Thermodynamic and kinetic studies on saccharide binding to soya-bean agglutinin.

The fluorescence of N-dansylgalactosamine [N-(5-dimethylaminonaphthalene-1-sulphonyl)galactosamine] was enhanced 11-fold with a 25 nm blue-shift in the emission maximum upon binding to soya-bean agglutinin (SBA). This change was used to determine the association constants and thermodynamic parameters for this interaction. The association constant of 1.51 X 10(6) M-1 at 20 degrees C indicated a very strong binding, which is mainly due to a relatively small entropy value, as revealed by the thermodynamic parameters: delta G = -34.7 kJ X mol-1, delta H = -37.9 kJ X mol-1 and delta S = -10.9 J X mol-1 X K-1. The specific binding of this sugar to SBA shows that the lectin can accommodate a large hydrophobic substituent on the C-2 of galactose. Binding of non-fluorescent ligands, studied by monitoring the fluorescence changes when they are added to a mixture of SBA and N-dansylgalactosamine, indicates that a hydrophobic substituent at the anomeric position increases the affinity of the interaction. The C-6 hydroxy group also stabilizes the binding considerably. Kinetics of binding of N-dansylgalactosamine to SBA studied by stopped-flow spectrofluorimetry are consistent with a single-step mechanism and yielded k+1 = 2.4 X 10(5) M-1 X s-1 and k-1 = 0.2 s-1 at 20 degrees C. The activation parameters indicate an enthalpicly controlled association process.

Chemical Modification Studies On Ricinus-Communis (Castor Bean) Agglutinin

Ricinus communis agglutinin was subjected to various chemical treatments and the effect on its hemagglutinating and saccharide-binding properties was studied. Acetylation, succinylation and citraconylation led to a complete loss in the activity of the agglutinin, whereas reductive methylation had no effect on the activity, showing that charged amino groups were involved in the hemagglutinating and saccharide-binding activity of Ricinus agglutinin. Modification of tryptophyl, arginyl and carboxyl-group-containing residues did not lead to any loss in the activity of the agglutinin. Acetylation of tyrosyl groups with N-acetylimidazole strongly reduced the hemagglutinating and saccharide-binding property of Ricinus agglutinin. The loss in activity was restored on deacetylation of the tyrosyl groups. Modification of tyrosyl residues also led to a change in the immunological properties of the agglutinin. The initial rate of modification of tyrosyl and amino groups and the concomitant loss of activity was reduced in the presence of lactose.

Proton NMR studies of dihalogenated phenyl benzamides: two-dimensional higher quantum methodologies

The scalar coupled proton NMR spectra of many organic molecules possessing more than one phenyl ring are generally complex due to degeneracy of transitions arising from the closely resonating protons, in addition to several short- and long- range couplings experienced by each proton. Analogous situations are generally encountered in derivatives of halogenated benzanilides. Extraction of information from such spectra is challenging and demands the differentiation of spectrum pertaining to each phenyl ring and the simplification of their spectral complexity. The present study employs the blend of independent spin system filtering and the spin-state selective detection of single quantum (SO) transitions by the two-dimensional multiple quantum (MQ) methodology in achieving this goal. The precise values of the scalar couplings of very small magnitudes have been derived by double quantum resolved experiments. The experiments also provide the relative signs of heteronuclear couplings. Studies on four isomers of dilhalogenated benzanilides are reported in this work.

Integrating sorghum whole genome sequence information with a compendium of sorghum QTL studies reveals uneven distribution of QTL and of gene-rich regions with significant implications for crop improvement.

