Identification of new human cadherin genes using a combination of protein motif search and gene finding methods

We have combined protein motif search and gene finding methods to identify genes encoding proteins containing specific domains. Particularly, we have focused on finding new human genes of the cadherin superfamily proteins, which represent a major group of cell-cell adhesion receptors contributing to embryonic neuronal morphogenesis. Models for three cadherin protein motifs were generated from over 100 already annotated cadherin domains and used to search the complete translated human genome. The genomic sequence regions containing motif "hits" were analyzed by eukaryotic GeneMark.hmm to identify the exon-intron structure of new genes. Three new genes CDH-J, PCDH-J and FAT-J were found. The predicted proteins PCDH-J and FAT-J were classified into protocadherin and FAT-like subfamilies, respectively, based on the number and organization of cadherin domains and presence of subfamily-specific conserved amino acid residues. Expression of FAT-J was shown in almost all tested tissues. The exon-intron organization of CDH-J was experimentally verified by PCR with specifically designed primers and its tissue-specific expression was demonstrated. The described methodology can be applied to discover new genes encoding proteins from families with well-characterized structural and functional domains.     

Structural differences among hemagglutinins of influenza A virus subtypes are reflected in their antigenic architecture: analysis of H9 escape mutants

We used a panel of monoclonal antibodies to H9 hemagglutinin to select 18 escape mutants of mouse-adapted influenza A/Swine/Hong Kong/9/98 (H9N2) virus. Cross-reactions of the mutants with the antibodies and the sequencing of hemagglutinin genes revealed two minimally overlapping epitopes. We mapped the amino acid changes to two areas of the recently reported three-dimensional structure of A/Swine/Hong Kong/9/98 hemagglutinin. The grouping of the antigenically relevant amino acid positions in H9 hemagglutinin differs from the pattern observed in H3 and H5 hemagglutinins. Several positions in site B of H3 hemagglutinin are distributed in two sites of H9 hemagglutinin. Unlike any subtype analyzed so far, H9 hemagglutinin does not contain an antigenic site corresponding to site A in H3 hemagglutinin. Positions 145 and 193 (H3 numbering), which in H3 hemagglutinin belong to sites A and B, respectively, are within one site in H9 hemagglutinin. This finding is consistent with the peculiarity of the three-dimensional structure of the H9 molecule, that is, the absence from H9 hemagglutinin of the lateral loop that forms site A in H3 and the equivalent site in H5 hemagglutinins. The escape mutants analyzed displayed phenotypic variations, including decreased virulence for mice and changes in affinity for sialyl substrates. Our results demonstrate a correlation between intersubtype differences in three-dimensional structure and variations among subtypes in the distribution of antigenic areas. Our findings also suggest that covariation and pleiotropic effects of antibody-selected mutations may be important in the evolution of H9 influenza virus, a possible causative agent of a future pandemic.     

Making noise: Emergent stochasticity in collective motion

Individual-based models of self-propelled particles (SPPs) are a popular and promising approach to explain features of the collective motion of animal aggregations. Many models that capture some features of group motion have been suggested but a common framework has yet to emerge. Key to all of these models is the inclusion of “noise” or stochastic errors in the individual behaviour of the SPPs. Here, we present a fully stochastic SPP model in one dimension that demonstrates a new way of introducing noise into SPP models whilst preserving emergent behaviours of previous models such as coherent groups and spontaneous direction switching. This purely individual-to-individual, local model is related to previous models in the literature and can easily be extended to higher dimensions. Its coarse-grained behaviour qualitatively reproduces recently reported locust movement data. We suggest that our approach offers an alternative to current reasoning about model construction and has the potential to offer mechanistic explanations for emergent properties of animal groups in nature.     

