Overexpression of eCLCA1 in small airways of horses with recurrent airway obstruction.

The human hCLCA1 and murine mCLCA3 (chloride channels, calcium-activated) have recently been identified as promising therapeutic targets in asthma. Recurrent airway obstruction in horses is an important animal model of human asthma. Here, we have cloned and characterized the first equine CLCA family member, eCLCA1. The 913 amino acids eCLCA1 polypeptide forms a 120-kDa transmembrane glycoprotein that is processed to an 80-kDa protein in vivo. Three single nucleotide polymorphisms were detected in the eCLCA1 coding region in 14 horses, resulting in two amino acid changes (485H/R and 490V/L). However, no functional differences were recorded between the channel properties of the two variants in transfected HEK293 cells. The eCLCA1 protein was detected immunohistochemically in mucin-producing cells in the respiratory and intestinal tracts, cutaneous sweat glands, and renal mucous glands. Strong overexpression of eCLCA1 was observed in the airways of horses with recurrent airway obstruction using Northern blot hybridization, Western blotting, immunohistochemistry, and real-time quantitative RT-PCR. The results suggest that spontaneous or experimental recurrent airway obstruction in horses may serve as a model to study the role of CLCA homologs in chronic airway disease with overproduction of mucins.     

Fusiogenic endogenous-retroviral syncytin-1 exerts anti-apoptotic functions in staurosporine-challenged CHO cells

Fusiogenic glycoprotein syncytin-1, expressed in human placenta, is a promising candidate for acquiring a basic knowledge of placental syncytialization. However, its cellular mode of action is unidentified. We investigated whether syncytin-1 may exert influence on apoptotic processes. Therefore, we incubated CHO cells after stable transfection with syncytin-1 (CHO-52) in the presence or absence of staurosporine (STS), a kinase inhibitor well characterized to induce apoptosis. When testing the phenotype of CHO-52 cells, we could demonstrate that the induction of apoptosis by STS was delayed over a period of up to 24 h. Furthermore, the cell death rate was decreased by approx 75% following transfection of syncytin-1 in CHO-52 compared to mock-treated cells. In detail, after 18h of incubation with 500 nM STS, 64 ± 2% of CHO-52 cells were viable compared to 16 ± 1% of CHO-mocks, after 24 h 43 ± 3% vs 5 ± 2%, respectively. CHO-52 cells exhibited a lower expression of active caspase 3 and anti-apoptotic Bcl-2 was found to be increased in CHO-52 cells at baseline and following STS treatment. Our study provides first evidence that syncytin-1 serves anti-apoptotic function under certain conditions. A lessened activation of caspase 3 and an increased expression of Bcl-2 are possible mechanisms.     

Light scattering from collagen fiber networks: micro-optical properties of normal and neoplastic stroma

Development of epithelial precancer and cancer leads to well-documented molecular and structural changes in the epithelium. Recently, it has been recognized that stromal biology is also altered significantly with preinvasive disease. We used the finite-difference time-domain method, a popular technique in computational electromagnetics, to model light scattering from heterogeneous collagen fiber networks and to analyze how neoplastic changes alter stromal scattering properties. Three-dimensional optical images from the stroma of fresh normal and neoplastic oral-cavity biopsies were acquired using fluorescence confocal microscopy. These optical sections were then processed to create realistic three-dimensional collagen networks as model input. Image analysis revealed that the volume fraction of collagen fibers in the stroma decreases with precancer and cancer progression, and fibers tend to be shorter and more disconnected in neoplastic stroma. The finite-difference time-domain modeling results showed that neoplastic fiber networks have smaller scattering cross sections compared to normal networks. Computed scattering-phase functions indicate that high-angle scattering probabilities tend to be higher for neoplastic networks. These results provide valuable insight into the micro-optical properties of normal and neoplastic stroma. Characterization of optical signals obtained from epithelial tissues can aid in development of optical spectroscopic and imaging techniques for noninvasive monitoring of early neoplastic changes.     

Elevated Levels of Plasma Phenylalanine in Schizophrenia: A Guanosine Triphosphate Cyclohydrolase-1 Metabolic Pathway Abnormality?

Background

Phenylalanine and tyrosine are precursor amino acids required for the synthesis of dopamine, the main neurotransmitter implicated in the neurobiology of schizophrenia. Inflammation, increasingly implicated in schizophrenia, can impair the function of the enzyme Phenylalanine hydroxylase (PAH; which catalyzes the conversion of phenylalanine to tyrosine) and thus lead to elevated phenylalanine levels and reduced tyrosine levels. This study aimed to compare phenylalanine, tyrosine, and their ratio (a proxy for PAH function) in a relatively large sample of schizophrenia patients and healthy controls.

Methods

We measured non-fasting plasma phenylalanine and tyrosine in 950 schizophrenia patients and 1000 healthy controls. We carried out multivariate analyses to compare log transformed phenylalanine, tyrosine, and phenylalanine:tyrosine ratio between patients and controls.

Results

Compared to controls, schizophrenia patients had higher phenylalanine (p<0.0001) and phenylalanine: tyrosine ratio (p<0.0001) but tyrosine did not differ between the two groups (p = 0.596).

Conclusions

Elevated phenylalanine and phenylalanine:tyrosine ratio in the blood of schizophrenia patients have to be replicated in longitudinal studies. The results may relate to an abnormal PAH function in schizophrenia that could become a target for novel preventative and interventional approaches.     

