New evidences on Tau-DNA interactions and relevance to neurodegeneration

Tau is mainly distributed in cytoplasm and also found to be localized in the nucleus. There is limited data on DNA binding potential of Tau.We provide novel evidence on nicking of DNA by Tau. Tau nicks the supercoiled DNA leading to open circular and linear forms. The metal ion magnesium (a co-factor for endonuclease) enhanced the Tau DNA nicking ability, while an endonuclease specific inhibitor,aurinetricarboxylic acid (ATA) inhibited the Tau DNA nicking ability Further, we also evidenced that Tau induces B-C-A mixed conformational transition in DNA and also changes DNA stability. Tau-scDNA complex is more sensitive to DNAse I digestion indicating stability changes in DNA caused by Tau. These findings indicate that Tau alters DNA helicity and integrity and also nicks the DNA. The relevance of these novel intriguing findings regarding the role Tau in neuronal dysfunction is discussed. (C) 2010 Elsevier Ltd. All rights reserved.

Structural alteration from non-B to B-form could reflect DNase I hypersensitivity

Preferential cleavage of active genes by DNase I has been correlated with a structurally altered conformation of DNA at the hypersensitive site in chromatin. To have a better understanding of the structural requirements for gene activation as probed by DNase I action, digestability by DNase I of synthetic polynucleotides having the ability to adopt B and non-B conformation (like Z-form) was studied which indicated a marked higher digestability of the B-form of DNA. Left handed Z form present within a natural sequence in supercoiled plasmid also showed marked resistance towards DNase I digestion. We show that alternating purine-pyrimidine sequences adopting Z-conformation exhibit DNAse I foot printing even in a protein free system. The logical deductions from the results indicate that 1) altered structure like Z-DNA is not a favourable substrate for DNase I, 2) both the ends of the alternating purine-pyrimidine insert showed hypersensitivity, 3) B-form with a minor groove of 12-13 A is a more favourable substrate for DNase I than an altered structure, 4) any structure of DNA deviating largely from B form with a capacity to flip over to the B-form are potential targets for the DNase I enzymic probes in naked DNA.

Real-Time PCR : A Molecular Approach to Investigate the Role of Intestinal Microbiota in the Pathophysiology of Irritable Bowel Syndrome

Irritable bowel syndrome (IBS) is a common multifactorial functional intestinal disorder, the pathogenesis of which is not completely understood. Increasing scientific evidence suggests that microbes are involved in the onset and maintenance of IBS symptoms. The microbiota of the human gastrointestinal (GI) tract constitutes a massive and complex ecosystem consisting mainly of obligate anaerobic microorganisms making the use of culture-based methods demanding and prone to misinterpretation. To overcome these drawbacks, an extensive panel of species- and group-specific assays for an accurate quantification of bacteria from fecal samples with real-time PCR was developed, optimized, and validated. As a result, the target bacteria were detectable at a minimum concentration range of approximately 10 000 bacterial genomes per gram of fecal sample, which corresponds to the sensitivity to detect 0.000001% subpopulations of the total fecal microbiota. The real-time PCR panel covering both commensal and pathogenic microorganisms was assessed to compare the intestinal microbiota of patients suffering from IBS with a healthy control group devoid of GI symptoms. Both the IBS and control groups showed considerable individual variation in gut microbiota composition. Sorting of the IBS patients according to the symptom subtypes (diarrhea, constipation, and alternating predominant type) revealed that lower amounts of Lactobacillus spp. were present in the samples of diarrhea predominant IBS patients, whereas constipation predominant IBS patients carried increased amounts of Veillonella spp. In the screening of intestinal pathogens, 17% of IBS samples tested positive for Staphylococcus aureus, whereas no positive cases were discovered among healthy controls. Furthermore, the methodology was applied to monitor the effects of a multispecies probiotic supplementation on GI microbiota of IBS sufferers. In the placebo-controlled double-blind probiotic intervention trial of IBS patients, each supplemented probiotic strain was detected in fecal samples. Intestinal microbiota remained stable during the trial, except for Bifidobacterium spp., which increased in the placebo group and decreased in the probiotic group. The combination of assays developed and applied in this thesis has an overall coverage of 300-400 known bacterial species, along with the number of yet unknown phylotypes. Hence, it provides good means for studying the intestinal microbiota, irrespective of the intestinal condition and health status. In particular, it allows screening and identification of microbes putatively associated with IBS. The alterations in the gut microbiota discovered here support the hypothesis that microbes are likely to contribute to the pathophysiology of IBS. The central question is whether the microbiota changes described represent the cause for, rather than the effect of, disturbed gut physiology. Therefore, more studies are needed to determine the role and importance of individual microbial species or groups in IBS. In addition, it is essential that the microbial alterations observed in this study will be confirmed using a larger set of IBS samples of different subtypes, preferably from various geographical locations.

