RESUMO
BACKGROUNDS: To characterize the acute phase clinical manifestations and visual outcomes of the patients with Vogt-Koyanagi Harada (VKH) disease in southern China. METHODS: In total, 186 patients with acute-onset VKH disease were recruited. The demographic data, clinical signs, ophthalmic examinations, and visual outcomes were analyzed. RESULTS: Among the 186 VKH patients, 3 were diagnosed as complete VKH, 125 as incomplete VKH, and 58 as probable VKH. All patients visited the hospital within 3 months of onset and complained of decreased vision. For the extraocular manifestations, 121 patients (65%) referred neurological symptoms. Anterior chamber activity was negative in most eyes within an onset of 7 days, which increased slightly with onset beyond 1 week. Exudative retinal detachment (366 eyes, 98%) and optic disc hyperaemia (314 eyes, 84%) were commonly observed at presentation. A typical ancillary examination helped with the diagnosis of VKH. Systemic corticosteroid therapy was prescribed. The logMAR best-corrected visual acuity improved significantly from 0.74 ± 0.54 at baseline to 0.12 ± 0.24 at the 1-year follow-up visit. The recurrence rate was 18% in the follow-up visits. Erythrocyte sedimentation rate and C-reactive protein were significantly correlated to VKH recurrences. CONCLUSION: Posterior uveitis, followed by mild anterior uveitis, is the typical initial manifestation in the acute phase of Chinese VKH patients. Visual outcome improvement is promising in most patients receiving systemic corticosteroid therapy in the acute phase. Detection of the clinical features at the initial onset of VKH could facilitate early treatment and better vision improvement.
Assuntos
Descolamento Retiniano , Uveíte Posterior , Síndrome Uveomeningoencefálica , Humanos , Síndrome Uveomeningoencefálica/diagnóstico , Síndrome Uveomeningoencefálica/tratamento farmacológico , Visão Ocular , Doença Aguda , Corticosteroides/uso terapêutico , Estudos RetrospectivosRESUMO
Amelogenesis imperfecta (AI) is a collection of isolated (non-syndromic) inherited diseases affecting dental enamel formation or a clinical phenotype in syndromic conditions. We characterized three consanguineous AI families with generalized irregular hypoplastic enamel with rapid attrition that perfectly segregated with homozygous defects in a novel gene: RELT that is a member of the tumor necrosis factor receptor superfamily (TNFRSF). RNAscope in situ hybridization of wild-type mouse molars and incisors showed specific Relt mRNA expression by secretory stage ameloblasts and by odontoblasts. Relt-/- mice generated by CRISPR/Cas9 exhibited incisor and molar enamel malformations. Relt-/- enamel had a rough surface and underwent rapid attrition. Normally unmineralized spaces in the deep enamel near the dentino-enamel junction (DEJ) were as highly mineralized as the adjacent enamel, which likely altered the mechanical properties of the DEJ. Phylogenetic analyses showed the existence of selective pressure on RELT gene outside of tooth development, indicating that the human condition may be syndromic, which possibly explains the history of small stature and severe childhood infections in two of the probands. Knowing a TNFRSF member is critical during the secretory stage of enamel formation advances our understanding of amelogenesis and improves our ability to diagnose human conditions featuring enamel malformations.
Assuntos
Amelogênese Imperfeita/diagnóstico , Amelogênese Imperfeita/genética , Genes Recessivos , Estudos de Associação Genética , Predisposição Genética para Doença , Mutação , Receptores do Fator de Necrose Tumoral/genética , Consanguinidade , Genótipo , Mutação em Linhagem Germinativa , Humanos , Hibridização In Situ , Linhagem , Fenótipo , Splicing de RNA , Sequenciamento do ExomaRESUMO
Dentin sialophosphoprotein (DSPP) is primarily expressed by differentiated odontoblasts (dentin-forming cells), and transiently expressed by presecretory ameloblasts (enamel-forming cells). Disease-causing DSPP mutations predominantly fall into two categories: 5' mutations affecting targeting and trafficking, and 3' - 1 frameshift mutations converting the repetitive, hydrophilic, acidic C-terminal domain into a hydrophobic one. We characterized the dental phenotypes and investigated the pathological mechanisms of DsppP19L and Dspp-1fs mice that replicate the two categories of human DSPP mutations. In DsppP19L mice, dentin is less mineralized but contains dentinal tubules. Enamel mineral density is reduced. Intracellular accumulation and ER retention of DSPP is observed in odontoblasts and ameloblasts. In Dspp-1fs mice, a thin layer of reparative dentin lacking dentinal tubules is deposited. Odontoblasts show severe pathosis, including intracellular accumulation and ER retention of DSPP, strong ubiquitin and autophagy activity, ER-phagy, and sporadic apoptosis. Ultrastructurally, odontoblasts show extensive autophagic vacuoles, some of which contain fragmented ER. Enamel formation is comparable to wild type. These findings distinguish molecular mechanisms underlying the dental phenotypes of DsppP19L and Dspp-1fs mice and support the recently revised Shields classification of dentinogenesis imperfecta caused by DSPP mutations in humans. The Dspp-1fs mice may be valuable for the study of autophagy and ER-phagy.
