A novel FRET pair for detection of parallel DNA triplexes by the LightCycler

Background  

Melting temperature of DNA structures can be determined on the LightCycler using quenching of FAM. This method is very suitable for pH independent melting point (Tm) determination performed at basic or neutral pH, as a high throughput alternative to UV absorbance measurements. At acidic pH quenching of FAM is not very suitable, since the fluorescence of FAM is strongly pH dependent and drops with acidic pH.     

Lipase-catalysed acylation of starch and determination of the degree of substitution by methanolysis and GC

Background  

Natural polysaccharides such as starch are becoming increasingly interesting as renewable starting materials for the synthesis of biodegradable polymers using chemical or enzymatic methods. Given the complexity of polysaccharides, the analysis of reaction products is challenging.     

Environmental changes affect ecosystem services of the intermittent Lake Cerknica

Lake Cerknica is an intermittent wetland with seasonal water level fluctuations. This paper discusses the possible changes of ecosystem services due to altered ecosystem functions caused by regional climate change. For this purpose, the lake's water regime was analysed and biomass production of common reed (Phragmites australis), the prevailing species, was related to ambient temperatures and water level. The effect of the latter was also examined for reed transpiration rate and plant diversity. The results revealed a gradual loss of seasonality of floods and droughts. High water level in winter months and temperatures at the beginning as well as the end of vegetation period were found to have a significant influence on reed biomass. The plant diversity research in six subsequent years in three different aquatic habitats revealed trends of a decrease due to increased water depth during the vegetation period. The complexity of the system and consequently ecosystem services might also be affected due to changes of other processes, which revealed to be related to water level; namely transpiration rate, plant mycorrhizal colonisation and soil mineralisation.     

Principles of determination and verification of muscle forces in the human musculoskeletal system: Muscle forces to minimise bending stress

While there are a growing number of increasingly complex methodologies available to model geometry and material properties of bones, these models still cannot accurately describe physical behaviour of the skeletal system unless the boundary conditions, especially muscular loading, are correct. Available in vivo measurements of muscle forces are mostly highly invasive and offer no practical way to validate the outcome of any computational model that predicts muscle forces. However, muscle forces can be verified indirectly using the fundamental property of living tissue to functional adaptation and finite element (FE) analysis. Even though the mechanisms of the functional adaptation are not fully understood, its result is clearly seen in the shape and inner structure of bones. The FE method provides a precise tool for analysis of the stress/strain distribution in the bone under given loading conditions. The present work sets principles for the determination of the muscle forces on the basis of the widely accepted view that biological systems are optimized light-weight structures with minimised amount of unloaded/underloaded material and hence evenly distributed loading throughout the structure. Bending loading of bones is avoided/compensated in bones under physiological loading. Thus, bending minimisation provides the basis for the determination of the musculoskeletal system loading. As a result of our approach, the muscle forces for a human femur during normal gait and sitting down (peak hip joint force) are obtained such that the bone is loaded predominantly in compression and the stress distribution in proximal and diaphyseal femur corresponds to the material distribution in bone.     

Palmitoylation of the S0-S1 Linker Regulates Cell Surface Expression of Voltage- and Calcium-activated Potassium (BK) Channels

S-Palmitoylation is rapidly emerging as an important post-translational mechanism to regulate ion channels. We have previously demonstrated that large conductance calcium- and voltage-activated potassium (BK) channels are palmitoylated within an alternatively spliced (STREX) insert. However, these studies also revealed that additional site(s) for palmitoylation must exist outside of the STREX insert, although the identity or the functional significance of these palmitoylated cysteine residues are unknown. Here, we demonstrate that BK channels are palmitoylated at a cluster of evolutionary conserved cysteine residues (Cys-53, Cys-54, and Cys-56) within the intracellular linker between the S0 and S1 transmembrane domains. Mutation of Cys-53, Cys-54, and Cys-56 completely abolished palmitoylation of BK channels lacking the STREX insert (ZERO variant). Palmitoylation allows the S0-S1 linker to associate with the plasma membrane but has no effect on single channel conductance or the calcium/voltage sensitivity. Rather, S0-S1 linker palmitoylation is a critical determinant of cell surface expression of BK channels, as steady state surface expression levels are reduced by ∼55% in the C53:54:56A mutant. STREX variant channels that could not be palmitoylated in the S0-S1 linker also displayed significantly reduced cell surface expression even though STREX insert palmitoylation was unaffected. Thus our work reveals the functional independence of two distinct palmitoylation-dependent membrane interaction domains within the same channel protein and demonstrates the critical role of S0-S1 linker palmitoylation in the control of BK channel cell surface expression.     