Structural Requirements of the Photoreceptor Phosphodiesterase ��-Subunit for Inhibition of Rod PDE6 Holoenzyme and for Its Activation by Transducin

The central enzyme of the visual transduction cascade, cGMP phosphodiesterase (PDE6), is regulated by its γ-subunit (Pγ), whose inhibitory constraint is released upon binding of activated transducin. It is generally believed that the last four or five C-terminal amino acid residues of Pγ are responsible for blocking catalysis. In this paper, we showed that the last 10 C-terminal residues (Pγ78–87) are the minimum required to completely block catalysis. The kinetic mechanism of inhibition by the Pγ C terminus depends on which substrate is undergoing catalysis. We also discovered a second mechanism of Pγ inhibition that does not require this C-terminal region and that is capable of inhibiting up to 80% of the maximal cGMP hydrolytic rate. Furthermore, amino acids 63–70 and/or the intact α2 helix of Pγ stabilize binding of C-terminal Pγ peptides by 100-fold. When PDE6 catalytic subunits were reconstituted with portions of the Pγ molecule and tested for activation by transducin, we found that the C-terminal region (Pγ63–87) by itself could not be displaced but that transducin could relieve inhibition of certain Pγ truncation mutants. Our results are consistent with two distinct mechanisms of Pγ inhibition of PDE6. One involves direct interaction of the C-terminal residues with the catalytic site. A second regulatory mechanism may involve binding of other regions of Pγ to the catalytic domain, thereby allosterically reducing the catalytic rate. Transducin activation of PDE6 appears to require interaction with both the C terminus and other regions of Pγ to effectively relieve its inhibitory constraint.     

Tissue and Cellular Expression Patterns of Porcine CFTR: Similarities to and Differences From Human CFTR

Emerging porcine models of cystic fibrosis (CF) are expected to mimic the human disease more closely than current mouse models do. However, little is known of the tissue and cellular expression patterns of the porcine CF transmembrane conductance regulator (pCFTR) and possible differences from human CFTR (hCFTR). Here, the expression pattern of pCFTR was systematically established on the mRNA and protein levels. Using specific anti-pCFTR antibodies, the majority of the protein was immunohistochemically detected on paraffin-embedded sections and on cryostate sections in the apical cytosol of intestinal crypt epithelial cells, nasal, tracheal, and bronchial epithelial cells, and other select, mostly glandular epithelial cells. Confocal laser scanning microscopy with co-localization of the Golgi marker 58K localized the protein in the cytosol between the Golgi apparatus and the apical cell membrane with occasional punctate or diffuse staining of the apical membrane. The tissue and cellular distribution patterns were confirmed by RT-PCR from whole tissue lysates or select cells after laser capture microdissection. Thus, expression of pCFTR was found to largely resemble that of hCFTR except for the kidney, brain, and cutaneous glands, which lack expression in pigs. Species-specific differences between pCFTR and hCFTR may become relevant for future interpretations of the CF phenotype in pig models. (J Histochem Cytochem 58:785–797, 2010)     

Quantifying and imaging NY-ESO-1/LAGE-1-derived epitopes on tumor cells using high affinity T cell receptors

Presentation of intracellular tumor-associated Ags (TAAs) in the context of HLA class I molecules offers unique cancer-specific cell surface markers for the identification and targeting of tumor cells. For most peptide Ags, the levels of and variations in cell surface presentation remain unknown, yet these parameters are of crucial importance when considering specific TAAs as targets for anticancer therapy. Here we use a soluble TCR with picomolar affinity for the HLA-A2-restricted 157-165 epitope of the NY-ESO-1 and LAGE-1 TAAs to investigate presentation of this immunodominant epitope on the surface of a variety of cancer cells. By single molecule fluorescence microscopy, we directly visualize HLA-peptide presentation for the first time, demonstrating that NY-ESO-1/LAGE-1-positive tumor cells present 10-50 NY-ESO-1/LAGE-1(157-165) epitopes per cell.     

