Architectural model for muscle growth during maturation

Biomechanics and Modeling in Mechanobiology - Muscle architecture, which includes parameters like fascicle length, pennation angle, and physiological cross-sectional area, strongly influences...     

HPV DNA/RNA detection in various oral and oropharyngeal biomaterials identifies active HPV infections also in non-neoplastic tonsils

Previous studies describe a correlation between HPV-positivity and non-smoking in TSCC; p16^INK4A-expression as surrogate-marker for HPV-DNA/RNA-positivity is discussed controversially. In the present study, these parameters are assessed prospectively. HPV-status of sputum and tonsillar-swabs was analyzed to determine their validity as surrogate-marker for tissue-HPV-status.TSCC- (n = 52) and non-neoplastic tonsillar tissue (n = 163) were analyzed. HPV-DNA- and HPV-RNA-status of total sputum, cellular fraction and supernatants, tonsillar-swabs and -tissue was determined by (RT)-PCR. Immunohistochemistry determined p16^INK4A-expression.23/163 (14.2%) non-neoplastic tonsils were HPV-DNA-positive; five patients (3 HPV16, 2 HPV11) had active HPV-infections (HPV-RNA-positive), in all biomaterials. 140/163 (85.9%) patients were either HPV-DNA-positive or HPV-DNA-negative in all samples. 21/52 (40.4%) TSCC-tonsils were HPV-DNA-positive; 17 patients were HPV-RNA-positive (14 HPV16; 4 HPV18). 40/52 (76.9%) TSCC-patients were congruent in all biomaterials. p16^INK4A-expression alone would have misclassified the HPV-status of 14/52 (26.2%) TSCC-patients.This prospective study confirms the discrepancy between HPV-status and p16^INK4A-expression and the significant correlation between non-smoking and HPV-DNA-positivity. HPV-sputum- and/or swab-results do not consistently match tissue-results, possibly having (detrimental) consequences if those were used to assess tissue-HPV-status. In the 5 patients with active HPV infection in the non-neoplasitic tonsils, tonsillectomy likely prevented subsequent development of TSCC.     

SOD3 R231G polymorphism associated with coronary artery disease and myocardial infarction. The Ludwigshafen Risk and Cardiovascular Health (LURIC) study

This study examined the superoxide dismutase 3 (SOD3) R231G polymorphism in relation to the severity of coronary artery disease (CAD) and the risk of myocardial infarction (MI) in 3211 individuals; 94.4% of study participants were homozygous for SOD3 231RR and 5.5% were heterozygous for SOD3 231RG. The odds ratios of the RG and GG genotype (adjusted for age, gender and for conventional cardiovascular risk factors) were 2.02 (95% CI, 1.23–3.33, p=0.005) for the highest vs the lowest Friesinger coronary score and 1.40 (95% CI, 1.02–1.92, p=0.037) for MI, respectively. Further the SOD3 RG and GG genotype was associated with lower alpha-tocopherol levels than the wild type SOD3 RR genotype. It is concluded that the SOD3 231RG and GG genotype is associated with lower alpha-tocopherol levels and the severity of CAD and the risk of MI.     

Discovery and Evaluation of Terephthalic Acid Derivatives as Potent α4β1 Integrin Antagonists

Terephthalic acid based derivatives containing β- and γ-amino acid residues were prepared as antagonists of the leukocyte cell adhesion process that is mediated through the interaction of the very late antigen 4 (VLA-4) and the vascular cell adhesion molecule 1 (VCAM-1). The compounds 2, 10–12, 14, and 16–17 inhibited the adhesion in a cell based assay in the low and sub micromolar range.     

DNA Duplexes Containing Alpha Anomeric Nucleotides and Polarity Reversals: Coexistence of Parallel and Antiparallel DNA

Abstract

Oligodeoxynucleotides that possess alpha anomeric nucleotides and polarity reversals show promise for application in the area of antisense therapy. Here we provide a survey of the spectroscopic, thermodynamic, and enzymatic techniques used in our laboratories to investigate model systems containing such unnatural features with the ultimate goal of designing a new class of more potent and effective antisense therapeutics.     

