RESUMEN

European eels (Anguilla anguilla) undertake an impressive 5 000 km long migration from European fresh waters through the North Atlantic Ocean to the Sargasso Sea. Along with sexual maturation, the eel skeleton undergoes a remarkable morphological transformation during migration, where a hitherto completely obscure bone loss phenomenon occurs. To unravel mechanisms of the maturation-related decay of the skeleton, we performed a multiscale assessment of eels' bones at different life-cycle stages. Accordingly, the skeleton reflects extensive bone loss that is mediated via multinucleated bone-resorbing osteoclasts, while other resorption mechanisms such as osteocytic osteolysis or matrix demineralization were not observed. Preserving mechanical stability and releasing minerals for energy metabolism are two mutually exclusive functions of the skeleton that are orchestrated in eels through the presence of two spatially segregated hard tissues: cellular bone and acellular notochord. The cellular bone serves as a source of mineral release following osteoclastic resorption, whereas the mineralized notochord sheath, which is inaccessible for resorption processes due to an unmineralized cover layer, ensures sufficient mechanical stability as a part of the notochord sheath. Clearly, an eel's skeleton is structurally optimized to meet the metabolic challenge of fasting and simultaneous sexual development during an exhausting journey to spawning areas, while the function of the vertebral column is maintained to achieve this goal.

Asunto(s)

Anguilla/anatomía & histología , Migración Animal , Resorción Ósea , Huesos/fisiología , Estadios del Ciclo de Vida , Anguilla/fisiología , Animales , Océano Atlántico , Calcificación Fisiológica

RESUMEN

Novel therapeutic concepts against cerebral ischemia focus on cell-based therapies in order to overcome some of the side effects of thrombolytic therapy. However, cell-based therapies are hampered because of restricted understanding regarding optimal cell transplantation routes and due to low survival rates of grafted cells. We therefore transplanted adult green fluorescence protein positive neural precursor cells (NPCs) either intravenously (systemic) or intrastriatally (intracerebrally) 6 hours after stroke in mice. To enhance survival of NPCs, cells were in vitro protein-transduced with TAT-heat shock protein 70 (Hsp70) before transplantation followed by a systematic analysis of brain injury and underlying mechanisms depending on cell delivery routes. Transduction of NPCs with TAT-Hsp70 resulted in increased intracerebral numbers of grafted NPCs after intracerebral but not after systemic transplantation. Whereas systemic delivery of either native or transduced NPCs yielded sustained neuroprotection and induced neurological recovery, only TAT-Hsp70-transduced NPCs prevented secondary neuronal degeneration after intracerebral delivery that was associated with enhanced functional outcome. Furthermore, intracerebral transplantation of TAT-Hsp70-transduced NPCs enhanced postischemic neurogenesis and induced sustained high levels of brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor, and vascular endothelial growth factor in vivo. Neuroprotection after intracerebral cell delivery correlated with the amount of surviving NPCs. On the contrary, systemic delivery of NPCs mediated acute neuroprotection via stabilization of the blood-brain-barrier, concomitant with reduced activation of matrix metalloprotease 9 and decreased formation of reactive oxygen species. Our findings imply two different mechanisms of action of intracerebrally and systemically transplanted NPCs, indicating that systemic NPC delivery might be more feasible for translational stroke concepts, lacking a need of in vitro manipulation of NPCs to induce long-term neuroprotection.

Asunto(s)

Isquemia Encefálica/terapia , Proteínas HSP70 de Choque Térmico/genética , Células-Madre Neurales/fisiología , Células-Madre Neurales/trasplante , Accidente Cerebrovascular/terapia , Animales , Isquemia Encefálica/metabolismo , Isquemia Encefálica/cirugía , Diferenciación Celular/fisiología , Modelos Animales de Enfermedad , Técnicas de Transferencia de Gen , Proteínas HSP70 de Choque Térmico/biosíntesis , Proteínas HSP70 de Choque Térmico/metabolismo , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Trasplante de Células Madre/métodos , Accidente Cerebrovascular/genética , Accidente Cerebrovascular/metabolismo , Accidente Cerebrovascular/cirugía , Transducción Genética

