Synthesis of tropomyosin in myogenic cultures and in RNA-directed cell-free systems: qualitative changes in the polypeptides

The synthesis of polypeptides with the properties of alpha and beta tropomyosin was investigated in differentiating cultures of a myogenic cell line and in a wheat germ cell-free system directed by purified RNA extracted at different stages of differentiation. The polypeptides co-migrate with tropomyosin in isoelectric focusing and SDS two-dimensional gel electrophoresis and SDS-urea/SDS two-dimensional gels. Like authentic tropomyosin, these polypeptides change their mobility greatly in the presence of urea and do not become labeled with proline. The beta tropomyosin synthesized in the intact cells and in the cell-free system can be separated by isoelectric focusing into at least two components. One component (designated beta1) is present in a small amount at all developmental stages examined, and a more basic component (beta2) is specific for differentiated cultures. The synthesis of beta2 in the intact cells and the capacity of purified RNA to direct its synthesis in a cell-free system become detectable and increase greatly during the period of fusion of the mononucleated cells into multinucleated fibers. The results suggest that the beta1 and beta2 tropomyosins are coded for by different genes.     

STING Millennium Suite: integrated software for extensive analyses of 3d structures of proteins and their complexes

Background  

The integration of many aspects of protein/DNA structure analysis is an important requirement for software products in general area of structural bioinformatics. In fact, there are too few software packages on the internet which can be described as successful in this respect. We might say that what is still missing is publicly available, web based software for interactive analysis of the sequence/structure/function of proteins and their complexes with DNA and ligands. Some of existing software packages do have certain level of integration and do offer analysis of several structure related parameters, however not to the extent generally demanded by a user.     

Solid-phase PCR in microwells: effects of linker length and composition on tethering, hybridization, and extension

During the solid-phase PCR (SP-PCR), DNA oligonucleotides complementary to a soluble template and immobilized on a surface are extended in situ. Although primarily used for pathogen detection, SP-PCR has the potential for much broader application, including disease diagnostics, genotyping, and expression studies. Current protocols for SP-PCR in microwells are suitable for enzymatic detection of immobilized products, but yields are generally insufficient for direct detection of products using conventional fluorescent probes. Here, we quantitatively measure the outcome of tethering, hybridization, and solid-phase extension, and examine the effect of composition and length of the spacer at the 5' end of tethered oligonucleotides. Our results indicate that steric hindrance primarily affects polymerase activity rather than the efficiency of hybridization between the template and the tethered oligonucleotide. SP-PCR yields are significantly higher for a five-unit hexaethyleneglycol (HEG) spacer than for the more commonly used 10-residue deoxythymidine spacer. The optimal 5' HEG spacer resulted in a 60-fold increase in extension efficiency relative to a previously reported value for SP-PCR on a glass surface. Thus, optimized spacers should allow direct quantification of SP-PCR products, providing a simple, quantitative, and cost effective means of sample analysis for a variety of applications.     

Coupling of dopamine receptors to G proteins: studies with chimeric D2/D3 dopamine receptors

D₂ and D₃ dopamine receptors belong to the superfamily of G protein-coupled receptors; they share a high degree of homology and are structurally similar. However, they differ from each other in their second messenger coupling properties. Previously, we have studied the differential coupling of these receptors to G proteins and found that while D₂ receptor couples only to inhibitory G proteins, D₃ receptor couples also to a stimulatory G protein, Gs. We aimed to investigate the molecular basis of these differences and to determine which domains in the receptor control its coupling to G proteins. For this purpose four chimeras were constructed, each composed of different segments of the original D₂ and D₃ receptors. We have demonstrated that chimeras with a third cytoplasmic loop of D₂ receptor couple to Gi protein in a pattern characteristic of D₂ receptor. On the other hand chimeras containing a third cytoplasmic loop of D₃ receptor have coupling characteristics like those of D₃ receptor, and they couple also to Gs protein. These findings demonstrate that the third cytoplasmic loop determines and accounts for the coupling of dopamine receptors D₂ and D₃ to G proteins.     

Development of a bioassay for detection of Wnt-binding affinities for individual frizzled receptors

Wnts are secreted lipid-modified glycoproteins that carry out various signaling functions during development and in adult tissue. Wnt signaling is mediated by frizzled receptors (Fzds) at the cell surface and can be modulated by the secreted frizzled-related proteins (SFRPs) and other molecular antagonists. Abnormal Wnt signaling has been implicated in several diseases. However, due to the complexity of the Wnt signal and the lack of knowledge pertaining to the binding properties of different Wnt ligands, no therapeutic agents that target this pathway exist. Using a novel enzyme-linked immunosorbent assay (ELISA)-based technique, we were able to determine the first measurements of binding affinity for specific Wnt interactions. This study shows that purified Wnt3a, Wnt7a, and Wnt5a have different binding specificities for Fzds and SFRPs.     

