Enhanced axon outgrowth and improved long‐distance axon regeneration in sprouty2 deficient mice

Sprouty (Spry) proteins are negative feedback inhibitors of receptor tyrosine kinase signaling. Downregulation of Spry2 has been demonstrated to promote elongative axon growth of cultured peripheral and central neurons. Here, we analyzed Spry2 global knockout mice with respect to axon outgrowth in vitro and peripheral axon regeneration in vivo. Neurons dissociated from adult Spry2 deficient sensory ganglia revealed stronger extracellular signal‐regulated kinase activation and enhanced axon outgrowth. Prominent axon elongation was observed in heterozygous Spry2^+/? neuron cultures, whereas homozygous Spry2^?/? neurons predominantly exhibited a branching phenotype. Following sciatic nerve crush, Spry2^+/? mice recovered faster in motor but not sensory testing paradigms (Spry2^?/? mice did not tolerate anesthesia required for nerve surgery). We attribute the improvement in the rotarod test to higher numbers of myelinated fibers in the regenerating sciatic nerve, higher densities of motor endplates in hind limb muscles and increased levels of GAP‐43 mRNA, a downstream target of extracellular regulated kinase signaling. Conversely, homozygous Spry2^?/? mice revealed enhanced mechanosensory function (von Frey's test) that was accompanied by an increased innervation of the epidermis, elevated numbers of nonmyelinated axons and more IB4‐positive neurons in dorsal root ganglia. The present results corroborate the functional significance of receptor tyrosine kinase signaling inhibitors for axon outgrowth during development and nerve regeneration and propose Spry2 as a novel potential target for pharmacological inhibition to accelerate long‐distance axon regeneration in injured peripheral nerves. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 217–231, 2015     

Pennsylvanian miospore assemblages from the Bèdero Section,Varese, Italian Southern Alps

Thin continental Carboniferous sequences crop out sparsely in the western Southern Alps (Alpe Logone, Mesenzana, Grantola, Bosco Valtravaglia-Fabiasco, Val Tresa, etc.) and are currently the subject of a detailed litho- and biostratigraphic revision, to reconsider their chronological position with respect to previously published age constraints. The age of these Upper Palaeozoic sedimentary successions, scattered over a wide area and strongly tectonized along major structural lineaments, has long been debated between Westphalian and Stephanian. The present work focuses mainly on the palynology of the Brezzo di Bèdero section (Luino, Lake Maggiore). The recovered palynoflora is assigned to 42 spore genera and 76 species of which one genus and 10 species are newly proposed. Fifty-seven spore species and 19 pollen species are described and illustrated. Qualitatively, the Bèdero palynoflora shows strong affinities to those of Western Europe. The most abundant palynomorphs are trilete spores known to be characteristics of the late Westphalian and early Stephanian assemblages. These are characterized by the remarkable presence of Florinites and subordinately Wilsonites species, low numbers of Potonieisporites, rare Limitisporites and Vesicaspora, and very rare Latensina-Cordaitina pollen. The occurrence of diverse monolete spores Laevigatosporites, Punctatosporites, Spinosporites, Thymospora and Torispora, with common Lundbladispora gigantea, L. simonii, Stenozonotriletes rubius n. sp. together with the absence of plicate and taeniate pollen grains suggests a strong resemblance to the late Westphalian–early Stephanian interval assigned to the OT Zone of Western Europe. Variations in the quantitative composition can be likely attributed to variations in the environmental setting with regards to non-forming peat deposits. The palynological suite is indicative of the existence of a well-established lowland Cordaiteans vegetation and well settled pterophytic, pteridosperm and subordinately sphenophytic and lycophytic, hygrophytic plant communities.     

The Mitochondrial Disulfide Relay System Protein GFER Is Mutated in Autosomal-Recessive Myopathy with Cataract and Combined Respiratory-Chain Deficiency

