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In critical nerve gap repair, decellularized nerve allografts are considered a promising tissue engineering strategy that can provide superior regeneration results compared to nerve conduits. Decellularized nerves offer a well-conserved... more
In critical nerve gap repair, decellularized nerve allografts are considered a promising tissue engineering strategy that can provide superior regeneration results compared to nerve conduits. Decellularized nerves offer a well-conserved extracellular matrix component that has proven to play an important role in supporting axonal guiding and peripheral nerve regeneration. Up to now, the known decellularized techniques are time and effort consuming. The present study, performed on rat sciatic nerves, aims at investigating a novel nerve decellularization protocol able to combine an effective decellularization in short time with a good preservation of the extracellular matrix component. To do this, a decellularization protocol proven to be efficient for tendons (DN-P1) was compared with a decellularization protocol specifically developed for nerves (DN-P2). The outcomes of both the decellularization protocols were assessed by a series of in vitro evaluations, including qualitative and q...
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Over the last 10 years, we have investigated a particular type of bioengineered nerve guide, the muscle-vein-combined tube, which is made by filling a vein with skeletal muscle. In our previous studies we have always used fresh skeletal... more
Over the last 10 years, we have investigated a particular type of bioengineered nerve guide, the muscle-vein-combined tube, which is made by filling a vein with skeletal muscle. In our previous studies we have always used fresh skeletal muscle to fill vein conduits. In the present study we compared the use of fresh and predegenerated (freeze-thawed) skeletal muscle for muscle-vein-combined nerve guides. In this study, a 10-mm-long rat median nerve defect was repaired using either type of nerve guide. The samples were analyzed 5 and 30 days after surgery by light and electron microscopy. In addition, reverse transcription polymerase chain reaction (RT-PCR) was carried out to investigate the expression of mRNAs coding for glial markers, as well as glial growth factor (NRG1) and its receptors (erbB2 and erbB3). Results showed differences between the two types of nerve guides at postoperative day 5; however, no difference was detected at day 30 suggesting that both types of tissue-engineered conduit are effective for repairing peripheral nerve defects in this experimental model.
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Hepatocyte growth factor/scatter factor (HGF/SF) is a growth factor with pleiotropic effects on different cell types. It acts as a mitogen and motility factor for many epithelial cells. HGF/SF and its receptor Met are present in the... more
Hepatocyte growth factor/scatter factor (HGF/SF) is a growth factor with pleiotropic effects on different cell types. It acts as a mitogen and motility factor for many epithelial cells. HGF/SF and its receptor Met are present in the developing and adult mammalian brain and control neuritogenesis of sympathetic and sensory neurons. We report that the striatal progenitor ST14A cells express the Met receptor, which is activated after binding with HGF/SF. The interaction between Met and HGF/SF triggers a signaling cascade that leads to increased levels of c-Jun, c-Fos, and Egr-1 proteins, in agreement with data reported on the signaling events evoked by HGF in other cellular types. We also studied the effects of the exposure of ST14A cells to HGF/SF. By time-lapse photography, we observed that a 24-hr treatment with 50 ng/ml HGF/SF induced modification in cell morphology, with a decrease in cell-cell interactions and increase of cell motility. In contrast, no effect on cell proliferation was observed. To investigate which intracellular pathway is primarily involved we used PD98059 and LY294002, two specific inhibitors of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAP-kinase/ERK-kinase) and phosphoinositide 3-OH kinase (PI3-K), respectively. Cell motility in HGF/SF treated cultures was inhibited by LY294002 but not by PD98059, suggesting that PI3-K plays a key role in mediating the HGF/SF-induced dissociation of ST14A cells. Previous evidence of HGF stimulation of motility in nervous system has been obtained on postmitotic neurons, which have already acquired their specificity. Data reported here of a motogenic response of ST14A cell line, which displays properties of neuronal progenitors, seem of interest because they suggest that HGF could play a role in very early steps of neurogenesis.
