RESUMO
The amyloid cascade hypothesis proposes that excessive accumulation of amyloid beta-peptides is the initiating event in Alzheimer's disease. These neurotoxic peptides are generated from the amyloid precursor protein via sequential cleavage by ß- and γ-secretases in the 'amyloidogenic' proteolytic pathway. Alternatively, the amyloid precursor protein can be processed via the 'non-amyloidogenic' pathway which, through the action of the α-secretase a disintegrin and metalloproteinase (ADAM) 10, both precludes amyloid beta-peptide formation and has the additional benefit of generating a neuroprotective soluble amyloid precursor protein fragment, sAPPα. In the current study, we investigated whether the orphan drug, dichloroacetate, could alter amyloid precursor protein proteolysis. In SH-SY5Y neuroblastoma cells, dichloroacetate enhanced sAPPα generation whilst inhibiting ß-secretase processing of endogenous amyloid precursor protein and the subsequent generation of amyloid beta-peptides. Over-expression of the amyloid precursor protein partly ablated the effect of dichloroacetate on amyloidogenic and non-amyloidogenic processing whilst over-expression of the ß-secretase only ablated the effect on amyloidogenic processing. Similar enhancement of ADAM-mediated amyloid precursor protein processing by dichloroacetate was observed in unrelated cell lines and the effect was not exclusive to the amyloid precursor protein as an ADAM substrate, as indicated by dichloroacetate-enhanced proteolysis of the Notch ligand, Jagged1. Despite altering proteolysis of the amyloid precursor protein, dichloroacetate did not significantly affect the expression/activity of α-, ß- or γ-secretases. In conclusion, dichloroacetate can inhibit amyloidogenic and promote non-amyloidogenic proteolysis of the amyloid precursor protein. Given the small size and blood-brain-barrier permeability of the drug, further research into its mechanism of action with respect to APP proteolysis may lead to the development of therapies for slowing the progression of Alzheimer's disease.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Ácido Dicloroacético/farmacologia , Proteólise/efeitos dos fármacos , Proteínas ADAM/metabolismo , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Proteína Jagged-1/genética , Proteína Jagged-1/metabolismoRESUMO
Global deployment of an effective and safe vaccine is necessary to curtail the coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we evaluated a Newcastle disease virus (NDV)-based vectored-vaccine in mice and hamsters for its immunogenicity, safety, and protective efficacy against SARS-CoV-2. Intranasal administration of recombinant (r)NDV-S vaccine expressing spike (S) protein of SARS-CoV-2 to mice induced high levels of SARS-CoV-2-specific neutralizing immunoglobulin A (IgA) and IgG2a antibodies and T-cell-mediated immunity. Hamsters immunized with two doses of vaccine showed complete protection from lung infection, inflammation, and pathological lesions following SARS-CoV-2 challenge. Importantly, administration of two doses of intranasal rNDV-S vaccine significantly reduced the SARS-CoV-2 shedding in nasal turbinate and lungs in hamsters. Collectively, intranasal vaccination has the potential to control infection at the site of inoculation, which should prevent both clinical disease and virus transmission to halt the spread of the COVID-19 pandemic.
