2 and suggest only a modest role of CYP3A4 in the elimination of lidocaine in vivo. The inhibition of CYP1A2 by fluvoxamine considerably reduces the elimination of lidocaine. Concomitant use of fluvoxamine and the CYP3A4 inhibitor erythromycin further increases lidocaine concentrations. The clinical implication of this work is that clinicians should be aware of the potentially increased toxicity of lidocaine when used together with inhibitors of CYP1A2 and particularly with the combination of drugs inhibiting both CYP1A2 and CYP3A4 enzymes.

Pannus invasion into cartilage and bone in rheumatoid arthritis

Rheumatoid arthritis is the most common of all types of arthritis and despite of intensive research etiology of the disease remains unclear. Distinctive features of rheumatic arthritis comprise continuous inflammation of synovium, in which synovial membrane expands on cartilage leading to pannus tissue formation. Pannus formation, appearance of proteolytic enzymes and osteoclast formation cause articular cartilage and bone destruction, which lead to erosions and permanent joint damage. Proteolytic pathways play major roles in the development of tissue lesions in rheumatoid arthritis. Degradation of extracellular matrix proteins is essential to pannus formation and invasion. Matrix metalloproteinases (MMP) form a large proteolytic enzyme family and in rheumatoid arthritis they contribute to pannus invasion by degrading extracellular matrix and to joint destruction by directly degrading the cartilage. MMP-1 and MMP-3 are shown to be increased during cell invasion and also involved in cartilage destruction. Increase of many cytokines has been observed in rheumatoid arthritis, especially TNF-α and IL-1β are studied in synovial tissue and are involved in rheumatoid inflammation and degradation of cartilage. Underlying bone resorption requires first demineralization of bone matrix with acid secreted by osteoclasts, which exposes the collagen-rich matrix for degradation. Cathepsin K is the best known enzyme involved in bone matrix degradation, however deficiency of this protein in pycnodysostosis patient did not prevent bone erosion and on the contrary pannus tissue invading to bone did not expressed much cathepsin K. These indicate that other proteinases are involved in bone degradation, perhaps also via their capability to replace the role of other enzymes especially in diseases like pycnodysostosis or during medication e.g. using cathepsin K inhibitors. Multinuclear osteoclasts are formed also in pannus tissue, which enable the invasion into underlying bone matrix. Pannus tissue express a receptor activator of nuclear factor kappa B ligand (RANKL), an essential factor for osteoclast differentiation and a disintegrin and a metalloproteinase 8 (ADAM8), an osteoclast-activating factors, involved in formation of osteoclast-like giant cells by promoting fusion of mononuclear precursor cells. The understanding of pannus invasion and degradation of extracellular matrix in rheumatic arthritis will open us new more specific methods to prevent this destructive joint disease.

Matrix proteinases in lung injury in the preterm infant

During inflammation, excess production and release of matrix proteinases, including matrix metalloproteinases (MMPs) and serine proteinases, may result in dysregulated extracellular proteolysis leading to development of tissue damage. Pulmonary inflammation may play an important role in the pathogenesis of lung injury in the preterm infant. The aims of this study were to evaluate involvement of MMPs and serine proteinase trypsin in acute and chronic lung injury in preterm infants and to study the role of these enzymes in acute lung injury by means of an animal model of hyperoxic lung injury. Molecular forms and levels of MMP-2, -8, and -9, and their specific inhibitor, tissue inhibitor of metalloproteinases (TIMP)-2, as well as trypsin were studied in tracheal aspirate fluid (TAF) samples collected from preterm infants with respiratory distress. Expression and distribution of trypsin-2 and proteinase-activated receptor 2 (PAR2) was examined in autopsy lung specimens from fetuses, from preterm infants with respiratory distress syndrome (RDS) or bronchopulmonary dysplasia (BPD), and from newborn infants without lung injury. We detected higher MMP-8 and trypsin-2 and lower TIMP-2 in TAF from preterm infants with more severe acute respiratory distress. Infants subsequently developing BPD had higher levels of MMP-8 and trypsin-2 early postnatally than did those who survived without this chronic lung injury. Immunohistochemically, trypsin-2 was mainly detectable in bronchial epithelium, but also in alveolar epithelium, and its expression was strongest in prolonged RDS. Since trypsin-2 is potent activator of PAR2, a G-protein coupled receptor involved in inflammation, we studied PAR2 expression in the lung. PAR2 co-localized with trypsin-2 in bronchoalveolar epithelium and its expression was significantly higher in bronchoalveolar epithelium in preterm infants with prolonged RDS than in newborn controls. In the experimental study, rats were exposed to >95% oxygen for 24, 48, and 60 hours, or room air. At 48 hours of hyperoxia, MMP-8 and trypsin levels sharply increased in bronchoalveolar lavage fluid, and expression of trypsin appeared in alveolar epithelium, and MMP-8 predominantly in macrophages. In conclusion, high pulmonary MMP-8 and trypsin-2 early postnatally are associated with severity of acute lung injury and subsequent development of BPD in preterm infants. In the injured preterm lung, trypsin-2 co-localizes with PAR2 in bronchoalveolar epithelium, suggesting that PAR2 activated by high levels of trypsin-2 is involved in lung inflammation associated with development of BPD. Marked increase in MMP-8 and trypsin early in the course of experimental hyperoxic lung injury suggests that these enzymes play a role in the pathogenesis of acute lung injury. Further exploration of the roles of trypsin and MMP-8 in lung injury may offer new targets for therapeutic intervention.