Lack of structure in the gene pool of the highly polyploid ornamental chrysanthemum

Selection of clonally propagated chrysanthemums is mostly performed on F1 hybrids using phenotypic characteristics without the use of molecular information. We applied 448 amplified fragment length polymorphism markers to a set of 81 accessions, mainly from the European gene pool, covering the different horticultural types (cut, pot and garden varieties) and originating from the most important European chrysanthemum breeders. The average pairwise genetic similarity of 0.69 was moderate to rather high. Neighbour-joining clustering resulted in no grouping of the accessions, either by their common origin or their horticultural type, or by similarities in important phenotypic characteristics. The structure of the dendrogram could not be supported by bootstrap analysis. Furthermore, network analysis using SplitsTree, principal coordinate analysis via DARwin or analysis of the population with structure did not differentiate reliable and invariable clusters. Therefore, we tested the marker saturation by plotting the mean coefficient of variation for every pairwise similarity of the bootstrap analysis against the different numbers of markers. We showed that the number of markers is sufficient for a precise estimate of genetic similarity and that the lack of bootstrap support is not due to a low genetic diversity or a lack of marker information, but most likely resulted from the breeding history of the cultivars, involving repeated backcrosses and the exchange of genotypes between breeders.     

Metal binding dictates conformation and function of the amyloid precursor protein (APP) E2 domain

The amyloid precursor protein (APP) and its neurotoxic cleavage product Aβ are key players in the development of Alzheimer's disease and appear essential for neuronal development and cell homeostasis in mammals. Proteolytic processing of APP is influenced by metal ions, protein ligands and its oligomerization state. However, the structural basis and functional mechanism of APP regulation are hitherto largely unknown. Here we identified a metal-dependent molecular switch located within the E2 domain of APP containing four evolutionary highly conserved histidine residues. Three X-ray structures of the metal-bound molecule were solved at 2.6–2.0 Å resolution. Using protein crystallographic and biochemical methods, we characterized this novel high-affinity binding site within the E2 domain that binds competitively to copper and zinc at physiological concentrations. Metal-specific coordination spheres induce large conformational changes and enforce distinct structural states, most likely regulating the physiological function of APP and its processing in Alzheimer's disease.     

Chromosomal assignment of the human smg GDP dissociation stimulator gene to human chromosome 4q21-q25

The 3-end of the cDNA encoding the smg GDP dissociation stimulator (smg GDS) protein shares 100% homology with the previously published expressed sequence tag 00038 site. This site extends the 3-end of the smg GDS gene by 212 bp. It has been localized to human chromosome 4. Here, we have refined the localization of smg GDP to human chromosome 4q21-q25 using a mapping panel of rodent/human somatic cell hybrids containing different parts of chromosome 4. This chromosomal localization of smg GDP to 4q21-25 overlaps with a region of allele loss in primary hepatocellular carcinoma (4q13-q26).HGM symbol: RAP1GDS1     

Efficient shRNA delivery into B and T lymphoma cells using lentiviral vector-mediated transfer

RNA interference is a powerful tool for the functional analysis of proteins by specific gene knockdown. In this study, we devised a rapid and efficient way to screen suitable siRNA sequences and subsequently employ them for specific gene knockdown in usually hard-to-transfect lymphoid cell lines, using a self-inactivating lentiviral vector. Two proteins with different half-lives were chosen, cyclin D1 and STAT3. A specific lacZ reporter fusion assay was used to identify highly effective siRNA sequences. Only siRNA molecules with more than 85% of knockdown efficiency were selected for the generation of lentiviral transfer vectors. Transduction rates of 75–99% were achieved in the lymphoma cell lines Granta 519 (mantle cell lymphoma), Karpas 299, and SUDHL-1 (anaplastic large T cell lymphoma), as demonstrated by green fluorescent protein expression in fluorescence-activated cell sorting analysis. The high level of transduction efficiency allows RNA interference studies to be performed on transduced cells without further manipulation, such as cell sorting or cloning. The LacZ reporter system together with the lentivirus technology is a very important tool in the hematology field, which enables experiments in lymphoid cells that were not possible before.

Electronic supplementary material

The online version of this article (doi:10.1007/s12308-008-0020-x) contains supplementary material, which is available to authorized users.     

Short-term progressive early diagenesis in density bands of recent corals:Porites Colonies,Mauritius Island,Indian Ocean

Summary The growth history of some recentPorites colonies of Mauritius Island (Indian Ocean) was dated by sclerochronological methods. Couples of high-density and low-density bands represent the annual growth rate of the corals and allow the growth pattern of every year in the corallum to be counted. The growth and structure of the skeletons ofPorites solida andPorites lutea were investigated. Older parts of the aragonitic skeleton in these 10 to 20 year old corals show various secondary microstructures resulting from alterations and thickenings of the elements of the skeleton. The primary needle-like aragonite crystals are absent in older parts of the corallum and blocky aragonitic cements can occur. Pores and primary skeletal elements are overgrown by new microstructures. These microstructures are caused by secondary cementation and exhibit frontal zones (Stirnzonen), zigzag-like and pseudolamellar-structures. The lamellar structures can be compared with similar structures in the exoskeleton of some Rugosa. A very short early diagenesis within the recent corals is responsible for the thickening and alteration of skeletal elements. It occurs only 4 to 5 years after the formation of the skeleton and tends to increase in importance in older parts of the corallum. Nevertheless, there is no proof for any alteration of aragonite to calcite.