Close linkage between X-linked ectodermal dysplasia and a cloned DNA sequence detecting a two allele restriction fragment length polymorphism in the region Xp11-q12

Summary EDA (ectodermal dysplasia, anhidrotic) is an X-linked recessive disorder characterized by hypohidrosis, hypoor anodontia, and hypotrichosis. A possible linkage between the gene for EDA and a number of restriction fragment length polymorphisms (RFLPs) spread over the X chromosome was investigated in two Danish families segregating EDA. No recombination between the gene for EDA and our probe pTAK8, which detects a two allele polymorphism in the region Xp11-q12, was found in nine informative meiotic events (seven of which are phase known), giving a maximal lod score of 2.41 at a recombination fraction of 0.00. This juxtacentromeric location of the gene for EDA agrees well with the linkage data obtained with the other markers used in this study.     

Survival in treated hypertension: follow up study after two decades

Objective: To compare survival and cause specific mortality in hypertensive men with non-hypertensive men derived from the same random population, and to study mortality and morbidity from cardiovascular diseases in the hypertensive men in relation to effects on cardiovascular risk factors during 22-23 years of follow up. Design: Prospective, population based observational study. Subjects and methods: 686 hypertensive men aged 47-55 at screening compared with 6810 non-hypertensive men. The hypertensive men were having stepped care treatment with either β adrenergic blocking drugs, thiazide diuretics, or combination treatment. Mortality, morbidity, and adverse effects were registered at yearly examinations and from death certificates. Main outcome measures: All cause mortality and cause specific mortality. Results: Treated hypertensive men had significantly impaired probability of total survival as well as survival from coronary heart disease and stroke. All cause mortality as well as coronary heart disease and stroke mortality were very similar in hypertensive men and normotensive men during the first decade, but increased steadily thereafter despite continuous good blood pressure control. Smoking, signs of target organ damage, and high serum cholesterol levels, but not blood pressure at screening, were significantly related to the incidence of coronary heart disease during follow up. In time dependent Cox’s regression analysis, the incidence of coronary heart disease was significantly related only to serum cholesterol concentrations in the study. Cancer mortality was almost similar in treated hypertensive men (61/686, 8.9%) and non-hypertensive men (732/6810, 10.8%). Conclusion: Treated hypertensive men had impaired survival and increased mortality from cardiovascular disease compared with non-hypertensive men of similar age. These differences were observed during the second decade of follow up. During an observation period of 22-23 years—about 15 000 patient years—hypertensive men receiving diuretics and β blockers had no increased risk of cancer or non-cardiovascular disease.

Key messages

• Hypertension is a prevalent (10-20%) and important risk factor for cardiovascular disease.
• In controlled trials over 3-5 years drug treatment for hypertension prevents these complications, but little is known about long term prognosis
• During 20-22 years treated hypertensive men had a significantly increased mortality, especially from coronary heart disease, compared with non-hypertensive men from the same population
• The high incidence of myocardial infarction was related to organ damage, smoking, and cholesterol at the time of entry to the study, and to achieved serum cholesterol concentrations during follow up
• The poor prognosis for mortality from coronary heart disease is dependent upon strict monitoring of serum cholesterol concentrations

     

New Approaches for Absolute Quantification of Stable‐Isotope‐Labeled Peptide Standards for Targeted Proteomics Based on a UV Active Tag

Targeted proteomics depends on the availability of stable isotope labeled (SIL) peptide standards, which for absolute protein quantification need to be absolutely quantified. In the present study, three new approaches for absolute quantification of SIL peptides are developed. All approaches rely on a quantification tag (Qtag) with a specific UV absorption. The Qtag is attached to the peptide during synthesis and is removed by tryptic digestion under standard proteomics workflow conditions. While one quantification method (method A) is designed to allow the fast and economic production of absolutely quantified SIL peptides, two other methods (methods B and C) are developed to enable the straightforward re‐quantification of SIL peptides after reconstitution to control and monitor known problems related to peptide solubility, precipitation, and adhesion to vials. All methods yield consistent results when compared to each other and when compared to quantification by amino acid analysis. The precise quantitation methods are used to characterize the in vivo specificity of the H3 specific histone methyltransferase EZH2.     

