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Muscle-derived mesenchymal stromal cells (mdMSCs) hold great promise in regenerative medicine due to their immunomodulatory properties, multipotent differentiation capacity and ease of collection. However, traditional in vitro expansion methods use fetal bovine serum (FBS) and have numerous limitations including ethical concerns, batch-to-batch variability, immunogenicity, xenogenic contamination and regulatory compliance issues. This study investigates the use of 10% equine platelet lysate (ePL) obtained by plasmapheresis as a substitute for FBS in the culture of mdMSCs in innovative 2D and 3D models. Using muscle microbiopsies as the primary cell source in both models showed promising results. Initial investigations indicated that small variations in heparin concentration in 2D cultures strongly influenced medium coagulation with an optimal proliferation observed at final heparin concentrations of 1.44 IU/mL. The two novel models investigated showed that expansion of mdMSCs is achievable. At the end of expansion, the 3D model revealed a higher total number of cells harvested (64.60 ± 5.32 million) compared to the 2D culture (57.20 ± 7.66 million). Trilineage differentiation assays confirmed the multipotency (osteoblasts, chondroblasts and adipocytes) of the mdMSCs generated in both models with no significant difference observed. Immunophenotyping confirmed the expression of the mesenchymal stem cell (MSC) markers CD-90 and CD-44, with low expression of CD-45 and MHCII markers for mdMSCs derived from the two models. The generated mdMSCs also had great immunomodulatory properties. Specific immunological extraction followed by enzymatic detection (SIEFED) analysis demonstrated that mdMSCs from both models inhibited myeloperoxidase (MPO) activity in a strong dose-dependent manner. Moreover, they were also able to reduce reactive oxygen species (ROS) activity, with mdMSCs from the 3D model showing significantly higher dose-dependent inhibition compared to the 2D model. These results highlighted for the first time the feasibility and efficacy of using 10% ePL for mdMSC expansion in novel 2D and 3D approaches and also that mdMSCs have strong immunomodulatory properties that can be exploited to advance the field of regenerative medicine and cell therapy instead of using FBS with all its drawbacks.
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Plaquetas , Diferenciação Celular , Imunomodulação , Células-Tronco Mesenquimais , Animais , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/imunologia , Cavalos , Plaquetas/metabolismo , Proliferação de Células/efeitos dos fármacos , Técnicas de Cultura de Células/métodos , Células Cultivadas , Músculos , ImunofenotipagemRESUMO
OBJECTIVES: To evaluate the ability of the Assessment of Different NEoplasias in the adneXa (ADNEX) model and the International Ovarian Tumour Analysis (IOTA) two-step strategy to predict malignancy in adnexal masses detected in an outpatient low-risk setting, and to estimate the risk of complications in masses with benign ultrasound morphology managed using clinical and ultrasound follow-up. METHODS: This single-center study was performed at Hospital Universitari Dexeus, Barcelona, Spain, using interim data from the ongoing prospective observational IOTA Phase-5 (IOTA5) study. The primary aim of the IOTA5 study is to describe the cumulative incidence of complications during follow-up of adnexal masses classified as benign on ultrasound examination. Consecutive patients with an adnexal mass detected between June 2012 and September 2016 in a private center offering screening for gynecological cancer were included and followed up until February 2020. Tumors were classified as benign or malignant based on histology (if patients underwent surgery) or the outcome of clinical and ultrasound follow-up at 12 (range, 10-14) months. Multiple imputation was used when outcomes were uncertain. The ability of the ADNEX model without CA125 and of the IOTA two-step strategy to distinguish benign from malignant masses was evaluated retrospectively using the prospectively collected data. We assessed performance with regard to discrimination (area under the receiver-operating-characteristics curve (AUC)), calibration, classification (sensitivity and specificity) and clinical utility (Net Benefit). In the group of patients with a mass judged to be benign who were selected for conservative management, we evaluated the occurrence of spontaneous resolution or any mass complication during the first 5 years of follow-up by assessing the cumulative incidence of malignancy, torsion, cyst rupture and minor mass complications (inflammation, infection or adhesions) and the time to occurrence of an event. RESULTS: A total of 2654 patients were recruited to the study. After application of exclusion criteria, 2039 patients with a newly detected mass were included for the model validation. Of those, 1684 (83%) masses were benign, 49 (2%) masses were malignant and, for 306 (15%) masses, the outcome was uncertain and therefore imputed. The AUC was 0.95 (95% CI, 0.89-0.98) for ADNEX without CA125 and 0.94 (95% CI, 0.88-0.97) for the two-step strategy. Calibration performance could not be meaningfully interpreted because the small number of malignancies resulted in very wide confidence intervals. The two-step strategy had better clinical utility than did the ADNEX model at malignancy risk thresholds < 3%. There were 1472 (72%) patients whose mass was judged to be benign based on pattern recognition by an experienced ultrasound examiner and were managed with clinical and ultrasound follow-up. In this group, the 5-year cumulative incidence was 66% (95% CI, 63-69%) for spontaneous resolution of the mass, 0% (95% CI, 0-0.2%) for torsion, 0.1% (95% CI, < 0.1-0.4%) for cyst rupture, 0.2% (95% CI, 0.1-0.6%) for a borderline tumor and 0.2% (95% CI, 0.1-0.6%) for invasive malignancy. CONCLUSIONS: The ADNEX model and IOTA two-step strategy performed well to distinguish benign from malignant adnexal masses detected in a low-risk population. Conservative management is safe for masses with a benign ultrasound appearance in this population. © 2024 International Society of Ultrasound in Obstetrics and Gynecology.