A comprehensive analysis was conducted using 48 sorghum QTL studies published from 1995 to 2010 to make information from historical sorghum QTL experiments available in a form that could be more readily used by sorghum researchers and plant breeders. In total, 771 QTL relating to 161 unique traits from 44 studies were projected onto a sorghum consensus map. Confidence intervals (CI) of QTL were estimated so that valid comparisons could be made between studies. The method accounted for the number of lines used and the phenotypic variation explained by individual QTL from each study. In addition, estimated centimorgan (cM) locations were calculated for the predicted sorghum gene models identified in Phytozome (JGI GeneModels SBI v1.4) and compared with QTL distribution genome-wide, both on genetic linkage (cM) and physical (base-pair/bp) map scales. QTL and genes were distributed unevenly across the genome. Heterochromatic enrichment for QTL was observed, with approximately 22% of QTL either entirely or partially located in the heterochromatic regions. Heterochromatic gene enrichment was also observed based on their predicted cM locations on the sorghum consensus map, due to suppressed recombination in heterochromatic regions, in contrast to the euchromatic gene enrichment observed on the physical, sequence-based map. The finding of high gene density in recombination-poor regions, coupled with the association with increased QTL density, has implications for the development of more efficient breeding systems in sorghum to better exploit heterosis. The projected QTL information described, combined with the physical locations of sorghum sequence-based markers and predicted gene models, provides sorghum researchers with a useful resource for more detailed analysis of traits and development of efficient marker-assisted breeding strategies.

Genetic studies on recurrent miscarriage

Recurrent miscarriage (RM) is defined as three consecutive pregnancy failures and is estimated to affect ~1% of couples trying to conceive. The cause of RM remains unknown in approximately 50% of cases. In this study, it was hypothesized that some of the underlying factors yet to be discovered are genetic. The aim was to search for mutations in genes AMN, EPCR, TM, and p53 known to cause miscarriage in mouse models and thereby find new genetic causes for unexplained miscarriages in humans. In addition, the mitochondrial genome was studied because mitochondria are involved in processes important in early development. Furthermore, sex chromosome characteristics suggested to underlie miscarriage were also studied. A total of 40 couples and 8 women with unexplained RM were collected for this study and screened for mutations in the candidate genes. Six interesting exonic or potential splice site disrupting variations were detected. However, their phenotypic effects cannot be determined without further investigations. Additionally, an association between the C11992A polymorphism of the p53 gene and RM was detected. The results indicate that women carrying the C/A or A/A genotype have a two-fold higher risk for RM than women with a C/C genotype. This strengthens the results of previous studies reporting that p53 sequence variations may cause miscarriage. The role of variation C11992A in embryonic development is, however, difficult to predict without further studies When screening the mitochondrial genome a heteroplasmic mtDNA variation was found in an unexpected high number of women, as heteroplasmic variations are reported to be rare. One novel variation and 18 previously reported polymorphisms were detected in the mitochondrial genome. Although the detected variations are likely to be neutral polymorphisms, a role in the aetiology of miscarriage cannot be excluded as some mtDNA variations may be pathogenic only when a threshold is reached. Recent publications have reported skewed X chromosome inactivation and Y chromosome microdeletions to be associated with RM. Therefore, these sex chromosome abnormalities in the context of RM were investigated. No associations between skewed X chromosome inactivation or Y chromosome microdeletions and RM in the Finnish patients were detected. Data on ancestral birthplaces of the patients were collected to study any possible geographic clustering, which would indicate a common predisposing factor. The results showed clustering of the birthplaces in eastern Finland in a subset of patients. This suggests a possibility of an enriched susceptibility gene which may contribute to RM.

Studies of the genetic epidemiology of cardiovascular disease: focus on inflammatory candidate genes