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Research Interests: Cognitive Science, Electron Microscopy, Immunohistochemistry, Medicine, Mice, and 15 moreAnimals, Laboratory Animals, Male, PARALYSIS, Nerve Regeneration, Genetically Modified, Functional Recovery, Motor Function, Biological markers, Biological Assay, Axons, Neurosciences, Median Nerve, Experimental Model, and Neuroscience Methods
Stable expression of the tyrosine kinase receptor ErbB4 confers increased migratory behavior to the neuronal progenitor cell line ST14A, in response to neuregulin 1 (NRG1) stimulation. We used gene expression profiling analysis to... more
Stable expression of the tyrosine kinase receptor ErbB4 confers increased migratory behavior to the neuronal progenitor cell line ST14A, in response to neuregulin 1 (NRG1) stimulation. We used gene expression profiling analysis to identify transcriptional changes associated with higher migratory activity caused by the activation of a specific ErbB4 isoform, and found constitutive up-regulation of the epidermal growth factor receptor pathway substrate 8 (Eps8), a multimodular regulator of actin dynamics. We confirmed the increase of Eps8, both at the mRNA and at the protein level, in stable clones expressing two different ErbB4 isoforms, both characterized by high migratory activity. Using Transwell assays and experimental manipulation of Eps8 expression level, we demonstrated that Eps8 synergizes with ErbB4 to increase both basal and ligand induced cell migration, whereas siRNA mediated Eps8 silencing strongly impairs cell motility and NRG1 induced actin cytoskeleton remodeling. By transient knockdown of Eps8 through in vivo siRNA electroporation, followed by explant primary cultures, we demonstrated that Eps8 down-regulation affects migration of normal neuronal precursors. In conclusion, our data demonstrate that Eps8 is a key regulator of motility of neuronal progenitor cells expressing ErbB4, both in basal conditions and in response to external motogenic cues.
Research Interests: Migration, Biology, Cell Migration, Cell Biology, Medicine, and 15 moreGene expression, Mice, Animals, Proteins, Actin Cytoskeleton, Actin Dynamics, Clinical Sciences, Epidermal Growth Factor Receptor, Microarray Analysis, Neural Stem Cells, Protein isoforms, Primary Culture, qRT PCR, Biochemistry and cell biology, and Cell Motility
Research Interests: Neuroscience, Cognitive Science, Metabolism, Biology, Immunohistochemistry, and 15 moreMedicine, Animals, Metabotropic Glutamate Receptors, Neurons, Denervation, European, Epithelium, Biosynthesis, Amino Acid Sequence, Messenger, Glutamate Receptor, Molecular Sequence Data, Blotting, Afferent, and deafferentation
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Research Interests: Biology, Cell Biology, Biological Chemistry, Biological Sciences, Cell Differentiation, and 15 moreAnimals, Biological, Chemotaxis, Phosphorylation, Plasmids, Neurons, Immunoprecipitation, CHEMICAL SCIENCES, Protein isoforms, Cell Proliferation, Protein Binding, Dimerization, Molecular Sequence Data, Cell Membrane, and Medical and Health Sciences
Over the last five years, we have used the rat forelimb model for investigating neuromuscular recovery after microsurgical nerve reconstruction of median and ulnar nerves by end-to-side neurorrhaphy and muscle-vein-combined tubulization... more
Over the last five years, we have used the rat forelimb model for investigating neuromuscular recovery after microsurgical nerve reconstruction of median and ulnar nerves by end-to-side neurorrhaphy and muscle-vein-combined tubulization (using both straight and Y-shaped guides). The outcome of nerve repair at different postoperative times was assessed by functional, morphological and biomolecular analysis. Results showed that both end-to-side and tubulization repair of rat median and ulnar nerves led to successful axonal regeneration along the severed nerve trunk as well as to a partial recovery of the lost function as assessed by grasping test. Biomolecular analysis by means of reverse transcription polymerase chain reaction (RT-PCR) demonstrated early overexpression during nerve regeneration of the gliotrophic factor NRG1 and two of its receptors: erbB2 and erbB3. Finally, our experience also suggests that the rat forelimb experimental model is particularly appropriate for the stu...