Identification of an autophagy defect in smokers' alveolar macrophages

Alveolar macrophages are essential for clearing bacteria from the alveolar surface and preventing microbe-induced infections. It is well documented that smokers have an increased incidence of infections, in particular lung infections. Alveolar macrophages accumulate in smokers' lungs, but they have a functional immune deficit. In this study, we identify an autophagy defect in smokers' alveolar macrophages. Smokers' alveolar macrophages accumulate both autophagosomes and p62, a marker of autophagic flux. The decrease in the process of autophagy leads to impaired protein aggregate clearance, dysfunctional mitochondria, and defective delivery of bacteria to lysosomes. This study identifies the autophagy pathway as a potential target for interventions designed to decrease infection rates in smokers and possibly in individuals with high environmental particulate exposure.     

Daughter Centriole Elongation Is Controlled by Proteolysis

The centrosome is the major microtubule-organizing center of most mammalian cells and consists of a pair of centrioles embedded in pericentriolar material. Before mitosis, the two centrioles duplicate and two new daughter centrioles form adjacent to each preexisting maternal centriole. After initiation of daughter centriole synthesis, the procentrioles elongate in a process that is poorly understood. Here, we show that inhibition of cellular proteolysis by Z-L₃VS or MG132 induces abnormal elongation of daughter centrioles to approximately 4 times their normal length. This activity of Z-L₃VS or MG132 was found to correlate with inhibition of intracellular protease-mediated substrate cleavage. Using a small interfering RNA screen, we identified a total of nine gene products that either attenuated (seven) or promoted (two) abnormal Z-L₃VS–induced daughter centriole elongation. Our hits included known regulators of centriole length, including CPAP and CP110, but, interestingly, several proteins involved in microtubule stability and anchoring as well as centrosome cohesion. This suggests that nonproteasomal functions, specifically inhibition of cellular proteases, may play an important and underappreciated role in the regulation of centriole elongation. They also highlight the complexity of daughter centriole length control and provide a framework for future studies to dissect the molecular details of this process.     

Insights into the mechanism of dihydropyrimidine dehydrogenase from site-directed mutagenesis targeting the active site loop and redox cofactor coordination

In mammals, the pyrimidines uracil and thymine are metabolised by a three-step reductive degradation pathway. Dihydropyrimidine dehydrogenase (DPD) catalyses its first and rate-limiting step, reducing uracil and thymine to the corresponding 5,6-dihydropyrimidines in an NADPH-dependent reaction. The enzyme is an adjunct target in cancer therapy since it rapidly breaks down the anti-cancer drug 5-fluorouracil and related compounds. Five residues located in functionally important regions were targeted in mutational studies to investigate their role in the catalytic mechanism of dihydropyrimidine dehydrogenase from pig. Pyrimidine binding to this enzyme is accompanied by active site loop closure that positions a catalytically crucial cysteine (C671) residue. Kinetic characterization of corresponding enzyme mutants revealed that the deprotonation of the loop residue H673 is required for active site closure, while S670 is important for substrate recognition. Investigations on selected residues involved in binding of the redox cofactors revealed that the first FeS cluster, with unusual coordination, cannot be reduced and displays no activity when Q156 is mutated to glutamate, and that R235 is crucial for FAD binding.     

Glycoprotein Capture and Quantitative Phosphoproteomics Indicate Coordinated Regulation of Cell Migration upon Lysophosphatidic Acid Stimulation

