Polymorphisms in the human surfactant protein-D (SFTPD) gene: strong evidence that serum levels of surfactant protein-D (SP-D) are genetically influenced

The collectin surfactant protein-D (SP-D) plays a significant role in innate immunity. Epidemiological studies described associations between single nucleotide polymorphisms (SNPs) of the human gene coding surfactant protein-D (SFTPD) and infectious pulmonary diseases. Studies on twins indicated very strong genetic dependence for serum levels of SP-D. The aim of this study was to determine the genetic influence of sequence variations within the SFTPD gene on the constitutional serum SP-D levels. We sequenced the 5 untranslated region (5UTR), the coding region and the 3 region of the SFTPD gene of 32 randomly selected blood donors. Six validated SNPs were genotyped with sequence-specific probes (TaqMan 7000) in 290 German blood donors. Serum SP-D levels were analysed by ELISA, and the association of SFTPD haplotype estimates with the quantitative phenotype serum SP-D level was determined. One single SFTPD haplotype (allele frequency 13.53%) revealed a negative association with serum SP-D levels (P<0.0001). This was confirmed in a second prospectively collected group of blood donors (n=160, P=0.0034). The discovery of a frequent negative variant of the SFTPD gene provides a basis for genetic analysis of the function of SP-D in the resistance against pulmonary infections and inflammatory disorders in humans.     

Developmentally regulated alterations in Polycomb repressive complex 1 proteins on the inactive X chromosome

Polycomb group (PcG) proteins belonging to the polycomb (Pc) repressive complexes 1 and 2 (PRC1 and PRC2) maintain homeotic gene silencing. In Drosophila, PRC2 methylates histone H3 on lysine 27, and this epigenetic mark facilitates recruitment of PRC1. Mouse PRC2 (mPRC2) has been implicated in X inactivation, as mPRC2 proteins transiently accumulate on the inactive X chromosome (Xi) at the onset of X inactivation to methylate histone H3 lysine 27 (H3-K27). In this study, we demonstrate that mPRC1 proteins localize to the Xi, and that different mPRC1 proteins accumulate on the Xi during initiation and maintenance of X inactivation in embryonic cells. The Xi accumulation of mPRC1 proteins requires Xist RNA and is not solely regulated by the presence of H3-K27 methylation, as not all cells that exhibit this epigenetic mark on the Xi show Xi enrichment of mPRC1 proteins. Our results implicate mPRC1 in X inactivation and suggest that the regulated assembly of PcG protein complexes on the Xi contributes to this multistep process.     

Beta-defensin-2 expression is regulated by TLR signaling in intestinal epithelial cells

The intestinal epithelium serves as a barrier to the intestinal flora. In response to pathogens, intestinal epithelial cells (IEC) secrete proinflammatory cytokines. To aid in defense against bacteria, IEC also secrete antimicrobial peptides, termed defensins. The aim of our studies was to understand the role of TLR signaling in regulation of beta-defensin expression by IEC. The effect of LPS and peptidoglycan on beta-defensin-2 expression was examined in IEC lines constitutively or transgenically expressing TLRs. Regulation of beta-defensin-2 was assessed using promoter-reporter constructs of the human beta-defensin-2 gene. LPS and peptidoglycan stimulated beta-defensin-2 promoter activation in a TLR4- and TLR2-dependent manner, respectively. A mutation in the NF-kappaB or AP-1 site within the beta-defensin-2 promoter abrogated this response. In addition, inhibition of Jun kinase prevents up-regulation of beta-defensin-2 protein expression in response to LPS. IEC respond to pathogen-associated molecular patterns with expression of the antimicrobial peptide beta-defensin-2. This mechanism may protect the intestinal epithelium from pathogen invasion and from potential invaders among the commensal flora.     

The DrosDel collection: a set of P-element insertions for generating custom chromosomal aberrations in Drosophila melanogaster

We describe a collection of P-element insertions that have considerable utility for generating custom chromosomal aberrations in Drosophila melanogaster. We have mobilized a pair of engineered P elements, p[RS3] and p[RS5], to collect 3243 lines unambiguously mapped to the Drosophila genome sequence. The collection contains, on average, an element every 35 kb. We demonstrate the utility of the collection for generating custom chromosomal deletions that have their end points mapped, with base-pair resolution, to the genome sequence. The collection was generated in an isogenic strain, thus affording a uniform background for screens where sensitivity to genetic background is high. The entire collection, along with a computational and genetic toolbox for designing and generating custom deletions, is publicly available. Using the collection it is theoretically possible to generate >12,000 deletions between 1 bp and 1 Mb in size by simple eye color selection. In addition, a further 37,000 deletions, selectable by molecular screening, may be generated. We are now using the collection to generate a second-generation deficiency kit that is precisely mapped to the genome sequence.     

