Development of both colorimetric and fluorescence heparinase activity assays using fondaparinux as substrate

Because tumors and other diseases are characterized by increased heparanase levels, human heparanase is a promising drug target and diagnostic marker. Therefore, methods are needed to determine heparanase activity and to examine potential inhibitors. Because of substrate comparability, we used the bacterial enzyme heparinase II (heparinase) for the assay development. Usually the substrate of heparanase assays is heparan sulfate, which has several disadvantages. Because of that, we used fondaparinux, which is being cleaved by both heparanase and heparinase. Two concepts to detect its degradation were examined: measurement of anti-factor Xa activity of fondaparinux and its direct quantification with the fluorescent sensor polymer-H. Using fondaparinux as substrate, the anti-factor Xa assay was shsown to be appropriate to determine heparinase activity. The detection with polymer-H was easier and even faster to perform. Linearity was given with fondaparinux as well as heparan sulfate, and heparin as substrates, but fondaparinux turned out to be most suitable. By modifications (incubation time, fondaparinux concentration, and polymer-H concentration), the limit of quantification and the linear range can be adapted to the respective requirements. In conclusion, a simple, accurate, and robust heparinase assay was developed. It is suitable for heparinase quality control and testing heparinase inhibitors and could be adapted to heparanase.     

Affinity pulldown of γ‐secretase and associated proteins from human and rat brain

γ‐Secretase is a transmembrane protease complex responsible for the processing of a multitude of type 1 transmembrane proteins, including amyloid precursor protein (APP) and Notch. A functional complex is dependent on the assembly of four proteins: presenilin (PS), nicastrin, Aph‐1 and Pen‐2. Little is known about how the substrates are selected by γ‐secretase, but it has been suggested that γ‐secretase associated proteins (GSAPs) could be of importance. For instance, it was recently reported from studies in cell lines that TMP21, a transmembrane protein involved in trafficking, binds to γ‐secretase and regulates the processing of APP‐derived substrates without affecting Notch cleavage. Here, we present an efficient and selective method for purification and analysis of γ‐secretase and GSAPs. Microsomal membranes were prepared from rat or human brain and incubated with a γ‐secretase inhibitor coupled to biotin via a long linker and a S‐S bridge. After pulldown using streptavidin beads, bound proteins were eluted under reducing conditions and digested by trypsin. The tryptic peptides were subjected to LC‐MS/MS analysis, and proteins were identified by sequence data from MS/MS spectra. All of the known γ‐secretase components were identified. Interestingly, TMP21 and the PS associated protein syntaxin1 were associated to γ‐secretase in rat brain. We suggest that the present method can be used for further studies on the composition of the γ‐secretase complex.     

A high-throughput system for genome-wide measurement of genetic recombination in Arabidopsis thaliana based on transgenic markers

A high-throughput system for the measurement of recombination frequencies in the genetic model plant, Arabidopsis thaliana, is described. It is based on 21 mono-transgenic isogenic lines harboring antibiotic resistance genes on all five chromosomes. Recombination between pairs of gene insertions in repulsion phase that confer resistance against kanamycin (kan) and hygromycin (hyg) is determined by a phenotypic assay of progeny (DART: Double Antibiotic Resistance Technique). DART allows testing for the influence of numerous environmental and genetic factors, including candidate genes, on recombination frequencies in specific genomic regions as well as the entire genome. Its usefulness is demonstrated by investigating the effects of UV treatment, different temperature and phosphorus supply regimes, and sex on recombination frequencies for all five chromosomes of A. thaliana. Electronic Publication     

Expression of a bacterial carotene hydroxylase gene (crtZ) enhances UV tolerance in tobacco

