21Ne and 131Xe NMR study of electric field gradients and multinuclear NMR study of the composition of a ferroelectric liquid crystal.

This study has two goals. First, the electric field gradient (EFG) present in the liquid-crystalline phases of ferroelectric FELIX-R&D is determined using NMR spectroscopy of noble gases 21Ne and 131Xe. The 21Ne and 131Xe NMR spectra were recorded over a temperature range, which covers all the mesophases of FELIX-R&D: nematic N*, smectic A, and smectic C*. The spin quantum number of both 21Ne and 131Xe is 3/2. Their electric quadrupole moment interacts with the EFG at the nuclear site, which in liquid-crystalline phases results in the NMR spectra of the triplet structure, instead of a singlet detectable in the isotropic phase. The total EFG experienced by the noble gas nuclei consists of two contributions; one arises from the quadrupole moments of the liquid crystal molecules (external contribution) and the other one from the deformation of the electron distribution of the atoms (deformational contribution). The total EFGs determined from the 131Xe and 21Ne quadrupole splittings are very similar in the nematic and smectic A phases but differ in the smectic C* phase, being about twice larger in the 21Ne case which stems from the larger deformation of the xenon electron cloud than that of neon. For the first time, EFG was determined also in the smectic C* phase applying noble gas NMR spectroscopy. Second, the structure of molecules which, as a mixture, compose the used ferroelectric liquid crystal, FELIX-R&D, is determined by applying a number of various NMR methods and sophisticated spectral analysis. In this part, NMR spectra were recorded from FELIX-R&D/CDCl3 solution. The NMR spectral analysis was divided into four subsystems with over 13 000 000 nonzero intensity transitions. It appeared that FELIX-R&D is composed of three phenyl pyrimidine derivatives and a chiral dopant with fluorine in the asymmetric carbon atom.

Using slaughterhouse waste in a biochemical-based biorefinery - results from pilot scale tests.

A novel biorefinery concept was piloted using protein-rich slaughterhouse waste, chicken manure and straw as feedstocks. The basic idea was to provide a proof of concept for the production of platform chemicals and biofuels from organic waste materials at non-septic conditions. The desired biochemical routes were 2,3-butanediol and acetone-butanol fermentation. The results showed that hydrolysis resulted only in low amounts of easily degradable carbohydrates. However, amino acids released from the protein-rich slaughterhouse waste were utilized and fermented by the bacteria in the process. Product formation was directed towards acidogenic compounds rather than solventogenic products due to increasing pH-value affected by ammonia release during amino acid fermentation. Hence, the process was not effective for 2,3-butanediol production, whereas butyrate, propionate, γ-aminobutyrate and valerate were predominantly produced. This offered fast means for converting tedious protein-rich waste mixtures into utilizable chemical goods. Furthermore, the residual liquid from the bioreactor showed significantly higher biogas production potential than the corresponding substrates. The combination of the biorefinery approach to produce chemicals and biofuels with anaerobic digestion of the residues to recover energy in form of methane and nutrients that can be utilized for animal feed production could be a feasible concept for organic waste utilization.

Volatile fatty acids as an added value from biowaste.

The aim of the present work was to provide proof of concept of employing a co-culture of K. mobilis and E. coli for producing short and medium chain volatile fatty acids (VFAs) from kitchen biowaste and potato peels. To this aim, experiments were carried out at pilot-scale installation with a bioreactor of 250L. Different feeding strategies were tested under microaerobic conditions, at pH 6.0-6.5 in order to enhance chain elongation. Acetic acid and ethanol were dominating products in the initial stages of the bioprocess, but in a relatively short time of approx. 20-22h from the process start accumulation of propionic acid took place followed by a chain elongation to butyric and valeric acids. The highest final products yield of 325mg/g TS was achieved for the substrate load of 99.1g TS/L (VS of 91.1g/L) and pH 6.5, with the productivity of 448mg/L/h. However, the highest average VFAs chain length (3.77C) was observed in the process run with the loading of 63.2g TS/L and pH 6.0. In this study, we demonstrated that the existing symbiosis of the co-culture of K. mobilis and E. coli favours formation and chain elongation of VFA, induced most likely by the enhanced ethanol formation. Our finding differs from the previous research which focus mostly on anaerobic conditions of VFAs production. The results provide good basis for further optimisation of VFAs production process.