Assuntos
Proteínas da Matriz Extracelular , Mutação da Fase de Leitura , Camundongos , Humanos , Animais , Proteínas da Matriz Extracelular/genética , Odontoblastos , Mutação , Fosfoproteínas/genética , Sialoglicoproteínas/genética , Dentina , Autofagia/genéticaRESUMO
Human ACP4 (OMIM*606362) encodes a transmembrane protein that belongs to histidine acid phosphatase (ACP) family. Recessive mutations in ACP4 cause non-syndromic hypoplastic amelogenesis imperfecta (AI1J, OMIM#617297). While ACP activity has long been detected in developing teeth, its functions during tooth development and the pathogenesis of ACP4-associated AI remain largely unknown. Here, we characterized 2 AI1J families and identified a novel ACP4 disease-causing mutation: c.774_775del, p.Gly260Aspfs*29. To investigate the role of ACP4 during amelogenesis, we generated and characterized Acp4R110C mice that carry the p.(Arg110Cys) loss-of-function mutation. Mouse Acp4 expression was the strongest at secretory stage ameloblasts, and the protein localized primarily at Tomes' processes. While Acp4 heterozygous (Acp4+/R110C) mice showed no phenotypes, incisors and molars of homozygous (Acp4R110C/R110C) mice exhibited a thin layer of aplastic enamel with numerous ectopic mineralized nodules. Acp4R110C/R110C ameloblasts appeared normal initially but underwent pathology at mid-way of secretory stage. Ultrastructurally, sporadic enamel ribbons grew on mineralized dentin but failed to elongate, and aberrant needle-like crystals formed instead. Globs of organic matrix accumulated by the distal membranes of defective Tomes' processes. These results demonstrated a critical role for ACP4 in appositional growth of dental enamel probably by processing and regulating enamel matrix proteins around mineralization front apparatus.
Assuntos
Amelogênese Imperfeita , Proteínas do Esmalte Dentário , Fosfatase Ácida/metabolismo , Ameloblastos/metabolismo , Amelogênese , Amelogênese Imperfeita/metabolismo , Animais , Proteínas do Esmalte Dentário/genética , Proteínas do Esmalte Dentário/metabolismo , Histidina/metabolismo , Humanos , Camundongos , MutaçãoRESUMO
Mutations of Odontogenesis-Associated Phosphoprotein (ODAPH, OMIM *614829) cause autosomal recessive amelogenesis imperfecta, however, the function of ODAPH during amelogenesis is unknown. Here we characterized normal Odaph expression by in situ hybridization, generated Odaph truncation mice using CRISPR/Cas9 to replace the TGC codon encoding Cys41 into a TGA translation termination codon, and characterized and compared molar and incisor tooth formation in Odaph+/+, Odaph+/C41*, and OdaphC41*/C41* mice. We also searched genomes to determine when Odaph first appeared phylogenetically. We determined that tooth development in Odaph+/+ and Odaph+/C41* mice was indistinguishable in all respects, so the condition in mice is inherited in a recessive pattern, as it is in humans. Odaph is specifically expressed by ameloblasts starting with the onset of post-secretory transition and continues until mid-maturation. Based upon histological and ultrastructural analyses, we determined that the secretory stage of amelogenesis is not affected in OdaphC41*/C41* mice. The enamel layer achieves a normal shape and contour, normal thickness, and normal rod decussation. The fundamental problem in OdaphC41*/C41* mice starts during post-secretory transition, which fails to generate maturation stage ameloblasts. At the onset of what should be enamel maturation, a cyst forms that separates flattened ameloblasts from the enamel surface. The maturation stage fails completely.
