16 research outputs found

    Nitrosative stress induces proliferation and viability changes in high glucose-exposed rat Schwannoma cells

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    OBJECTIVES: Schwann cells may be involved in the pathogenesis of several neuropathies, such as those linked to an excess of d-glucose. Indeed, hyperglicemic condition can often result in the production of high reactive/nitrosative oxygen species concentration and possible damage of several cell structures. In the present work attention has been focused on the possible nitrosative effect of hyperglycemia on RT4 Schwannoma cell lines. METHODS: Cells were cultured for 72hrs in the presence of 180 mM D-glucose. Morphology, growth rate, cell viability, catalase evaluation and Western blot were performed. RESULTS: In D-glucose-exposed cells, 3-Nitrotyrosine increase and subsequent modifications in cell morphology, growth rate, viability and catalase activity were found. CONCLUSION: Our findings suggested a possible primary role played by Schwann cells in the hyperglicemic neuropathy pathogenesis, through the excessive production of RNS and a decrease in antioxidant defense systems, bearing out the importance of the "nitrosative hypothesis" in the hyperglicemic-induced nervous system complications.</br

    Microtubular Assessment of C6 Rat Glioma Cell Spheroids Developed in Transparent Liquid Marbles or Hanging Drops

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    Glioblastoma is a brain tumour frequently used as an experimental model to exploit innovative therapeutic approaches due to its high lethality and refractoriness to therapies. Part of these innovative anticancer therapies address cytoskeletal microtubules (MTs) since specific tubulin post-translational modifications (PTMs) are considered markers of tumour plasticity. In vitro studies, which traditionally employ two-dimensional (2D) culture systems, are now being replaced by three-dimensional (3D) systems that more closely mimic in vivo physiological conditions and allow a better understanding of the signalling between cells. In this work, we compared 2 liquid base 3D methods for the generation of spheroids from C6 rat glioma cells (RGCs) using 30 &micro;L of liquid marble (LM) or the hanging drops (HDs), which contained 2 different cell numbers (5000 or 15,000). After 24 or 48 h of in vitro culture (IVC), the morphology of the spheroids was observed and the behaviour of the two main tubulin PTMs, tyrosinated &alpha;-tubulin (Tyr-T) and acetylated &alpha;-tubulin (Ac-T), was evaluated by fluorescence and Western blot (WB). RGCs spontaneously formed spherical agglomerates more rapidly in the LM than in the HD system. Cell density influenced the size of the spheroids, which reached a larger size (&gt; of 300 &micro;m &Oslash;), with 15,000 cells compared to 5000 cells (150 &micro;m &Oslash;). Moreover, an increase in Tyr-T and Ac-T was observed in both the HD and LM system from 24 to 48 h, with the highest values shown in the 48 h/LM spheroids of 5000 cells (p &lt; 0.05). In conclusion, by comparing the morphology and microtubular architecture of spheroids from C6 rat glioma cells developed by LM or HD methodology, our findings demonstrate that the use of a fumed silica microbioreactor boosts the induction and maintenance of a high plasticity state in glioma cells. RGCs cultured in LM express levels of tubulin PTMs that can be used to evaluate the efficacy of new anticancer therapies

    Growth-associated protein expression in the frontal and occipital cortices of callosotomized rats

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    Neurons confined to the central nervous system usually fail to regenerate their axons after injury, although evidence of axonal re-growth has been reported. In this study, rat transcallosal fibres were severed in the midline in order to investigate the reactive plasticity responses in the frontal and occipital cortices of both sides. The expression of growth-associated proteins, tyrosinated alpha-tubulin and GAP-43, was monitored at different time-points post-injury. Protein levels dropped during the first days post-axotomy, but subsequently returned to control levels. This initial decrease could be due to degeneration, and the subsequent increase connected to the reactive neosynaptogenesis and fibre sprouting from surrounding ipsilateral neurons, which is responsible for the reinnervation of the denervated area. Although transcallosal neurons are usually considered poorly regenerative, their axotomy may therefore induce reactive events

    Morphological and functional changes induced by the amino acid analogue 3-nitrotyrosine in mouse neuroblastoma and rat glioma cell lines

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    The amino acid analogue 3-nitrotyrosine (3-NT) is formed in neural cells as a result of the intense stimulation of NMDA glutamate receptors. 3-NT is involved in the pathology of diverse neurodegenerative disorders. The aim of our work is to investigate the sensitivity of cultured neural and glial cells to 3-NT. We report the morphologicalchanges detected on mouse neuroblastoma (C1300) and rat glioma (C6) celllines cultured in a medium supplemented with different 3-NT concentrations. Western blot displayed a selective incorporation of 3-NT into a single protein that co-migrated with tubulin. Both celllines showed morphologicalchanges, nuclear suffering, decreased viability and growth inhibition (starting from 90 and 360 μM for C1300 and C6, respectively). Such effects were dose-dependent, though gliomacells showed severe alterations at higher 3-NT concentrations. Our results point out a higher 3-NT sensitivity in the neural cells studied in comparison with those of glial origin. The dramatic toxicity of 3-NT in neural cells suggests further investigations focused on the biochemical mechanisms at the roots of neurodegenerative diseases