Quantitative Quantum Mechanical Spectral Analysis (qQMSA) of (1)H NMR spectra of complex mixtures and biofluids.

The quantitative interpretation of (1)H NMR spectra of mixtures like the biofluids is a demanding task due to spectral complexity and overlap. Complications may arise also from water suppression, T2-editing, protein interactions, relaxation differences of the species, experimental artifacts and, furthermore, the spectra may contain unknown components and macromolecular background which cannot be easily separated from baseline. In this work, tools and strategies for quantitative Quantum Mechanical Spectral Analysis (qQMSA) of (1)H NMR spectra from complex mixtures were developed and systematically assessed. In the present approach, the signals of well-defined, stoichiometric components are described by a QM model, while the background is described by a multiterm baseline function and the unknown signals using optimizable and adjustable lines, regular multiplets or any spectral structures which can be composed from spectral lines. Any prior knowledge available from the spectrum can also be added to the model. Fitting strategies for weak and strongly overlapping spectral systems were developed and assessed using two basic model systems, the metabolite mixtures without and with macromolecular (serum) background. The analyses show that if the spectra are measured in high-throughput manner, the consistent absolute quantification demands some calibration to compensate the different response factors of the protons and compounds. On the other hand, the results show that also the T2-edited spectra can be measured so that they obey well the QM rules. In general, qQMSA exploits and interprets the spectral information in maximal way taking full advantage from the QM properties of the spectra and, at the same time, offers chemical confidence which means that individual components can be identified with high confidence on the basis of their accurate spectral parameters.

Preclinical pharmacology of FL442, a novel nonsteroidal androgen receptor modulator.

The preclinical profiles of two most potent compounds of our recently published cycloalkane[d]isoxazole pharmacophore-based androgen receptor (AR) modulators, FL442 (4-(3a,4,5,6,7,7a-hexahydro-benzo[d]isoxazol-3-yl)-2-(trifluoromethyl)benzonitrile) and its nitro analog FL425 (3-(4-nitro-3-(trifluoromethyl)phenyl)-3a,4,5,6,7,7a-hexahydrobenzo[d]isoxazole), were explored to evaluate their druggability for the treatment of AR dependent prostate cancer. The studies revealed that both compounds are selective to AR over other closely related steroid hormone receptors and that FL442 exhibits equal inhibition efficiency towards the androgen-responsive LNCaP prostate cancer cell line as the most widely used antiandrogen bicalutamide and the more recently discovered enzalutamide. Notably, FL442 maintains antiandrogenic activity with enzalutamide-activated AR mutant F876L. In contrast to bicalutamide, FL442 does not stimulate the VCaP prostate cancer cells which express elevated levels of the AR. Distribution analyses showed that [(14)CN]FL442 accumulates strongly in the mouse prostate. In spite of its low plasma concentration obtained by intraperitoneal administration, FL442 significantly inhibited LNCaP xenograft tumor growth. These findings provide a preclinical proof for FL442 as a promising AR targeted candidate for a further optimization.

Comprehensive strategy for proton chemical shift prediction: linear prediction with nonlinear corrections.

A fast 3D/4D structure-sensitive procedure was developed and assessed for the chemical shift prediction of protons bonded to sp3carbons, which poses the maybe greatest challenge in the NMR spectral parameter prediction. The LPNC (Linear Prediction with Nonlinear Corrections) approach combines three well-established multivariate methods viz. the principal component regression (PCR), the random forest (RF) algorithm, and the k nearest neighbors (kNN) method. The role of RF is to find nonlinear corrections for the PCR predicted shifts, while kNN is used to take full advantage of similar chemical environments. Two basic molecular models were also compared and discussed: in the MC model the descriptors are computed from an ensemble of the conformers found by conformational search based on Metropolis Monte Carlo (MMC) simulation; in the 4D model the conformational space was further expanded to the fourth dimension (time) by adding molecular dynamics to the MC conformers. An illustrative case study about the application and interpretation of the 4D prediction for a conformationally flexible structure, scopolamine, is described in detail.