Research Interests: Microsurgery, Medicine, Female, Animals, Glycoproteins, and 15 moreNerve Regeneration, Clinical Sciences, Research Purpose, Rats, Functional Recovery, Guided Tissue Regeneration, Peripheral Nerve, Neuromuscular Junction, Axons, Neurosciences, Median Nerve, Experimental Model, Recovery of Function, Reverse transcription polymerase chain reaction, and axon regeneration
Previously, we have shown that erbB-3 expression is restricted to the ensheathing cells of the olfactory nerve layer, while erbB-4 is found in the periglomerular and mitral/tufted cells of the olfactory bulb and in cells coming out from... more
Previously, we have shown that erbB-3 expression is restricted to the ensheathing cells of the olfactory nerve layer, while erbB-4 is found in the periglomerular and mitral/tufted cells of the olfactory bulb and in cells coming out from the rostral migratory stream of the subependymal layer. In the present work, we have treated adult mice with zinc sulfate intranasal irrigation and analyzed erbB-3 and erbB-4 expression in the deafferented olfactory bulb. Following treatment, olfactory axons undergo degeneration, as indicated by the loss of OMP expression in the deafferented olfactory bulb. The thickness of the olfactory nerve layer is reduced, but the specific intensity of erbB-3 labeling in the remaining olfactory nerve layer is increased with respect to control. Interestingly, following deafferentation, erbB-4 immunoreactivity decreases specifically in cell types that normally make synaptic contacts with primary olfactory neurons in the glomeruli, i.e. periglomerular and mitral/tu...
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The role of prostaglandins (PGs) in the mechanism of action of acetylcholine (ACh) on frog adrenocortical cells has been examined. Administration of a single dose of ACh (5 X 10(-5) M) to perifused frog interrenal fragments, for 20 min,... more
The role of prostaglandins (PGs) in the mechanism of action of acetylcholine (ACh) on frog adrenocortical cells has been examined. Administration of a single dose of ACh (5 X 10(-5) M) to perifused frog interrenal fragments, for 20 min, stimulated the production of corticosterone, aldosterone, PGE2 and 6-keto-PGF1 alpha. In contrast ACh did not significantly alter TXB2 production. The effect of ACh could be mimicked by muscarine (10(-5) M). Conversely, nicotine (10(-6) to 10(-4) M) was totally inactive. The increase in PG biosynthesis preceded the peak of corticosteroid release. Repeated 20-min pulses of ACh (5 X 10(-5) M) or muscarine (10(-5) M) given at 130-min intervals induced a desensitization phenomenon. In presence of indomethacin (5 X 10(-6) M), the effect of ACh on PG and steroid secretion was totally abolished. In calcium-free medium, the effect of ACh on PG and corticosteroid production was completely blocked. These results indicate that, in the frog, ACh stimulates corticosteroid secretion through a PG-dependent mechanism.
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Cell migration from the olfactory neuroepithelium to the brain has been widely studied during vertebrate development. Immunocytochemical analysis has revealed that many of the migrating cells contain GnRH (Gonadotropin-Releasing Hormone).... more
Cell migration from the olfactory neuroepithelium to the brain has been widely studied during vertebrate development. Immunocytochemical analysis has revealed that many of the migrating cells contain GnRH (Gonadotropin-Releasing Hormone). The GnRH positive cells migrate from the medial olfactory placode, steam along the nasal septum, cross the basal forebrain and reach the hypothalamic and septal areas from where they control the release of hypophyseal gonadotropic peptides. A peculiar feature of these cells is that they start expressing GnRH during migration. We have analysed the presence of immunoreactivity for peptides typically expressed in olfactory neurones, along the migratory pathway followed by GnRH neurones. We have used polyclonal antibodies raised against carnosine and olfactory marker protein (OMP), and performed double immunolabelling on mouse embryos and on early neonatal Brazilian opossum (Monodelphis domestica) tissues. Beside the GnRH neurones we observed other mig...