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OBJECTIVES: Timely and accurate preoperative diagnosis of uterine sarcoma will increase patient survival. The primary aim of this study was to describe the ultrasound features of uterine sarcoma compared with those of uterine leiomyoma based on the terms and definitions of the Morphological Uterus Sonographic Assessment (MUSA) group. A secondary aim was to assess the interobserver agreement for reporting on ultrasound features according to MUSA terminology. METHODS: This was a retrospective cohort study of patients with uterine sarcoma or uterine leiomyoma treated in a single tertiary center during the periods 1997-2019 and 2016-2019, respectively. Demographic characteristics, presenting symptoms and surgical outcomes were extracted from patients' files. Ultrasound images were re-evaluated independently by two sonologists using MUSA terms and definitions. Descriptive statistics were calculated and interobserver agreement was assessed using Cohen's κ (with squared weights) or intraclass correlation coefficient, as appropriate. RESULTS: A total of 107 patients were included, of whom 16 had a uterine sarcoma and 91 had a uterine leiomyoma. Abnormal uterine bleeding was the most frequent presenting symptom (69/107 (64%)). Compared with leiomyoma cases, patients with uterine sarcoma were older (median age, 65 (interquartile range (IQR), 60-70) years vs 48 (IQR, 43-52) years) and more likely to be postmenopausal (13/16 (81%) vs 15/91 (16%)). In the uterine sarcoma cohort, leiomyosarcoma was the most frequent histological type (6/16 (38%)), followed by adenosarcoma (4/16 (25%)). On ultrasound evaluation, according to Observers 1 and 2, the tumor border was irregular in most sarcomas (11/16 (69%) and 13/16 (81%) cases, respectively), but regular in most leiomyomas (65/91 (71%) and 82/91 (90%) cases, respectively). Lesion echogenicity was classified as non-uniform in 68/91 (75%) and 51/91 (56%) leiomyomas by Observers 1 and 2, respectively, and 15/16 (94%) uterine sarcomas by both observers. More than 60% of the uterine sarcomas showed acoustic shadows (11/16 (69%) and 10/16 (63%) cases by Observers 1 and 2, respectively), whereas calcifications were reported in a small minority (0/16 (0%) and 2/16 (13%) cases by Observers 1 and 2, respectively). In uterine sarcomas, intralesional vascularity was reported as moderate to abundant in 13/16 (81%) cases by Observer 1 and 15/16 (94%) cases by Observer 2, while circumferential vascularity was scored as moderate to abundant in 6/16 (38%) by both observers. Interobserver agreement for the presence of cystic areas, calcifications, acoustic shadow, central necrosis, color score (overall, intralesional and circumferential) and maximum diameter of the lesion was moderate. The agreement for shape of lesion, tumor border and echogenicity was fair. CONCLUSIONS: A postmenopausal patient presenting with abnormal uterine bleeding and a new or growing mesenchymal mass with irregular tumor borders, moderate-to-abundant intralesional vascularity, cystic areas and an absence of calcifications on ultrasonography is at a higher risk of having a uterine sarcoma. Interobserver agreement for most MUSA terms and definitions is moderate. Future studies should validate the abovementioned clinical and ultrasound findings on uterine mesenchymal tumors in a prospective multicenter fashion. © 2023 International Society of Ultrasound in Obstetrics and Gynecology.
