Purification and crystallization of the hydroxylase component of the methanesulfonate monooxygenase from Methylosulfonomonas methylovora strain M2

The aim of the work described in this paper was two-fold: (1) the purification of the hydroxylase component of the MSAMO to electrophoretic homogeneity using a four-step chromatographic strategy and (2) the crystallization of the two-component hydroxylase of the MSAMO in order to enhance our understanding of the precise three-dimensional structure of the MSAMO, thus yielding an insight into the nature of the active site of this enzyme. Optimised crystallization conditions were identified allowing growth of crystals of the hydroxylase component of the MSAMO within five days. Crystals exhibited a brown colour suggesting the presence on an intact Rieske-iron sulfur centre and diffracted to 7.0 Å when a few degrees of data were evaluated on a beam line X11. © 2006 Elsevier Inc. All rights reserved.

The effect of BCMO1 gene variants on macular pigment optical density in young healthy Caucasians

Background Serum lutein (L) and zeaxanthin (Z) positively correlate with macular pigment optical density (MPOD), hence the latter is a valuable indirect tool for measuring L and Z content in the macula. L and Z have been attributed antioxidant capacity and protection from certain retinal diseases but their uptake within the eye is thought to depend on genetic, age and environmental factors. In particular gene variants within beta-carotene monooxygenase (BCMO1) are thought to modulate MPOD in the macula. Objectives: To determine the effect of BCMO1 single nucleotide polymorphisms (SNPs) rs11645428, rs6420424 and rs6464851 on macular pigment optical density (MPOD) in a cohort of young healthy participants of Caucasian origin with normal ocular health. Design In this cohort study, MPOD was assessed in 46 healthy participants (22 male and 24 female) with a mean age of 24 ± 4.0 years (range 19-33). The three SNPs, rs11645428, rs6420424, rs6564851 that have established associations with MPOD were determined using MassEXTEND (hME) Sequenom assay. One-way analysis of variance (ANOVA) was performed on groups segregated into homozygous and heterozygous BCMO1 genotypes. Correlations between body mass index (BMI), iris colour, gender, central retinal thickness (CRT), diet and MPOD were investigated. Results MPOD did not significantly vary with BCMO1 rs11645428 (F2,41 = 0.700, p = 0.503), rs6420424 (F2,41 = 0.210, p = 0.801) nor rs6464851 homozygous or heterozygous genotypes (F2,41 = 0,13, p = 0.88), in this young healthy cohort. The combination of these three SNPs into triple genotypes based on plasma conversion efficiency did not affect MPOD (F2,41 = 0.07, p = 0.9). There was a significant negative correlation with MPOD and central retinal thickness (r = - 0.39, p = 0.01) but no significant correlation between BMI, iris colour, gender and MPOD. Conclusion Our results indicate that macular pigment deposition within the central retina is not dependent on BCMO1 gene variants in young healthy people. We propose that MPOD is saturated in younger persons and/or other gene variant combinations determine its deposition.

Genome-wide association identifies genetic variants associated with lentiform nucleus volume in N = 1345 young and elderly subjects

Deficits in lentiform nucleus volume and morphometry are implicated in a number of genetically influenced disorders, including Parkinson's disease, schizophrenia, and ADHD. Here we performed genome-wide searches to discover common genetic variants associated with differences in lentiform nucleus volume in human populations. We assessed structural MRI scans of the brain in two large genotyped samples: the Alzheimer's Disease Neuroimaging Initiative (ADNI; N = 706) and the Queensland Twin Imaging Study (QTIM; N = 639). Statistics of association from each cohort were combined meta-analytically using a fixed-effects model to boost power and to reduce the prevalence of false positive findings. We identified a number of associations in and around the flavin-containing monooxygenase (FMO) gene cluster. The most highly associated SNP, rs1795240, was located in the FMO3 gene; after meta-analysis, it showed genome-wide significant evidence of association with lentiform nucleus volume (PMA = 4. 79 × 10-8). This commonly-carried genetic variant accounted for 2. 68 % and 0. 84 % of the trait variability in the ADNI and QTIM samples, respectively, even though the QTIM sample was on average 50 years younger. Pathway enrichment analysis revealed significant contributions of this gene to the cytochrome P450 pathway, which is involved in metabolizing numerous therapeutic drugs for pain, seizures, mania, depression, anxiety, and psychosis. The genetic variants we identified provide replicated, genome-wide significant evidence for the FMO gene cluster's involvement in lentiform nucleus volume differences in human populations.

