Haplotype-matched controls as a tool to discriminate polymorphisms from pathogenic mutations in mtDNA

We have studied the pathogenic role of 10044A-->G, a heteroplasmic mitochondrial DNA (mtDNA) mutation that has been suggested to be pathogenic in one family with severe pediatric morbidity. We found the mutation at an average frequency of 1.9% among 259 individuals including healthy controls. The mutation appeared to be heteroplasmic by restriction fragment analysis but analysis of subcloned polymerase chain reaction fragments confirmed homoplasmy. The polymorphic nature of 10044A-->G was verified by demonstrating exclusive association with a rare mtDNA haplotype within haplogroup H. We suggest that the evaluation of putatively pathogenic mutations in mtDNA should include the analysis of a sufficient number of haplotype-matched control samples and that the heteroplasmy should be verified by cloning.     

Finding local structural similarities among families of unrelated protein structures: a generic non-linear alignment algorithm

We have developed a generic tool for the automatic identification of regions of local structural similarity in unrelated proteins having different folds, as well as for defining more global similarities that result from homologous protein structures. The computer program GENFIT has evolved from the genetic algorithm-based three-dimensional protein structure comparison program GA_FIT. GENFIT, however, can locate and superimpose regions of local structural homology regardless of their position in a pair of structures, the fold topology, or the chain direction. Furthermore, it is possible to restrict the search to a volume centered about a region of interest (e.g., catalytic site, ligand-binding site) in two protein structures. We present a number of examples to illustrate the function of the program, which is a parallel processing implementation designed for distribution to multiple machines over a local network or to run on a single multiprocessor computer.     

Sand goby (Pomatoschistus minutus) males exposed to an endocrine disrupting chemical fail in nest and mate competition

Endocrine disrupting chemicals (EDCs) are a widely studied group of chemicals that interfere with the endocrinology of organisms. So far, few studies have demonstrated the effect of EDCs on the reproductive behavior of aquatic wildlife. Here we show that sand goby males' (Pomatoschistus minutus) success in mating competition greatly decreases after an exposure for 7 to 24 days to 17α-ethinyl estradiol (EE2, measured concentration 4 ng L^− 1). The sand goby exhibits a polygynous mating system with male parental care, in which males compete for nest sites and females. The aim of this study was to test how EE2 exposure affects the ability of males to compete for breeding resources, i.e. nest sites and mates. First, EE2 exposed males competed over a nest site against a non-exposed, control male of the same size. Secondly, we examined male courtship behavior and female mate preferences for EE2 exposed males and similar-sized non-exposed, control males. In addition to the behavioral experiments we determined the zona radiata protein (Zrp) mRNA gene expression and measured morphometric indicators of sexual maturation. Our study revealed that EE2 treated males were not able to acquire or defend a nest site. Additionally, EE2 treated males spent significantly less time in active courtship and nest leading behavior than control males. As a result, females clearly preferred to mate with control males. However, we found no significant differences in Zrp mRNA expression or the morphometric indicators between treatments. Our study illustrates that exposure to this EDC can greatly reduce the chances of an individual reproducing successfully. Moreover, it demonstrates that severe behavioral effects can be seen before any effects are detectable at the molecular or morphometric level.     

Determination of endocannabinoids in nematodes and human brain tissue by liquid chromatography electrospray ionization tandem mass spectrometry

A simple and highly sensitive liquid chromatography/tandem mass spectrometric (LC/MS/MS) method was developed to compare endogenous cannabinoid levels in nematodes and in brains of rats and humans, with and without prior exposure to ethanol. After liquid-liquid extraction of the lipid fraction from homogenized samples, a reversed-phase sub 2 μm column was used for separating analytes with an isocratic mobile phase. Deuterated internal standards were used in the analysis, and detection was made by triple quadrupole mass spectrometer with multiple reaction monitoring (MRM). Ionization was performed with positive electrospray ionization (ESI). The nematode Caenorhabditis elegans fat-3 mutant, that lacks the necessary enzyme to produce arachidonic acid, the biologic precursor to 2-arachidonoyl glycerol and anandamide, was used as an analyte-free surrogate material for selectivity and calibration studies. The matrix effect was further investigated by in-source multiple reaction monitoring (IS-MRM) and standard addition studies. Selectivity studies demonstrated that the method was free from matrix effects. Good accuracy and precision were obtained for concentrations within the calibration range of 0.4-70 nM and 40-11,000 nM for monitored N-acylethanolamides (NAEs) and acyl glycerols, respectively.     

Toll-like receptor 9 ligands enhance mesenchymal stem cell invasion and expression of matrix metalloprotease-13

Human mesenchymal stem cells (hMSCs) are multipotent cells that are found in the bone marrow. Inflammation and tissue damage mobilize MSCs and induce their migration towards the damaged site through mechanisms that are not well defined. Toll-like receptor-9 (TLR9) is a cellular receptor for microbial and vertebrate DNA. Stimulation of TLR9 induces inflammatory and invasive responses in TLR9-expressing cells. We studied here the expression of TLR9 in human MSCs and the effects of synthetic TLR9-agonists on their invasion. Constitutive expression of TLR9 was detected in human MSCs but the expression was suppressed when MSCs were induced to differentiate into osteoblasts. Using standard invasion assays and a novel organotypic culture model based on human myoma tissue, we discovered that stimulation with the TLR9 agonistic, CpG oligonucleotides increased the invasion capacity of undifferentiated MSCs. Simultaneously, an increase in MMP-13 synthesis and activity was detected in the CpG-activated MSCs. Addition of anti-MMP-13 antibody significantly diminished the CpG-induced hMSC invasion. We conclude that treatment with TLR9-ligands increases MSC invasiveness, and this process is at least partially MMP-13-mediated.     