Purification and properties of trehalase from the thermophilic fungus Humicola lanuginosa

Trehalase (?,?-Trehalosee gludohydrolase, EC 3.2.1.28) was partially solubilized from the thermophilic fungus Humicola lanuginosa RM-B, and purified 184-fold. The purified enzyme was optimally active at 50°C in acetate buffer at pH 5.5. It was highly specific for ?,?-trehalose and had an apparent Km = 0.4 mM at 50°C. None of the other disaccharides tested either inhibited or activated the enzyme. The molecular weight of the enzyme was around 170000. Trehalase from mycelium grown at 40 and 50°C had similar properties. The purified enzyme, in contrast to that in the crude-cell free extract, was less stable. At low concentration, purified trehalase was afforded protection against heat-inactivation by �protective factor(s)� present in mycelial extracts. The �protective factor(s)� was sensitive to proteolytic digestion. It was not diffusable and was stable to boiling for at least 30 min. Bovine serum albumin and casein also protected the enzyme from heat-inactivation.

Proteolytic susceptibility and methionine modification of monodeamidated ribonuclease A

The susceptibility of a monodeamidated RNAaseA (RNAaseAa1) towards carboxypeptidaseA , alpha-chymotrypsin and pepsin has been studied. Similar to RNAaseA, the C-terminal of RNAaseAa1 is not available for carboxypeptidaseA hydrolysis. The thermal stability of RNAaseAa1 as probed through chymotryptic digestion is found to be less than that of RNAaseA. Preliminary chromatographic analysis of the digested material, however, suggests that the nature of thermal transition might be the same in the two proteins. Pepsin inactivates RNAaseAa1 more slowly than does RNAaseA. Accordingly, less peptide bonds, almost half that of RNAaseA, are cleaved by pepsin in RNAaseAa1. The accumulation of RNAase-P type intermediates is not evident during peptic digestion of RNAaseAa1. Reaction with O-benzoquinone at low pH shows that methionines of the deamidated protein seem to have higher reactivities. These observations indicate a different structure for RNAaseAa1 at elevated temperature and low pH.

Isolation and characterization of a naturally occurring inhibitor from mung bean (Vigna radiata) seedlings for serine hydroxymethyltransferase

A naturally occurring inhibitor of serine hydroxymethyltransferase (EC2.1.2.1) in mung bean seedlings extracts was purified by ammonium sulphate precipitation, phenyl-Sepharose chromatography followed by heating to release the inhibitor bound to the protein. The inhibitor had an absorption maximum at 200 nm, was not precipitated by trichloroacetic acid, was dialysable and resistant to inactivation by heating at 98-degrees-C for 4 hr, protease and ribonuclease digestion; but was acid labile. The chromatographically pure preparation inhibited both mung bean and sheep liver SHMT. Qualitative and quantitative analyses indicated that it contained a carbohydrate moiety, an O-amino and vicinal diol groups. Paper electrophoresis at pH 4.3 suggested that the inhibitor was positively charged.