Use of bioluminescence for monitoring the viability of individual Pseudomonas putida KT2442 cells

Exponential phase cells of Pseudomonas putida KT2442 rapidly lost viability when incubated at 0°C without entering a viable but non-culturable state. The majority of dead cells retained their cellular integrity and contained DNA. However, their cellular rRNA content was substantially reduced. By employing a luciferase-marked derivative of P. putida KT2442 in combination with a highly sensitive low-light imaging system, live and dead cells could be distinguished.     

Structure and dynamics of small soluble Aβ(1-40) oligomers studied by top-down hydrogen exchange mass spectrometry

Aβ peptides can assemble into amyloid fibrils, which represent one of the hallmarks of Alzheimer's disease. Recent studies, however, have focused on the behavior of small soluble Aβ oligomers that possess a much greater neurotoxicity than mature fibrils. The structural characterization of these oligomers remains difficult because of their highly dynamic and polymorphic nature. This work explores the behavior of Aβ(1-40) in a slightly basic solution (pH 9.3) at a low salt concentration (10 mM ammonium acetate). These conditions lead to the formation of small oligomers, without any signs of fibrillation for several hours. The structure and dynamics of these oligomers were characterized by circular dichroism spectroscopy, size exclusion chromatography, and millisecond time-resolved hydrogen exchange mass spectrometry (MS). Our results reveal rapid interconversion between Aβ(1-40) oligomers and monomers. The mole fraction of monomeric molecules is on the order of 40%. Oligomers consist of ~4 Aβ(1-40) molecules on average, and the resulting assemblies have a predominantly β-sheet secondary structure. Hydrogen exchange proceeds in the EX1 regime. This feature allows the application of conformer-specific top-down MS. Electron capture dissociation is used for interrogating the deuteration behavior of the Aβ(1-40) oligomers. This approach provides a spatial resolution of ~2 residues. The backbone amide deuteration pattern uncovered in this way is consistent with a β-turn-β motif for L17-M35. The N-terminus is involved in hydrogen bonding, as well, whereas protection gradually tapers off for C-terminal residues 35-40. Our data are consistent with earlier proposals, according to which Aβ(1-40) oligomers adopt a β-barrel structure. In general terms, this study demonstrates how top-down MS with precursor ion selection can be employed for structural studies of specific protein conformers within a heterogeneous mix.     

Human small cell lung cancer NYH cells resistant to the bisdioxopiperazine ICRF-187 exhibit a functional dominant Tyr165Ser mutation in the Walker A ATP binding site of topoisomerase II alpha

Bisdioxopiperazine anti-cancer agents are catalytic inhibitors of topoisomerase II which by unknown means lock the enzyme in a closed clamp form and inhibit its ATPase activity. In order to demarcate a putative pharmacophore, we here describe a novel Tyr165Ser mutation in the enzyme's Walker A ATP binding site leading to specific bisdioxopiperazine resistance when transformed into a temperature-conditional yeast system. The Tyr165Ser mutation differed from a previously described Arg162Gln by being heterozygous and by purified Tyr165Ser enzyme being drug-resistant in a kinetoplast DNA decatenation enzymatic assay. This suggested dominant nature of Tyr165Ser was supported by co-transformation studies in yeast of plasmids carrying wild type and mutant genes. These results enable a model of the bisdioxopiperazine pharmacophore using the proposed asymmetric ATP hydrolysis of the enzyme.     

Complete genome sequence of Desulfocapsa sulfexigens,a marine deltaproteobacterium specialized in disproportionating inorganic sulfur compounds

Desulfocapsa sulfexigens SB164P1 (DSM 10523) belongs to the deltaproteobacterial family Desulfobulbaceae and is one of two validly described members of its genus. This strain was selected for genome sequencing, because it is the first marine bacterium reported to thrive on the disproportionation of elemental sulfur, a process with a unresolved enzymatic pathway in which elemental sulfur serves both as electron donor and electron acceptor. Furthermore, in contrast to its phylogenetically closest relatives, which are dissimilatory sulfate-reducers, D. sulfexigens is unable to grow by sulfate reduction and appears metabolically specialized in growing by disproportionating elemental sulfur, sulfite or thiosulfate with CO₂ as the sole carbon source. The genome of D. sulfexigens contains the set of genes that is required for nitrogen fixation. In an acetylene assay it could be shown that the strain reduces acetylene to ethylene, which is indicative for N-fixation. The circular chromosome of D. sulfexigens SB164P1 comprises 3,986,761 bp and harbors 3,551 protein-coding genes of which 78% have a predicted function based on auto-annotation. The chromosome furthermore encodes 46 tRNA genes and 3 rRNA operons.     

