In vivo detection of free radicals in mouse septic encephalopathy using molecular MRI and immuno-spin trapping

Free radicals are known to play a major role in sepsis. Combined immuno-spin trapping and molecular magnetic resonance imaging (MRI) was used to detect in vivo and in situ levels of free radicals in murine septic encephalopathy after cecal ligation and puncture (CLP). DMPO (5,5-dimethyl pyrroline N-oxide) was injected over 6 h after CLP, before administration of an anti-DMPO probe (anti-DMPO antibody bound to albumin–gadolinium–diethylene triamine pentaacetic acid–biotin MRI targeting contrast agent). In vitro assessment of the anti-DMPO probe in oxidatively stressed mouse astrocytes significantly decreased T₁ relaxation (p < 0.0001) compared to controls. MRI detected the presence of anti-DMPO adducts via a substantial decrease in %T₁ change within the hippocampus, striatum, occipital, and medial cortex brain regions (p < 0.01 for all) in septic animals compared to shams, which was sustained for over 60 min (p < 0.05 for all). Fluorescently labeled streptavidin was used to target the anti-DMPO probe biotin, which was elevated in septic brain, liver, and lungs compared to sham. Ex vivo DMPO adducts (qualitative) and oxidative products, including 4-hydroxynonenal and 3-nitrotyrosine (quantitative, p < 0.05 for both), were elevated in septic brains compared to shams. This is the first study that has reported on the detection of in vivo and in situ levels of free radicals in murine septic encephalopathy.     

OKN-007 decreases free radical levels in a preclinical F98 rat glioma model

Free radicals are associated with glioma tumors. Here, we report on the ability of an anticancer nitrone compound, OKN-007 [Oklahoma Nitrone 007; a disulfonyl derivative of α-phenyl-tert-butyl nitrone (PBN)] to decrease free radical levels in F98 rat gliomas using combined molecular magnetic resonance imaging (mMRI) and immunospin-trapping (IST) methodologies. Free radicals are trapped with the spin-trapping agent, 5,5-dimethyl-1-pyrroline N-oxide (DMPO), to form DMPO macromolecule radical adducts, and then further tagged by immunospin trapping by an antibody against DMPO adducts. In this study, we combined mMRI with a biotin–Gd-DTPA–albumin-based contrast agent for signal detection with the specificity of an antibody for DMPO nitrone adducts (anti-DMPO probe), to detect in vivo free radicals in OKN-007-treated rat F98 gliomas. OKN-007 was found to significantly decrease (P < 0.05) free radical levels detected with an anti-DMPO probe in treated animals compared to untreated rats. Immunoelectron microscopy was used with gold-labeled antibiotin to detect the anti-DMPO probe within the plasma membrane of F98 tumor cells from rats administered anti-DMPO in vivo. OKN-007 was also found to decrease nuclear factor erythroid 2-related factor 2, inducible nitric oxide synthase, 3-nitrotyrosine, and malondialdehyde in ex vivo F98 glioma tissues via immunohistochemistry, as well as decrease 3-nitrotyrosine and malondialdehyde adducts in vitro in F98 cells via ELISA. The results indicate that OKN-007 effectively decreases free radicals associated with glioma tumor growth. Furthermore, this method can potentially be applied toward other types of cancers for the in vivo detection of macromolecular free radicals and the assessment of antioxidants.     

Nitrite correlates with 3-nitrotyrosine but not with the F2-isoprostane 15(S)-8-iso-PGF2α in urine of rheumatic patients

