The tyrosine gate as a potential entropic lever in the receptor-binding site of the bacterial adhesin FimH

Uropathogenic Escherichia coli (UPEC) are the major causative agents of urinary tract infections. During infection, UPEC adhere to mannosylated glycoreceptors on the urothelium via the FimH adhesin located at the tip of type 1 pili. Synthetic FimH antiadhesives such as alkyl and phenyl α-D-mannopyranosides are thus ideal candidates for the chemical interception of this crucial step in pathogenesis. The crystal structures of the FimH lectin domain in its ligand-free form and in complexes with eight medium- and high-affinity mannopyranoside inhibitors are presented. The thermodynamic profiles of the FimH-inhibitor interactions indicate that the binding of FimH to α-D-mannopyranose is enthalpy-driven and has a negative entropic change. Addition of a hydrophobic aglycon influences the binding enthalpy and can induce a favorable entropic change. The alleviation of the entropic cost is at least in part explained by increased dynamics in the tyrosine gate (Tyr48 and Tyr137) of the FimH receptor-binding site upon binding of the ligand. Ligands with a phenyl group directly linked to the anomeric oxygen of α-D-mannose introduce the largest dynamics into the Tyr48 side chain, because conjugation with the anomeric oxygen of α-D-mannose forces the aromatic aglycon into a conformation that comes into close contact (≈2.65 ?) with Tyr48. A propargyl group in this position predetermines the orientation of the aglycon and significantly decreases affinity. FimH has the highest affinity for α-D-mannopyranosides substituted with hydrophobic aglycons that are compatible in shape and electrostatic properties to the tyrosine gate, such as heptyl α-D-mannose.     

Genome-wide promoter methylation analysis in neuroblastoma identifies prognostic methylation biomarkers

Background

Accurate outcome prediction in neuroblastoma, which is necessary to enable the optimal choice of risk-related therapy, remains a challenge. To improve neuroblastoma patient stratification, this study aimed to identify prognostic tumor DNA methylation biomarkers.

Results

To identify genes silenced by promoter methylation, we first applied two independent genome-wide methylation screening methodologies to eight neuroblastoma cell lines. Specifically, we used re-expression profiling upon 5-aza-2''-deoxycytidine (DAC) treatment and massively parallel sequencing after capturing with a methyl-CpG-binding domain (MBD-seq). Putative methylation markers were selected from DAC-upregulated genes through a literature search and an upfront methylation-specific PCR on 20 primary neuroblastoma tumors, as well as through MBD- seq in combination with publicly available neuroblastoma tumor gene expression data. This yielded 43 candidate biomarkers that were subsequently tested by high-throughput methylation-specific PCR on an independent cohort of 89 primary neuroblastoma tumors that had been selected for risk classification and survival. Based on this analysis, methylation of KRT19, FAS, PRPH, CNR1, QPCT, HIST1H3C, ACSS3 and GRB10 was found to be associated with at least one of the classical risk factors, namely age, stage or MYCN status. Importantly, HIST1H3C and GNAS methylation was associated with overall and/or event-free survival.

Conclusions

This study combines two genome-wide methylation discovery methodologies and is the most extensive validation study in neuroblastoma performed thus far. We identified several novel prognostic DNA methylation markers and provide a basis for the development of a DNA methylation-based prognostic classifier in neuroblastoma.     

The long QT syndrome: a novel missense mutation in the S6 region of the KVLQT1 gene

The Romano Ward long QT syndrome (LQTS) has an autosomal dominant mode of inheritance. Patients suffer from syncopal attacks often resulting in sudden cardiac death. The main diagnostic parameter is a prolonged QT_(c) interval as judged by electro-cardiographic investigation. LQTS is a genetically heterogeneous disease with four loci having been identified to date: chromosome 11p15.5 (LQT1), 7q35–36 (LQT2), 3p21–24 (LQT3) and 4q25–26 (LQT4). The corresponding genes code for potassium channels KVLQT1 (LQT1)and HERG (LQT2) and the sodium channel SCN5A (LQT3). The KVLQT1 gene is characterized by six transmembrane domains (S1– S6), a pore region situated between the S5 and S6 domains and a C-terminal domain accounting for approximately 60% of the channel. This domain is thought to be co-associated with another protein, viz. minK (minimal potassium channel). We have studied a Romano Ward family with several affected individuals showing a severe LQTS phenotype (syncopes and occurrence of sudden death). Most affected individuals had considerable prolongations of QT_(c). By using haplotyping with a set of markers covering the four LQT loci, strong linkage was established to the LQT1 locus, whereas the other loci (LQT2, LQT3 and LQT4) could be excluded. Single-strand conformation polymorphism analysis and direct sequencing were used to screen the KVLQT1 gene for mutations in the S1–S6 region, including the pore domain. We identified a Gly-216-Arg substitution in the S6 transmembrane domain of KVLQT1. The mutation was present in all affected family members but absent in normal control individuals, providing evidence that the mutated KVLQT1-gene product indeed caused LQTS in this family. The mutated KVLQT1-gene product thus probably results in a dominant negative suppression of channel activity. Received: 25 March 1997 / Accepted: 21 April 1997     

Human mast cells express leptin and leptin receptors

Mast cells are immune cells that produce and secrete a variety of mediators and cytokines that influence various inflammatory and immune processes. Leptin is a cytokine regulating metabolic, endocrine as well as immune functions via the leptin receptor which is expressed by many immune cells. However, there are no data about leptin receptor expression in mast cells. Immunohistochemical and immunofluorescent double stainings showed the expression of leptin and leptin receptors in mast cells in human skin and several parts of the respiratory, gastrointestinal and urogenital tract. Leptin was expressed in mast cells expressing the classification marker chymase, whereas a variable expression was observed in tryptase positive mast cells. For leptin receptors, the expression pattern was tissue dependent and not related to tryptase or chymase expression. Our results demonstrate the expression of leptin and leptin receptors on mast cells, suggesting paracrine and/or autocrine immunomodulatory effects of leptin on mast cells.     

methBLAST and methPrimerDB: web-tools for PCR based methylation analysis

Background  

DNA methylation plays an important role in development and tumorigenesis by epigenetic modification and silencing of critical genes. The development of PCR-based methylation assays on bisulphite modified DNA heralded a breakthrough in speed and sensitivity for gene methylation analysis. Despite this technological advancement, these approaches require a cumbersome gene by gene primer design and experimental validation. Bisulphite DNA modification results in sequence alterations (all unmethylated cytosines are converted into uracils) and a general sequence complexity reduction as cytosines become underrepresented. Consequently, standard BLAST sequence homology searches cannot be applied to search for specific methylation primers.