Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 12 de 12
Filtrar
1.
J Virol ; 95(3)2021 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-33208443

RESUMO

Leishmania parasites cause a variety of symptoms, including mucocutaneous leishmaniasis, which results in the destruction of the mucous membranes of the nose, mouth, and throat. The species of Leishmania carrying Leishmania RNA virus 1 (LRV1), from the family Totiviridae, are more likely to cause severe disease and are less sensitive to treatment than those that do not contain the virus. Although the importance of LRV1 for the severity of leishmaniasis was discovered a long time ago, the structure of the virus remained unknown. Here, we present a cryo-electron microscopy reconstruction of the virus-like particle of LRV1 determined to a resolution of 3.65 Å. The capsid has icosahedral symmetry and is formed by 120 copies of a capsid protein assembled in asymmetric dimers. RNA genomes of viruses from the family Totiviridae are synthetized, but not capped at the 5' end, by virus RNA polymerases. To protect viral RNAs from degradation, capsid proteins of the L-A totivirus cleave the 5' caps of host mRNAs, creating decoys to overload the cellular RNA quality control system. Capsid proteins of LRV1 form positively charged clefts, which may be the cleavage sites for the 5' cap of Leishmania mRNAs. The putative RNA binding site of LRV1 is distinct from that of the related L-A virus. The structure of the LRV1 capsid enables the rational design of compounds targeting the putative decapping site. Such inhibitors may be developed into a treatment for mucocutaneous leishmaniasis caused by LRV1-positive species of LeishmaniaIMPORTANCE Twelve million people worldwide suffer from leishmaniasis, resulting in more than 30 thousand deaths annually. The disease has several variants that differ in their symptoms. The mucocutaneous form, which leads to disintegration of the nasal septum, lips, and palate, is caused predominantly by Leishmania parasites carrying Leishmania RNA virus 1 (LRV1). Here, we present the structure of the LRV1 capsid determined using cryo-electron microscopy. Capsid proteins of a related totivirus, L-A virus, protect viral RNAs from degradation by cleaving the 5' caps of host mRNAs. Capsid proteins of LRV1 may have the same function. We show that the LRV1 capsid contains positively charged clefts that may be sites for the cleavage of mRNAs of Leishmania cells. The structure of the LRV1 capsid enables the rational design of compounds targeting the putative mRNA cleavage site. Such inhibitors may be used as treatments for mucocutaneous leishmaniasis.


Assuntos
Proteínas do Capsídeo/química , Capsídeo/química , Leishmaniavirus/química , RNA Viral/metabolismo , Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Microscopia Crioeletrônica , Genoma Viral , Leishmaniavirus/genética , Leishmaniavirus/metabolismo , RNA Viral/genética
2.
Proc Natl Acad Sci U S A ; 114(9): E1641-E1650, 2017 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-28196895

RESUMO

The formation of mineralized tissues is governed by extracellular matrix proteins that assemble into a 3D organic matrix directing the deposition of hydroxyapatite. Although the formation of bones and dentin depends on the self-assembly of type I collagen via the Gly-X-Y motif, the molecular mechanism by which enamel matrix proteins (EMPs) assemble into the organic matrix remains poorly understood. Here we identified a Y/F-x-x-Y/L/F-x-Y/F motif, evolutionarily conserved from the first tetrapods to man, that is crucial for higher order structure self-assembly of the key intrinsically disordered EMPs, ameloblastin and amelogenin. Using targeted mutations in mice and high-resolution imaging, we show that impairment of ameloblastin self-assembly causes disorganization of the enamel organic matrix and yields enamel with disordered hydroxyapatite crystallites. These findings define a paradigm for the molecular mechanism by which the EMPs self-assemble into supramolecular structures and demonstrate that this process is crucial for organization of the organic matrix and formation of properly structured enamel.


