RESUMO
Human AUTS2 mutations are linked to a syndrome of intellectual disability, autistic features, epilepsy, and other neurological and somatic disorders. Although it is known that this unique gene is highly expressed in developing cerebral cortex, the molecular and developmental functions of AUTS2 protein remain unclear. Using proteomics methods to identify AUTS2 binding partners in neonatal mouse cerebral cortex, we found that AUTS2 associates with multiple proteins that regulate RNA transcription, splicing, localization, and stability. Furthermore, AUTS2-containing protein complexes isolated from cortical tissue bound specific RNA transcripts in RNA immunoprecipitation and sequencing assays. Deletion of all major functional isoforms of AUTS2 (full-length and C-terminal) by conditional excision of exon 15 caused breathing abnormalities and neonatal lethality when Auts2 was inactivated throughout the developing brain. Mice with limited inactivation of Auts2 in cerebral cortex survived but displayed abnormalities of cerebral cortex structure and function, including dentate gyrus hypoplasia with agenesis of hilar mossy neurons, and abnormal spiking activity on EEG. Also, RNA transcripts that normally associate with AUTS2 were dysregulated in mutant mice. Together, these findings indicate that AUTS2 regulates RNA metabolism and is essential for development of cerebral cortex, as well as subcortical breathing centers.
Assuntos
Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/fisiologia , Giro Denteado/crescimento & desenvolvimento , Giro Denteado/metabolismo , RNA/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Animais , Animais Recém-Nascidos , Córtex Cerebral/anormalidades , Córtex Cerebral/metabolismo , Eletroencefalografia , Éxons/genética , Deleção de Genes , Regulação da Expressão Gênica , Deficiência Intelectual/genética , Camundongos , Camundongos Endogâmicos C57BL , RNA-Seq , RespiraçãoRESUMO
The cortical area map is initially patterned by transcription factor (TF) gradients in the neocortical primordium, which define a "protomap" in the embryonic ventricular zone (VZ). However, mechanisms that propagate regional identity from VZ progenitors to cortical plate (CP) neurons are unknown. Here we show that the VZ, subventricular zone (SVZ), and CP contain distinct molecular maps of regional identity, reflecting different gene expression gradients in radial glia progenitors, intermediate progenitors, and projection neurons, respectively. The "intermediate map" in the SVZ is modulated by Eomes (also known as Tbr2), a T-box TF. Eomes inactivation caused rostrocaudal shifts in SVZ and CP gene expression, with loss of corticospinal axons and gain of corticotectal projections. These findings suggest that cortical areas and connections are shaped by sequential maps of regional identity, propagated by the Pax6 â Eomes â Tbr1 TF cascade. In humans, PAX6, EOMES, and TBR1 have been linked to intellectual disability and autism.
Assuntos
Córtex Cerebral/anatomia & histologia , Córtex Cerebral/metabolismo , Proteínas com Domínio T/metabolismo , Animais , Transtorno Autístico/genética , Transtorno Autístico/metabolismo , Transtorno Autístico/patologia , Padronização Corporal , Mapeamento Encefálico , Córtex Cerebral/crescimento & desenvolvimento , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Feminino , Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Deficiência Intelectual/genética , Deficiência Intelectual/metabolismo , Deficiência Intelectual/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Vias Neurais/citologia , Vias Neurais/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , Gravidez , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais , Proteínas com Domínio T/deficiência , Proteínas com Domínio T/genéticaRESUMO
Areas and layers of the cerebral cortex are specified by genetic programs that are initiated in progenitor cells and then, implemented in postmitotic neurons. Here, we report that Tbr1, a transcription factor expressed in postmitotic projection neurons, exerts positive and negative control over both regional (areal) and laminar identity. Tbr1 null mice exhibited profound defects of frontal cortex and layer 6 differentiation, as indicated by down-regulation of gene-expression markers such as Bcl6 and Cdh9. Conversely, genes that implement caudal cortex and layer 5 identity, such as Bhlhb5 and Fezf2, were up-regulated in Tbr1 mutants. Tbr1 implements frontal identity in part by direct promoter binding and activation of Auts2, a frontal cortex gene implicated in autism. Tbr1 regulates laminar identity in part by downstream activation or maintenance of Sox5, an important transcription factor controlling neuronal migration and corticofugal axon projections. Similar to Sox5 mutants, Tbr1 mutants exhibit ectopic axon projections to the hypothalamus and cerebral peduncle. Together, our findings show that Tbr1 coordinately regulates regional and laminar identity of postmitotic cortical neurons.