Calculation of crystal optical properties from molecular electron density

We have recently developed a method to obtain distributed atomic polarizabilities adopting a partitioning of the molecular electron density (for example, the Quantum Theory of Atoms in Molecules, [1]), calculated with or without an applied electric field. The procedure [2] allows to obtained atomic polarizability tensors, which are perfectly exportable, because quite representative of an atom in a given functional group. Among the many applications of this idea, the calculation of crystal susceptibility is easily available, either from a rough estimation (the polarizability of the isolated molecule is used) or from a more precise estimation (the polarizability of a molecule embedded in a cluster representing the first coordination sphere is used). Lorentz factor is applied to include the long range effect of packing, which is enhancing the molecular polarizability. Simple properties like linear refractive index or the gyration tensor can be calculated at relatively low costs and with good precision. This approach is particularly useful within the field of crystal engineering of organic/organometallic materials, because it would allow a relatively easy prediction of a property as a function of the packing, thus allowing "reverse crystal engineering". Examples of some amino acid crystals and salts of amino acids [3] will be illustrated, together with other crystallographic or non-crystallographic applications. For example, the induction and dispersion energies of intermolecular interactions could be calculated with superior precision (allowing anisotropic van der Waals interactions). This could allow revision of some commonly misunderstood intermolecular interactions, like the halogen bonding (see for example the recent remarks by Stone or Gilli [4]). Moreover, the chemical reactivity of coordination complexes could be reinvestigated, by coupling the conventional analysis of the electrostatic potential (useful only in the circumstances of hard nucleophilic/electrophilic interaction) with the distributed atomic polarizability. The enhanced reactivity of coordinated organic ligands would be better appreciated. [1] R. F. W. Bader, Atoms in Molecules: A Quantum Theory. Oxford Univ. Press, 1990. [2] A. Krawczuk-Pantula, D. Pérez, K. Stadnicka, P. Macchi, Trans. Amer. Cryst. Ass. 2011, 1-25 [3] A. S. Chimpri1, M. Gryl, L. H.R. Dos Santos1, A. Krawczuk, P. Macchi Crystal Growth & Design, in the press. [4] a) A. J. Stone, J. Am. Chem. Soc. 2013, 135, 7005−7009; b) V. Bertolasi, P. Gilli, G. Gilli Crystal Growth & Design, 2013, 12, 4758-4770.

Pharmacological characterization of nipecotic acid ethyl ester: A novel cholinergic muscarinic agonist

The aim of this dissertation was to examine the hypothesis that (R)-nipecotic acid ethyl ester ((R)-NAEE) is a cholinergic agonist that is selective for a particular subclass (M$\sb1$ or M$\sb2$) of muscarinic receptors.^ Ligand binding studies indicated that like cholinergic agonists (R)-NAEE selectively interacts with rat heart (M$\sb2$) and brain (M$\sb1$) muscarinic binding sites. Physiological studies revealed that unlike cholinergic agonists (R)-NAEE stimulated only those responses coupled to M$\sb2$ muscarinic receptors (acid secretion, negative inotropic response, smooth muscle contraction). Moreover, in rat brain (R)-NAEE differentiated between M$\sb2$ receptors negatively coupled to adenylate cyclase activity and M$\sb1$ receptors mediating PI turnover, being a weak competitive antagonist at these latter sites. In isolated rat gastric mucosal cells (R)-NAEE also differentiated between two M$\sb2$ coupled responses where it potentiated acid secretion but could not stimulate PI turnover. Atropine, a selective antimuscarinic agent, competitively antagonized all agonist effects of (R)-NAEE.^ Unlike (R)-NAEE, the muscarinic agonist arecoline, which is structurally similar to (R)-NAEE, stimulates both M$\sb1$ and M$\sb2$ receptors. Structure activity studies revealed that saturation of the piperidine ring and the length of the ester side chain of (R)-NAEE are the most important determinants for both M$\sb2$ efficacy and selectivity.^ The results of this dissertation establish that (R)-NAEE is a cholinergic muscarinic receptor agonist that displays greater efficacy at M$\sb2$ than at M$\sb1$ receptors, being a weak antagonist at the M$\sb1$ site. With such selectivity, (R)-NAEE may be regarded as a prototype for a unique class of cholinergic muscarinic M$\sb2$ receptor agonists. Because of these unique properties, (R)-NAEE should be useful in the further characterization of muscarinic receptors, and could lead to the development of a new class of therapeutic agents. ^

