Microarray-based detection of mannose-binding lectin 2 (MBL2) polymorphisms in a routine clinical setting

The assessment of allelic variants in the human mannose-binding lectin 2 (MBL2) gene is of great clinical importance in newborns or immune-suppressed patients at high risk for a variety of infections. Here, we present a study on the genotyping accuracy of a DNA microarray-based on-chip PCR method suited for the detection of five different polymorphisms in the MBL2 gene. We tested 153 genomic DNA samples, prepared from archival blood spots on Guthrie cards, for the presence of allelic variants in the human MBL2 gene by the on-chip PCR method and compared the obtained results of three variants to standard DNA capillary sequencing. The genotyping power of the described assay was readily comparable to DNA sequencing (453/459 correct genotype calls in 153 DNA samples; 98.7% accuracy), mainly due to intrinsic technical benefits of microarrays such as high number of test replicates and automated data analysis. This study demonstrates, for the first time, the accuracy and reliability of a microarray-based on-chip PCR genotyping assay for measuring allelic variants in a routine clinical setting.     

pIQPNNI: parallel reconstruction of large maximum likelihood phylogenies

SUMMARY: IQPNNI is a program to infer maximum-likelihood phylogenetic trees from DNA or protein data with a large number of sequences. We present an improved and MPI-parallel implementation showing very good scaling and speed-up behavior.     

A preliminary study of the metabolic stability of a series of benzoxazinone derivatives as potent neuropeptide Y5 antagonists

The metabolic stability of benzoxazinone derivatives, a potent series of NPY Y5 antagonists, has been investigated. This study resulted in the identification of the structural moieties prone to metabolic transformations and which strongly influenced the in vitro half-life. This provides opportunities to optimize the structure of this new class of NPY Y5 antagonists.     

Heterozygous deficiency of manganese superoxide dismutase results in severe lipid peroxidation and spontaneous apoptosis in murine myocardium in vivo

To circumvent the early lethality of manganese superoxide dismutase (SOD2)-deficient mice, we have used a skin-specific strategy with introduction of loxP sites flanking exon 3 of the SOD2 gene. To our surprise, when breeding a female keratin 14 Cre transgenic mouse to a SOD2 "floxed" male mouse, due to keratin 14 promoter-driven Cre expression in the oocytes, all offspring were heterozygous for SOD2. In sharp contrast to initial publications on SOD2(+/-) mice, the herein reported mice on a mixed genetic background (C57BL/6 x 129/Ola) in their heterozygous state (SOD(+/-)) revealed distinct ultrastructural damage of the myocard, with swelling and disruption of mitochondria and accumulation of lipid droplets, increased nitrotyrosine formation, and lipid peroxidation as well as activation of apoptosis signaling pathways in the heart in vivo. Strikingly, and so far unreported, we found a substantial decrease in the activity of the cytosolic copper, zinc superoxide dismutase (SOD1) in the heart tissue of SOD2(+/-) mice, suggesting that the breakdown of mitochondrial membranes in the heart of SOD2(+/-) mice results in the enhanced release of superoxide anion radicals or derivatives thereof with subsequent inactivation of cytosolic SOD1. This model may be particularly suited to long-term studies on age-related heart failure as well as other age-related diseases and the polygenic base of tissue-specific responses to oxidative injury.     

N-acetyl-D-galactosamine/N-acetyl-D-glucosamine--recognizing lectin from the snail Cepaea hortensis: purification, chemical characterization, cloning and expression in E. coli

From the albumin gland of the snail Cepaea hortensis we isolated and characterized a new N-acetyl-D-galactosamine/N-acetyl-D-glucosamine (GalNAc/GlcNAc) specific lectin (CHA-II) which was purified by a combination of affinity chromatography on GalNAc-agarose and gel filtration. The purified native lectin was found to be a multimeric protein, as revealed by SDS-PAGE and MALDI-TOF analysis. In SDS-PAGE the denatured and reduced lectin showed two bands of molecular masses with 17 and 15.5 kDa which reacted equally with anti-CHA-II rabbit antiserum. The lectin was O- and N-glycosylated with [(Gal)2-Man]2-Man-GlcNAc-GlcNAc-Asn as a probable structure for the oligosaccharide. Isoelectric focusing revealed a heterogeneous protein of at least four bands around pH 8.7. Tryptic peptides of CHA-II were N-terminally sequenced and highly degenerated gene specific oligonucleotide primers (GSPs) had been constructed. Using total RNA isolated from albumin glands, cDNAs were produced by the running race technique. Specific PCR fragments were obtained by PCR using GSPs, the universal primer and 5'- or 3'-RACE-cDNAs. The amplified fragments were cloned into the vector pDrive and were sequenced. The resulting total cDNA sequence consisted of 496 base pairs including an open reading frame of 360 base pairs which encoded a protein of 120 amino acids. The protein carried a putative signal peptide. The mature protein was predicted to comprise 99 amino acid residues with a calculated molecular weight of 11,239 Da. The PCR fragment encoding the mature protein was cloned into the vector pQE30 and expressed in E. coli. Recombinant CHA-II lectin was produced as inclusion bodies and extracted by 6 M guanidine hydrochloride. After refolding, the recombinant CHA-II agglutinated specifically human red blood cells of groups A and AB. In immunodiffusion experiments using rabbit antiserum raised against the native lectin, the protein showed a precipitation line of identity with the native lectin.     