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OBJECTIVE: Previous work has suggested that the ultrasound-based benign simple descriptors (BDs) can reliably exclude malignancy in a large proportion of women presenting with an adnexal mass. This study aimed to validate a modified version of the BDs and to validate a two-step strategy to estimate the risk of malignancy, in which the modified BDs are followed by the Assessment of Different NEoplasias in the adneXa (ADNEX) model if modified BDs do not apply. METHODS: This was a retrospective analysis using data from the 2-year interim analysis of the International Ovarian Tumor Analysis (IOTA) Phase-5 study, in which consecutive patients with at least one adnexal mass were recruited irrespective of subsequent management (conservative or surgery). The main outcome was classification of tumors as benign or malignant, based on histology or on clinical and ultrasound information during 1 year of follow-up. Multiple imputation was used when outcome based on follow-up was uncertain according to predefined criteria. RESULTS: A total of 8519 patients were recruited at 36 centers between 2012 and 2015. We excluded patients who were already in follow-up at recruitment and all patients from 19 centers that did not fulfil our criteria for good-quality surgical and follow-up data, leaving 4905 patients across 17 centers for statistical analysis. Overall, 3441 (70%) tumors were benign, 978 (20%) malignant and 486 (10%) uncertain. The modified BDs were applicable in 1798/4905 (37%) tumors, of which 1786 (99.3%) were benign. The two-step strategy based on ADNEX without CA125 had an area under the receiver-operating-characteristics curve (AUC) of 0.94 (95% CI, 0.92-0.96). The risk of malignancy was slightly underestimated, but calibration varied between centers. A sensitivity analysis in which we expanded the definition of uncertain outcome resulted in 1419 (29%) tumors with uncertain outcome and an AUC of the two-step strategy without CA125 of 0.93 (95% CI, 0.91-0.95). CONCLUSION: A large proportion of adnexal masses can be classified as benign by the modified BDs. For the remaining masses, the ADNEX model can be used to estimate the risk of malignancy. This two-step strategy is convenient for clinical use. © 2022 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.
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Doenças dos Anexos , Neoplasias Ovarianas , Feminino , Humanos , Estudos Retrospectivos , Neoplasias Ovarianas/patologia , Doenças dos Anexos/patologia , Ultrassonografia/métodos , Antígeno Ca-125 , Sensibilidade e Especificidade , Diagnóstico DiferencialRESUMO
OBJECTIVE: To estimate the diagnostic value of tumor and immune related proteins in the discrimination between benign and malignant adnexal masses, and between different subgroups of tumors. METHODS: In this exploratory diagnostic study, 254 patients with an adnexal mass scheduled for surgery were consecutively enrolled at the University Hospitals Leuven (128 benign, 42 borderline, 22 stage I, 55 stage II-IV, and 7 secondary metastatic tumors). The quantification of 33 serum proteins was done preoperatively, using multiplex high throughput immunoassays (Luminex) and electrochemiluminescence immuno-assay (ECLIA). We calculated univariable areas under the Receiver Operating Characteristic Curves (AUCs). To discriminate malignant from benign tumors, multivariable ridge logistic regression with backward elimination was performed, using bootstrapping to validate the resulting AUCs. RESULTS: CA125 had the highest univariable AUC to discriminate malignant from benign tumors (0.85, 95% confidence interval 0.79-0.89). Combining CA125 with CA72.4 and HE4 increased the AUC to 0.87. For benign vs borderline tumors, CA125 had the highest univariable AUC (0.74). For borderline vs stage I malignancy, no proteins were promising. For stage I vs II-IV malignancy, CA125, HE4, CA72.4, CA15.3 and LAP had univariable AUCs ≥0.80. CONCLUSIONS: The results confirm the dominant role of CA125 for identifying malignancy, and suggest that other markers (HE4, CA72.4, CA15.3 and LAP) may help to distinguish between stage I and stage II-IV malignancies. However, further research is needed, also to investigate the added value over clinical and ultrasound predictors of malignancy, focusing on the differentiation between subtypes of malignancy.
