Spirocyclic nonpeptide glycoprotein IIb-IIIa antagonists. Part 1: design of potent and specific 3,9-diazaspiro[5.5]undecanes

The synthesis and biological activity of novel glycoprotein IIb-IIla antagonists containing the 3,9-diazaspiro[5.5]undecane nucleus are described. The potent activity of these compounds as platelet aggregation inhibitors demonstrates the utility of the spirocyclic structures as a central template for nonpeptide RGD mimics.     

Cutting edge: intravenous soluble antigen is presented to CD4 T cells by CD8- dendritic cells, but cross-presented to CD8 T cells by CD8+ dendritic cells

Mouse spleen contains three distinct mature dendritic cell (DC) populations (CD4(+)8(-), CD4(-)8(-), and CD4(-)8(+)) which retain a capacity to take up particulate and soluble AGS: Although the three splenic DC subtypes showed similar uptake of injected soluble OVA, they differed markedly in their capacity to present this Ag and activate proliferation in OVA-specific CD4 or CD8 T cells. For class II MHC-restricted presentation to CD4 T cells, the CD8(-) DC subtypes were more efficient, but for class I MHC-restricted presentation to CD8 T cells, the CD8(+) DC subtype was far more effective. This differential persisted when the DC were activated with LPS. The CD8(+) DC are therefore specialized for in vivo cross-presentation of exogenous soluble Ags into the class I MHC presentation pathway.     

Fetal gene transfer by transuterine injection of cationic liposome-DNA complexes

In utero injection of cationic liposome-DNA complexes (CLDCs) containing chloramphenicol acetyltransferase, beta-galactosidase (beta-gal), or human granulocyte colony-stimulating factor (hG-CSF) expression plasmids produced high-level gene expression in fetal rats. Tissues adjacent to the injection site exhibited the highest levels of gene expression. Chloramphenicol acetyltransferase expression persisted for at least 14 days and was reexpressed following postnatal reinjection of CLDCs. Intraperitoneal administration of the hG-CSF gene produced high serum hG-CSF levels. X-gal staining demonstrated widespread beta-gal expression in multiple fetal tissues and cell types. No toxic or inflammatory responses were observed, nor was there evidence of fetal-maternal or maternal-fetal gene transfer, suggesting that CLDCs may provide a useful alternative to viral vectors for in utero gene transfer.     

Helminth parasites of the wild rabbit Oryctolagus cuniculus near Malham Tarn, Yorkshire, UK

Between 1992 and 1996, 95 rabbits from the immediate locality of Malham Tarn, North Yorkshire, UK were examined for the presence of helminth parasites. All the examinations took place in late September or October. Three species of nematodes, Graphidium strigosum, Passalurus ambiguus and Trichostrongylus retortaeformis and two species of cestodes, Taenia pisiformis and Cittotaenia pectinata were identified. There were no associations between helminth species richness and year of sampling, host weight or sex. A logistic model was fitted to the prevalence data from these helminths as was an over-dispersed Poisson model to the worm burden data. Graphidium strigosum was the most frequently identified species with an average prevalence of 78%. The mean prevalence and intensity of Graphidium infection were significantly effected by sampling year. The lower than normal rainfall recorded at the Tarn during the years 1995 and 1996 may have be one reason for this pattern. The worm burden of G. strigosum was significantly positively associated with rabbit body weight. The intensity of infection with P. ambiguus was significantly higher in female rabbits. There was a significant non-linear relationship between P. ambiguus worm burden and rabbit weight (P = 0.002) with worm burdens being highest in the 1000 g to 1499 g weight cohort. Trichostrongylus retortaeformis was only identified in 1994 and male rabbits harboured significantly higher worm burdens than females (48 vs. 7, P = 0.022). Over the five years, the average Taenia pisiformis prevalence was 31% and there was a significant positive association between worm burden and rabbit weight (P = 0.001). Cittotaenia pectinata had a prevalence of 37% over the whole study period with no interactions between prevalence or intensity and body weight, year of sampling or rabbit sex. All five helminths showed an overdispersed distribution with k values less than 1.     