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Mitose , Neocórtex/citologia , Neocórtex/embriologia , Neurônios/citologia , Animais , Biomarcadores/metabolismo , Proteínas do Citoesqueleto , Proteínas de Ligação a DNA/genética , Regulação para Baixo/genética , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Mutação/genética , Neocórtex/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Especificidade de Órgãos , Ligação Proteica , Proteínas com Domínio T , Fatores de Transcrição , Ativação Transcricional , Regulação para Cima/genéticaRESUMO
BACKGROUND: Coronavirus disease 2019 (COVID-19) is associated with adverse maternal and neonatal outcomes. Early studies suggested that COVID-19 was associated with a higher incidence of hypotension following neuraxial anesthesia in parturients. We explored the hemodynamic response to spinal anesthesia for cesarean delivery in pregnant severe respiratory distress syndrome-coronavirus-2 (SARS-CoV-2) positive patients, using a retrospective case-control design. METHODS: We searched our electronic medical records for patients who received spinal anesthesia for cesarean delivery, and were SARS-CoV-2 positive or recovered at delivery, and used historical and SARS-CoV-2 negative controls from two tertiary care hospitals. We compared the demographic, clinical, and hemodynamic variables between patients who were SARS-CoV-2 positive at delivery, those who were positive during pregnancy and recovered before delivery, and controls. Analyses were stratified by normotensive versus hypertensive status of the patients at delivery. RESULTS: We identified 22 SARS-CoV-2 positive, 73 SARS-CoV-2 recovered, and 1517 controls. The SARS-CoV-2 positive, and recovered pregnant patients, had on average 5.6 and 2.2â¯mmHg, respectively, higher post-spinal mean arterial pressures (MAPs) than control patients, adjusting for covariates. Additionally, the lowest post-spinal MAP was negatively correlated with the number of daysbetween the onset of COVID-19 symptoms and delivery in patients with hypertension (correlation -0.55, 95% CI -0.81 to -0.09). CONCLUSIONS: Patients with SARS-CoV-2 infection during pregnancy exhibit less spinal hypotension than non-infected patients. While the clinical significance of this finding is unknown, it points to important cardiovascular effects of the virus.
Assuntos
Raquianestesia , COVID-19 , Hipotensão , Complicações Infecciosas na Gravidez , Gravidez , Recém-Nascido , Feminino , Humanos , Estudos Retrospectivos , Estudos de Casos e Controles , SARS-CoV-2 , Raquianestesia/efeitos adversos , Hipotensão/etiologia , Hemodinâmica , Complicações Infecciosas na Gravidez/diagnósticoRESUMO
The developing cerebral cortex contains apical and basal types of neurogenic progenitor cells. Here, we investigated the cellular properties and neurogenic output of basal progenitors, also called intermediate neuronal progenitors (INPs). We found that basal mitoses expressing transcription factor Tbr2 (an INP marker) were present throughout corticogenesis, from embryonic day 10.5 through birth. Postnatally, Tbr2(+) progenitors were present in the dentate gyrus, subventricular zone (SVZ), and posterior periventricle (pPV). Two morphological subtypes of INPs were distinguished in the embryonic cortex, "short radial" in the ventricular zone (VZ) and multipolar in the SVZ, probably corresponding to molecularly defined INP subtypes. Unexpectedly, many short radial INPs appeared to contact the apical (ventricular) surface and some divided there. Time-lapse video microscopy suggested that apical INP divisions produced daughter INPs. Analysis of neurogenic divisions (Tis21-green fluorescent protein [GFP](+)) indicated that INPs may produce the majority of projection neurons for preplate, deep, and superficial layers. Conversely, proliferative INP divisions (Tis21-GFP(-)) increased from early to middle corticogenesis, concomitant with SVZ growth. Our findings support the hypothesis that regulated amplification of INPs may be an important factor controlling the balance of neurogenesis among different cortical layers.