X-ray and neutron scattering studies on some nanoscale structures in molecular biology

Scattering of X-rays and neutrons has been applied to the study of nanostructures with interesting biological functions. The systems studied were the protein calmodulin and its complexes, bacterial virus bacteriophage phi6, and the photosynthetic antenna complex from green sulfur bacteria, chlorosome. Information gathered using various structure determination methods has been combined to the low resolution information obtained from solution scattering. Conformational changes in calmodulin-ligand complex were studied by combining the directional information obtained from residual dipole couplings in nuclear magnetic resonance to the size information obtained from small-angle X-ray scattering from solution. The locations of non-structural protein components in a model of bacteriophage phi6, based mainly on electron microscopy, were determined by neutron scattering, deuterium labeling and contrast variation. New data are presented on the structure of the photosynthetic antenna complex of green sulfur bacteria and filamentous anoxygenic phototrophs, also known as the chlorosome. The X-ray scattering and electron cryomicroscopy results from this system are interpreted in the context of a new structural model detailed in the third paper of this dissertation. The model is found to be consistent with the results obtained from various chlorosome containing bacteria. The effect of carotenoid synthesis on the chlorosome structure and self-assembly are studied by carotenoid extraction, biosynthesis inhibition and genetic manipulation of the enzymes involved in carotenoid biosynthesis. Carotenoid composition and content are found to have a marked effect on the structural parameters and morphology of chlorosomes.

Two ex situ fungal technologies to treat contaminated soil

Wood-degrading fungi are able to degrade a large range of recalcitrant pollutants which resemble the lignin biopolymer. This ability is attributed to the production of lignin-modifying enzymes, which are extracellular and non-specific. Despite the potential of fungi in bioremediation, there is still an understanding gap in terms of the technology. In this thesis, the feasibility of two ex situ fungal bioremediation methods to treat contaminated soil was evaluated. Treatment of polycyclic aromatic hydrocarbons (PAHs)-contaminated marsh soil was studied in a stirred slurry-phase reactor. Due to the salt content in marsh soil, fungi were screened for their halotolerance, and the white-rot fungi Lentinus tigrinus, Irpex lacteus and Bjerkandera adusta were selected for further studies. These fungi degraded 40 - 60% of a PAH mixture (phenanthrene, fluoranthene, pyrene and chrysene) in a slurry-phase reactor (100 ml) during 30 days of incubation. Thereafter, B. adusta was selected to scale-up and optimize the process in a 5 L reactor. Maximum degradation of dibenzothiophene (93%), fluoranthene (82%), pyrene (81%) and chrysene (83%) was achieved with the free mycelium inoculum of the highest initial biomass (2.2 g/l). In autoclaved soil, MnP was the most important enzyme involved in PAH degradation. In non-sterile soil, endogenous soil microbes together with B. adusta also degraded the PAHs extensively, suggesting a synergic action between soil microbes and the fungus. A fungal solid-phase cultivation method to pretreat contaminated sawmill soil with high organic matter content was developed to enhance the effectiveness of the subsequent soil combustion. In a preliminary screening of 146 fungal strains, 28 out of 52 fungi, which extensively colonized non-sterile contaminated soil, were litter-decomposing fungi. The 18 strains further selected were characterized by their production of lignin-modifying and hydrolytic enzymes, of which MnP and endo-1,4-β-glucanase were the main enzymes during cultivation on Scots pine (Pinus sylvestris) bark. Of the six fungi selected for further tests, Gymnopilus luteofolius, Phanerochaete velutina, and Stropharia rugosoannulata were the most active soil organic matter degraders. The results showed that a six-month pretreatment of sawmill soil would result in a 3.5 - 9.5% loss of organic matter, depending on the fungus applied. The pretreatment process was scaled-up for a 0.56 m3 reactor, in which perforated plastic tubes filled with S. rugosoannulata growing on pine bark were introduced into the soil. The fungal pretreatment resulted in a soil mass loss of 30.5 kg, which represents 10% of the original soil mass (308 kg). Despite the fact that Scots pine bark contains several antimicrobial compounds, it was a suitable substrate for fungal growth and promoter of the production of oxidative enzymes, as well as an excellent and cheap natural carrier of fungal mycelium. This thesis successfully developed two novel fungal ex situ bioremediation technologies and introduce new insights for their further full-scale application. Ex situ slurry-phase fungal reactors might be applied in cases when the soil has a high water content or when the contaminant bioavailability is low; for example, in wastewater treatment plants to remove pharmaceutical residues. Fungal solid-phase bioremediation is a promising remediation technology to ex situ or in situ treat contaminated soil.