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Antígeno Ca-125/sangue , Proteínas de Membrana/sangue , Neoplasias Ovarianas/diagnóstico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos Glicosídicos Associados a Tumores/sangue , Antígenos Glicosídicos Associados a Tumores/imunologia , Antígeno Ca-125/imunologia , Diagnóstico Diferencial , Feminino , Humanos , Proteínas de Membrana/imunologia , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neoplasias Ovarianas/sangue , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/cirurgia , Ovário/patologia , Ovário/cirurgia , Período Pré-Operatório , Estudos Prospectivos , Curva ROC , Proteína 2 do Domínio Central WAP de Quatro Dissulfetos/análise , Adulto JovemRESUMO
Enset (Ensete ventricosum) provides staple food for 15 million people in Ethiopia after fermentation into kocho. The fermentation process has hardly been investigated and is prone to optimization. The aim of this study was to investigate the physicochemical and microbial dynamics of fermentation practices in the Gamo highlands. These practices show local variation, but two steps were omnipresent: scraping of the pseudostem and fermenting it in a pit or a bamboo basket. Enset plants were fragmented and fermented for two months in order to investigate the physicochemical (temperature, moisture content, pH and titratable acidity) and microbial dynamics (total viable aerobic counts, counts of Enterobacteriaceae, lactic acid bacteria, yeasts and moulds and Clostridium spores counts, and Illumina Miseq sequencing). Samples were taken on days 1, 7, 15, 17, 31 and 60. The pH decreased, whereas the titratable acidity increased during fermentation. Of all counts those of lactic acid bacteria and Clostridium spores increased during fermentation. Leuconostoc mesenteroides initiated the fermentation. Later on, Prevotella paludivivens, Lactobacillus sp. and Bifidobacterium minimum dominated. These three species are potential candidates for the development of a starter culture.
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Lactobacillales/isolamento & purificação , Musaceae/microbiologia , Biodiversidade , Clostridium/classificação , Clostridium/genética , Clostridium/isolamento & purificação , Clostridium/metabolismo , Etiópia , Fermentação , Concentração de Íons de Hidrogênio , Lactobacillales/classificação , Lactobacillales/metabolismo , Musaceae/química , Musaceae/metabolismo , TemperaturaRESUMO
Bone marrow and adipose tissue represent the two most commonly exploited sources of adult mesenchymal stem cells for musculoskeletal applications. Unfortunately the sampling of bone marrow and fat tissue is invasive and does not always lead to a sufficient number of cells. The present study describes a novel sampling method based on microbiopsy of skeletal muscle in man, pigs, dogs and horses. The process includes explant of the sample, Percoll density gradient for isolation and subsequent culture of the cells. We further characterized the cells and identified their clonogenic and immunomodulatory capacities, their immune-phenotyping behavior and their capability to differentiate into chondroblasts, osteoblasts and adipocytes. In conclusion, this report describes a novel and easy-to-use technique of skeletal muscle-derived mesenchymal stem cell harvest, culture, characterization. This technique is transposable to a multitude of different animal species.
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Músculo Esquelético/citologia , Células-Tronco/citologia , Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Diferenciação Celular , Células Cultivadas , Cães , Cavalos , Humanos , Imunomodulação , Imunofenotipagem , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Fenótipo , Células-Tronco/metabolismo , SuínosRESUMO
REASONS FOR PERFORMING STUDY: Intensive exercise induces a systemic inflammatory response characterised by an increase of blood neutrophil count and myeloperoxidase (MPO) release. Neutrophil elastase (NE) could also contribute to tissues lesions by its proteinase activities. OBJECTIVE: To compare plasmatic NE concentrations before and after different forms of intensive exercise. MATERIALS AND METHODS: EDTA blood samples were taken from 51 eventing horses (EvH) and 32 endurance horses (EndH) were sampled before the race (T0). Blood sampling was performed 2 h (T1) after completing either phase D of a one or 2 star eventing competition (n = 51), or a 120 or 160 km endurance race (n = 32). Plasmatic NE and MPO were measured by a specific equine ELISA. Neutrophil counts and creatine kinase (CK) levels were also measured. A Wilcox on test for paired samples was used to compare mean values of neutrophils, CK, MPO and NE at T0 and T1 in EvH and in EndH. Correlations were calculated between all the 4 parameters in EvH and EndH. RESULTS: At T0, mean NE levels were 14.43 ± 3.63 ng/ml for EvH and 11.7 ± 2.11 ng/ml for EndH. The competition induced a significant increase of NE levels in (58.57 ± 24.06 ng/ml) EvH and (95.74 ± 22.70 ng/ml) EndH (P < 0.05). NE was significantly (P < 0.0001) correlated to MPO in EvH (r = 0.293) and EndH (r = 0.594) and to CK (r = 0.297) in EndH (P < 0.0001). Neutrophils, CK and MPO were significantly increased between T0 and T1 in both types of horses. CONCLUSIONS: Significant increase of NE (EndH) was observed after intense exercise with a significant correlation between NE and MPO. The huge variability in MPO and NE indicates that not all horses show the same intensity of systemic inflammatory response.
