吗啡经阿片μ受体增强坐骨神经损伤大鼠脊髓Ⅱ板层抗氟化物酸性磷酸酶活性

目的　探讨吗啡增强受损伤坐骨神经大鼠脊髓Ⅱ板层抗氟化物酸性磷酸酶 (fluoride resistantacidphosphatase ,FRAP)阳性反应物面积的可能途径。方法　用显示FRAP的组织化学方法 ,借助图像分析仪检测损伤SD大鼠坐骨神经后 ,15d和 30d生理盐水组、吗啡组和纳络酮 +吗啡组大鼠脊髓Ⅱ板层内FRAP阳性反应物的面积。结果　损伤坐骨神经后 ,生理盐水组、吗啡组和纳络酮 +吗啡组大鼠脊髓Ⅱ板层FRAP阳性反应区均有不同程度的缺失。 3组大鼠损伤坐骨神经后 30d的层FRAP阳性反应面积较坐骨神经损伤后 15d的FRAP阳性反应物面积呈恢复性增大。但纳络酮 +吗啡组损伤坐骨神经后脊髓Ⅱ板层FRAP阳性反应物面积较吗啡组的明显减少 ,与生理盐水组的FRAP阳性反应物面积相似。结论　吗啡是通过阿片 μ受体的介导促进受损伤坐骨神经大鼠脊髓Ⅱ板层FRAP阳性反应物面积的增大。     

Fluoride-resistant acid phosphatase-containing neurones in dorsal root ganglia are separate from those containing substance P or somatostatin

The distribution of fluoride-resistant acid phosphatase, substance P and somatostatin were investigated in the dorsal horn of the spinal cord and in dorsal root ganglia. In the dorsal horn, the distribution of fluoride-resistant acid phosphatase closely paralleled that of somatostatin and only partly overlapped with that of substance P. In sensory ganglia, none of the fluoride-resistant acid phosphatase-containing neurones contained either substance P or somatostatin. The results suggest the existence of a population of fluoride-resistant phosphatase-positive sensory neurones which is distinct from neurones containing either of these peptides.     

心理干预在儿童功能性再发性腹痛治疗中的应用体会

[目的]探讨心理干预在儿童功能性再发性腹痛治疗中的作用。[方法]应用心理干预结合中药治疗儿童功能性再发性腹痛38例,并与单纯中药治疗30例进行对照分析。[结果]2组有效率比较,治疗组优于对照组,治疗组明显高于对照组(P<0·01)。[结论]心理干预结合中药治疗儿童功能性再发性腹痛疗效显著。     

Polyphenols,methylxanthines, and antioxidant capacity of chocolates produced in Serbia

Different kinds of chocolates produced in Serbia were analyzed regarding total polyphenol, flavonoid and proanthocyanidin content using spectrophotometric methods. Flavan-3ols and methylxanthines in all samples were determined with RP-HPLC. DPPH, FRAP, ABTS and ORAC assays were applied for measuring antioxidant capacity. The average of all four antioxidant tests for each cocoa product was used for calculating antioxidant potency composite index (ACI). Obtained results for all four assays have shown that antioxidant capacity of analyzed chocolate/cocoa extracts followed cocoa, polyphenol, flavonoid, and proanthocyanidin contents. Although the addition of raspberries to dark chocolates had no significant influence on their total polyphenol, flavonoid and proanthocyanidin contents, statistical analysis showed that there was significant increase in the antioxidant capacity of dark chocolates with raspberry compared to plain dark chocolates (p = 0.007). Overall range for theobromine content varied from 5.5 to 22.3 mg/g depending on the product type, while the content of caffeine was 13–30 times lower in all analyzed cocoa products. In addition, correlation between antioxidant potency composite index and declared percentage of cocoa was high (R² = 0.798, p < 0.05) and indicated that declared cocoa content was a reliable indication for antioxidant capacity of chocolates produced in Serbia.     