RESUMEN

The stock of the catadromous European eel (Anguilla anguilla L.) continues to decline and there is growing evidence that poor health status due to contaminants might be a key element in this decrease. Organic contaminants such as polycyclic aromatic hydrocarbons (PAHs) belong to the major threats to yellow eel in their growth habitat and their metabolites are detectable in the bile. Starting the silvering process eels undergo physiological and morphological changes including cessation of feeding and downstream migration back to their spawning grounds. Reduced feed intake results in a diminishment of bile production and induces accumulation of e.g. PAH-metabolites in bile. Therefore, the aim of the present study was to demonstrate the impact of silvering on biliary PAH metabolite concentrations and to utilize normalization procedures to overcome silvering related accumulation effects of PAH-metabolites in eel bile. We investigated the hydroxyl-metabolites of pyrene (1-OH Pyr) and phenantrene (1-OH Phen) in the bile of different maturation stages of eels (silvering index I-V) from nine German rivers. We detected increasing absolute PAH metabolite levels in bile during the silvering process. The highest rise could be observed at the transition from pre migration stage III to the migrating stage IV, suggesting the onset of cessation of feeding at this stage. A cessation bias in PAH metabolite measurement could be diminished by normalization of absolute values against bile pigments (A(380), biliverdin). In conclusion, we demonstrated the impact of silvering on PAH metabolite concentrations in eel bile and present suitable normalization procedures to overcome silvering related accumulation effects. Thus, for a future eel monitoring we recommend (1) to regularly monitor PAH metabolites in bile, (2) to determine silvering index of eel and (3) to normalize PAH metabolite values in bile based on maturation/silvering stages. The knowledge of the silvering stage is mandatory for an unbiased evaluation of PAH contamination of European eel towards an international harmonized eel monitoring program.

Asunto(s)

Anguilla/fisiología , Bilis/metabolismo , Monitoreo del Ambiente , Hidrocarburos Policíclicos Aromáticos/metabolismo , Contaminantes Químicos del Agua/metabolismo , Animales , Femenino , Alemania

RESUMEN

OBJECTIVES: The objective of this in vitro study was to investigate the behavior of different composite restorative materials under the load of cast circumferential clasps for removable dental prostheses (RDPs). METHODS: In 60 human molars, standardized mesial-occlusal-distal cavities were prepared. The cavities were restored with the following materials: Definite, Tetric Ceram, SureFil, Heliomolar RO, Ariston pHc, and Oralloy, and provided with a rest seat. The rest seats were subjected to 5,000 cycles of thermal cycling and 1,200,000 masticatory cycles in a mastication simulator via cobalt-chromium circumferential clasps cast to standardized frameworks in a laboratory model designed to simulate the biomechanics of a free-end denture base. Fracture analysis of the restorations was performed by light microscopy. Before and after loading, material wear was measured with a 3D-laser scanner, and an analysis of the marginal quality was performed in an SEM at ×200 applying the replica technique. RESULTS: No significant differences in the fracture behavior among the composite materials were found; the amalgam control group showed a significantly higher fracture resistance. Regarding the wear of the materials, the composites Definite and SureFil exhibited a behavior similar to that of amalgam. The other composites demonstrated higher wear rates. The initial marginal quality was significantly worse for Ariston pHc. The marginal adaptation decreased significantly after thermal and mechanical loading for Definite and Ariston pHc. CONCLUSIONS: In terms of the investigated aspects of mechanical performance, the tested composites seemed to be inferior to amalgam. Further clinical studies are needed to evaluate the ability of composite restorations to provide support for RDP clasps. CLINICAL RELEVANCE: The use of composites as direct restoration materials should be avoided in teeth, which serve as abutments for clasp-retained RDPs.