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Over the last five years, we have used the rat forelimb model for investigating neuromuscular recovery after microsurgical nerve reconstruction of median and ulnar nerves by end-to-side neurorrhaphy and muscle-vein-combined tubulization... more
Over the last five years, we have used the rat forelimb model for investigating neuromuscular recovery after microsurgical nerve reconstruction of median and ulnar nerves by end-to-side neurorrhaphy and muscle-vein-combined tubulization (using both straight and Y-shaped guides). The outcome of nerve repair at different postoperative times was assessed by functional, morphological and biomolecular analysis. Results showed that both end-to-side and tubulization repair of rat median and ulnar nerves led to successful axonal regeneration along the severed nerve trunk as well as to a partial recovery of the lost function as assessed by grasping test. Biomolecular analysis by means of reverse transcription polymerase chain reaction (RT-PCR) demonstrated early overexpression during nerve regeneration of the gliotrophic factor NRG1 and two of its receptors: erbB2 and erbB3. Finally, our experience also suggests that the rat forelimb experimental model is particularly appropriate for the study of microsurgical reconstruction of major mixed nerve trunks. Furthermore, since the forelimb model is less compromising for the animal, it should be preferred to the hindlimb model for many research purposes.
Research Interests: Microsurgery, Female, Animals, Glycoproteins, Nerve Regeneration, and 15 moreClinical Sciences, Research Purpose, Rats, Functional Recovery, Guided Tissue Regeneration, Peripheral Nerve, Neuromuscular Junction, Axons, Schwann Cell, Neurosciences, Median Nerve, Experimental Model, Recovery of Function, Reverse transcription polymerase chain reaction, and axon regeneration
Olfactory neuroepithelial (OE) cells were dissociated from late stage embryonic mice and analysed for carnosine expression. The yield of carnosine neurones was twice as high when the OE cells were seeded along with the olfactory bulb... more
Olfactory neuroepithelial (OE) cells were dissociated from late stage embryonic mice and analysed for carnosine expression. The yield of carnosine neurones was twice as high when the OE cells were seeded along with the olfactory bulb cells. Carnosine neurones resulted from both in vitro survival and neurogenesis, and were associated with clusters of underlying flat cells immunopositive for keratin. Our results demonstrate that olfactory neurones expressing their neurotransmitter carnosine can be studied in culture, and the close association with keratin-immunopositive basal cells suggests that they are dependent on these cells for survival and/or differentiation.
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The role of prostacyclin (PGI2) on amphibian adrenal steroidogenesis was studied in vitro in perifused interrenal fragments from adult male frogs. Exogenous PGI2 (3 X 10(-8) M to 3 X 10(-5) M) and, in a lesser extent, 6-keto-PGF1 alpha... more
The role of prostacyclin (PGI2) on amphibian adrenal steroidogenesis was studied in vitro in perifused interrenal fragments from adult male frogs. Exogenous PGI2 (3 X 10(-8) M to 3 X 10(-5) M) and, in a lesser extent, 6-keto-PGF1 alpha increased both corticosterone and aldosterone production in a dose-related manner. Short pulses (20 min) of 0.88 microM PGI2 administered at 90 min intervals within the same experiment did not induce any desensitization phenomenon. A prolonged administration (6 h) of PGI2 gave rise to an important increase in steroid production followed by a decline of corticosteroidogenesis. Indomethacin (IDM, 5 microM) induced a marked reduction of the spontaneous secretion of corticosteroid which confirmed the involvement of endogenous PGs in the process of corticosteroid biosynthesis. The IDM-induced blockade of corticosterone and aldosterone secretion was totally reversed by administration of exogenous PGI2 in our model. Angiotensin II (AII) induced a massive release of 6-keto-PGF1 alpha, the stable metabolite of PGI2. The increase of 6-keto-PGF1 alpha preceded the stimulation of corticosterone and aldosterone secretions. In contrast, the administration of ACTH did not modify the release of 6-keto-PGF1 alpha. These results indicate that PGI2 might be an important mediator of adrenal steroidogenesis in frog. They confirm that the corticosteroidogenic actions of ACTH and AII are mediated by different mechanisms.
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Over the last five years, we have used the rat forelimb model for investigating neuromuscular recovery after microsurgical nerve reconstruction of median and ulnar nerves by end-to-side neurorrhaphy and muscle-vein-combined tubulization... more
Over the last five years, we have used the rat forelimb model for investigating neuromuscular recovery after microsurgical nerve reconstruction of median and ulnar nerves by end-to-side neurorrhaphy and muscle-vein-combined tubulization (using both straight and Y-shaped guides). The outcome of nerve repair at different postoperative times was assessed by functional, morphological and biomolecular analysis. Results showed that both end-to-side and tubulization repair of rat median and ulnar nerves led to successful axonal regeneration along the severed nerve trunk as well as to a partial recovery of the lost function as assessed by grasping test. Biomolecular analysis by means of reverse transcription polymerase chain reaction (RT-PCR) demonstrated early overexpression during nerve regeneration of the gliotrophic factor NRG1 and two of its receptors: erbB2 and erbB3. Finally, our experience also suggests that the rat forelimb experimental model is particularly appropriate for the study of microsurgical reconstruction of major mixed nerve trunks. Furthermore, since the forelimb model is less compromising for the animal, it should be preferred to the hindlimb model for many research purposes.
