Notch signalling in the regulation of peripheral T-cell function

The Notch signalling pathway plays a highly-conserved role in regulating the cellular differentiation and proliferation events that characterise pattern formation in the embryo. As cells in the embryo respond to environmental signals, similarly T-cells in the peripheral immune system must monitor their environment for antigens and respond accordingly by entering one of several potential differentiation pathways. Recent studies have identified a role for the Notch pathway in regulating the responses of T-cells in the periphery. In this review, we discuss these findings in the context of the Notch signalling pathway's role as an orchestrator of cellular differentiation, and propose a central role for Notch as a regulator of immune system function.     

Virulence factors of the human opportunistic pathogen Serratia marcescens identified by in vivo screening

The human opportunistic pathogen Serratia marcescens is a bacterium with a broad host range, and represents a growing problem for public health. Serratia marcescens kills Caenorhabditis elegans after colonizing the nematode's intestine. We used C.elegans to screen a bank of transposon-induced S.marcescens mutants and isolated 23 clones with an attenuated virulence. Nine of the selected bacterial clones also showed a reduced virulence in an insect model of infection. Of these, three exhibited a reduced cytotoxicity in vitro, and among them one was also markedly attenuated in its virulence in a murine lung infection model. For 21 of the 23 mutants, the transposon insertion site was identified. This revealed that among the genes necessary for full in vivo virulence are those that function in lipopolysaccharide (LPS) biosynthesis, iron uptake and hemolysin production. Using this system we also identified novel conserved virulence factors required for Pseudomonas aeruginosa pathogenicity. This study extends the utility of C.elegans as an in vivo model for the study of bacterial virulence and advances the molecular understanding of S.marcescens pathogenicity.     

Late cytoplasmic maturation of the small ribosomal subunit requires RIO proteins in Saccharomyces cerevisiae

Numerous nonribosomal trans-acting factors involved in pre-rRNA processing have been characterized, but few of them are specifically required for the last cytoplasmic steps of 18S rRNA maturation. We have recently demonstrated that Rrp10p/Rio1p is such a factor. By BLAST analysis, we identified the product of a previously uncharacterized essential gene, YNL207W/RIO2, called Rio2p, that shares 43% sequence similarity with Rrp10p/Rio1p. Rio2p homologues were identified throughout the Archaea and metazoan species. We show that Rio2p is a cytoplasmic-nuclear protein and that its depletion blocks 18S rRNA production, leading to 20S pre-rRNA accumulation. In situ hybridization reveals that in Rio2p-depleted cells, 20S pre-rRNA localizes in the cytoplasm, demonstrating that its accumulation is not due to an export defect. We also show that both Rio1p and Rio2p accumulate in the nucleus of crm1-1 cells at the nonpermissive temperature. Nuclear as well as cytoplasmic Rio2p and Rio1p cosediment with pre-40S particles. These results strongly suggest that Rio2p and Rrp10p/Rio1p are shuttling proteins which associate with pre-40S particles in the nucleus and they are not necessary for export of the pre-40S complexes but are absolutely required for the cytoplasmic maturation of 20S pre-rRNA at site D, leading to mature 40S ribosomal subunits.     

The expanding interleukin-1 family and its receptors: do alternative IL-1 receptor/signaling pathways exist in the brain?

Interleukin-1 (IL-1) has been implicated in neuroimmune responses and has pleiotropic actions in the brain. Compelling evidence has shown that IL-1 is a major mediator of inflammation and the progression of cell death in response to brain injury and cerebral ischemia. Its expression is strongly increased in these pathological conditions, and central administration of exogenous IL-1 significantly exacerbates ischemic brain damage. In contrast, inhibiting IL-1 actions (by intracerebroventricular [icv] injection of IL-1ra, neutralizing antibody to IL-1 or caspase-1 inhibitor) significantly reduces ischemic brain damage. IL-1 acts by binding to the IL-1 type-1 receptor (IL-1RI), which is to date, the only known functional receptor for IL-1. However, our recent investigations suggest that IL-1 can act independently of IL-1RI, raising the possibility that additional, as yet undiscovered, receptor(s) for IL-1 exist in the brain. The recent characterization of putative, new IL-1 ligands and new IL-1 receptor-related molecules leads to the hypothesis that there might be alternative IL-1 signaling pathway(s) in the central nervous system (CNS).     

2-(3,4-Dihydro-1H-isoquinolin-2yl)-pyridines as a novel class of NR1/2B subtype selective NMDA receptor antagonists

Recently, we disclosed 4-aminoquinolines as structurally novel NR1/2B subtype selective NMDA receptor antagonists. We would now like to report our findings on structurally related pyridine analogues. The SAR developed in this series resulted in the discovery of high affinity antagonists which are selective (vs alpha1 and M1 receptors) and active in vivo.     

