Quantification of bioactive acylethanolamides in rat plasma by electrospray mass spectrometry

We developed a high-performance liquid chromatography/mass spectrometry (HPLC/MS) method for the identification and quantification of anandamide, an endogenous cannabinoid substance, and other fatty acid ethanolamides (AEs) in biological samples. Using a mobile-phase system of methanol/water and gradient elution, we achieved satisfactory resolution of all major AEs, including anandamide, palmitylethanolamide (PEA), and oleylethanolamide (OEA). Electrospray-generated quasi-molecular species were used as diagnostic ions and detected by selected ion monitoring (SIM). Synthetic deuterium-labeled AEs were used as internal standards, and quantification was carried out by isotope dilution. A linear correlation (r2 = 0.99) was observed in the calibration curves for standard AEs over the range 0-0.5 nmol. Detection limits between 0.1 and 0.3 pmol per sample and quantification limits between 0.5 and 1.2 pmol per sample were obtained. The method was applied to the quantification of anandamide, PEA, and OEA in plasma prepared from rat blood collected either by cardiac puncture or by decapitation. After cardiac puncture, AE levels were in the low-nanomolar range: anandamide, 3.1 +/- 0.6 pmol/ml; PEA, 9.4 +/- 1.6 pmol/ml; OEA, 9.2 +/- 1.8 pmol/ml (mean +/- SE, n = 9). By contrast, after decapitation AEs were dramatically elevated (anandamide, 144 +/- 13 pmol/ml; PEA, 255 +/- 55 pmol/ml; OEA, 175 +/- 48 pmol/ml). Thus, disruptive procedures of blood collection may result in gross overestimates in the concentrations of circulating AEs.     

In vitro regeneration from leaf explants of Neoregelia cruenta (R. Graham) L.B. Smith,an endemic bromeliad from Eastern Brazil

An efficient plant regeneration system was developed for the induction of direct shoot formation from leaves derived from seedlings of Neoregelia cruenta, an endemic Bromeliaceae of Eastern Brazil. Shoot differentiation occurred directly from the leaf bases. In vitro responses were influenced by seedling age and growth regulator combinations. Highest regeneration rates were obtained from explants excised from 7-week-old seedlings cultured in the presence of 22 μM BA and 2.5 μM NAA. Shoot conversion to whole plants was most effective in shoots formed in response to 4.4 or 8.8 μM BA combined with 2.5 μM NAA. This revised version was published online in June 2006 with corrections to the Cover Date.     

Enhanced in vitro and in vivo antioxidant activity and mobilization of free phenolic compounds of soybean flour fermented with different β-glucosidase-producing fungi

Aims:  To evaluate the soybean polyphenol glucosides bioconversion to aglycone forms by different β-glucosidases-producing filamentous fungi to enhance their antioxidant activity.
Methods and Results:  Soybean defatted flour was submitted to solid-state fermentation with Aspergillus niger , Aspergillus niveus and Aspergillus awamori . The fungi studied produced approximately the same β-glucosidase activity units amount when p- nitrophenyl-β- d -glucopyranoside was used as substrate for the assay. However, electrophoretic analysis, using 4-methylumbellipheryl-β- d -glucopyranoside as substrate, showed that β-glucosidase produced by A.   niveus was more active. Fermented methanolic extracts showed an increase in polyphenol and genistein contents and antioxidant activities. The highest genistein content was found in soybean fermented by A. niveus . Methanolic extracts of the soybean fermented by the different fungi showed a similar capacity of scavenging H₂O₂ generated in vivo by the tumour promoter 12- O- tetradecanoyl phorbol-13-acetate.
Conclusions:  A.   niveus synthesized a β-glucosidase with higher specificity to hydrolyse genistin β-glycosidic bond than those produced by A .  awamori and A. niger .
Significance and Impact of the Study:  The utilization of these β-glucosidases-producing fungi in soybean fermentation processes resulted in the obtaining of methanolic extracts with different antioxidant potentials that could be used either therapeutically or as an antioxidant in nonphysiological oxidative stress conditions, as the one induced in skin by UV radiation.     