BIOCHEMICAL PROPERTIES OF GABA (B) RECEPTORS (BACLOFEN, ADENYLATE CYCLASE, CYCLIC-AMP, PHOSPHOLIPASE, PROTEIN KINASE C)

(gamma)-Aminobutyric acid (GABA), a neurotransmitter in the mammalian central nervous system, influences neuronal activity by interacting with at least two pharmacologically and functionally distinct receptors. GABA(,A) receptors are sensitive to blockade by bicuculline, are associated with benzodiazepine and barbiturate binding sites, and mediate chloride flux. The biochemical and pharmacolocal properties of GABA(,B) receptors, which are stereoselectively activated by (beta)-p-chlorophenyl GABA (baclofen), are less well understood. The aim of this study was to define these features of GABA(,B) receptors, with particular emphasis on their possible relationship to the adenylate cyclase system in brain.^ By themselves, GABA agonists have no effect on cAMP accumulation in rat brain slices. However, some GABA agonists markedly enhance the cAMP accumulation that results from exposure to norepinephrine, adenosine, VIP, and cholera toxin. Evidence that this response is mediated by the GABA(,B) system is provided by the finding that it is bicuculline-insensitive, and by the fact that only those agents that interact with GABA(,B) binding sites are active in this regard. GABA(,B) agonists are able to enhance neurotransmitter-stimulated cAMP accumulation in only certain brain regions, and the response is not influenced by phosphodiesterase inhibitors, although is totally dependent on the availability of extracellular calcium. Furthermore, data suggest that inhibition of phospholipase A(,2), a calcium-dependent enzyme, decreases the augmenting response to baclofen, although inhibitors of arachidonic acid metabolism are without effect. These findings indicate that either arachidonic acid or lysophospholipid, products of PLA(,2)-mediated degradation of phospholipids, mediates the augmentation. Moreover, phorbol esters, compounds which directly activate protein kinase C, were also found to enhance neurotransmitter-stimulated cAMP accumulation in rat brain slices. Since this enzyme is known to be stimulated by unsaturated fatty acids such as arachidonate, it is proposed that GABA(,B) agonists enhance cAMP accumulation by fostering the production of arachidonic acid which stimulates protein kinase C, leading to the phosphorylation of some component of the adenylate cyclase system. Thus, GABA, through an interaction with GABA(,B) receptors, modulates neurotransmitter receptor responsiveness in brain. The pharmocological manipulation of this response could lead to the development of therapeutic agents having a more subtle influence than current drugs on central nervous system function. ^

Effects of Lipophilic Extract of Viscum album L. and Oleanolic Acid on Migratory Activity of NIH/3T3 Fibroblasts and on HaCat Keratinocytes

Viscum album L. lipophilic extract (VALE) contains pharmacologically active pentacyclic triterpenes that are known to exhibit immunomodulatory, antitumor, and wound healing activity. Preliminary clinical observations indicate that VALE was able to influence cutaneous wound healing in vivo. The objective of this study was to investigate wound closure related properties of VALE in vitro. As measured in a wound healing assay, VALE and its predominant triterpene oleanolic acid (OA) significantly and dose dependently promoted the migration of NIH/3T3 fibroblasts in vitro, thereby leading to an enhanced wound closure. Compared to the negative control, maximal stimulation by 26.1% and 26.2%, respectively, was attained with 10 μg/mL VALE and 1 μg/mL OA. Stimulation of proliferation in NIH/3T3 fibroblasts by VALE and OA could be excluded. At higher concentrations both substances affected proliferation and viability of NIH/3T3 fibroblasts and HaCat keratinocytes. In the toxic range of concentrations of VALE and OA, migration of NIH/3T3 fibroblasts was suppressed. The extent of the stimulatory effect on cell migration of VALE quite closely corresponded to the effect expected by the concentrations of OA contained in the crude extract VALE. These data support the casual observation that Viscum album L. lipophilic extract might modulate wound healing related processes in vivo.