Assuntos
Ameloblastos/fisiologia , Amelogênese , Proteínas da Matriz Extracelular/metabolismo , Fosfoproteínas/metabolismo , Amelogênese Imperfeita/genética , Amelogênese Imperfeita/patologia , Animais , Esmalte Dentário/crescimento & desenvolvimento , Esmalte Dentário/ultraestrutura , Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/genética , Técnicas de Introdução de Genes , Hibridização In Situ , Incisivo/anatomia & histologia , Camundongos , Dente Molar/anatomia & histologia , Odontogênese , Fosfoproteínas/química , Fosfoproteínas/genéticaRESUMO
Non-syndromic inherited defects of tooth dentin are caused by two classes of dominant negative/gain-of-function mutations in dentin sialophosphoprotein (DSPP): 5' mutations affecting an N-terminal targeting sequence and 3' mutations that shift translation into the - 1 reading frame. DSPP defects cause an overlapping spectrum of phenotypes classified as dentin dysplasia type II and dentinogenesis imperfecta types II and III. Using CRISPR/Cas9, we generated a Dspp-1fs mouse model by introducing a FLAG-tag followed by a single nucleotide deletion that translated 493 extraneous amino acids before termination. Developing incisors and/or molars from this mouse and a DsppP19L mouse were characterized by morphological assessment, bSEM, nanohardness testing, histological analysis, in situ hybridization and immunohistochemistry. DsppP19L dentin contained dentinal tubules but grew slowly and was softer and less mineralized than the wild-type. DsppP19L incisor enamel was softer than normal, while molar enamel showed reduced rod/interrod definition. Dspp-1fs dentin formation was analogous to reparative dentin: it lacked dentinal tubules, contained cellular debris, and was significantly softer and thinner than Dspp+/+ and DsppP19L dentin. The Dspp-1fs incisor enamel appeared normal and was comparable to the wild-type in hardness. We conclude that 5' and 3' Dspp mutations cause dental malformations through different pathological mechanisms and can be regarded as distinct disorders.
Assuntos
Dentinogênese Imperfeita/genética , Proteínas da Matriz Extracelular/genética , Fosfoproteínas/genética , Sialoglicoproteínas/genética , Animais , Esmalte Dentário/metabolismo , Dentina/metabolismo , Dentinogênese Imperfeita/metabolismo , Dentinogênese Imperfeita/fisiopatologia , Modelos Animais de Doenças , Proteínas da Matriz Extracelular/metabolismo , Feminino , Mutação da Fase de Leitura/genética , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Fenótipo , Fosfoproteínas/metabolismo , Sialoglicoproteínas/metabolismo , Dente/metabolismoRESUMO
The respiration flow pattern plays a key role in fluid flow, heat and mass transfer in human lung airway. To reveal the complex flow pattern within human lung multiple-generation airway, both the steady inspiration and expiration flows are comprehensively studied using laser Doppler velocimetry technique and computational fluid dynamics method for an idealized human tracheobronchial three-generation airway model at two flow rates, corresponding to an adult male breathing under light activity and moderate exercise conditions, respectively. The comparison of mainstream velocity between the measurements and simulations are generally good. Both of the inspiration and expiration flows are heavily influenced by the combination of geometrical bifurcating/merging, local wall curvature, limited generation length and multi-generation interaction. The mainstream flow is non-uniform and behaves as skewed, double-peaked and M-shaped patterns. The secondary flow is complex and characteristic of Dean-type two-vortex, four-vortex, six-vortex and eight-vortex patterns. This work is of scientific significance for a deep understanding of respiratory flow physics and of certain application values for clinical diagnosis and remedy of respiratory deceases.