Melanocortin-4 receptor gene variant I103 is negatively associated with obesity

Several rare mutations in the melanocortin-4 receptor gene (MC4R) predispose to obesity. For the most common missense variant V103I (rs2229616), however, the previously reported similar carrier frequencies in obese and nonobese individuals are in line with in vitro studies, which have not shown a functional implication of this variant. In the present study, we initially performed a transmission/disequilibrium test on 520 trios with obesity, and we observed a lower transmission rate of the I103 allele (P=.017), which was an unexpected finding. Therefore, we initiated two large case-control studies (N=2,334 and N=661) and combined the data with those from 12 published studies, for a total of 7,713 individuals. The resulting meta-analysis provides evidence for a negative association of the I103 allele with obesity (odds ratio 0.69; 95% confidence interval 0.50-0.96; P=.03), mainly comprising samples of European origin. Additional screening of four other ethnic groups showed comparable I103 carrier frequencies well below 10%. Genomic sequencing of the MC4R gene revealed three polymorphisms in the noncoding region that displayed strong linkage disequilibrium with V103I. In our functional in vitro assays, the variant was indistinguishable from the wild-type allele, as was the result in previous studies. This report on an SNP/haplotype that is negatively associated with obesity expands the successful application of meta-analysis of modest effects in common diseases to a variant with a carrier frequency well below 10%. The respective protective effect against obesity implies that variation in the MC4R gene entails both loss and gain of function.     

Evaluation of methods for storage of marine macroorganisms with optimal recovery of bacteria

Marine macroorganisms are a potential source for new bioactive substances. In many cases marine microorganisms--especially bacteria--associated with these macroorganisms are actually producing the bioactive substances. One often is not able to immediately isolate microorganisms from collected macroorganismic materials; we therefore evaluated different methods for storage of such material, e.g., on board research vessels. These methods were the following: storage of macerates in sintered glass beads and 5% trehalose at -20 degrees C (SGT method); storage of sections in 5% dimethyl sulfoxide at -70 degrees C (SD method); storage of macerates at -20 degrees C using the commercial ROTI-STORE system (RS method); storage of macerates at -20 degrees C in 50% glycerol (GC method); and storage of macerates covered by mineral oil at 4 degrees C (MO method). The SGT and SD methods resulted in numbers of and especially diversity of recoverable bacteria that were higher than for the other methods. Data for the RS method indicated its potential usefulness, too. The MO method resulted in growth during storage, thereby enriching a few selected microorganisms; the GC method resulted in a survival and diversity of recovered bacteria that was too low.     

AtToc90, a New GTP-Binding Component of the Arabidopsis Chloroplast Protein Import Machinery

AtToc159 is a GTP-binding chloroplast protein import receptor. In vivo, atToc159 is required for massive accumulation of photosynthetic proteins during chloroplast biogenesis. Yet, in mutants lacking atToc159 photosynthetic proteins still accumulate, but at strongly reduced levels whereas non-photosynthetic proteins are imported normally: This suggests a role for the homologues of atToc159 (atToc132, -120 and -90). Here, we show that atToc90 supports accumulation of photosynthetic proteins in plastids, but is not required for import of several constitutive proteins. Part of atToc90 associates with the chloroplast surface in vivo and with the Toc-complex core components (atToc75 and atToc33) in vitro suggesting a function in chloroplast protein import similar to that of atToc159. As both proteins specifically contribute to the accumulation of photosynthetic proteins in chloroplasts they may be components of the same import pathway.     

Scaffolding by ERK3 regulates MK5 in development

Extracellular-regulated kinase 3 (ERK3, MAPK6) is an atypical member of the ERKs, lacking the threonine and tyrosine residues in the activation loop, carrying a unique C-terminal extension and being mainly regulated by its own protein stability and/or by autophosphorylation. Here we show that ERK3 specifically interacts with the MAPK-activated protein kinase 5 (MK5 or PRAK) in vitro and in vivo. Expression of ERK3 in mammalian cells leads to nuclear-cytoplasmic translocation and activation of MK5 and to phosphorylation of both ERK3 and MK5. Remarkably, activation of MK5 is independent of ERK3 enzymatic activity, but depends on its own catalytic activity as well as on a region in the C-terminal extension of ERK3. In mouse embryonic development, mRNA expression patterns of ERK3 and MK5 suggest spatiotemporal coexpression of both kinases. Deletion of MK5 leads to strong reduction of ERK3 protein levels and embryonic lethality at about stage E11, where ERK3 expression in wild-type mice is maximum, indicating a role of this signalling module in development.