High affinity amino acid transporters specifically expressed in xylem parenchyma and developing seeds of Arabidopsis

Arabidopsis amino acid transporters (AAPs) show individual temporal and spatial expression patterns. A new amino acid transporter, AAP8 was isolated by reverse transcription-PCR. Growth and transport assays in comparison to AAP1-5 characterize AAP8 and AAP6 as high affinity amino acid transport systems from Arabidopsis. Histochemical promoter-beta-glucuronidase (GUS) studies identified AAP6 expression in xylem parenchyma, cells requiring high affinity transport due to the low amino acid concentration in xylem sap. AAP6 may thus function in uptake of amino acids from xylem. Histochemical analysis of AAP8 revealed stage-dependent expression in siliques and developing seeds. Thus AAP8 is probably responsible for import of organic nitrogen into developing seeds. The only missing transporter of the family AAP7 was nonfunctional in yeast with respect to amino acid transport, and expression was not detectable. Therefore, AAP6 and -8 are the only members of the family able to transport aspartate with physiologically relevant affinity. AAP1, -6 and -8 are the closest AAP paralogs. Although AAP1 and AAP8 originate from a duplicated region on chromosome I, biochemical properties and expression pattern diverged. Overlapping substrate specificities paired with individual properties and expression patterns point to specific functions of each of the AAP genes in nitrogen distribution rather than to mere redundancy.

Biotic and Abiotic Properties Mediating Plant Diversity Effects on Soil Microbial Communities in an Experimental Grassland

Plant diversity drives changes in the soil microbial community which may result in alterations in ecosystem functions. However, the governing factors between the composition of soil microbial communities and plant diversity are not well understood. We investigated the impact of plant diversity (plant species richness and functional group richness) and plant functional group identity on soil microbial biomass and soil microbial community structure in experimental grassland ecosystems. Total microbial biomass and community structure were determined by phospholipid fatty acid (PLFA) analysis. The diversity gradient covered 1, 2, 4, 8, 16 and 60 plant species and 1, 2, 3 and 4 plant functional groups (grasses, legumes, small herbs and tall herbs). In May 2007, soil samples were taken from experimental plots and from nearby fields and meadows. Beside soil texture, plant species richness was the main driver of soil microbial biomass. Structural equation modeling revealed that the positive plant diversity effect was mainly mediated by higher leaf area index resulting in higher soil moisture in the top soil layer. The fungal-to-bacterial biomass ratio was positively affected by plant functional group richness and negatively by the presence of legumes. Bacteria were more closely related to abiotic differences caused by plant diversity, while fungi were more affected by plant-derived organic matter inputs. We found diverse plant communities promoted faster transition of soil microbial communities typical for arable land towards grassland communities. Although some mechanisms underlying the plant diversity effect on soil microorganisms could be identified, future studies have to determine plant traits shaping soil microbial community structure. We suspect differences in root traits among different plant communities, such as root turnover rates and chemical composition of root exudates, to structure soil microbial communities.

The histological features and physical properties of eroded dental hard tissues.