Mechanism of pH-independent Cleavage and Isomerization of 3′-Ribonucleotide Phosphodiesters

Abstract

The detailed mechanisms of pH-independent cleavage and isomerization reactions of the phosphodiester bond are evaluated by examining kinetics of hydrolytic reactions of selected mono- and dialkyl esters of 3′-uridylic acid. The mechanistic choices are made by comparing LFER's between reaction rates and acidity of the alkyl group of phosphodi- and triesters.     

Profiling Protein Kinases and Other ATP Binding Proteins in Arabidopsis Using Acyl-ATP Probes

Many protein activities are driven by ATP binding and hydrolysis. Here, we explore the ATP binding proteome of the model plant Arabidopsis thaliana using acyl-ATP (AcATP)¹ probes. These probes target ATP binding sites and covalently label lysine residues in the ATP binding pocket. Gel-based profiling using biotinylated AcATP showed that labeling is dependent on pH and divalent ions and can be competed by nucleotides. The vast majority of these AcATP-labeled proteins are known ATP binding proteins. Our search for labeled peptides upon in-gel digest led to the discovery that the biotin moiety of the labeled peptides is oxidized. The in-gel analysis displayed kinase domains of two receptor-like kinases (RLKs) at a lower than expected molecular weight, indicating that these RLKs lost the extracellular domain, possibly as a result of receptor shedding. Analysis of modified peptides using a gel-free platform identified 242 different labeling sites for AcATP in the Arabidopsis proteome. Examination of each individual labeling site revealed a preference of labeling in ATP binding pockets for a broad diversity of ATP binding proteins. Of these, 24 labeled peptides were from a diverse range of protein kinases, including RLKs, mitogen-activated protein kinases, and calcium-dependent kinases. A significant portion of the labeling sites could not be assigned to known nucleotide binding sites. However, the fact that labeling could be competed with ATP indicates that these labeling sites might represent previously uncharacterized nucleotide binding sites. A plot of spectral counts against expression levels illustrates the high specificity of AcATP probes for protein kinases and known ATP binding proteins. This work introduces profiling of ATP binding activities of a large diversity of proteins in plant proteomes. The data have been deposited in ProteomeXchange with the identifier PXD000188.ATP binding and hydrolysis are the driving processes in all living organisms. Hundreds of cellular proteins are able to bind and hydrolyze ATP to unfold proteins, transport molecules over membranes, or phosphorylate small molecules or proteins. Proteins with very different structures are able to bind ATP. A large and important class of ATP binding proteins is that of the kinases, which transfer the gamma phosphate from ATP to substrates. Kinases, and particularly protein kinases, play pivotal roles in signaling and protein regulation.The genome of the model plant Arabidopsis thaliana encodes for over 1099 protein kinases and hundreds of other ATP binding proteins (1, 2). Protein kinases are involved in nearly all signaling cascades and regulate processes ranging from cell cycle to flowering and from immunity to germination. Many protein kinases in plants are receptor-like kinases (RLKs), often carrying extracellular leucine-rich repeats (LRRs). The RLK class contains at least 610 members (3), including famous examples such as receptors involved in development (e.g. BRI1, ER, CLV1) and immunity (e.g. FLS2, EFR). Other important classes are mitogen-activated protein (MAP) kinases (MPKs) (20 different members), MPK kinase kinase kinases (MAP3Ks) (60 different members (4)), and calcium-dependent protein kinases (CPKs) (34 different members (5)). Because of their diverse and important roles, protein kinases have been intensively studied in plant science. The current approach is to study protein kinases individually—a daunting task, considering the remaining hundreds of uncharacterized protein kinases. New approaches are necessary in order to study protein kinases and other ATP binding proteins globally rather than individually.ATP binding activities of protein kinases and other proteins can be detected globally by acyl-ATP (AcATP) probes (6, 7) (Fig. 1A). AcATP