Carotenoids are essential components of the photosynthetic apparatus involved in plant photoprotection. To investigate the protective role of zeaxanthin under high light and UV stress we have increased the capacity for its biosynthesis in tobacco plants (Nicotiana tabacum L. cv. Samsun) by transformation with a heterologous carotenoid gene encoding -carotene hydroxylase (crtZ) from Erwinia uredovora under constitutive promoter control. This enzyme is responsible for the conversion of -carotene into zeaxanthin. Although the total pigment content of the transgenics was similar to control plants, the transformants synthesized zeaxanthin more rapidly and in larger quantities than controls upon transfer to high-intensity white light. Low-light-adapted tobacco plants were shown to be susceptible to UV exposure and therefore chosen for comparative analysis of wild-type and transgenics. Overall effects of UV irradiation were studied by measuring bioproductivity and pigment content. The UV exposed transformed plants maintained a higher biomass and a greater amount of photosynthetic pigments than controls. For revelation of direct effects, photosynthesis, pigment composition and chlorophyll fluorescence were examined immediately after UV treatment. Low-light-adapted plants of the crtZ transgenics showed less reduction in photosynthetic oxygen evolution and had higher chlorophyll fluorescence levels in comparison to control plants. After 1 h of high-light pre-illumination and subsequent UV exposure a greater amount of xanthophyll cycle pigments was retained in the transformants. In addition, the transgenic plants suffered less lipid peroxidation than the wild-type after treatment with the singlet-oxygen generator rose bengal. Our results indicate that an enhancement of zeaxanthin formation in the presence of a functional xanthophyll cycle contributes to UV stress protection and prevention of UV damage.     

Body adiposity index and other indexes of body composition in the SAPHIR study: Association with cardiovascular risk factors

Objective:

The accuracy of anthropometric surrogate markers such as the body adiposity index (BAI) and other common indexes like the body mass index (BMI), waist‐to‐hip ratio (WHR) and waist‐to‐height ratio (WHtR) to predict metabolic sequelae is essential for its use in clinical practice.

Design and Methods:

Thus, we evaluated the strength of BAI and other indexes to relate with anthropometric parameters, adipocytokines, blood lipids, parameters of glucose‐homeostasis and blood pressure in 1,770 patients from the Salzburg Atherosclerosis Prevention Program in Subjects at High Individual Risk (SAPHIR) study in a crosssectional design. Measurements were BAI, BMI, WHR, WHtR, abdominal subcutaneous and visceral adipose tissue (aSAT and VAT), total body adipose tissue mass, body weight, waist‐ and hip circumference (WC and HC), leptin, adiponectin, high‐density lipoprotein‐cholesterol (HDL‐C), low‐density lipoprotein‐cholesterol (LDL‐C), triglycerides (TG), fasting plasma glucose, fasting plasma insulin, the homeostasis model assessment of insulin resistance (HOMAIR), systolic and diastolic blood pressure.

Results and Conclusions:

BAI was significantly associated with leptin and HC. We conclude that BAI was the best calculator for leptin. BAI was inferior to BMI to predict anthropometric parameters other than HC, adiponectin, blood lipids, parameters of glucose homeostasis, and blood pressure in this cross‐sectional study.     

Redox regulation of chloroplast enzymes in Galdieria sulphuraria in view of eukaryotic evolution

Redox modulation is a general mechanism for enzyme regulation, particularly for the post-translational regulation of the Calvin cycle in chloroplasts of green plants. Although red algae and photosynthetic protists that harbor plastids of red algal origin contribute greatly to global carbon fixation, relatively little is known about post-translational regulation of chloroplast enzymes in this important group of photosynthetic eukaryotes. To address this question, we used biochemistry, phylogenetics and analysis of recently completed genome sequences. We studied the functionality of the chloroplast enzymes phosphoribulokinase (PRK, EC 2.7.1.19), NADP-dependent glyceraldehyde 3-phosphate dehydrogenase (NADP-GAPDH, GapA, EC 1.2.1.13), fructose 1,6-bisphosphatase (FBPase, EC 3.1.3.11) and glucose 6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49), as well as NADP-malate dehydrogenase (NADP-MDH, EC 1.1.1.37) in the unicellular red alga Galdieria sulphuraria (Galdieri) Merola. Despite high sequence similarity of G. sulphuraria proteins to those of other photosynthetic organisms, we found a number of distinct differences. Both PRK and GAPDH co-eluted with CP12 in a high molecular weight complex in the presence of oxidized glutathione, although Galdieria CP12 lacks the two cysteines essential for the formation of the N-terminal peptide loop present in higher plants. However, PRK inactivation upon complex formation turned out to be incomplete. G6PDH was redox modulated, but remained in its tetrameric form; FBPase was poorly redox regulated, despite conservation of the two redox-active cysteines. No indication for the presence of plastidic NADP-MDH (and other components of the malate valve) was found.     