Erosive demineralisation causes characteristic histological features. In enamel, mineral is dissolved from the surface, resulting in a roughened structure similar to an etching pattern. If the acid impact continues, the initial surface mineral loss turns into bulk tissue loss and with time a visible defect can develop. The microhardness of the remaining surface is reduced, increasing the susceptibility to physical wear. The histology of eroded dentine is much more complex because the mineral component of the tissue is dissolved by acids whereas the organic part is remaining. At least in experimental erosion, a distinct zone of demineralised organic material develops, the thickness of which depends on the acid impact. This structure is of importance for many aspects, e.g. the progression rate or the interaction with active agents and physical impacts, and needs to be considered when quantifying mineral loss. The histology of experimental erosion is increasingly well understood, but there is lack of knowledge about the histology of in vivo lesions. For enamel erosion, it is reasonable to assume that the principal features may be similar, but the fate of the demineralised dentine matrix in the oral cavity is unclear. As dentine lesions normally appear hard clinically, it can be assumed that it is degraded by the variety of enzymes present in the oral cavity. Erosive tooth wear may lead to the formation of reactionary or reparative dentine.

Identification of amino acid substitutions with compensational effects in the attachment protein of canine distemper virus.

The hemagglutinin (H) gene of canine distemper virus (CDV) encodes the receptor-binding protein. This protein, together with the fusion (F) protein, is pivotal for infectivity since it contributes to the fusion of the viral envelope with the host cell membrane. Of the two receptors currently known for CDV (nectin-4 and the signaling lymphocyte activation molecule [SLAM]), SLAM is considered the most relevant for host susceptibility. To investigate how evolution might have impacted the host-CDV interaction, we examined the functional properties of a series of missense single nucleotide polymorphisms (SNPs) naturally accumulating within the H-gene sequences during the transition between two distinct but related strains. The two strains, a wild-type strain and a consensus strain, were part of a single continental outbreak in European wildlife and occurred in distinct geographical areas 2 years apart. The deduced amino acid sequence of the two H genes differed at 5 residues. A panel of mutants carrying all the combinations of the SNPs was obtained by site-directed mutagenesis. The selected mutant, wild type, and consensus H proteins were functionally evaluated according to their surface expression, SLAM binding, fusion protein interaction, and cell fusion efficiencies. The results highlight that the most detrimental functional effects are associated with specific sets of SNPs. Strikingly, an efficient compensational system driven by additional SNPs appears to come into play, virtually neutralizing the negative functional effects. This system seems to contribute to the maintenance of the tightly regulated function of the H-gene-encoded attachment protein. Importance: To investigate how evolution might have impacted the host-canine distemper virus (CDV) interaction, we examined the functional properties of naturally occurring single nucleotide polymorphisms (SNPs) in the hemagglutinin gene of two related but distinct strains of CDV. The hemagglutinin gene encodes the attachment protein, which is pivotal for infection. Our results show that few SNPs have a relevant detrimental impact and they generally appear in specific combinations (molecular signatures). These drastic negative changes are neutralized by compensatory mutations, which contribute to maintenance of an overall constant bioactivity of the attachment protein. This compensational mechanism might reflect the reaction of the CDV machinery to the changes occurring in the virus following antigenic variations critical for virulence.

Placental Uric Acid Transport System: Glucose Transporter 9 (SLC2A9)

Placental Uric Acid Transport System: Glucose Transporter 9 (SLC2A9). INTRODUCTION: Pre-eclampsia, a pregnancy-specific disease, contributes substantially to perinatal morbidity and mortality of both the mother and her child. Pre-eclampsia is often associated with high maternal urate serum levels, which in turn has been shown to play a role in the pathogenesis of this disease. The aim of this study was to investigate the glucose transporter GLUT9-mediated placental uric acid transport system. METHODS: In this study western blot, immunofluorescence techniques as well as a transepithelial transport (Transwell) model were used to assess GLUT9 protein expression and, respectively, uric acid transport activity. Electrophysiological techniques and transmission electron microscopy (TEM) were used to characterize the properties and the structure of GLUT9. RESULTS: Uric acid is transported across a BeWo choriocarcinoma cell monolayer with 530 pmol/min. We could successfully overexpress and for the first time purify the GLUT9b isoform using the Xenopus laevis oocytes expression system. Chloride seems to modulate the urate transport system. TEM revealed that GLUT9b isoform is present as monomer and dimmer in the Xenopus laevis overexpression model. A class average of all the particles allowed us to develop a first model of human GLUT9b structure, which was derived from the published crystal structure of the bacterial homologue of GLUT1-4. CONCLUSIONS: In vitro the “materno-fetal” transport of uric acid is slow indicating that in vivo the fetus might be protected from short-term fluctuations of maternal urate serum levels. The low-resolution structure obtained from TEM validates the proposed homology model regarding the structure of human GLUT9b. In ongoing studies this model is used to perform virtual screening to identify novel modulators of the urate transport system enabling the development of novel therapies in pregnancy complications.

