Tempo and mode of concerted evolution in the L1 repeat family of mice

A 300-bp DNA sequence has been determined for 30 (10 from each of three species of mice) random isolates of a subset of the long interspersed repeat family L1. From these data we conclude that members of the L1 family are evolving in concert at the DNA sequence level in Mus domesticus, Mus caroli, and Mus platythrix. The mechanism responsible for this phenomenon may be either duplicative transposition, gene conversion, or a combination of the two. The amount of intraspecies divergence averages 4.4%, although between species base substitutions accumulate at the rate of approximately 0.85%/Myr to a maximum divergence of 9.1% between M. platythrix and both M. domesticus and M. caroli. Parsimony analysis reveals that the M. platythrix L1 family has evolved into a distinct clade in the 10-12 Myr since M. platythrix last shared a common ancestor with M. domesticus and M. caroli. The parsimony tree also provides a means to derive the average half-life of L1 sequences in the genome. The rates of gain and loss of individual copies of L1 were estimated to be approximately equal, such that approximately one-half of them turn over every 3.3 Myr.      

Isolation of phosphoglycerate kinases by affinity chromatography

A variety of Sepharose derivatives containing DL-O-phosphorylserine or adenosine nucleotides with different points of attachment, has been synthesized and tested for affinity to phosphoglycerate kinase. The most effective gels contained periodate-oxidized ATP or ADP bound via the ribose by hydrazone formation to adipoyl-dihydrazo-Sepharose. The effect of pH, magnesium and buffer ions on the binding capacity of the ATP derivative of Sepharose has been examined. Optimal elution of phosphoglycerate kinase was investigated using different combinations of adenosine nucleotides, 3-phosphogylcerate and magnesium ions. A method is presented giving conditions for the purification of phosphoglycerate kinase from different sources (spinach, human erythrocytes, human, rabbit and trout muscle). It includes extract preparation, affinity chromatography and gel filtration. The method is greatly superior to known isolation procedures by virtue of its technical simplicity, excellent yield (85-100%) and reproducability. The capacity of the ATP-ribosyl-adipoyl-dihydrazo-Sepharose was 5 mg phosphoglycerate kinase per 1 g of matrix. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate indicated that the final products are homogeneous. The phosphoglycerate kinases from different sources appear to have the same affinity for this ATP derivative of Sepharose, the same molecular weight and the same specific activity.     

The Fate of Chromosomes and Alleles in an Allohexaploid Brassica Population

Production of allohexaploid Brassica (2n = AABBCC) is a promising goal for plant breeders due to the potential for hybrid heterosis and useful allelic contributions from all three of the Brassica genomes present in the cultivated diploid (2n = AA, 2n = BB, 2n = CC) and allotetraploid (2n = AABB, 2n = AACC, and 2n = BBCC) crop species (canola, cabbages, mustards). We used high-throughput SNP molecular marker assays, flow cytometry, and fluorescent in situ hybridization (FISH) to characterize a population of putative allohexaploids derived from self-pollination of a hybrid from the novel cross (B. napus × B. carinata) × B. juncea to investigate whether fertile, stable allohexaploid Brassica can be produced. Allelic segregation in the A and C genomes generally followed Mendelian expectations for an F₂ population, with minimal nonhomologous chromosome pairing. However, we detected no strong selection for complete 2n = AABBCC chromosome complements, with weak correlations between DNA content and fertility (r² = 0.11) and no correlation between missing chromosomes or chromosome segments and fertility. Investigation of next-generation progeny resulting from one highly fertile F₂ plant using FISH revealed general maintenance of high chromosome numbers but severe distortions in karyotype, as evidenced by recombinant chromosomes and putative loss/duplication of A- and C-genome chromosome pairs. Our results show promise for the development of meiotically stable allohexaploid lines, but highlight the necessity of selection for 2n = AABBCC karyotypes.     

Bronchial epithelial cells are rendered insensitive to glucocorticoid transactivation by transforming growth factor-β1

Background

We have previously shown that transforming growth factor-beta (TGF-beta) impairs glucocorticoid (GC) function in pulmonary epithelial cell-lines. However, the signalling cascade leading to this impairment is unknown. In the present study, we provide the first evidence that TGF-beta impairs GC action in differentiated primary air-liquid interface (ALI) human bronchial epithelial cells (HBECs). Using the BEAS-2B bronchial epithelial cell line, we also present a systematic examination of the known pathways activated by TGF-beta, in order to ascertain the molecular mechanism through which TGF-beta impairs epithelial GC action.

Methods

GC transactivation was measured using a Glucocorticoid Response Element (GRE)–Secreted embryonic alkaline phosphatase (SEAP) reporter and measuring GC-inducible gene expression by qRT-PCR. GC transrepression was measured by examining GC regulation of pro-inflammatory mediators. TGF-beta signalling pathways were investigated using siRNA and small molecule kinase inhibitors. GRα level, phosphorylation and sub-cellular localisation were determined by western blotting, immunocytochemistry and localisation of GRα–Yellow Fluorescent Protein (YFP). Data are presented as the mean ± SEM for n independent experiments in cell lines, or for experiments on primary HBEC cells from n individual donors. All data were statistically analysed using GraphPad Prism 5.0 (Graphpad, San Diego, CA). In most cases, two-way analyses of variance (ANOVA) with Bonferroni post-hoc tests were used to analyse the data. In all cases, P <0.05 was considered to be statistically significant.