Studies in biogas technology. Part III. Thermal analysis of biogas plants

A thermal model for a conventional biogas plant has been developed in order to understand the heat transfer from the slurry and the gas holder to the surrounding earth and air respectively. The computations have been performed for two conditions : (i) when the slurry is at an ambient temperature of 20°C, and (ii) when it is at 35°C, the optimum temperature for anaerobic fermentation. Under both these conditions, the gas holder is the major “culprit” with regard to heat losses from the biogas plant. The calculations provide an estimate for the heat which has to be supplied by external means to compensate for the net heat losses which occur if the slurry is to be maintained at 35°C. Even if this external supply of heat is realised through (the calorific value of) biogas, there is a net increase in the biogas output, and therefore a net benefit, by operating the plant at 35°C. At this elevated temperature, the cooling effect of adding the influent at ambient temperature is not insignificant. In conclusion, the results of the thermal analysis are used to define a strategy for operating biogas plants at optimum temperatures, or at higher temperatures than the ambient.

Enhanced photodynamic effect of cobalt(III) dipyridophenazine complex on thyrotropin receptor expressing HEK293 cells

Ternary cobalt(III) complexes CoL(B)] (1-3) of a trianionic tetradentate phenolate-based ligand (L) and phenanthroline bases (B), viz. 1,10-phenanthroline (phen in 1), dipyridoquinoxaline (dpq in 2) and dipyridophenazine (dppz in 3) are synthesized, characterized from X-ray crystallographic, analytical and spectral techniques, and their utility in photodynamic therapy (PDT) of thyroid diseases caused by TSH receptor dysfunction is probed. The complexes display a visible spectral band within the PDT spectral window at similar to 690 nm. Photodynamic potential was estimated through DNA cleavage activity of the dpq and dppz complexes in UV-A light of 365 nm and red light of 676 nm. The reactions proceed via the hydroxyl radical pathway. The complexes retain their DNA photocleavage activity in red light under anaerobic conditions, a situation normally prevails in hypoxic tumor core. Investigation into the photocytotoxic potential of these complexes showed that the dppz complex 3 is approximately 4-fold more active in the HEK293 cells expressing human thyrotropin receptor (HEK293-hTSHR) than in the parental cell line and has an insignificant effect on an unrelated human cervical carcinoma cell line (HeLa). Photoexcitation of complex 3 in HEK293-hTSHR cells leads to damage hTSHR as evidenced from the decrease in cAMP formation both in absence and presence of hTSH and decrease in the TSHR immunofluorescence with a concomitant cytoplasmic translocation of the membrane protein, cadherin. The involvement of hTSHR is evidenced from the ability of complex 3 to bind to the extracellular domain of hTSHR (hTSHR-ECD) with a K-d value of 81 nM and from the photocleavage of hTSHR-ECD.

Crystal structures of Salmonella typhimurium propionate kinase and its complex with Ap4A: evidence for a novel Ap4A synthetic activity

Propionate kinase catalyses the last step in the anaerobic breakdown of L-threonine to propionate in which propionyl phosphate and ADP are converted to propionate and ATR Here we report the structures of propionate kinase (TdcD) in the native form as well as in complex with diadenosine 5 ',5 '''-P-1,P-4-tetraphosphate (AP(4)A) by X-ray crystallography. Structure of TdcD obtained after cocrystallization with ATP showed Ap(4)A bound to the active site pocket suggesting the presence of Ap(4)A synthetic activity in TdcD. Binding of Ap(4)A to the enzyme was confirmed by the structure determination of a TdcD-Ap(4)A complex obtained after cocrystallization of TdcD with commercially available Ap(4)A. Mass spectroscopic studies provided further evidence for the formation of Ap(4)A by propionate kinase in the presence of ATP. In the TdcD-Ap(4)A complex structure, Ap(4)A is present in an extended conformation with one adenosine moiety present in the nucleotide binding site and other in the proposed propionate binding site. These observations tend to support direct in-line transfer of phosphoryl group during the kinase reaction.

Molekyylibiologisten menetelmien kehittäminen ihmisen suoliston sulfaattia pelkistävien bakteerien määrittämiseksi

