Fluoride complexes of oncogenic Ras mutants to study the Ras-RasGap interaction

Down-regulation of Ras signalling is mediated by specific GTPase-activating proteins (GAPs), which stimulate the very slow GTPase reaction of Ras by 10(5)-fold. The basic features of the GAP activity involve the stabilisation of both switch regions of Ras in the transition state, and the insertion of an arginine finger. In the case of oncogenic Ras mutations, the features of the active site are disturbed. To understand these features in more detail, we have investigated the effects of oncogenic mutations of Ras and compared the GAP-stimulated GTPase reaction with the ability to form GAP-mediated aluminium or beryllium fluoride complexes. In general we find a correlation between the size of the amino acid at position 12, the GTPase activity and ability to form aluminium fluoride complexes. While Gly12 is very sensitive to even the smallest possible structural change, Gly13 is much less sensitive to steric hindrance, but is sensitive to charge. Oncogenic mutants of Ras defective in the GTPase activity can however form ground-state GppNHp complexes with GAP, which can be mimicked by beryllium fluoride binding. We show that beryllium fluoride complexes are less sensitive to structural changes and report on a state close to but different from the ground state of the GAP-stimulated GTPase reaction.     

Antiproliferative activity of chloroformic extract of Persian Shallot, Allium hirtifolium, on tumor cell lines

Allium hirtifolim (Persian Shallot) belongs to Allium genus (Alliaceae family). We investigated the in vitro effects of chloroformic extract of A. hirtifolium and its Allicin on the proliferation of HeLa (cervical cancer), MCF7 (human, caucasion, breast, adenocarcinoma) and L929 (mouse, C3H/An, connective) cell lines. Our results showed that components of A. hirtifolium might inhibit proliferation of tumor cell lines. This inhibition in HeLa and MCF-7 cells was dose-dependent. The presence of Allicin was evaluated by TLC method in bulbs and the extract of A. hirtifolium was analyzed by HPLC. MTT test was performed 24, 48 and 72 h after cell culture. A significant decrease in cell lines was observed in HeLa and MCF-7 as compared to L929 cell lines. DNA fragmentation analysis revealed a large number of apoptotic cells in treated HeLa and MCF-7 cell groups, but no effects in L929 cells. Therefore A. hirtifolium might be a candidate for tumor suppression.     

Nano-composition of riboflavin-nafion functional film and its application in biosensing

A novel nafion-riboflavin membrane was constructed and characterized by the scanning electron microscopy (SEM), transmission electron microscopy (TEM), UV-visible spectroscopy and cyclic voltammetric techniques. The estimated average diameter of the designed nanoparticles was about 60 nm. The functional membrane showed a quasi-reversible electrochemical behaviour with a formal potential of -562 +/- 5 mV (vs Ag/AgCl) on the gold electrode. Some electrochemical parameters were estimated, indicating that the system has good and stable electron transfer properties. Moreover, horseradish peroxidase (HRP) was immobilized on the riboflavin-nafion functional membrane. The electrochemical behaviour of HRP was quasi-reversible with a formal potential of 80 +/- 5 mV (vs Ag/AgCl). The HRP in the film exhibited good catalytic activity towards the reduction of H2O2. It shows a linear dependence of its cathodic peak current on the concentration of H2O2, ranging from 10 to 300 (micro)M.     

Viral and microbial genotyping by a combination of multiplex competitive hybridization and specific extension followed by hybridization to generic tag arrays

Detection and identification of microbial pathogens are important for disease diagnosis, treatment and prophylaxis measurements. By introducing an innovative technique, we show a robust, reliable and accurate microarray-based method for identification of microbial pathogens. The technique utilizes a unique combination of multiplex competitive hybridization, which enhances hybridization accuracy of oligonucleotides to the specific target, and apyrase-mediated allele-specific extension, which improves specific extension. As a model system, different clinically relevant human papillomaviruses were selected for this study. The method generated accurate results and proves to be promising for specific and correct microbial and viral typing.     

Structural fingerprints of the Ras-GTPase activating proteins neurofibromin and p120GAP

Ras specific GTPase activating proteins (GAPs), neurofibromin and p120GAP, bind GTP bound Ras and efficiently complement its active site. Here we present comparative data from mutations and fluorescence-based assays of the catalytic domains of both RasGAPs and interpret them using the crystal structures. Three prominent regions in RasGAPs, the arginine-finger loop, the phenylalanine-leucine-arginine (FLR) region and alpha7/variable loop contain structural fingerprints governing the GAP function. The finger loop is crucial for the stabilization of the transition state of the GTPase reaction. This function is controlled by residues proximal to the catalytic arginine, which are strikingly different between the two RasGAPs. These residues specifically determine the orientation and therefore the positioning of the arginine finger in the Ras active site. The invariant FLR region, a hallmark for RasGAPs, indirectly contributes to GTPase stimulation by forming a scaffold, which stabilizes Ras switch regions. We show that a long hydrophobic side-chain in the FLR region is crucial for this function. The alpha7/variable loop uses several conserved residues including two lysine residues, which are involved in numerous interactions with the switch I region of Ras. This region determines the specificity of the Ras-RasGAP interaction.     