Research Interests: Microsurgery, Female, Animals, Glycoproteins, Nerve Regeneration, and 15 moreClinical Sciences, Research Purpose, Rats, Functional Recovery, Guided Tissue Regeneration, Peripheral Nerve, Neuromuscular Junction, Axons, Schwann Cell, Neurosciences, Median Nerve, Experimental Model, Recovery of Function, Reverse transcription polymerase chain reaction, and axon regeneration
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In chick parasympathetic ciliary ganglion the neuronal birthdate is well defined, between 2.5 and 5.5 days of embryonic development, and neuronal precursor cells that are able to differentiate into neurons in vitro can be isolated from... more
In chick parasympathetic ciliary ganglion the neuronal birthdate is well defined, between 2.5 and 5.5 days of embryonic development, and neuronal precursor cells that are able to differentiate into neurons in vitro can be isolated from E4.5 ganglia. In this report, using bromodeoxyuridine incorporation and Maplb immunostaining, we demonstrate that these cells can be isolated from E7-E8 chick embryos as well, suggesting that neuronal precursor cells are still present in the ciliary ganglion after the end of the in vivo neurogenesis. These precursor cells retain the ability to divide and generate newly differentiated neurons in vitro when cultured in a chemically defined medium. Such a capacity is highly stimulated by bFGF but not by CNTF.
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ErbBs are a family of receptors involved in the trophic maintenance of Schwann cells. Little is known about their expression changes during peripheral nerve regeneration. The aim of this study was thus to investigate variations in ErbBs... more
ErbBs are a family of receptors involved in the trophic maintenance of Schwann cells. Little is known about their expression changes during peripheral nerve regeneration. The aim of this study was thus to investigate variations in ErbBs after end-to-end and end-to-side nerve regeneration in the rat median nerve model. Expression of ErbBs was assessed at 7, 14, and 28 days postoperatively by real-time PCR. Results showed that expression of ErbB1 and ErbB4 mRNAs was downregulated, whereas ErbB3 mRNA was upregulated. No significant changes in ErbB2 mRNA were detected. Our results suggest that ErbBs changes are involved in the molecular response to peripheral nerve injuries.
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Soluble and transmembrane neuregulin 1 isoforms can act as short-range and long-range attractants for migration of cortical and olfactory interneurons expressing ErbB4, a tyrosine kinase receptor whose characteristics are strongly... more
Soluble and transmembrane neuregulin 1 isoforms can act as short-range and long-range attractants for migration of cortical and olfactory interneurons expressing ErbB4, a tyrosine kinase receptor whose characteristics are strongly affected by alternative splicing. Here, we have investigated the expression of the four ErbB4 isoforms and we found that all of them are expressed by neural progenitor cells migrating from the subventricular zone toward the olfactory bulb through the rostral migratory stream. We quantified the absolute expression of the different ErbB4 isoforms and found that all of them are highly expressed in the regions characterized by high interneuron migration, whereas in the olfactory bulb regions, where migration stops, ErbB4 isoforms containing exon JMb and lacking exon cyt1 (called 'cyt2 isoforms') are expressed more than isoforms containing exons JMa and cyt1. Indeed, we have shown previously that neural progenitor cells stably expressing ErbB4-JMb-cyt2 have a very low migratory activity. To investigate whether the different ErbB4 isoforms confer a distinct adhesion preference for transmembrane neuregulin 1, neural progenitor cells expressing these were tested in vitro in the stripe choice assay. We found that each of the four ErbB4 isoforms is able to confer cells with an adhesion preference for cells expressing the transmembrane neuregulin 1 type III.