Patterns of time since last meal revealed by sparse PCA in an observational LC–MS based metabolomics study

In metabolomics studies, liquid chromatography mass spectrometry (LC–MS) provides comprehensive information on biological samples. However, extraction of few relevant metabolites from this large and complex data is cumbersome. To resolve this issue, we have employed sparse principal component analysis (SPCA) to capture the underlying patterns and select relevant metabolites from LC–MS plasma profiles. The study involves a small pilot cohort with 270 subjects where each subject’s time since last meal (TSLM) has been recorded prior to plasma sampling. Our results have demonstrated that both PCA and SPCA can capture the TSLM patterns. Nevertheless, SPCA provides more easily interpretable loadings in terms of selection of relevant metabolites, which are identified as amino acids and lyso-lipids. This study demonstrates the utility of SPCA as a pattern recognition and variable selection tool in metabolomics. Furthermore, amino acids and lyso-lipids are determined as dominating compounds in response to TSLM.     

A novel alkyne cholesterol to trace cellular cholesterol metabolism and localization

Cholesterol is an important lipid of mammalian cells and plays a fundamental role in many biological processes. Its concentration in the various cellular membranes differs and is tightly regulated. Here, we present a novel alkyne cholesterol analog suitable for tracing both cholesterol metabolism and localization. This probe can be detected by click chemistry employing various reporter azides. Alkyne cholesterol is accepted by cellular enzymes from different biological species (Brevibacterium, yeast, rat, human) and these enzymes include cholesterol oxidases, hydroxylases, and acyl transferases that generate the expected metabolites in in vitro and in vivo assays. Using fluorescence microscopy, we studied the distribution of cholesterol at subcellular resolution, detecting the lipid in the Golgi and at the plasma membrane, but also in the endoplasmic reticulum and mitochondria. In summary, alkyne cholesterol represents a versatile, sensitive, and easy-to-use tool for tracking cellular cholesterol metabolism and localization as it allows for manifold detection methods including mass spectrometry, thin-layer chromatography/fluorography, and fluorescence microscopy.     

Characterization of the interaction of interleukin-8 with hyaluronan, chondroitin sulfate, dermatan sulfate and their sulfated derivatives by spectroscopy and molecular modeling

The interactions between glycosaminoglycans (GAGs), important components of the extracellular matrix, and proteins such as growth factors and chemokines play critical roles in cellular regulation processes. Therefore, the design of GAG derivatives for the development of innovative materials with bio-like properties in terms of their interaction with regulatory proteins is of great interest for tissue engineering and regenerative medicine. Previous work on the chemokine interleukin-8 (IL-8) has focused on its interaction with heparin and heparan sulfate, which regulate chemokine function. However, the extracellular matrix contains other GAGs, such as hyaluronic acid (HA), dermatan sulfate (DS) and chondroitin sulfate (CS), which have so far not been characterized in terms of their distinct molecular recognition properties towards IL-8 in relation to their length and sulfation patterns. NMR and molecular modeling have been in great part the methods of choice to study the structural and recognition properties of GAGs and their protein complexes. However, separately these methods have challenges to cope with the high degree of similarity and flexibility that GAGs exhibit. In this work, we combine fluorescence spectroscopy, NMR experiments, docking and molecular dynamics simulations to study the configurational and recognition properties of IL-8 towards a series of HA and CS derivatives and DS. We analyze the effects of GAG length and sulfation patterns in binding strength and specificity, and the influence of GAG binding on IL-8 dimer formation. Our results highlight the importance of combining experimental and theoretical approaches to obtain a better understanding of the molecular recognition properties of GAG-protein systems.