RESUMO
DrosophilaAcer (Angiotensin-converting enzyme-related) encodes a member of the angiotensin-converting enzyme (ACE) family of metallopeptidases that in mammals play roles in the endocrine regulation of blood homeostasis. ACE is also expressed in adipose tissue, where it is thought to play a role in metabolic regulation. Drosophila ACER is expressed in the adult fat body of the head and abdomen and is secreted into the haemolymph. Acer null mutants have previously been found to have reduced night-time sleep and greater sleep fragmentation. ACER may thus be part of a signalling system linking metabolism with sleep. To further understand the role of ACER in response to diet, we measured sleep and other nutrient-responsive phenotypes in Acer null flies under different dietary conditions. We show that loss of Acer disrupts the normal response of sleep to changes in nutrition. Other nutrient-sensitive phenotypes, including survival and glycogen storage, were also altered in the Acer mutant but lipid storage was not. Although the physiological substrate of the ACER peptidase has not been identified, an alteration of the normal nutrient-dependent control of Drosophila insulin-like peptide 5 protein in the Acer mutant suggests insulin/IGF-like signalling as a candidate pathway modulated by ACER in the nutrient-dependent control of sleep, survival and metabolism.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/fisiologia , Metaloendopeptidases/genética , Nutrientes/metabolismo , Sono , Fenômenos Fisiológicos da Nutrição Animal , Animais , Dieta , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Comportamento Alimentar , Feminino , Masculino , Metaloendopeptidases/metabolismoRESUMO
INTRODUCTION: Suppressor of cytokine signaling 3 (SOCS3) is a tumour suppressor, limiting intestinal epithelial cell (IEC) proliferation in acute inflammation, and tumour growth, but little is known regarding its role in mucosal homeostasis. Resistance to the intestinal helminth Trichuris muris relies on an "epithelial escalator" to expel the parasite. IEC turnover is restricted by parasite-induced indoleamine 2,3-dioxygenase (IDO). METHODS: Mice with or without conditional knockout of SOCS3 were infected with T. muris. Crypt depth, worm burden, and proliferating cells and IDO were quantified. SOCS3 knockdown was also performed in human IEC cell lines. RESULTS: Chronic T. muris infection increased expression of SOCS3 in wild-type mice. Lack of IEC SOCS3 led to a modest increase in epithelial turnover. This translated to a lower worm burden, but not complete elimination of the parasite suggesting a compensatory mechanism, possibly IDO, as seen in SOCS3 knockdown. CONCLUSIONS: We report that SOCS3 impacts on IEC turnover following T. muris infection, potentially through enhancement of IDO. IDO may dampen the immune response which can drive IEC hyperproliferation in the absence of SOCS3, demonstrating the intricate interplay of immune signals regulating mucosal homeostasis, and suggesting a novel tumour suppressor role of SOCS3.
Assuntos
Homeostase/imunologia , Imunidade nas Mucosas , Mucosa Intestinal/imunologia , Modelos Imunológicos , Proteína 3 Supressora da Sinalização de Citocinas/imunologia , Animais , Linhagem Celular , Homeostase/genética , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Indolamina-Pirrol 2,3,-Dioxigenase/imunologia , Inflamação/genética , Inflamação/imunologia , Inflamação/parasitologia , Inflamação/patologia , Mucosa Intestinal/parasitologia , Mucosa Intestinal/patologia , Camundongos , Camundongos Knockout , Proteína 3 Supressora da Sinalização de Citocinas/genética , Tricuríase/genética , Tricuríase/imunologia , Tricuríase/patologia , Trichuris/imunologiaRESUMO
We applied surface-enhanced Raman spectroscopy (SERS) to immunolabeled endothelial cells to derive enhanced spectra of the biomolecular makeup of the cellular surface. A two-step immunolabeling protocol with gold-conjugated antibodies coupled with silver enhancement to attach silver nanoparticles to the cell surface was employed. This approach generated â¼50-fold SERS enhancement of spectral signals. The SERS spectra exhibited several SERS-enhanced peaks associated with cell membrane components. The SERS detection of silver nanoparticles proved more far more sensitive than conventional light microscopy techniques. The SERS enhancement allowed us to carry out spectral mapping using wavenumbers associated with membrane components that correlated directly with the distribution of silver nanoparticles. SERS has the potential to detect immunolabeling at lower levels than is possible using conventional immunolabeling methods while simultaneously providing unique, spatially defined, biochemical information.