In vitro studies suggested that nitrite may play a cytoprotective role in inflammation. The aim of the present clinical study was to investigate the relationship between the NO metabolites nitrite and nitrate and the biomarkers of oxidative stress 3-nitrotyrosine (3-NT) and 15(S)-iso-PGF_2α in patients suffering from chronic inflammatory rheumatic diseases. In morning urine from 28 patients with different chronic inflammatory rheumatic diseases (23–82 years of age) and from 41 healthy persons of both genders, nitrite and nitrate were quantitated by GC-MS, and 3-NT and 15(S)-iso-PGF_2α by GC-MS/MS. Mean creatinine-corrected urinary excretion rates of nitrite (1.1 versus 0.19 μmol/mmol, P = 0.00012) and 3-NT (1.2 versus 0.39 nmol/mmol, P = 0.01629), but not of nitrate (105 versus 106 μmol/mmol), were significantly elevated in rheumatism as compared to health. Urinary excretion rate of 15(S)-iso-PGF_2α did not differ between patients and healthy subjects (65 versus 69 pmol/mmol creatinine, P = 0.48). In rheumatism, urinary 3-NT correlated closely with nitrite (R = 0.788, P < 0.0001) and moderately with nitrate (R = 0.45, P < 0.016), but did not correlate with 15(S)-iso-PGF_2α (R = ?0.083, P = 0.68). In healthy persons there was no correlation between urinary 3-NT and nitrite or nitrate. Our study suggests that urinary nitrite may represent a novel specific biomarker of nitrative stress in chronic inflammatory rheumatic disease. In another eight patients with chronic inflammatory rheumatic diseases we found higher nitrite concentrations in synovial fluid as compared to serum (1.30 versus 0.35 μM). We hypothesize that in chronic inflammatory rheumatic diseases nitrite concentration is elevated in the inflamed joint and contributes to the inactivation of myeloperoxidase-catalyzed production of hypochloric acid by forming nitryl chloride which eventually nitrates tyrosine to form 3-NT.     

Unchanged H-reflex during a sustained isometric submaximal plantar flexion performed with an EMG biofeedback

The aim of this study was to assess H-reflex plasticity and activation pattern of the plantar flexors during a sustained contraction where voluntary EMG activity was controlled via an EMG biofeedback. Twelve healthy males (28.0 ± 4.8 yr) performed a sustained isometric plantar flexion while instructed to maintain summed EMG root mean square (RMS) of gastrocnemius lateralis (GL) and gastrocnemius medialis (GM) muscles fixed at a target corresponding to 80% maximal voluntary contraction torque via an EMG biofeedback. Transcutaneous electrical stimulation of the posterior tibial nerve was evoked during the contraction to obtain the maximal H-reflex amplitude to maximal M-wave amplitude ratio (H_sup/M_sup ratio) from GL, GM and soleus (SOL) muscles. Neuromuscular function was also assessed before and immediately after exercise. Results showed a decrease in SOL activation during sustained flexion (from 65.5 ± 6.4% to 42.3 ± 3.8% maximal EMG, p < 0.001), whereas summed EMG RMS of GL and GM remained constant (59.7 ± 4.8% of maximal EMG on average). No significant change in the H_sup/M_sup ratio was found for SOL, GL and GM muscles. Furthermore, it appears that the decrease in maximal voluntary contraction torque (?20.4 ± 2.9%, p < 0.001) was related to both neural and contractile impairment. Overall, these findings indicate that the balance between excitation and inhibition affecting the motoneuron pool remains constant during a sustained contraction where myoelectrical activity is controlled via an EMG biofeedback or let free to vary.     

Design,synthesis and anti-Alzheimer properties of dimethylaminomethyl-substituted curcumin derivatives

Eight dimethylaminomethyl-substituted curcumin derivatives were designed and synthesized. The antioxidant test revealed that the synthesized compounds had higher free radical scavenging activity towards both 2,2-diphenyl-1-picrylhydrazyl free radicals (DPPH) (IC₅₀ 1.5–29.9 μM) and galvinoxyl radicals (IC₅₀ 4.9–41.1 μM) than the lead compound curcumin. Besides, compound 3a could effectively inhibit the Aβ self-aggregation in vitro. Investigated in phosphate-buffered solutions (pH = 7.4) in the presence or absence of 0.1% FBS 3a showed a good stability while curcumin did not. Furthermore, 3a showed a good lipophilicity (log P = 3.48), suggesting a potential ability to penetrate the blood–brain-barrier. The aqueous solubility of the hydrochloride salt of 3a (16.7 mg/mL) has also been significantly improved as compared with curcumin (<0.1 mg/mL).     