Asunto(s)

Abrazadera Dental , Materiales Dentales/química , Dentadura Parcial Removible , Resinas Acrílicas/química , Fenómenos Biomecánicos , Fuerza de la Mordida , Aleaciones de Cromo/química , Resinas Compuestas/química , Pilares Dentales , Amalgama Dental/química , Preparación de la Cavidad Dental/clasificación , Adaptación Marginal Dental , Fracaso de la Restauración Dental , Alisadura de la Restauración Dental , Restauración Dental Permanente/clasificación , Análisis del Estrés Dental/instrumentación , Humanos , Imagenología Tridimensional/métodos , Rayos Láser , Ensayo de Materiales , Metacrilatos/química , Microscopía Electrónica de Rastreo , Cerámicas Modificadas Orgánicamente/química , Poliuretanos/química , Siloxanos/química , Estrés Mecánico , Propiedades de Superficie , Temperatura , Terpenos/química

RESUMEN

Toxins such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 6-hydroxydopamine (6-OHDA), or rotenone have been used to induce degeneration of dopaminergic (DA) neurons in the nigrostriatal pathway and to reproduce pathological characteristics of Parkinson's disease (PD). DA neurons are also present in the retina, and visual impairments in PD patients have been reported. We examined the vulnerability of TH-positive (TH(+)) amacrine interneurons in the retina against MPTP, 6-OHDA, or rotenone-induced cell death. We intraperitoneally (i.p.) injected mice with MPTP, which induced degeneration of DA neurons in the midbrain. However, no death of TH(+) amacrine cells was detectable in the same mice. HPLC analysis revealed a 9 times lower level of the toxic metabolite of MPTP, MPP(+), in the eye compared with the striatum. Another membrane-permeable compound (Tat-Hsp70) could be delivered into the retina after i.p. application, suggesting that the blood-retina barrier (BRB) could be overcome after systemic application. Possible reason for the survival of retinal amacrine cells after systemic MPTP application was a less efficient conversion into toxic MPP(+) in the retina or a general higher resistance against toxic insults of retinal DA neurons compared with DA neurons in the substantia nigra pars compacta (SNpc). Therefore, we directly injected high doses of MPP(+), 6-OHDA, or rotenone into the eye. No loss of TH(+) amacrine cells in the retina was observed, suggesting different properties and less vulnerability of amacrine neurons compared with DA neurons in the midbrain.

Asunto(s)

Células Amacrinas/efectos de los fármacos , Células Amacrinas/fisiología , Intoxicación por MPTP , Oxidopamina/toxicidad , Rotenona/toxicidad , Tirosina 3-Monooxigenasa/metabolismo , Animales , Astrocitos/efectos de los fármacos , Astrocitos/fisiología , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Dopamina/metabolismo , Ojo/efectos de los fármacos , Ojo/metabolismo , Ojo/patología , Gliosis/inducido químicamente , Gliosis/patología , Masculino , Mesencéfalo/efectos de los fármacos , Mesencéfalo/fisiología , Ratones , Ratones Endogámicos C57BL , Neuronas/efectos de los fármacos , Neuronas/fisiología

RESUMEN

Cerebral ischemia stimulates endogenous neurogenesis within the subventricular zone and the hippocampal dentate gyrus of the adult rodent brain. However, such newly generated cells soon die after cerebral ischemia. To enhance postischemic survival of neural precursor cells (NPC) and long-lasting neural regeneration, we applied the antiapoptotic chaperone heat shock protein 70 (Hsp70) fused to a cell-penetrating peptide derived from the HIV TAT to ensure delivery across the blood-brain barrier and the cell membrane. After transient focal cerebral ischemia in mice, TAT-Hsp70 was intravenously injected concomitant with reperfusion and additionally on day 14 after stroke. TAT-Hsp70 treatment resulted in smaller infarct size (27.1+/-9.0 versus 109.0+/-14.0 and 88.5+/-26.0 mm(3) in controls) and in functional improvement as assessed by the rota rod, tight rope, and water maze tests when compared with saline- and TAT-hemagglutinin-treated controls. In addition, postischemic survival of endogenous doublecortin (Dcx)-positive NPC was improved within the lesioned striatum of TAT-Hsp70-treated animals for up to 4 weeks after stroke without changing overall cell proliferation of BrdU(+) cells. Thus, TAT-Hsp70 treatment after stroke may be a promising tool to act neuroprotective and improve postischemic functional outcome, and also to increase survival of endogenous NPC after stroke.