Assuntos
Córtex Cerebral/embriologia , Células-Tronco Multipotentes/fisiologia , Neurogênese/fisiologia , Neurônios/fisiologia , Animais , Contagem de Células , Córtex Cerebral/metabolismo , Imunofluorescência , Corantes Fluorescentes , Processamento de Imagem Assistida por Computador , Hibridização in Situ Fluorescente , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Microscopia de Vídeo , Proteínas com Domínio T/metabolismoRESUMO
BACKGROUND: In a genome-wide scan (GWS) of 175 multiplex prostate cancer (PCa) families from the University of Michigan Prostate Cancer Genetics Project (PCGP), linkage was observed to markers on chromosome 17q21-24, a region that includes two breast cancer susceptibility genes, BRCA1 and BRIP1. BRIP1 is a Fanconi anaemia gene (FANCJ) that interacts with the BRCT domain of BRCA1 and has a role in DNA damage repair. Protein truncating mutations in BRIP1 have been identified in hereditary breast and ovarian cancer families, and a recent report suggested that a recurrent truncating mutation (R798X) may have a role in PCa susceptibility. METHODS: We examined the role of BRIP1 mutations in hereditary PCa through sequence analysis of 94 individuals from PCGP families showing linkage to 17q. RESULTS: A total of 24 single-nucleotide polymorphisms, including 7 missense variants but no protein truncating mutations, were observed. CONCLUSION: The data presented here suggest that BRIP1 truncating mutations are uncommon in PCa cases and do not account for the linkage to chromosome 17q observed in our GWS. Additional investigation is needed to determine the significance, if any, of the observed BRIP1 missense variants in hereditary PCa.
Assuntos
Cromossomos Humanos Par 17/genética , Proteínas de Ligação a DNA/genética , Mutação , Neoplasias da Próstata/genética , RNA Helicases/genética , Idoso , Proteínas de Grupos de Complementação da Anemia de Fanconi , Feminino , Ligação Genética , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Unipolar brush cells (UBCs) are glutamatergic interneurons in the cerebellar cortex and dorsal cochlear nucleus. We studied the development of UBCs, using transcription factor Tbr2/Eomes as a marker for UBCs and their progenitors in embryonic and postnatal mouse cerebellum. Tbr2+ UBCs appeared to migrate out of the upper rhombic lip via two cellular streams: a dorsal pathway into developing cerebellar white matter, where the migrating cells dispersed widely before entering the internal granular layer, and a rostral pathway along the cerebellar ventricular zone toward the brainstem. Ablation of the rhombic lip in organotypic slice cultures substantially reduced the production of Tbr2+ UBCs. In coculture experiments, Tbr2+ UBCs migrated from rhombic lip explants directly into the developing white matter of adjacent cerebellar slices. The origin of Tbr2+ UBCs was confirmed by colocalization with beta-galactosidase expressed from the Math1 locus, a molecular marker of rhombic lip lineages. Moreover, the production of Tbr2+ UBCs was Math1 dependent, as Tbr2+ UBCs were severely reduced in Math1-null cerebellum. In reeler mutant mice, Tbr2+ UBCs accumulated near the rhombic lip, consistent with impaired migration through developing white matter. Our results suggest that UBCs arise from the rhombic lip and migrate via novel pathways to their final destinations in the cerebellum and dorsal cochlear nucleus. Our findings support a model of cerebellar neurogenesis, in which glutamatergic and GABAergic neurons are produced from separate progenitor pools located mainly in the rhombic lip and the cerebellar ventricular zone, respectively.