Synaptic Localization and Restricted Diffusion of a Drosophila Neuronal Synaptobrevin - Green Fluorescent Protein Chimera in Vivo

Fluorescent markers for subcellular compartments in Drosophila neurons should allow one to combine genetic mutant analysis with visualization of subcellular structures in vivo. Here we describe an analysis of two markers which may be used to observe different compartments of live Drosophila synapses. Soluble jellyfish green fluorescent protein (GFP) expressed at high levels in neurons diffuses freely in the neuronal cytosol as evidenced by confocal microscopy and fluorescence recovery from photobleaching experiments. Thus, the distribution pattern of soluble GFP in motor axons and larval motor terminals indicates the expected distribution for diffusible presynaptic molecules. In contrast to GFP. a neurally expressed neuronal synaptobrevin-GFP chimera (n-syb GFP) is transported down axons and specifically localized to nerve terminals. We demonstrate that n-syb GFP labels synaptic-vesicle membrane at larval motor terminals by documenting its restriction to presynaptic varicosities, its colocalization with synaptic vesicle antigens, and its redistribution in Drosophila shi^ts1 mutant nerve terminals transiently depleted of synaptic vesicles. Surprisingly, n-syb GFP expressed in muscle is concentrated at the subsynaptic reticulum (SSR). postsynaptic infoldings of muscle plasma membrane. We suggest, using different membrane markers, that this apparent postsynaptic enrichment simply reflects a concentration of plasma membrane in the SSR, rather than a selective targeting of n-syb GFP to postsynaptic sites. Utilities and implications of these studies are demonstrated or discussed.     

Monitoring nutrient transport in tissue‐engineered grafts

Limited nutrient diffusion in three‐dimensional (3D) constructs is a major concern in tissue engineering. Therefore, monitoring nutrient availability and diffusion within a scaffold is an important asset. Since nutrients come in various forms, we have investigated the diffusion of the oxygen, luciferin and dextran molecules within tissue‐engineered constructs using optical imaging technologies. First, oxygen availability and diffusion were investigated, using transgenic cell lines in which a hypoxia‐responsive element drives expression of the green fluorescent protein gene. Using confocal imaging, we observed oxygen limitation, starting at around 200 µm from the periphery in the context of agarose gel with 1 million CHO cells. Diffusion of luciferin was monitored real‐time in agarose gels using a cell line in which the luciferase gene was driven by a constitutively active CMV promoter. Gel concentration affected the diffusion rate of luciferin. Furthermore, we assessed the diffusion rates of fluorescent dextran molecules of different molecular weights in biomaterials by fluorescence recovery after photobleaching (FRAP) and observed that diffusion depended on both molecular size and gel concentration. In conclusion, we have validated a set of efficient tools to investigate molecular diffusion of a range of molecules and to optimize biomaterials design in order to improve nutrient delivery. Copyright © 2013 John Wiley & Sons, Ltd.     

Silica and Silica–Titania Xerogels Doped with Iron(III) for Total Antioxidant Capacity Determination

In order to design a sensor material for total antioxidant capacity determination we have prepared silica and silica–titania xerogels doped with iron(III) and modified with 1,10-phenanthroline. Titanium(IV) tetraethoxyde content in the precursors (titanium(IV) tetraethoxyde and tetraethyl orthosilicate) mixtures has been varied from 0 to 12.5% vol. Iron(III) concentrations in sol has been varied from 1 to 100 mM. The increase of titanium(IV) content has led to a decrease in BET surface area and average pore diameter and an increase of micropore surface area and volume, which has resulted in better iron(III) retention in the xerogels. Iron(III), immobilized in the xerogel matrix, retains its ability to form complexes with 1,10-phenanthroline and to be reduced to iron(II). Static capacities for 1,10-phenanthroline have been determined for all the iron(III) doped xerogels (0.207 mmol/g–0.239 mmol/g) and they are not dependent on the iron(III) content. Sensor materials—xerogels doped with iron(III) and modified with 1,10-phenanthroline—have been used for antioxidants (catechol, gallic and ascorbic acids, and sulphite) solid phase spectrophotometric determination. Limits of detection for catechol, gallic and ascorbic acids, and sulphite equal 7.8 × 10⁻⁶ M, 5.4 × 10⁻⁶ M, 1.2 × 10⁻⁵ M, and 3.1 × 10⁻⁴ M, respectively. The increase of titanium(IV) content in sensor material has led to an increase of the reaction rate and the sensitivity of determination. Proposed sensor materials have been applied for total antioxidant capacity (in gallic acid equivalents) determination in soft beverages, have demonstrated high stability, and can be stored up to 6 months at room temperature.     