Results

TGF-beta impaired Glucocorticoid Response Element (GRE) activation and the GC induction of several anti-inflammatory genes, but did not broadly impair the regulation of pro-inflammatory gene expression in A549 and BEAS-2B cell lines. TGF-beta-impairment of GC transactivation was also observed in differentiated primary HBECs. The TGF-beta receptor (ALK5) inhibitor SB431541 fully prevented the GC transactivation impairment in the BEAS-2B cell line. However, neither inhibitors of the known downstream non-canonical signalling pathways, nor knocking down Smad4 by siRNA prevented the TGF-beta impairment of GC activity.

Conclusions

Our results indicate that TGF-beta profoundly impairs GC transactivation in bronchial epithelial cells through activating ALK5, but not through known non-canonical pathways, nor through Smad4-dependent signalling, suggesting that TGF-beta may impair GC action through a novel non-canonical signalling mechanism.     

Monitoring the Cry1Ab susceptibility of European corn borer in Germany

The European corn borer, Ostrinia nubilalis (Hübner), is one of the most important insect pests in corn, Zea mays L. Transgenic corn cultivars expressing Bacillus thuringiensis (Bt) toxin provide a promising crop protection strategy against European corn borer; however, management is needed to avoid resistance development of the target pest species. The aim of this work was to establish the baseline susceptibility of different European corn borer populations in Germany to be able to forecast a possible development of resistance at an early stage. To standardize test procedures for future resistance management, the efficiency of Cry1Ab toxins from different suppliers and different production was assessed. Furthermore, two different test methods, surface treatment and the incorporation method, were compared with regard to their practicability and efficiency. Neither method provided significant differences in the baseline susceptibility of populations from different German regions. Overall, the data suggested little differentiation among German populations in terms of their susceptibility to Bt toxin and their genetic background. Future monitoring could therefore use a single European corn borer population as a representative for southwestern Germany. However, toxins from different suppliers and different production batches produced a vast range of LC50 values. Changes because of different toxin batches may be mistaken as a change in baseline susceptibility or even as the start of a resistance development. Thus, it is important throughout insect resistance management that the same toxin batches will be available for baseline susceptibility bioassays and for future tests.     

A Disease Model for Wheezing Disorders in Preschool Children Based on Clinicians' Perceptions

Background

Wheezing disorders in childhood vary widely in clinical presentation and disease course. During the last years, several ways to classify wheezing children into different disease phenotypes have been proposed and are increasingly used for clinical guidance, but validation of these hypothetical entities is difficult.

Methodology/Principal Findings

The aim of this study was to develop a testable disease model which reflects the full spectrum of wheezing illness in preschool children. We performed a qualitative study among a panel of 7 experienced clinicians from 4 European countries working in primary, secondary and tertiary paediatric care. In a series of questionnaire surveys and structured discussions, we found a general consensus that preschool wheezing disorders consist of several phenotypes, with a great heterogeneity of specific disease concepts between clinicians. Initially, 24 disease entities were described among the 7 physicians. In structured discussions, these could be narrowed down to three entities which were linked to proposed mechanisms: a) allergic wheeze, b) non-allergic wheeze due to structural airway narrowing and c) non-allergic wheeze due to increased immune response to viral infections. This disease model will serve to create an artificial dataset that allows the validation of data-driven multidimensional methods, such as cluster analysis, which have been proposed for identification of wheezing phenotypes in children.

Conclusions/Significance

While there appears to be wide agreement among clinicians that wheezing disorders consist of several diseases, there is less agreement regarding their number and nature. A great diversity of disease concepts exist but a unified phenotype classification reflecting underlying disease mechanisms is lacking. We propose a disease model which may help guide future research so that proposed mechanisms are measured at the right time and their role in disease heterogeneity can be studied.     

Molecular analysis of the fibrinogen gene cluster in 16 patients with congenital afibrinogenemia: novel truncating mutations in the FGA and FGG genes

Congenital afibrinogenemia is an autosomal recessive disorder characterized by the complete absence of detectable fibrinogen. We previously identified the first causative mutations for this disease in a non-consanguineous Swiss family. These were homozygous deletions of approximately 11 kb of the fibrinogen alpha chain gene (FGA). Our subsequent study revealed that the majority of cases were attributable to truncating mutations in FGA, with the most common mutation affecting the donor splice site in FGA intron 4 (IVS4+1 G-->T). Here, we report 13 further unrelated patients with mutations in FGA, confirming the relative importance of this gene compared with FGG and FGB in the molecular aetiology of afibrinogenemia. Three other patients were homozygous for mutations in FGG. Eight novel mutations were identified: five in FGA and three in FGG. Sufficient mutation data is now available to permit an effective strategy for the genetic diagnosis of congenital afibrinogenemia.     

Tolerability and efficacy of single dose albendazole,diethylcarbamazine citrate (DEC) or co-administration of albendazole with DEC in the clearance of <Emphasis Type="Italic">Wuchereria bancrofti</Emphasis>in asymptomatic microfilaraemic volunteers in Pondicherry,South India: a hospital-based study

Background  

The tolerability and efficacy of single dose albendazole (400 mg), diethylcarbamazine citrate (DEC) (6 mg/kg bodyweight) or co-administration of albendazole (400 mg) + DEC (6 mg/kg bodyweight) was studied in 54 asymptomatic Wuchereria bancrofti microfilaraemic volunteers in a double blind hospital-based clinical study.