Cardiorespiratory Responses and Prediction of Peak Oxygen Uptake during the Shuttle Walking Test in Healthy Sedentary Adult Men

BackgroundThe application of the Shuttle Walking Test (SWT) to assess cardiorespiratory fitness and the intensity of this test in healthy participants has rarely been studied. This study aimed to assess and correlate the cardiorespiratory responses of the SWT with the cardiopulmonary exercise testing (CEPT) and to develop a regression equation for the prediction of peak oxygen uptake (VO2 peak) in healthy sedentary adult men.MethodsIn the first stage of this study, 12 participants underwent the SWT and the CEPT on a treadmill. In the second stage, 53 participants underwent the SWT twice. In both phases, the VO2 peak, respiratory exchange ratio (R), and heart rate (HR) were evaluated.ResultsSimilar results in VO2 peak (P>0.05), R peak (P>0.05) and predicted maximum HR (P>0.05) were obtained between the SWT and CEPT. Both tests showed strong and significant correlations of VO2 peak (r = 0.704, P = 0.01) and R peak (r = 0.737, P<0.01), as well as the agreement of these measurements by Bland-Altman analysis. Body mass index and gait speed were the variables that explained 40.6% (R2 = 0.406, P = 0.001) of the variance in VO2 peak. The results obtained by the equation were compared with the values obtained by the gas analyzer and no significant difference between them (P>0.05) was found.ConclusionsThe SWT produced maximal cardiorespiratory responses comparable to the CEPT, and the developed equation showed viability for the prediction of VO2 peak in healthy sedentary men.     

Characterization of Bacterial,Archaeal and Eukaryote Symbionts from Antarctic Sponges Reveals a High Diversity at a Three-Domain Level and a Particular Signature for This Ecosystem

Sponge-associated microbial communities include members from the three domains of life. In the case of bacteria, they are diverse, host specific and different from the surrounding seawater. However, little is known about the diversity and specificity of Eukarya and Archaea living in association with marine sponges. This knowledge gap is even greater regarding sponges from regions other than temperate and tropical environments. In Antarctica, marine sponges are abundant and important members of the benthos, structuring the Antarctic marine ecosystem. In this study, we used high throughput ribosomal gene sequencing to investigate the three-domain diversity and community composition from eight different Antarctic sponges. Taxonomic identification reveals that they belong to families Acarnidae, Chalinidae, Hymedesmiidae, Hymeniacidonidae, Leucettidae, Microcionidae, and Myxillidae. Our study indicates that there are different diversity and similarity patterns between bacterial/archaeal and eukaryote microbial symbionts from these Antarctic marine sponges, indicating inherent differences in how organisms from different domains establish symbiotic relationships. In general, when considering diversity indices and number of phyla detected, sponge-associated communities are more diverse than the planktonic communities. We conclude that three-domain microbial communities from Antarctic sponges are different from surrounding planktonic communities, expanding previous observations for Bacteria and including the Antarctic environment. Furthermore, we reveal differences in the composition of the sponge associated bacterial assemblages between Antarctic and tropical-temperate environments and the presence of a highly complex microbial eukaryote community, suggesting a particular signature for Antarctic sponges, different to that reported from other ecosystems.     

Decrease of CD68 Synovial Macrophages in Celastrol Treated Arthritic Rats

Background

Rheumatoid arthritis (RA) is a chronic immune-mediated inflammatory disease characterized by cellular infiltration into the joints, hyperproliferation of synovial cells and bone damage. Available treatments for RA only induce remission in around 30% of the patients, have important adverse effects and its use is limited by their high cost. Therefore, compounds that can control arthritis, with an acceptable safety profile and low production costs are still an unmet need. We have shown, in vitro, that celastrol inhibits both IL-1β and TNF, which play an important role in RA, and, in vivo, that celastrol has significant anti-inflammatory properties. Our main goal in this work was to test the effect of celastrol in the number of sublining CD68 macrophages (a biomarker of therapeutic response for novel RA treatments) and on the overall synovial tissue cellularity and joint structure in the adjuvant-induced rat model of arthritis (AIA).

Methods

Celastrol was administered to AIA rats both in the early (4 days after disease induction) and late (11 days after disease induction) phases of arthritis development. The inflammatory score, ankle perimeter and body weight were evaluated during treatment period. Rats were sacrificed after 22 days of disease progression and blood, internal organs and paw samples were collected for toxicological blood parameters and serum proinflammatory cytokine quantification, as well as histopathological and immunohistochemical evaluation, respectively.

Results

Here we report that celastrol significantly decreases the number of sublining CD68 macrophages and the overall synovial inflammatory cellularity, and halted joint destruction without side effects.

Conclusions

Our results validate celastrol as a promising compound for the treatment of arthritis.     