Assuntos
Expiração , Inalação , Pulmão , Modelos Biológicos , Adulto , Simulação por Computador , Expiração/fisiologia , Humanos , Inalação/fisiologia , Pulmão/anatomia & histologia , Pulmão/fisiologia , MasculinoRESUMO
BACKGROUND AND OBJECTIVE: Liver disease is a multifactorial complex disease with high global prevalence and poor long-term clinical efficacy and liver disease patients with different comorbidities often incorporate multiple phenotypes in the clinic. Thus, there is a pressing need to improve understanding of the complexity of clinical liver population to help gain more accurate disease subtypes for personalized treatment. METHODS: Individualized treatment of the traditional Chinese medicine (TCM) provides a theoretical basis to the study of personalized classification of complex diseases. Utilizing the TCM clinical electronic medical records (EMRs) of 6475 liver inpatient cases, we built a liver disease comorbidity network (LDCN) to show the complicated associations between liver diseases and their comorbidities, and then constructed a patient similarity network with shared symptoms (PSN). Finally, we identified liver patient subgroups using community detection methods and performed enrichment analyses to find both distinct clinical and molecular characteristics (with the phenotype-genotype associations and interactome networks) of these patient subgroups. RESULTS: From the comorbidity network, we found that clinical liver patients have a wide range of disease comorbidities, in which the basic liver diseases (e.g. hepatitis b, decompensated liver cirrhosis), and the common chronic diseases (e.g. hypertension, type 2 diabetes), have high degree of disease comorbidities. In addition, we identified 303 patient modules (representing the liver patient subgroups) from the PSN, in which the top 6 modules with large number of cases include 51.68% of the whole cases and 251 modules contain only 10 or fewer cases, which indicates the manifestation diversity of liver diseases. Finally, we found that the patient subgroups actually have distinct symptom phenotypes, disease comorbidity characteristics and their underlying molecular pathways, which could be used for understanding the novel disease subtypes of liver conditions. For example, three patient subgroups, namely Module 6 (M6, nâ¯=â¯638), M2 (nâ¯=â¯623) and M1 (nâ¯=â¯488) were associated to common chronic liver disease conditions (hepatitis, cirrhosis, hepatocellular carcinoma). Meanwhile, patient subgroups of M30 (nâ¯=â¯36) and M36 (nâ¯=â¯37) were mostly related to acute gastroenteritis and upper respiratory infection, respectively, which reflected the individual comorbidity characteristics of liver subgroups. Furthermore, we identified the distinct genes and pathways of patient subgroups and the basic liver diseases (hepatitis b and cirrhosis), respectively. The high degree of overlapping pathways between them (e.g. M36 with 93.33% shared enriched pathways) indicates the underlying molecular network mechanisms of each patient subgroup. CONCLUSIONS: Our results demonstrate the utility and comprehensiveness of disease classification study based on community detection of patient network using shared TCM symptom phenotypes and it can be used to other more complex diseases.
Assuntos
Hepatopatias/diagnóstico , Hepatopatias/metabolismo , Avaliação de Sintomas , Adulto , Idoso , Doença Crônica , Comorbidade , Registros Eletrônicos de Saúde , Feminino , Estudos de Associação Genética , Humanos , Fígado/metabolismo , Masculino , Medicina Tradicional Chinesa , Pessoa de Meia-Idade , FenótipoRESUMO
BACKGROUND: ENAM mutations cause autosomal dominant or recessive amelogenesis imperfecta (AI) and show a dose effect: enamel malformations are more severe or only penetrant when both ENAM alleles are defective. METHODS: Whole exome sequences of recruited AI probands were initially screened for mutations in known AI candidate genes. Sanger sequencing was used to confirm sequence variations and their segregation with the disease phenotype. The co-occurrence of ENAM and LAMA3 mutations in one family raised the possibility of digenic inheritance. Enamel formed in Enam+/+ Ambn+/+ , Enam+/- , Ambn+/- , and Enam+/- Ambn+/- mice was characterized by dissection and backscattered scanning electron microscopy (bSEM). RESULTS: ENAM mutations segregating with AI in five families were identified. Two novel ENAM frameshift mutations were identified. A single-nucleotide duplication (c.395dupA/p.Pro133Alafs*13) replaced amino acids 133-1142 with a 12 amino acid (ATTKAAFEAAIT*) sequence, and a single-nucleotide deletion (c.2763delT/p.Asp921Glufs*32) replaced amino acids 921-1142 with 31 amino acids (ESSPQQASYQAKETAQRRGKAKTLLEMMCPR*). Three families were heterozygous for a previously reported single-nucleotide ENAM deletion (c.588+1delG/p.Asn197Ilefs*81). One of these families also harbored a heterozygous LAMA3 mutation (c.1559G>A/p.Cys520Tyr) that cosegregated with both the AI phenotype and the ENAM mutation. In mice, Ambn+/- maxillary incisors were normal. Ambn+/- molars were also normal, except for minor surface roughness. Ambn+/- mandibular incisors were sometimes chalky and showed minor chipping. Enam+/- incisor enamel was thinner than normal with ectopic mineral deposited laterally. Enam+/- molars were sometimes chalky and rough surfaced. Enam+/- Ambn+/- enamel was thin and rough, in part due to ectopic mineralization, but also underwent accelerated attrition. CONCLUSION: Novel ENAM mutations causing AI were identified, raising to 22 the number of ENAM variations known to cause AI. The severity of the enamel phenotype in Enam+/- Ambn+/- double heterozygous mice is caused by composite digenic effects. Digenic inheritance should be explored as a cause of AI in humans.
