The retinoid ligand 4-oxo-retinoic acid is a highly active modulator of positional specification

Retinoids (vitamin A and its metabolites) are suspected of regulating diverse aspects of growth, differentiation, and patterning during embryogenesis, but many questions remain about the identities and functions of the endogenous active retinoids involved. The pleiotropic effects of retinoids may be explained by the existence of complex signal transduction pathways involving diverse nuclear receptors of the retinoic acid receptor (RAR) and retinoid X receptor (RXR) families, and at least two types of cellular retinoic acid binding proteins (CRABP-I and -II). The different RARs, RXRs, and CRABPs have different expression patterns during vertebrate embryogenesis, suggesting that they each have particular functions. Another level at which fine tuning of retinoid action could occur is the metabolism of vitamin A to active metabolites, which may include all-trans-retinoic acid, all-trans-3,4-didehydroretinoic acid, 9-cis-retinoic acid, and 14-hydroxy-4,14-retroretinol. Formation of the metabolite all-trans-4-oxo-retinoic acid from retinoic acid was considered to be an inactivation pathway during growth and differentiation. We report here that, in contrast, 4-oxo-retinoic acid is a highly active metabolite which can modulate positional specification in early embryos. We also show that this retinoid binds avidly to and activates RAR beta, and that it is available in early embryos. The different activities of 4-oxo-retinoic acid and retinoic acid in modulating positional specification on the one hand, and growth and differentiation on the other, interest us in the possibility that specific retinoid ligands regulate different physiological processes in vivo.     

In vitro folding of Escherichia coli outer-membrane phospholipase A

Recombinant outer-membrane phospholipase A (OMPLA) of Escherichia coli was expressed without its signal sequence from the T7 phi 10 promoter. As a result of the cloning strategy the protein had an N-terminal extension of six amino acid residues. The protein accumulated in the cytosol in inclusion bodies. Conditions were established for the efficient folding of OMPLA in vitro in the presence of Triton X-100. After in vitro folding, the protein was present as a mixture of folded and unfolded forms. Ion-exchange chromatography was used for the purification of OMPLA and the separation of correctly folded, enzymically active enzyme from unfolded inactive protein. The final protein preparation was pure and fully heat-modifiable based on SDS/PAGE. The recombinant enzyme had a specific activity of 71 U/mg, which is similar to the value of the wild-type enzyme, purified from the membrane. The final yield of active enzyme was 35 mg protein/l culture of an A600 of 6. Circular dichroism spectroscopy revealed a high content of beta strand, in good agreement with a predicted beta-barrel structure of this outer-membrane protein.     

The sulphoxidation of thioanisole catalysed by lactoperoxidase and Coprinus cinereus peroxidase: evidence for an oxygen-rebound mechanism

Using both stopped-flow and conventional spectroscopy, the oxygenation of methyl phenyl sulphide by both lactoperoxidase (LPO) and Coprinus cinereus peroxidase (CiP) was monitored. Controlled continuous addition of H2O2 during turnover and monitoring the presence of native enzymes, compounds I, II and III, led to formation of the sulphoxide in high yield and enantioselectivity. Under those conditions, LPO catalysed the formation of (R) methyl phenyl sulphoxide with a yield of 85% and an enantiomeric excess (e.e.) of 80%. CiP catalysed the formation of (S) methyl phenyl sulphoxide with a yield of 84% and an e.e. of 73%. The enantioselective performance was markedly influenced by the purity of the enzymes used. Presence of compound III during turnover led to rapid inactivation of the peroxidases and, therefore, to both a lower yield of the sulphoxides and a lower enantioselectivity. Stopped-flow kinetic data show that, for both LPO and CiP, the transition of compound I to compound II depends on the concentration of the methyl phenyl sulphide, suggesting an oxygen-rebound mechanism. In line with this mechanism, a methyl phenyl sulphide radical cation was detected by EPR during turnover for LPO.     