Firefly luciferase inhibitor-conjugated peptide quenches bioluminescence: a versatile tool for real time monitoring cellular uptake of biomolecules.

In this paper, novel firefly luciferase-specific inhibitor compounds (FLICs) are evaluated as potential tools for cellular trafficking of transporter conjugates. As a proof-of-concept, we designed FLICs that were suitable for solid phase peptide synthesis and could be covalently conjugated to peptides via an amide bond. The spacer between inhibitor and peptide was optimized to gain efficient inhibition of recombinant firefly luciferase (FLuc) without compromising the activity of the model peptides. The hypothesis of using FLICs as tools for cellular trafficking studies was ensured with U87Fluc glioblastoma cells expressing firefly luciferase. Results show that cell penetrating peptide (penetratin) FLIC conjugate 9 inhibited FLuc penetrated cells efficiently (IC50 = 1.6 µM) and inhibited bioluminescence, without affecting the viability of the cells. Based on these results, peptide-FLIC conjugates can be used for the analysis of cellular uptake of biomolecules in a new way that can at the same time overcome some downsides seen with other methods. Thus, FLICs can be considered as versatile tools that broaden the plethora of methods that take advantage of the bioluminescence phenomena.

MetIDB: a publicly accessible database of predicted and experimental 1H NMR spectra of flavonoids.

Identification of natural compounds, especially secondary metabolites, has been hampered by the lack of easy to use and accessible reference databases. Nuclear magnetic resonance (NMR) spectroscopy is the most selective technique for identification of unknown metabolites. High quality (1)H NMR (proton nuclear magnetic resonance) spectra combined with elemental composition obtained from mass spectrometry (MS) are essential for the identification process. Here, we present MetIDB, a reference database of experimental and predicted (1)H NMR spectra of 6000 flavonoids. By incorporating the stereochemistry, intramolecular interactions, and solvent effects into the prediction model, chemical shifts and couplings were predicted with great accuracy. A user-friendly web-based interface for MetIDB has been established providing various interfaces to the data and data-mining possibilities. For each compound, additional information is available comprising compound annotation, a (1)H NMR spectrum, 2D and 3D structure with correct stereochemistry, and monoisotopic mass as well as links to other web resources. The combination of chemical formula and (1)H NMR chemical shifts proved to be very efficient in metabolite identification, especially for isobaric compounds. Using this database, the process of flavonoid identification can then be significantly shortened by avoiding repetitive elucidation of already described compounds.

¹H-NMR fingerprinting of Vaccinium vitis-idaea flavonol glycosides.

INTRODUCTION: The fruits of Vaccinium vitis-idaea L. are a valuable source of biologically active flavonoid derivatives. For studies focused on the purification of its quercetin glycosides (QGs) and related glycosides from plants and for the purpose of biological studies, the availability of numeric datasets from computer-assisted ¹H iterative full spin analysis (HiFSA), that is, ¹H-NMR fingerprinting, can replace and assist the repetitive and tedious two-dimensional NMR identification protocol required for both known and new compounds, respectively. OBJECTIVE: To fully interpret the complex ¹H-NMR fingerprints of eight QGs obtained from the berries of V. vitis-idaea and provide complete and unambiguous signal assignments. METHODS: Vaccinium vitis-idaea QGs were purified in a single run by long-bed gel permeation chromatography and identified by comparison with commercially available compounds using LC-MS combining ion-trap and time-of-flight detection and one- or two-dimensional NMR. The HiFSA analysis yielded full sets of ¹H chemical shifts and proton-proton coupling constants, allowing for field-independent spectral simulation. RESULTS: Signal assignments were achieved for the reference standards and the QGs that dominated in purified fractions. However, even mixtures of two to three QGs could be fitted using the HiFSA approach. In the case of the overlapped sugar resonances, the initial fitting of the ¹H spectra of reference compounds, together with values extracted from the two-dimensional NMR data and literature data, assisted in the process. CONCLUSION: The HiFSA method revealed for the first time the presence of Q-3-O-ß-glucopyranoside and Q-3-O-ß-glucuronopyranoside in the berries of V. vitis-idaea, and unambiguously confirmed the structures of Q-3-O-[4″-(3-hydroxy-3-methylglutaroyl)]-α-rhamnopyranoside, Q-3-O-α-rhamnopyranoside, Q-3-O-ß-galactopyranoside, Q-3-O-α-arabinofuranoside, Q-3-O-ß-xylopyranoside and Q-3-O-α-arabinopyranoside.