A disulfide relay system (DRS) was recently identified in the yeast mitochondrial intermembrane space (IMS) that consists of two essential components: the sulfhydryl oxidase Erv1 and the redox-regulated import receptor Mia40. The DRS drives the import of cysteine-rich proteins into the IMS via an oxidative folding mechanism. Erv1p is reoxidized within this system, transferring its electrons to molecular oxygen through interactions with cytochrome c and cytochrome c oxidase (COX), thereby linking the DRS to the respiratory chain. The role of the human Erv1 ortholog, GFER, in the DRS has been poorly explored. Using homozygosity mapping, we discovered that a mutation in the GFER gene causes an infantile mitochondrial disorder. Three children born to healthy consanguineous parents presented with progressive myopathy and partial combined respiratory-chain deficiency, congenital cataract, sensorineural hearing loss, and developmental delay. The consequences of the mutation at the level of the patient''s muscle tissue and fibroblasts were 1) a reduction in complex I, II, and IV activity; 2) a lower cysteine-rich protein content; 3) abnormal ultrastructural morphology of the mitochondria, with enlargement of the IMS space; and 4) accelerated time-dependent accumulation of multiple mtDNA deletions. Moreover, the Saccharomyces cerevisiae erv1^R182H mutant strain reproduced the complex IV activity defect and exhibited genetic instability of the mtDNA and mitochondrial morphological defects. These findings shed light on the mechanisms of mitochondrial biogenesis, establish the role of GFER in the human DRS, and promote an understanding of the pathogenesis of a new mitochondrial disease.     

Biomarkers for oxidative stress in acute lung injury induced in rabbits submitted to different strategies of mechanical ventilation

Oxidative damage has been said to play an important role in pulmonary injury, which is associated with the development and progression of acute respiratory distress syndrome (ARDS). We aimed to identify biomarkers to determine the oxidative stress in an animal model of acute lung injury (ALI) using two different strategies of mechanical ventilation. Rabbits were ventilated using either conventional mechanical ventilation (CMV) or high-frequency oscillatory ventilation (HFOV). Lung injury was induced by tracheal saline infusion (30 ml/kg, 38°C). In addition, five healthy rabbits were studied for oxidative stress. Isolated lymphocytes from peripheral blood and lung tissue samples were analyzed by alkaline single cell gel electrophoresis (comet assay) to determine DNA damage. Total antioxidant performance (TAP) assay was applied to measure overall antioxidant performance in plasma and lung tissue. HFOV rabbits had similar results to healthy animals, showing significantly higher antioxidant performance and lower DNA damage compared with CMV in lung tissue and plasma. Total antioxidant performance showed a significant positive correlation (r = 0.58; P = 0.0006) in plasma and lung tissue. In addition, comet assay presented a significant positive correlation (r = 0.66; P = 0.007) between cells recovered from target tissue and peripheral blood. Moreover, antioxidant performance was significantly and negatively correlated with DNA damage (r = -0.50; P = 0.002) in lung tissue. This study indicates that both TAP and comet assay identify increased oxidative stress in CMV rabbits compared with HFOV. Antioxidant performance analyzed by TAP and oxidative DNA damage by comet assay, both in plasma, reflects oxidative stress in the target tissue, which warrants further studies in humans.     

Use of poly(DL-lactide-ε-caprolactone) membranes and mesenchymal stem cells from the Wharton's jelly of the umbilical cord for promoting nerve regeneration in axonotmesis: In vitro and in vivo analysis

Cellular systems implanted into an injured nerve may produce growth factors or extracellular matrix molecules, modulate the inflammatory process and eventually improve nerve regeneration. In the present study, we evaluated the therapeutic value of human umbilical cord matrix MSCs (HMSCs) on rat sciatic nerve after axonotmesis injury associated to Vivosorb® membrane. During HMSCs expansion and differentiation in neuroglial-like cells, the culture medium was collected at 48, 72 and 96 h for nuclear magnetic resonance (NMR) analysis in order to evaluate the metabolic profile. To correlate the HMSCs ability to differentiate and survival capacity in the presence of the Vivosorb® membrane, the [Ca²⁺]i of undifferentiated HMSCs or neuroglial-differentiated HMSCs was determined by the epifluorescence technique using the Fura-2AM probe. The Vivosorb® membrane proved to be adequate and used as scaffold associated with undifferentiated HMSCs or neuroglial-differentiated HMSCs. In vivo testing was carried out in adult rats where a sciatic nerve axonotmesis injury was treated with undifferentiated HMSCs or neuroglial differentiated HMSCs with or without the Vivosorb® membrane. Motor and sensory functional recovery was evaluated throughout a healing period of 12 weeks using sciatic functional index (SFI), extensor postural thrust (EPT), and withdrawal reflex latency (WRL).     