Pharmacological Blockade of Cannabinoid CB1 Receptors in Diet-Induced Obesity Regulates Mitochondrial Dihydrolipoamide Dehydrogenase in Muscle

Cannabinoid CB₁ receptors peripherally modulate energy metabolism. Here, we investigated the role of CB₁ receptors in the expression of glucose/pyruvate/tricarboxylic acid (TCA) metabolism in rat abdominal muscle. Dihydrolipoamide dehydrogenase (DLD), a flavoprotein component (E3) of α-ketoacid dehydrogenase complexes with diaphorase activity in mitochondria, was specifically analyzed. After assessing the effectiveness of the CB₁ receptor antagonist AM251 (3 mg kg^-1, 14 days) on food intake and body weight, we could identified seven key enzymes from either glycolytic pathway or TCA cycle—regulated by both diet and CB₁ receptor activity—through comprehensive proteomic approaches involving two-dimensional electrophoresis and MALDI-TOF/LC-ESI trap mass spectrometry. These enzymes were glucose 6-phosphate isomerase (GPI), triosephosphate isomerase (TPI), enolase (Eno3), lactate dehydrogenase (LDHa), glyoxalase-1 (Glo1) and the mitochondrial DLD, whose expressions were modified by AM251 in hypercaloric diet-induced obesity. Specifically, AM251 blocked high-carbohydrate diet (HCD)-induced expression of GPI, TPI, Eno3 and LDHa, suggesting a down-regulation of glucose/pyruvate/lactate pathways under glucose availability. AM251 reversed the HCD-inhibited expression of Glo1 and DLD in the muscle, and the DLD and CB₁ receptor expression in the mitochondrial fraction. Interestingly, we identified the presence of CB₁ receptors at the membrane of striate muscle mitochondria. DLD over-expression was confirmed in muscle of CB ₁ ^-/- mice. AM251 increased the pyruvate dehydrogenase and glutathione reductase activity in C₂C₁₂ myotubes, and the diaphorase/oxidative activity in the mitochondria fraction. These results indicated an up-regulation of methylglyoxal and TCA cycle activity. Findings suggest that CB₁ receptors in muscle modulate glucose/pyruvate/lactate pathways and mitochondrial oxidative activity by targeting DLD.     

A novel colorimetric assay of β‐D‐glucans in basidiomycete strains by alcian blue dye in a 96‐well microtiter plate

Basidiomycete strains synthesize several types of β‐d ‐glucans, which play a major role in the medicinal properties of mushrooms. Therefore, the specific quantification of these β‐d ‐glucans in mushroom strains is of great biochemical importance. Because published assay methods for these β‐d ‐glucans present some disadvantages, a novel colorimetric assay method for β‐d ‐glucan with alcian blue dye was developed. The complex formation was detected by following the decrease in absorbance in the range of 620 nm and by hypsochromic shift from 620 to 606 nm (～14 nm) in UV‐Vis spectrophotometer. Analysis of variance was used for optimization of the slope of the calibration curve by using the assay mixture containing 0.017% (w/v) alcian blue in 2% (v/v) acetic acid at pH 3.0. The high‐throughput colorimetric assay method on microtiter plates was used for quantification of β‐d ‐glucans in the range of 0–0.8 μg, with a slope of 44.15 × 10^?2 and a limit of detection of 0.017 μg/well. Recovery experiments were carried out by using a sample of Hericium erinaceus, which exhibited a recovery of 95.8% for β‐1,3‐d ‐glucan. The present assay method exhibited a 10‐fold higher sensitivity and a 59‐fold lower limit of detection compared with the published method with congo red. β‐d ‐glucans of several mushrooms strains were isolated from fruiting bodies and mycelia, and they were quantified by this assay method. This assay method is fast, specific, simple, and it can be used to quantify β‐d ‐glucans from other biological sources. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1526–1535, 2015     

VMS-based fishing effort and population demographics for the European squid (Loligo vulgaris) off the Portuguese coast

This study presents a 1-year synopsis of the trawl fishery for the European squid Loligo vulgaris in Portuguese waters, integrating length-structured landings with corresponding geo-referenced fishing activities. From vessel monitoring system (VMS) data, landings and biological sampling, a “status-report” was obtained for 2003. Fishing pressure was found to be most intense in inshore areas of the northwest and the south coasts. Population size structure, classified into three categories, was not uniform throughout the fishing areas. Larger squid are found offshore in the northwest (International Council for the Exploration of the Sea—ICES rectangle 10E0) and in the south (rectangles 2E1 and 3E1), whereas all inshore western coast rectangles showed larger proportions of small squid relative to the southern rectangles. The analysis carried out in this study provides an insight into the possible impact of the fishing intensity pattern on the population structure in various zones. The results demonstrate the benefits of combining geo-referenced fisheries information with landings and size data, to produce explicit spatio-temporal information that can contribute to integrated planning and management for the sustainable exploitation of this resource.