Assuntos
Membrana Celular/metabolismo , Epitélio Corneano/metabolismo , Mapeamento de Epitopos/métodos , Imuno-Histoquímica/métodos , Proteínas de Membrana/metabolismo , Análise Espectral Raman/métodos , Membrana Celular/ultraestrutura , Células Cultivadas , Epitélio Corneano/citologia , HumanosRESUMO
PURPOSE: Safe and prolonged drug delivery to the retina is a key obstacle to overcome in the development of new medicines aimed at treating progressive retinal disease. We took advantage of the ability of embryonic stem cells to survive long-term in foreign tissue and used these cells to deliver neuroprotectant molecules to the retina of the rhodopsin TgN S334ter-4 rat model of retinitis pigmentosa (RP). METHODS: Mouse embryonic stem (mES) cells, derived from the pluripotent embryonic stem cell line E14TG2a, were genetically engineered to oversecrete the glial cell-derived neurotrophic factor (GDNF). Cell suspensions, containing approximately 200,000 cells and expressing approximately 35ng/10(6) cells/24 h GDNF, were injected into the vitreous cavity of TgN S334ter rat eyes at postnatal day 21 (P21) without immunosuppression. Histological and immunofluorescence imaging was used to evaluate photoreceptor survival up to P90. Local (vitreous) and systemic (serum) concentrations of GDNF were determined and ocular side effects were monitored. RESULTS: Green fluorescent protein (GFP)-expressing mES cells were observed on the inner limiting membrane of the retina in retinal flatmounts up to P90. In cryostat sections at P45, some GFP-expressing cells had integrated into the inner retina, but did not migrate into the outer nuclear layer. After an initial lag period, the photoreceptor cell counts were significantly higher (p< or =0.05) in animals treated with GDNF-secreting mES cells than in untreated animals, principally in the peripheral retina. Several adverse side effects such as tractional detachments and areas of hyperplasia were seen in a minimal number of treated eyes. Abnormally high levels of GDNF in the peripheral circulation were also observed. CONCLUSIONS: ES cells engineered to secrete GDNF exerted a neuroprotective effect for at least three months on retinal structure in the TgN S334ter rat model of retinal degeneration. Immunosuppression was not required for this. Several adverse effects were identified which require further investigation to make cell-based delivery of neuroprotection a viable clinical strategy.
Assuntos
Células-Tronco Embrionárias/metabolismo , Terapia Genética/métodos , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Retina/metabolismo , Degeneração Retiniana/terapia , Animais , Diferenciação Celular , Sobrevivência Celular , Modelos Animais de Doenças , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/transplante , Fator Neurotrófico Derivado de Linhagem de Célula Glial/análise , Fator Neurotrófico Derivado de Linhagem de Célula Glial/sangue , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Histocitoquímica , Injeções , Camundongos , Fármacos Neuroprotetores/análise , Fármacos Neuroprotetores/sangue , Ratos , Retina/ultraestrutura , Degeneração Retiniana/patologia , Retinose Pigmentar/metabolismo , Retinose Pigmentar/patologia , Retinose Pigmentar/terapia , Estatísticas não Paramétricas , Transplante de Células-Tronco , Transfecção , Corpo Vítreo/químicaRESUMO
We ascertained three different families affected with oto-dental syndrome, a rare but severe autosomal-dominant craniofacial anomaly. All affected patients had the unique phenotype of grossly enlarged molar teeth (globodontia) segregating with a high-frequency sensorineural hearing loss. In addition, ocular coloboma segregated with disease in one family (oculo-oto-dental syndrome). A genome-wide scan was performed using the Affymetrix GeneChip10K 2.0 Array. Parametric linkage analysis gave a single LOD score peak of 3.9 identifying linkage to chromosome 11q13. Haplotype analysis revealed three obligatory recombination events defining a 4.8 Mb linked interval between D11S1889 and SNP rs2077955. Higher resolution mapping and Southern blot analysis in each family identified overlapping hemizygous microdeletions. SNP expression analysis and real-time quantitative RT-PCR in patient lymphoblast cell lines excluded a positional effect on the flanking genes ORAOV1, PPFIA1 and CTTN. The smallest 43 kb deletion resulted in the loss of only one gene, FGF3, which was also deleted in all other otodental families. These data suggest that FGF3 haploinsufficiency is likely to be the cause of otodental syndrome. In addition, the Fas-associated death domain (FADD) gene was also deleted in the one family segregating ocular coloboma. Spatiotemporal in situ hybridization in zebrafish embryos established for the first time that fadd is expressed during eye development. We therefore propose that FADD haploinsufficiency is likely to be responsible for ocular coloboma in this family. This study therefore implicates FGF3 and FADD in human craniofacial disease.