Ihmisen ruuansulatuskanavan bakteeriston kehitys alkaa syntymästä, jolloin ensimmäiset bakteerit kansoittavat steriilin ruuansulatuskanavan. Bakteeristo kehittyy perimän, ympäristön ja varhaisen ruokavalion vaikutuksesta kohti monimuotoisempaa bakteeripopulaatiota. Aikuisen ruuansulatuskanavan normaalibakteeristo on varsin muuttumaton, mutta siihen vaikuttavat monet tekijät, kuten ikä, terveydentila, ruokavalio ja antibioottien käyttö. Bakteeriston koostumus vaihtelee ruuansulatuskanavan eri osissa ja bakteerimäärä kasvaa kohti paksusuolta, ollen paksusuolessa ja ulosteessa peräti 1010-1012 pmy/ml. Suurin osa ruuansulatuskanavan bakteereista on anaerobeja. Ruuansulatuskanavan bakteeristo vaikuttaa muun muassa suoliston kehittymiseen ja hiilihydraattien ja proteiinien hajotukseen sekä toimii osana immuunipuolustusta. Sulfaattia pelkistävät bakteerit (SRB) ovat monimuotoinen ryhmä pääosin anaerobisia bakteereita, jotka käyttävät aineenvaihdunnassaan elektronin vastaanottajana sulfaattia muuttaen sen lopulta sulfidiksi. SRB:t ovat sopeutuneet useisiin erilaisiin ympäristöihin. Niitä tavataan mm. vesistöjen sedimenteissä sekä ihmisen ruuansulatuskanavassa. Ihmisen ruuansulatuskanavassa on SRB:ta n. 105-108 pmy/g, ja niitä on löydetty erityisesti anaerobisista osista kuten suun ientaskuista ja paksusuolesta. SRB:t voivat olla haitaksi ruuansulatuskanavalle tuottamansa sulfidin vuoksi, joka esiintyy vesiliuoksessa vetysulfidina. Tämän on havaittu olevan toksista suoliston epiteelisoluille. Viimeaikoina on kiinnostuttu sulfaatinpelkistäjien yhteydestä suoliston sairaustiloihin, kuten tulehduksellisiin suolistosairauksiin (IBD). Pro gradu -tutkimukseni tavoitteena oli kehittää PCR-DGGE- ja qPCR-menetelmät ulosteen sulfaattia pelkistävien bakteerien määritykseen. Kohdegeeninä menetelmänkehityksessä käytettiin dsrAB-geeniä, joka koodaa dissimilatorista sulfiitinpelkistysentsyymiä. dsrAB-geeni on sulfaatinpelkistäjille ominainen konservoitunut geenialue, johon perustuvia tutkimuksia ei vielä ole paljon ihmispuolelta. qPCR-menetelmä saatiin optimoitua herkäksi ja spesifiseksi käyttäen dsrA-geenispesifisiä alukkeita, mutta PCR-DGGE-menetelmää ei saatu optimoitua käytössä olleilla alukkeilla, jotka monistivat PCR-DGGE:ssa myös negatiivikontrollikantoja. Tutkittaessa qPCR:lla IBD:tä (Crohn ja ulseratiivinen koliitti) sairastavien lasten ja terveiden kontrollihenkilöiden ulostenäytteistä eristettyä DNA:ta, merkittävää eroa SRB-määrissä ei havaittu eri ryhmien välillä. Crohnin tautia sairastavien aktiivisen vaiheen ja oireettoman vaiheen näytteiden välillä oli kuitenkin tilastollisesti merkitsevä ero (SRB-määrät; oireeton vaihe>oireellinen vaihe) (P <0,05).

Seasonal variation in nitrification and nitrate-reduction pathways in coastal sediments in the Gulf of Finland, Baltic Sea

The Baltic Sea is one of the most eutrophic marine areas in the world. The role of nitrogen as a eutrophicating nutrient in the Baltic Sea has remained controversial, due to lack of understanding of nitrogen cycling in the area. We investigated the seasonal variation in sediment nitrification, denitrification, anaerobic ammonium oxidation (anammox), and dissimilatory nitrate reduction to ammonium (DNRA) at two coastal sites in the Gulf of Finland. In addition to the in situ rates, we assessed the potential for these processes in different seasons. The nitrification and nitrogen removal processes were maximal during the warm summer months, when the sediment organic content was highest. In colder seasons, the in situ rates of the nitrification and nitrate reduction processes decreased, but the potential for nitrification remained equal to or higher than that during the warm months. The denitrification and nitrification rates were usually higher in the accumulation basin, where the organic content of the sediment was higher, but the transportation area, despite lower denitrification rates and potential, typically had higher potential for nitrification than the accumulation basin. Anammox and DNRA were not significant nitrate sinks in any of the seasons sampled. The results also show that the denitrification rates in the coastal Gulf of Finland sediment have decreased, and that benthic denitrification might be a less important sink for fixed nitrogen than previously assumed.