Opposite changes in cannabinoid CB1 and CB2 receptor expression in human gliomas

Gliomas are the most important group of malignant primary brain tumors and one of the most aggressive forms of cancer. During the last years, several studies have demonstrated that cannabinoids induce apoptosis of glioma cells and inhibit angiogenesis of gliomas in vivo. As the effects of cannabinoids rely on CB₁ and CB₂ receptors activation, the aim of the present study was to investigate both receptors protein expression in cellular membrane homogenates of human glial tumors using specific antibodies raised against these proteins. Additionally, we studied the functionality of the cannabinoid receptors in glioblastomas by using WIN 55,212-2 stimulated [³⁵S]GTPγS binding.Western blot analysis showed that CB₁ receptor immunoreactivity was significantly lower in glioblastoma multiforme (?43%, n = 10; p < 0.05) than in normal post-mortem brain tissue (n = 16). No significant differences were found for astrocytoma (n = 6) and meningioma (n = 8) samples. Conversely, CB₂ receptor immunoreactivity was significantly greater in membranes of glioblastoma multiforme (765%, n = 9; p < 0.05) and astrocytoma (471%, n = 4; p < 0.05) than in control brain tissue (n = 10). Finally, the maximal stimulation of [³⁵S]GTPγS binding by WIN 55,212-2 was significantly lower in glioblastomas (134 ± 4%) than in control membranes (183 ± 2%; p < 0.05). The basal [³⁵S]GTPγS binding and the EC₅₀ values were not significantly different between both groups.The present results demonstrate opposite changes in CB₁ and CB₂ receptor protein expression in human gliomas. These changes may be of interest for further research about the therapeutic effects of cannabinoids in glial tumors.     

In vivo detection of inducible nitric oxide synthase in rodent gliomas

Increased iNOS expression is often found in brain tumors, such as gliomas. The goal of this study was to develop and assess a novel molecular MRI (mMRI) probe for in vivo detection of iNOS in rodent models for gliomas (intracerebral implantation of rat C6 or RG2 cells or ethyl nitrosourea-induced glioma). The probe we used incorporated a Gd-DTPA (gadolinium(III) complex of diethylenetriamine-N,N,N′,N″,N″-pentaacetate) backbone with albumin and biotin moieties and covalent binding of an anti-iNOS antibody (Ab) to albumin (anti-iNOS probe). We used mMRI with the anti-iNOS probe to detect in vivo iNOS levels in gliomas. Nonimmune normal rat IgG coupled to albumin–Gd-DTPA–biotin was used as a control nonspecific contrast agent. By targeting the biotin component of the anti-iNOS probe with streptavidin Cy3, fluorescence imaging confirmed the specificity of the probe for iNOS in glioma tissue. iNOS levels in glioma tumors were also confirmed via Western blots and immunohistochemistry. The presence of plasma membrane-associated iNOS in glioma cells was established by transmission electron microscopy and gold-labeled anti-iNOS Ab. The more aggressive RG2 glioma was not found to have higher levels of iNOS compared to C6. Differences in glioma vascularization and blood–brain barrier permeability between the C6 and the RG2 gliomas are discussed. In vivo assessment of iNOS levels associated with tumor development is quite feasible in heterogeneous tissues with mMRI.     

Plasma levels of intermedin (adrenomedullin-2) in healthy human volunteers and patients with heart failure

Intermedin/adrenomedullin-2 (IMD) is a member of the adrenomedullin/CGRP peptide family. Less is known about the distribution of IMD than for other family members within the mammalian cardiovascular system, particularly in humans. The aim was to evaluate plasma IMD levels in healthy subjects and patients with chronic heart failure. IMD and its precursor fragments, preproIMD_25–56 and preproIMD_57–92, were measured by radioimmunoassay in 75 healthy subjects and levels of IMD were also compared to those of adrenomedullin (AM) and mid-region proadrenomedullin_45–92 (MRproAM_45–92) in 19 patients with systolic heart failure (LVEF < 45%). In healthy subjects, plasma levels (mean + SE) of IMD (6.3 + 0.6 pg ml⁻¹) were lower than, but correlated with those of AM (25.8 + 1.8 pg ml⁻¹; r = 0.49, p < 0.001). Plasma preproIMD_25–56 (39.6 + 3.1 pg ml⁻¹), preproIMD_57–92 (25.9 + 3.8 pg ml⁻¹) and MRproAM_45–92 (200.2 + 6.7 pg ml⁻¹) were greater than their respective bioactive peptides. IMD levels correlated positively with BMI but not age, and were elevated in heart failure (9.8 + 1.3 pg ml⁻¹, p < 0.05), similarly to MRproAM_45–92 (329.5 + 41.9 pg ml⁻¹, p < 0.001) and AM (56.8 + 10.9 pg ml⁻¹, p < 0.01). IMD levels were greater in heart failure patients with concomitant renal impairment (11.3 + 1.8 pg ml⁻¹) than those without (6.5 + 1.0 pg ml⁻¹; p < 0.05). IMD and AM were greater in patients receiving submaximal compared with maximal heart failure drug therapy and were decreased after 6 months of cardiac resynchronization therapy. In conclusion, IMD is present in the plasma of healthy subjects less abundantly than AM, but is similarly correlated weakly with BMI. IMD levels are elevated in heart failure, especially with concomitant renal impairment, and tend to be reduced by high intensity drug or pacing therapy.     