Genome-wide association study identifies eight loci associated with blood pressure

Elevated blood pressure is a common, heritable cause of cardiovascular disease worldwide. To date, identification of common genetic variants influencing blood pressure has proven challenging. We tested 2.5 million genotyped and imputed SNPs for association with systolic and diastolic blood pressure in 34,433 subjects of European ancestry from the Global BPgen consortium and followed up findings with direct genotyping (N 71,225 European ancestry, N 12,889 Indian Asian ancestry) and in silico comparison (CHARGE consortium, N = 29,136). We identified association between systolic or diastolic blood pressure and common variants in eight regions near the CYP17A1 (P = 7 × 10 24), CYP1A2 (P = 1 × 10 23), FGF5 (P = 1 × 10 21), SH2B3 (P = 3 × 10 18), MTHFR (P = 2 × 10 13), c10orf107 (P = 1 × 10 9), ZNF652 (P = 5 × 10 9) and PLCD3 (P = 1 × 10 8) genes. All variants associated with continuous blood pressure were associated with dichotomous hypertension. These associations between common variants and blood pressure and hypertension offer mechanistic insights into the regulation of blood pressure and may point to novel targets for interventions to prevent cardiovascular disease.

Spiroacetal biosynthesis in insects from diptera to hymenoptera: The Giant Ichneumon wasp Megarhyssa nortoni nortoni Cresson

The volatile components of the mandibular gland secretion generated by the Giant Ichneumon parasitoid wasp Megarhyssa nortoni nortoni Cresson are mainly spiroacetals and methyl ketones, and all have an odd number of carbon atoms. A biosynthetic scheme rationalizing the formation of these diverse components is presented. This scheme is based on the results of incorporation studies using 2H-labeled precursors and [18O]dioxygen. The key steps are postulated to be decarboxylation of β-ketoacid equivalents, β-oxidation (chain shortening), and monooxygenase-mediated hydroxylation leading to a putative ketodiol that cyclizes to spiroacetals. The generality of the role of monooxygenases in spiroacetal formation in insects is considered, and overall, a cohesive, internally consistent theory of spiroacetal generation by insects is presented, against which future hypotheses will have to be compared.

Processing of Stimulus Repetition and Change in the Somatosensory System: Recordings of Electrical and Magnetic Brain Responses

In the present work, effects of stimulus repetition and change in a continuous stimulus stream on the processing of somatosensory information in the human brain were studied. Human scalp-recorded somatosensory event-related potentials (ERPs) and magnetoencephalographic (MEG) responses rapidly diminished with stimulus repetition when mechanical or electric stimuli were applied to fingers. On the contrary, when the ERPs and multi-unit a ctivity (MUA) were directly recorded from the primary (SI) and secondary (SII) somatosensory cortices in a monkey, there was no marked decrement in the somatosensory responses as a function of stimulus repetition. These results suggest that this rate effect is not due to the response diminution in the SI and SII cortices. Obviously the responses to the first stimulus after a long "silent" period are nhanced due to unspecific initial orientation, originating in more broadly distributed and/or deeper neural structures, perhaps in the prefrontal cortices. With fast repetition rates not only the late unspecific but also some early specific somatosensory ERPs were diminished in amplitude. The fast decrease of the ERPs as a function of stimulus repetition is mainly due to the disappearance of the orientation effect and with faster repetition rates additively due to stimulus specific refractoriness. A sudden infrequent change in the continuous stimulus stream also enhanced somatosensory MEG responses to electric stimuli applied to different fingers. These responses were quite similar to those elicited by the deviant stimuli alone when the frequent standard stimuli were omitted. This enhancement was obviously due to the release from refractoriness because the neural structures generating the responses to the infrequent deviants had more time to recover from the refractoriness than the respective structures for the standards. Infrequent deviant mechanical stimuli among frequent standard stimuli also enhanced somatosensory ERPs and, in addition, they elicited a new negative wave which did not occur in the deviants-alone condition. This extra negativity could be recorded to deviations in the stimulation site and in the frequency of the vibratory stimuli. This response is probably a somatosensory analogue of the auditory mismatch negativity (MMN) which has been suggested to reflect a neural mismatch process between the sensory input and the sensory memory trace.