Assuntos
Mapeamento Cromossômico , Cromossomos Humanos Par 11 , Coloboma/genética , Proteína de Domínio de Morte Associada a Fas/genética , Fator 3 de Crescimento de Fibroblastos/genética , Doenças do Labirinto/genética , Polimorfismo de Nucleotídeo Único , Doenças Estomatognáticas/congênito , Doenças Estomatognáticas/genética , Animais , Anormalidades Craniofaciais/genética , Análise Mutacional de DNA , Embrião não Mamífero , Olho/embriologia , Feminino , Deleção de Genes , Ligação Genética , Humanos , Doenças do Labirinto/congênito , Perda de Heterozigosidade , Masculino , Organogênese/genética , Linhagem , Síndrome , Peixe-Zebra/embriologiaRESUMO
OBJECTIVES: Previous studies have identified loss of chromosomal regions 7p12-q11.2 and 8p12-p21 in choriocarcinoma suggesting that suppressor genes involved in tumour development may be located within these regions. Our objectives were to refine the regions of loss and evaluate these deletions as prognostic indicators of trophoblastic tumour development following molar pregnancy. METHODS: Fluorescent microsatellite genotyping was used to perform deletion mapping in a series of thirty-nine gestational trophoblastic tumours (GTT) including both choriocarcinoma and placental site trophoblastic tumours. RESULTS: Significant loss of heterozygosity (LOH) was found for both regions in GTT that originated in non-molar pregnancies. Although no common interval of loss was found in those GTT with LOH for the 7q11.2 region, for the 8p12-p21 locus, markers D8S1731 and NEFL defined a minimal region of loss in all tumours showing LOH. However, complete LOH of either region occurred in only a minority of tumours (20%; chromosome 7: 24%; chromosome 8) suggesting that loss of neither region is likely to be a primary event in the development of GTT. This was further supported by the observation that no deletions were found in either region for the fourteen GTT that followed complete molar pregnancies. CONCLUSIONS: While we have defined a minimal interval in 8p12-p21 in which tumour suppressor genes involved in GTT are likely to be located, the data suggest that deletions in 7q11.2 or 8p12-p21 are unlikely to be useful prognostic indicators in the management of patients with molar pregnancies.
Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 7/genética , Cromossomos Humanos Par 8/genética , Doença Trofoblástica Gestacional/genética , Mola Hidatiforme/genética , Neoplasias Uterinas/genética , Adulto , Coriocarcinoma/genética , Coriocarcinoma/patologia , Mapeamento Cromossômico , Feminino , Doença Trofoblástica Gestacional/patologia , Humanos , Mola Hidatiforme/patologia , Pessoa de Meia-Idade , Gravidez , Neoplasias Uterinas/patologiaRESUMO
Identification of the genes underlying complex phenotypes and the definition of the evolutionary forces that have shaped eukaryotic genomes are among the current challenges in molecular genetics. Variation in gene copy number is increasingly recognized as a source of inter-individual differences in genome sequence and has been proposed as a driving force for genome evolution and phenotypic variation. Here we show that copy number variation of the orthologous rat and human Fcgr3 genes is a determinant of susceptibility to immunologically mediated glomerulonephritis. Positional cloning identified loss of the newly described, rat-specific Fcgr3 paralogue, Fcgr3-related sequence (Fcgr3-rs), as a determinant of macrophage overactivity and glomerulonephritis in Wistar Kyoto rats. In humans, low copy number of FCGR3B, an orthologue of rat Fcgr3, was associated with glomerulonephritis in the autoimmune disease systemic lupus erythematosus. The finding that gene copy number polymorphism predisposes to immunologically mediated renal disease in two mammalian species provides direct evidence for the importance of genome plasticity in the evolution of genetically complex phenotypes, including susceptibility to common human disease.