Assuntos
Cerebelo/embriologia , Cerebelo/fisiologia , Interneurônios/citologia , Interneurônios/fisiologia , Fibras Nervosas Mielinizadas/ultraestrutura , Rombencéfalo/embriologia , Rombencéfalo/fisiologia , Animais , Diferenciação Celular , Movimento Celular/fisiologia , Células Cultivadas , Cerebelo/citologia , Camundongos , Camundongos Mutantes Neurológicos , Fibras Nervosas Mielinizadas/fisiologia , Rombencéfalo/citologiaRESUMO
The deep cerebellar nuclei (DCN) are the main output centers of the cerebellum, but little is known about their development. Using transcription factors as cell type-specific markers, we found that DCN neurons in mice are produced in the rhombic lip and migrate rostrally in a subpial stream to the nuclear transitory zone (NTZ). The rhombic lip-derived cells express transcription factors Pax6, Tbr2, and Tbr1 sequentially as they enter the NTZ. A subset of rhombic lip-derived cells also express reelin, a key regulator of Purkinje cell migrations. In organotypic slice cultures, the rhombic lip was necessary and sufficient to produce cells that migrate in the subpial stream, enter the NTZ, and express Pax6, Tbr2, Tbr1, and reelin. In later stages of development, the subpial stream is replaced by the external granular layer, and the NTZ organizes into distinct DCN nuclei. Tbr1 expression persists to adulthood in a subset of medial DCN projection neurons. In reeler mutant mice, which have a severe cerebellar malformation, rhombic lip-derived cells migrated to the NTZ, despite reelin deficiency. Studies in Tbr1 mutant mice suggested that Tbr1 plays a role in DCN morphogenesis but is not required for reelin expression, glutamatergic differentiation, or the initial formation of efferent axon pathways. Our findings reveal underlying similarities in the transcriptional programs for glutamatergic neuron production in the DCN and the cerebral cortex, and they support a model of cerebellar neurogenesis in which glutamatergic and GABAergic neurons are produced from separate progenitor compartments.
Assuntos
Núcleos Cerebelares/citologia , Proteínas do Tecido Nervoso/biossíntese , Rombencéfalo/citologia , Fatores de Transcrição/biossíntese , Animais , Transporte Axonal , Biomarcadores , Moléculas de Adesão Celular Neuronais/biossíntese , Moléculas de Adesão Celular Neuronais/genética , Linhagem da Célula , Movimento Celular , Núcleos Cerebelares/anormalidades , Núcleos Cerebelares/embriologia , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Vias Eferentes/embriologia , Vias Eferentes/fisiologia , Proteínas da Matriz Extracelular/biossíntese , Proteínas da Matriz Extracelular/genética , Proteínas do Olho/biossíntese , Proteínas do Olho/genética , Idade Gestacional , Ácido Glutâmico/fisiologia , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes Neurológicos , Microscopia de Fluorescência , Morfogênese , Proteínas do Tecido Nervoso/genética , Neurônios/citologia , Neurônios/metabolismo , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/biossíntese , Fatores de Transcrição Box Pareados/genética , Núcleo Rubro/citologia , Núcleo Rubro/embriologia , Proteína Reelina , Proteínas Repressoras/biossíntese , Proteínas Repressoras/genética , Rombencéfalo/embriologia , Rombencéfalo/metabolismo , Serina Endopeptidases/biossíntese , Serina Endopeptidases/genética , Proteínas com Domínio T/biossíntese , Proteínas com Domínio T/genética , Fatores de Transcrição/genéticaRESUMO
The developing neocortex contains two types of progenitor cells for glutamatergic, pyramidal-projection neurons. The first type, radial glia, produce neurons and glia, divide at the ventricular surface, and express Pax6, a homeodomain transcription factor. The second type, intermediate progenitor cells, are derived from radial glia, produce only neurons, and divide away from the ventricular surface. Here we show that the transition from radial glia to intermediate progenitor cell is associated with upregulation of Tbr2, a T-domain transcription factor, and downregulation of Pax6. Accordingly, Tbr2 expression in progenitor compartments (the subventricular zone and ventricular zone) rises and falls with cortical plate neurogenesis. The subsequent transition from intermediate progenitor cell to postmitotic neuron is marked by downregulation of Tbr2 and upregulation of Tbr1, another T-domain transcription factor. These findings delineate the transcription factor sequence Pax6 --> Tbr2 --> Tbr1 in the differentiation of radial glia --> intermediate progenitor cell --> postmitotic projection neuron. This transcription factor sequence is modified in preplate neurons, in which Tbr2 is transiently coexpressed with Tbr1, and in the direct differentiation pathway from radial glia --> postmitotic projection neuron, in which Tbr2 is expressed briefly or not at all.
