Increased amylosucrase activity and specificity, and identification of regions important for activity, specificity and stability through molecular evolution

Amylosucrase is a transglycosidase which belongs to family 13 of the glycoside hydrolases and transglycosidases, and catalyses the formation of amylose from sucrose. Its potential use as an industrial tool for the synthesis or modification of polysaccharides is hampered by its low catalytic efficiency on sucrose alone, its low stability and the catalysis of side reactions resulting in sucrose isomer formation. Therefore, combinatorial engineering of the enzyme through random mutagenesis, gene shuffling and selective screening (directed evolution) was applied, in order to generate more efficient variants of the enzyme. This resulted in isolation of the most active amylosucrase (Asn387Asp) characterized to date, with a 60% increase in activity and a highly efficient polymerase (Glu227Gly) that produces a longer polymer than the wild-type enzyme. Furthermore, judged from the screening results, several variants are expected to be improved concerning activity and/or thermostability. Most of the amino acid substitutions observed in the totality of these improved variants are clustered around specific regions. The secondary sucrose-binding site and beta strand 7, connected to the important Asp393 residue, are found to be important for amylosucrase activity, whereas a specific loop in the B-domain is involved in amylosucrase specificity and stability.     

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

     

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.     

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.     

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.     

A longitudinal study to assess the persistence of vancomycin-resistant Enterococcus faecium (VREF) on an intensive broiler farm in the United Kingdom

Seven years after the ban of avoparcin, VREF could still be isolated within sectors of the UK broiler industry. The aim of this study was to assess whether there is a carryover of VREF between consecutive flocks of birds, to conduct a preliminary investigation of possible routes of entry of VREF into broiler houses and to follow the dynamics of VREF shed by growing birds. A series of nine visits were made to two of six houses on a conventional broiler farm. A total of 343 vanA VREF were recovered from environmental (95/843) and faecal (248/416) samples. Significant differences were observed in the carryover of VREF between pre- and postcohort postcleaning and disinfection visits (RR 0.57, P=0.006). Ninety-nine percent of the VREF isolates were resistant to more than five antimicrobials, with 42 isolates (n=49) positive for erm(B) and 32 (n=40) for vat(E). Pulsed field gel electrophoresis (PFGE) typing identified 50 PFGE types within 15 different PFGE clusters of 90% similarity, demonstrating a high level of genetic diversity within VREF populations from epidemiologically related broiler flocks and broiler houses. Further characterization of Tn1546 from different clones showed a low diversity of Tn-types, suggesting horizontal transfer of resistance determinants between different genetic clones. Thus, this study does not only show the persistence of VREF but also of multi-drug resistant lineages of VREF.     

Building blood vessels--stem cell models in vascular biology

Spheroids of differentiating embryonic stem cells, denoted embryoid bodies, constitute a high-quality model for vascular development, particularly well suited for loss-of-function analysis of genes required for early embryogenesis. This review examines vasculogenesis and angiogenesis in murine embryoid bodies and discusses the promise of stem cell-based models for the study of human vascular development.