Androgenetic alopecia: Identification of four genetic risk loci and evidence for the contribution of WNT signaling to its etiology

The pathogenesis of androgenetic alopecia (AGA, male-pattern baldness) is driven by androgens, and genetic predisposition is the major prerequisite. Candidate gene and genome-wide association studies have reported that single-nucleotide polymorphisms (SNPs) at eight different genomic loci are associated with AGA development. However, a significant fraction of the overall heritable risk still awaits identification. Furthermore, the understanding of the pathophysiology of AGA is incomplete, and each newly associated locus may provide novel insights into contributing biological pathways. The aim of this study was to identify unknown AGA risk loci by replicating SNPs at the 12 genomic loci that showed suggestive association (5 x 10(-8)

Engineering the pentose phosphate pathway of Saccharomyces cerevisiae for production of ethanol and xylitol

The baker s yeast Saccharomyces cerevisiae has a long tradition in alcohol production from D-glucose of e.g. starch. However, without genetic modifications it is unable to utilise the 5-carbon sugars D-xylose and L arabinose present in plant biomass. In this study, one key metabolic step of the catabolic D-xylose pathway in recombinant D-xylose-utilising S. cerevisiae strains was studied. This step, carried out by xylulokinase (XK), was shown to be rate-limiting, because overexpression of the xylulokinase-encoding gene XKS1 increased both the specific ethanol production rate and the yield from D xylose. In addition, less of the unwanted side product xylitol was produced. Recombinant D-xylose-utilizing S. cerevisiae strains have been constructed by expressing the genes coding for the first two enzymes of the pathway, D-xylose reductase (XR) and xylitol dehydrogenase (XDH) from the D-xylose-utilising yeast Pichia stipitis. In this study, the ability of endogenous genes of S. cerevisiae to enable D-xylose utilisation was evaluated. Overexpression of the GRE3 gene coding for an unspecific aldose reductase and the ScXYL2 gene coding for a xylitol dehydrogenase homologue enabled growth on D-xylose in aerobic conditions. However, the strain with GRE3 and ScXYL2 had a lower growth rate and accumulated more xylitol compared to the strain with the corresponding enzymes from P. stipitis. Use of the strictly NADPH-dependent Gre3p instead of the P. stipitis XR able to utilise both NADH and NADPH leads to a more severe redox imbalance. In a S. cerevisiae strain not engineered for D-xylose utilisation the presence of D-xylose increased xylitol dehydrogenase activity and the expression of the genes SOR1 or SOR2 coding for sorbitol dehydrogenase. Thus, D-xylose utilisation by S. cerevisiae with activities encoded by ScXYL2 or possibly SOR1 or SOR2, and GRE3 is feasible, but requires efficient redox balance engineering. Compared to D-xylose, D-glucose is a cheap and readily available substrate and thus an attractive alternative for xylitol manufacture. In this study, the pentose phosphate pathway (PPP) of S. cerevisiae was engineered for production of xylitol from D-glucose. Xylitol was formed from D-xylulose 5-phosphate in strains lacking transketolase activity and expressing the gene coding for XDH from P. stipitis. In addition to xylitol, ribitol, D-ribose and D-ribulose were also formed. Deletion of the xylulokinase-encoding gene increased xylitol production, whereas the expression of DOG1 coding for sugar phosphate phosphatase increased ribitol, D-ribose and D-ribulose production. Strains lacking phosphoglucose isomerase (Pgi1p) activity were shown to produce 5 carbon compounds through PPP when DOG1 was overexpressed. Expression of genes encoding glyceraldehyde 3-phosphate dehydrogenase of Bacillus subtilis, GapB, or NAD-dependent glutamate dehydrogenase Gdh2p of S. cerevisiae, altered the cellular redox balance and enhanced growth of pgi1 strains on D glucose, but co-expression with DOG1 reduced growth on higher D-glucose concentrations. Strains lacking both transketolase and phosphoglucose isomerase activities tolerated only low D-glucose concentrations, but the yield of 5-carbon sugars and sugar alcohols on D-glucose was about 50% (w/w).

