Influence of ethanol on the release of growth factors in human blood-derived platelet gels

Platelet gels (PG) are new topical single-donor blood products which are attracting great interest in regenerative medicine. They are obtained by mixing a platelet-rich plasma fraction with thrombin to generate a fibrin gel enriched in platelet growth factors (GF). The type of thrombin preparation may affect PG reproducibility. We have determined the impact of 14.6% (v/v) ethanol-stabilized thrombin (EHT) on the release of GF by platelets. Various ratios of EHT and platelet concentrates were mixed to obtain from 2.43 to 7.96% ethanol concentration. Platelet-derived growth factor-AB (PDGF-AB), transforming growth factor-ß1 (TGF-ß1), vascular endothelium growth factor (VEGF), epidermal growth factor (EGF), and insulin-like growth factor-1 (IGF-1) were assessed at 5, 120, and 300 min after PG formation. Protein profiles of thrombin and PG releasates were analyzed by SDS-PAGE. The amount of PDGF-AB, TGF-ß1, and VEGF released per platelet decreased significantly (p < 0.05) with increasing ethanol concentrations but, however, not that of EGF. IGF-1 content was stable, consistent with its presence mostly in plasma. SDS-PAGE indicated that ethanol did not affect fibrin formation. In conclusion, ethanol has a significant impact on the amount of GF released by platelets and should be strictly controlled to standardize PG and optimize clinical benefits.     

Early Changes of β-Catenins and Menins in Spinal Cord Dorsal Horn after Peripheral Nerve Injury

Injury to the peripheral nervous system can lead to spontaneous pain, hyperalgesia and allodynia. Previous studies have shown sprouting of Aβ-fibres into lamina II of the spinal cord dorsal horn after nerve injury and the formation of new synapses by these sprouts. β-Catenin and menin as synaptogenic factors are critically involved in synapse formation. However, the roles of β-catenin and menin in neuropathic pain are still unclear. Using Western blot analysis we investigated the changes of β-catenin and menin in the spinal dorsal horn after unilateral spared nerve injury (SNI). We demonstrated an increase in both β-catenin and menin protein levels in the ipsilateral spinal dorsal horn at days 1 and 3 following spared nerve injury (P < 0.05). These increases were associated with changes in paw withdrawal threshold to mechanical stimuli and weight bearing deficit suggestive of pain behavior and spontaneous ongoing pain respectively. However, the injury-associated increases in β-catenins and menins levels returned to control levels at day 14. In conclusion, these results indicate that peripheral nerve injury induces upregulation of β-catenins and menins in the dorsal horn of the spinal cord, which may contribute to the development of chronic neuropathic pain. Antagonists of these molecules may serve as new therapeutic agents.     

Systems genetics analyses predict a transcription role for P2P-R: Molecular confirmation that P2P-R is a transcriptional co-repressor

Background  

The 250 kDa P2P-R protein (also known as PACT and Rbbp6) was cloned over a decade ago and was found to bind both the p53 and Rb1 tumor suppressor proteins. In addition, P2P-R has been associated with multiple biological functions, such as mitosis, mRNA processing, translation and ubiquitination. In the current studies, the online GeneNetwork system was employed to further probe P2P-R biological functions. Molecular studies were then performed to confirm the GeneNetwork evaluations.     

Invertebrates in field margins: taxonomic group diversity and functional group abundance in relation to age

Sown, temporary field margins are a common agri-environment scheme (AES) in the Netherlands. Despite their wide application, though, there has been scarcely any long-term monitoring of the succession of invertebrates. In the field margins of 40 farms, invertebrate diversity and the abundance of three functional groups were assessed in relation to age. The diversity in terms of number of species groups was found to increase with the age of the margins. The abundance of herbivores and detritivores also showed a positive correlation with the age of the margins. However, the abundance of predators decreased with increasing age. Older margins showed a higher total vegetation cover and fewer plant species, also resulting in lower plant species evenness. We suggest several changes to the current AES regulations. For the conservation of invertebrate diversity, longer-lasting field margins are desirable. In addition, old margins are favoured by detritivores, a group that has particular difficulty finding suitable habitats in agricultural landscapes. However, such margins are less favourable from an agricultural perspective, as they appear unsuitable for high abundances of potentially useful predators and the high vegetation cover attracts many potentially harmful herbivores. To circumvent this, the AES might be extended by incorporating hay-making, which would reduce standing biomass and might lead to more predators and fewer herbivores.     

Antioxidant Properties of Selected <Emphasis Type="Italic">Boletus</Emphasis> Mushrooms

Considering the growing interest for mushrooms and the demand search of natural antioxidants sources, the aim of this study was to investigate the antioxidant properties of two edible widely used Boletus species, Boletus edulis, and Boletus auranticus, collected from Istra region in Croatia in late summer 2007. To evaluate the antioxidant properties and content of antioxidant compounds, scavenging capacity on DPPH˙, OH˙, and O₂˙⁻ radicals, reducing power and capacity to inhibit lipid peroxidation has been investigated. It is determined that content of total phenols (41.82 ± 0.08 mg gallic acid equivalent per gram of dry extract) was higher for B. edulis. Using high performance liquid chromatography/diode array detector analysis, the main antioxidant compound, variegatic acid, has been detected and quantified. 1,1-Diphenyl-2-picryl-hydrazyl-hydrate assay was used as a preliminary free radical–scavenging evaluation. By this assay, it has been found that B. edulis dry mushroom extract exhibits 50% of inhibition value at the extract concentration of 0.016 ± 0.0003 mg/ml. The extracts were capable of reducing iron(III) and, thus, are capable of donating electrons. Using electron paramagnetic resonance spin-trapping and spin-probing techniques, activity against relevant reactive species, ˙OH and O₂˙⁻ radical, was analyzed for both mushroom extracts. Both investigated extracts are determined as good inhibitors for ˙OH radical reduction, and both exhibited significant capacity for scavenging O₂˙⁻ radical and for that could help to prevent or meliorate oxidative damage. Only B. edulis extract prevents lipid peroxidation. Investigated mushroom extracts could represent easily accessible natural antioxidant resource.     