Assuntos
Neocórtex/embriologia , Neocórtex/metabolismo , Proteínas do Tecido Nervoso/biossíntese , Neurônios/metabolismo , Células-Tronco/metabolismo , Fatores de Transcrição/biossíntese , Animais , Células Cultivadas , Proteínas de Ligação a DNA/biossíntese , Proteínas do Olho/biossíntese , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/biossíntese , Camundongos , Mitose , Neocórtex/citologia , Neurônios/citologia , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados , Proteínas Repressoras/biossíntese , Proteínas com Domínio T/biossíntese , Fatores de TempoRESUMO
Glutamatergic, pyramidal-projection neurons are produced in the embryonic cerebral cortex by a series of genetically programmed fate choices, implemented in large part by developmental transcription factors. Our work has focused on Pax6, Tbr2/Eomes, NeuroD, and Tbr1, which are expressed sequentially during the neurogenesis of pyramidal-projection neurons. Recently, we have found that the same transcription factors are expressed, in the same order, during glutamatergic neurogenesis in the adult dentate gyrus, and (with modifications) in the developing cerebellum. While the precise functional significance of this transcription factor expression sequence is unknown, its common appearance in embryonic and adult neurogenesis, and in different brain regions, suggests it is part of a conserved genetic program that specifies general properties of glutamatergic neurons in these regions. Subtypes of glutamatergic neurons (e.g., layer-specific fates in the cortex) are further determined by combinations of transcription factors, superimposed on general sequential programs. These new perspectives on neurogenesis add to the conceptual framework for strategies to engineer neural stem cells for the repair of specific brain circuits.
Assuntos
Diferenciação Celular/genética , Cerebelo/embriologia , Ácido Glutâmico/metabolismo , Hipocampo/metabolismo , Neocórtex/embriologia , Fatores de Transcrição/genética , Animais , Proliferação de Células , Cerebelo/citologia , Cerebelo/metabolismo , Evolução Molecular , Regulação da Expressão Gênica no Desenvolvimento/genética , Hipocampo/citologia , Humanos , Neocórtex/citologia , Neocórtex/metabolismo , Neurônios/citologia , Neurônios/metabolismoRESUMO
BACKGROUND: Linkage studies have provided evidence for a prostate cancer susceptibility locus on chromosome 17q. The mitochondrial protein prohibitin (PHB) is a plausible candidate gene based on its chromosomal location (17q21) and known function. METHODS: All coding regions and intron/exon junctions of the PHB gene were sequenced in 32 men from families participating in the University of Michigan Prostate Cancer Genetics Project that demonstrated evidence of linkage to 17q markers. RESULTS: Although a number of nucleotide variants were identified, no coding region substitutions were identified in any of the 32 men with prostate cancer from 32 unrelated multiplex prostate cancer families. CONCLUSIONS: PHB mutations do not appear to account for the linkage signal on 17q21-22 detected in PCGP families. Fine mapping of this region is in progress to refine the candidate region and highlight additional candidate prostate cancer susceptibility genes for sequence analysis.
Assuntos
Cromossomos Humanos Par 17 , Predisposição Genética para Doença , Neoplasia Prostática Intraepitelial/genética , Neoplasias da Próstata/genética , Proteínas Repressoras/genética , Adulto , Idoso , Mapeamento Cromossômico/métodos , Família , Ligação Genética , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Proibitinas , Próstata/metabolismo , Neoplasia Prostática Intraepitelial/metabolismo , Neoplasias da Próstata/metabolismoRESUMO
A major problem with the use of serum prostate-specific antigen (PSA) in predicting prostate cancer risk is the considerable variability of such measurements. Cramer et al. identified a set of single-nucleotide polymorphisms (SNPs) in the upstream regulatory region of the PSA gene that were each associated with increased promoter activity and serum PSA, further suggesting that genotyping these SNPs could be useful in improving the predictive value of PSA screening. In order to replicate this finding, DNA samples from 475 African-American men were genotyped for the same SNPs and no association was observed with either serum PSA level or prostate cancer diagnosis.
Assuntos
Polimorfismo de Nucleotídeo Único/genética , Regiões Promotoras Genéticas/genética , Antígeno Prostático Específico/sangue , Antígeno Prostático Específico/genética , Neoplasias da Próstata/sangue , Neoplasias da Próstata/genética , Adulto , Negro ou Afro-Americano/etnologia , Idoso , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/imunologia , Predisposição Genética para Doença , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Neoplasias da Próstata/etnologiaRESUMO
Intermediate progenitors (IPs) amplify the production of pyramidal neurons, but their role in selective genesis of cortical layers or neuronal subtypes remains unclear. Using genetic lineage tracing in mice, we find that IPs destined to produce upper cortical layers first appear early in corticogenesis, by embryonic day 11.5. During later corticogenesis, IP laminar fates are progressively limited to upper layers. We examined the role of Tbr2, an IP-specific transcription factor, in laminar fate regulation using Tbr2 conditional mutant mice. Upon Tbr2 inactivation, fewer neurons were produced by immediate differentiation and laminar fates were shifted upward. Genesis of subventricular mitoses was, however, not reduced in the context of a Tbr2-null cortex. Instead, neuronal and laminar differentiation were disrupted and delayed. Our findings indicate that upper-layer genesis depends on IPs from many stages of corticogenesis and that Tbr2 regulates the tempo of laminar fate implementation for all cortical layers.