Cardiovascular disease (CVD) is a complex disease with multifactorial aetiology. Both genetic and environmental factors contribute to the disease risk. The lifetime risk for CVD differs markedly between men and women, men being at increased risk. Inflammatory reaction contributes to the development of the disease by promoting atherosclerosis in artery walls. In the first part of this thesis, we identified several inflammatory related CVD risk factors associating with the amount of DNA from whole blood samples, indicating a potential source of bias if a genetic study selects the participants based on the available amount of DNA. In the following studies, this observation was taken into account by applying whole genome amplification to samples otherwise subjected to exclusion due to very low DNA yield. We continued by investigating the contribution of inflammatory genes to the risk for CVD separately in men and women, and looked for sex-genotype interaction. In the second part, we explored a new candidate gene and its role in the risk for CVD. Selenoprotein S (SEPS1) is a membrane protein residing in the endoplasmic reticulum where it participates in retro-translocation of unfolded proteins to cytosolic protein degradation. Previous studies have indicated that SEPS1 protects cells from oxidative stress and that variations in the gene are associated with circulating levels of inflammatory cytokines. In our study, we identified two variants in the SEPS1 gene, which associated with coronary heart disease and ischemic stroke in women. This is, to our knowledge, the first study suggesting a role of SEPS1 in the risk for CVD after extensively examining the variation within the gene region. In the third part of this thesis, we focused on a set of seven genes (angiotensin converting enzyme, angiotensin II receptor type I, C-reactive protein (CRP), and fibrinogen alpha-, beta-, and gamma-chains (FGA, FGB, FGG)) related to inflammatory cytokine interleukin 6 (IL6) and their association with the risk for CVD. We identified one variant in the IL6 gene conferring risk for CVD in men and a variant pair from IL6 and FGA genes associated with decreased risk. Moreover, we identified and confirmed an association between a rare variant in the CRP gene and lower CRP levels, and found two variants in the FGA and FGG genes associating with fibrinogen. The results from this third study suggest a role for the interleukin 6 pathway genes in the pathogenesis of CVD and warrant further studies in other populations. In addition to the IL6 -related genes, we describe in this thesis several sex-specific associations in other genes included in this study. The majority of the findings were evident only in women encouraging other studies of cardiovascular disease to include and analyse women separately from men.

Studies on immune activation in rheumatoid arthritis and reactive arthritis

Rheumatoid arthritis (RA) is an autoimmune disease characterized by synovitis, progressive joint destruction, and disability. Reactive arthritis (ReA) is a sterile joint inflammation following a distant mucosal infection. The clinical course of these diseases is variable and cannot be predicted with reasonable accuracy by clinical and laboratory markers. The predictive value of circulating soluble interleukin-2 receptor (sIL-2R), a marker of lymphocyte activation, measured by Immulite® automated immunoassay analyzer, was evaluated in two cohorts of RA patients. In 175 patients with active early RA randomized to treatment with either on disease-modifying antirheumatic drug (DMARD) or a combination of 3 DMARDs and prednisolone, low baseline sIL-2R level predicted remission after 6 months in patients treated with a single DMARD. In 24 patients with active RA refractory to DMARDs, low baseline sIL-2R level predicted rapid clinical response to treatment with infliximab, an anti-tumour necrosis factor antibody. Furthermore, in a cohort of 26 patients with acute ReA, high baseline sIL-2R level predicted remission after 6 months. Levels of circulating soluble E-selectin (sE-selectin), a marker of endothelial activation, were measured annually by enzyme-linked immunosorbent assay (ELISA) in a cohort of 85 patients with early RA. During a five-year follow-up, sE-selectin levels were associated with activity and outcome of RA. The levels of neutrophil and monocyte CD11b/CD18 expression measured by flow cytometry, and circulating levels of sE-selectin measured by ELISA, and procalcitonin by immunoluminometric assay, were compared in 28 patients with acute ReA and 16 patients with early RA. The levels of the markers were comparable in ReA, RA, and healthy control subjects. In conlusion, sIL-2R may provide a new predictive marker in early RA treated with a single DMARD and refractory RA treated with infliximab. In addition, sIL-2R level predicts remission in acute ReA.