Assuntos
Amelogênese Imperfeita/patologia , Proteínas da Matriz Extracelular/genética , Amelogênese Imperfeita/genética , Feminino , Mutação da Fase de Leitura , Deleção de Genes , Heterozigoto , Humanos , Laminina/genética , Masculino , Linhagem , Fenótipo , Polimorfismo de Nucleotídeo Único , Sequenciamento do ExomaRESUMO
Replicative senescence/crisis is thought to act as a tumor suppressor mechanism. Although recent data indicate that normal human cells cannot be converted into cancer cells without telomerase, the original concept of senescence as a tumor suppressor mechanism is that senescence/crisis would act to limit the growth of telomerase-negative tumors. We show here that this concept is valid when oncogene-expressing human and bovine cells are introduced into immunodeficient mice using tissue reconstruction techniques, as opposed to conventional subcutaneous injection. Primary human and bovine adrenocortical cells were transduced with retroviruses encoding Ha-Ras(G12V) and SV40 large T antigen and transplanted in immunodeficient mice using tissue reconstruction techniques. Transduced cells were fully malignant (invasive and metastatic) in this model. They had negligible telomerase activity both before transplantation and when recovered from tumors. When serially transplanted, tumors showed progressively slower growth, decreased invasion and metastasis, shortened telomeres, and morphological features of crisis. Whereas telomerase was not essential for malignant behavior, expression of human telomerase reverse transcriptase enabled cells from serially transplanted tumors that had ceased growth to reacquire tumorigenicity. Moreover, telomerase-negative oncogene-expressing cells were tumorigenic only when transplanted using tissue reconstruction techniques; human telomerase reverse transcriptase was required for cells to form tumors when cells were injected subcutaneously. This work provides a new model to study crisis in an in vivo setting and its effects on malignancy; despite having invasive and metastatic properties, cells are eventually driven into crisis by proliferation in the absence of a telomere maintenance mechanism.
Assuntos
Neoplasias do Córtex Suprarrenal/enzimologia , Neoplasias do Córtex Suprarrenal/patologia , Córtex Suprarrenal/enzimologia , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Telomerase/deficiência , Telomerase/metabolismo , Córtex Suprarrenal/patologia , Córtex Suprarrenal/fisiologia , Neoplasias do Córtex Suprarrenal/genética , Animais , Antígenos Transformantes de Poliomavirus/fisiologia , Bovinos , Divisão Celular/fisiologia , Transformação Celular Neoplásica/genética , Proteínas de Ligação a DNA , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Camundongos SCID , Transplante de Neoplasias , Telomerase/genética , Telômero/fisiologia , Transdução Genética , Transplante Heterólogo , Proteínas ras/fisiologiaRESUMO
The main physiological function of respiratory system is exchange of oxygen and carbon dioxide between atmosphere and blood. Its physiological process is closely related with air flow and transport in respiratory airway. This paper studies numerically the inspiratory flows in a two generation and a three generation bronchial airway. The numerical results of the two generation bronchi show that the present computations fit the available experiments very well. For three generation bronchi, no separation appears within the whole airway under normal breathing rate, which is contrary to the occurrence of separation at even low Reynolds number by the previous two dimensional models. Strong secondary flow phenomenon, skew and m-shaped main flow velocity profiles are found in the airways due to geometrical curvature and bifurcation. These increase shear stress acting on the inner wall as well as on the anterior and posterior wall in the bifurcating airways. In the end bronchi of the three generation airway, the mass flow rates in medial and lateral bronchi are unequal, and the mass flow ratio between the medial and the lateral bronchi is 1.2 at the flow conditions considered.