Combinatorial effect of fish oil (Maxepa) and 1α,25-dihydroxyvitamin D3 in the chemoprevention of DMBA-induced mammary carcinogenesis in rats

The present study demonstrates the anti-tumor effects of combined supplementations of dietary fish oil (Maxepa) and 1α,25-dihydroxyvitamin D₃ (vitamin D₃) on 7,12-dimethylbenz(α)anthracene (DMBA)-induced rat mammary carcinogenesis. Female Sprague–Dawley rats at 50 days of age were treated with 7,12-dimethylbenz(α)anthracene (DMBA; 0.5 mg/100 g body weight) by a single tail vein injection in an oil emulsion. Both fish oil (rich in EPA and DHA) and vitamin D₃ were administered orally at a dose of 0.5 ml/day/rat and 0.3 μg/100 μL propylene glycol twice a week respectively and continued to 35 weeks after DMBA administration. Fish oil in combination with vitamin D₃ resulted in a significant reduction in incidence, multiplicity and volume of mammary tumors. These supplementation also inhibited DMBA-induced mammary 7-methylguanine DNA adducts formation, which was measured by HPLC-fluorescence assay (at four sequential time points; ANOVA, F = 42.56, P < 0.0001). Immunohistochemical analysis revealed that the effect of fish oil and vitamin D₃ occurred through suppression of cell proliferation (BrdU-LI: P < 0.0001). Fish oil and vitamin D₃ together also reduced the mRNA expression of iNOS (84%, P < 0.05). In view of their natural availability, non-toxicity and acceptability; combined supplementation of fish oil and vitamin D₃ might be effective for chemoprevention of mammary carcinogenesis.     

Does the presence of a vaginal probe alter pelvic floor muscle activation in young,continent women?

Vaginal probes may induce changes in pelvic floor muscle (PFM) recruitment by the very presence of the probes. Fine-wire electrodes allow us to detect muscle activation parameters without altering the natural position and shape of the PFMs. The purpose of this study was to determine whether PFM activation is altered by changes in sensory feedback, muscle length or tissue position caused by two different vaginal probes used to record surface electromyography (EMG). Twelve continent women (30.1 ± 5.4 years), performed PFM maximal voluntary contractions (MVCs) in supine while fine-wire EMG was recorded bilaterally from the PFMs under three conditions: (a) without any probe inserted into the vagina, (b) while a Femiscan? probe was in situ, and (c) while a Periform? vaginal probe was in situ. The reliability of the fine wire EMG data was assessed using intra-class correlation coefficients (ICCs) and coefficients of variation (CV). A repeated measures analysis of variance (ANOVA) model was used to determine if there were differences in EMG amplitude recorded when the different vaginal probes were in situ. For each condition the between-trial reliability was excellent, ICC_(3,1) = 0.93–0.96, (p < 0.001) and CV = 11.2–21.8%. There were no differences in peak EMG amplitude recorded during the MVCs across the three conditions (no probe 63.4 ± 48.4 μV, Femiscan? 55.3 ± 42.4 μV, Periform? 59.4 ± 42.2 μV, p = 0.178). These results suggest that women produce consistent MVCs over multiple contractions, and that PFM muscle activation is not affected by different probes inserted into the vagina.     