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Glutamate (Glu) released by olfactory nerve axons acts on postsynaptic ionotropic and metabotropic glutamate receptors expressed by principal neurones and interneurones of the olfactory bulb (OB). Using ZnSO4 lesioning of the rat... more
Glutamate (Glu) released by olfactory nerve axons acts on postsynaptic ionotropic and metabotropic glutamate receptors expressed by principal neurones and interneurones of the olfactory bulb (OB). Using ZnSO4 lesioning of the rat olfactory mucosa and semiquantitative RT-PCR, we examined the effect of removal of the glutamatergic input to the OB on the expression of mGluR1a, mGluR1b and GluR1 mRNAs. Two days after lesioning, mGluR1a mRNA levels in OB increased by 45%. At this time, the expression of tyrosine hydroxylase (TH) mRNA, which is strictly dependent on olfactory nerve input, was still unchanged. In contrast, 16 days after lesioning, deafferented OB exhibited a decrease in both mGluR1a (-30%) and TH (-40%) mRNAs. GluR1 and mGluR1b mRNA levels were not affected at either time point. These results suggest that alterations in glutamatergic input to OB selectively modulate the expression of the mGluR1 splicing form possessing a longer C-terminal domain.
Research Interests: Cognitive Science, Immunohistochemistry, Animals, Metabotropic Glutamate Receptors, Male, and 12 morePolymerase Chain Reaction, Neuronal Plasticity, Denervation, Rats, Zinc Sulfate, Olfactory Bulb, Wistar Rats, Base Sequence, Olfactory Pathways, Neurosciences, Glutamic Acid, and Molecular Sequence Data
In this study we have investigated the presence of nerve fibers containing dopamine, thyrotropin-releasing hormone (TRH) and neuropeptide Y (NPY) in the pars intermedia of the crested newt and we have examined the possible effect of these... more
In this study we have investigated the presence of nerve fibers containing dopamine, thyrotropin-releasing hormone (TRH) and neuropeptide Y (NPY) in the pars intermedia of the crested newt and we have examined the possible effect of these neurohormones on the release of alpha-melanotropin (alpha-MSH) by neurointermediate lobes in vitro. By means of immunohistochemistry, we observed the presence of tyrosine hydroxylase (TH)-immunoreactive fibers in the pars intermedia of the crested newt. Using a specific antiserum to dopamine, these fibers appeared to be mainly dopaminergic in nature. Unlike anurans, urodele amphibians do not exhibit TRH or NPY-like immunoreactivity in the pars intermedia. A perifusion system technique for newt pituitaries was developed to investigate the effect of dopamine, TRH and NPY on alpha-MSH secretion. Administration of increasing concentrations of dopamine (from 10(-9) to 10(-5)M) induced a dose-related inhibition of alpha-MSH release. This inhibitory effect was mimicked by the dopamine agonist apomorphine (10(-6)M). In contrast, the secretory activity of the newt pars intermedia was not affected by administration of synthetic TRH or NPY (up to 10(-7) and 10(-6)M, respectively). These results indicate that the neurotransmitter dopamine likely plays a pivotal role in the regulation of melanotropin secretion in urodele amphibians.
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Gelatin (GL) nanofibrous matrices mimicking the complex biological structure of the natural extracellular matrix (ECM) were prepared from aqueous solutions by electrospinning technique. GL nanofibres with a diameter size of around 300nm... more
Gelatin (GL) nanofibrous matrices mimicking the complex biological structure of the natural extracellular matrix (ECM) were prepared from aqueous solutions by electrospinning technique. GL nanofibres with a diameter size of around 300nm were obtained optimising the process and solution parameters. To increase the GL stability in aqueous environment γ-glycidoxypropyltrimethoxysilane (GPTMS) was used as GL crosslinker. GPTMS crosslinking did not modify the nanofibrous matrix morphology: fibre diameter and membrane pores size were 327±45 nm and 1.64±0.37 μm, respectively. The produced GPTMS crosslinked GL nanofibres (GL/GPTMS_NF) were found to support the in vitro adhesion, proliferation and survival of neonatal olfactory bulb ensheating cells (NOBECs).