Technical knockout, a Drosophila model of mitochondrial deafness

Mutations in mtDNA-encoded components of the mitochondrial translational apparatus are associated with diverse pathological states in humans, notably sensorineural deafness. To develop animal models of such disorders, we have manipulated the nuclear gene for mitochondrial ribosomal protein S12 in Drosophila (technical knockout, tko). The prototypic mutant tko(25t) exhibits developmental delay, bang sensitivity, impaired male courtship, and defective response to sound. On the basis of a transgenic reversion test, these phenotypes are attributable to a single substitution (L85H) at a conserved residue of the tko protein. The mutant is hypersensitive to doxycyclin, an antibiotic that selectively inhibits mitochondrial protein synthesis, and mutant larvae have greatly diminished activities of mitochondrial redox enzymes and decreased levels of mitochondrial small-subunit rRNA. A second mutation in the tko gene, Q116K, which is predicted to impair the accuracy of mitochondrial translation, results in the completely different phenotype of recessive female sterility, based on three independent transgenic insertions. We infer that the tko(25t) mutant provides a model of mitochondrial hearing impairment resulting from a quantitative deficiency of mitochondrial translational capacity.     

Increased variation in mtDNA in patients with familial sensorineural hearing impairment

Analyses of mitochondrial DNA (mtDNA) sequences have revealed non-neutral patterns, suggesting that many amino acid mutations in animal mtDNA may be mildly deleterious, but this has not been verified in human clinical series. Since sensorineural hearing impairment (SNHI) is a common manifestation in many of the syndromes caused by mutations in mtDNA, this may be regarded as the phenotype of choice in attempts to detect mutations that may have a mildly deleterious effect on mitochondrial function. We selected 32 subjects from among 117 unrelated SNHI patients with SNHI in maternal relatives by means of family history, determined the entire coding region sequence of mtDNA and compared the sequence variation with that in 32 haplogroup-matched controls taken at random from 192 Finnish sequences. The 32 control sequences differed from the remaining 160 sequences by 36±9 substitutions (mean ± SD), while the difference for the 32 patients was 58±4 substitutions (P=0.005 for difference; Wilcoxon signed rank test). Differences were also found in the number of new haplotypes and new non-synonymous mutations or mutations in tRNA or rRNA genes. A total of 12 rare mtDNA variants were detected in the patients, and only 3 of these were considered to be neutral in effect. It is proposed that increased sequence variation in mtDNA may be a genetic risk factor for SNHI, and the increased frequency of rare haplotypes in these patients points to the presence of mildly deleterious mutations in mtDNA.     

Candidate genes for colour and vision exhibit signals of selection across the pied flycatcher (Ficedula hypoleuca) breeding range

The role of natural selection in shaping adaptive trait differentiation in natural populations has long been recognized. Determining its molecular basis, however, remains a challenge. Here, we search for signals of selection in candidate genes for colour and its perception in a passerine bird. Pied flycatcher plumage varies geographically in both its structural and pigment-based properties. Both characteristics appear to be shaped by selection. A single-locus outlier test revealed 2 of 14 loci to show significantly elevated signals of divergence. The first of these, the follistatin gene, is expressed in the developing feather bud and is found in pathways with genes that determine the structure of feathers and may thus be important in generating variation in structural colouration. The second is a gene potentially underlying the ability to detect this variation: SWS1 opsin. These two loci were most differentiated in two Spanish pied flycatcher populations, which are also among the populations that have the highest UV reflectance. The follistatin and SWS1 opsin genes thus provide strong candidates for future investigations on the molecular basis of adaptively significant traits and their co-evolution.     

CD2-associated protein is widely expressed and differentially regulated during embryonic development

CD2-associated protein (CD2AP) is an adapter protein that is involved in various signaling and vesicular trafficking processes and also functions as a linker between plasma membrane proteins and the actin cytoskeleton. The protein is known to have important functions in T cells and glomerular podocytes, but it is also expressed by many other adult-type tissues and cells. Here we analyzed the expression of the protein during early embryonic development and organogenesis of the mouse. The results showed differential tissue-specific regulation of CD2AP in developing and maturing organs. In oocytes and pre-implantation embryos, CD2AP was located diffusely in the cytoplasm, whereas in late blastocysts it was concentrated to the intercellular contacts. During organogenesis, CD2AP was distinctly upregulated upon, e.g., the pretubular aggregation of metanephric mesenchyme cells and the appearance of the osteoblastic rim around cartilages during endochondral ossification. High CD2AP expression was also observed during epithelial-like conversion of some highly specialized secretory cell types such as the odontoblasts, the cells of the choroid plexus and the decidualized cells of the endometrial stroma. In other instances, such as the development of the proximal tubuli of the kidney and the flat alveolar epithelium of the lung, the protein was downregulated upon differentiation and maturation of the cells. Finally, certain cells, e.g., glomerular podocytes, those forming the collecting ducts of the kidney, and the urothelium of the kidney pelvis, expressed CD2AP throughout their differentiation and maturation. Multiple molecules and complex pathways regulate embryogenesis, and scaffolding proteins apparently have pivotal roles in targeting and finetuning, e.g., growth factor- or hormone-induced processes. The cell-type specific spatio-temporal regulation of CD2AP during development suggests that this adapter protein is a key regulatory partner in many signaling pathways and cellular processes governing differentiation and morphogenesis.