Discovery of 5-benzyl-3-phenyl-4,5-dihydroisoxazoles and 5-benzyl-3-phenyl-1,4,2-dioxazoles as potent firefly luciferase inhibitors.

Luciferase reporter assays are commonly used in high-throughput screening methods. Here, we report new firefly luciferase (FLuc) inhibitors based on 5-benzyl-3-phenyl-4,5-dihydroisoxazoles and 5-benzyl-3-phenyl-1,4,2-dioxazoles, which showed up as "false positives" in a luciferase reporter gene-based assay for nuclear receptor antagonists. The inhibition was shown to be noncompetitive for both natural enzyme substrates (d-luciferin and ATP) and selective to FLuc and proven to arise from a direct interaction between the enzyme and the inhibitor. Of the 63 evaluated compounds, 28 showed significantly better inhibition potency than the well-known inhibitor resveratrol (IC(50) = 59 nM), with five compounds having distinctly subnanomolar IC(50) values. The most efficient compounds inhibited the luminescence at concentrations lower than (1)/(100) in comparison to resveratrol (lowest IC(50) = 0.26 nM) and can thus be considered to belong to the most potent FLuc inhibitors reported thus far. Overall, the novel inhibitors form a unique molecular library for structure-activity relationship (SAR) analyses.

Design, synthesis, and biological evaluation of nonsteroidal cycloalkane[d]isoxazole-containing androgen receptor modulators.

We report here the design, preparation, and systematic evaluation of a novel cycloalkane[d]isoxazole pharmacophoric fragment-containing androgen receptor (AR) modulators. Cycloalkane[d]isoxazoles form new core structures that interact with the hydrophobic region of the AR ligand-binding domain. To systematize and rationalize the structure-activity relationship of the new fragment, we used molecular modeling to design a molecular library containing over 40 cycloalkane[d]isoxazole derivatives. The most potent compound, 4-(3a,4,5,6,7,7a-hexahydrobenzo[d]isoxazol-3-yl)-2-(trifluoromethyl)benzonitrile (6a), exhibits antiandrogenic activity significantly greater than that of the most widely used antiandrogenic prostate cancer drugs bicalutamide (1) and hydroxyflutamide (2) in reporter gene assays measuring the transcriptional activity of AR (decreasing approximately 90% of the total AR activity) and in competitive AR ligand-binding assays (showing over four times higher potency to inhibit radioligand binding in comparison to bicalutamide). Notably, 6a maintains its antiandrogenic activity with AR mutants W741L and T877A commonly observed and activated by bicalutamide and hydroxyflutamide, respectively, in prostate cancer patients.

(1) H NMR spectral analysis and conformational behavior of n-alkanes in different chemical environments.

