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The development of pharmacological therapies for mitochondrial diseases is hampered by the lack of tissue-level and circulating biomarkers reflecting effects of compounds on endothelial and mitochondrial function. This phase 0 study aimed to identify biomarkers differentiating between patients with mitochondrial disease and healthy volunteers (HVs). In this cross-sectional case-control study, eight participants with mitochondrial disease and eight HVs matched on age, sex, and body mass index underwent study assessments consisting of blood collection for evaluation of plasma and serum biomarkers, mitochondrial function in peripheral blood mononuclear cells (PBMCs), and an array of imaging methods for assessment of (micro)circulation. Plasma biomarkers GDF-15, IL-6, NT-proBNP, and cTNI were significantly elevated in patients compared to HVs, as were several clinical chemistry and hematology markers. No differences between groups were found for mitochondrial membrane potential, mitochondrial reactive oxygen production, oxygen consumption rate, or extracellular acidification rate in PBMCs. Imaging revealed significantly higher nicotinamide-adenine-dinucleotide-hydrogen (NADH) content in skin as well as reduced passive leg movement-induced hyperemia in patients. This study confirmed results of earlier studies regarding plasma biomarkers in mitochondrial disease and identified several imaging techniques that could detect functional differences at the tissue level between participants with mitochondrial disease and HVs. However, assays of mitochondrial function in PBMCs did not show differences between participants with mitochondrial disease and HVs, possibly reflecting compensatory mechanisms and heterogeneity in mutational load. In future clinical trials, using a mix of imaging and blood-based biomarkers may be advisable, as well as combining these with an in vivo challenge to disturb homeostasis.

Diversification of the structures and the applications possible for foldamers rely on expansion of the building block library available for their synthesis. In this work, we describe the synthesis of a range of three dimensional heteroaromatic monomers, based on iptycene scaffolds, that are suitable for the synthesis of aromatic oligoamide foldamers. These units can be obtained in gram quantities in up to 80 % yield through [4+2] cycloaddition between diester, diamine, and amino acid derivatives of 1,8-diazaanthracenes and a variety of dienophiles. X-ray structural studies of the monomers and an oligomer show that the new motif orients the two heterocyclic rings and attached groups at an angle of approximately 120° to each other, opening new geometric considerations for the design of this class of foldamer.

The TPM domain constitutes a family of recently characterized protein domains that are present in most living organisms. Although some progress has been made in understanding the cellular role of TPM-containing proteins, the relationship between structure and function is not clear yet. We have recently solved the solution and crystal structure of one TPM domain (BA42) from the Antarctic bacterium Bizionia argentinensis. In this work, we demonstrate that BA42 has phosphoric-monoester hydrolase activity. The activity of BA42 is strictly dependent on the binding of divalent metals and retains nearly 70% of the maximum at 4 °C, a typical characteristic of cold-adapted enzymes. From HSQC, 15 N relaxation measurements, and molecular dynamics studies, we determine that the flexibility of the crossing loops was associated to the protein activity. Thermal unfolding experiments showed that the local increment in flexibility of Mg2+ -bound BA42, when compared with Ca2+ -bound BA42, is associated to a decrease in global protein stability. Finally, through mutagenesis experiments, we unambiguously demonstrate that the region comprising the metal-binding site participates in the catalytic mechanism. The results shown here contribute to the understanding of the relationship between structure and function of this new family of TPM domains providing important cues on the regulatory role of Mg2+ and Ca2+ and the molecular mechanism underlying enzyme activity at low temperatures. Fil: Pellizza, Leonardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Smal, Clara. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Ithuralde, Raúl Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Turjanski, Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Cicero, Daniel Oscar. Universita Tor Vergata; Italia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Aran, Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina

The structure of the BA42 protein belonging to the Antarctic flavobacterium Bizionia argentinensis was determined by nuclear magnetic resonance and X-ray crystallography. This is the first structure of a member of the PF04536 family comprised of a stand-alone TPM domain. The structure reveals a new topological variant of the four β-strands constituting the central β-sheet of the αβα architecture and a double metal binding site stabilizing a pair of crossing loops, not observed in previous structures of proteins belonging to this family. BA42 shows differences in structure and dynamics in the presence or absence of bound metals. The affinity for divalent metal ions is close to that observed in proteins that modulate their activity as a function of metal concentration, anticipating a possible role for BA42.