Beta-casein-derived peptides, produced by bacteria, stimulate cancer cell invasion and motility

In colon cancer, enteric bacteria and dietary factors are major determinants of the microenvironment but their effect on cellular invasion is not known. We therefore incubated human HCT-8/E11 colon cancer cells with bacteria or bacterial conditioned medium on top of collagen type I gels. Listeria monocytogenes stimulate cellular invasion through the formation of a soluble motility-promoting factor, identified as a 13mer beta-casein-derived peptide (HKEMPFPKYPVEP). The peptide is formed through the combined action of Mpl, a Listeria thermolysin-like metalloprotease, and a collagen-associated trypsin-like serine protease. The 13mer peptide was also formed by tumour biopsies isolated from colon cancer patients and incubated with a beta-casein source. The pro- invasive 13mer peptide-signalling pathway implicates activation of Cdc42 and inactivation of RhoA, linked to each other through the serine/threonine p21- activated kinase 1. Since both changes are necessary but not sufficient, another pathway might branch upstream of Cdc42 at phosphatidylinositol 3-kinase. Delta opioid receptor (deltaOR) is a candidate receptor for the 13mer peptide since naloxone, an deltaOR antagonist, blocks both deltaOR serine phosphorylation and 13mer peptide-mediated invasion.     

Computational fluid dynamics modeling,a novel,and effective approach for developing scalable cell therapy manufacturing processes

Induced pluripotent stem cells (iPSCs) hold great potential to generate novel, curative cell therapy products. However, current methods to generate these novel therapies lack scalability, are labor-intensive, require a large footprint, and are not suited to meet clinical and commercial demands. Therefore, it is necessary to develop scalable manufacturing processes to accommodate the generation of high-quality iPSC derivatives under controlled conditions. The current scale-up methods used in cell therapy processes are based on empirical, geometry-dependent methods that do not accurately represent the hydrodynamics of 3D bioreactors. These methods require multiple iterations of scale-up studies, resulting in increased development cost and time. Here we show a novel approach using computational fluid dynamics modeling to effectively scale-up cell therapy manufacturing processes in 3D bioreactors. Using a GMP-compatible iPSC line, we translated and scaled-up a small-scale cardiomyocyte differentiation process to a 3-L computer-controlled bioreactor in an efficient manner, showing comparability in both systems.     

Regulation of triglyceride metabolism. IV. Hormonal regulation of lipolysis in adipose tissue

Triacylglycerol (TAG) stored in adipose tissue can be rapidly mobilized by the hydrolytic action of lipases, with the release of fatty acids (FA) that are used by other tissues during times of energy deprivation. Unlike synthesis of TAG, which occurs not only in adipose tissue but also in other tissues such as liver for very-low-density lipoprotein formation, hydrolysis of TAG, lipolysis, predominantly occurs in adipose tissue. Until recently, hormone-sensitive lipase was considered to be the key rate-limiting enzyme responsible for regulating TAG mobilization. However, recent studies on hormone-sensitive lipase-null mice have challenged such a concept. A novel lipase named desnutrin/ATGL has been recently discovered to play a key role in lipolysis in adipocytes. Lipolysis is under tight hormonal regulation. Although opposing regulation of lipolysis in adipose tissue by insulin and catecholamines is well understood, autocrine/paracrine factors may also participate in its regulation. Intricate cooperation of these endocrine and autocrine/paracrine factors leads to a fine regulation of lipolysis in adipocytes, needed for energy homeostasis. In this review, we summarize and discuss the recent progress made in the regulation of adipocyte lipolysis.     

Visual DNA -- identification of DNA sequence variations by bead trapping

In this paper we describe a method that uses the nearly covalent strength biotin-streptavidin interaction to attach a paramagnetic bead of micrometer size to a DNA molecule of nanometer size, scaling up the spatial size of a query DNA strand by a factor of 1000, making it visible to the human eye. The use of magnetic principles enables rapid binding and washing of detector beads, facilitating a readout of amplified DNA sequences in a few minutes. Here we exemplify the method on mitochondrial DNA variations using an array platform. Visual identification and documentation can be performed with an ordinary mobile phone equipped with a built-in camera.