Separation and identification of neuropeptide Y, two of its fragments and their degradation products using capillary electrophoresis–mass spectrometry

This paper describes the development of an analytical method for the separation and identification of neuropeptide Y (NPY) and two important NPY fragments by capillary electrophoresis (CE) and mass spectrometry (MS). A satisfactory separation and the highest sensitivity were obtained with formic acid at high concentrations (250 mM, pH 2.75). The addition of 25 or 50 mM triethylamine (TEA) improved the separation. When applying full scan CE–MS, the separated peptides could be detected and identified using the spectra of each peak. The use of TEA as an additive to the formic acid slightly decreased the sensitivity but was compensated by the improved efficiency. The best compromise for optimal separation and MS detection was found to be 50 mM formic acid to which 50 mM TEA was added. CE–MS could be used for identification of the decomposition products of NPY. Decomposition products with one amino acid difference, which could not be distinguished with CE–UV, could be distinguished with CE–MS.     

A New Auditory Multi-Class Brain-Computer Interface Paradigm: Spatial Hearing as an Informative Cue

Most P300-based brain-computer interface (BCI) approaches use the visual modality for stimulation. For use with patients suffering from amyotrophic lateral sclerosis (ALS) this might not be the preferable choice because of sight deterioration. Moreover, using a modality different from the visual one minimizes interference with possible visual feedback. Therefore, a multi-class BCI paradigm is proposed that uses spatially distributed, auditory cues. Ten healthy subjects participated in an offline oddball task with the spatial location of the stimuli being a discriminating cue. Experiments were done in free field, with an individual speaker for each location. Different inter-stimulus intervals of 1000 ms, 300 ms and 175 ms were tested. With averaging over multiple repetitions, selection scores went over 90% for most conditions, i.e., in over 90% of the trials the correct location was selected. One subject reached a 100% correct score. Corresponding information transfer rates were high, up to an average score of 17.39 bits/minute for the 175 ms condition (best subject 25.20 bits/minute). When presenting the stimuli through a single speaker, thus effectively canceling the spatial properties of the cue, selection scores went down below 70% for most subjects. We conclude that the proposed spatial auditory paradigm is successful for healthy subjects and shows promising results that may lead to a fast BCI that solely relies on the auditory sense.     

A genetic analysis of human minor histocompatibility antigens demonstrates Mendelian segregation independent of HLA

An analysis of the genetic traits of human minor histocompatibility (mH) antigens is, unlike with inbred mice, rather complicated. Moreover, the fact that mH antigens are recognized in the context of MHC molecules creates an additional complication for reliable segregation analysis. To gain insight into the mode of inheritance of the mH antigens, we relied upon a series of HLA-A2-restricted cytotoxic T-cell (CTL) clones specific for four mH antigens. To perform segregation analysis independent of HLA-A2 gene: we transfected HLA-A2-negative cells with the HLA-A2 gene: this results in the cell surface expression of the HLA-A2 gene product and, if present, mH antigen recognition. The mode of inheritance of the HLA-A2-restricted mH antigens HA-1, -2, -4, and -5 was analysed in 25 families whoese members either naturally expressed positive. Analysis of distribution of the mH antigens in the parent population among the mating types, together with their inheritance patterns in the families, demonstrated that the four mH antigens behaved as Mendelian traits, whereby each can be considered a product of a gene with two alleles, one expressing and one not expressing the detected specificity. We also showed that the loci encoding the HA-1 and HA-2 antigens are not closely linked to HLa (lod scores Z (0 = 0.05) <–4.0). Some indication was obtained that the HA-4- and HA-5-encoding loci may be losely linked to HLA. While we are aware of the limited results of this nonetheless comprehensive study, we feel the similarity in immunogenetic traits between human and mouse mH antigens is at least striking.     

The genus Pyrus L. (Rosaceae) in south-west Europe and North Afkica

A multivariate morphometric study of the genus Pyrus in south-west Europe and North Africa shows that five species may be recognized in the area: P. bourgaeana Decne., P. communis L., P. cordata Dew., P. spinosa Forssk, and P. nivalis Jacq. Some valuable characters for identification of these species are proposed. In particular the width of fruit peduncle, petal size, leaf width and petiole length served to discriminate the taxa. Several names such as P. gharbiona Trab., P. cossonii Rehder (|M= P. longipes Balansa ex Coss. & Durieu) and P. boisseriana Buhse, are regarded as synonyms of P. cordata , while P. marnormis Trab. of P. bourgaeana. Consequently a check-list and a key to these species are provided.     