Molecular genetic studies on nemaline myopathy and related disorders

Nemaline myopathy (NM) is a rare muscle disorder characterised by muscle weakness and nemaline bodies in striated muscle tissue. Nemaline bodies are derived from sarcomeric Z discs and may be detected by light microscopy. The disease can be divided into six subclasses varying from very severe, in some cases lethal forms to milder forms. NM is usually the consequence of a gene mutation and the mode of inheritance varies between NM subclasses and different families. Mutations in six genes are known to cause NM; nebulin (NEB), alpha-actin, alpha-tropomyosin (TPM3), troponin T1, beta-tropomyosin (TPM2) and cofilin 2, of which nebulin and -actin are the most common. One of the main interests of my research is NEB. Nebulin is a giant muscle protein (600-900 kDa) expressed mainly in the thin filaments of striated muscle. Mutations in NEB are the main cause of autosomal recessive NM. The gene consists of 183 exons. Thus being gigantic, NEB is very challenging to investigate. NEB was screened for mutations using denaturing High Performance Liquid Chromatography (dHPLC) and sequencing. DNA samples from 44 families were included in this study, and we found and published 45 different mutations in them. To date, we have identified 115 mutations in NEB in a total of 96 families. In addition, we determined the occurrence in a world-wide sample cohort of a 2.5 kb deletion containing NEB exon 55 identified in the Ashkenazi Jewish population. In order to find the seventh putative NM gene a genome-wide linkage study was performed in a series of Turkish families. In two of these families, we identified a homozygous mutation disrupting the termination signal of the TPM3 gene, a previously known NM-causing gene. This mutation is likely a founder mutation in the Turkish population. In addition, we described a novel recessively inherited distal myopathy, named distal nebulin myopathy, caused by two different homozygous missense mutations in NEB in six Finnish patients. Both mutations, when combined in compound heterozygous form with a more disruptive mutation, are known to cause NM. This study consisted of molecular genetic mutation analyses, light and electron microscopic studies of muscle biopsies, muscle imaging and clinical examination of patients. In these patients the distribution of muscle weakness was different from NM. Nemaline bodies were not detectable with routine light microscopy, and they were inconspicuous or absent even using electron microscopy. No genetic cause was known to underlie cap myopathy, a congenital myopathy characterised by cap-like structures in the muscle fibres, until we identified a deletion of one codon of the TPM2 gene, in a 30-year-old cap myopathy patient. This mutation does not change the reading frame of the gene, but a deletion of one amino acid does affect the conformation of the protein produced. In summary, this thesis describes a novel distal myopathy caused by mutations in the nebulin gene, several novel nebulin mutations associated with nemaline myopathy, the first molecular genetic cause of cap myopathy, i.e. a mutation in the beta-tropomyosin gene, and a founder mutation in the alpha-tropomyosin gene underlying autosomal recessive nemaline myopathy in the Turkish population.

Molecular Pathways of Disturbed Sleep and Depression: Studies on Adenosine and Gene Expression Patterns

Background: Adenosine is a potent sleep-promoting substance, and one of its targets is the basal forebrain. Fairly little is known about its mechanism of action in the basal forebrain and about the receptor subtype mediating its regulating effects on sleep homeostasis. Homeostatic deficiency might be one of the causes of the profoundly disturbed sleep pattern in major depressive disorder, which could explain the reduced amounts of delta-activity-rich stages 3 and 4. Since major depression has a relatively high heritability, and on the other hand adenosine regulates sleep homeostasis and might also be involved in mood modulation, adenosine-related genes should be considered for their possible contribution to a predisposition for depression and disturbed sleep in humans. Depression is a complex disorder likely involving the abnormal functioning of several genes. Novel target genes which could serve as the possible common substrates for depression and comorbid disturbed sleep should be identified. In this way specific brain areas related to sleep regulation should be studied by using animal model of depression which represents more homogenous phenotype as compared to humans. It is also important to study these brain areas during the development of depressive-like features to understand how early changes could facilitate pathophysiological changes in depression. Aims and methods: We aimed to find out whether, in the basal forebrain, adenosine induces recovery non-rapid eye movement (NREM) sleep after prolonged waking through the A1 or/and A2A receptor subtype. A1 and A2A receptor antagonists were perfused into the rat basal forebrain during 3 h of sleep deprivation, and the amount of NREM sleep and delta power during recovery NREM sleep were analyzed. We then explored whether polymorphisms in genes related to the metabolism, transport and signaling of adenosine could predispose to depression accompanied by signs of disturbed sleep. DNA from 1423 individuals representative of the Finnish population and including controls and cases with depression, depression accompanied by early morning awakenings and depression accompanied by fatigue, was used in the study to investigate the possible association between polymorphisms from adenosine-related genes and cases. Finally to find common molecular substrates of depression and disturbed sleep, gene expression changes were investigated in specific brain areas in the rat clomipramine model of depression. We focused on the basal forebrain of 3-week old clomipramine-treated rats which develop depressive-like symptoms later in adulthood and on the hypothalamus of adult female clomipramine-treated rats. Results: Blocking of the A1 receptor during sleep deprivation resulted in a reduction of the recovery NREM sleep amount and delta power, whereas A2A receptor antagonism had no effect. Polymorphisms in adenosine-related genes SLC29A3 (equilibrative nucleoside transporter type 3) in women and SLC28A1 (concentrative nucleoside transporter type 1) in men associated with depression alone as well as when accompanied by early morning awakenings and fatigue. In Study III the basal forebrain of postnatal rats treated with clomipramine displayed disturbances in gamma-aminobutyric acid (GABA) receptor type A signaling, in synaptic transmission and possible epigenetic changes. CREB1 was identified as a common transcription denominator which also mediates epigenetic regulation. In the hypothalamus the major changes included the expression of genes in GABA-A receptor pathway, K+ channel-related, glutamatergic and mitochondrial genes, as well as an overexpression of genes related to RNA and mRNA processing. Conclusions: Adenosine plays an important role in sleep homeostasis by promoting recovery NREM sleep via the A1 receptor subtype in the basal forebrain. Also adenosine levels might contribute to the risk of depression with disturbed sleep, since the genes encoding nucleoside transporters showed the strongest associations with depression alone and when accompanied by signs of disturbed sleep in both women and men. Sleep and mood abnormalities in major depressive disorder could be a consequence of multiple changes at the transcriptional level, GABA-A receptor signaling and synaptic transmission in sleep-related basal forebrain and the hypothalamus.