Assuntos
Brônquios/fisiologia , Inalação/fisiologia , Modelos Biológicos , Brônquios/anatomia & histologia , Análise de Elementos Finitos , HumanosRESUMO
BACKGROUND: It is documented that in tumor cell lines, the hTERT gene exhibits prominent methylation at a CpG island rich region about -600 bp upstream of the transcription start site, but mixed or allelically absent around ±150 bp region. Given the potential clinical implications of the findings in breast cancer diagnostics, we set out to investigate if such findings are reproducible on primary surgically resected invasive breast carcinomas. METHODS: The cohort consisted of 50 cases of freshly sampled and formalin-fixed paraffin embedded (FFPE) invasive breast cancers and normal tissue. A modified quasi-quantitative methylation specific polymerase chain reaction was used to determine methylation status in cancer relative to normal tissue at the 2 CpG island-rich regions. RESULTS: A global hypermethylation is evident at the -600 bp region in both fresh and parallel FFPE breast cancers as compared to normal tissue. In contrast, most of the tumor and normal tissue remains unmethylated around the ±150 bp region. CONCLUSIONS: Our results support further development of hTERT hypermethylation in the -600 bp region as a biomarker for breast cancer diagnostics. The unmethylated status of ±150 bp region in normal breast tissue does not support the suggestion that unmethylation is required for hTERT gene expression.
Assuntos
Biomarcadores Tumorais/análise , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/patologia , Metilação de DNA , Telomerase/genética , Estudos de Coortes , Ilhas de CpG , Feminino , Humanos , Reação em Cadeia da Polimerase , Reprodutibilidade dos TestesRESUMO
BACKGROUND: DNA hypermethylation has been documented to be prominent at a CpG island rich region about 600 bp upstream the transcription start site of the hTERT gene using qualitative methylation specific PCR on DNA isolated from tumor cell lines. In order to assess the potential significance of this biomarker in breast cancer research and diagnosis, we explored if such findings are reproducible on surgically resected fresh breast tumor cells. METHODS: Using quantitative pyrosequencing technology, we investigated and present methylation status of four CpG islands of this region in a cohort of 77 invasive breast carcinomas using normal breast tissue as controls. RESULTS: Globally, a significant hypermethylation in tumor cells was observed in the four CpG islands as a sum, in comparison to methylation of the normal breast tissue. Individually, certain CpG islands displayed methylation greater than 50% in about 3/4 of the 77 breast cancers, but in none of the normal breast tissue. Our results highlight the value of DNA hypermethylation in the -600 bp region of the hTERT gene as a potential marker for breast cancer diagnosis. CONCLUSIONS: We believe that integration of this novel, malignancy-associated molecular testing with morphology is of significant value in the accurate interpretation of small tumor sample size obtained via fine needle aspiration biopsy, ductal lavage, and nipple fluid aspirates both in clinical practice and in breast cancer research. Diagn. Cytopathol. 2016;44:670-675. © 2016 Wiley Periodicals, Inc.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Mama/diagnóstico , Metilação de DNA , Telomerase/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/genética , Ilhas de CpG , Feminino , Humanos , Pessoa de Meia-IdadeRESUMO
OBJECTIVES: KRAS mutations are frequent in pancreatic ductal adenocarcinoma, chronic pancreatitis, and mucinous neoplasms. In animal studies, KRAS mutations in acinar and Langerhans islets are associated with pancreatic intraepithelial neoplasia. Clinically, KRAS mutation is sometimes requested on cytology/biopsy specimens and negative results are helpful to rule out pancreatic ductal adenocarcinoma. This study set out to further elucidate these issues. METHODS: Surgical specimens with pancreatic ductal adenocarcinoma, premalignant lesions, and chronic pancreatitis were reviewed. Tissue microdissections on 53 such areas of 21 cases were performed followed by polymerase chain reaction and pyrosequencing. RESULTS: KRAS codon 12 mutations were detected in 100% pancreatic ductal adenocarcinomas. No KRAS codon 12 and 13 mutations were detected in benign acinar and Langerhans islets that lie adjacent to or away from the tumor. Variable mutation frequencies were seen in premalignant lesions. CONCLUSIONS: The results support such clinical practice that negative KRAS mutation helps rule out pancreatic ductal adenocarcinomas on small cytology/biopsy specimens. Negative KRAS mutations, however, cannot rule out pancreatic premalignant lesions. Additionally, the results that benign pancreas are negative for KRAS mutations complement the findings of other relevant study that KRAS mutation-associated premalignant lesions do not appear to arise from acinar cells or Langerhans islets.