binds to the ATP pocket of ATP binding proteins and places the acyl group in close proximity to conserved lysine residues in the ATP binding pocket. The acyl phosphonate moiety serves as an electrophilic warhead that can be nucleophilically attacked by the amino group of the lysine, resulting in a covalent attachment of the acyl reporter of the AcATP probe on the lysine and a concomitant release of ATP. The reporter tag is usually a biotin to capture and identify the labeled proteins. Labeled proteins can be displayed on protein blots using streptavidin-HRP. However, because AcATP labels many ATP binding proteins and protein kinases are of relatively low abundance, mass spectrometry is more often used to identify and quantify labeling with AcATP probes. The analysis is preferably done using Xsite, a procedure that involves trypsination of the entire labeled proteome, followed by analysis of the biotinylated peptides rather than the biotinylated proteins (8). This “KiNativ ” approach provides enough depth and resolving power to monitor ∼160 protein kinases in a crude mammalian proteome (7). Of the 518 human protein kinases (9), 394 (76%) have been detected via AcATP labeling (6).Open in a separate windowFig. 1.Structure and mechanism of labeling with BHAcATP. A, BHAcATP contains ATP, an acyl phosphate reactive group, and a biotin tag. When BHAcATP binds to the ATP binding pocket of a protein, the amino group of the nearby lysine reacts with the carbonyl carbon, which results in the covalent binding of the biotin tag to the protein while ATP is released. B, typical BHAcATP labeling profile of Arabidopsis leaf proteome. Arabidopsis leaf extracts were labeled with BHAcATP and the biotinylated proteins were detected on protein blots using streptavidin-HRP. Coomassie Brilliant Blue staining indicates equal loading. Asterisks indicate endogenously biotinylated proteins MCCA and BCCP. White, black, and gray arrowheads indicate bands containing ATBP+RBCL, PGK1, and a mix of ATP binding proteins, respectively. Abbreviations: MCCA, 3-methylcrotonyl-CoA carboxylase; BCCP, biotin carboxyl carrier protein; ATPB, chloroplastic ATPase; RBCL, ribulose-bisphosphate carboxylase; PGK1, phosphoglycerate kinase-1.KiNativ has mostly been used to validate targets of human drugs that target protein kinases using competitive labeling experiments. This approach has been used to identify selective inhibitors of, for example, Parkinson''s disease protein kinase LRRK2 (10), the BMK1 and JNK MAP kinases (11, 12), and the mTOR kinase (13). Importantly, the correlation of the biological activity of protein-kinase-inhibiting drugs with inhibitor affinity detected using KiNativ is better than that achieved when affinities are determined by assays using heterologously expressed protein kinases (7). This improved correlation illustrates that assays in the native environment provide a more realistic measure of protein kinase function.In addition to characterizing inhibitors selectively, AcATP probes can also display differential ATP binding activities of protein kinases. For example, labeling with AcATP probes during infection with dengue virus displayed a 2- to 8-fold activation of a DNA-dependent protein kinase (14) Similarly, AcATP labeling revealed an unexpected Raf kinase activation in extracts upon protein kinase inhibitor treatment (7). In conclusion, profiling with AcATP probes is a powerful approach for monitoring protein kinases and offers unprecedented opportunities to identify selective protein kinase inhibitors and discover protein kinases with differential ATP binding activities.In this work, we introduce AcATP profiling of plant proteomes. In addition to the analysis of labeled peptides, we characterized labeling using gel-based approaches and discovered that biotin is often oxidized in this procedure. We also performed an in-depth analysis of labeling sites in proteins other than protein kinases, which had not been done before. We discuss labeling outside known nucleotide binding pockets and investigate the correlation of labeling sites with protein abundance. We describe 63 labeling sites of known nucleotide binding pockets, of which 24 represent a remarkable diversity of protein kinases, including several LRR-RLKs. This work launches a new approach to study ATP binding proteins in plant science.