Genome-scale metabolic reconstruction and <Emphasis Type="Italic">in silico</Emphasis> analysis of methylotrophic yeast <Emphasis Type="Italic">Pichia pastoris</Emphasis> for strain improvement

Background  

Pichia pastoris has been recognized as an effective host for recombinant protein production. A number of studies have been reported for improving this expression system. However, its physiology and cellular metabolism still remained largely uncharacterized. Thus, it is highly desirable to establish a systems biotechnological framework, in which a comprehensive in silico model of P. pastoris can be employed together with high throughput experimental data analysis, for better understanding of the methylotrophic yeast's metabolism.     

Rhomboid cleaves Star to regulate the levels of secreted Spitz

Intracellular trafficking of the precursor of Spitz (Spi), the major Drosophila EGF receptor (EGFR) ligand, is facilitated by the chaperone Star, a type II transmembrane protein. This study identifies a novel mechanism for modulating the activity of Star, thereby influencing the levels of active Spi ligand produced. We demonstrate that Star can efficiently traffic Spi even when present at sub-stoichiometric levels, and that in Drosophila S(2)R(+) cells, Spi is trafficked from the endoplasmic reticulum to the late endosome compartment, also enriched for Rhomboid, an intramembrane protease. Rhomboid, which cleaves the Spi precursor, is now shown to also cleave Star within its transmembrane domain both in cell culture and in flies, expanding the repertoire of known Rhomboid substrates to include both type I and type II transmembrane proteins. Cleavage of Star restricts the amount of Spi that is trafficked, and may explain the exceptional dosage sensitivity of the Star locus in flies.     

Apoptosis,cytokine and chemokine induction by non-structural 1 (NS1) proteins encoded by different influenza subtypes

Background

Influenza pandemic remains a serious threat to human health. Viruses of avian origin, H5N1, H7N7 and H9N2, have repeatedly crossed the species barrier to infect humans. Recently, a novel strain originated from swine has evolved to a pandemic. This study aims at improving our understanding on the pathogenic mechanism of influenza viruses, in particular the role of non-structural (NS1) protein in inducing pro-inflammatory and apoptotic responses.

Methods

Human lung epithelial cells (NCI-H292) was used as an in-vitro model to study cytokine/chemokine production and apoptosis induced by transfection of NS1 mRNA encoded by seven infleunza subtypes (seasonal and pandemic H1, H2, H3, H5, H7, and H9), respectively.

Results

The results showed that CXCL-10/IP10 was most prominently induced (> 1000 folds) and IL-6 was slightly induced (< 10 folds) by all subtypes. A subtype-dependent pattern was observed for CCL-2/MCP-1, CCL3/MIP-1α, CCL-5/RANTES and CXCL-9/MIG; where induction by H5N1 was much higher than all other subtypes examined. All subtypes induced a similar temporal profile of apoptosis following transfection. The level of apoptosis induced by H5N1 was remarkably higher than all others. The cytokine/chemokine and apoptosis inducing ability of the 2009 pandemic H1N1 was similar to previous seasonal strains.

Conclusions

In conclusion, the NS1 protein encoded by H5N1 carries a remarkably different property as compared to other avian and human subtypes, and is one of the keys to its high pathogenicity. NCI-H292 cells system proves to be a good in-vitro model to delineate the property of NS1 proteins.

     

Secreted frizzled-related protein 4 regulates two Wnt7a signaling pathways and inhibits proliferation in endometrial cancer cells

In the endometrium, hormonal effects on epithelial cells are often elicited through stromal hormone receptors via unknown paracrine mechanisms. Several lines of evidence support the hypothesis that Wnts participate in stromal-epithelial cell communication. Wnt7a is expressed in the luminal epithelium, whereas the extracellular modulator of Wnt signaling, secreted frizzled-related protein 4 (SFRP4), is localized to the stroma. Studies have reported that SFRP4 expression is significantly decreased in endometrial carcinoma and that both SFRP4 and Wnt7a genes are differentially regulated in response to estrogenic stimuli. Aberrant Wnt7a signaling irrevocably causes organ defects and infertility and contributes to the onset of disease. However, specific frizzled receptors (Fzd) that bind Wnt7a and the particular signal transduction pathway each Wnt7a-Fzd pair activates have not been identified. Additionally, the function of SFRP4 in the endometrium has not been addressed. We show here that Wnt7a coimmunoprecipitates with Fzd5, Fzd10, and SFRP4 in Ishikawa cells. Wnt7a binding to Fzd5 was shown to activate beta-catenin/canonical Wnt signaling and increase cellular proliferation. Conversely, Wnt7a signaling mediated by Fzd10 induced a noncanonical c-Jun NH2-terminal kinase-responsive pathway. SFRP4 suppresses activation of Wnt7a signaling in both an autocrine and paracrine manner. Stable overexpression of SFRP4 and treatment with recombinant SFRP4 protein inhibited endometrial cancer cell growth in vitro. These findings support a mechanism by which the nature of the Wnt7a signal in the endometrium is dependent on the Fzd repertoire of the cell and can be regulated by SFRP4.