Geographic variation in diapause incidence, life-history traits, and climatic adaptation in Drosophila melanogaster

In Drosophila melanogaster, exposure of females to low temperature and shortened photoperiod can induce the expression of reproductive quiescence or diapause. Diapause expression is highly variable within and among natural populations and has significant effects on life-history profiles, including patterns of longevity, fecundity, and stress resistance. We hypothesized that if diapause expression is associated with overwintering mechanisms and adaptation to temperate environments, the frequency of diapause incidence would exhibit a latitudinal cline among natural populations. Because stress resistance and reproductive traits are also clinal in this species, we also examined how patterns of fecundity and longevity varied with geography and how stress resistance and associated traits differed constitutively between diapause and nondiapause lines. Diapause incidence was shown to vary predictably with latitude, ranging from 35% to 90% among natural populations in the eastern United States Survivorship under starvation stress differed between diapause and nondiapause lines; diapause phenotypes were also distinct for total body triglyceride content and the developmental distribution of oocytes in the ovary following stress exposure. Patterns of longevity, fecundity, and ovariole number also varied with geography. The data suggest that, for North American populations, diapause expression is functionally associated with overwintering mechanisms and may be an integral life-history component in natural populations.     

The cell-cell adhesion molecule EpCAM interacts directly with the tight junction protein claudin-7

We recently described that in the metastasizing rat pancreatic carcinoma line BSp73ASML the cell-cell adhesion molecule EpCAM, CD44 variant isoforms and the tetraspanins D6.1A and CD9 form a complex that is located in glycolipid-enriched membrane microdomains. This complex contains, in addition, an undefined 20 kDa protein. As such complex formation influenced cell-cell adhesion and apoptosis resistance, it became of interest to identify the 20 kDa polypeptide. This 20 kDa protein, which co-precipitated with EpCAM in BSp73ASML lysates, was identified as the tight junction protein claudin-7. Correspondingly, an association between EpCAM and claudin-7 was noted in rat and human tumors and in non-transformed tissues of the gastrointestinal tract. Co-localization of the two molecules was most pronounced at basolateral membranes, but was also observed in tight junctions. Evidence for direct protein-protein interactions between EpCAM and claudin-7 was obtained by co-immunoprecipitation after treatment of tumor cells with a membrane-permeable chemical cross-linker. The complex, which is located in glycolipid-enriched membrane microdomains, is not disrupted by partial cholesterol depletion, but claudin-7 phosphorylation is restricted to the localization in glycolipid-enriched membrane microdomains. This is the first report on an association between EpCAM and claudins in both non-transformed tissues and metastasizing tumor cell lines.     

Sequential analysis of surfactant,lung function and inflammation in cystic fibrosis patients

Background

In a cross-sectional analysis of cystic fibrosis (CF) patients with mild lung disease, reduced surfactant activity was correlated to increased neutrophilic airway inflammation, but not to lung function. So far, longitudinal measurements of surfactant function in CF patients are lacking and it remains unclear how these alterations relate to the progression of airway inflammation as well as decline in pulmonary function over time.

Methods

As part of the BEAT trial, a longitudinal study to assess the course of airway inflammation in CF, we studied lung function, surfactant function and endobronchial inflammation using bronchoalveolar lavage fluid from 20 CF patients with normal pulmonary function (median FEV₁ 94% of predicted) at three times over a three year period.

Results

There was a progressive loss of surfactant function, assessed as minimal surface tension. The decline in surfactant function was negatively correlated to an increase in neutrophilic inflammation and a decrease in lung function, assessed by FEV₁, MEF_75/25%VC, and MEF_25%VC. The concentrations of the surfactant specific proteins A, C and D did not change, whereas SP-B increased during this time period.

Conclusion

Our findings suggest a link between loss of surfactant function driven by progressive airway inflammation and loss of small airway function in CF patients with limited lung disease.