USING FLUORESCENT AND NON-FLUORESCENT STEROLS TO STUDY RAFTS AND INTRACELLULAR CHOLESTEROL TRANSPORT IN MAMMALIAN CELLS

Cholesterol is an essential component in the membranes of most eukaryotic cells, in which it mediates many functions including membrane fluidity, permeability and the formation of ordered membrane domains. In this work a fluorescent and a non-fluorescent cholesterol analog were characterized as tools to study cholesterol. Next, these analogs were used to study two specific cell biological processes that involve cholesterol, i.e. the structure and function of ordered membrane domains/rafts and intracellular cholesterol transport. The most common method for studying ordered membrane domains is by disrupting them by cholesterol depletion. Because cholesterol depletion affects many cellular functions besides those mediated by membrane domains, this procedure is highly unspecific. The cellular exchange of cholesterol by desmosterol as a tool to study ordered membrane domains was characterized. It turned out that the ability of desmosterol to form and stabilize membrane domains in vitro was weaker compared to cholesterol. This result was reinforced by atomistic scale simulations that indicated that desmosterol has a lower ordering effect on phospholipid acyl chains. Three procedures were established for exchanging cellular cholesterol by desmosterol. In cells in which desmosterol was the main sterol, insulin signaling was attenuated. The results suggest that this was caused by desmosterol destabilizing membrane rafts. Contrary to its effect on ordered membrane domains it was found that replacing cholesterol by desmosterol does not change cell growth/viability, subcellular sterol distribution, Golgi integrity, secretory pathway, phospholipid composition and membrane fluidity. Together these results suggest that exchanging cellular cholesterol by desmosterol provides a selective tool for perturbing rafts. Next, the importance of cholesterol for the structure and function of caveolae was analyzed by exchanging the cellular cholesterol by desmosterol. The sterol exchange reduced the stability of caveolae as determined by detergent resistance of caveolin-1 and heat resistance of caveolin-1 oligomers. Also the sterol exchange led to aberrations in the caveolar structure; the morphology of caveolae was altered and there was a larger variation in the amount of caveolin-1 molecules per caveola. These results demonstrate that cholesterol is important for caveolar stability and structural homogeneity. In the second part of this work a fluorescent cholesterol analog was characterized as a tool to study cholesterol transport. Tight control of the intracellular cholesterol distribution is essential for many cellular processes. An important mechanism by which cells regulate their membrane cholesterol content is by cholesterol traffic, mostly from the plasma membrane to lipid droplets. The fluorescent sterol probe BODIPY-cholesterol was characterized as a tool to analyze cholesterol transport between the plasma membrane, the endoplasmic reticulum (ER) and lipid droplets. The behavior of BODIPY-cholesterol was compared to that of natural sterols, using both biochemical and live-cell microcopy assays. The results show that the transport kinetics of BODIPY-cholesterol between the plasma membrane, the ER and lipid droplets is similar to that of unesterified cholesterol. Next, BODIPY-cholesterol was utilized to analyze the importance of oxysterol binding protein related proteins (ORPs) for cholesterol transport between the plasma membrane, the ER, and lipid droplets in mammalian cells. By overexpressing all human ORPs it turned out that especially ORP1S and ORP2 enhanced sterol transport from the plasma membrane to lipid droplets. Our results suggest that the increased sterol transport takes place between the plasma membrane and ER and not between the ER and lipid droplets. Simultaneous knockdown of ORP1S and ORP2 resulted in a moderate but significant inhibition of sterol traffic from the plasma membrane to ER and lipid droplets, suggesting a physiological role for these ORPs in this process. The two phenylalanines in an acidic tract (FFAT) motif in ORPs, which mediates interaction with vesicle associated membrane protein associated proteins (VAPs) in the ER, was not necessary for mediating sterol transport. However, VAP silencing slowed down sterol transport, most likely by destabilizing ORPs containing a FFAT motif.