    Seasonal Effect on Developmental Competence, Oxidative Status and Tubulin Assessment of Prepubertal Ovine Oocyte

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    The reproductive seasonality of domestic animals is often manipulated in order to have more reproductive periods for commercial purposes related to the production of milk and meat. It is scientifically proven that such an alteration of the reproductive activity in sheep entails a deterioration in oocyte quality, leading to an inability to generate embryos. Since oocytes obtained from prepubertal ewes can be incorporated into an in vitro embryo production system and considering that their quality is crucial to the success of in vitro procedures, the aim of this work was to investigate the effect of seasons on the quality of prepubertal ovine oocytes collected in autumn and spring. Ovaries were collected from a local slaughterhouse from 30–40-day-old suckling lambs during both seasons. Following 24 h of in vitro maturation, oocytes developmental competence, reactive oxygen species (ROS) intracellular levels, and mitochondrial activity were evaluated, and a tubulin assessment was performed. The results on embryo production, as a percentage of first divisions and number of blastocysts obtained, were significantly higher in oocytes collected in the spring. Mitochondrial activity in oocytes was higher, and ROS production significantly lower, in spring than in autumn. Tubulin PTMs (tyrosinated and acetylated α-tubulin) showed a higher immunoreactivity in oocytes collected in spring compared with autumn sampling. Our data showed that seasons may affect the developmental competence, energetic status, and tubulin assessment of oocytes recovered from prepubertal ewes. Therefore, special care should be taken when choosing the period of the year for prepuberal ovine oocytes collection aimed at in vitro embryo reproduction programs

    Testosterone induces neuroprotection from oxidative stress: effects on catalase activity and 3-nitro-L-tyrosine incorporation into alpha-tubulin in a mouse neuroblastoma cell line

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    3-nitro-L-tyrosine is formed by nitric oxide following different pathways such as NADPH oxidase, xanthine oxidase or glutamate NMDA receptor activation and is involved in the pathology of different neurological disorders. Unlike estradiol, a neuroprotective role of androgens against oxidative cell injury has not been fully investigated. This work targets the possible effects of testosterone on neuroblastoma cells exposed to 3-nitro-L-tyrosine. C1300 mouse undifferentiated neuroblastoma cells exposed to 3-nitro-L-tyrosine were cultured in the presence of testosterone. Morphological examination, proliferation and nuclear viability assays were performed. The expression of tyrosinated alpha-tubulin and incorporation of 3-nitro-L-tyrosine into protein were also estimated. Cells exposed to 3-nitro-L-tyrosine showed globular shape, reduced cytoplasmic processes and growth inhibition in comparison with controls. When testosterone was added to the medium, these changes were not evident. In addition, testosterone induced an upregulation of tyrosinated alpha-tubulin, a marker of neuronal plasticity, and a decrease in 3-nitro-L-tyrosine incorporation into tubulin. Our results suggest that testosterone exposure can diminish 3-nitro-L-tyrosine toxic effects on the morphology and growth rate of neuroblastoma cells. The upregulation of tyrosinated alpha-tubulin in testosterone-exposed cells would be consistent with concurrent plasticity events. Failure in alpha-tubulin nitration detected in cells exposed to both 3-nitro-L-tyrosine and testosterone, may support the idea that testosterone interferes with 3-nitro-L-tyrosine protein incorporation. Moreover, testosterone-induced neuroprotection likely entails a linkage with the androgen receptor as is suggested by the flutamide-induced inhibition of the hormone activity. Finally, the neuroprotective effects of testosterone in neuroblastoma cells could deal with the cellular antioxidant defence system, as shown by testosterone-induced increase in catalase activity

    Morphology, morphometry and spatial distribution of secondary osteons in equine femur

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    A high number of differences exist in bone histological features depending on the species, breed, age and bone. Moreover, osteon distribution may vary in the different sides of a bone as a consequence of different biomechanical strains. The aim of this work was to study the distribution and morphology of osteons in different sides of the equine femoral diaphysis with the attempt to correlate them to the main strains operating on them. The following parameters of secondary osteons and Haversian canals were measured in the transverse sections of diaphyses: perimeter, area, minimum and maximum diameter, eccentricity and osteon population density. A typical Haversian tissue was observed with elliptic secondary osteons consisting in about 10 well-defined lamellae surrounding a circular Haversian canal. Quantitative analysis displays a different population density of secondary osteons depending on the side. The caudal and medial sides, where compression strains are higher, have more secondary osteons in comparison with the cranial and lateral sides, where tension strains are prevalent. These data suggest that secondary osteon population density may depend on the predominant strains. Even the elliptical shape of secondary osteons may be related to biomechanical strains, as their major axes are oriented cranio-caudally parallel to prevalent strains