Assuntos
Córtex Cerebral/citologia , Neurônios/citologia , Células-Tronco/citologia , Proteínas com Domínio T/metabolismo , Animais , Contagem de Células , Diferenciação Celular , Linhagem da Célula , Embrião de Mamíferos/citologia , Regulação da Expressão Gênica , Camundongos Knockout , Mitose , Atividade Motora , Neurogênese , Proteínas com Domínio T/deficiência , Fatores de Transcrição/metabolismoRESUMO
1. Earlier optimization of structure-activity relationships in a novel series of 4-(benzoylamino)-benzopyrans, led to the discovery of SB-204269 (trans-(+)-6-acetyl-4S-(4-fluorobenzoylamino)-3,4-dihydro-2, 2-dimethyl-2H-benzo[b]pyran-3R-ol, hemihydrate), a potent orally-active anticonvulsant in the mouse maximal electroshock seizure threshold (MEST) test. 2. Studies have now been undertaken to determine the effects of SB-204269 in a range of seizure models and tests of neurological deficits in rats. In addition, the compound has been evaluated in a series of in vitro mechanistic assays. 3. SB-204269 proved to be an orally-effective anticonvulsant agent, at doses (0.1-30 mg Kg-1) devoid of overt behavioural depressant properties, in models of both electrically (MEST and maximal electroshock (MEST)) and chemically (i.v. pentylenetetrazol (PTZ) infusion)-evoked tonic extension seizures. However, the compound did not inhibit PTZ-induced myoclonic seizures at doses up to 30 mg kg-1, p.o. 4. SB-204269 also selectively reduced focal electrographic seizure activity in an in vitro elevated K+ rat hippocampal slice model at concentrations (0.1-10 microM) that had no effect on normal synaptic activity and neuronal excitability. 5. In all of these seizure models, SB-204269 was equivalent or better than the clinically established antiepileptic drugs carbamazepine and lamotrigine, in terms of anticonvulsant potency and efficacy. 6. Unlike SB-204269, the corresponding trans 3S,4R enantiomer, SB-204268, did not produce marked anticonvulsant effects, an observation in accord with previous findings for other related pairs of trans enantiomers in the benzopyran series. 7. In the rat accelerating rotarod test, a sensitive paradigm for the detection of neurological deficits such as sedation and motor incoordination, SB-204269 was inactive even at doses as high as 200 mg kg-1, p.o. This was reflected in the excellent therapeutic index (minimum significantly effective dose in the rotarod test/ED50 in the MES test) for SB-204269 of > 31, as compared to equivalent values of only 7 and 13 for carbamazepine and lamotrigine, respectively. 8. At concentrations (> or = 10 microM) well above those required to produce anticonvulsant activity in vivo (i.e. 0.1 microM in brain), SB-204269 did not interact with many of the well known mechanistic targets for established antiepileptic drugs (e.g. Na+ channels or GABAergic neurotransmission). Subsequent studies have shown that the anticonvulsant properties of SB-204269 are likely to be mediated by a novel stereospecific binding site present in the CNS. 9. The overall efficacy profile in rodent seizure models, together with a minimal liability for inducing neurological impairment and an apparently unique mechanism of action, highlight the therapeutic potential of SB-204269 for the treatment of refractory partial and generalized tonic-clonic seizures.
