Synthesis and immunological evaluation of self-adjuvanting glycolipopeptide vaccine candidates

Synthesis of four glycolipids with different number of lauroyl groups on glucose or cellobiose as scaffolds is described. Their immunological evaluations either admixed with or covalently linked to J8, a peptide antigen derived from the C-terminus of the antiphagocytic M-protein of group A streptococcus, are also investigated. Administration of mixtures of J8 and glycolipids to B10BR (H-2(k)) mice induced low-levels of J8-specific IgG antibodies. While glycolipopeptides, in which J8 was covalently linked to the synthetic glycolipids, demonstrated high-levels of antibody titers comparable with the co-administration of these glycolipopeptides with complete Freund's adjuvant, suggesting clearly the strong potency of the synthesized glycolipids as self-adjuvanting moieties.     

Imidazolyl benzimidazoles and imidazo[4,5-b]pyridines as potent p38alpha MAP kinase inhibitors with excellent in vivo antiinflammatory properties

Herein we report investigations into the p38alpha MAP kinase activity of trisubstituted imidazoles that led to the identification of compounds possessing highly potent in vivo activity. The SAR of a novel series of imidazopyridines is demonstrated as well, resulting in compounds possessing cellular potency and enhanced in vivo activity in the rat collagen-induced arthritis model of chronic inflammation.     

Sensitive detection of GFP utilizing tyramide signal amplification to overcome gene silencing

The green fluorescent protein (GFP) is among the most commonly used expression markers in biology. GFP-tagged cells have played a particularly important role in studies of cell lineage. Sensitive detection of GFP is crucially important for such studies to be successful, and problems with detection may account for discrepancies in the literature regarding the possible fate choices of stem cells. Here we describe a very sensitive technique for visualization of GFP. Using it we can detect about 90% of cells of donor origin while we could only see about 50% of these cells when we employ the methods that are in general use in other laboratories. In addition, we provide evidence that some cells permanently silence GFP expression. In the case of the progeny of bone marrow stem cells, it appears that the more distantly related they are to their precursors, the more likely it is that they will turn off the lineage marker.     

Lipopolysaccharide effects on neuronal activity in rat basal forebrain and hypothalamus during sleep and waking

Peripheral administration of lipopolysaccharide (LPS) is associated with alterations in sleep and the electroencephalogram. To evaluate potential neuronal mechanisms for the somnogenic effects of LPS administration, we used unanesthetized rats to survey the firing patterns of neurons in various regions of rat basal forebrain (BF) and hypothalamus during spontaneous sleep and waking and during the epochs of sleep and waking that occurred after the intraperitoneal administration of LPS. In the brain regions studied, LPS administration was associated with altered firing rates in 39% of the neurons examined. A larger proportion of LPS-responsive units showed vigilance-related alterations in firing rates compared with nonresponsive units. Approximately equal proportions of LPS-responsive neurons showed increased and decreased firing rates after LPS administration, with some units in the lateral preoptic area of the hypothalamus showing particularly robust increases. These findings are consistent with other studies showing vigilance-related changes in neuronal activity in various regions of BF and hypothalamus and further demonstrate that peripheral LPS administration alters neuronal firing rates in these structures during both sleep and waking.     

The anxiety-like phenotype of 5-HT receptor null mice is associated with genetic background-specific perturbations in the prefrontal cortex GABA-glutamate system

A deficit in the serotonin 5-HT(1A) receptor has been found in panic and post-traumatic stress disorders, and genetic inactivation of the receptor results in an anxiety-like phenotype in mice on both the C57Bl6 and Swiss-Webster genetic backgrounds. Anxiety is associated with increased neuronal activity in the prefrontal cortex and here we describe changes in glutamate and GABA uptake of C57Bl6 receptor null mice. Although these alterations were not present in Swiss-Webster null mice, we have previously reported reductions in GABA(A) receptor expression in these but not in C57Bl6 null mice. This demonstrates that inactivation of the 5-HT(1A) receptor elicits different and genetic background-dependent perturbations in the prefrontal cortex GABA/glutamate system. These perturbations can result in a change in the balance between excitation and inhibition, and indeed both C57Bl6 and Swiss-Webster null mice show signs of increased neuronal excitability. Because neuronal activity in the prefrontal cortex controls the extent of response to anxiogenic stimuli, the genetic background-specific perturbations in glutamate and GABA neurotransmission in C57Bl6 and Swiss-Webster 5-HT(1A) receptor null mice may contribute to their shared anxiety phenotype. Our study shows that multiple strains of genetically altered mice could help us to understand the common and individual features of anxiety.     

Adaptive Changes in Postsynaptic Dopamine Receptors Despite Unaltered Dopamine Dynamics in Mice Lacking Monoamine Oxidase B

Monoamine oxidase (MAO) B is considered a key enzyme in dopamine metabolism. The present studies, conducted in MAO B knockout mice, show that lack of MAO B does not alter extracellular levels of dopamine in striatum. Similarly, the synthesis, storage, uptake, and release of dopamine are also unaltered. However, autoradiography revealed a significant up-regulation of the D2-like dopamine receptors in the striatum of MAO B knockout mice. Mutant mice also exhibit a functional supersensitivity of D1-dopamine receptors in the nucleus accumbens. Thus, the agonist SKF 38,393-induced c-Fos immunoreactivity was significantly increased in knockout mice as compared with wild-type controls. In view of the apparently normal basal dopamine dynamics observed in MAO B knockout mice, we hypothesize that a dopamine-independent mechanism underlies adaptations in dopamine receptor function that occur as a consequence of MAO B depletion. Finally, these findings suggest that chronic administration of MAO inhibitors, as occurs in the treatment of Parkinson's disease and depression, may be associated with an increased responsiveness of CNS neurons to dopamine receptor ligands.     

Use of a pooled transposon mutation grid to demonstrate roles in disease development for Erwinia carotovora subsp. atroseptica putative type III secreted effector (DspE/A) and helper (HrpN) proteins

Soft rot Erwinia spp., like other closely related plant pathogens, possess a type III secretion system (TTSS) (encoded by the hrp gene cluster) implicated in disease development. We report the sequence of the entire hrp gene cluster and adjacent dsp genes in Erwinia carotovora subsp. atroseptica SCRI1039. The cluster is similar in content and structural organization to that in E. amylovora. However, eight putative genes of unknown function located within the E. carotovora subsp. atroseptica cluster do not have homologues in the E. amylovora cluster. An arrayed set of Tn5 insertional mutants (mutation grid) was constructed and pooled to allow rapid isolation of mutants for any given gene by polymerase chain reaction screening. This novel approach was used to obtain mutations in two structural genes (hrcC and hrcV), the effector gene dspE/A, and the helper gene hrpN. An improved pathogenicity assay revealed that these mutations led to significantly reduced virulence, showing that both the putative E. carotovora subsp. atroseptica TTSS-delivered effector and helper proteins are required for potato infection.     

The discovery of 3-(N-alkyl)aminopropylphosphonic acids as potent S1P receptor agonists

3-(N-Alkyl)aminopropylphosphonic acids are potent agonists of four of the five known sphingosine-1-phosphate (S1P) receptor subtypes.