Deoxycytidine kinase (dCK) is a critical enzyme for activation of anticancer nucleoside analogs. Its activity is controlled via Ser-74 phosphorylation. Here, we investigated which Ser/Thr phosphatase dephosphorylates Ser-74. In cells, the PP1/PP2A inhibitor okadaic acid increased both dCK activity and Ser-74 phosphorylation at concentrations reported to specifically target PP2A. In line with this, purified PP2A, but not PP1, dephosphorylated recombinant pSer-74-dCK. In cell lysates, the Ser-74-dCK phosphatase activity was found to be latent, Mn2+-activated, responsive to PP2A inhibitors, and diminished after PP2A-immunodepletion. Use of siRNAs allowed concluding definitively that PP2A constitutively dephosphorylates dCK in cells and negatively regulates its activity.

The pharmacokinetic properties of the developmental Amino-Acetonitrile Derivative (AAD), monepantel and its sulfone metabolite, monepantel sulfone were investigated in sheep following intravenous (i.v.) and oral administrations. The sulfone metabolite was rapidly formed and predominated over monepantel 4 h after dosing, irrespective of the route of administration. The steady-state volume of distribution, total body clearance and mean residence time of monepantel were 7.4 L/kg, 1.49 L/(kg x h) and 4.9 h, respectively and 31.2 L/kg, 0.28 L/(kg x h) and 111 h, respectively for monepantel sulfone. The overall bioavailability of monepantel was 31%, but it was demonstrated that approximately the same amount of monepantel sulfone was produced whether monepantel was given intravenously or orally (AUC((0-infinity)) oral/AUC((0-infinity)) i.v. of 94% for monepantel sulfone), making oral administration a very efficient route of administration for monepantel in terms of the amount of sulfone metabolite generated. Because monepantel sulfone is the main chemical entity present in sheep blood after monepantel administration and because it is also an active metabolite, its pharmacokinetic properties are of primary importance for the interpretation of future residue and efficacy studies. Overall, these pharmacokinetic data aid in the evaluation of monepantel as an oral anthelmintic in sheep.

The combining site specificities of IgE antibodies that react with the oral antibacterial agent trimethoprim and found in the sera of two subjects who experienced anaphylaxis after taking the drug, were investigated. Hapten inhibition studies with some close analogues of trimethoprim and a range of other structurally related compounds showed that the allergenic determinant complementary to the IgE antibodies in the serum of one of the subjects was the 3,4-dimethoxybenzyl group. The complementary allergenic structure recognized by the IgE antibodies in the serum from the second subject comprised both the trimethoxybenzyl and diaminopyrimidine rings of trimethoprim. Thus, as with thiopentone, but unlike the neuromuscular blocking drugs, the trimethoprim molecule has more than one determinant each with the capacity to provoke IgE formation, interact with the antibody combining site and provoke drug-induced allergic reactions. The general approach set out here employing carefully selected structural analogues in hapten inhibition studies should be invaluable for confirming specificity and identifying allergenic determinants in IgE antibody-mediated allergic drug reactions.

1. The European Water Framework Directive requires the determination of ecological status in European fresh and saline waters. This is to be through the establishment of a typology of surface water bodies, the determination of reference (high status) conditions in each element (ecotype) of the typology and of lower grades of status (good, moderate, poor and bad) for each ecotype. It then requires classification of the status of the water bodies and their restoration to at least 'good status' in a specified period. 2. Though there are many methods for assessing water quality, none has the scope of that defined in the Directive. The provisions of the Directive require a wide range of variables to be measured and give only general guidance as to how systems of classification should be established. This raises issues of comparability across States and of the costs of making the determinations. 3. Using expert workshops and subsequent field testing, a practicable pan-European typology and classification system has been developed for shallow lakes, which can easily be extended to all lakes. It is parsimonious in its choice of determinands, but based on current limnological understanding and therefore as cost-effective as possible. 4. A core typology is described, which can be expanded easily in particular States to meet local conditions. The core includes 48 ecotypes across the entire European climate gradient and incorporates climate, lake area, geology of the catchment and conductivity. 5. The classification system is founded on a liberal interpretation of Annexes in the Directive and uses variables that are inexpensive to measure and ecologically relevant. The need for taxonomic expertise is minimized. 6. The scheme has been through eight iterations, two of which were tested in the field on tranches of 66 lakes. The final version, Version 8, is offered for operational testing and further refinement by statutory authorities.