Assuntos
Anticonvulsivantes/farmacologia , Benzamidas/farmacologia , Benzopiranos/farmacologia , Epilepsias Parciais/tratamento farmacológico , Epilepsia Generalizada/tratamento farmacológico , Animais , Relação Dose-Resposta a Droga , Feminino , Hipocampo/efeitos dos fármacos , Hipocampo/fisiologia , Técnicas In Vitro , Masculino , Ratos , Ratos Sprague-Dawley , EstereoisomerismoRESUMO
Previous studies have shown the effectiveness of an injectable bone substitute (IBS) composed of biphasic calcium phosphate in 2% hydroxypropyl methylcellulose gel (50/50 w/w). A therapeutic agent in the form of a drug can be added to the biomaterial by encapsulation into microparticles to protect the active agent, control its release and preserve the material rheological properties. Poly(epsilon-caprolactone) was used in this study because of its biocompatibility and resorbability, as tested in orthopaedic implants and surgical sutures. Particles (80-200 microm) were manufactured by a solvent evaporation-extraction process (1 g of polymer, 11-15 ml methylene chloride, with a stirring speed of 400-600 rpm) and introduced into the IBS in a 5-50% (V/V) range. Injectability was evaluated by texture analysis. With less than 45% of particles, the material had rheological properties similar to those of the reference IBS, whereas injectability decreased markedly with more than 45% of particles. A preliminary in vitro release study showed that this type of triphasic IBS could be efficient for drug delivery systems with osteoconduction properties.
Assuntos
Materiais Biocompatíveis , Substitutos Ósseos , Fosfatos de Cálcio , Poliésteres , Antibacterianos/administração & dosagem , Microscopia Eletrônica de Varredura , Tamanho da Partícula , Vancomicina/administração & dosagemRESUMO
Vancomycin encapsulation in biodegradable poly(epsilon-caprolactone) microparticles (200 microm mean diameter) was most efficient with a simple emulsion technique that dispersed 122.5 mg/g of polymer. Scanning electron micrographs showed smooth or pitted particles. Dissolution studies were correlated with microparticle morphology, indicating higher release with pitted particles when vancomycin was encapsulated in a dissolved state. The cytocompatibility of these poly(epsilon-caprolactone) microparticles was demonstrated by a direct contact cytotoxic assay. This material can be considered as an efficient drug delivery system for bone implantation.
Assuntos
Materiais Biocompatíveis/farmacologia , Caproatos/química , Caproatos/farmacologia , Sistemas de Liberação de Medicamentos , Lactonas/química , Lactonas/farmacologia , Vancomicina/farmacologia , Implantes Absorvíveis , Animais , Antibacterianos/administração & dosagem , Antibacterianos/farmacologia , Substitutos Ósseos , Linhagem Celular , Fibroblastos/metabolismo , Cinética , Camundongos , Microscopia Eletrônica de Varredura , Espectrofotometria , Fatores de Tempo , Vancomicina/administração & dosagemRESUMO
We have explored the neuroprotective efficacy of the cell penetrant caspase inhibitor, Ac-YVAD-cmk, in a hippocampal slice model of neuronal cell death induced by oxygen and glucose deprivation. Organotypic hippocampal slice cultures were prepared from 8 to 10-day-old rats and maintained for 10 to 12 days in vitro. Pre-treatment with Ac-YVAD-cmk prior to 45 min oxygen and glucose deprivation was neuroprotective as measured by propidium iodide uptake, with an EC(50) between 1 and 10 micromol/l. Ac-YVAD-cmk was also able to preserve synaptic function in the organotypic hippocampal slice cultures 24 h after oxygen and glucose deprivation. Ac-YVAD-cmk prevented the increase in histone-associated DNA fragmentation induced by oxygen and glucose deprivation. Interleukin-1beta did not reverse the protective effect of Ac-YVAD-cmk, and interleukin-1 receptor antagonist alone was not protective. These results show that caspase inhibitors are neuroprotective in a hippocampal slice culture system, using structural, biochemical and electrophysiological endpoints, and that this effect is not a result of inhibition of interleukin-1beta production.