Application of distance geometry to 3D visualization of sequence relationships

SUMMARY: We describe the application of distance geometry methods to the three-dimensional visualization of sequence relationships, with examples for mumps virus SH gene cDNA and prion protein sequences. Sequence-sequence distance measures may be obtained from either a multiple sequence alignment or from sets of pairwise alignments. AVAILABILITY: C/Perl code and HTML/VRML files from http://www.nibsc.ac.uk/dg3dseq/     

Two divergent plasma membrane syntaxin-like SNAREs, nsyn1 and nsyn2, contribute to hyphal tip growth and other developmental processes in Neurospora crassa

Highly polarized exocytosis of vesicles at hyphal apices is an essential requirement of tip growth. This requirement may be met by the localization and/or activation of an apical SNARE-based machinery. We have cloned nsyn1 and nsyn2, SNAREs predicted to function at the plasma membrane in Neurospora crassa. Transformation of extra copies of nsyn1 into wild-type strains displayed effects consistent with quelling of nsyn1 expression, which was lethal in most transformants. All surviving transformants grew slowly, conidiated poorly, and were male sterile. In addition, antisense nsyn1 strains grew slowly, with abnormal hyphal diameters and polarity and defective conidiation. For nsyn2, several repeat induced point mutation (RIP) crosses produced no, or poorly germinating ascospores. Those that germinated produced slow-growing hyphae with abnormal branching. The defects in nsyn1 and nsyn2 mutants are consistent with differential impaired vesicle fusion in hyphal tips and other developmental stages.     

Endogenous redox activity in mouse spermatozoa and its role in regulating the tyrosine phosphorylation events associated with sperm capacitation

We investigated the role of endogenous redox activity in regulating the signal transduction pathway leading to tyrosine phosphorylation in mouse spermatozoa. Endogenous redox activity was monitored using a luminol-peroxidase chemiluminescent probe. Chemiluminescence increased in spermatozoa that were actively undergoing cAMP-mediated tyrosine phosphorylation events associated with capacitation and was inhibited in a dose-dependent manner by addition of catalase or diphenylene iodonium, both of which also inhibited tyrosine phosphorylation within the cell at points downstream of cAMP. Excluding bicarbonate from the incubation medium reduced the redox activity of sperm by 80-90% and dramatically reduced tyrosine phosphorylation. This study provides the first evidence that tyrosine phosphorylation associated with capacitation in mouse spermatozoa is redox regulated by a flavinoid-containing enzyme involving mediation by hydrogen peroxide. Bicarbonate regulated the redox activity of mouse spermatozoa, and this regulation may contribute to the impact of this anion on tyrosine phosphorylation during capacitation of mouse spermatozoa.     

Mutations in the immunoglobulin-like domain of gp190, the leukemia inhibitory factor (LIF) receptor,increase or decrease its affinity for LIF

The leukemia inhibitory factor (LIF) receptor comprises the low affinity binding chain gp190 and the high affinity converter gp130. The ectodomain of gp190 is among the most complex in the hematopoietin receptor family, because it contains two typical cytokine receptor homology domains separated by an immunoglobulin-like (Ig-like) domain. Human and murine gp190 proteins share 76% homology, but murine gp190 binds human LIF with a much higher affinity, a property attributed to the Ig-like domain. Using alanine-scanning mutagenesis of the Ig-like domain, we mapped a LIF binding site at its carboxyl terminus, mainly involving residue Phe-328. Mutation of selected residues into their orthologs in the murine receptor (Q251E and N321D) significantly increased the affinity for human LIF. Interestingly, these residues, although localized at both the amino and carboxyl terminus, make a spatially unique LIF binding site in a structural model of the Ig-like module. These results demonstrate definitively the role of the Ig-like domain in LIF binding and the potential to modulate receptor affinity in this family with very limited amino acid changes.     

Clustering mass spectrometry data using order statistics

Mass spectrometry data is inherently uncertain. Rather than compare peak heights across samples, a comparison can be made of the relative ordering of the peak height across samples. Order statistics are used to provide a distance metric between each ordered list of peak heights from the samples. A principal component analysis is performed on the set of distance vectors to highlight to important components.