P2X7 receptor as predictor gene for glioma radiosensitivity and median survival

Glioblastoma multiforme (GBM) is considered the most lethal intracranial tumor and the median survival time is approximately 14 months. Although some glioma cells present radioresistance, radiotherapy has been the mainstay of therapy for patients with malignant glioma. The activation of P2X7 receptor (P2X7R) is responsible for ATP-induced death in various cell types. In this study, we analyzed the importance of ATP-P2X7R pathway in the radiotherapy response P2X7R silenced cell lines, in vivo and human tumor samples. Both glioma cell lines used in this study present a functional P2X7R and the P2X7R silencing reduced P2X7R pore activity by ethidium bromide uptake. Gamma radiation (2 Gy) treatment reduced cell number in a P2X7R-dependent way, since both P2X7R antagonist and P2X7R silencing blocked the cell cytotoxicity caused by irradiation after 24 h. The activation of P2X7R is time-dependent, as EtBr uptake significantly increased after 24 h of irradiation. The radiotherapy plus ATP incubation significantly increased annexin V incorporation, compared with radiotherapy alone, suggesting that ATP acts synergistically with radiotherapy. Of note, GL261 P2X7R silenced-bearing mice failed in respond to radiotherapy (8 Gy) and GL261 WT-bearing mice, that constitutively express P2X7R, presented a significant reduction in tumor volume after radiotherapy, showing in vivo that functional P2X7R expression is essential for an efficient radiotherapy response in gliomas. We also showed that a high P2X7R expression is a good prognostic factor for glioma radiosensitivity and survival probability in humans. Our data revealed the relevance of P2X7R expression in glioma cells to a successful radiotherapy response, and shed new light on this receptor as a useful predictor of the sensitivity of cancer patients to radiotherapy and median survival.     

Uptake of advanced glycation end products by proximal tubule epithelial cells via macropinocytosis

Chronic hyperglycaemia during diabetes leads to non-enzymatic glycation of proteins to form advanced glycation end products (AGEs) that contribute to nephropathy. We describe AGE uptake in LLC-PK1 and HK2 proximal tubule cell lines by macropinocytosis, a non-specific, endocytic mechanism. AGE–BSA induced dorsal circular actin ruffles and amiloride-sensitive dextran–TRITC uptake, significantly increased AGE–BSA–FITC uptake (167 ± 20% vs BSA control, p < 0.01) and was ezrin-dependent. AGE–BSA–FITC uptake was significantly inhibited by amiloride and inhibitors of Arf6, Rac1, racGEF Tiam1, PAK1 and actin polymerisation. AGE–BSA–FITC, Arf6 and PIP2 co-localised within dorsal circular actin ruffles. AGE–BSA increased PAK1 kinase activity (212 ± 41% vs control, p < 0.05) and protein levels of Tiam1, a Rac1 activator. AGE–BSA significantly increased TGF-β₁ protein levels (160 ± 6%, p < 0.001 vs BSA), which were significantly inhibited by inhibitors of Arf6 (82 ± 19%, p < 0.001 vs AGE) and actin polymerisation (107 ± 11%, p < 0.001 vs AGE), suggesting AGEs partially exert their profibrotic effects via macropinocytosis. PAK1 and PIP5Kγ siRNA significantly decreased AGE–BSA–FITC uptake (81 ± 6% and 64 ± 7%, respectively, p < 0.05 vs control for both), and AGE-stimulated TGF-β₁ protein release (99 ± 15% and 49 ± 8% of control, p < 0.05 and p < 0.001, respectively). Inhibition of AGE uptake by macropinocytosis inhibitors and a neutralising TGF-β antibody, reversed the AGE-induced decrease in surface Na⁺K⁺ATPase, suggesting AGE uptake by macropinocytosis may contribute to diabetic kidney fibrosis and/or EMT by modulating this pump. Understanding methods of cellular uptake and signalling by AGEs may lead to novel therapies for diabetic nephropathy.     