Alkyl chains are common structural units, for example in lipids, and their (1) H NMR spectral parameters offer valuable information about their conformational behavior in solvent environment. Even the spectra of short n-alkanes are complex, which is obviously a reason why their accurate spectral analyses have not been reported before. The present study reports the quantum mechanical analysis of (1) H NMR spectra of n-butane, n-pentane, n-hexane, and n-heptane. The spectral parameters were used to characterize the conformational behavior of n-alkanes. The temperature dependence analysis of coupling constants suggests that the enthalpy difference between the gauche (g) and trans (t) conformations (ΔH(g) ) of n-butane in chloroform is 2.55-2.85 kJ mol(-1) . The difference between the trans-gauche (tg) and all-trans (tt) conformers of n-pentane (ΔH(tg) ) seems to be 0.1-0.2 kJ mol(-1) higher. The coupling constant information shows that the t(n) conformations become more favored with longer chains, although not only for energetic reasons but also partly because the g(+) g(-) arrangements become sterically unfavorable, which decreases the number of favorable g(n) -type conformations. The analysis of the (1) H NMR spectra of n-pentane and n-hexane in solvents representing different chemical environments indicates that polar and spherical dimethyl sulfoxide favors clearly the g conformations, whereas n-hexane-d(14) favors slightly the extended t(n) conformation. In addition to the intrinsic scientific importance for NMR spectral parameter prediction and molecular modeling in solution, the results provide some insights to behavior of hydrocarbon chains and their spectra in different chemical environments.

NMR protocol for determination of oxidation susceptibility of serum lipids and application of the protocol to a chocolate study.

A protocol for determination of oxidation susceptibility of serum lipids based on proton nuclear magnetic resonance ((1)H NMR) spectroscopy is presented and compared to the commonly used spectrophotometric method. Even though there are methodological differences between these two methods, the NMR-based oxidation susceptibility correlates well (r(2) = 0.73) with the lag time determined spectrophotometrically. In addition to the oxidizability of serum lipids, the NMR method provides also information about the lipid profile. The NMR oxidation assay was applied to the chocolate study including fasting serum samples (n = 45) from subjects who had consumed white (WC), dark (DC) or high-polyphenol chocolate (HPC) daily for 3 weeks. The oxidation susceptibility of serum lipids decreased in the HPC group, and there was a significant difference between the WC and HPC groups (P = 0.031). According to the random forest analysis, the consumption of the HPC chocolate induced changes to the amounts of HDL, phosphatidylcholine, sphingomyelin, and nervonic, docosahexaenoic and myristic acids. Furthermore, arachidonic, docosahexaenoic, docosapentaenoic and palmitic acids, gamma-glutamyl transferase, hemoglobin, HDL, phosphatidylcholine and choline containing phospholipids explained about 60% of the oxidation susceptibility values. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11306-011-0323-2) contains supplementary material, which is available to authorized users.

High-throughput quantification of circulating metabolites improves prediction of subclinical atherosclerosis.

AIMS: High-throughput metabolite quantification holds promise for cardiovascular risk assessment. Here, we evaluated whether metabolite quantification by nuclear magnetic resonance (NMR) improves prediction of subclinical atherosclerosis in comparison to conventional lipid testing. METHODS AND RESULTS: Circulating lipids, lipoprotein subclasses, and small molecules were assayed by NMR for 1595 individuals aged 24-39 years from the population-based Cardiovascular Risk in Young Finns Study. Carotid intima-media thickness (IMT), a marker of subclinical atherosclerosis, was measured in 2001 and 2007. Baseline conventional risk factors and systemic metabolites were used to predict 6-year incidence of high IMT (≥ 90 th percentile) or plaque. The best prediction of high intima-media thickness was achieved when total and HDL cholesterol were replaced by NMR-determined LDL cholesterol and medium HDL, docosahexaenoic acid, and tyrosine in prediction models with risk factors from the Framingham risk score. The extended prediction model improved risk stratification beyond established risk factors alone; area under the receiver operating characteristic curve 0.764 vs. 0.737, P =0.02, and net reclassification index 17.6%, P =0.0008. Higher docosahexaenoic acid levels were associated with decreased risk for incident high IMT (odds ratio: 0.74; 95% confidence interval: 0.67-0.98; P = 0.007). Tyrosine (1.33; 1.10-1.60; P = 0.003) and glutamine (1.38; 1.13-1.68; P = 0.001) levels were associated with 6-year incident high IMT independent of lipid measures. Furthermore, these amino acids were cross-sectionally associated with carotid IMT and the presence of angiographically ascertained coronary artery disease in independent populations. CONCLUSION: High-throughput metabolite quantification, with new systemic biomarkers, improved risk stratification for subclinical atherosclerosis in comparison to conventional lipids and could potentially be useful for early cardiovascular risk assessment.

