NASCENT: an automatic protein interaction network generation tool for non-model organisms.

Large quantity of reliable protein interaction data are available for model organisms in public depositories (e.g., MINT, DIP, HPRD, INTERACT). Most data correspond to experiments with the proteins of Saccharomyces cerevisiae, Drosophila melanogaster, Homo sapiens, Caenorhabditis elegans, Escherichia coli and Mus musculus. For other important organisms the data availability is poor or non-existent. Here we present NASCENT, a completely automatic web-based tool and also a downloadable Java program, capable of modeling and generating protein interaction networks even for non-model organisms. The tool performs protein interaction network modeling through gene-name mapping, and outputs the resulting network in graphical form and also in computer-readable graph-forms, directly applicable by popular network modeling software. AVAILABILITY: http://nascent.pitgroup.org

A Note on the PageRank of Undirected Graphs

The PageRank is a widely used scoring function of networks in general and of the World Wide Web graph in particular. The PageRank is defined for directed graphs, but in some special cases applications for undirected graphs occur. In the literature it is widely noted that the PageRank for undirected graphs are proportional to the degrees of the vertices of the graph. We prove that statement for a particular personalization vector in the definition of the PageRank, and we also show that in general, the PageRank of an undirected graph is not exactly proportional to the degree distribution of the graph: our main theorem gives an upper and a lower bound to the L_1 norm of the difference of the PageRank and the degree distribution vectors

Binding of the Bacillus subtilis LexA protein to the SOS operator

The Bacillus subtilis LexA protein represses the SOS response to DNA damage by binding as a dimer to the consensus operator sequence 5′-CGAACN(4)GTTCG-3′. To characterize the requirements for LexA binding to SOS operators, we determined the operator bases needed for site-specific binding as well as the LexA amino acids required for operator recognition. Using mobility shift assays to determine equilibrium constants for B.subtilis LexA binding to recA operator mutants, we found that several single base substitutions within the 14 bp recA operator sequence destabilized binding enough to abolish site-specific binding. Our results show that the AT base pairs at the third and fourth positions from the 5′ end of a 7 bp half-site are essential and that the preferred binding site for a LexA dimer is 5′-CGAACATATGTTCG-3′. Binding studies with LexA mutants, in which the solvent accessible amino acid residues in the putative DNA binding domain were mutated, indicate that Arg-49 and His-46 are essential for binding and that Lys-53 and Ala-48 are also involved in operator recognition. Guided by our mutational analyses as well as hydroxyl radical footprinting studies of the dinC and recA operators we docked a computer model of B.subtilis LexA on the preferred operator sequence in silico. Our model suggests that binding by a LexA dimer involves bending of the DNA helix within the internal 4 bp of the operator

A Flagellar A-Kinase Anchoring Protein with Two Amphipathic Helices Forms a Structural Scaffold in the Radial Spoke Complex

A-kinase anchoring proteins (AKAPs) contain an amphipathic helix (AH) that binds the dimerization and docking (D/D) domain, RIIa, in cAMP-dependent protein kinase A (PKA). Many AKAPs were discovered solely based on the AH–RIIa interaction in vitro. An RIIa or a similar Dpy-30 domain is also present in numerous diverged molecules that are implicated in critical processes as diverse as flagellar beating, membrane trafficking, histone methylation, and stem cell differentiation, yet these molecules remain poorly characterized. Here we demonstrate that an AKAP, RSP3, forms a dimeric structural scaffold in the flagellar radial spoke complex, anchoring through two distinct AHs, the RIIa and Dpy-30 domains, in four non-PKA spoke proteins involved in the assembly and modulation of the complex. Interestingly, one AH can bind both RIIa and Dpy-30 domains in vitro. Thus, AHs and D/D domains constitute a versatile yet potentially promiscuous system for localizing various effector mechanisms. These results greatly expand the current concept about anchoring mechanisms and AKAPs

Demonstration of a Melanoma-Specific CD44 Alternative Splicing Pattern That Remains Qualitatively Stable, but Shows Quantitative Changes during Tumour Progression

The role of CD44 in the progression of human melanoma has mostly been characterised by qualitative changes in expression of its individual variable exons. These exons however, may be expressed to form a number of molecules, the alternative splice variants of CD44, which may be structurally and functionally different. Using real-time PCR measurements with variable exon specific primers we have determined that all are expressed in human melanoma. To permit comparison between different tumours we identified a stable CD44 variable exon (CD44v) expression pattern, or CD44 ‘fingerprint’. This was found to remain unchanged in melanoma cell lines cultured in different matrix environments. To evaluate evolution of this fingerprint during tumour progression we established a scid mouse model, in which the pure expression pattern of metastatic primary tumours, circulating cells and metastases, non-metastatic primary tumours and lung colonies could be studied. Our analyses demonstrated, that although the melanoma CD44 fingerprint is qualitatively stable, quantitative changes are observed suggesting a possible role in tumour progression

Comparative evaluation of acetaminophen form (I) in commercialized paracetamol brands