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Cavalos/fisiologia , Elastase de Leucócito/sangue , Condicionamento Físico Animal/fisiologia , Resistência Física/fisiologia , Esportes , Animais , Elastase de Leucócito/metabolismo , Peroxidase/metabolismoRESUMO
REASONS FOR PERFORMING STUDY: Limited information exists about the muscle mitochondrial respiratory function changes that occur in horses during an endurance season. OBJECTIVES: To determine effects of training and racing on muscle oxidative phosphorylation (OXPHOS) and electron transport system (ETS) capacities in horses with high resolution respirometry (HRR). METHODS: Mitochondrial respiration was measured in microbiopsies taken from the triceps brachii (tb) and gluteus medius (gm) muscles in 8 endurance horses (7 purebred Arabians and 1 crossbred Arabian) before training (T0), after two 10 week training periods (T1, T2) and after 2 CEI** endurance races (R1, R2). Muscle OXPHOS capacity was determined using 2 titration protocols without (SUIT 1) or with pyruvate (SUIT 2) as substrate. Electrons enter at the level of Complex I, Complex II or both complexes simultaneously (Complexes I+II). Muscle ETS capacity was obtained by uncoupling Complexes I+II sustained respiration. RESULTS: T1 improved OXPHOS and ETS capacities in the tb as demonstrated by the significant increase of oxygen fluxes vs. T0 (Complex I: +67%; ETS: +37%). Training improved only OXPHOS in the gm (Complex I: +34%). Among horses that completed the race, a significant decrease in OXPHOS (Complex I: â¼ -35%) and ETS (-22%) capacities was found in the tb with SUIT 2 indicating a reduced aerobic glycolysis. Significant correlations between CK activities and changes in OXPHOS were found suggesting a relationship between exercise-induced muscle damage and depression of mitochondrial respiration. CONCLUSIONS: For the first time, OXPHOS and ETS capacities in equine muscle at different steps of an endurance season have been determined by HRR. Significant alterations in mitochondrial respiratory function in response to endurance training and endurance racing have been observed although these changes appeared to be muscle group specific.
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Cavalos/fisiologia , Mitocôndrias Musculares/fisiologia , Consumo de Oxigênio/fisiologia , Condicionamento Físico Animal/fisiologia , Resistência Física/fisiologia , Animais , Feminino , Masculino , EsportesRESUMO
REASONS FOR PERFORMING STUDY: Intense physical exercise can induce the degranulation of neutrophils leading to an increase in plasma concentration of the neutrophil marker enzymes myeloperoxidase (MPO) and elastase (ELT). These enzymes have pro-oxidative and pro-inflammatory properties and may play a role in the exercised-induced muscular damage. OBJECTIVES: To measure MPO and ELT concentrations in plasma and muscles of endurance horses and to correlate them to the extent of exercise-induced muscular damage. METHODS: Seven endurance horses qualified on 120 km races were tested in this study. Neutrophil count, serum creatine kinase (CK), plasmatic and muscular MPO and ELT concentrations were measured before and 2 h after a 120 km endurance race. RESULTS: The race produced a significant increase of neutrophils, CK, and plasma MPO and ELT levels. A significant correlation was observed between the MPO and ELT values in plasma (r(2) = 0.92, P < 0.01) and in muscles (r(2) = 0.89, P < 0.01) while plasmatic concentrations of MPO and ELT were not significantly correlated to muscular ones. An increase of mean concentrations (± s.e.) of MPO (T0: 9.85 ± 3.9, T1: 228.9 ± 95.9 ng/mg proteins) and ELT (T0: 8.4 ± 2.4, T1: 74.5 ± 39.7 ng/mg proteins) in the muscles were observed after the race. Interestingly, the individual data showed large differences between the horses. Muscular MPO and ELT concentrations were significantly correlated to plasma CK levels. The coefficient of correlation (r(2)) was 0.69 (P < 0.01) for MPO and 0.66 (P < 0.01) for ELT, respectively. CONCLUSIONS: Results underline the possible role of MPO and ELT in exercise-induced muscular damage. POTENTIAL RELEVANCE: Further studies should investigate the effect of exercise type and intensity, as well as the role of the training state on MPO and ELT involvement in muscular damage. The assessment of the intensity of exercise-induced neutrophilic degranulation may have a potential role in the monitoring of the athletic career.