Protein exchange dynamics at chemoreceptor clusters in Escherichia coli

Signal processing in bacterial chemotaxis relies on large sensory complexes consisting of thousands of protein molecules. These clusters create a scaffold that increases the efficiency of pathway reactions and amplifies and integrates chemotactic signals. The cluster core in Escherichia coli comprises a ternary complex composed of receptors, kinase CheA, and adaptor protein CheW. All other chemotaxis proteins localize to clusters by binding either directly to receptors or to CheA. Here, we used fluorescence recovery after photobleaching (FRAP) to investigate the turnover of chemotaxis proteins at the cluster and their mobility in the cytoplasm. We found that cluster exchange kinetics were protein-specific and took place on several characteristic time scales that correspond to excitation, adaptation, and cell division, respectively. We further applied analytical and numerical data fitting to analyze intracellular protein diffusion and to estimate the rate constants of cluster equilibration in vivo. Our results indicate that the rates of protein turnover at the cluster have evolved to ensure optimal performance of the chemotaxis pathway.     

Anthocyanin profile and antioxidant capacity of black carrots (Daucus carota L. ssp. sativus var. atrorubens Alef.) from Cuevas Bajas,Spain

The present work deals with the study of the anthocyanin profile of two different black carrots (Daucus carota L. ssp. sativus var. atrorubens Alef.) cultivars, associated with Antonina and Purple Haze varieties, from Cuevas Bajas (Málaga, Spain) and some of their antioxidant features. The main anthocyanins detected by LC–MS were found to correspond to five cyanidin-based anthocyanins: cyanidin 3-xylosylglucosylgalactoside, cyanidin 3-xylosylgalactoside and the sinapic, ferulic and coumaric acids derivative of cyanidin 3-xylosylglucosylgalactoside. The anthocyanins present in the black carrots were essentially acylated and their levels were found to correspond to 25% and 50% of the total phenolic content for the Purple Haze and Antonina varieties, respectively. Moreover, the reducing capacity of the two black carrots extracts (86.4 ± 8.0 and 182.0 ± 27 μM TE/100 g fw) and the radical scavenging ability (17.6 ± 9.0 and 240.0 ± 54.0 μM TE/100 g fw) expressed in Trolox equivalents units were determined. The antioxidant features of the black carrot extracts were shown to be significantly higher than those of orange carrots used herein for comparison. Overall, this work highlights the Cuevas Bajas black carrots as rich sources of anthocyanins with significant antioxidant capacities and good nutritional value.     

Quantifying transient binding of ISWI chromatin remodelers in living cells by pixel-wise photobleaching profile evolution analysis

Interactions between nuclear proteins and chromatin frequently occur on the time scale of seconds and below. These transient binding events are important for the fast identification of target sites as concluded from our previous analysis of the human chromatin remodelers Snf2H and Snf2L from the imitation switch (ISWI) family. Both ATP-driven molecular motor proteins are able to translocate nucleosomes along the DNA and appear to exert this activity only on a small number of nucleosomes to which they bind more tightly. For mechanistic studies, one needs to distinguish such translocation reactions or other long-lived interactions associated with conformational changes and/or ATP hydrolysis from nonproductive chromatin sampling during target search. These processes can be separated by measuring the duration of nucleosome binding with subsecond time resolution. To reach this goal, we have developed a fluorescence bleaching technique termed pixel-wise photobleaching profile evolution analysis (3PEA). It exploits the inherent time structure of confocal microscopy images and yields millisecond resolution. 3PEA represents a generally applicable approach to quantitate transient chromatin interactions in the 2- to 500-ms time regime within only ∼1 s needed for a measurement. The green autofluorescent protein (GFP)-tagged Snf2H and Snf2L and the inactive Snf2L+13 splice variant were studied by 3PEA in comparison to the isolated GFP or red autofluorescent protein and a GFP pentamer. Our results reveal that the residence time for transient chromatin binding of Snf2H and Snf2L is <2 ms, and strongly support the view that ISWI-type remodelers are only rarely active in unperturbed cells during G1 phase.