A cold inducible multidomain cystatin from winter wheat inhibits growth of the snow mold fungus, Microdochium nivale

A novel cold-induced cystatin cDNA clone (TaMDC1) was isolated from cold acclimated winter wheat crown tissue by using a macroarray-based differential screening method. The deduced amino acid sequence consisted of a putative N-terminal secretory signal peptide of 37 amino acids and a mature protein (mTaMDC1) with a molecular mass of 23 kDa. The mTaMDC1 had a highly conserved N-terminal cystatin domain and a long C-terminal extension containing a second region, which exhibited partial similarity to the cystatin domain. The recombinant mTaMDC1 was purified from Escherichia coli and its cysteine proteinase inhibitory activity against papain was analyzed. The calculated Ki value of 5.8×10⁻⁷ M is comparable to those reported for other phytocystatins. Northern and western blot analyses showed elevated expression of TaMDC1 mRNA and protein during cold acclimation of wheat. In addition to cold, accumulation of the TaMDC1 message was induced by other abiotic stresses including drought, salt and ABA treatment. Investigation of in vitro antifungal activity of mTaMDC1 showed strong inhibition on the mycelium growth of the snow mold fungus Microdochium nivale. Hyphae growth was totally inhibited in the presence of 50 μg/ml mTaMDC1 and morphological changes such as swelling, fragmentation and sporulation of the fungus were observed. The mechanisms of the in vitro antifungal effects and the possible involvement of TaMDC1 in cold induced snow mold resistance of winter wheat are discussed.     

Cosecretion of protease inhibitor stabilizes antibodies produced by plant roots

A plant-based system for continuous production of monoclonal antibodies based on the secretion of immunoglobulin complexes from plant roots into a hydroponic medium (rhizosecretion) was engineered to produce high levels of single-chain and full-size immunoglobulins. Replacing the original signal peptides of monoclonal antibodies with a plant-derived calreticulin signal increased the levels of antibody yield 2-fold. Cosecretion of Bowman-Birk Ser protease inhibitor reduced degradation of the immunoglobulin complexes in the default secretion pathway and further increased antibody production to 36.4 microg/g root dry weight per day for single-chain IgG1 and 21.8 microg/g root dry weight per day for full-size IgG4 antibodies. These results suggest that constitutive cosecretion of a protease inhibitor combined with the use of the plant signal peptide and the antibiotic marker-free transformation system offers a novel strategy to achieve high yields of complex therapeutic proteins secreted from plant roots.     

Childhood thyroid cancer, radiation dose from Chernobyl, and dose uncertainties in Bryansk Oblast, Russia: a population-based case-control study

A population-based case-control study was conducted to estimate the radiation-related risk of thyroid cancer in persons who were exposed in childhood to (131)I from the Chernobyl accident of April 26, 1986 and to investigate the impact of uncertainties in individual dose estimates. Included were all 66 confirmed cases of primary thyroid cancer diagnosed from April 26, 1986 through September 1998 in residents of Bryansk Oblast, Russia, who were 0-19 years old at the time of the accident, along with two individually matched controls for each case. Thyroid radiation doses, estimated using a semi-empirical model based on environmental contamination data and individual characteristics, ranged from 0.00014 Gy to 2.73 Gy and had large uncertainties (median geometric standard deviation 2.2). The estimated excess relative risk (ERR) associated with radiation exposure, 48.7/Gy, was significantly greater than 0 (P = 0.00013) but had an extremely wide 95% confidence interval (4.8 to 1151/Gy). Adjusting for dose uncertainty nearly tripled the ERR to 138/Gy, although this was likely an overestimate due to limitations in the modeling of dose uncertainties. The radiation-related excess risk observed in this study is quite large, especially if the uncertainty of dose estimation is taken into account, but is not inconsistent with estimates previously reported for risk after (131)I exposure or acute irradiation from external sources.     

Different transformation pathways of murine fibroblast NIH 3T3 cells by hepatitis C virus core and NS3 proteins

The oncogenic potential of both Hepatitis C virus (HCV) core and HCV NS3 proteins has been demonstrated, but these proteins induce transformation of immortal murine fibroblasts NIH 3T3 via different pathways. As long-term expression (50-100 passages) of HCV core triggers neoplastic transformation of NIH 3T3 through crisis of growth, HCV NS3 induces transformation shortly after transfection. We explain this distinction by different effects of core and NS3 on p53-mediated transactivation: inhibition by NS3 and activation by core protein.