In vivo and in vitro SAR of tetracyclic MAPKAP-K2 (MK2) inhibitors. Part II

Spirocyclopropane- and spiroazetidine-substituted tetracycles 13D–E and 16A are described as orally active MK2 inhibitors. The spiroazetidine derivatives are potent MK2 inhibitors with IC₅₀ <3 nM and inhibit the release of TNFα (IC₅₀<0.3 μM) from hPBMCs and hsp27 phosphorylation in anisomycin stimulated THP-1 cells. The spirocyclopropane analogues are less potent against MK2 (IC₅₀ = 0.05–0.23 μM), less potent in cells (IC₅₀ <1.1 μM), but show good oral absorption. Compound 13E (100 mg/kg po; bid) showed oral activity in rAIA and mCIA, with significant reduction of swelling and histological score.     

Urease immobilization on biotinylated polypyrrole coated ChemFEC devices for urea biosensor development

In this report, we describe a novel strategy for the design of a clinical urea biosensor using a process based on assembled multilayer systems. Biotinylated enzyme (urease–subiotin) was immobilized on the biotinylated polypyrrole coated Chemical field effect capacitance (ChemFEC) devices using the high avidin–biotin affinity. The immobilized enzyme activity was checked by its catalytic conversion of urea into carbon dioxide and ammonia. Electrochemical response of the bridge system constructed on Si/SiO₂/Si₃N₄ electrodes to urea addition was evaluated using the capacity–potential measurements. In addition, contact-angle measurements were carried out to control the change of surface energy and their components before and after each layer formation. The developed urea biosensor demonstrates high performances: a good sensitivity of 50 mV/pUrea in the linear urea concentration range from 10⁻⁴ to 10⁻¹ M and an excellent operational stability after three weeks of continuous use. The immobilized urease was characterised through its apparent Michaelis–Menten constant (5 mM) and the activation energy of the enzymatic reaction (20 kJ mol⁻¹). It was also shown that capacitive measurements can be used to quantify the interaction between molecular systems, based on avidin and biotin molecules.     

Absence of an effect of vitamin E on protein and lipid radical formation during lipoperoxidation of LDL by lipoxygenase

Low-density lipoprotein (LDL) oxidation is the primary event in atherosclerosis, and LDL lipoperoxidation leads to modifications in apolipoprotein B-100 (apo B-100) and lipids. Intermediate species of lipoperoxidation are known to be able to generate amino acid-centered radicals. Thus, we hypothesized that lipoperoxidation intermediates induce protein-derived free radical formation during LDL oxidation. Using DMPO and immuno-spin trapping, we detected the formation of protein free radicals on LDL incubated with Cu²⁺ or the soybean lipoxidase (LPOx)/phospholipase A₂ (PLA₂). With low concentrations of DMPO (1 mM), Cu²⁺ dose-dependently induced oxidation of LDL and easily detected apo B-100 radicals. Protein radical formation in LDL incubated with Cu²⁺ showed maximum yields after 30 min. In contrast, the yields of apo B-100 radicals formed by LPOx/PLA₂ followed a typical enzyme-catalyzed kinetics that was unaffected by DMPO concentrations of up to 50 mM. Furthermore, when we analyzed the effect of antioxidants on protein radical formation during LDL oxidation, we found that ascorbate, urate, and Trolox dose-dependently reduced apo B-100 free radical formation in LDL exposed to Cu²⁺. In contrast, Trolox was the only antioxidant that even partially protected LDL from LPOx/PLA₂. We also examined the kinetics of lipid radical formation and protein radical formation induced by Cu²⁺ or LPOx/PLA₂ for LDL supplemented with α-tocopherol. In contrast to the potent antioxidant effect of α-tocopherol on the delay of LDL oxidation induced by Cu²⁺, when we used the oxidizing system LPOx/PLA₂, no significant protection was detected. The lack of protection of α-tocopherol on the apo B-100 and lipid free radical formation by LPOx may explain the failure of vitamin E as a cardiovascular protective agent for humans.     