Assuntos
Motivos de Aminoácidos/fisiologia , Esmalte Dentário/metabolismo , Proteínas Intrinsicamente Desordenadas/metabolismo , Amelogenina/metabolismo , Sequência de Aminoácidos , Animais , Evolução Biológica , Proteínas do Esmalte Dentário/metabolismo , Durapatita/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Masculino , Camundongos , Ligação Proteica/fisiologia
3.
Hum Mol Genet ; 24(15): 4340-52, 2015 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-25954033

RESUMO

Cleft lip and/or palate (CL/P) are common structural birth defects in humans. We used exome sequencing to study a patient with bilateral CL/P and identified a single nucleotide deletion in the patient and her similarly affected son­c.546_546delG, predicting p.Gln183Argfs*57 in the Distal-less 4 (DLX4) gene. The sequence variant was absent from databases, predicted to be deleterious and was verified by Sanger sequencing. In mammals, there are three Dlx homeobox clusters with closely located gene pairs (Dlx1/Dlx2, Dlx3/Dlx4, Dlx5/Dlx6). In situ hybridization showed that Dlx4 was expressed in the mesenchyme of the murine palatal shelves at E12.5, prior to palate closure. Wild-type human DLX4, but not mutant DLX4_c.546delG, could activate two murine Dlx conserved regulatory elements, implying that the mutation caused haploinsufficiency. We showed that reduced DLX4 expression after short interfering RNA treatment in a human cell line resulted in significant up-regulation of DLX3, DLX5 and DLX6, with reduced expression of DLX2 and significant up-regulation of BMP4, although the increased BMP4 expression was demonstrated only in HeLa cells. We used antisense morpholino oligonucleotides to target the orthologous Danio rerio gene, dlx4b, and found reduced cranial size and abnormal cartilaginous elements. We sequenced DLX4 in 155 patients with non-syndromic CL/P and CP, but observed no sequence variants. From the published literature, Dlx1/Dlx2 double homozygous null mice and Dlx5 homozygous null mice both have clefts of the secondary palate. This first finding of a DLX4 mutation in a family with CL/P establishes DLX4 as a potential cause of human clefts.


Assuntos
Encéfalo/anormalidades , Fenda Labial/genética , Fissura Palatina/genética , Proteínas de Homeodomínio/genética , Anormalidades Maxilomandibulares/genética , Fatores de Transcrição/genética , Proteínas de Peixe-Zebra/genética , Animais , Proteína Morfogenética Óssea 4/genética , Encéfalo/patologia , Fenda Labial/patologia , Fissura Palatina/patologia , Exoma/genética , Regulação da Expressão Gênica no Desenvolvimento , Células HeLa , Sequenciamento de Nucleotídeos em Larga Escala , Proteínas de Homeodomínio/biossíntese , Humanos , Anormalidades Maxilomandibulares/patologia , Mesoderma/metabolismo , Camundongos , Camundongos Knockout , Morfolinos , Fatores de Transcrição/biossíntese , Peixe-Zebra
4.
Front Endocrinol (Lausanne) ; 15: 1286365, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39129916

RESUMO

Introduction: Fibroblast growth factor 20 (Fgf20), a member of the Fgf9 subfamily, was identified as an important regulator of bone differentiation and homeostasis processes. However, the role of Fgf20 in bone physiology has not been approached yet. Here we present a comprehensive bone phenotype analysis of mice with functional ablation of Fgf20. Methods: The study conducts an extensive analysis of Fgf20 knockout mice compared to controls, incorporating microCT scanning, volumetric analysis, Fgf9 subfamily expression and stimulation experiment and histological evaluation. Results: The bone phenotype could be detected especially in the area of​ the lumbar and caudal part of the spine and in fingers. Regarding the spine, Fgf20-/- mice exhibited adhesions of the transverse process of the sixth lumbar vertebra to the pelvis as well as malformations in the distal part of their tails. Preaxial polydactyly and polysyndactyly in varying degrees of severity were also detected. High resolution microCT analysis of distal femurs and the fourth lumbar vertebra showed significant differences in structure and mineralization in both cortical and trabecular bone. These findings were histologically validated and may be associated with the expression of Fgf20 in chondrocytes and their progenitors. Moreover, histological sections demonstrated increased bone tissue formation, disruption of Fgf20-/- femur cartilage, and cellular-level alterations, particularly in osteoclasts. We also observed molar dysmorphology, including root taurodontism, and described variations in mineralization and dentin thickness. Discussion: Our analysis provides evidence that Fgf20, together with other members of the Fgf9 subfamily, plays a crucial regulatory role in skeletal development and bone homeostasis.