Effects of gender, and SLCO1B1 and CYP7A1 polymorphisms on plasma bile acids in humans and the pharmacokinetics of therapeutic ursodeoxycholic acid

Bile acids are important steroid-derived molecules essential for fat absorption in the small intestine. They are produced in the liver and secreted into the bile. Bile acids are transported by bile flow to the small intestine, where they aid the digestion of lipids. Most bile acids are reabsorbed in the small intestine and return to the liver through the portal vein. The whole recycling process is referred to as the enterohepatic circulation, during which only a small amount of bile acids are removed from the body via faeces. The enterohepatic circulation of bile acids involves the delicate coordination of a number of bile acid transporters expressed in the liver and the small intestine. Organic anion transporting polypeptide 1B1 (OATP1B1), encoded by the solute carrier organic anion transporter family, member 1B1 (SLCO1B1) gene, mediates the sodium independent hepatocellular uptake of bile acids. Two common SNPs in the SLCO1B1 gene are well known to affect the transport activity of OATP1B1. Moreover, bile acid synthesis is an important elimination route for cholesterol. Cholesterol 7α-hydroxylase (CYP7A1) is the rate-limiting enzyme of bile acid production. The aim of this thesis was to investigate the effects of SLCO1B1 polymorphism on the fasting plasma levels of individual endogenous bile acids and a bile acid synthesis marker, and the pharmacokinetics of exogenously administered ursodeoxycholic acid (UDCA). Furthermore, the effects of CYP7A1 genetic polymorphism and gender on the fasting plasma concentrations of individual endogenous bile acids and the bile acid synthesis marker were evaluated. Firstly, a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for the determination of bile acids was developed (Study I). A retrospective study examined the effects of SLCO1B1 genetic polymorphism on the fasting plasma concentrations of individual bile acids and a bile acid synthesis marker in 65 healthy subjects (Study II). In another retrospective study with 143 healthy individuals, the effects of CYP7A1 genetic polymorphism and gender as well as SLCO1B1 polymorphism on the fasting plasma levels of individual bile acids and the bile acid synthesis marker were investigated (Study III). The effects of SLCO1B1 polymorphism on the pharmacokinetics of exogenously administered UDCA were evaluated in a prospective genotype panel study including 27 healthy volunteers (Study IV). A robust, sensitive and simple HPLC-MS/MS method was developed for the simultaneous determination of 16 individual bile acids in human plasma. The method validation parameters for all the analytes met the requirements of the FDA (Food and Drug Administration) bioanalytical guidelines. This HPLC-MS/MS method was applied in Studies II-IV. In Study II, the fasting plasma concentrations of several bile acids and the bile acid synthesis marker seemed to be affected by SLCO1B1 genetic polymorphism, but these findings were not replicated in Study III with a larger sample size. Moreover, SLCO1B1 polymorphism had no effect on the pharmacokinetic parameters of exogenously administered UDCA. Furthermore, no consistent association was observed between CYP7A1 genetic polymorphism and the fasting plasma concentrations of individual bile acids or the bile acid synthesis marker. In contrast, gender had a major effect on the fasting plasma concentrations of several bile acids and also total bile acids. In conclusion, gender, but not SLCO1B1 or CYP7A1 polymorphisms, has a major effect on the fasting plasma concentrations of individual bile acids. Moreover, the common genetic polymorphism of CYP7A1 is unlikely to influence the activity of CYP7A1 under normal physiological conditions. OATP1B1 does not play an important role in the in vivo disposition of exogenously administered UDCA.