Synthesis, structure and magnetic properties of cobalt(II) and copper(II) coordination polymers assembled by phthalate and 4-methylimidazole

New coordination polymers [M(Pht)(4-MeIm)2(H2O)]n (M=Co (1), Cu (2); Pht2−=dianion of o-phthalic acid; 4-MeIm=4-methylimidazole) have been synthesized and characterized by IR spectroscopy, X-ray crystallography, thermogravimetric analysis and magnetic measurements. The crystal structures of 1 and 2 are isostructural and consist of [M(4-MeIm)2(H2O)] building units linked in infinite 1D helical chains by 1,6-bridging phthalate ions which also act as chelating ligands through two O atoms from one carboxylate group in the case of 1. In complex 1, each Co(II) atom adopts a distorted octahedral N2O4 geometry being coordinated by two N atoms from two 4-MeIm, three O atoms of two phthalate residues and one O atom of a water molecule, whereas the square-pyramidal N2O3 coordination of the Cu(II) atom in 2 includes two N atoms of N-containing ligands, two O atoms of two carboxylate groups from different Pht, and a water molecule. An additional strong O–H⋯O hydrogen bond between a carboxylate group of the phthalate ligand and a coordinated water molecule join the 1D helical chains to form a 2D network in both compounds. The thermal dependences of the magnetic susceptibilities of the polymeric helical Co(II) chain compound 1 were simulated within the temperature range 20–300 K as a single ion case, whereas for the Cu(II) compound 2, the simulations between 25 and 300 K, were made for a linear chain using the Bonner–Fisher approximation. Modelling the experimental data of compound 1 with MAGPACK resulted in: g=2.6, |D|=62 cm−1. Calculations using the Bonner–Fisher approximation gave the following result for compound 2: g=2.18, J=–0.4 cm−1.

Nickel(II)-, cobalt(II)-, copper(II)-, and zinc(II)-phthalate and 1-methylimidazole coordination compounds: synthesis, crystal structures and magnetic properties

Three new coordination polymers [M(Pht)(1-MeIm)2]n (where M=Cu (1), Zn (2), Co (3); Pht2−=dianion of o-phthalic acid; 1-MeIm=1-methylimidazole) and two compounds [M(1-MeIm)6](HPht)2 · 2H2O (M=Co (4), Ni (5)) have been synthesized and characterized by X-ray crystallography. The structures of 1–3 (2 is isostructural to 3) consist of [M(1-MeIm)2] building units connected by 1,6-bridging phthalate ions to form infinite chains. In complex 1, each copper(II) center adopts a square coordination mode of N2O2 type by two O atoms from different phthalate ions and two N atoms of 1-MeIm, whereas in 3 two independent metal atoms are tetrahedrally (N2O2) coordinated to a pair of Pht ligands and a pair of 1-MeIm molecules. There are only van der Waals interactions between the chains in 1, while the three-dimensional network in 3 is assembled by C–H⋯O contacts. In contrast to polymers 1–3 the structures of 4 and 5 (complexes are also isostructural) are made up of the [M(1-MeIm)6]2+ cation, two hydrogen phthalate anions (HPht−) and two H2O solvate molecules. The coordination around each metal(II) atom is octahedral with six nitrogen atoms of 1-MeIm. Extended hydrogen bonding networks embracing the solvate water molecules and a phthalate residue as well as the weak C–H⋯O interactions stabilize the three-dimensional structures. Magnetic studies clearly show that the magnetic ions do not interact with each other. Furthermore, in compound 4 we have another example of a highly anisotropic Co2+ ion with a rhombic g-tensor and large zero-field-splitting. The complexes were also characterized by IR and 1H NMR spectroscopy, thermogravimetric analysis, and all data are discussed in the terms of known structures.