Chronic conventional resistance exercise reduces blood pressure in stage 1 hypertensive men

To investigate the antihypertensive effects of conventional resistance exercise (RE) on the blood pressure (BP) of hypertensive subjects, 15 middle-aged (46 ± 3 years) hypertensive volunteers, deprived of antihypertensive medication (reaching 153 ± 6/93 ± 2 mm Hg systolic/diastolic BP after a 6-week medication washout period) were submitted to a 12-week conventional RE training program (3 sets of 12 repetitions at 60% 1 repetition maximum, 3 times a week on nonconsecutive days). Blood pressure was measured in all phases of the study (washout, training, detraining). Additionally, the plasma levels of several vasodilators or vasoconstrictors that potentially could be involved with the effects of RE on BP were evaluated pre- and posttraining. Conventional RE significantly reduced systolic, diastolic, and mean BP, respectively, by an average of 16 (p < 0.001), 12 (p < 0.01), and 13 mm Hg (p < 0.01) to prehypertensive values. There were no significant changes of vasoactive factors from the kallikrein-kinin or renin-angiotensin systems. After the RE training program, the BP values remained stable during a 4-week detraining period. Taken together, this study shows for the first time that conventional moderate-intensity RE alone is able to reduce the BP of stage 1 hypertensive subjects free of antihypertensive medication. Moreover, the benefits of BP reduction achieved with RE training remained unchanged for up to 4 weeks without exercise.     

Following the Fate In Vivo of COPI Vesicles Generated In Vitro

COPI vesicles are a class of transport carriers that function in the early secretory pathway. Their fate and function are still controversial. This includes their contribution to bidirectional transport within the Golgi apparatus and their role during cell division. Here we describe a method that should address several open questions about the fate and function of COPI vesicles in vivo . To this end, fluorescently labeled COPI vesicles were generated in vitro from isolated rat liver Golgi membranes, labeled with the fluorescent dyes Alexa-488 or Alexa-568. These vesicles appeared to be active and colocalized with endogenous Golgi membranes within 30 min after microinjection into mammalian cells. The COPI vesicle-derived labeled membrane proteins could be classified into two types that behaved like endogenous proteins after Brefeldin A treatment.     

Growth hormone therapy in GH-deficient adults: continuous vs alternate-days treatment.

In the present report, we have compared 12 months of rhGH therapy given daily (D) at the beginning and then on alternate days (A) to 20 subjects with severe adult-onset GH deficiency (GHD). Aim of the study was to establish whether the lower frequency injection regimen is as effective as the daily dose. Measurements included: IGF-I levels, body composition (BF%), lipid profile, insulin sensitivity by homeostasis model assessment (HOMA-IR) and quantitative insulin check index (QUICKI), as well as thyroid function. Evaluation on A therapy was performed both 12 and 36 hours after the last rhGH injection. The final rhGH dose was 0.3 +/- 0.1mg/day. During A, the dose used in D was doubled and given on alternate days. Recombinant hGH given during the A period induced changes in IGF-I levels, BF% and lipid profile comparable to daily treatment. HOMA-IR increased similarly after both regimens, though QUICKI did not significantly change. A significant reduction in serum FT4 levels occurred after both D and A therapy, so that an adjustment of L-T4 replacement dose in 5 of 20 patients was necessary. No differences were found in the various parameters after 12 and 36 hours post rhGH injection. In conclusion, rhGH therapy given on alternate days is clinically effective and may result in improved patient compliance. Monitoring glucose tolerance and thyroid function while on rhGH is essential.     

Isolation of the epithiospecifier protein from oil-rape (Brassica napus ssp. oleifera) seed and its characterization

The epithiospecifier protein (ESP) is a myrosinase (MYR) cofactor, which is necessary to drive the MYR-catalyzed hydrolysis of some specific glucosinolates towards the production of cyanoepithioalkanes instead of isothiocyanates and nitriles. ESP was isolated from Brassica napus seeds by anionic exchange and gel filtration chromatography. ESP showed a molecular weight of about 39 kDa and pI 5.3. The amino acid sequence of several tryptic peptides of ESP (accounting for about 50% of the total sequence) made it possible to establish the high similarity (81% identity) with a hypothetical 37 kDa protein (TrEMBL data base accession number Q39104) and several jasmonate-inducible proteins from Arabidopsis thaliana. This observation suggests that ESP is likely to be involved in jasmonate-mediated defence and disease resistance mechanisms.