Design and fabrication of an automated thermal desorption gas-solid reaction system: Oxidation of ammonia and methanol over YBa2Cu3O7−δ(123) oxide systems

A fully automated, versatile Temperature Programmed Desorption (TDP), Temperature Programmed Reaction (TPR) and Evolved Gas Analysis (EGA) system has been designed and fabricated. The system consists of a micro-reactor which can be evacuated to 10−6 torr and can be heated from 30 to 750°C at a rate of 5 to 30°C per minute. The gas evolved from the reactor is analysed by a quadrupole mass spectrometer (1–300 amu). Data on each of the mass scans and the temperature at a given time are acquired by a PC/AT system to generate thermograms. The functioning of the system is exemplified by the temperature programmed desorption (TPD) of oxygen from YBa2Cu3−xCoxO7 ± δ, catalytic ammonia oxidation to NO over YBa2Cu3O7−δ and anaerobic oxidation of methanol to CO2, CO and H2O over YBa2Cu3O7−δ (Y123) and PrBa2Cu3O7−δ (Pr123) systems.

Inhibitory effects of wood extracts on the spoilage flora of marinated poultry meat

Polyphenolic compounds occurring naturally in knotwood of plants are known to have antimicrobial effects. The knots (i.e. the branch bases inside tree stems) and outer branches in pine trees contain a remarkably high concentration of phenolic stilbenes, while lignans are the major phenolic constituents of spruce knots. Large amount of these phenolic compounds can be extracted from wood knots at pulp and paper mills where their presence is undesirable. In Finland, marinating of broiler meat is done not only to increase or add value to the meat, but also to enhance the safety and shelf-life. These products are usually packed under a modified atmosphere for further protection against spoilage microorganisms. However, studies have revealed that addition of marinades to poultry products do not have an inhibitory effect on either some psychrotrophic anaerobic bacteria, such as Brochothrix thermosphacta or lactic acid bacteria associated with spoilage. Also, the activity of pathogenic Campylobacter jejuni is not affected by marinating. The objective of this study was to investigate the inhibitory and lethal activities of extracts from spruce (Picea spp.) and pine (Pinus spp.) knotwood and outer branches that are dissolved in ethanol against the spoilage microorganisms in modified atmosphere packaged marinated broiler products. Modified atmosphere packaged broiler products were separately inoculated with ‘normal’ marinades, marinades with 70% ethanol, marinades with a mixture of spruce and pine extracts dissolved in 70% ethanol or mixture of spruce and pine extracts in powder form. The bacterial colony forming units per gram obtained from each of the samples were analysed on de Man Rogosa and Sharpe agar at days 1, 6, 12 and 15. The results showed that there were significant differences in bacterial colony forming units per gram (P <0.05) between packages with ‘normal’ marinades and packages with extracts added to their marinades on the 12th and 15th day. It can be concluded that the addition of extracts from spruce and pine knotwood to marinades significantly retarded growth of spoilage microorganisms during the 15 day test period. However further research is warranted to characterise and establish the safety and suitability of the compound(s) in spruce and pine knotwood extracts that are responsible for inhibitory or lethal activity against the microbes that may be present in marinated poultry meat.

Architecture of Physalis Mottle Tymovirus as Probed by Monoclonal Antibodies and Cross-Linking Studies

Physalis mottle tymovirus (previously named belladonna mottle virus, Iowa strain) RNA was cross-linked to its coat protein by exposure of the intact virus to ultraviolet light. The site of cross-linking of the coat protein with the RNA was identified as Lys-10 by sequencing the oligonucleotide-linked tryptic peptide obtained upon HPLC separation subsequent to enzymetic digestion of the cross-linked and dissociated virus. Three monoclonal antibodies PA3B2, PB5G9, and PF12C9, obtained using denatured coat protein as antigen, cross-reacted effectively with the intact virus indicating that the epitopes recognized by these monoclonals are on the surface of the virus. Using the peptides generated by digestion with CNBr, clostripain, V-8 protease, or trypsin and a recombinant protein lacking the N-terminal 21 residues expressed from a cDNA clone, it was shown that PA3B2 recognizes the sequence 22-36 on the coat protein while PB5G9 and PF12C9 recognize region 75-110. These results suggest that Lys-10 is one of the specific sites through which the RNA interacts in the intact virus. The polypeptide segment (region 22-36) following this buried portion as well as the epitope within the region 75-110 are exposed in the intact virus. These observations are consistent with the canonical β-barrel structure observed in certain other plant viruses.