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Research Interests: Cognitive Science, Transmission Electron Microscopy, Female, Animals, Morphological Analysis, and 13 moreNeurons, Nerve Regeneration, Rats, Functional Recovery, Analysis of Variance, Rat, Peripheral Nerve, Wistar Rats, Neurosciences, Median Nerve, Motor Skills, Experimental Model, and Neuroscience Methods
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A partial prolactin (PRL) cDNA was specifically PCR amplified from a cDNA library constructed from pituitary mRNAs of the newt (Cynops pyrrhogaster) and cloned into plasmid vectors. One clone thus obtained contained a 739-bp insert... more
A partial prolactin (PRL) cDNA was specifically PCR amplified from a cDNA library constructed from pituitary mRNAs of the newt (Cynops pyrrhogaster) and cloned into plasmid vectors. One clone thus obtained contained a 739-bp insert encoding the C-terminal amino acid sequence of the mature hormone molecule. Using this clone as a probe, the full-length newt PRL cDNA was screened from the cDNA library. The PRL cDNA clone thus obtained consisted of 1024 bp encoding the entire sequence of the mature PRL molecule in addition to its signal peptide. The amino acid sequence of newt PRL deduced from its nucleotide sequence showed higher homologies with those PRL sequences of tetrapod animals than with those of teleosts. Northern blot analysis revealed the newt PRL mRNA size to be approximately 1 kb. In situ hybridization using the newt PRL cDNA as a probe revealed that the pituitary region expressing PRL mRNA corresponded to that immunoreactive with antiserum against PRL. PRL mRNA levels in the pituitary of newts subjected to room and low temperatures were determined by Northern analysis employing the PRL cDNA as a probe. PRL mRNA levels were significantly higher in the pituitaries of newts subjected to 10 degrees than in those of newts kept at 23 degrees. Likewise, immunoassayable plasma PRL levels were higher in animals subjected to 10 degrees than in those kept at 23 degrees.
Research Interests: Physiology, Immunohistochemistry, In Situ Hybridization, Prolactin, General, and 15 moreAnimals, Temperature, Plasmids, Pituitary Gland, Molecular cloning, Salamandridae, Low Temperature, Veterinary Sciences, SIGNAL PEPTIDE, Amino Acid Sequence, cDNA library, Radioimmunoassay, Molecular Sequence Data, cDNA cloning, and Nucleotide sequence
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Research Interests: Physiology, Kinetics, Somatostatin, Progesterone, General, and 15 moreAldosterone, Animals, Corticosterone, Male, Temperature, Perfusion, Pituitary Gland, Adrenocorticotropic Hormone, Veterinary Sciences, Structure activity Relationship, Alpha MSH, Radioimmunoassay, Adrenal glands, Rana Ridibunda, and In Vitro Techniques
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A perifusion technique using frog adrenal glands has been applied to investigate the effects of long-term administration of a new aldosterone antagonist (potassium prorenoate; SC 23992) on mineralocorticoid production. Whatever the... more
A perifusion technique using frog adrenal glands has been applied to investigate the effects of long-term administration of a new aldosterone antagonist (potassium prorenoate; SC 23992) on mineralocorticoid production. Whatever the duration of administration of potassium prorenoate, at a constant concentration of 5 X 10(-4) M, a significant inhibition of aldosterone output occurred during the passage of the compound. The inhibition was immediate (lag period less than 10 min); the amplitude of the inhibition was constant during the whole experiment and ranged from 77 to 89%; the aldosterone output returned to a regular basal value 80-100 min after the end of infusion of potassium prorenoate. We have also investigated the effect of a concentration gradient of potassium prorenoate (similar to the concentration gradient of aldosterone antagonist observed in plasma after a single oral administration of the molecule) upon aldosterone production over 12 h. From this study, we have established the existence of a highly significant correlation between the extent of the inhibition of aldosterone production and the concentration of the aldosterone antagonist. Finally we have observed that potassium prorenoate blocked the stimulation of aldosterone secretion induced by synthetic ACTH and significantly reduced the angiotensin-induced aldosterone stimulation. The present results indicate that, besides the well-known competitive inhibition of aldosterone binding exerted by potassium prorenoate at the renal receptor site, a direct inhibition of aldosterone biosynthesis also accounts for the pharmacological activity of this aldosterone antagonist.