Noninvasive biomarkers for early smoking related lung disease

Chronic obstructive pulmonary disease (COPD) is a slowly progressive disease characterized by airway inflammation and largely irreversible airflow limitation. One major risk factor for COPD is cigarette smoking. Since the inflammatory process starts many years prior to the onset of clinical symptoms and still continues after smoking cessation, there is an urgent need to find simple non-invasive biomarkers that can be used in the early diagnosis of COPD and which could help in predicting the disease progression. The first aim of the present study was to evaluate the involvement of different oxidative/nitrosative stress markers, matrix metalloproteinases (MMPs) and their tissue inhibitor-1 (TIMP-1) in smokers and in COPD. Elevated numbers of inducible nitric oxide synthase (iNOS), nitrotyrosine, myeloperoxidase (MPO) and 4-hydroxy-2-nonenal (4-HNE) positive cells and increased levels of 8-isoprostane and lactoferrin were found in sputum of non-symptomatic smokers compared to non-smokers, and especially in subjects with stable mild to moderate COPD, and they correlated with the severity of airway obstruction. This suggests that an increased oxidant burden exists already in the airways of smokers with normal lung function values. However, none of these markers could differentiate healthy smokers from symptomatic smokers with normal lung function values i.e. those individuals who are at risk of developing COPD. In contrast what is known about asthma exhaled nitric oxide (FENO) was lower in smokers than in non-smokers, the reduced FENO value was significantly associated with neutrophilic inflammation and the elevated oxidant burden (positive cells for iNOS, nitrotyrosine and MPO). The levels of sputum MMP-8 and plasma MMP-12 appeared to differentiate subjects who have a risk for COPD development but these finding require further investigations. The levels of all studied MMPs correlated with the numbers of neutrophils, and MMP-8 and MMP-9 with markers of neutrophil activation (MPO, lactoferrin) suggesting that especially neutrophil derived oxidants may stimulate the tissue destructive MMPs already in lungs of smokers who are not yet experiencing any airflow limitation. When investigating the role of neutrophil proteases (neutrophil elastase, MMP-8, MMP-9) during COPD exacerbation and its recovery period, we found that levels of all these proteases were increased in sputum of patients with COPD exacerbation as compared to stable COPD and controls, and decreased during the one-month recovery period, giving evidence for a role of these enzymes in COPD exacerbations. In the last study, the effects of subject`s age and smoking habits were evaluated on the plasma levels of surfactant protein A (SP-A), SP-D, MMP-9 and TIMP-1. Long-term smoking increased the levels of all of these proteins. SP-A most clearly correlated with age, pack years and lung function decline (FEV1/FVC), and based on the receiver operating characteristic curve analysis, SP-A was the best marker for discriminating subjects with COPD from controls. In conclusion, these findings support the hypothesis that especially neutrophil derived oxidants may activate MMPs and induce an active remodeling process already in the lungs of smokers with normal lung function values. The marked increase of sputum levels of neutrophil proteases in smokers, stable COPD and/or during its exacerbations suggest that these enzymes play a role in the development and progression of COPD. Based on the comparison of various biomarkers, SP-A can be proposed to serve as sensitive biomarker in COPD development.

ATP hydrolysis is required for DNA cleavage by EcoPI restriction enzyme

The type III restriction endonuclease EcoPI, coded by bacteriophage Fl, cleaves unmodified DNA in the presence of ATP and magnesium ions. We show that purified EcoPI restriction enzyme fails to cleave DNA in the presence of non-hydrolyzable ATP analogs. More importantly, this study demonstrates that EcoPI restriction enzyme has an inherent ATPase activity, and ATP hydrolysis is necessary for DNA cleavage. Furthermore, we show that the progress curve of the reaction with Eco PI restriction enzyme exhibits a lag which is dependent on the enzyme concentration. Kinetic analysis of the progress curves of the reaction suggest slow transitions that can occur during the reaction, characteristic of hysteretic enzymes. The role of ATP in the cleavage mechanism of type III restriction enzymes is discussed.