Synthetic, spectroscopic, magnetic, and x-ray structural studies on a vitamin B6-amino acid Schiff base complex, aqua(5'-phosphopyridoxylidenetyrosinato)copper(II) tetrahydrate

A Schiff base metal complex, [Cu(II)(PLP-DL-tyrosinato)(H2O)].4H2O (PLP = pyridoxal phosphate), with the molecular formula CuC17O13N2H27P has been prepared and characterized by magnetic, spectral, and X-ray structural studies. The compound crystallizes in the triclinic space group P1BAR with a = 8.616 (2) angstrom, b = 11.843 (3) angstrom, c = 12.177 (3) angstrom, alpha = 103.40 (2)degrees, beta = 112.32 (2)degrees, gamma = 76.50 (1)degrees, and Z = 2. The structure was solved by the heavy-atom method and refined by least-squares techniques to a final R value of 0.057 for 3132 independent reflections. The coordination geometry around Cu(II) is distorted square pyramidal with phenolic oxygen, imino nitrogen, and carboxylate oxygen from the Schiff base ligand and water oxygen as basal donor atoms. The axial site is occupied by a phosphate oxygen from a neighboring molecule, thus resulting in a one-dimensional polymer. The structure reveals pi-pi interaction of the aromatic side chain of the amino acid with the pyridoxal pi system. A comparative study is made of this complex with similar Schiff base complexes. The variable-temperature magnetic behavior of this compound shows a weak antiferromagnetic interaction.

Metal-insulator transitions in Cr-doped V2O3 by x-ray and ultraviolet photoelectron spectroscopy

X-ray and ultraviolet photoelectron spectroscopy have been employed to investigate the high temperature metal-insulator transitions in V2O3 and (V0.99Cr0.01)2O3. The high temperature transitions are associated with more gradual changes in the 3d bands compared to the low-temperature transitions

HDL subspecies : association with low HDL-C, obesity, and metabolic syndrome

AIMS An independent, powerful coronary heart disease (CHD) predictor is a low level of high-density lipoprotein cholesterol (HDL-C). Discoidal preβ-HDL particles and large HDL2 particles are the primary cholesterol acceptors in reverse cholesterol transport, a key anti-atherogenic HDL mechanism. The quality of HDL subspecies may provide better markers of HDL functionality than does HDL-C alone. We aimed I) to study whether alterations in the HDL subspecies profile exist in low-HDL-C subjects II) to explore the relationship of any changes in HDL subspecies profile in relation to atherosclerosis and metabolic syndrome; III) to elucidate the impact of genetics and acquired obesity on HDL subspecies distribution. SUBJECTS The study consisted of 3 cohorts: A) Finnish families with low HDL-C and premature CHD (Study I: 67 subjects with familial low HDL-C and 64 controls; Study II: 83 subjects with familial low HDL-C, 65 family members with normal HDL-C, and 133 controls); B) a cohort of 113 low- and 133 high-HDL-C subjects from the Health 2000 Health Examination Survey carried out in Finland (Study III); and C) a Finnish cohort of healthy young adult twins (52 monozygotic and 89 dizygotic pairs) (Study IV). RESULTS AND CONCLUSIONS The subjects with familial low HDL-C had a lower preβ-HDL concentration than did controls, and the low-HDL-C subjects displayed a dramatic reduction (50-70%) in the proportion of large HDL2b particles. The subjects with familial low HDL-C had increased carotid atherosclerosis measured as intima-media-thickness (IMT), and HDL2b particles correlated negatively with IMT. The reduction in both key cholesterol acceptors, preβ-HDL and HDL2 particles, supports the concept of impaired reverse cholesterol transport contributing to the higher CHD risk in low-HDL-C subjects. The family members with normal HDL-C and the young adult twins with acquired obesity showed a reduction in large HDL2 particles and an increase in small HDL3 particles, which may be the first changes leading to the lowering of HDL-C. The low-HDL-C subjects had a higher serum apolipoprotein E (apoE) concentration, which correlated positively with the metabolic syndrome components (waist circumference, TG, and glucose), highlighting the need for a better understanding of apoE metabolism in human atherosclerosis. In the twin study, the increase in small HDL3b particles was associated with obesity independent of genetic effects. The heritability estimate, of 73% for HDL-C and 46 to 63% for HDL subspecies, however, demonstrated a strong genetic influence. These results suggest that the relationship between obesity and lipoproteins depends on different elements in each subject. Finally, instead of merely elevating HDL-C, large HDL2 particles and discoidal preβ-HDL particles may provide beneficial targets for HDL-targeted therapy.