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Cavalos/fisiologia , Elastase de Leucócito/metabolismo , Músculo Esquelético/enzimologia , Peroxidase/metabolismo , Condicionamento Físico Animal/fisiologia , Resistência Física/fisiologia , Animais , Regulação Enzimológica da Expressão Gênica/fisiologia , Cavalos/sangue , Elastase de Leucócito/genética , Peroxidase/genéticaRESUMO
REASONS FOR PERFORMING STUDY: Intense exercise in horses induces an increase of creatine kinase (CK) and stimulation of neutrophils which release the strong oxidant enzyme, myeloperoxidase (MPO) into the blood. It is not known whether active MPO is found in equine muscles and whether oxidant activity of neutrophils could affect muscular tissues and mitochondrial activity. OBJECTIVES: Specific immuno-extraction followed by enzymatic detection (SIEFED) methods will be employed for the first time to assess both the normal range of MPO and mitochondrial complex I (MCI) activities in equine muscular microbiopsies and to study the variation of these activities induced by endurance races. MATERIALS AND METHODS: Forty-six microbiopsies were taken from 8 endurance Arabian horses (age: 10 ± 2 years) in the triceps brachii (n = 23) or in the gluteus medius muscle (n = 23). Myeloperoxidase and MCI activities were measured in muscle extracts by enzyme immunocapture assays or SIEFED methods. Further, 7 endurance horses were sampled in the triceps brachii muscle before (T0) and after (T1) a 120 km endurance race (mean speed: 15.4 ± 1.4 km/h). RESULTS: The 46 microbiopsies from 8 horses revealed mean values for active MPO concentration and MCI activity of 21 ± 14 ng/mg proteins and 0.0172 ± 0.0066 mOD/min/µg proteins, respectively. No significant difference was observed between the 2 muscles. In 3 out of the 7 horses sampled after exercise, the 120 km endurance race induced a severe increase of muscle MPO activity (+118 ± 45% vs. T0), a large decrease of MCI activity (-63 ± 18% vs. T0) associated with a high mean plasma CK level (4642 ± 658 iu/l). In the 4 remaining horses, the 120 km endurance race did not modify the MPO and MCI activities and moderately increased the plasma CK level. CONCLUSIONS: Preliminary observations showed a possible link between MPO activity and mitochondrial functions.
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Biópsia/veterinária , Complexo I de Transporte de Elétrons/metabolismo , Cavalos/metabolismo , Técnicas Imunoenzimáticas/veterinária , Peroxidase/metabolismo , Resistência Física/fisiologia , Animais , Técnicas Imunoenzimáticas/métodos , Músculo Esquelético/enzimologia , Condicionamento Físico Animal/fisiologiaRESUMO
BACKGROUND AND AIMS: The deployment of temporally separated carboxylation pathways for net CO(2) uptake in CAM plants provides plasticity and thus uncertainty on how species with this photosynthetic pathway will respond to life in a higher-CO(2) world. The present study examined how long-term exposure to elevated CO(2) influences the relative contributions that C(3) and C(4) carboxylation make to net carbon gain and to establish how this impacts on the availability of carbohydrates for export and growth and on water use efficiency over the day/night cycle. METHODS: Integrated measurements of leaf gas exchange and diel metabolite dynamics (e.g. malate, soluble sugars, starch) were made in leaves of the CAM bromeliad Aechmea 'Maya' after exposure to 700 micromol mol(-1) CO(2) for 5 months. KEY RESULTS: There was a 60 % increase in 24-h carbon gain under elevated CO(2) due to a stimulation of daytime C(3) and C(4) carboxylation in phases II and IV where water use efficiency was comparable with that measured at night. The extra CO(2) taken up under elevated CO(2) was largely accumulated as hexose sugars during phase IV and net daytime export of carbohydrate was abolished. Under elevated CO(2) there was no stimulation of dark carboxylation and nocturnal export and respiration appeared to be the stronger sinks for carbohydrate. CONCLUSIONS: Despite the increased size of the soluble sugar storage pool under elevated CO(2), there was no change in the net allocation of carbohydrates between provision of substrates for CAM and export/respiration in A. 'Maya'. The data imply the existence of discrete pools of carbohydrate that provide substrate for CAM or sugars for export/respiration. The 2-fold increase in water-use efficiency could be a major physiological advantage to growth under elevated CO(2) in this CAM bromeliad.