Influence of ankle functional instability on the ankle electromyography during landing after volleyball blocking

The purpose of this study was to describe, interpret and compare the EMG activation patterns of ankle muscles – tibialis anterior (TA), peroneus longus (PL) and gastrocnemius lateralis (GL) – in volleyball players with and without ankle functional instability (FI) during landing after the blocking movement. Twenty-one players with FI (IG) and 19 controls (CG) were studied. The cycle of movement analyzed was the time period between 200 ms before and 200 ms after the time of impact determined by ground reaction forces. The variables were analyzed for two different phases: pre-landing (200 ms before impact) and post-landing (200 ms after impact). The RMS values and the timing of onset activity were calculated for the three studied muscles, in both periods and for both groups. The co-activation index for TA and PL, TA and GL were also calculated. Individuals with FI presented a lower RMS value pre-landing for PL (CG = 43.0 ± 22.0; IG = 26.2 ± 8.4, p < 0.05) and higher RMS value post-landing (CG = 47.5 ± 13.3; IG = 55.8 ± 21.6, p < 0.10). Besides that, in control group PL and GL activated first and simultaneously, and TA presented a later activation, while in subjects with FI all the three muscles activated simultaneously. There were no significant differences between groups for co-activation index. Thus, the rate of contraction between agonist and antagonist muscles is similar for subjects with and without FI but the activation individually was different. Volleyball players with functional instability of the ankle showed altered patterns of the muscles that play an important role in the stabilization of the foot–ankle complex during the performance of the blocking movement, to the detriment of the ligament complex, and this fact could explain the usual complaints in these subjects.     

Structural changes of the myofibrillar proteins in common carp (Cyprinus carpio) muscle exposed to a hydroxyl radical-generating system

Oxidation is a leading cause for quality deterioration during processing and storage of food. The objective of the present study was to examine the sensitivity of common carp (Cyprinus carpio) myofibrillar protein (MP) to oxidising radicals produced by a hydroxyl radical-generating system. Both structural and functional changes of common carp MP were evaluated. With increasing H₂O₂ concentrations and oxidation time, the protein carbonyl content, surface hydrophobicity and turbidity of MP increased (P < 0.05), while total sulfhydryl groups decreased (P < 0.05). Sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed protein polymerisation in oxidised MP. The oxidative process destroyed (P < 0.05) the texture (springiness and hardness) of MP gels and decreased their water-binding capacity and whiteness. The thermal gelation profile analysis indicated that oxidation led to a great reduction in the elasticity of samples. Taken together, proteins are susceptible to free radical attack, and oxidative stress had a detrimental effect on protein structure and the general functionality of MP.     

Extent estimates and land cover relationships for functional indicators in non-wadeable rivers

Functional indicators are being increasingly used to assess waterway health but their responses to pressure in non-wadeable rivers have not been widely documented or applied in modern survey designs that provide unbiased estimates of extent. This study tests the response of river metabolism and loss in cotton strip tensile strength across a land use pressure gradient in non-wadeable rivers of northern New Zealand, and reports extent estimates for river metabolism and decomposition rates. Following adjustment for probability of selection, ecosystem respiration (ER) and gross primary production (GPP) for the target population of order 5–7 non-wadeable rivers averaged −7.3 and 4.8 g O₂ m⁻² d⁻¹, respectively, with average P/R < 1 indicating dominance by heterotrophic processes. Ecosystem respiration was <−3.3 g O₂ m⁻² d⁻¹ for 75% of non-wadeable river length with around 20% of length between −10 and −20 g O₂ m⁻² d⁻¹. Cumulative distribution functions of cotton strength loss estimates indicated a more-or-less linear relationship with river km reflecting an even spread of decay rates (range in k 0.0007–0.2875 d⁻¹) across non-wadeable rivers regionally. A non-linear relationship with land cover was detected for GPP which was typically <5 g O₂ m⁻² d⁻¹ where natural vegetation cover was below 20% and greater than 80% of upstream catchment area. For cotton strength loss, the relationship with land cover was wedge-shaped such that sites with >60% natural cover had low decay rates (<0.02 d⁻¹) with variability below this increasing as natural cover declined. Using published criteria for assessing waterway health based on ER and GPP, 232–298 km (20–29%) of non-wadeable river length was considered to have severely impaired ecosystem functioning, and 436–530 km (42–50%) had no evidence of impact on river metabolism.     