Helicobacter pylori in Vammala, Finland: seroepidemiological studies and a population-based ‘screen-and-treat’ programme

Helicobacter pylorin (helikobakteeri) tartunta saadaan yleensä lapsena ja tauti jää tavallisesti pysyväksi ilman täsmähoitoa. Onnistunut hoito parantaa pysyvästi helikobakteerista aiheutuvan mahan haavataudin ja näyttää ehkäisevän mahalaukun pahanlaatuisten muutosten kehittymistä. Aloitimme Vammalassa terveyskeskuksessa toteutetun kansainvälisesti ainutlaatuisen väestöpohjaisen helikobakteeritulehduksen seulonta- ja hoito-ohjelman pilottitutkimuksella 1994. 1996 kaikki 15-40-vuotiaat ja 1997-2000 15- ja 45-vuotiaat vammalalaiset kutsuttiin verinäyteseulontaan. Yhteensä 4626 henkilöä (75% kutsutuista) osallistui seulontaan. Vasta-ainepositiivisille tarjottiin helikobakteeritulehduksen lopettava lääkekuuri. Toiminnan seurauksena helikobakteeritulehduksen esiintyvyyden laskettiin vähentyneen 12%:sta 4%:iin 15-40-vuotiaiden ikäryhmässä. Tutkimme myös helikobakteerivasta-ainepositiivisten ja -negatiivisten eroja sekä helikobakteeritulehduksen riskitekijöitä kyselytutkimuksella. Lapsuudenkodin asumisahtauden, äidin matalan koulutusasteen, tupakoinnin, alkoholinkäytön, huonojen asunto-olojen ja ylävatsavaivoista johtuvien sairauslomien todettiin liittyvän helikobakteeritulehdukseen monimuuttuja-analyysissa. Tutkimme seulontaohjelmassa käyttämiemme IgG- ja IgA-luokan helikobakteeri-vasta-ainetestien luotettavuutta eri ikäryhmissä ottaen huomioon atrofisen gastriitin esiintyvyyden. 561 kliinisin perustein gastroskopoidun potilaan aineistossa IgG-testi osoittautui erittäin herkäksi kaikissa ikäryhmissä (99%). Tarkkuus oli myös vanhemmissa ikäryhmissä hyvä (97-93%), kun atrofista gastriittia sairastavat suljettiin pois. IgA- ja CagA-helikobakteerivasta-aineiden on todettu liittyvän lisääntyneeseen mahahaava- ja mahasyöpäriskiin. Analysoimme 560 henkilön pariseeruminäytteet, jotka oli otettu kahden vuosikymmenen välein, ja totesimme, että IgA-vasta-aineiden esiintyvyyden lisääntyyminen iän myötä johtuu paitsi syntymäajankohdasta ja uusista infektioista myös IgA-vasta-ainetasojen kohoamisesta helikobakteeritulehduksen aikana. Selvitimme myös CagA-vasta-ainetasojen muuttumista analysoimalla seeruminäytteet, jotka oli otettu kahden vuosikymmenen välein. Totesimme, että samanaikaisesti kun helikobakteerin esiintyvyys väestössä on alentunut, erityisesti CagA-positiiviset infektiot ovat vähentyneet. Tutkimuksemme osoittaa, että Suomessa terveyskeskuksen yhteydessä voidaan toteuttaa näin laajamittainen seulonta- ja hoito-ohjelma, johon suomalaiset osallistuvat aktiivisesti. Nähtäväksi jää, kuinka paljon ohjelma kykeni vähentämään helikobakteeritulehdukseen liittyviä myöhäisseuraamuksia.