Fluorinated peptide nucleic acid

The fluorinated olefinic peptide nucleic acid analogue (F-OPA) monomer containing the base thymine was synthesised in 13 steps. PNAs containing this unit were prepared and their pairing properties assessed by means of UV-melting experiments

Olefinic peptide nucleic acid (OPA)

The olefinic peptide nucleic acid analogues (OPA) monomers containing the bases thymine and adenine were synthesised in 11 steps. Fully modified oligomers containing these units were prepared and their pairing properties assessed by means of UV-melting experiments

Stability and kinetics of nucleic acid triplexes with chimaeric DNA/RNA third strands

A series of chimaeric DNA/RNA triplex-forming oligonucleotides (TFOs) with identical base-sequence but varying sequential composition of the sugar residues were prepared. The structural, kinetic and thermodynamic properties of triplex formation with their corresponding double-helical DNA target were investigated by spectroscopic methods. Kinetic and thermodynamic data were obtained from analysis of non-equilibrium UV-melting- and annealing curves in the range of pH 5.1 to 6.7 in a 10 mM citrate/phosphate buffer containing 0.1M NaCl and 1 mM EDTA. It was found that already single substitutions of ribo- for deoxyribonucleotides in the TFOs greatly affect stability and kinetics of triplex formation in a strongly sequence dependent manner. Within the sequence context investigated, triplex stability was found to increase when deoxyribonucleotides were present at the 5'-side and ribonucleotides in the center of the TFO. Especially the substitution of thymidines for uridines in the TFO was found to accelerate both, the association and dissociation process, in a strongly position-dependent way. Differential structural information on triplexes and TFO single-strands was obtained from CD-spectroscopy and gel mobility experiments. Only minor changes were observed in the CD spectra of the triplexes at all pH values investigated, and the electrophoretic mobility was nearly identical in all cases, indicating a high degree of structural similarity. In contrast, the single-stranded TFOs showed high structural variability as determined in the same way. The results are discussed in the context of the design of TFOs for therapeutic or biochemical applications.

Characterization and performance of carbonaceous materials obtained from exhausted sludges for the anaerobic biodecolorization of the azo dye Acid Orange II.

This work presents the preliminary study of new carbonaceous materials (CMs) obtained from exhausted sludge, their use in the heterogeneous anaerobic process of biodecolorization of azo dyes and the comparison of their performance with one commercial active carbon. The preparation of carbonaceous materials was conducted through chemical activation and carbonization. Chemical activation was carried out through impregnation of sludge-exhausted materials with ZnCl2 and the activation by means of carbonization at different temperatures (400, 600 and 800°C). Their physicochemical and surface characteristics were also investigated. Sludge based carbonaceous (SBC) materials SBC400, SBC600 and SBC800 present values of 13.0, 111.3 and 202.0m(2)/g of surface area. Biodecolorization levels of 76% were achieved for SBC600 and 86% for SBC800 at space time (τ) of 1.0min, similar to that obtained with commercial activated carbons in the continuous anaerobic up-flow packed bed reactor (UPBR). The experimental data fit well to the first order kinetic model and equilibrium data are well represented by the Langmuir isotherm model. Carbonaceous materials show high level of biodecolorization even at very short space times. Results indicate that carbonaceous materials prepared from sludge-exhausted materials have outstanding textural properties and significant degradation capacity for treating textile effluents.