Generation of DNA nanocircles containing mismatched bases

The DNA mismatch repair (MMR) system recognizes and repairs errors that escaped the proofreading function of DNA polymerases. To study molecular details of the MMR mechanism, in vitro biochemical assays require specific DNA substrates carrying mismatches and strand discrimination signals. Current approaches used to generate MMR substrates are time-consuming and/or not very flexible with respect to sequence context. Here we report an approach to generate small circular DNA containing a mismatch (nanocircles). Our method is based on the nicking of PCR products resulting in single-stranded 3' overhangs, which form DNA circles after annealing and ligation. Depending on the DNA template, one can generate mismatched circles containing a single hemimethylated GATC site (for use with the bacterial system) and/or nicking sites to generate DNA circles nicked in the top or bottom strand (for assays with the bacterial or eukaryotic MMR system). The size of the circles varied (323 to 1100 bp), their sequence was determined by the template DNA, and purification of the circles was achieved by ExoI/ExoIII digestion and/or gel extraction. The quality of the nanocircles was assessed by scanning-force microscopy and their suitability for in vitro repair initiation was examined using recombinant Escherichia coli MMR proteins.     

One Enzyme,Two Functions: PON2 PREVENTS MITOCHONDRIAL SUPEROXIDE FORMATION AND APOPTOSIS INDEPENDENT FROM ITS LACTONASE ACTIVITY*

The human enzyme paraoxonase-2 (PON2) has two functions, an enzymatic lactonase activity and the reduction of intracellular oxidative stress. As a lactonase, it dominantly hydrolyzes bacterial signaling molecule 3OC12 and may contribute to the defense against pathogenic Pseudomonas aeruginosa. By its anti-oxidative effect, PON2 reduces cellular oxidative damage and influences redox signaling, which promotes cell survival. This may be appreciated but also deleterious given that high PON2 levels reduce atherosclerosis but may stabilize tumor cells. Here we addressed the unknown mechanisms and linkage of PON2 enzymatic and anti-oxidative function. We demonstrate that PON2 indirectly but specifically reduced superoxide release from the inner mitochondrial membrane, irrespective whether resulting from complex I or complex III of the electron transport chain. PON2 left O₂^˙̄ dismutase activities and cytochrome c expression unaltered, and it did not oxidize O₂^˙̄ but rather prevented its formation, which implies that PON2 acts by modulating quinones. To analyze linkage to hydrolytic activity, we introduced several point mutations and show that residues His¹¹⁴ and His¹³³ are essential for PON2 activity. Further, we mapped its glycosylation sites and provide evidence that glycosylation, but not a native polymorphism Ser/Cys³¹¹, was critical to its activity. Importantly, none of these mutations altered the anti-oxidative/anti-apoptotic function of PON2, demonstrating unrelated activities of the same protein. Collectively, our study provides detailed mechanistic insight into the functions of PON2, which is important for its role in innate immunity, atherosclerosis, and cancer.     

The zebrafish mutant bumper shows a hyperproliferation of lens epithelial cells and fibre cell degeneration leading to functional blindness

The development of the eye lens is one of the classical paradigms of induction during embryonic development in vertebrates. But while there have been numerous studies aimed at discovering the genetic networks controlling early lens development, comparatively little is known about later stages, including the differentiation of secondary lens fibre cells. The analysis of mutant zebrafish isolated in forward genetic screens is an important way to investigate the roles of genes in embryogenesis. In this study we describe the zebrafish mutant bumper (bum), which shows a transient, tumour-like hyperproliferation of the lens epithelium as well as a progressively stronger defect in secondary fibre cell differentiation, which results in a significantly reduced lens size and ectopic location of the lens within the neural retina. Interestingly, the initial hyperproliferation of the lens epithelium in bum spontaneously regresses, suggesting this mutant as a valuable model to study the molecular control of tumour progression/suppression. Behavioural analyses demonstrate that, despite a morphologically normal retina, larval and adult bum^?/? zebrafish are functionally blind. We further show that these fish have defects in their craniofacial skeleton with normal but delayed formation of the scleral ossicles within the eye, several reduced craniofacial bones resulting in an abnormal skull shape, and asymmetric ectopic bone formation within the mandible. Genetic mapping located the mutation in bum to a 4 cM interval on chromosome 7 with the closest markers located at 0.2 and 0 cM, respectively.     

Hydrostatic pressurization and depletion of trapped lubricant pool during creep contact of a rippled indenter against a biphasic articular cartilage layer

This study presents an analysis of the contact of a rippled rigid impermeable indenter against a cartilage layer, which represents a first simulation of the contact of rough cartilage surfaces with lubricant entrapment. Cartilage was modeled with the biphasic theory for hydrated soft tissues, to account for fluid flow into or out of the lubricant pool. The findings of this study demonstrate that under contact creep, the trapped lubricant pool gets depleted within a time period on the order of seconds or minutes as a result of lubricant flow into the articular cartilage. Prior to depletion, hydrostatic fluid load support across the contact interface may be enhanced by the presence of the trapped lubricant pool, depending on the initial geometry of the lubricant pool. According to friction models based on the biphasic nature of the tissue, this enhancement in fluid load support produces a smaller minimum friction coefficient than would otherwise be predicted without a lubricant pool. The results of this study support the hypothesis that trapped lubricant decreases the initial friction coefficient following load application, independently of squeeze-film lubrication effects.     

