Sterols and oxysterols

The ingredients of animal feed formulation, especially the fatty sources, play a significant role in the production of high-quality food of animal origin. Industrial fat by-products are a major source of feeding fats in Europe. The main objectives of the project were firstly to assess feed fats and oils for their composition and content of sterols (cholesterol and phytosterols) and oxysterols, and secondly to assess the levels of cholesterol and oxycholesterols of chicken and rabbit tissues after feeding with especially formulated feeds containing trans fatty acids and oxidized lipids. The lack of standardized analytical procedure prompted the evaluation of an in-house purification method by comparing it with a number of commonly used methods for the analysis of oxysterols. The saponification and transesterification steps showed rather comparable results. A two-dimensional capillary GC column with different stationary phases (a 35% phenyl column coupled to an apolar 5% phenyl column) was used for better resolution of a large number of oxysterols. This new system improved the separation efficiency and reduced the analytical time for a wide range of oxysterols. The satisfactory purification method and the reliable separation of oxysterols facilitated the qualitative and quantitative assessment of sterols and oxysterols in samples of by-products from chemical and physical refining. A large variation in the levels of sterols and oxysterols was observed in the fat by-products from chemical and physical refining processes for edible fats and oils. The observed variations in the contents and composition of sterols and oxysterols were mainly due to the origins, production facility and different processing conditions of the by-product samples. The highly oxidized lipid and trans fatty acid feeds significantly increased the contents of cholesterol and oxycholesterols in edible parts, e.g. the muscles and livers of chickens and rabbits (0.01< p ≤0.05). Hence, the consumption of products from animals fed with higher levels of trans fatty acids and oxidized lipid feeds may contribute to higher ingestion of cholesterol and oxycholesterols by humans

Az ingázás térbeli jellegzetességeinek változása az Észak-Dunántúlon, különös tekintettel Győr munkaügyi vonzáskörzetére

A tanulmányban megvizsgáljuk a KSH által 2001-ben és 2011-ben elvégzett népszámlálás ingázásra vonatkozó főbb adatainak és a két felvétel összehasonlítása során tapasztalt eltérések területi konzekvenciáit. A vizsgálat célja elsősorban az volt, hogy Győr köré szerveződő ingázó, munkaügyi vonzásterek jellegzetességeit megismerjük. Ezért az országos szintű vizsgálatok mellett részletesebben koncentráltunk az észak-Dunántúlra, melynek központrendszerét tártuk fel, s még részletesebben vizsgáltuk ezen belül Győr, s a városkörnyéki tér jellegzetességeit

CYPome of the conifer pathogen Heterobasidion irregulare : Inventory, phylogeny, and transcriptional analysis of the response to biocontrol

The molecular mechanisms underlying the interaction of the pathogen, Heterobasidion annosum s.l., the conifer tree and the biocontrol fungus, Phlebiopsis gigantea have not been fully elucidated. Members of the cytochrome P450 (CYP) protein family may contribute to the detoxification of components of chemical defence of conifer trees by H. annosum during infection. Additionally, they may also be involved in the interaction between H. annosum and P. gigantea. A genome-wide analysis of CYPs in Heterobasidion irregulare was carried out alongside gene expression studies. According to the Standardized CYP Nomenclature criteria, the H. irregulare genome has 121 CYP genes and 17 CYP pseudogenes classified into 11 clans, 35 families, and 64 subfamilies. Tandem CYP arrays originating from gene duplications and belonging to the same family and subfamily were found. Phylogenetic analysis showed that all the families of H. irregulare CYPs were monophyletic groups except for the family CYP5144. Microarray analysis revealed the transcriptional pattern for 130 transcripts of CYP-encoding genes during growth on culture filtrate produced by P. gigantea. The high level of P450 gene diversity identified in this study could result from extensive gene duplications presumably caused by the high metabolic demands of H. irregulare in its ecological niches. (C) 2016 British Mycological Society. Published by Elsevier Ltd. All rights reserved.Peer reviewe

Concentrations of canine prostate specific esterase, CPSE, at baseline are associated with the relative size of the prostate at three-year follow-up