TFF3 and EGF induce different migration patterns of intestinal epithelial cells in vitro and trigger increased internalization of E-cadherin.

BACKGROUND/AIMS: TFF3, a member of the TFF (trefoil factor family) peptides, and epidermal growth factor (EGF) actively support the repair of mucosal barriers, particularly during restitution. The aim of this study was to compare the motogenic effects of TFF3 and EGF. METHODS: The influence of recombinant human TFF3 (dimeric form) and EGF on the migration of IEC-18 cells was characterized in an in vitro restitution model (scratch wound assay) with the help of time-lapse video microscopy, morphometry, and immunocytochemistry including confocal laser scanning microscopy. RESULTS: TFF3- and EGF-treated cells re-populated the wounded area via different migration patterns; TFF3 treatment resulted in the formation of continuous sheets of migrating cells with only a few gaps. In contrast, EGF-treated cells formed a network of migrating cells (often with a fibroblast-like morphology) with numerous gaps and only punctual contacts. TFF3 and EGF treatment also changed the localization of E-cadherin indicating endocytotic recycling and/or degradation of E-cadherin. CONCLUSION: TFF3, in contrast to EGF, enhanced a collective cell migration ensuring a precise coverage of the re-populated area avoiding gaps.     

Domestic Dogs Use Contextual Information and Tone of Voice when following a Human Pointing Gesture

Domestic dogs are skillful at using the human pointing gesture. In this study we investigated whether dogs take contextual information into account when following pointing gestures, specifically, whether they follow human pointing gestures more readily in the context in which food has been found previously. Also varied was the human''s tone of voice as either imperative or informative. Dogs were more sustained in their searching behavior in the ‘context’ condition as opposed to the ‘no context’ condition, suggesting that they do not simply follow a pointing gesture blindly but use previously acquired contextual information to inform their interpretation of that pointing gesture.Dogs also showed more sustained searching behavior when there was pointing than when there was not, suggesting that they expect to find a referent when they see a human point. Finally, dogs searched more in high-pitched informative trials as opposed to the low-pitched imperative trials, whereas in the latter dogs seemed more inclined to respond by sitting. These findings suggest that a dog''s response to a pointing gesture is flexible and depends on the context as well as the human''s tone of voice.     

A role for MCP-1/CCR2 in interstitial lung disease in children

Background

Particulate matter (PM) can exacerbate allergic airway diseases. Although health effects of PM with a diameter of less than 100 nm have been focused, few studies have elucidated the correlation between the sizes of particles and aggravation of allergic diseases. We investigated the effects of nano particles with a diameter of 14 nm or 56 nm on antigen-related airway inflammation.

Methods

ICR mice were divided into six experimental groups. Vehicle, two sizes of carbon nano particles, ovalbumin (OVA), and OVA + nano particles were administered intratracheally. Cellular profile of bronchoalveolar lavage (BAL) fluid, lung histology, expression of cytokines, chemokines, and 8-hydroxy-2'-deoxyguanosine (8-OHdG), and immunoglobulin production were studied.

Results

Nano particles with a diameter of 14 nm or 56 nm aggravated antigen-related airway inflammation characterized by infiltration of eosinophils, neutrophils, and mononuclear cells, and by an increase in the number of goblet cells in the bronchial epithelium. Nano particles with antigen increased protein levels of interleukin (IL)-5, IL-6, and IL-13, eotaxin, macrophage chemoattractant protein (MCP)-1, and regulated on activation and normal T cells expressed and secreted (RANTES) in the lung as compared with antigen alone. The formation of 8-OHdG, a proper marker of oxidative stress, was moderately induced by nano particles or antigen alone, and was markedly enhanced by antigen plus nano particles as compared with nano particles or antigen alone. The aggravation was more prominent with 14 nm of nano particles than with 56 nm of particles in overall trend. Particles with a diameter of 14 nm exhibited adjuvant activity for total IgE and antigen-specific IgG₁ and IgE.

Conclusion

Nano particles can aggravate antigen-related airway inflammation and immunoglobulin production, which is more prominent with smaller particles. The enhancement may be mediated, at least partly, by the increased local expression of IL-5 and eotaxin, and also by the modulated expression of IL-13, RANTES, MCP-1, and IL-6.