Rapid and sensitive GC/MS characterization of glycolipid released Galalpha1,3Gal-terminated oligosaccharides from small organ specimens of a single pig

Pig to human xenotransplantation is considered a possible solution to the prevailing chronic lack of human donor organs for allotransplantation. The Galalpha1,3Gal determinant is the major porcine xenogeneic epitope causing hyperacute rejection following human antibody binding and complement activation. In order to characterize the tissue distribution of Galalpha1,3Gal-containing and blood group- type glycosphingolipids in pig, acid and nonacid glycosphingolipids were isolated from the kidney, small intestine, spleen, salivary gland, liver, and heart of a single pig obtained from a semi-inbred strain homozygous at the SLA locus. Glycolipids were analyzed by thin-layer immunostaining using monoclonal antibodies, and following ceramide glycanase cleavage as permethylated oligosaccharides by gas chromatography, gas chromatography-mass spectrometry, and matrix- assisted laser desorption/ionization mass spectrometry. The kidney contained large amounts of Galalpha1,3Gal-containing penta- and hexasaccharides having carbohydrate sequences consistent with the Galalpha1,3nLc4and Galalpha1,3Lexstructures, respectively. The former structure was tentatively identified in all organs by GC/MS. The presence of extended Galalpha1,3Gal-terminated structures in the kidney and heart was suggested by antibody binding, and GC/MS indicated the presence of a Galalpha1,3nLc6structure in the heart. The kidney, spleen, and heart contained blood group H pentaglycosylceramides based on type 1 (H-5-1) and type 2 (H-5-2) chains, and H hexaglycosylceramides based on the type 4 chain (H-6-4). In the intestine H-5-1 and H-6-4 were expressed, in the salivary gland H-5-1 and H-5-2, whereas only the H-5-1 structure was identified in the liver. Blood group A structures were identified in the salivary gland and the heart by antibody binding and GC/MS, indicating an organ- specific expression of blood group AH antigens in the pig.      

Detection and quantification of Lyme spirochetes using sensitive and specific molecular beacon probes

Background  

Lyme disease, caused by Borrelia burgdorferi, affects a large number of people in both the USA and Europe. The mouse is a natural host for this spirochete and is widely used as a model system to study Lyme pathogenesis mechanisms. Since disease manifestations often depend upon the spirochete burden in a particular tissue, it is critical to accurately measure the bacterial number in infected tissues. The current methods either lack sensitivity and specificity (SYBR Green), or require independent analysis of samples in parallel to quantitate host and bacterial DNA (TaqMan). We have developed a novel molecular beacon-based convenient multiplex real-time quantitative PCR assay to identify and detect small numbers of B. burgdorferi in infected mouse tissues.     

Challenges in the delimitation of morphologically similar species: a case study of <Emphasis Type="Italic">Tuber brumale</Emphasis> agg. (Ascomycota,Pezizales)

Tuber brumale (winter truffle) is one of the most controversial true truffles, not only in regard to its ecological and economical role but also its taxonomy. Multilocus phylogenetic analyses have revealed that specimens identified earlier as T. brumale belong to two species. These species were deemed cryptic right away, because preliminary morphological measurements did not show any phenotypical differences. In this study, we measured the morphology of 119 T. brumale agg. specimens, identified by DNA-based phylogenetic tools. We found several continuous morphological characters which show strong statistical differences between the two species, albeit not without overlap. Using a combination of these characters, we show that efficient separation of the two species is possible. We describe T. cryptobrumale sp. nov. and present the environmental demands and the potential area reconstruction of both species. We argue that non-representative sampling is a major culprit in most failures to detect both the existence of morphologically similar species and their morphological differences. Our findings illustrate the benefits of integrative taxonomy: the use of a combination of molecular, morphological and ecological tools.     