Lower Gastrointestinal Microbiota in Health and Irritable Bowel Syndrome : Characterisation and Effect of Probiotic Intervention

The human gastrointestinal (GI) microbiota is a complex ecosystem that lives in symbiosis with its host. The growing awareness of the importance of the microbiota to the host as well as the development of culture-free laboratory techniques and computational methods has enormously expanded our knowledge of this microbial community. Irritable bowel syndrome (IBS) is a common functional bowel disorder affecting up to a fifth of the Western population. To date, IBS diagnosis has been based on GI symptoms and the exclusion of organic diseases. The GI microbiota has been found to be altered in this syndrome and probiotics can alleviate the symptoms, although clear links between the symptoms and the microbiota have not been demonstrated. The aim of the present work was to characterise IBS related alterations in the intestinal microbiota, their relation to IBS symptoms and their responsiveness to probiotic theraphy. In this thesis research, the healthy human microbiota was characterised by cloning and sequencing 16S rRNA genes from a faecal microbial community DNA pool that was first profiled and fractionated according to its guanine and cytosine content (%G+C). The most noticeable finding was that the high G+C Gram-positive bacteria (the phylum Actinobacteria) were more abundant compared to a corresponding library constructed from the unfractionated DNA pool sample. Previous molecular analyses of the gut microbiota have also shown comparatively low amounts of high G+C bacteria. Furthermore, the %G+C profiling approach was applied to a sample constructed of faecal DNA from diarrhea-predominant IBS (IBS-D) subjects. The phylogenetic microbial community comparison performed for healthy and IBS-D sequence libraries revealed that the IBS-D sample was rich in representatives of the phyla Firmicutes and Proteobacteria whereas Actinobacteria and Bacteroidetes were abundant in the healthy subjects. The family Lachnospiraceae within the Firmicutes was especially prevalent in the IBS-D sample. Moreover, associations of the GI microbiota with intestinal symptoms and the quality of life (QOL) were investigated, as well as the effect of probiotics on these factors. The microbial targets that were analysed with the quantitative real-time polymerase chain reaction (qPCR) in this study were phylotypes (species definition according to 16S rRNA gene sequence similarity) previously associated with either health or IBS. With a set of samples, the presence or abundance of a phylotype that had 94% 16S rRNA gene sequence similarity to Ruminococcus torques (R. torques 94%) was shown to be associated with the severity of IBS symptoms. The qPCR analyses for selected phylotypes were also applied to samples from a six-month probiotic intervention with a mixture of Lactobacillus rhamnosus GG, L. rhamnosus Lc705, Propionibacterium freudenreichii ssp. shermanii JS and Bifidobacterium breve Bb99. The intervention had been previously reported to alleviate IBS symptoms, but no associations with the analysed microbiota representatives were shown. However, with the phylotype-specific assays applied here, the abundance of the R. torques 94% -phylotype was shown to be lowered in the probiotic-receiving group during the probiotic supplementation, whereas a Clostridium thermosuccinogenes 85% phylotype, previously associated with a healthy microbiota, was found to be increased compared to the placebo group. To conclude, with the combination of methods applied, higher abundance of Actinobacteria was detected in the healthy gut than found in previous studies, and significant phylum-level microbiota alterations could be shown in IBS-D. Thus, the results of this study provide a detailed overview of the human GI microbiota in healthy subjects and in subjects with IBS. Furthermore, the IBS symptoms were linked to a particular clostridial phylotype, and probiotic supplementation was demonstrated to alter the GI microbiota towards a healthier state with regard to this and an additional bacterial phylotype. For the first time, distinct phylotype-level alterations in the microbiota were linked to IBS symptoms and shown to respond to probiotic therapy.