Electron- Spin-Resonance studies of phase transitions in double propionates

Detailed ESR investigations of Mn2+ substituting for Ca2+ in Ca2Sr(C2H5COO)6, (DSP) and Ca2Pb(C2H5COO)6, (DLP) and Ca2Ba(C2H5COO)6, (DBP), in single crystals and powders, over the temperature range from 300°C to -180°C have been carried out to study the successive phase transitions in these compounds. Spectra have been analyzed in terms of axial spin Hamiltonians and the temperature dependences of the parameters studied. Across the I-II transition, new physically and chemically inequivalent sites appear indicating the disappearance of the diad axes on which the propionate groups are located, bringing out the connection between the motional states of the propionate groups and the occurrence of ferroelectricity. The II-III transition also causes chemically inequivalent sites to develop, indicating that the transitions may not be isomorphous as believed previously. Similarities and dissimilarities of the ESR spectra of DLP, DSP and DBP are discussed in relation to the phase transitions.

Thermal studies on melting and crystallization of polyamide fibres

Effect of heating rate on melting and crystallization of polyamide fibres has been examined using differential scanning calorimetric (DSC) technique. Peak temperature for melting (T m) and crystallization (T k) get suppressed with the increase in the heating rate which has been explained on the basis of chain orientation. Heat of melting (DeltaH m) and crystallization (DeltaH k) have been measured.DeltaH m vs. T m shows a nonlinear dependence which has been explained on the basis of entropy change. Quantitative difference inDeltaH m andDeltaH k values has been explained on the basis of orientation and degradation of the polymer.

Structural studies of analgesics and their interactions. VII. Stereoisomerism and disorder in the structure of oxyphenbutazone monohydrate

Oxyphenbutazone, C19H20N203, a metabolite and perhaps the active form of phenylbutazone, is a widely used non-narcotic analgesic and anti-inflammatory pyrazolidinedione derivative. The monohydrate of the compound crystallizes in the triclinic space group Pi with two molecules in a unit cell of dimensions a -- 9.491 (4), b = 10.261 (5), c = 11.036 (3)A and ¢~ = 72.2 (1), fl = 64.3 (1), 7 = 73.0 (1) °. The structure was solved by direct methods and refined to an R value of 0.107 for 1498 observed reflections. The butyl group in the molecule is disordered. The hydroxyl group occupies two sites with unequal occupancies. On account of the asymmetry at the two N atoms and one of the C atoms in the central five-membered ring, the molecule can exist in eight isomeric states, of which four are sterically unfavourable. The disorder in the position of the hydroxyl group can be readily explained on the basis of the existence, with unequal abundances, of all four sterically favourable isomers.The bond lengths and angles in the molecule are similar to those in phenylbutazone. The crystal structure is stabilized by van der Waals interactions, and O-H... O hydrogen bonds involving the carbonyl and the hydroxyl groups as well as a water molecule.