Automated DNA mutation detection using universal conditions direct sequencing: application to ten muscular dystrophy genes

Background

Developing a greater understanding of population genetic structure in lowland tropical plant species is highly relevant to our knowledge of increasingly fragmented forests and to the conservation of threatened species. Specific studies are particularly needed for taxa whose population dynamics are further impacted by human harvesting practices. One such case is the fishtail or xaté palm (Chamaedorea ernesti-augusti) of Central America, whose wild-collected leaves are becoming progressively more important to the global ornamental industry. We use microsatellite markers to describe the population genetics of this species in Belize and test the effects of climate change and deforestation on its recent and historical effective population size.

Results

We found high levels of inbreeding coupled with moderate or high allelic diversity within populations. Overall high gene flow was observed, with a north and south gradient and ongoing differentiation at smaller spatial scales. Immigration rates among populations were more difficult to discern, with minimal evidence for isolation by distance. We infer a tenfold reduction in effective population size ca. 10,000 years ago, but fail to detect changes attributable to Mayan or contemporary deforestation.

Conclusion

Populations of C. ernesti-augusti are genetically heterogeneous demes at a local spatial scale, but are widely connected at a regional level in Belize. We suggest that the inferred patterns in population genetic structure are the result of the colonization of this species into Belize following expansion of humid forests in combination with demographic and mating patterns. Within populations, we hypothesize that low aggregated population density over large areas, short distance pollen dispersal via thrips, low adult survival, and low fruiting combined with early flowering may contribute towards local inbreeding via genetic drift. Relatively high levels of regional connectivity are likely the result of animal-mediated long-distance seed dispersal. The greatest present threat to the species is the potential onset of inbreeding depression as the result of increased human harvesting activities. Future genetic studies in understory palms should focus on both fine-scale and landscape-level genetic structure.     

A laminar flow model of aerosol survival of epidemic and non-epidemic strains of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> isolated from people with cystic fibrosis

Background  

Cystic fibrosis (CF) is an inherited multi-system disorder characterised by chronic airway infection with pathogens such as Pseudomonas aeruginosa.     

Genomic organization and single-nucleotide polymorphism map of desmuslin, a novel intermediate filament protein on chromosome 15q26.3

Background  

Desmuslin is an α-dystrobrevin-interacting protein expressed primarily in heart and skeletal muscle. The desmuslin protein interacts with and is closely related to desmin, a protein encoded by a locus mutated in some forms of hereditary distal myopathy. As a muscle-specific intermediate filament protein, desmuslin is also a candidate for myopathies of unknown etiology.     

T cell responses to allogeneic human mesenchymal stem cells: immunogenicity,tolerance, and suppression

Human mesenchymal stem cells (MSCs) were evaluated for their ability to activate allogeneic T cells in cell mixing experiments. Phenotypic characterization of MSCs by flow cytometry showed expression of MHC Class I alloantigens, but minimal expression of Class II alloantigens and costimulatory molecules, including CD80 (B7-1), CD86 (B7-2), and CD40. T cells purified from peripheral blood mononuclear cells (PBMCs) did not proliferate to allogeneic MSCs. Lack of response was not due to a deficiency of costimulation, since retroviral transduction of MSCs with either B7-1 or B7-2 costimulatory molecules did not result in lymphoproliferation. Although these results suggested that MSCs were immunologically inert or potentially tolerogenic, T cells cultured with MSCs produced IFN- and displayed secondary kinetics to restimulation with PBMCs, indicating alloantigen priming rather than tolerance induction by the MSCs. To determine whether MSCs suppressed alloreactive T cells, MSCs were added to primary mixed lymphocyte reaction (MLR) cultures. MSCs suppressed cell proliferation when added at the initiation of culture or when added to an ongoing MLR culture. Suppression was dose-dependent, genetically unrestricted, and occurred whether or not MSCs were pretreated with IFN-. MSCs in transwell chambers suppressed primary MLR cultures, indicating that suppression was mediated by soluble molecules. Analysis of cytokines in suppressed MLR cultures demonstrated up-regulation of IFN- and IL-10, and down-regulation of TNF- production relative to control cultures. We conclude that MSCs can initiate activation of alloreactive T cells, but do not elicit T cell proliferative responses due to active suppressive mechanisms.     