BackgroundEnlargement of the prostate is associated with prostatic diseases in dogs, and an estimation of prostatic size is a central part in the diagnostic workup. Ultrasonography is often the method of choice, but biomarkers constitute an alternative. Canine prostate specific esterase (CPSE) shares many characteristics with human prostate specific antigen (PSA) and is related to prostate size. In men with clinical symptoms of prostatic disease, PSA concentrations are related to prostate growth. The aims of the present follow-up study were to evaluate if the concentration of CPSE is associated with future growth of the prostate, and if analysis of a panel of 16 steroids gives further information on prostatic growth. Owners of dogs included in a previous study were 3 years later contacted for a follow-up study that included an interview and a clinical examination. The prostate was examined by ultrasonography. Serum concentrations of CPSE were measured, as was a panel of steroids.ResultsOf the 79 dogs included at baseline, owners of 77 dogs (97%) were reached for an interview, and 22 were available for a follow-up examination. Six of the 79 dogs had clinical signs of prostatic disease at baseline, and eight of the remaining 73 dogs (11%) developed clinical signs between baseline and follow-up, information was lacking for two dogs. Development of clinical signs was significantly more common in dogs with a relative prostate size of >= 2.5 at baseline (n=20) than in dogs with smaller prostates (n=51). Serum concentrations of CPSE at baseline were not associated with the change in prostatic size between baseline and follow-up. Serum concentrations of CPSE at baseline and at follow-up were positively associated with the relative prostatic size (S-rel) at follow-up. Concentrations of corticosterone (P = 0.024), and the class corticosteroids (P = 0.0035) were positively associated with the difference in S-rel between baseline and follow-up.ConclusionsThe results support the use of CPSE for estimating present and future prostatic size in dogs >= 4years, and the clinical usefulness of prostatic size for predicting development of clinical signs of prostatic disease in the dog. The association between corticosteroids and prostate growth warrants further investigation

N-glycans of Human Protein C Inhibitor: Tissue-Specific Expression and Function

Protein C inhibitor (PCI) is a serpin type of serine protease inhibitor that is found in many tissues and fluids in human, including blood plasma, seminal plasma and urine. This inhibitor displays an unusually broad protease specificity compared with other serpins. Previous studies have shown that the N-glycan(s) and the NH2-terminus affect some blood-related functions of PCI. In this study, we have for the first time determined the N-glycan profile of seminal plasma PCI, by mass spectrometry. The N-glycan structures differed markedly compared with those of both blood-derived and urinary PCI, providing evidence that the N-glycans of PCI are expressed in a tissue-specific manner. The most abundant structure (m/z 2592.9) had a composition of Fuc3Hex5HexNAc4, consistent with a core fucosylated bi-antennary glycan with terminal Lewisx. A major serine protease in semen, prostate specific antigen (PSA), was used to evaluate the effects of N-glycans and the NH2-terminus on a PCI function related to the reproductive tract. Second-order rate constants for PSA inhibition by PCI were 4.3±0.2 and 4.1±0.5 M−1s−1 for the natural full-length PCI and a form lacking six amino acids at the NH2-terminus, respectively, whereas these constants were 4.8±0.1 and 29±7 M−1s−1 for the corresponding PNGase F-treated forms. The 7–8-fold higher rate constants obtained when both the N-glycans and the NH2-terminus had been removed suggest that these structures jointly affect the rate of PSA inhibition, presumably by together hindering conformational changes of PCI required to bind to the catalytic pocket of PSA

A Novel Targeted Analysis of Peripheral Steroids by Ultra-Performance Supercritical Fluid Chromatography Hyphenated to Tandem Mass Spectrometry

Ultra-performance supercritical fluid chromatography-tandem mass spectrometry (UPSFC-MS/MS) is an alternative method for steroid analysis. Continuous development of analytical methodologies for steroid profiling is of major importance in the clinical environment to provide useful and more comprehensive data. The aim of this study was to identify and quantify a large number of endogenous steroids from the four major classes (estrogens, androgens, progestogens and corticosteroids) simultaneously within a short analytical time. This novel UPSFC-MS/MS method with electrospray in positive ionisation (ESI+) mode is robust, selective and present sufficiently high sensitivity to profile nineteen steroids in 50 mu L human plasma. Under optimised conditions, nineteen different steroids were separated with high efficiency in the multiple reaction monitoring (MRM) mode. The linearity of the method was good with correlation coefficients (R-2) in the range of 0.9983-0.9999 and with calibration range from 0.05-500 ng/mL in human plasma. The intraday and interday precision of the method, as RSD, was less than 15%. The accuracy of the nineteen analytes varied between 80 to 116%. Finally, the novel method was successfully applied for the determination of nineteen steroids within 5 minutes providing the possibility to use it for research as well as routine healthcare practice

Functional analyses of the chitin-binding domains and the catalytic domain of Brassica juncea chitinase BjCHI1