Asunto(s)

Isquemia Encefálica/tratamiento farmacológico , Isquemia Encefálica/patología , Productos del Gen tat/uso terapéutico , Proteínas HSP70 de Choque Térmico/uso terapéutico , Fármacos Neuroprotectores/uso terapéutico , Animales , Bromodesoxiuridina/farmacología , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Proteína Doblecortina , Productos del Gen tat/genética , Productos del Gen tat/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Fármacos Neuroprotectores/metabolismo , Ratas

RESUMEN

Cell-penetrating peptides (CPPs), such as the one derived from the human immunodeficiency virus Tat protein, facilitate the delivery of cargoes across cellular membranes. However, questions about the therapeutic potential of CPP-mediated delivery remain. For instance, the impact of the purification procedure on the functionality of Tat-fusion proteins has not been systematically examined. Here, we isolated fusion proteins of the chaperone heat shock protein 70 (Hsp70) and the Tat CPP under denaturing or native conditions. To investigate the therapeutic potential of different recombinant protein preparations, we examined Tat-Hsp70 transduction efficiency and quantified Tat-Hsp70-mediated folding of a chaperone-dependent yellow fluorescent protein in vitro. Transduction efficiency and chaperone activity of Tat-Hsp70-treated cells was significantly higher compared to cells treated with Hsp70. The application of native isolated Tat-Hsp70 had the strongest effect. This chaperone activity correlates with increased viability of cells treated with the recombinant protein after cell death induction with 6-hydroxydopamine. This suggests that the method of recombinant Tat-fusion protein purification influences its functionality. For Tat-Hsp70, the method of choice seems to be isolation under native conditions, for which we present a purification protocol. Our results may contribute to improve Tat-fusion protein application in basic research and may facilitate its use as therapeutic tool, for instance in Parkinson's disease.

Asunto(s)

Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP70 de Choque Térmico/farmacología , Animales , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Productos del Gen tat/genética , Productos del Gen tat/metabolismo , Productos del Gen tat/farmacología , Proteínas HSP70 de Choque Térmico/genética , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Neuroblastoma , Oxidopamina/toxicidad , Transporte de Proteínas , Ratas , Proteínas Recombinantes/metabolismo , Simpaticolíticos/toxicidad , Transfección

RESUMEN

Parkinson's disease is characterized by the progressive loss of dopaminergic (DA) neurons in the substantia nigra. The heat-shock protein 70 (Hsp70) reduces protein misfolding and aggregation. It has been shown to protect cells against oxidative stress and apoptotic stimuli in various neurodegenerative disease models. To deliver Hsp70 across cellular membranes and into the brain, we linked it to a cell-penetrating peptide derived from the HIV trans-activator of transcription (Tat) protein. In vitro, Tat-Hsp70 transduced neuroblastoma cells and protected primary mesencephalic DA neurons and their neurites against MPP+-mediated degeneration. In vivo, the systemic application of cell-permeable Hsp70 protected DA neurons of the substantia nigra pars compacta against subacute toxicity of MPTP. Furthermore, Tat-Hsp70 diminished the MPTP induced decrease in DA striatal fiber density. Thus, we demonstrate that systemically applied Tat-Hsp70 effectively prevents neuronal cell death in in vitro and in vivo models of Parkinson's disease. The use of Tat-fusion proteins might therefore be a valuable tool to deliver molecular chaperones like Hsp70 into the brain and may be the starting point for new protective strategies in neurodegenerative diseases.

Asunto(s)

Productos del Gen tat/genética , Terapia Genética/métodos , Proteínas HSP70 de Choque Térmico/metabolismo , Estrés Oxidativo/genética , Proteínas Recombinantes de Fusión/metabolismo , Sustancia Negra/metabolismo , Animales , Línea Celular Tumoral , Células Cultivadas , Cuerpo Estriado/metabolismo , Cuerpo Estriado/patología , Cuerpo Estriado/fisiopatología , Citoprotección/genética , Dopamina/metabolismo , Productos del Gen tat/química , Proteínas HSP70 de Choque Térmico/genética , Humanos , Masculino , Ratones , Trastornos Parkinsonianos/genética , Trastornos Parkinsonianos/metabolismo , Trastornos Parkinsonianos/terapia , Fragmentos de Péptidos/genética , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/uso terapéutico , Sustancia Negra/citología , Sustancia Negra/fisiopatología , Transducción Genética/métodos , Resultado del Tratamiento , Degeneración Walleriana/genética , Degeneración Walleriana/metabolismo , Degeneración Walleriana/terapia