The lipid mediator lysophosphatidic acid (LPA) is a serum component that regulates cellular functions such as proliferation, migration, and survival via specific G protein-coupled receptors. The underlying signaling mechanisms are still incompletely understood, including those that operate at the plasma membrane to modulate cell-cell and cell-matrix interactions in LPA-promoted cell migration. To explore LPA-evoked phosphoregulation with a focus on cell surface proteins, we combined glycoproteome enrichment by immobilized lectins with SILAC-based quantitative phosphoproteomics. We performed biological replicate analyses in SCC-9 squamous cell carcinoma cells and repeatedly quantified the effect of 1.5- and 5-min LPA treatment on more than 700 distinct phosphorylations in lectin-purified proteins. We detected many regulated phosphorylation events on various types of plasma membrane proteins such as cell adhesion molecules constituting adherens junctions, desmosomes, and hemidesmosomes. Several of these LPA-regulated phosphorylation sites have been characterized in a biological context other than G protein-coupled receptor signaling, and the transfer of this functional information suggests coordinated and multifactorial cell adhesion control in LPA-induced cell migration. Additionally, we identified LPA-mediated activation loop phosphorylation of the serine/threonine kinase Wnk1 and verified a role of Wnk1 for LPA-induced cell migration in knock-down experiments. In conclusion, the glycoproteome phosphoproteomics strategy described here sheds light on incompletely understood mechanisms in LPA-induced cell migratory behavior.The plasma membrane separates the interior of a mammalian cell from the environment. To respond to external signals such as growth factors, cells possess various types of plasma membrane-spanning receptors that communicate to the intracellular signaling machinery in a ligand-regulated manner. G protein-coupled receptors (GPCRs),1 which are integral membrane proteins with seven transmembrane helices, constitute the largest superfamily of cell surface receptors. GPCRs mediate intracellular activation of heterotrimeric G proteins in response to extracellular ligand binding. A plethora of different factors are known to act on GPCRs, including peptide ligands, proteases, nucleotides as well as bioactive lipid molecules such as lysophosphatidic acid (LPA). LPA induces various biological responses including proliferation and migration in a wide range of mammalian cell types and has been implicated in the progression of several human cancers (1, 2). Upon LPA binding to its cognate receptors, heterotrimeric G proteins from the G_i, G_q, and G_12/13 families are activated by guanine nucleotide exchange factors resulting in their dissociation into activated Gα and Gβγ subunits. Activated G protein subunits interact with various effector proteins including phospholipase C and adenylate cyclase isoforms as well as guanine nucleotide exchange factors for Rho family GTPases, which either directly or via second messenger production communicate to cellular kinase signaling. GPCR activation by LPA is also known to trigger the proteolytic activity of ADAM transmembrane metalloproteases, such as ADAM17, which processes epidermal growth factor receptor (EGFR) ligand precursors on the extracellular side to release mature growth factors triggering EGFR activation (3–8). The molecular mechanisms involved in the control of ADAM metalloprotease activity are not clear yet. The resulting transactivation of the EGFR tyrosine kinase provides a link to signaling modules such as mitogen-activated protein kinase cascades and has been implicated in the control of cell proliferation and migration upon LPA treatment (9). Regarding the induction of cell motility upon LPA, previous studies have reported several signaling elements in addition to EGFR transactivation that contribute to this complex cellular behavior. In particular, RhoGTPase-dependent signals that activate downstream effectors such as Rho kinase and focal adhesion kinase are involved in the control of cytoskeletal organization and cell attachment to the surrounding extracellular matrix (ECM) (10, 11). The coordinated regulation of such integrin-mediated interactions is required to enable cell movement and occurs in dedicated macromolecular assemblies such as focal adhesion complexes and hemidesmosomes (12, 13). Despite the key role of integrins, the molecular mechanisms that underlie their functional modulation upon GPCR activation are poorly understood. Moreover, cell-cell contacts such as adherens junctions and desmosomes have to dissociate prior to cell migration. Likewise, it is unclear how the components of these structures, such as members of the cadherin family, might be regulated by GPCR-mediated signaling pathways. Both LPA levels and LPA_1–3 receptor expression are often elevated in cancer patients, and the bioactive lipid acts as a potent inducer of cell migration and invasion in vitro. Due to the key role of protein phosphorylation in LPA-induced signal transmission, comprehensive phosphorylation analysis of regulated proteins might generate new insights into pro-migratory signaling mechanisms in cancer cells. Mass spectrometry (MS)-based analysis has emerged as the key method for unbiased protein phosphorylation studies due to various technological advances in recent years (14, 15). Because of the substoichiometric nature of many site-specific phosphorylation events, phosphopeptides constitute only a small fraction in total peptide samples. Therefore, they need to be efficiently enriched prior to MS analysis, which has become routinely possible by phosphate group-selective purification strategies employing capture reagents such as immobilized metal ion affinity chromatography or titanium dioxide beads (16). Moreover, phosphopeptide analysis has benefited enormously from the availability of hybrid mass spectrometers that combine the sensitivity and speed of linear ion traps with the high resolution and accuracy of orbitrap mass analyzers (17). These advances together with quantitative approaches such as stable isotope labeling by amino acids in cell cultures (SILAC) (18, 19) and substantial progress in computational proteomics (20) now allow for concomitant identification and quantification of several thousand phosphorylation sites from single cellular extracts (21–23).We previously analyzed cell signaling responses in A498 kidney carcinoma cells upon LPA and heparin-binding EGF-like growth factor treatment by monitoring phosphorylation changes in total cell lysate and protein kinase-enriched fractions (24). In a complementary approach, we now aimed for a systematic survey of LPA-induced phosphorylation changes on plasma membrane proteins and their interaction partners. Furthermore, we were interested in time-resolved analysis of LPA-induced phosphorylation changes on ADAM17 and the EGFR to gain further insights into possible mechanisms underlying the still enigmatic EGFR transactivation process. In our present study, we therefore analyzed SCC-9 squamous carcinoma cells due to their pronounced EGFR transactivation response upon LPA. As plasma membrane proteins usually contain covalently attached carbohydrate structures, we performed lectin affinity enrichment of glycosylated proteins prior to SILAC-based quantitative phosphoproteomics (25). This experimental strategy enabled us to acquire in-depth data about LPA regulation of diverse glycoproteins and revealed coordinated phosphoregulation of cell adhesion proteins as likely mechanism underlying cell migratory behavior.