Purification and properties of 4-hydroxyphenylacetic acid 3-hydroxylase from pseudomonas putida

4-Hydroxyphenylacetic acid 3-hydroxylase is a key enzyme in the pathway for the microbial degradation of phenylalanine, tyrosine and many aromatic amines. This enzyme was purified to homogeneity from Image by affinity chromatography. The protein had a molecular weight of 91,000 and was a dimer of identical subunits. It was a typical external flavoprotein monooxygenase and showed an absolute requirement of NADH for activity. The enzyme had a pH optimum of 7.5 and the Km values for 4-hydroxyphenylacetic acid and NADH were 2×10−4 M and 5.9×10−5 M respectively. It was strongly inhibited by heavy metal ions and thiol reagents, suggesting the possible involvement of -SH group(s) in enzyme reaction.

Affinity purification and characterization of 2,4-dichlorophenol hydroxylase from Pseudomonas cepacia

2,4-Dichlorophenol hydroxylase, a flavoprotein monooxygenase from Pseudomonas cepacia grown on 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole source of carbon, was purified to homogeneity by a single-step affinity chromatography on 2,4-DCP-Sepharose CL-4B. The enzyme was eluted from the affinity matrix with the substrate 2,4-dichlorophenol. The enzyme has a molecular weight of 275,000 consisting of four identical subunits of molecular weight 69,000 and requires exogenous addition of FAD for its complete catalytic activity. The enzyme required an external electron donor NADPH for hydroxylation of 2,4-dichlorophenol to 3,5-dicholorocatechol. NADPH was preferred over NADH. The enzyme had Km value of 14 μImage for 2,4-dichlorophenol, and 100 μImage for NADPH. The enzyme activity was significantly inhibited by heavy metal ions like Hg2+ and Zn2+ and showed marked inhibition with thiol reagents. Trichlorophenols inhibited the enzyme competitively. The hydroxylase activity decreased as a function of increasing concentrations of Cibacron blue and Procion red dyes. The apoenzyme prepared showed complete loss of FAD when monitored spectrophotometrically and had no enzymatic activity. The inactive apoenzyme was reconstituted with exogenous FAD which completely restored the enzyme activity.

Hantavirus infection: Insights into entry, assembly and pathogenesis

Hantaviruses (family Bunyaviridae, genus Hantavirus) are enveloped viruses incorporating a segmented, negative-sense RNA genome. Each hantavirus is carried by its specific host, either a rodent or an insectivore (shrew), in which the infection is asymptomatic and persistent. In humans, hantaviruses cause Hemorrhagic fever with renal syndrome (HFRS) in Eurasia and Hantavirus cardiopulmonary syndrome (HCPS) in the Americas. In Finland, Puumala virus (genus Hantavirus) is the causative agent of NE, a mild form of HFRS. The HFRS-type diseases are often associated with renal failure and proteinuria that might be mechanistically explained by infected kidney tubular cell degeneration in patients. Previously, it has been shown that non-pathogenic hantavirus, Tula virus (TULV), could cause programmed cell death, apoptosis, in cell cultures. This suggested that the infected kidney tubular degeneration could be caused directly by virus replication. In the first paper of this thesis the molecular mechanisms involved in TULV-induced apoptosis was further elucidated. A virus replication-dependent down-regulation of ERK1/2, concomitantly with the induced apoptosis, was identified. In addition, this phenomenon was not restricted to TULV or to non-pathogenic hantaviruses in general since also a pathogenic hantavirus, Seoul virus, could inhibit ERK1/2 activity. Hantaviruses consist of membrane-spanning glycoproteins Gn and Gc, RNA-dependent RNA polymerase (L protein) and nucleocapsid protein N, which encapsidates the viral genome, and thus forms the ribonucleoprotein (RNP). Interaction between the cytoplasmic tails of viral glycoproteins and RNP is assumed to be the only means how viral genetic material is incorporated into infectious virions. In the second paper of this thesis, it was shown by immunoprecipitation that viral glycoproteins and RNP interact in the purified virions. It was further shown that peptides derived from the cytoplasmic tails (CTs) of both Gn and Gc could bind RNP and recombinant N protein. In the fourth paper the cytoplamic tail of Gn but not Gc was shown to interact with genomic RNA. This interaction was probably rather unspecific since binding of Gn-CT with unrelated RNA and even single-stranded DNA were also observed. However, since the RNP consists of both N protein and N protein-encapsidated genomic RNA, it is possible that the viral genome plays a role in packaging of RNPs into virions. On the other hand, the nucleic acid-binding activity of Gn may have importance in the synthesis of viral RNA. Binding sites of Gn-CT with N protein or nucleic acids were also determined by peptide arrays, and they were largely found to overlap. The Gn-CT of hantaviruses contain a conserved zinc finger (ZF) domain with an unknown function. Some viruses need ZFs in entry or post-entry steps of the viral life cycle. Cysteine residues are required for the folding of ZFs by coordinating zinc-ions, and alkylation of these residues can affect virus infectivity. In the third paper, it was shown that purified hantavirions could be inactivated by treatment with cysteine-alkylating reagents, especially N-ethyl maleimide. However, the effect could not be pin-pointed to the ZF of Gn-CT since also other viral proteins reacted with maleimides, and it was, therefore, impossible to exclude the possibility that other cysteines besides those that were essential in the formation of ZF are required for hantavirus infectivity.