Combining NMR ensembles and molecular dynamics simulations provides more realistic models of protein structures in solution and leads to better chemical shift prediction.

While chemical shifts are invaluable for obtaining structural information from proteins, they also offer one of the rare ways to obtain information about protein dynamics. A necessary tool in transforming chemical shifts into structural and dynamic information is chemical shift prediction. In our previous work we developed a method for 4D prediction of protein (1)H chemical shifts in which molecular motions, the 4th dimension, were modeled using molecular dynamics (MD) simulations. Although the approach clearly improved the prediction, the X-ray structures and single NMR conformers used in the model cannot be considered fully realistic models of protein in solution. In this work, NMR ensembles (NMRE) were used to expand the conformational space of proteins (e.g. side chains, flexible loops, termini), followed by MD simulations for each conformer to map the local fluctuations. Compared with the non-dynamic model, the NMRE+MD model gave 6-17% lower root-mean-square (RMS) errors for different backbone nuclei. The improved prediction indicates that NMR ensembles with MD simulations can be used to obtain a more realistic picture of protein structures in solutions and moreover underlines the importance of short and long time-scale dynamics for the prediction. The RMS errors of the NMRE+MD model were 0.24, 0.43, 0.98, 1.03, 1.16 and 2.39 ppm for (1)Hα, (1)HN, (13)Cα, (13)Cß, (13)CO and backbone (15)N chemical shifts, respectively. The model is implemented in the prediction program 4DSPOT, available at http://www.uef.fi/4dspot.

Molecular dynamics simulations for human CAR inverse agonists.

Constitutive androstane receptor (CAR), along with pregnane x receptor (PXR), is an important metabolic sensor in the hepatocytes. Like all other nuclear receptors (NRs), CAR works in concert with coregulator proteins, coactivators, and corepressors which bind to the NRs. The main basis for the receptor to distinguish between coactivators and corepressors is the position of the C-terminal helix 12 (H12), which is determined by the bound NR ligand. CAR, having constitutive activity, can be repressed or further activated by its ligands. Crystal structure of human CAR bound to an agonist and a coactivator peptide is available, but no structural information on an inverse agonist-bound human CAR and a corepressor exists. In our previous molecular dynamics (MD) studies, no corepressor peptide was included. Therefore, probably due to the strong interactions which keep the relatively short H12 of CAR in the active position, the structural changes elicited by inverse agonists were very subtle, and H12 of CAR seemed to more or less retain its active conformation. Here, we have run a series of MD simulations to study the movement of H12 in the presence of both activating and repressing ligands as well as a corepressor peptide. The presence of the corepressor on the coregulator surface of CAR induced a clear shift of H12 of the inverse agonists-bound CAR. In general, H12 moved toward H10 and not away from the ligand binding domain, as seen in some other NRs. However, H12 of CAR is short enough that this movement seems to be adequate to accommodate the binding of the corepressor.

Metabolic diversity of progressive kidney disease in 325 patients with type 1 diabetes (the FinnDiane Study).

Type 1 diabetic patients with varying severity of kidney disease were investigated to create multimetabolite models of the disease process. Urinary albumin excretion rate was measured for 3358 patients with type 1 diabetes. Prospective records were available for 1051 patients, of whom 163 showed progression of albuminuria (8.3-year follow-up), and 162 were selected as stable controls. At baseline, serum lipids, lipoprotein subclasses, and low-molecular weight metabolites were quantified by NMR spectroscopy (325 samples). The data were analyzed by the self-organizing map. In cross-sectional analyses, patients with no complications had low serum lipids, less inflammation, and better glycemic control, whereas patients with advanced kidney disease had high serum cystatin-C and sphingomyelin. These phenotype extremes shared low unsaturated fatty acids (UFAs) and phospholipids. Prospectively, progressive albuminuria was associated with high UFAs, phospholipids, and IDL and LDL lipids. Progression at longer duration was associated with high HDL lipids, whereas earlier progression was associated with poor glycemic control, increased saturated fatty acids (SFAs), and inflammation. Diabetic kidney disease consists of diverse metabolic phenotypes: UFAs, phospholipids, IDL, and LDL may be important in the subclinical phase, high SFAs and low HDL suggest accelerated progression, and the sphingolipid pathway in advanced kidney injury deserves further research.