Assuntos
Fatores de Crescimento de Fibroblastos , Camundongos Knockout , Animais , Camundongos , Fatores de Crescimento de Fibroblastos/metabolismo , Fatores de Crescimento de Fibroblastos/genética , Microtomografia por Raio-X , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/anormalidades , Calcificação Fisiológica , Masculino , Osteogênese , Feminino , Camundongos Endogâmicos C57BL , Fenótipo
5.
Mol Pain ; 9: 24, 2013 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-23668392

RESUMO

BACKGROUND: Odontoblasts are specialized cells that form dentin and they are believed to be sensors for tooth pain. Transforming growth factor-ß1 (TGF-ß1), a pro-inflammatory cytokine expressed early in odontoblasts, plays an important role in the immune response during tooth inflammation and infection. TGF-ß1 is also known to participate in pain signaling by regulating cyclin-dependent kinase 5 (Cdk5) in nociceptive neurons of the trigeminal and dorsal root ganglia. However, the precise role of TGF-ß1 in tooth pain signaling is not well characterized. The aim of our present study was to determine whether or not in odontoblasts Cdk5 is functionally active, if it is regulated by TGF-ß1, and if it affects the downstream pain receptor, transient receptor potential vanilloid-1 (TRPV1). RESULTS: We first determined that Cdk5 and p35 are indeed expressed in an odontoblast-enriched primary preparation from murine teeth. For the subsequent analysis, we used an odontoblast-like cell line (MDPC-23) and found that Cdk5 is functionally active in these cells and its kinase activity is upregulated during cell differentiation. We found that TGF-ß1 treatment potentiated Cdk5 kinase activity in undifferentiated MDPC-23 cells. SB431542, a specific inhibitor of TGF-ß1 receptor 1 (Tgfbr1), when co-administered with TGF-ß1, blocked the induction of Cdk5 activity. TGF-ß1 treatment also activated the ERK1/2 signaling pathway, causing an increase in early growth response-1 (Egr-1), a transcription factor that induces p35 expression. In MDPC-23 cells transfected with TRPV1, Cdk5-mediated phosphorylation of TRPV1 at threonine-407 was significantly increased after TGF-ß1 treatment. In contrast, SB431542 co-treatment blocked TRPV1 phosphorylation. Moreover, TGF-ß1 treatment enhanced both proton- and capsaicin-induced Ca²âº influx in TRPV1-expressing MDPC-23 cells, while co-treatment with either SB431542 or roscovitine blocked this effect. CONCLUSIONS: Cdk5 and p35 are expressed in a murine odontoblast-enriched primary preparation of cells from teeth. Cdk5 is also functionally active in odontoblast-like MDPC-23 cells. TGF-ß1 sensitizes TRPV1 through Cdk5 signaling in MDPC-23 cells, suggesting the direct involvement of odontoblasts and Cdk5 in dental nociceptive pain transduction.


Assuntos
Quinase 5 Dependente de Ciclina/metabolismo , Odontoblastos/metabolismo , Transdução de Sinais , Canais de Cátion TRPV/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Animais , Linhagem Celular , Quinase 5 Dependente de Ciclina/genética , Camundongos , Nociceptores/metabolismo , Dor/metabolismo , Fosforilação , Fosfotransferases/metabolismo , Canais de Cátion TRPV/genética
6.
Sci Rep ; 13(1): 1471, 2023 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-36702824

RESUMO

Highly specialized enamel matrix proteins (EMPs) are predominantly expressed in odontogenic tissues and diverged from common ancestral gene. They are crucial for the maturation of enamel and its extreme complexity in multiple independent lineages. However, divergence of EMPs occured already before the true enamel evolved and their conservancy in toothless species suggests that non-canonical functions are still under natural selection. To elucidate this hypothesis, we carried out an unbiased, comprehensive phenotyping and employed data from the International Mouse Phenotyping Consortium to show functional pleiotropy of amelogenin, ameloblastin, amelotin, and enamelin, genes, i.e. in sensory function, skeletal morphology, cardiovascular function, metabolism, immune system screen, behavior, reproduction, and respiratory function. Mice in all KO mutant lines, i.e. amelogenin KO, ameloblastin KO, amelotin KO, and enamelin KO, as well as mice from the lineage with monomeric form of ameloblastin were affected in multiple physiological systems. Evolutionary conserved motifs and functional pleiotropy support the hypothesis of role of EMPs as general physiological regulators. These findings illustrate how their non-canonical function can still effect the fitness of modern species by an example of influence of amelogenin and ameloblastin on the bone physiology.