Synthesis of a novel family of diterpenes and their evaluation as anti-inflammatory agents

The synthesis and biological evaluation of a new family of diterpenes, represented by structures 2 and 3, is presented. These compounds constitute isomeric analogues of acanthoic acid (1) and were examined as potent anti-inflammatory agents. Among them, methyl ester 12 exhibited a low non-specific cytotoxicity, inhibited TNF-alpha synthesis and displayed good specificity in suppressing cytokine expression.     

Novel synthesis of 2'-O-methylguanosine

An efficient and chemoselective synthesis of 2'-O-methylguanosine (6) has been accomplished in high yield without protection of the guanine base. The salient feature of the synthesis of 6 lies in the application of methylene-bis-(diisopropylsilyl chloride), (MDPSCl(2), 2) as a new 3',5'-O-protecting group for nucleosides. Use of CH(3)Cl as a weak electrophile and NaHMDS as a mild base was crucial to the success of the 2'-O-methylation of 3',5'-O-protected guanosine.     

CFTR gene mutations – including three novel nucleotide substitutions – and haplotype background in patients with asthma, disseminated bronchiectasis and chronic obstructive pulmonary disease

In order to investigate the incidence of cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations and unclassified variants in chronic pulmonary disease in children and adults, we studied 20 patients with asthma, 19 with disseminated bronchiectasis (DB) of unknown aetiology, and 12 patients with chronic obstructive pulmonary disease (COPD), and compared the results to 52 subjects from the general Greek population. Analysis of the whole coding region of the CFTR gene and its flanking intronic regions revealed that the proportion of CFTR mutations was 45% in asthma (P<0.05), 26.3% in DB (P>0.05), 16.7% in COPD (P>0.05), compared to 15.4% in the general population. Seventeen different molecular defects involved in disease predisposition were identified in 16 patients. Three potentially disease-causing mutations, T388 M, M1R and V11I, are novel, found so far only in three asthma patients. The hyperactive M470 allele was found more frequently in COPD patients (frequency 70.8%, P<0.01) than in the controls. The study of the TGmTnM470 V polyvariant CFTR allele revealed the presence of CFTR function-modulating haplotypes TG13/T5/M470, TG11/T5/M470, TG12/T5/V470 and TG12/T7, combined with M470 or V470, in six asthma patients, four DB patients (P<0.01), and two COPD patients (P<0.05). These results confirm the involvement of the CFTR gene in asthma, DB and possibly in COPD.     

Technical advance: a high throughput system for transposon tagging and promoter trapping in tomato

We describe new tools for functional analysis of the tomato genome based on insertional mutagenesis with the maize Ac/Ds transposable elements in the background of the miniature cultivar Micro-Tom. 2932 F3 families, in which Ds elements transposed and were stabilized, were screened for phenotypic mutations. Out of 10 families that had a clear mutant phenotype, only one mutant was Ds-tagged. In addition, we developed promoter trapping using the firefly luciferase reporter gene and enhancer trapping, using beta-glucuronidase (GUS). We show that luciferase can be used as a non-invasive reporter to identify, isolate and regenerate somatic sectors, to study the time course of mutant expression, and to identify inducible genes. Out of 108 families screened for luciferase activity 55% showed expression in the flower, 11% in the fruit and 4% in seedlings, suggesting a high rate of Ds insertion into genes. Preferential insertion into genes was supported by the analysis of Ds flanking sequences: 28 out of 50 sequenced Ds insertion sites were similar to known genes or to ESTs. In summary, the 2932 lines described here contain 2-3 Ds inserts per line, representing a collection of approximately 7500 Ds insertions. This collection has potential for use in high-throughput functional analysis of genes and promoter isolation in tomato.     

Disturbance and Seasonal Dynamics of Mycorrhizae in a Tropical Deciduous Forest in Mexico1

Mycorrhizal fungi were sampled in a deciduous tropical forest on the Pacific coast of Mexico during different seasons and in natural treefall gaps and pastures. All 12 plant species sampled in the forest were arbuscular mycorrhizal. The percent root infection and spore production were closely related to the phenology of the plants. Most tree species and all herbaceous species had the highest infection in the summer rainy season, but two species, Opuntia excelsa and Jacquinia pungens, had highest infection in the dry season. Unusually high rainfall during the dry season was associated with increased infection but not increased spore production. Spore density was low for all species at all sample times, except at the beginning of the July 1993 rainy season in, when we observed up to 28 spores/g soil. The percent cover of shrubs or herbs did not increase in gaps after two years, and we observed no colonizing seedlings. No plant species with cover higher than 2.7 percent occurred exclusively in gaps or forest. The percent mycorrhizal infection did not differ significantly between gaps and forest. Spore counts were as high in the gaps as in the forest in two of the three gaps but lower in the third gap. The lack of significant response of plants in these gaps after two years differed from the rapid response in tropical rainforests. It is likely related to the small size of the gaps and to light infiltration to the forest floor. Pastures were dominated by two species of exotic grasses and one species of mycorrhizal fungus, whereas forests had 15 fungal species. The slow regrowth of vegetation in gaps was not limited by mycorrhizal fungi, since they were still abundant after the treefalls, but recovery in pastures could be affected by low fungal diversity and dominance of grasses.