Congenital fibrosis of the extraocular muscles (autosomal dominant congenital external ophthalmoplegia): genetic homogeneity, linkage refinement, and physical mapping on chromosome 12.

Congenital fibrosis of the extraocular muscles (CFEOM) is an autosomal dominant syndrome of congenital external ophthalmoplegia and bilateral ptosis. We previously reported linkage of this disorder in two unrelated families to an 8-cM region near the centromere of human chromosome 12. We now present refinement of linkage in the original two families, linkage analysis of five additional families, and a physical map of the critical region for the CFEOM gene. In each of the seven families the disease gene is linked to the pericentromeric region of chromosome 12. D12S345, D12S59, D12S331, and D12S1048 do not recombine with the disease gene and have combined lod scores of 35.7, 35.6, 16.0, and 31.4, respectively. AFM136xf6 and AFMb320wd9 flank the CFEOM locus, defining a critical region of 3 cM spanning the centromere of chromosome 12. These data support the concept that this may be a genetically homogeneous disorder. We also describe the generation of a YAC contig encompassing the critical region of the CFEOM locus. This interval has been assigned cytogenetically to 12p11.2-q12 and spans the centromere of chromosome 12. These results provide the basis for further molecular analyses of the structure and organization of the CFEOM locus and will help in the identification of candidate genes.     

Quantitative analysis of sugar constituents of glycoproteins by capillary electrophoresis

A method for quantitative analysis of monosaccharides including N- acetylneuraminic acid derived from sialic acid-containing oligosaccharides and glycoproteins is presented. The analysis is based on the combination of chemical and enzymatic methods coupled with capillary electrophoretic (CE) separation and laser-induced fluorescence (LIF) detection. The present method utilizes a simplified acid hydrolysis procedure consisting of mild hydrolysis (0.1 M TFA) to release sialic acid and strong acid hydrolysis (2.0 N TFA) to produce amino and neutral sugars. Amino sugars released from strong acid hydrolysis of oligosaccharides and glycoproteins were reacetylated and derivatized with 8-aminopyrene-1,3,6-trisulfonate (APTS) along with neutral sugars in the presence of sodium cyanoborohydride to yield quantitatively the highly stable fluorescent APTS adducts. N- acetylneuraminic acid (Neu5Ac), a major component of most mammalian glycoproteins, was converted in a fast specific reaction by the action of neuraminic acid aldolase (N-acylneuraminate pyruvate-lyase EC 4.1.3.3) to N-acetylmannosamine (ManNAc) and pyruvate. ManNAc was then derivatized with APTS in the same manner as the other monosaccharides. This method was demonstrated for the quantitation of pure Neu5Ac and the species derived from mild acid hydrolysis of 6'-sialyl-N- acetyllactosamine and bovine fetuin glycan. Quantitative recovery of the N-acetylmannosamine was obtained from a known amount of Neu5Ac in a mixture of seven other monosaccharides or from the sialylated oligosaccharides occurring in glycoproteins. The sequence of procedures consists of acid hydrolysis, enzymatic conversion and APTS derivatization which produced quantitative recovery of APTS- monosaccharide adducts. The detection limits for sugars derivatized with APTS and detected by CE-LIF are 100 pmol for Neu5Ac and 50 pmol for the other sugars.      

Polypeptide components of Drosophila small nuclear ribonucleoprotein particles.

In eukaryotes splicing of pre-mRNAs is mediated by the spliceosome, a dynamic complex of small nuclear ribonucleoprotein particles (snRNPs) that associate transiently during spliceosome assembly and the splicing reaction. We have purified snRNPs from nuclear extracts of Drosophila cells by affinity chromatography with an antibody specific for the trimethylguanosine (m3G) cap structure of snRNAs U1-U5. The polypeptide components of Drosophila snRNPs have been characterized and shown to consist of a number of proteins shared by all the snRNPs, and some proteins which appear to be specific to individual snRNP particles. On the basis of their apparent molecular weight and antigenicity many of these common and particle specific Drosophila snRNP proteins are remarkably conserved between Drosophila and human spliceosomes. By probing western blots of the Drosophila snRNP polypeptides with a number of antisera raised against human snRNP proteins, Drosophila polypeptides equivalent to many of the HeLa snRNP-common proteins have been identified, as well as candidates for a number of U1, U2 and U5-specific proteins.