    Effects of testosterone on differentiation and oxidative stress resistance in C1300 neuroblastoma cells

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    OBJECTIVES: Using undifferentiated mouse neuroblastoma cells (C1300), we have previously observed that testosterone (T) exerts a neuroprotective action against oxidative stress. Nitrogen intermediates induce the production of 3-nitro-L-tyrosine (3NT), an amino acid analogue involved in many neurodegenerative disorders. The aim of our work is to investigate T capability on C1300 cell differentiation. It is also evaluated whether differentiation could mitigate the nitrosative effects of 3NT. METHODS: The effects of both T and 3NT were studied on an undifferentiated cell line of neural origin (C1300). For this purpose, cell cultures underwent morphometric investigation, blot analyses and catalase activity assay. All data obtained were expressed as mean+/-SD and tested by one-way ANOVA or Student's t test. RESULTS: The results were compared with those gathered by means of N6,2'-O-dibutyryl-adenosine-3',5'-cyclic-mono-phosphate (db-cAMP), a well-known differentiating agent. T-exposed cells showed an irregular shape and exhibited long branching cytoplasmic extensions, which were longer than in db-cAMP cells. Moreover, T-exposure induced an increase in the expression of tyrosinated and acetylated alpha-tubulin while 3NT-incorporation into tubulin was markedly reduced. The action of antioxidant defence systems, namely catalase activity, was enhanced in cells exposed to T. CONCLUSION: This work highlighted the effects of db-cAMP on differentiation and neuroprotection, but even indicated that T exposure induced differentiation in C1300 cells and this process matches a significant neuroprotective effect. This action seemed to be more effective than in db-cAMP-treated cells. T is suggested, like other substances having antioxidant properties, to be of potential interest in the experimental therapy of neuropathological conditions.</br

    D-glucose induces microtubular changes in C1300 neuroblastoma cell line through the incorporation of 3-nitro-L-tyrosine into tubulin

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    The microtubular network of neurons is involved in several functions such as formation and tropism of cellular processes, cell division and intracellular transport. A lot of evidences testify that the microtubular network of neurons can be impaired by oxidative stress. A condition of oxidative stress is often possible when D-glucose overloads its metabolic pathway, resulting in an increase in reactive oxygen species and subsequent neurological disorders. The aim of this work was to check in undifferentiated mouse neuroblastoma cells (C1300) the possible oxidative effects of D-glucose on microtubules. Using a concentration of 110mM D-glucose, cell morphology, growth rate, viability and catalase activity were seriously altered. Noteworthy, an increase in 3-nitro-L-tyrosine and a downregulation of tubulins was found in D-glucose-exposed cells, whereas another cytoskeletal proteins, namely actin, did not show any changes. In conclusion, microtubular network can be impaired by D-glucose through specific nitrosative effects, suggesting a possible mechanism at the basis of hyperglycemia-induced neuronal damage

    Prophylactic gene therapy with human tissue kallikrein ameliorates limb ischemia recovery in type 1 diabetic mice

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    Diabetes macro- and microvascular disease causes tissue hypoperfusion. This deficit, together with a failure to mount an adequate angiogenic response, might explain why vascular occlusion evolves more severely among diabetic patients. The present study investigated whether prophylactic gene therapy with human tissue kallikrein (hTK) may protect diabetic limbs from the consequences of supervening ischemia. Vehicle (saline) or an adenovirus carrying the gene for either hTK (Ad.hTK) or luciferase (Ad.Luc) was injected into left adductor muscles of streptozotocin-induced type 1 diabetic mice 2 weeks before operative occlusion of the ipsilateral femoral artery. Saline-injected nondiabetic mice served as controls. Hindlimb blood flow recovery was analyzed sequentially over the 2 weeks after ischemia induction. At necroscopy, microvessel density and endothelial cell proliferation and apoptosis were quantified in skeletal muscles. We found that limb perfusion recovery of saline-injected type 1 diabetic mice is delayed because of insufficient reparative neovascularization and excessive activation of endothelial cell apoptosis. By contrast, prophylactic Ad.hTK renewed the ability to mount an appropriate neovascularization response to ischemia, suppressed apoptosis, and upregulated endothelial nitric oxide synthase expression. Ultimately, correction of diabetic endotheliopathy by Ad.hTK allowed proper perfusion recovery as seen in nondiabetic mice. These discoveries disclose new therapeutic options for the treatment of diabetic complications
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