Five years investigation of female and male genotypes in périgord black truffle (Tuber melanosporum Vittad.) revealed contrasted reproduction strategies

The Périgord black truffle (Tuber melanosporum Vittad.) is a heterothallic ascomycete that establishes ectomycorrhizal symbiosis with trees and shrubs. Small‐scale genetic structures of female genotypes in truffle orchards are known, but it has not yet been studied in male genotypes. In this study, our aim was to characterize the small‐scale genetic structure of both male and female genotypes over five years in an orchard to better understand the T. melanosporum sexual reproduction strategy, male genotype dynamics, and origins. Two‐hundred forty‐one ascocarps, 475 ectomycorrhizas, and 20 soil cores were harvested and genotyped using microsatellites and mating type genes. Isolation by distance analysis revealed pronounced small‐scale genetic structures for both female and male genotypes. The genotypic diversity was higher for male than female genotypes with numerous small size genotypes suggesting an important turnover due to ascospore recruitment. Larger and perennial female and male genotypes were also detected. Only three genotypes (1.5%) were found as both female and male genotypes (hermaphrodites) while most were detected only as female or male genotype (dioecy). Our results suggest that germinating ascospores act as male genotypes, but we also proposed that soil mycelium could be a reservoir of male genotypes.     

Protein structure prediction force fields: Parametrization with quasi-newtonian dynamics

We present an unusual method for parametrizing low-resolution force fields of the type used for protein structure prediction. Force field parameters were-determined by assigning each a fictitious mass and using a quasi-molecular dynamics algorithm in parameter space. The quasi-energy term favored folded native structures and specifically penalized folded nonnative structures. The force field was generated after optimizing less than 70 adjustable parameters, but shows a strong ability to discriminate between native structures and compact misfolded-alternatives. The functional form of the force field was chosen as in molecular mechanics and is not table-driven. It is continuous with continuous derivatives and is thus suitable for use with algorithms such as energy minimization or newtonian dynamics. Proteins 27:367–384, 1997. © 1997 Wiley-Liss, Inc.     

Optimization methods for conformational sampling using a Boltzmann-weighted mean field approach

An optimization protocol is proposed that combines a mean field simulation approach with Boltzmann-weighted sampling. This is done by including Boltzmann probabilities of multiple conformations in the optimization procedure. The method is demonstrated on a simple model system and on the side-chain conformations of phenylalanines in a small hexapeptide. For comparison, calculations were performed using classical stochastic dynamics simulations [M. Saunders, K. N. Houk, Y. Wu, C. Still, M. Lipton, G. Chang, and W. C. Guida (1990), Journal of the American Chemical Society, Vol. 12, pp. 1419], iterative optimization of probabilities on a fixed set of basis conformations [P. Koehl and M. Delaure (1994), Journal of Molecular Biology, Vol. 239, pp. 249–275], and simulations with locally enhanced sampling [A. Roitberg and R. Elber (1991), Journal of Chemical Physics, Vol. 95, pp. 9277–9287]. Although approximations are used in our method, the results may be more physically meaningful than those of the other procedures discussed. Furthermore, the approximate Boltzmann distribution allows generalization of the results. © 1996 John Wiley & Sons, Inc.     

On newborn hypothalamic phenylethanolamine-N-methyltransferase

Phenylethanolamine-N-methyltransferase activity of rat hypothalami was assayed. The enzyme was present at birth, in traces, and gradually increased during the first 2 postnatal months. Exposure to recurrent stressful situations increased PNMT activity in a statistically significant manner. Persistence of exposure to stressful events resulted in higher adult PNMT activity. Assays of hypothalamic tissue cultures revealed that part of PNMT activity increase was due to temporary potentiation by local factors, and partly to increase of tissue concentration of enzyme by increased protein synthesis. One of the submolecular chain reactions generated by stress (and able to induce protein synthesis) was identified as: release of ACTH during stress, activation of local adenylate cyclase by ACTH to synthesize cyclic AMP. When released, this cyclic AMP increased the local cyclic AMP: cyclic GMP ratio, a process known to induce protein synthesis. A potent and selective competitive inhibitor, SK&F 64139, when added to tissue cultures, prevented increase of PNMT activity by prolonged stimulation.     

Inhibition of thyroid monoamineoxidase by thyroxine and some specific inhibitors.

Rat thyroid monoamine oxidase (MAO) was inhibited by the non-hydrazine derivatives paragyline and tranylcypromine to a higher degree than the hydrazine derivative iproniazide. MAO was also inhibited by monoiodotyrosine and thyroxine. These results indicate that MAO may be an important enzyme in the metabolism of biogenic amines, iodotyrosines and tyronines in the thyroid gland.