Assuntos
Antígenos CD/genética , Dosagem de Genes/genética , Predisposição Genética para Doença/genética , Nefrite Lúpica/genética , Polimorfismo Genético/genética , Receptores de IgG/genética , Animais , Sequência de Bases , Éxons/genética , Proteínas Ligadas por GPI , Duplicação Gênica , Haplótipos , Humanos , Nefrite Lúpica/imunologia , Nefrite Lúpica/patologia , Dados de Sequência Molecular , Ratos , Ratos Endogâmicos WKY , Deleção de Sequência/genéticaRESUMO
This article reviews published data on familial recurrent hydatidiform mole with particular reference to the genetic basis of this condition, the likely outcome of subsequent pregnancies in affected women and the risk of persistent trophoblastic disease following molar pregnancies in these families. Familial recurrent hydatidiform mole is characterized by recurrent complete hydatidiform moles of biparental, rather than the more usual androgenetic, origin. Although the specific gene defect in these families has not been identified, genetic mapping has shown that in most families the gene responsible is located in a 1.1 Mb region on chromosome 19q13.4. Mutations in this gene result in dysregulation of imprinting in the female germ line with abnormal development of both embryonic and extraembryonic tissue. Subsequent pregnancies in women diagnosed with this condition are likely to be complete hydatidiform moles. In 152 pregnancies in affected women, 113 (74%) were complete hydatidiform moles, 26 (17%) were miscarriages, 6 (4%) were partial hydatidiform moles, and 7 (5%) were normal pregnancies. Molar pregnancies in women with familial recurrent hydatidiform mole have a risk of progressing to persistent trophoblastic disease similar to that of androgenetic complete hydatidiform mole.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Predisposição Genética para Doença , Mola Hidatiforme/genética , Mola Hidatiforme/patologia , Neoplasias Uterinas/genética , Neoplasias Uterinas/patologia , Aborto Espontâneo , Adulto , Cromossomos Humanos Par 19 , Análise Mutacional de DNA , Desenvolvimento Embrionário , Feminino , Humanos , Linhagem , Gravidez , Resultado da Gravidez , Prognóstico , RecidivaRESUMO
Hydatidiform mole (HM) is an abnormal gestation characterized by trophoblast hyperplasia and overgrowth of placental villi. The genetic basis in the vast majority of cases is an excess of paternal to maternal genomes, suggesting that global misexpression of imprinted genes is the common molecular mechanism underlying the genesis of this condition. Although most complete HM are androgenetic in origin, a rare, frequently familial, biparental variant has been described. Here we evaluate the expression of p57(KIP2), the product of CDKN1C, an imprinted, maternally expressed gene in a series of these rare, biparental complete HM (BiCHM). We observed dramatic underexpression of p57(KIP2) in BiCHM, identical to that seen in complete HM of androgenetic origin (AnCHM). The series included two sisters, both of whom had BiCHM. Genotyping of this family identified a 15 cM region of homozygosity for 19q13.3-13.4 similar to that found in three other families with recurrent BiCHM. These results demonstrate that BiCHM, like AnCHM, result from abnormal expression of imprinted genes. In addition we provide further evidence for a major control gene on 19q13.3-13.4 which regulates expression of imprinted genes on other chromosomes.
Assuntos
Impressão Genômica , Mola Hidatiforme/genética , Proteínas Nucleares/genética , Neoplasias Uterinas/genética , Cromossomos Humanos Par 19 , Inibidor de Quinase Dependente de Ciclina p57 , Feminino , Humanos , Mola Hidatiforme/metabolismo , Imuno-Histoquímica , Masculino , Proteínas Nucleares/metabolismo , Linhagem , Gravidez , Neoplasias Uterinas/metabolismoRESUMO
We have previously shown that there is a temporal difference in human CRX: gene expression compared with that of mouse Crx. We have now characterized these genes at the genomic and transcriptional levels and here we expand on this earlier report. Human CRX: spans 25 kb and has six exons, and mouse CRX: spans 15 kb and has four exons. We isolated seven human and two mouse mRNAs generated by alternative splicing of a variable 5' untranslated region. The human and mouse genes share an evolutionarily conserved promoter, which contains OTX/CRX type and SP1/AP2 binding sites and drives expression of two conserved transcripts in both species. Additionally, the human gene has a second human-specific promoter, which has OTX/CRX type binding sites and drives expression of five other transcripts. Band shift assays have shown that six of the seven candidate OTX/CRX elements bind CRX in vitro, possibly implying that the gene can regulate its own expression. These data may account for the differences in temporal expression IN VIVO: we have previously reported between these two species.