Synthesis and biological evaluation of phenolic 4,5-dihydroisoxazoles and 3-hydroxy ketones as estrogen receptor alpha and beta agonists.

In this work, 52 diphenyl-4,5-dihydroisoxazoles and -3-hydroxy ketones were prepared and their estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta) activities were explored in order to systematize and maximize their biological activity. The biological activity was firstly screened by using ERE reporter assay to find out how aromatic hydroxylation and methylation of the chiral centers of the compounds affect the ability of ER to mediate biological responses. For selected 19 compounds, the relative binding affinities (RBA, relative to 3,17beta-estradiol) and ability to induce transcription of primary E2 target gene pS2 in human MCF-7 breast cancer cells were determined. In the reporter assay, many compounds showed even stronger activity than E2 and some of them showed RBA larger than 1%. The highest RBAs were determined for the enantiomers of 1-hydroxy-6-(4-hydroxy-phenyl)-1-phenyl-hexan-3-one (50a and 50b). Isomer 50a showed high binding affinity both to ERalpha (with RBA approximately 200%) and ERbeta (with RBA approximately 60%), while the RBAs of 50b were ca. 40% of those. Some of the other compounds (with RBA approximately 1-16%) showed also notable ERalpha binding selectivity. When four most promising ligands (50a, 50b, 45a, and 45b) were studied with respect to their ability to induce the transcription of primary E2 target gene pS2, the compounds acted as agonists or partial agonists. Computer modeling was used to predict receptor binding conformations and to rationalize the RBA differences of the compounds.

Screening of binding activity of Streptococcus pneumoniae, Streptococcus agalactiae and Streptococcus suis to berries and juices.

Antiadhesion therapy is a promising approach to the fight against pathogens. Antibiotic resistance and the lack of effective vaccines have increased the search for new methods to prevent infectious diseases. Previous studies have shown the antiadhesion activity of juice from cultivated cranberries (Vaccinium macrocarpon Ait.) against bacteria, especially E. coli. In this study, the binding of two streptococcal strains, Streptococcus pneumoniae and Streptococcus agalactiae, to molecular size fractions (FI, FII and FIII, <10 kDa, 10-100 kDa, and >100 kDa, respectively) of berries and berry and fruit juices from 12 plant species were studied using a microtiter well assay. For Streptococcus suis a hemagglutination inhibition assay was used. In general, binding activity was detected especially to wild cranberry (Vaccinium oxycoccos L.) and to other Vaccinium species. S. pneumoniae cells bound most to cranberry juice fraction FI and S. agalactiae cells to cranberry fraction FIII. Hemagglutination induced by S. suis was most effectively inhibited by cranberry fraction FII. NMR spectra of some characteristic active and non-active fractions were also measured. They indicate that fractions FII and FIII contained proanthocyanidins and/or other phenolic compounds. The results suggest Vaccinium berries as possible sources of antiadhesives against bacterial infections.

13C isotope effects on 1H chemical shifts: NMR spectral analysis of 13C-labelled D-glucose and some 13C-labelled amino acids.

The one- and two-bond (13)C isotope shifts, typically -1.5 to -2.5 ppb and -0.7 ppb respectively, in non-cyclic aliphatic systems and up to -4.4 ppb and -1.0 ppb in glucose cause effects that need to be taken into account in the adaptive NMR spectral library-based quantification of the isotopomer mixtures. In this work, NMR spectral analyses of some (13)C-labelled amino acids, D-glucose and other small compounds were performed in order to obtain rules for prediction of the (13)C isotope effects on (1)H chemical shifts. It is proposed that using the additivity rules, the isotope effects can be predicted with a sufficient accuracy for amino acid isotopomer applications. For glucose the effects were found strongly non-additive. The complete spectral analysis of fully (13)C-labelled D-glucose made it also possible to assign the exocyclic proton signals of the glucose.