Assuntos
Clorometilcetonas de Aminoácidos/farmacologia , Inibidores de Caspase , Morte Celular/efeitos dos fármacos , Inibidores de Cisteína Proteinase/farmacologia , Neurônios/citologia , Fármacos Neuroprotetores/farmacologia , Animais , Isquemia Encefálica/tratamento farmacológico , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular Transformada , Fragmentação do DNA/efeitos dos fármacos , Eletrofisiologia , Glucose/farmacologia , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Hipocampo/fisiologia , Camundongos , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Técnicas de Cultura de Órgãos , Oxigênio/farmacologia , Ratos , Acidente Vascular Cerebral/tratamento farmacológicoRESUMO
Cajal-Retzius cells are reelin-secreting neurons found in the marginal zone of the mammalian cortex during development. Recently, it has been proposed that Cajal-Retzius cells may be generated both early and late in corticogenesis, and may migrate into the cortex from proliferative zones in the subpallium (lateral ganglionic eminence and medial ganglionic eminence) or cortical hem. In the present study, we used reelin as a marker to study the properties of Cajal-Retzius cells, including their likely origins, neurotransmitters, and birthdates. In double labeling experiments, Cajal-Retzius cells (reelin(+)) expressed transcription factors characteristic of pallial neurons (Tbr1 and Emx2), contained high levels of glutamate, were usually calretinin(+), and were born early in corticogenesis, on embryonic days (E)10.5 and E11.5. Tbr1(+) cells in the marginal zone were almost always reelin(+). The first Cajal-Retzius cells (Tbr1(+)/reelin(+)) appeared in the preplate on E10.5. In contrast, interneurons expressed a subpallial transcription factor (Dlx), contained high levels of GABA, were frequently calbindin(+), and were born throughout corticogenesis (from E10.5 to E16.5). Interneurons (Dlx(+)) first appeared in the cortex on E12.5. Our results suggest that the marginal zone contains two main types of neurons: Cajal-Retzius cells derived from the pallium, and migrating interneurons derived from the subpallium.
Assuntos
Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Linhagem da Célula/fisiologia , Movimento Celular/fisiologia , Córtex Cerebral/embriologia , Neurônios/metabolismo , Células-Tronco/metabolismo , Animais , Animais Recém-Nascidos , Biomarcadores , Calbindina 2 , Calbindinas , Moléculas de Adesão Celular Neuronais/metabolismo , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Feminino , Feto , Ácido Glutâmico/metabolismo , Proteínas de Homeodomínio/metabolismo , Interneurônios/citologia , Interneurônios/metabolismo , Camundongos , Camundongos Endogâmicos , Proteínas do Tecido Nervoso , Neurônios/citologia , Neurotransmissores/metabolismo , Gravidez , Proteína Reelina , Proteína G de Ligação ao Cálcio S100/metabolismo , Serina Endopeptidases , Células-Tronco/citologia , Proteínas com Domínio T , Fatores de Transcrição/metabolismo , Ácido gama-Aminobutírico/metabolismoRESUMO
In order to study the tissue distribution of biodegradable nanoparticles after oral administration in animals, end-chain-radiolabeled poly(D,L-lactides) were prepared. Two groups of polymers (Mn = 7500, I = 2.4 and Mn = 28000, I = 1.4 as determined by organic size-exclusion chromatography) were chemically modified by reaction of [14C]acetic anhydride with hydroxyl end-chain groups. The activities of both resulting radioactive poly(D,L-lactides) varied from 57 to 1140 microCi/g. Poly(D,L-lactide) or poly(D,L-lactide-co-glycolide) nanoparticles containing various amounts of radioactive polymer were prepared according to the solvent evaporation process with acetone as cosolvent with methylene chloride in the organic phase. Their mean diameter was 133 +/- 25 nm, measured by photon correlation spectroscopy. The radiolabeled-end-group stability of these particles in buffer solutions was found to be greater when the matrix was made from the radiolabeled poly(D,L-lactide) having the highest molecular weight and the lowest polydispersity index. The polymer-chain stability was totally retained for at least 1 week in a phosphate buffer, pH 7.4, i.e. for the selected experiment time.
Assuntos
Poliésteres/química , Radioisótopos de Carbono , Portadores de Fármacos , Estabilidade de Medicamentos , Peso MolecularRESUMO
We report a case of cervicofacial necrotizing fasciitis that developed after blepharoplasty, an occurrence that, to our knowledge, has not previously been reported in the medical literature. A patient who presented to our institution 3 days after undergoing blepharoplasty of the upper eyelid was diagnosed as having fulminant fasciitis involving extensive areas of the face, scalp, and neck. We review the case in detail and discuss clinical and radiological clues to diagnosis, surgical and medical management, wound care, and subsequent scar contracture. This case emphasizes the need for individualized, appropriate postoperative care and for an awareness of this rare, potentially fatal complication. Early recognition and aggressive treatment of cervicofacial fasciitis can arrest its rapid progression and prevent devastating sequelae.