On the base of experimental spontaneous emission spectra measured at laser threshold the temperature dependencies of the radiative Rsp and nonradiative Q recombination rates as well as the spontaneous emission internal quantum efficiency ηsp for optically pumped wurtzite GaN/Al2O3 epilayers have been calculated. The recombination rate induced by the amplified luminescence Rlum was taken into account. It is shown that the main reason of the increasing threshold pump rate Rp with temperature T for the GaN-based compounds is a substantial rise of the nonradiative recombination rate. The portion of Q in Rp exceeds 80–90%. The contributions of Rsp and Rlum in Rp amount to several percents. The ηsp value is of 0.12–0.18 within the temperature interval of 300–450 K. Further increase in T leads to an abrupt decrease in ηsp down to 0.06 at 517 K.

The structure of the BA42 protein belonging to the Antarctic flavobacterium Bizionia argentinensis was determined by nuclear magnetic resonance and X-ray crystallography. This is the first structure of a member of the PF04536 family comprised of a stand-alone TPM domain. The structure reveals a new topological variant of the four β-strands constituting the central β-sheet of the αβα architecture and a double metal binding site stabilizing a pair of crossing loops, not observed in previous structures of proteins belonging to this family. BA42 shows differences in structure and dynamics in the presence or absence of bound metals. The affinity for divalent metal ions is close to that observed in proteins that modulate their activity as a function of metal concentration, anticipating a possible role for BA42. Fil: Aran, Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; Argentina Fil: Smal, Clara. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; Argentina Fil: Pelliza, Leonardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; Argentina Fil: Gallo, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; Argentina Fil: Otero, Lisandro Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Plataforma Argentina de Biología Estructural y Metabolómica PLABEM; Argentina. Fundación Instituto Leloir; Argentina Fil: Klinke, Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; Argentina. Plataforma Argentina de Biología Estructural y Metabolómica PLABEM; Argentina Fil: Goldbaum, Fernando Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Plataforma Argentina de Biología Estructural y Metabolómica PLABEM; Argentina. Fundación Instituto Leloir; Argentina Fil: Ithurralde, Esteban. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina Fil: Bercovich, Andrés. Biosidus S.a.; Argentina Fil: Mac Cormack, Walter P.. Ministerio de Relaciones Exteriores, Comercio Interno y Culto. Dirección Nacional del Antártico. Instituto Antártico Argentino; Argentina Fil: Turjanski, Adrian G.. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina Fil: Cicero, Daniel Oscar. Universita Di Roma; Italia

The ability of bioassay, enzyme-linked immunosorbent assay (ELISA) and high-performance liquid chromatography (hplc) methods to detect sulfonylurea herbicides in soil was evaluated as part of a project studying the leaching and persistence of these herbicides in the alkaline soils of south-eastern Australia. Soil samples with known concentrations between 0. 1 and 10 μg a.i. kg -1 chlorsulfuron, metsulfuron-methyl or triasulfuron were prepared by an independent laboratory and supplied in coded bags to separate laboratories for testing. The accuracy of the results was analysed, and the merits of each method are discussed. Bioassay was suitable for measuring biologically active residues from 0.1 to 1.0 μg a.i. kg -1 . ELISA accurately measured residues in the range of 0.1- 10 μg a.i. kg -1 , making it the most widely adaptable assay tested. It will be useful for measuring residues in sodic subsoils where bioassay plants grow poorly, There was good reproducibility between the bioassay and ELISA. The hplc technique used in this study was not as accurate as bioassay or ELISA at quantifying residues of 3.0-10 μg a.i. kg -1 and could not detect residues at or below 1.0 μg a.i. kg -1 .