The Tim core complex defines the number of mitochondrial translocation contact sites and can hold arrested preproteins in the absence of matrix Hsp70-Tim44

Preprotein import into mitochondria is mediated by translocases located in the outer and inner membranes (Tom and Tim) and a matrix Hsp70-Tim44 driving system. By blue native electrophoresis, we identify an approximately 90K complex with assembled Tim23 and Tim17 as the core of the inner membrane import site for presequence-containing preproteins. Preproteins spanning the two membranes link virtually all Tim core complexes with one in four Tom complexes in a stable 600K supercomplex. Neither mtHsp70 nor Tim44 are present in stoichiometric amounts in the 600K complex. Preproteins in transit stabilize the Tim core complex, preventing an exchange of subunits. Our studies define a central role for the Tim core complexes in mitochondrial protein import; they are not passive diffusion channels, but can stably interact with preproteins and determine the number of translocation contact sites. We propose the hypothesis that mtHsp70 functions in protein import not only by direct interaction with preproteins, but also by exerting a regulatory effect on the Tim channel.     

Determination of pectic substances in plant material

Pectic substances have been extracted from plant material (a pasture legume, Stylosanthes humilis) by homogenisation with a solution of ammonium oxalate and oxalic acid and subsequently hydrolysed with polygalacturonase. The galacturonic acid produced was determined by a modified carbazole reaction. The enzymic hydrolysis step may be omitted by relying on the acid in the carbazole reaction partially to hydrolyse the pectic substances, but in doing so the sensitivity of the method is reduced sevenfold. Attempts to carry out the hydrolysis with sulphuric acid before the carbazole reaction gave incomplete hydrolysis and probably decarboxylation. Attempts to extract the pectic substances with alkali gave low values, probably because of incomplete extraction.     

Lithographically fabricated nanopore-based electrodes for electrochemistry

We report a new technique for fabricating electrodes for electrochemical applications with lateral dimensions in the range 15-200 nm and a reproducible, well-defined geometry. This technique allows determining the electrode size by electron microscopy prior to electrochemical measurements and without contamination of the metal electrode. We measured the diffusion-limited current with stepped-current voltammetry and showed that its dependence on electrode size can be quantitatively understood if the known geometry of the electrodes is explicitly taken into account.     

Determination of starch in plant material

Starch has been determined in the pasture legume Stylosanthes humilis (Stylo) by hydrolysis with amylo-glucosidase and subsequent analysis for glucose with glucose oxidase. Prior gelatinisation of starch granules within the plant sample by water at 130°c gave low values and it was concluded that the complete solubilisation of starch by alkali was necessary, with subsequent neutralisation of the extract with acetic acid. It was shown that the alkali also extracted material which inhibited one or more of the enzymes involved in the subsequent glucose analysis, but this inhibition was removed by treatment of the extract with charcoal. Under the above conditions the high acetate concentration decreased the efficiency of the final glucose analysis, but this effect was readily corrected by use of appropriate blank solutions. The proposed method of starch analysis is relatively economical in time compared with previous methods and is believed to give more meaningful values for plants with low starch content.     

Digestion of the dry matter, nitrogen, phosphorus, sulphur, calcium and detergent-fibre fractions of the seed and pod of Stylosanthes humilis contained in terylene bags in the bovine rumen

The rates of digestion of the separated seed and pods of Stylosanthes humilis in Terylene bags in the rumen of cattle were studied. The rates of disappearance of dry matter, N, P, S, Ca and detergent fibre fractions and lignin were examined. The magnitude of the errors of determination were assessed statistically for dry matter digestion. Four bag replications were necessary to detect differences of 20% between samples. The cell contents constituted 80% of the seed dry matter, but only 20% of the pod dry matter. Lignin content of seed was negligible (0.5%), but was high in pods (18%). The seed was rapidly digested (77% of dry matter after 48 h). This was largely due to almost complete digestion of the cell contents. The pod was only slightly digested (14% at 48 h). Protein and phosphorus appeared to be selectively solubilised, but calcium was only slowly solubilised in both seed and pod. The cell-wall constituents present were compared to the concentrations of hemicellulose and cellulose found in seed and pod by more definitive chemical methods.     

Reliability of the assessment of impairments, disabilities and handicaps in survey research on speech therapy

An important aspect to the practice of speech therapy is the diagnostic assessment. This assessment is complementary to the medical diagnosis. The International Classification of Impairments, Disabilities and Handicaps (ICIDH) is regarded as a good starting point for the classification of speech therapy assessments. Based on the ICIDH, a form was developed for the registration of impairments, disabilities and handicaps in survey research on speech therapy. The aim of this study was to evaluate the reliability of the assessment of impairments, disabilities and handicaps based on clinical observations and examinations by speech therapists. On the basis of the percentage of agreement and kappa values it is concluded that the reliability of the assessment of the impairments, disabilities and handicaps was satisfactory to excellent for most of the categories.