Isolation and characterization of lactic acid bacteria strains with ornithine producing capacity from natural sea salt

Two lactic acid bacteria (LAB) having ornithine-producing capacity were isolated from Korean natural sea salt. They were Gram-positive, short rod-type bacteria, and able to grow anaerobically with CO₂ production. The isolates grew well on MRS broth at 30–37°C and a pH of 6.5–8.0. The optimum temperature and pH for growth are 37°C and pH 7.0. The isolates fermented D-ribose, D-galactose, D-lactose, D-maltose, Dcellobiose, D-tagatose, D-trehalose, sucrose, D-melezitose, gentiobiose, D-glucose but not D-melibiose, inositol, and L-sorbose. The 16S rDNA sequences of the two isolates showed 99.5% and 99.6% homology with the Weissella koreensis S5623 16S rDNA (Access no. AY035891). They were accordingly identified and named as Weissella koreensis MS1-3 and Weissella koreensis MS1-14, and produced intracellular ornithine at levels of 72 mg/100 g cell F.W. and 105 mg/100 g cell F.W. and extracellular ornithine at levels of 4.5 mg/100 ml and 4.6 mg/100 ml medium, respectively, by culturing in MRS broth supplemented with 1% arginine. High cell growth was maintained in MRS broth with a NaCl concentration of 0–6%. These results show for the first time that Korean natural sea salts contain lactic acid bacteria Weissella koreensis strains having ornithine producing capacity.     

Identification and characterization of adipose triglyceride lipase (ATGL) gene in birds

Adipose triglyceride lipase (ATGL) is a triglyceride hydrolysis lipase and is generally related to lipid metabolism in animals. The ATGL gene was well studied in mammals, however very less was known in birds that differed significantly with mammals for lipid metabolism. In this study, cloning, mRNA real time and association analysis was performed to characterize the ATGL gene in birds. Results showed that the obtained ATGL gene cDNA of parrot, quail, duck were 1,651 bp (NCBI accession number: GQ221784), 1,557 bp (NCBI accession number: GQ221783) and 1,440 bp each, encoded 481-, 482- and 279-amino acid (AA) peptide, respectively. The parrot ATGL (pATGL) gene was found to predominantly express in breast muscle and leg muscle, and very higher ATGL mRNA level was also found in heart, abdominal fat and subcutaneous fat. The quail ATGL (qATGL) gene was also predominantly expressed in breast muscle and leg muscle, and then to a much lesser degree in heart. The duck ATGL (dATGL) gene was found to predominantly express in subcutaneous fat and abdominal fat, quite higher ATGL mRNA was also found in heart, spleen, breast muscle and leg muscle. Blast analyses indicated the high homology of ATGL and its patatin region, and moreover, and the active serine hydrolase motif (“GASAG” for “GXSXG”) and the glycine rich motif (“GCGFLG” for “GXGXXG”) were completely conservative among 14 species. Association analyses showed that c.950+24C>A, c.950+45C>G, c.950+73G>A, c.950+83C>T and c.950+128delA of chicken ATGL gene (cATGL) were all significantly or highly significantly with cingulated fat width (CFW) (P < 0.05 or P < 0.01), and c.777−26C>A, c.950+45C>G, c.950+73G>A and c.950+118C>T were all significantly or highly significantly with pH value of breast muscle (BMPH) (P < 0.05).     

Lack of apoE causes alteration of cytokines expression in young mice liver

To investigate the effect of apolipoprotein E (apoE) on cytokine expression profile of the liver of young mice, quantitative RT-PCR (qRT-PCR) assay and cytokine antibody array for multiplex analysis of 62 cytokines have been used to analyze characteristics of expression of cytokines in the liver of 6-week-old apoE-null (apoE^−/−) mice. The levels of plasma cytokines were also analyzed. The mRNA level of IL-1β, IL-2, IL-6, ICAM-1, VCAM-1, MCP-1, NF-κB (p65), IFN-γ and IκB-α were increased significantly in apoE^−/− mice comparative to wild-type (WT) mice. IL-4, IL-10 and GM-CSF, however, were slightly decreased. Compared with WT, levels of 21 cytokines altered twofold or more in apoE^−/− mice, including 10 cytokines increased and 11 decreased. Expression patterns of IL-1β, IL-2, IL-4, IL-6, IL-10, GM-CSF, IFN-γ and VCAM-1 showed identical trend between cytokine antibody array and qRT-PCR analysis. Moreover, levels of IL-1β, IFN-γ and IL-6 in the plasma were elevated, while IL-4 was lightly decreased in apoE^−/− mice compared to those in WT mice. These results implied that promotion of type I immune response in the liver of young apoE^−/− mice due to alteration of these cytokines, and the phenotypes may be caused by the regulation of NF-κB. The inflammation and lipid metabolism dysfunction in the liver cooperated in dysfunction of the liver in young apoE^−/− mice.