The vibrational spectroscopy (FT-IR/Raman) and X-ray diffraction techniques are combined alongside the principal component analysis (PCA) as novel integrated analytical techniques to comparatively investigate latent chemical information and quality discrepancies regarding twelve (12) commercialized paracetamol (APAP) brands. This research aim is to present an advanced computational screening approach using spectroscopic and X-ray diffraction techniques with PCA as a tool to investigate the structural properties of pharmaceutical solid drugs by vibrational mode and diffraction pattern analyzes. Herein, the acquired vibrational, absorption, and diffraction datasets of APAP functionalities were collected at spectra and diffraction regions of intense peaks to develop predictive PCA models. Interestingly, the PCA models invalidate drug falsification in all the brands and predicted dissimilarities arising from observed differences in the vibrational/absorption modes of APAP form (I) in some brands due to excessive use of cheap (fillers and hydrocolloid alternatives) excipients. The PCA-PXRD model unveils discrepancies regarding the contrasting diffraction patterns (structure-property relationships) observed for APAP form (I) in the brands, which suggests differences in their pharmacokinetic properties cause an unapparent structural modification. Nevertheless, the comparative drug release studies present a%CDR between 93 and 98% in 30 min for all the brands, thus, structural modifications of APAP form (I) as observed in some brands show no serious effects on the%CDR and/ or solubility. Finally, it is expected that this work will contribute to the advances in screening techniques toward addressing the global drug challenges, especially in developing countries

The regulatory subunit of PKA-I remains partially structured and undergoes β-aggregation upon thermal denaturation

Background: The regulatory subunit (R) of cAMP-dependent protein kinase (PKA) is a modular flexible protein that responds with large conformational changes to the binding of the effector cAMP. Considering its highly dynamic nature, the protein is rather stable. We studied the thermal denaturation of full-length RIα and a truncated RIα(92-381) that contains the tandem cyclic nucleotide binding (CNB) domains A and B. Methodology/Principal Findings: As revealed by circular dichroism (CD) and differential scanning calorimetry, both RIα proteins contain significant residual structure in the heat-denatured state. As evidenced by CD, the predominantly α-helical spectrum at 25°C with double negative peaks at 209 and 222 nm changes to a spectrum with a single negative peak at 212-216 nm, characteristic of β-structure. A similar α→β transition occurs at higher temperature in the presence of cAMP. Thioflavin T fluorescence and atomic force microscopy studies support the notion that the structural transition is associated with cross-β-intermolecular aggregation and formation of non-fibrillar oligomers. Conclusions/Significance: Thermal denaturation of RIα leads to partial loss of native packing with exposure of aggregation-prone motifs, such as the B' helices in the phosphate-binding cassettes of both CNB domains. The topology of the β-sandwiches in these domains favors inter-molecular β-aggregation, which is suppressed in the ligand-bound states of RIα under physiological conditions. Moreover, our results reveal that the CNB domains persist as structural cores through heat-denaturation. © 2011 Dao et al

Overexpression of specific CD44 isoforms is associated with aggressive cell features in acquired endocrine resistance

While endocrine therapy is the mainstay of ER+ breast cancer, the clinical effectiveness of these agents is limited by the phenomenon of acquired resistance that is associated with disease relapse and poor prognosis. Our previous studies revealed that acquired resistance is accompanied by a gain in cellular invasion and migration and also that CD44 family proteins are overexpressed in the resistant phenotype. Given the association of CD44 with tumor progression, we hypothesized that its overexpression may act to promote the aggressive behavior of endocrine-resistant breast cancers. Here, we have investigated further the role of two specific CD44 isoforms, CD44v3 and CD44v6, in the endocrine-resistant phenotype. Our data revealed that overexpression of CD44v6, but not CD44v3, in endocrine-sensitive MCF-7 cells resulted in a gain in EGFR signaling, enhanced their endogenous invasive capacity, and attenuated their response to endocrine treatment. Suppression of CD44v6 in endocrine-resistant cell models was associated with a reduction in their invasive capacity. Our data suggest that upregulation of CD44v6 in acquired resistant breast cancer may contribute to a gain in the aggressive phenotype of these cells and loss of endocrine response through transactivation of the EGFR pathway. Future therapeutic targeting of CD44v6 may prove to be an effective strategy alongside EGFR-targeted agents in delaying/preventing acquired resistance in breast cancer

Controlled synthesis and structure characterization of a new fluconazole polymorph using analytical techniques and multivariate method

In the crystallization and search for higher multicomponent forms of fluconazole (FLZ), a metastable FLZ polymorph (concomitant) that manifests in the same crystallization system and transforms into the stable FLZ form (II) after the lyophilization process was observed. In this report, we demonstrated and showed how this FLZ polymorph 10 (Mw = 306.79 g/mol) of the monoclinic C2 space group was detected and reproduced through a controlled lyophilized experiment, and modeled differentiation between vibrational and absorption modes of FLZ functionalities like C=O, OH, –CH2 and –NH. The FLZ polymorph shows strong O–H···N and weak C−H···X (X = N, and F) hydrogen bond and the presence of pi-pi bond interactions in the overlapping triazole rings. The combination of vibrational spectroscopic techniques (Raman/FT-IR) and principal component analysis (PCA) aid the development of important models for polymorph screening and identification. In addition, X-ray diffraction (powder and single crystal) techniques support the polymorph characterization and structure depiction. The PCA models and X-ray diffraction analyses confirmed the newness of FLZ polymorph 10, and further solid-state characterization using thermal techniques (DSC and TGA) affirmed its uniqueness and novelty. Finally, the thermal stability and solubility studies on the new FLZ polymorph were determined to understand its structure properties and compare these with previously reported polymorphs of FLZ