Medetomidiinin glukuronidaation inhibitio

Lääkeainemetabolialla tarkoitetaan entsymaattisia reaktioita, jotka muuttavat lääkeaineita paremmin elimistöstä poistuvaan muotoon. Lääkeaineet voivat vaikuttaa toistensa metaboliaan inhiboimalla tai indusoimalla metaboloivia entsyymejä. Tällaisten interaktioiden seurauksena lääkeaineen pitoisuus elimistössä voi kasvaa jopa toksiseksi tai vähentyä merkittävästi. Tämä on erityisesti ongelmana silloin, kun käytössä on useita lääkkeitä samanaikaisesti. Lääketutkimuksessa onkin keskitytty tällaisten interaktioiden ennustamiseen ja niitä yritetään välttää tai ainakin vähentää. Työssä tutkittiin medetomidiinia, jonka on äskettäin havaittu metaboloituvan UDP-glukuronosyylitransferaasien (UGT) välityksellä. Työn tarkoituksena oli löytää medetomidiinin glukuronidaatiota inhiboivia yhdisteitä. Lisäksi haluttiin selvittää mahdollisen inhibition mekanismeja. On yleistä tutkia tietyn entsyymin substraatin interaktioita muiden saman perheen entsyymien kanssa. On kuitenkin harvinaisempaa tutkia tällaisia interaktioita kahden eri entsyymiperheen välillä. Tässä työssä tutkittiin inhiboivatko mahdolliset sytokromi P450 -entsyymiä (CYP) inhiboivat yhdisteet myös medetomidiinia glukuronoivia UDP-glukuronosyylitransferaaseja. Glukuronidaation inhibitiota tutkittiin HPLC-menetelmällä, joka on kehitetty aiemmin medetomidiinin glukuronidaation tutkimiseen. Aluksi glukuronidaatiota tutkittiin ilman inhibiittoreita. Tämän jälkeen tutkittiin kolmen mahdollisen inhibiittoriyhdisteen vaikutuksia medetomidiinin glukuronidaatioon ja tuloksia verrattiin ilman inhibiittoria saatuihin tuloksiin. Kolmen tutkitun yhdisteen havaittiin inhiboivan medetomidiinin glukuronidaatiota. Tutkimuksessa havaittiin myös mielenkiintoinen ilmiö, jossa inhibiittoriyhdisteen sitoutuminen aiheutti entsyymikineettisiä muutoksia UDP-glukuronosyylitransferaasin toiminnassa. On mielenkiintoista, että samat yhdisteet inhiboivat sekä CYP- että UGT-metaboliaa. Tulosten perusteella voidaan päätellä, että jos CYP ja UGT metaboloivat samaa yhdistettä, on mahdollista että yhdisteen rakenteelliset analogit aiheuttavat interaktioita molempien entsyymien kanssa. Uusia lääkeaineita kehitettäessä onkin otettava huomioon yleisesti tunnettujen CYP-entsyymien lisäksi myös UGT:t ja niiden mahdolliset yhteisvaikutukset.