Assuntos
Proteínas do Esmalte Dentário , Animais , Camundongos , Amelogenina/metabolismo , Proteínas do Esmalte Dentário/genética
7.
Virology ; 577: 149-154, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36371873

RESUMO

The presence of Leishmania RNA virus 1 (LRV1) enables Leishmania protozoan parasites to cause more severe disease than the virus-free strains. The structure of LRV1 virus-like particles has been determined previously, however, the structure of the LRV1 virion has not been characterized. Here we used cryo-electron microscopy and single-particle reconstruction to determine the structures of the LRV1 virion and empty particle isolated from Leishmania guyanensis to resolutions of 4.0 Å and 3.6 Å, respectively. The capsid of LRV1 is built from sixty dimers of capsid proteins organized with icosahedral symmetry. RNA genomes of totiviruses are replicated inside the virions by RNA polymerases expressed as C-terminal extensions of a sub-population of capsid proteins. Most of the virions probably contain one or two copies of the RNA polymerase, however, the location of the polymerase domains in LRV1 capsid could not be identified, indicating that it varies among particles. Importance. Every year over 200 000 people contract leishmaniasis and more than five hundred people die of the disease. The mucocutaneous form of leishmaniasis produces lesions that can destroy the mucous membranes of the nose, mouth, and throat. Leishmania parasites carrying Leishmania RNA virus 1 (LRV1) are predisposed to cause aggravated symptoms in the mucocutaneous form of leishmaniasis. Here, we present the structure of the LRV1 virion determined using cryo-electron microscopy.

8.
Front Genet ; 9: 542, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30505318

RESUMO

Members of the fibroblast growth factor (FGF) family have myriad functions during development of both non-vertebrate and vertebrate organisms. One of these family members, FGF10, is largely expressed in mesenchymal tissues and is essential for postnatal life because of its critical role in development of the craniofacial complex, as well as in lung branching. Here, we review the function of FGF10 in morphogenesis of craniofacial organs. Genetic mouse models have demonstrated that the dysregulation or absence of FGF10 function affects the process of palate closure, and FGF10 is also required for development of salivary and lacrimal glands, the inner ear, eye lids, tongue taste papillae, teeth, and skull bones. Importantly, mutations within the FGF10 locus have been described in connection with craniofacial malformations in humans. A detailed understanding of craniofacial defects caused by dysregulation of FGF10 and the precise mechanisms that underlie them offers new opportunities for development of medical treatments for patients with birth defects and for regenerative approaches for cancer patients with damaged gland tissues.

9.
J Bone Miner Res ; 32(11): 2219-2231, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28650075

RESUMO

Rodents are characterized by continuously renewing incisors whose growth is fueled by epithelial and mesenchymal stem cells housed in the proximal compartments of the tooth. The epithelial stem cells reside in structures known as the labial (toward the lip) and lingual (toward the tongue) cervical loops (laCL and liCL, respectively). An important feature of the rodent incisor is that enamel, the outer, highly mineralized layer, is asymmetrically distributed, because it is normally generated by the laCL but not the liCL. Here, we show that epithelial-specific deletion of the transcription factor Islet1 (Isl1) is sufficient to drive formation of ectopic enamel by the liCL stem cells, and also that it leads to production of altered enamel on the labial surface. Molecular analyses of developing and adult incisors revealed that epithelial deletion of Isl1 affected multiple, major pathways: Bmp (bone morphogenetic protein), Hh (hedgehog), Fgf (fibroblast growth factor), and Notch signaling were upregulated and associated with liCL-generated ectopic enamel; on the labial side, upregulation of Bmp and Fgf signaling, and downregulation of Shh were associated with premature enamel formation. Transcriptome profiling studies identified a suite of differentially regulated genes in developing Isl1 mutant incisors. Our studies demonstrate that ISL1 plays a central role in proper patterning of stem cell-derived enamel in the incisor and indicate that this factor is an important upstream regulator of signaling pathways during tooth development and renewal. © 2017 American Society for Bone and Mineral Research.