Crystal structure of the p-hydroxybenzoate hydroxylase-substrate complex refined at 1.9 A resolution. Analysis of the enzyme-substrate and enzyme-product complexes

Using synchrotron radiation, the X-ray diffraction intensities of crystals of p-hydroxy-benzoate hydroxylase, complexed with the substrate p-hydroxybenzoate, were measured to a resolution of 1.9 A. Restrained least-squares refinement alternated with rebuilding in electron density maps yielded an atom model of the enzyme-substrate complex with a crystallographic R-factor of 15.6% for 31,148 reflections between 6.0 and 1.9 A. A total of 330 solvent molecules was located. In the final model, only three residues have deviating phi-psi angle combinations. One of them, the active site residue Arg44, has a well-defined electron density and may be strained to adopt this conformation for efficient catalysis. The mode of binding of FAD is distinctly different for the different components of the coenzyme. The adenine ring is engaged in three water-mediated hydrogen bonds with the protein, while making only one direct hydrogen bond with the enzyme. The pyrophosphate moiety makes five water-mediated versus three direct hydrogen bonds. The ribityl and ribose moieties make only direct hydrogen bonds, in all cases, except one, with side-chain atoms. The isoalloxazine ring also makes only direct hydrogen bonds, but virtually only with main-chain atoms. The conformation of FAD in p-hydroxybenzoate hydroxylase is strikingly similar to that in glutathione reductase, while the riboflavin-binding parts of these two enzymes have no structural similarity at all. The refined 1.9 A structure of the p-hydroxybenzoate hydroxylase-substrate complex was the basis of further refinement of the 2.3 A structure of the enzyme-product complex. The result was a final R-factor of 16.7% for 14,339 reflections between 6.0 and 2.3 A and an improved geometry. Comparison between the complexes indicated only small differences in the active site region, where the product molecule is rotated by 14 degrees compared with the substrate in the enzyme-substrate complex. During the refinements of the enzyme-substrate and enzyme-product complexes, the flavin ring was allowed to bend or twist by imposing planarity restraints on the benzene and pyrimidine ring, but not on the flavin ring as a whole. The observed angle between the benzene ring and the pyrimidine ring was 10 degrees for the enzyme-substrate complex and 19 degrees for the enzyme-product complex. Because of the high temperature factors of the flavin ring in the enzyme-product complex, the latter value should be treated with caution. Six out of eight peptide residues near the flavin ring are oriented with their nitrogen atom pointing towards the ring.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cultured endothelial cells produce heparinlike inhibitor of smooth muscle cell growth

Using cultured cells from bovine and rat aortas, we have examined the possibility that endothelial cells might regulate the growth of vascular smooth muscle cells. Conditioned medium from confluent bovine aortic endothelial cells inhibited the proliferation of growth-arrested smooth muscle cells. Conditioned medium from exponential endothelial cells, and from exponential or confluent smooth muscle cells and fibroblasts, did not inhibit smooth muscle cell growth. Conditioned medium from confluent endothelial cells did not inhibit the growth of endothelial cells or fibroblasts. In addition to the apparent specificity of both the producer and target cell, the inhibitory activity was heat stable and not affected by proteases. It was sensitive flavobacterium heparinase but not to hyaluronidase or chondroitin sulfate ABC lyase. It thus appears to be a heparinlike substance. Two other lines of evidence support this conclusion. First, a crude isolate of glycosaminoglycans (TCA-soluble, ethanol-precipitable material) from endothelial cell-conditioned medium reconstituted in 20 percent serum inhibited smooth muscle cell growth; glycosaminoglycans isolated from unconditioned medium (i.e., 0.4 percent serum) had no effect on smooth muscle cell growth. No inhibition was seen if the glycosaminoglycan preparation was treated with heparinase. Second, exogenous heparin, heparin sulfate, chondroitin sulfate B (dermatan sulfate), chondroitin sulfate ABC, and hyaluronic acid were added to 20 percent serum and tested for their ability to inhibit smooth muscle cell growth. Heparin inhibited growth at concentrations as low as 10 ng/ml. Other glycosaminoglycans had no effect at doses up to 10 μg/ml. Anticoagulant and non- anticoagulant heparin were equally effective at inhibiting smooth muscle cell growth, as they were in vivo following endothelial injury (Clowes and Karnovsk. Nature (Lond.). 265:625-626, 1977; Guyton et al. Circ. Res. 46:625-634, 1980), and in vitro following exposure of smooth muscle cells to platelet extract (Hoover et al. Circ. Res. 47:578-583, 1980). We suggest that vascular endothelial cells may secrete a heparinlike substance in vivo which may regulate the growth of underlying smooth muscle cells.