Purification,structural and immunological characterization of a timothy grass (Phleum pratense) pollen allergen,Phl p 4, with cross-reactive potential

Almost 500 million people worldwide suffer from Type I allergy, a genetically determined immunodisorder which is based on the production of IgE antibodies against per se harmless antigens (allergens). Due to their worldwide distribution and heavy pollen production, grasses represent a major allergen source for approximately 40% of allergic patients. We purified Phl p 4, a major timothy grass (Phleum pratense) pollen allergen with a molecular mass of 61.3 kDa and a pl of 9.6 to homogeneity. Circular dichroism spectroscopical analysis indicates that Phl p 4 contains a mixed alpha-helical/beta-pleated secondary structure and, unlike many other allergens, showed no reversible unfolding after thermal denaturation. We show that Phl p 4 is a major allergen which reacts with IgE antibodies of 75% of grass pollen allergic patients (n=150) and induces basophil histamine release as well as immediate type skin reactions in sensitized individuals. Phl p 4-specific IgE from three patients as well as two rabbit-anti Phl p 4 antisera cross-reacted with allergens present in pollen of trees, grasses, weeds as well as plant-derived food. Rabbit antibodies raised against Phl p 4 also inhibited the binding of allergic patients IgE to Phl p 4. Phl p 4 may thus be used for diagnosis and treatment of sensitized allergic patients.     

Golgi complex fragmentation in G2/M transition: An organelle-based cell-cycle checkpoint

In mammalian cells, the Golgi complex is organized into a continuous membranous system known as the Golgi ribbon, which is formed by individual Golgi stacks that are laterally connected by tubular bridges. During mitosis, the Golgi ribbon undergoes extensive fragmentation through a multistage process that is required for its correct partitioning into the daughter cells. Importantly, inhibition of this Golgi disassembly results in cell-cycle arrest at the G2 stage, suggesting that accurate inheritance of the Golgi complex is monitored by a "Golgi mitotic checkpoint." Here, we discuss the mechanisms and regulation of the Golgi ribbon breakdown and briefly comment on how Golgi partitioning may inhibit G2/M transition.     

Restrained and External-Emotional Eating Patterns in Young Overweight Children–Results of the Ulm Birth Cohort Study

Childhood obesity is one of the greatest public health challenges in Western countries. Abnormal eating behavior is thought to be a developmental trajectory to obesity. The Eating Pattern Inventory for Children (EPI-C) has not been used for children as young as eight years, and possible associations with body weight have not yet been established. Five hundred and twenty-one children of the Ulm Birth Cohort Study (UBCS; age eight) filled out the EPI-C and BMI was assessed. Adequacy of the scales was tested with confirmatory factor analysis and a MANOVA and cluster analysis established associations between eating patterns and BMI. The factor structure of the EPI-C was confirmed (GFI = .968) and abnormal eating behavior was associated with overweight (χ²(8) = 79.29, p<.001). The EPI-C is a valid assessment tool in this young age group. Overweight children consciously restrain their eating.     

Transcriptomic and reverse genetic analyses of branched-chain fatty acid and acyl sugar production in Solanum pennellii and Nicotiana benthamiana

Acyl sugars containing branched-chain fatty acids (BCFAs) are exuded by glandular trichomes of many species in Solanaceae, having an important defensive role against insects. From isotope-feeding studies, two modes of BCFA elongation have been proposed: (1) fatty acid synthase-mediated two-carbon elongation in the high acyl sugar-producing tomato species Solanum pennellii and Datura metel; and (2) alpha-keto acid elongation-mediated one-carbon increments in several tobacco (Nicotiana) species and a Petunia species. To investigate the molecular mechanisms underlying BCFAs and acyl sugar production in trichomes, we have taken a comparative genomic approach to identify critical enzymatic steps followed by gene silencing and metabolite analysis in S. pennellii and Nicotiana benthamiana. Our study verified the existence of distinct mechanisms of acyl sugar synthesis in Solanaceae. From microarray analyses, genes associated with alpha-keto acid elongation were found to be among the most strongly expressed in N. benthamiana trichomes only, supporting this model in tobacco species. Genes encoding components of the branched-chain keto-acid dehydrogenase complex were expressed at particularly high levels in trichomes of both species, and we show using virus-induced gene silencing that they are required for BCFA production in both cases and for acyl sugar synthesis in N. benthamiana. Functional analysis by down-regulation of specific KAS I genes and cerulenin inhibition indicated the involvement of the fatty acid synthase complex in BCFA production in S. pennellii. In summary, our study highlights both conserved and divergent mechanisms in the production of important defense compounds in Solanaceae and defines potential targets for engineering acyl sugar production in plants for improved pest tolerance.