Angiotensin-(1–7) abolishes AGE-induced cellular hypertrophy and myofibroblast transformation via inhibition of ERK1/2

Angiotensin-(1–7) (Ang-(1–7))/AT₇-Mas receptor axis is an alternative pathway within the renin–angiotensin system (RAS) that generally opposes the actions of Ang II/AT₁ receptor pathway. Advanced glycated end product (AGEs) including glucose- and methylglyoxal-modified albumin (MGA) may contribute to the development and progression of diabetic nephropathy in part through activation of the Ang II/AT₁ receptor system; however, the influence of AGE on the Ang-(1–7) arm of the RAS within the kidney is unclear. The present study assessed the impact of AGE on the Ang-(1–7) axis in NRK-52E renal epithelial cells. MGA exposure for 48 h significantly reduced the intracellular levels of Ang-(1–7) approximately 50%; however, Ang I or Ang II expression was not altered. The reduced cellular content of Ang-(1–7) was associated with increased metabolism of the peptide to the inactive metabolite Ang-(1–4) [MGA: 175 ± 9 vs. Control: 115 ± 11 fmol/min/mg protein, p < 0.05, n = 3] but no change in the processing of Ang I to Ang-(1–7). Treatment with Ang-(1–7) reversed MGA-induced cellular hypertrophy and myofibroblast transition evidenced by reduced immunostaining and protein expression of α-smooth muscle actin (α-SMA) [0.4 ± 0.1 vs. 1.0 ± 0.1, respectively, n = 3, p < 0.05]. Ang-(1–7) abolished AGE-induced activation of the MAP kinase ERK1/2 to a similar extent as the TGF-β receptor kinase inhibitor SB58059; however, Ang-(1–7) did not attenuate the MGA-stimulated release of TGF-β. The AT₇-Mas receptor antagonist D-Ala⁷-Ang-(1–7) abolished the inhibitory actions of Ang-(1–7). In contrast, AT₁ receptor antagonist losartan did not attenuate the MGA-induced effects. We conclude that Ang-(1–7) may provide an additional therapeutic approach to the conventional RAS blockade regimen to attenuate AGE-dependent renal injury.     

Rumen microbial responses in fermentation characteristics and production of CLA and methane to linoleic acid in associated with malate or fumarate

An in vitro incubation in batch was conducted to investigate the effect of propionate precursor (malate or fumarate) on fermentation characteristics, and production of CLA and methane by rumen microbes when incubated with linoleic acid (C_18:2). Sixty milligrams of C_18:2 alone (LA), 60 mg C_18:2 with 24 mM malic acid (M-LA), or 60 mg C_18:2 with 24 mM fumaric acid (F-LA) was added to 150 ml culture solution consisting of 75 ml strained rumen fluid and 75 ml McDougall's artificial saliva. Culture solution for incubation was also made without malate, fumarate, and C_18:2 (control). Two grams of feed consisting of 1.4 g concentrate and 0.6 g ground alfalfa (DM basis) was also added to the culture solution of each treatment. An in vitro incubation in batch was made anaerobically in a shaking incubator for up to 12 h at 39 °C.The pH of the culture solution was increased (P<0.0001) in M-LA or F-LA treatments from 3 h to 12 h compared with the control and LA treatments. At 12 h incubation, the concentration of total VFA in the culture solution was higher (P<0.01) in M-LA and F-LA than in control and LA treatments. Concentration of C₃ by M-LA and F-LA was increased at 3 h (P<0.01), 6 h (P<0.01) and 12 h (P<0.01) compared with control and LA. However, no difference in C₃ concentration was observed between control and LA, or between M-LA and F-LA. Accumulated total gas produced for up to 12 h incubation was increased (P<0.01) by M-LA or F-LA compared with the control. Accumulated total methane produced for up to 12 h incubation, however, was greatly reduced (P<0.01) by all the supplements compared with control, and its production from M-LA or F-LA was smaller than the LA. The M-LA or F-LA also increased (P<0.05–<0.001) the concentrations of cis9, trans11-CLA for all incubation times and trans10, cis12-CLA at 1 h (P<0.01), 3 h (P<0.05), and 12 h (P<0.05) incubation times compared with LA.It can be concluded that malate and fumarate, as propionate precursors, act as alternative electron sinks and may compete with CH₄ generation and bio-hydrogenation of C_18:2 in the utilization of metabolic H₂. The highest CLA concentration at the early incubation stage (1 h) was accompanied by reduced propionate proportion. Linoleic acid is also considered one of the potential alternatives to suppress CH₄ generation.