Molecular genetics of Usher syndrome -inherited deafness and blindness

Usher syndrome (USH) is an inherited blindness and deafness disorder with variable vestibular dysfunction. The syndrome is divided into three subtypes according to the progression and severity of clinical symptoms. The gene mutated in Usher syndrome type 3 (USH3), clarin 1 (CLRN1), was identified in Finland in 2001 and two mutations were identified in Finnish patients at that time. Prior to this thesis study, the two CLRN1 gene mutations were the only USH mutations identified in Finnish USH patients. To further clarify the Finnish USH mutation spectrum, all nine USH genes were studied. Seven mutations were identified: one was a previously known mutation in CLRN1, four were novel mutations in myosin VIIa (MYO7A) and two were a novel and a previously known mutation in usherin (USH2A). Another aim of this thesis research was to further study the structure and function of the CLRN1 gene, and to clarify the effects of mutations on protein function. The search for new splice variants resulted in the identification of eight novel splice variants in addition to the three splice variants that were already known prior to this study. Studies of the possible promoter regions for these splice variants showed the most active region included the 1000 bases upstream of the translation start site in the first exon of the main three exon splice variant. The 232 aa CLRN1 protein encoded by the main (three-exon) splice variant was transported to the plasma membrane when expressed in cultured cells. Western blot studies suggested that CLRN1 forms dimers and multimers. The CLRN1 mutant proteins studied were retained in the endoplasmic reticulum (ER) and some of the USH3 mutations caused CLRN1 to be unstable. During this study, two novel CLRN1 sequence alterations were identified and their pathogenicity was studied with cell culture protein expression. Previous studies with mice had shown that Clrn1 is expressed in mouse cochlear hair cells and spiral ganglion cells, but the expression profile in mouse retina remained unknown. The Clrn1 knockout mice display cochlear cell disruption/death, but do not have a retinal phenotype. The zebrafish, Danio rerio, clrn1 was found to be expressed in hair cells associated with hearing and balance. Clrn1 expression was also found in the inner nuclear layer (INL), photoreceptor layer and retinal pigment epithelium layer (RPE) of the zebrafish retina. When Clrn1 production was knocked down with injected morpholino oligonucleotides (MO) targeting Clrn1 translation or correct splicing, the zebrafish larvae showed symptoms similar to USH3 patients. These larvae had balance/hearing problems and reduced response to visual stimuli. The knowledge this thesis research has provided about the mutations in USH genes and the Finnish USH mutation spectrum are important in USH patient diagnostics. The extended information about the structure and function of CLRN1 is a step further in exploring USH3 pathogenesis caused by mutated CLRN1 as well as a step in finding a cure for the disease.

X-ray studies on crystalline complexes involving amino acids and peptides XIX. Effects of change in chirality in the complexes of succinic acid with dl- and l-arginine

Crystals of dl-arginine hemisuccinate dihydrate (I)(monoclinic; P21/c; a = 5.292, b = 16.296, c = 15.203 Å; α= 92.89°; Z = 4) and l-arginine hemisuccinate hemisuccinic acid monohydrate (II) (triclinic; P1; a = 5.099; b = 10.222, c = 14.626 Å; α= 77.31, β= 89.46, γ= 78.42°; Z = 2) were grown under identical conditions from aqueous solutions of the components in molar proportions. The structures were solved by direct methods and refined to R = 0.068 for 2585 observed reflections in the case of (I) and R = 0.036 for 2154 observed reflections in the case of (11). Two of the three crystallographically independent arginine molecules in the complexes have conformations different from those observed so far in the crystal structures containing arginine. The succinic acid molecules and the succinate ions in the structures are centrosymmetric and planar. The crystal structure of (II) is highly pseudosymmetric. Arginine-succinate interactions in both the complexes involve specific guanidyl-carboxylate interactions. The basic elements of aggregation in both the structures are ribbons made up of alternating arginine dimers and succinate ions. However, the ribbons pack in different ways in the two structures. (II) presents an interesting case in which two ionisation states of the same molecule coexist in a crystal. The two complexes provide a good example of the effect of change in chirality on stoichiometry, conformation, aggregation, and ionisation state in the solid state.

X-ray studies on crystalline complexes involving amino acids and peptides. XXI. Structure of a (1:1) complex between L-phenylalanine and D-valine

CsHllNO2.C9HilNO2, Mr = 282.3, P1, a = 5.245 (1), b = 5.424 (1), c = 14.414 (2) A, a = 97.86 (1), fl = 93-69 (2), y = 70-48 (2) °, V= 356 A 3, Z = 1, O m = 1-32 (2), Dx = 1.32 g cm-3, h(Mo Ka) = 0-7107 A, g = 5-9 cm-1, F(000) = 158, T= 298 K, R=0.035 for 1518 observed reflections with I>2tr(I). The molecules aggregate in double layers, one ayer made up of L-phenylalanine molecules and the other of D-valine molecules. Each double layer is stabilized by interactions involving main-chain atoms of both types of molecules. The interactions include hydrogen bonds which give rise to two head-to-tail sequences. The arrangement of molecules in the complex is almost the same as that in the structure of DL-valine (and DL-leucine and DL-isoleucine) except for the change in the side chain of L molecules. The molecules in crystals containing an equal number of L and O hydrophobic amino-acid molecules thus appear to aggregate in a similar fashion, irrespective of the precise details of the side chain.