Objectives The importance of professional attitudes in medical care has long been recognized; however, medical training has not stressed attitude development until recently. In previous studies among medical students, we found that gender and specialty preference are important factors in attitudes. In this study, patient-centredness of trainees in general practice and surgery and of final-year clerks preferring one of these specialties was assessed in one medical school in The Netherlands. The effect of gender, specialty and training level on attitude was investigated. Design In 1995, attitudes of 37 general practice trainees, 31 surgery trainees and 120 clerks were measured anonymously using questionnaires containing the Doctor–Patient Scale. This attitude scale measures patient-centredness vs. doctor-centredness. Response rates were 78%, 58% and 84%, respectively. Setting University of Utrecht. Subjects Medical students. Results Attitudes were related to specialty. General practice trainees showed more patient-centredness than surgery trainees. In accordance with previous findings among younger students, no differences were found between final-year clerks and vocational trainees. In contrast to previous studies, gender was not related to patient-centredness. Conclusions Professional attitudes, in particular patient-centredness, seem to be related to specialty preference in the final year of graduate medical training and specialty as a career choice. It remains unclear whether professional socialization reinforces existing attitudes or whether existing attitudes result in specialty preference.

The structure of luminescence spectra of free excitons in CdS single crystals at T = 77 K is investigated. The dip formation is found in the top part of free A and B exciton resonant peaks of photoluminescence (PL) spectra after heating of the CdS crystals followed by fast cooling down to 77 K. Similar effects are observed when an external electric field is applied. It is shown that the structure of exciton PL spectra is due to self-absorption of resonance radiation in the near-surface layer with low exciton concentration. Our calculations demonstrate that the formation of an exciton concentration gradient may be caused by the influence of a surface electric field on the binding of electron-hole pairs to excitons. It is assumed that the increase of surface electric fields after heat treatment is caused by the thermostimulated adsorption of oxygen, which results in the formation of electron trap levels. [Russian Text Ignored].

The object of this study was to examine whether prolonged-release hard gelatin capsule formulations could be developed for dogs. Different viscosity grades of hydroxypropyl methylcellulose (HPMC) and sodium carboxymethylcellulose (NaCMC) were used to control drug release. Furosemide was chosen because of its wide use in the management of heart failure in dogs. In vitro, selecting different viscosity grades allowed good control of drug release, whereas in vivo the difference between formulations was clearly smaller. Although all formulations gave prolonged release, both inter- and intra-individual variation in the plasma concentration-time curves was high. It is difficult to develop prolonged-release formulations for drugs such as furosemide with highly variable pharmacokinetic properties. However, hard gelatin capsules containing hydrophilic polymers could still be a suitable choice for some drugs.

Observations on the state of the environment, soil properties, soil hazards and some possible changes in soil parameters are discussed. Sandy and light loamy soils cover more than 50 per cent of the arable land in Poland. About 58 per cent of soils are acid (pH

A homologous protein, recombinant trout somatotropin (rtST) and its heterologous counterpart, native bovine somatotropin (bST), were administered anally to juvenile rainbow trout (Oncorhynchus mykiss). Plasma levels of rtST, determioned by radio-immunoassay, peaked between 15 and 60 min and remained high until 2 h after administration. Immunogold labelling was used to follow the routes of transfer of rtST and bST, and to observe potential interaction between the hormones and the cells constituting the first line of non-specific defence, ie the macrophages infiltrated between epithelial cells, or dispersed in the subepithelial lamina propria, and eosinophilic granulocytes (EGCs) of the lamina propria, whose properties have been considered to be similar to mammalian mast cells. Macrophages were immunolabelled for the homologous and heterologous proteins. EGCs took up to the heterologous but not the homologous protein. This finding was confirmed using indirect immunofluorescence assay. EGCs could internalize foreign proteins transferred from the intestinal lumen to the lamina propria.