We previously isolated a Brassica juncea cDNA encoding BjCHI1, a novel chitinase with two chitin-binding domains. Synthesis of its mRNA is induced by wounding, methyl jasmonate treatment, Aspergillus niger infection and caterpillar (Pieris rapae) feeding, suggesting that the protein has a role in defense. In that it possesses two chitin-binding domains, BjCHI1 resembles the precursor of Urtica dioica agglutinin but unlike that protein, BjCHI1 retains its chitinase catalytic domain after post-translational processing. To explore the properties of multi-domain BjCHI1, we have expressed recombinant BjCHI1 and two derivatives, which lack one (BjCHI2) or both (BjCHI3) chitin-binding domains, as secreted proteins in Pichia pastoris. Recombinant BjCHI1 and BjCHI2 showed apparent molecular masses on SDS-PAGE larger than calculated, and could be deglycosylated using α-mannosidase. Recombinant BjCHI3, without the proline/ threonine-rich linker region containing predicted O-glycosylation sites, did not appear to be processed by α-mannosidase. BjCHI1's ability to agglutinate rabbit erythrocytes is unique among known chitinases. Both chitin-binding domains are essential for agglutination; this property is absent in recombinant BjCHI2 and BjCHI3. To identify potential catalytic residues, we generated site-directed mutations in recombinant BjCHI3. Mutation E212A showed the largest effect, exhibiting 0% of wild-type specific activity. H211N and R361A resulted in considerable (>91%) activity loss, implying these charged residues are also important in catalysis. E234A showed 36% retention of activity and substitution Y269D, 50%. The least affected mutants were E349A and D360A, with 73% and 68% retention, respectively. Like Y269, E349 and D360 are possibly involved in substrate binding rather than catalysis.link_to_subscribed_fulltex

The first crystal structures of a family 19 class IV chitinase: The enzyme from Norway spruce

Chitinases help plants defend themselves against fungal attack, and play roles in other processes, including development. The catalytic modules of most plant chitinases belong to glycoside hydrolase family 19. We report here x-ray structures of such a module from a Norway spruce enzyme, the first for any family 19 class IV chitinase. The bi-lobed structure has a wide cleft lined by conserved residues; the most interesting for catalysis are Glu113, the proton donor, and Glu122, believed to be a general base that activate a catalytic water molecule. Comparisons to class I and II enzymes show that loop deletions in the class IV proteins make the catalytic cleft shorter and wider; from modeling studies, it is predicted that only three N-acetylglucosamine-binding subsites exist in class IV. Further, the structural comparisons suggest that the family 19 enzymes become more closed on substrate binding. Attempts to solve the structure of the complete protein including the associated chitin-binding module failed, however, modeling studies based on close relatives indicate that the binding module recognizes at most three N-acetylglucosamine units. The combined results suggest that the class IV enzymes are optimized for shorter substrates than the class I and II enzymes, or alternatively, that they are better suited for action on substrates where only small regions of chitin chain are accessible. Intact spruce chitinase is shown to possess antifungal activity, which requires the binding module; removing this module had no effect on measured chitinase activity. © 2009 Springer Science+Business Media B.V.link_to_subscribed_fulltex

Functional properties of low-modulus PMMA bone cements containing linoleic acid

Acrylic bone cements modified with linoleic acid are a promising low-modulus alternative to traditional high-modulus bone cements. However, several key properties remain unexplored, including the effect of autoclave sterilization and the potential use of low-modulus cements in other applications than vertebral augmentation. In this work, we evaluate the effect of sterilization on the structure and stability of linoleic acid, as well as in the handling properties, glass transition temperature, mechanical properties, and screw augmentation potential of low-modulus cement containing the fatty acid. Neither 1H NMR nor SFC-MS/MS analysis showed any detectable differences in autoclaved linoleic acid compared to fresh one. The peak polymerization temperature of the low-modulus cement was much lower (28–30 °C) than that of the high-modulus cement (67 °C), whereas the setting time remained comparable (20–25 min). The Tg of the low-modulus cement was lower (75–78 °C) than that of the high-stiffness cement (103 °C). It was shown that sterilization of linoleic acid by autoclaving did not significantly affect the functional properties of low-modulus PMMA bone cement, making the component suitable for sterile production. Ultimately, the low-modulus cement exhibited handling and mechanical properties that more closely match those of osteoporotic vertebral bone with a screw holding capacity of under 2000 N, making it a promising alternative for use in combination with orthopedic hardware in applications where high-stiffness augmentation materials can result in undesired effects.Authors in thesis list of papers: C. Robo, D. Wenner, M. Nilsson, J. Hilborn, C. Öhman-Mägi, C. Persson</p