Assuntos
Padronização Corporal , Calcificação Fisiológica , Esmalte Dentário/embriologia , Esmalte Dentário/metabolismo , Incisivo/embriologia , Incisivo/metabolismo , Proteínas com Homeodomínio LIM/metabolismo , Fatores de Transcrição/metabolismo , Animais , Epitélio/embriologia , Epitélio/metabolismo , Feminino , Deleção de Genes , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Proteínas com Homeodomínio LIM/genética , Mandíbula/metabolismo , Camundongos , Mutação/genética , Especificidade de Órgãos , Análise de Sequência de RNA , Transdução de Sinais , Fatores de Transcrição/genética
10.
J Bone Miner Res ; 31(1): 152-62, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26179131

RESUMO

The continuously growing rodent incisor is an emerging model for the study of renewal of mineralized tissues by adult stem cells. Although the Bmp, Fgf, Shh, and Wnt pathways have been studied in this organ previously, relatively little is known about the role of Notch signaling during incisor renewal. Notch signaling components are expressed in enamel-forming ameloblasts and the underlying stratum intermedium (SI), which suggested distinct roles in incisor renewal and enamel mineralization. Here, we injected adult mice with inhibitory antibodies against several components of the Notch pathway. This blockade led to defects in the interaction between ameloblasts and the SI cells, which ultimately affected enamel formation. Furthermore, Notch signaling inhibition led to the downregulation of desmosome-specific proteins such as PERP and desmoplakin, consistent with the importance of desmosomes in the integrity of ameloblast-SI attachment and enamel formation. Together, our data demonstrate that Notch signaling is critical for proper enamel formation during incisor renewal, in part by regulating desmosome-specific components, and that the mouse incisor provides a model system to dissect Jag-Notch signaling mechanisms in the context of mineralized tissue renewal.


Assuntos
Ameloblastos/metabolismo , Esmalte Dentário/metabolismo , Incisivo/metabolismo , Receptores Notch , Transdução de Sinais , Ameloblastos/patologia , Animais , Esmalte Dentário/patologia , Desmossomos/metabolismo , Desmossomos/patologia , Incisivo/patologia , Camundongos , Doenças Dentárias
11.
Dev Cell ; 35(6): 713-24, 2015 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-26702830

RESUMO

The proper positioning of organs during development is essential, yet little is known about the regulation of this process in mammals. Using murine tooth development as a model, we have found that cell migration plays a central role in positioning of the organ primordium. By combining lineage tracing, genetic cell ablation, and confocal live imaging, we identified a migratory population of Fgf8-expressing epithelial cells in the embryonic mandible. These Fgf8-expressing progenitors furnish the epithelial cells required for tooth development, and the progenitor population migrates toward a Shh-expressing region in the mandible, where the tooth placode will initiate. Inhibition of Fgf and Shh signaling disrupted the oriented migration of cells, leading to a failure of tooth development. These results demonstrate the importance of intraepithelial cell migration in proper positioning of an initiating organ.


Assuntos
Movimento Celular/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Dente Molar/embriologia , Morfogênese/fisiologia , Dente/citologia , Animais , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Mesoderma/citologia , Mesoderma/metabolismo , Camundongos , Dente Molar/citologia , Dente Molar/metabolismo , Odontogênese/fisiologia , Dente/embriologia
12.
Int J Oral Sci ; 7(1): 23-6, 2015 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-25634121

RESUMO

Continuously growing incisors are common to all rodents, which include the Microtus genus of voles. However, unlike many rodents, voles also possess continuously growing molars. Here, we report spontaneous molar defects in a population of Prairie voles (Microtus ochrogaster). We identified bilateral protuberances on the ventral surface of the mandible in several voles in our colony. In some cases, the protuberances broke through the cortical bone. The mandibular molars became exposed and infected, and the maxillary molars entered the cranial vault. Visualisation upon soft tissue removal and microcomputed tomography (microCT) analyses confirmed that the protuberances were caused by the overgrowth of the apical ends of the molar teeth. We speculate that the unrestricted growth of the molars was due to the misregulation of the molar dental stem cell niche. Further study of this molar phenotype may yield additional insight into stem cell regulation and the evolution and development of continuously growing teeth.


Assuntos
Arvicolinae/anatomia & histologia , Dente Molar/crescimento & desenvolvimento , Animais , Arvicolinae/genética , Feminino , Humanos , Masculino , Dente Molar/diagnóstico por imagem , Linhagem , Microtomografia por Raio-X
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA