RESUMEN
Ischemic stroke is a disturbance in cerebral blood flow caused by brain tissue ischemia and hypoxia. We optimized a multifactorial in vitro model of acute ischemic stroke using rat primary neural cultures. This model was exploited to investigate the pro-viable activity of cell-penetrating peptides: arginine-rich Tat(49-57)-NH2 (R49KKRRQRRR57-amide) and its less basic analogue, PTD4 (Y47ARAAARQARA57-amide). Our model included glucose deprivation, oxidative stress, lactic acidosis, and excitotoxicity. Neurotoxicity of these peptides was excluded below a concentration of 50 µm, and PTD4-induced pro-survival was more pronounced. Circular dichroism spectroscopy and molecular dynamics (MD) calculations proved potential contribution of the peptide conformational properties to neuroprotection: in MD, Tat(49-57)-NH2 adopted a random coil and polyproline type II helical structure, whereas PTD4 adopted a helical structure. In an aqueous environment, the peptides mostly adopted a random coil conformation (PTD4) or a polyproline type II helical (Tat(49-57)-NH2) structure. In 30% TFE, PTD4 showed a tendency to adopt a helical structure. Overall, the pro-viable activity of PTD4 was not correlated with the arginine content but rather with the peptide's ability to adopt a helical structure in the membrane-mimicking environment, which enhances its cell membrane permeability. PTD4 may act as a leader sequence in novel drugs for the treatment of acute ischemic stroke.
Asunto(s)
Isquemia Encefálica/prevención & control , Péptidos de Penetración Celular/farmacología , Modelos Animales de Enfermedad , Accidente Cerebrovascular Isquémico/prevención & control , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Animales , Isquemia Encefálica/etiología , Isquemia Encefálica/patología , Permeabilidad de la Membrana Celular , Femenino , Accidente Cerebrovascular Isquémico/etiología , Accidente Cerebrovascular Isquémico/patología , Ratas , Ratas WistarRESUMEN
Regeneration and wound healing are vital to tissue homeostasis and organism survival. One of the biggest challenges of today's science and medicine is finding methods and factors to stimulate these processes in the human body. Effective solutions to promote regenerative responses will accelerate advances in tissue engineering, regenerative medicine, transplantology, and a number of other clinical specialties. In this study, we assessed the potential efficacy of a synthetic hexapeptide, RDKVYR, for the stimulation of tissue repair and wound healing. The hexapeptide is marketed under the name "Imunofan" (IM) as an immunostimulant. IM displayed stability in aqueous solutions, while in plasma it was rapidly bound by albumins. Structural analyses demonstrated the conformational flexibility of the peptide. Tests in human fibroblast and keratinocyte cell lines showed that IM exerted a statistically significant (p < 0.05) pro-proliferative activity (30-40% and 20-50% increase in proliferation of fibroblast and keratinocytes, respectively), revealed no cytotoxicity over a vast range of concentrations (p < 0.05), and had no allergic properties. IM was found to induce significant transcriptional responses, such as enhanced activity of genes involved in active DNA demethylation (p < 0.05) in fibroblasts and activation of genes involved in immune responses, migration, and chemotaxis in adipose-derived stem cells derived from surgery donors. Experiments in a model of ear pinna injury in mice indicated that IM moderately promoted tissue repair (8% in BALB/c and 36% in C57BL/6 in comparison to control).
Asunto(s)
Proliferación Celular/efectos de los fármacos , Oligopéptidos/farmacología , Piel/patología , Cicatrización de Heridas , Albúminas/metabolismo , Animales , Basófilos/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Línea Celular , Quimiotaxis/efectos de los fármacos , Citocinas/metabolismo , Metilación de ADN/efectos de los fármacos , Oído/patología , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Células HaCaT/citología , Células HaCaT/efectos de los fármacos , Humanos , Inyecciones Subcutáneas , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Oligopéptidos/sangre , Oligopéptidos/química , Oligopéptidos/metabolismo , Estabilidad Proteica/efectos de los fármacos , Células Madre/citología , Células Madre/efectos de los fármacos , Transcripción Genética/efectos de los fármacosRESUMEN
Dietary nucleic acids (dietNAs) are being increasingly recognized as important food components with nutritional value. However, the precise dietary recommendations for dietNAs are limited, because established methods for determining the quantity and nutritional role of dietNAs are still lacking. One of the tools to narrow this gap could be microscopic imaging, as a convenient approach to visualize the abundance and distribution of dietNAs in food products. With the aid of appropriate bioinformatic elaboration, such images may in future enable the direct semiquantitative estimation of these macromolecules in food products. In the presented study, two methods of preparing microscopic sections and staining them with DNA-specific fluorochromes were used for microscopic imaging of dietNAs in food products of plant and animal origin. Procedures for preparing formalin-fixed paraffin-embedded sections and cryosections were compared in terms of their usefulness for routine food analysis. Both methods turned out equally suitable for visualizing dietNA distribution in animal and plant products. However, the use of cryosections allowed a significantly shorter analysis time and reduced the consumption of organic solvents. Both of these advantages make the cryosection method preferable while establishing a dedicated methodology for routine assessment of dietNAs in the food industry.
RESUMEN
In the adult brain, new neurons are constitutively derived from postnatal neural stem cells/progenitors located in two neurogenic regions: the subventricular zone (SVZ) of the lateral ventricles (migrating and differentiating into different subtypes of the inhibitory interneurons of the olfactory bulbs), and the subgranular layer of the hippocampal dentate gyrus. Cyclin D2 knockout (cD2-KO) mice exhibit reduced numbers of new hippocampal neurons; however, the proliferation deficiency and the dysregulation of adult neurogenesis in the SVZ required further investigation. In this report, we characterized the differentiation potential of each subpopulation of the SVZ neural precursors in cD2-KO mice. The number of newly generated cells in the SVZs was significantly decreased in cD2-KO mice compared to wild type mice (WT), and was not accompanied by elevated levels of apoptosis. Although the number of B1-type quiescent precursors (B1q) and the overall B1-type activated precursors (B1a) were not affected in the SVZ neurogenic niche, the number of transit-amplifying progenitors (TaPs) was significantly reduced. Additionally, the subpopulations of calbindin D28k and calretinin interneurons were diminished in the olfactory bulbs of cD2-KO mice. Our results suggest that cyclin D2 might be critical for the proliferation of neural precursors and progenitors in the SVZ-the transition of B1a into TaPs and, thereafter, the production of newly generated interneurons in the olfactory bulbs. Untangling regulators that functionally modulate adult neurogenesis provides a basis for the development of regenerative therapies for injuries and neurodegenerative diseases.
Asunto(s)
Ciclina D2/metabolismo , Ventrículos Laterales/metabolismo , Animales , Diferenciación Celular , Proliferación Celular , Genotipo , Ratones , Ratones NoqueadosRESUMEN
The ear pinna is a complex tissue consisting of the dermis, cartilage, muscles, vessels, and nerves. Ear pinna healing is a model of regeneration in mammals. In some mammals, including rabbits, punch wounds in the ear pinna close spontaneously; in common-use laboratory mice, they remain for life. Agents inducing ear pinna healing are potential regenerative drugs. We tested the effects of selected bioactive agents on 2 mm ear pinna wound closure in BALB/c mice. Our previous research demonstrated that a DNA methyltransferase inhibitor, zebularine, remarkably induced ear pinna regeneration. Although experiments with two other demethylating agents, RG108 and hydralazine, were unsuccessful, a histone deacetylase inhibitor, valproic acid, was another epigenetic agent found to increase ear hole closure. In addition, we identified a pro-regenerative activity of 4-ketoretinoic acid, a retinoic acid metabolite. Attempts to counteract the regenerative effects of the demethylating agent zebularine, with folates as methyl donors, failed. Surprisingly, a high dose of methionine, another methyl donor, promoted ear hole closure. Moreover, we showed that the regenerated areas of ear pinna were supplied with nerve fibre networks and blood vessels. The ear punch model proved helpful in testing the pro-regenerative activities of small-molecule compounds and observations of peripheral nerve regeneration.
RESUMEN
L1 cell adhesion molecule (L1CAM) supports spinal cord cellular milieu after contusion and compression lesions, contributing to neuroprotection, promoting axonal outgrowth, and reducing outgrowth-inhibitory molecules in lesion proximity. We extended investigations into L1CAM molecular targets and explored long-distance effects of L1CAM rostral and caudal to complete spinal cord transection (SCT) in adult rats. L1CAM overexpression in neurons and glia after Th10/Th11 SCT was achieved using adeno-associated viral vector serotype 5 (AAV5) injected into an L1-lumbar segment immediately after transection. At 5 weeks, a L1CAM mRNA profound decrease detected rostral and caudal to the transection site was alleviated by AAV5-L1CAM treatment, with increased endogenous L1CAM rostral to the SCT. Transected corticospinal tract fibers showed attenuated retraction after treatment, accompanied by a multi-segmental increase of lesion-reduced expression of adenylate cyclase 1 (Adcy1), synaptophysin, growth-associated protein 43, and myelin basic protein genes caudal to transection, and Adcy1 rostral to transection. In parallel, chondroitin sulfate proteoglycan phosphacan elevated after SCT was downregulated after treatment. Low-molecular L1CAM isoforms generated after spinalization indicated the involvement of sheddases in L1CAM processing and long-distance effects. A disintegrin and metalloproteinase (ADAM)10 sheddase immunoreactivity, stronger in AAV5-L1CAM than AAV5- enhanced green fluorescent protein (EGFP)-transduced motoneurons indicated local ADAM10 upregulation by L1CAM. The results suggest that increased L1CAM availability and penetration of diffusible L1CAM fragments post-lesion induce both local and long-distance neuronal and glial responses toward better neuronal maintenance, neurite growth, and myelination. Despite the fact that intervention promoted beneficial molecular changes, kinematic analysis of hindlimb movements showed minor improvement, indicating that spinalized rats require longer L1CAM treatment to regain locomotor functions.
Asunto(s)
Regulación hacia Abajo/fisiología , Molécula L1 de Adhesión de Célula Nerviosa/biosíntesis , Plasticidad Neuronal/fisiología , Proteínas Tirosina Fosfatasas Clase 5 Similares a Receptores/biosíntesis , Traumatismos de la Médula Espinal/metabolismo , Regulación hacia Arriba/fisiología , Animales , Expresión Génica , Masculino , Molécula L1 de Adhesión de Célula Nerviosa/genética , Ratas , Ratas Wistar , Proteínas Tirosina Fosfatasas Clase 5 Similares a Receptores/antagonistas & inhibidores , Proteínas Tirosina Fosfatasas Clase 5 Similares a Receptores/genética , Traumatismos de la Médula Espinal/genética , Vértebras Torácicas/lesionesRESUMEN
Nitric oxide (NO) plays an important role in prostaglandin secretion and angiogenesis in the reproductive system. In the present study, the roles of the NO donor spermine NONOate and tumour necrosis factor-alpha (TNF; as a positive control) in prostaglandin production and angiogenic activity of equine endometria during the oestrous cycle were evaluated. In addition, the correlation between NO production and the expression of key prostaglandin synthase proteins was determined. The protein expression of prostaglandin F synthase (PGFS) increased in early and mid-luteal stages, whereas that of prostaglandin E synthase (PGES) was increased in the early luteal stage. The in vitro release of NO was highest after ovulation. There was a high correlation between NO production and PGES expression, as well as NO release and PGFS expression. There were no differences detected in prostaglandin H synthase 2 (PTGS-2) throughout the oestrous cycle and there was no correlation between PTGS-2 expression and NO. In TNF- or spermine-treated endometria, the expression of prostaglandin (PG) E(2) increased in the early and mid-luteal phases, whereas that of PGF(2alpha) increased in the follicular and late luteal phases. Bovine aortic endothelial cell (BAEC) proliferation was stimulated in TNF-treated follicular-phase endometria. However, in spermine-treated endometria, NO delivered from its donor had no effect, or even an inhibitory effect, on BAEC proliferation. In conclusion, despite no change in PTGS-2 expression throughout the oestrous cycle in equine endometrial tissue, there were changes observed in the expression of PGES and PGFS, as well as in the production of PGE(2) and PGF(2alpha). In the mare, NO is involved in the secretory function of the endometrium, modulating PGE(2) and PGF(2alpha) production. Even though TNF caused an increase in the production of angiogenic factors and prostaglandins, its complex action in mare uterus should be elucidated.
Asunto(s)
Endometrio/efectos de los fármacos , Endometrio/metabolismo , Caballos/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , Donantes de Óxido Nítrico/farmacología , Prostaglandinas/biosíntesis , Animales , Bovinos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Células Endoteliales/fisiología , Ciclo Estral/efectos de los fármacos , Femenino , Neovascularización Fisiológica/fisiología , Óxido Nítrico/farmacología , Prostaglandina-Endoperóxido Sintasas/metabolismo , Prostaglandinas/fisiología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Beneficial effects of locomotor training on the functional recovery after complete transection of the spinal cord indicate that in chronic spinal animals spontaneous recovery processes are enhanced and shaped by the training. The mechanisms of that use-dependent improvement are still not fully understood. This review tackles three aspects of this issue: (1) neurochemical attributes of functional improvement showing that concentrations of excitatory and inhibitory amino acids in the lumbar spinal segments, which were changed after transection, normalize after the training, or even raise beyond normal. As it does not translate to functional equilibrium between excitatory and inhibitory neurotransmission and may lead to hyperexcitability, the postsynaptic mechanisms which might be responsible for the hyperexcitability are discussed, including (i) dysfunction of K(+)-Cl(-) cotransporter KCC2, which controls the strength and robustness of inhibition, and (ii) altered function of 5-HT2 receptors, which may be targeted to restore KCC2 activity and intrinsic inhibition; (2) morphological changes of lumbar motoneurons and their inputs related to functional improvement of spinal animals, pointing to use-dependent diminution/reversal of the atrophy of the dendritic tree of the hindlimb motoneurons and of their synaptic impoverishment, which in paraplegic animals differs depending on the degree of disuse of the muscles; (3) the role of neurotrophins in motor improvement of spinal animals showing, that increases in neurotrophins due to training or due to efficient viral vector-based transgene expression, that might be responsible for the enrichment of the dendritic tree, elongation of processes and influence neurotransmitter systems in the areas subjected to plastic modifications after injury, correlate with improvement of locomotor functions.