Driving the equilibrium between selenides and osmium(VIII) reagents with selenoxides and osmium(VI) by a subsequent reaction (rearrangement of allyl selenoxides to allyl alcohols or addition of osmium(VIII) species on CC double bonds) to one side, allows the transformation of methyl geranyl selenides to linalool and of methyl citronellyl selenoxide to 6,7-dihydroxy citronellyl selenide.

Platelet activating factor (PAF), a naturally occurring phospholipid with many potent physiological and pharmacological activities, is implicated as a mediator of many diseases. An immunoassay for PAF would greatly improve quantitation, and hence PAF-specific antibodies were required. Chemically-reactive analogs of PAF, containing an aldehyde group at the end of the 1-O-alkyl chain (hexyl or dodecyl), were synthesized from readily available materials. During the multi-step synthetic procedure, the aldehyde group was protected as an acetal, which was converted by mild acidic hydrolysis to the aldehyde immediately prior to protein coupling. These analogs were coupled to methylated bovine serum albumin and the resultant conjugates were injected into rabbits. Antibodies to PAF were detected using a solid phase radioimmunoassay based on Protein A-Sepharose. The dodecyl PAF conjugate proved to be the more immunogenic conjugate with more than half of the rabbits producing significant levels of antibodies (at least a 10-fold increase in radioactive uptake over pre-immune levels). Results from solid phase immunoassays employing nitrocellulose discs impregnated with PAF, lysoPAF, lecithin, lysolecithin and 2-O-methyl-lysoPAF indicated that the antibodies recognized only PAF. PAF-specific antibodies were isolated by affinity chromatography using a column of PAF-poly(lysine) conjugated to carboxy-activated polyacrylamide. The antibodies may be employed in a sensitive and specific immunoassay for PAF and for many other studies involving PAF.

Populations of feral American mink Mustela vison were investigated in several study areas in Ireland, principally by capture-recapture techniques. The populations were found to be essentially self-regulating, with stress, weight loss and mortality evident during the winter and spring. The highest mean density recorded, in terms of animals per unit length of river or lake shore, was 1.4 individuals km−1 with a peak density of 2.3 individuals km−1, but population densities were considerably lower in most areas. Trappability and population structure, in age, sex and residency classes, varied between habitats, but population turnover was high, leading to complete replacement of individuals in two populations monitored for two years. The availability of the preferred prey, crayfish Austropotamobius pallipes appeared to be a major factor determining population size and preferred breeding areas. Control methods are either unnecessary or are likely to fail in most situations, and may be counterproductive.

Quantitative hapten inhibition experiments employing sheep anti-PAF antibodies and selected PAF analogues were undertaken with the aim of defining the antigenic determinant structures complementary to the antibody combining sites. The most important fine structural features for inhibition of antibody binding to PAF were shown to be an acetyl group at position 2 of the phospholipid glycerol backbone and an ether group at position 1. Of the naturally occurring compounds, C16- and C18:1-PAF proved to be the most potent inhibitors and more active than C18-PAF while phospholipids with a propionyl, butyryl or hexanoyl group at position 2 showed either weak or no inhibitory activity. The 1-acyl, thioether and deoxy analogues proved inactive. Variations in the polar head group of PAF were found to be less critical with, for example, the dimethyl and ethanolamine derivatives retaining some activity. This antibody recognition pattern is very similar to that of the PAF receptor, although the antibodies appear to have a more specific requirement for an acyl linkage at position 2.

Recent studies indicate that deoxycytidine kinase (dCK), which activates various nucleoside analogues used in antileukemic therapy, can be regulated by post-translational modification, most probably through reversible phosphorylation. To further unravel its regulation, dCK was overexpressed in HEK-293 cells as a His-tag fusion protein. Western blot analysis showed that purified overexpressed dCK appears as doublet protein bands. The slower band disappeared after treatment with protein phosphatase lambda (PP lambda) in parallel with a decrease of dCK activity, providing additional arguments in favor of both phosphorylated and unphosphorylated forms of dCK.