Protein Hydration and Water Structure: X-ray Analysis of a Closely Packed Protein Crystal with Very Low Solvent Content

Low-humidity monoclinic lysozyme, resulting from a water-mediated transformation, has one of the lowest solvent contents (22% by volume) observed in a protein crystal. Its structure has been solved by the molecular replacement method and refined to an R value of 0.175 for 7684 observed reflections in the 10–1.75 Å resolution shell. 90% of the solvent in the well ordered crystals could be located. Favourable sites of hydration on the protein surface include side chains with multiple hydrogen-bonding centres, and regions between short hydrophilic side chains and the main-chain CO or NH groups of the same or nearby residues. Major secondary structural features are not disrupted by hydration. However, the free CO groups at the C terminii and, to a lesser extent, the NH groups at the N terminii of helices provide favourable sites for water interactions, as do reverse turns and regions which connect β-structure and helices. The hydration shell consists of discontinuous networks of water molecules, the maximum number of molecules in a network being ten. The substrate-binding cleft is heavily hydrated, as is the main loop region which is stabilized by water interactions. The protein molecules are close packed in the crystals with a molecular coordination number of 14. Arginyl residues are extensively involved in intermolecular hydrogen bonds and water bridges. The water molecules in the crystal are organized into discrete clusters. A distinctive feature of the clusters is the frequent occurrence of three-membered rings. The protein molecules undergo substantial rearrangement during the transformation from the native to the low-humidity form. The main-chain conformations in the two forms are nearly the same, but differences exist in the side-chain conformation. The differences are particularly pronounced in relation to Trp 62 and Trp 63. The shift in Trp 62 is especially interesting as it is also known to move during inhibitor binding.

X-ray studies on crystalline complexes involving amino acids and peptides. XXV. Structures of DL-proline hemisuccinic acid and glycyl-L-histidinium semisuccinate monohydrate and a comparative study of amino-acid and peptide complexes of succinic acid

DL-Proline hemisuccinic acid, C5H9NO2.1/2C4H6O4, M(r) = 174.2, P2(1/c) a = 5.254 (1), b = 17.480 (1), c = 10.230 (i) angstrom, beta = 119.60 (6)-degrees Z = 4, D(m) = 1.41 (4), D(x) = 1.42 g cm-3, R = 0.045 for 973 observed reflections. Glycyl-L-histidinium semisuccinate monohydrate, C8H13N4O3+.C4H5O4-.H2O, M(r) = 348.4, P2(1), a = 4.864 (1), b = 17.071 (2), c = 9.397 (1) angstrom, beta = 90.58-degrees, Z = 2, D(m) = 1.45 (1), D(x) = 1.48 g cm-3, R = 0.027 for 1610 observed reflections. Normal amino-acid and dipeptide aggregation patterns are preserved in the structures in spite of the presence of succinic acid/semisuccinate ions. In both the structures, the amino-acid/dipeptide layers stack in such a way that the succinic acid molecules/semisuccinate ions are enclosed in voids created during stacking. Substantial variability in the ionization state and the stoichiometry is observed in amino-acid and peptide complexes of succinic acid. Succinic acid molecules and succinate ions appear to prefer a planar centro-symmetric conformation with the two carboxyl (carboxylate) groups trans with respect to the central C=C bond. Considerable variation is seen in the departure from and modification of normal amino-acid aggregation patterns produced by the presence of succinic acid. Some of the complexes can be described as inclusion compounds with the amino acid/dipeptide as the 'host' and succinic acid/semisuccinate/succinate as the 'guest'. The effects of change in chirality, though very substantial, are not the same in different pairs of complexes involving DL and L isomers of the same amino acid.

A2MoTiO7−x (Atriple bond; length as m-dashrare earth or Y): a new series of oxide pyrochlores prepared by topochemical reduction of A2MoTiO8 scheelites

A series of anion-deficient pyrochlore oxides of the formula A2MoTiO7−x (xless-than-or-equals, slant0.5), where Atriple bond; length as m-dashSm, Gd, Tb, Dy, Ho, Er, Lu and Y, has been prepared by reduction of A2MoTiO8 scheelites. The scheelite-to-pyrochlore conversion is reversible, indicating that the reaction is likely to be topochemical. The oxidation states of molybdenum and titanium are most probably Mo(III) and Ti(IV) for the limiting composition of the pyrochlores A2MoTiO6.5. The new pyrochlores are non-metallic and paramagnetic as expected.