Assuntos
Células Acinares/metabolismo , Carcinoma Ductal Pancreático/genética , Ilhotas Pancreáticas/metabolismo , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Células Acinares/patologia , Códon/genética , Análise Mutacional de DNA/métodos , Humanos , Ilhotas Pancreáticas/patologia , Mutação , Reação em Cadeia da PolimeraseRESUMO
The identification of genes important in the pathogenesis of Lyme disease Borrelia has been hampered by exceedingly low transformation rates in low-passage, infectious organisms. Using the infectious, moderately transformable B. burgdorferi derivative 5A18NP1 and signature-tagged versions of the Himar1 transposon vector pGKT, we have constructed a defined transposon library for the efficient genome-wide investigation of genes required for wild-type pathogenesis, in vitro growth, physiology, morphology, and plasmid replication. To facilitate analysis, the insertion sites of 4,479 transposon mutants were determined by sequencing. The transposon insertions were widely distributed across the entire B. burgdorferi genome, with an average of 2.68 unique insertion sites per kb DNA. The 10 linear plasmids and 9 circular plasmids had insertions in 33 to 100 percent of their predicted genes. In contrast, only 35% of genes in the 910 kb linear chromosome had incapacitating insertions; therefore, the remaining 601 chromosomal genes may represent essential gene candidates. In initial signature-tagged mutagenesis (STM) analyses, 434 mutants were examined at multiple tissue sites for infectivity in mice using a semi-quantitative, Luminex-based DNA detection method. Examples of genes found to be important in mouse infectivity included those involved in motility, chemotaxis, the phosphoenolpyruvate phosphotransferase system, and other transporters, as well as putative plasmid maintenance genes. Availability of this ordered STM library and a high-throughput screening method is expected to lead to efficient assessment of the roles of B. burgdorferi genes in the infectious cycle and pathogenesis of Lyme disease.
Assuntos
Borrelia burgdorferi/genética , Mutação , Animais , Quimiotaxia , Elementos de DNA Transponíveis , Biblioteca Gênica , Doença de Lyme/microbiologia , Camundongos , Camundongos Endogâmicos C3H , Modelos Biológicos , Modelos Genéticos , Mutagênese , Plasmídeos/metabolismo , Análise de Sequência de DNA , CarrapatosRESUMO
Proteolytic processing and degradation of enamel matrix proteins appears to be an essential feature of dental enamel formation. The source and character of proteolytic activity in the enamel matrix of developing teeth changes as enamel formation progresses. Two proteinases have been isolated from the extracellular enamel matrix of developing teeth: enamelysin (MMP-20), a matrix metalloproteinase. and kallikrein-4 (KLK4), a serine proteinase. Here, we ask if the expression of MMP-20 and KLK4 correlate with the stage-associated changes in the digestion of enamel proteins. Using in situ hybridization, we localized MMP-20 and KLK4 mRNA in mouse maxillary first molars on postnatal days 1, 2, 3, 5, 6, 7, 9, 11, and 14. Enamelysin signal was first detected in preameloblasts, ameloblasts, and odontoblasts on day 2, but not in ameloblasts covering the enamel-free zone. Enamelysin signal declined in ameloblasts on day 6 but persisted in the dental pulp. In contrast, KLK4 transcripts were first observed on day 3 in pulp and on day 6 in ameloblasts covering the enamel-free zone. the KLK4 signal was present in maturation-stage ameloblasts on days 9, 11, and 14. The expression patterns of MMP-20 and KLK4 by ameloblasts in mouse molars are stage-specific and complementary.
Assuntos
Amelogênese/fisiologia , Proteínas do Esmalte Dentário/biossíntese , Esmalte Dentário/enzimologia , Órgão do Esmalte/enzimologia , Calicreínas/biossíntese , Metaloproteinases da Matriz/biossíntese , Animais , Autorradiografia , Polpa Dentária/enzimologia , Matriz Extracelular/enzimologia , Hibridização In Situ , Metaloproteinase 20 da Matriz , Camundongos , Dente Molar/enzimologia , Sondas RNA , RNA Mensageiro/biossínteseRESUMO
Dentin sialophosphoprotein (DSPP) is a chimeric glycoprotein with dentin sialoprotein (DSP) on its N-terminus and dentin phosphoprotein (DPP) on its C-terminus. We have constructed and screened a unidirectional cDNA library derived from the pulp organ of developing pig teeth, and isolated cDNA clones encoding DSP-only, as well as two DSPP clones with alternative sequences in their 3' coding regions. The DSP-only transcript has an open reading frame of 386 codons, and is generated through the use of a polyadenylation signal within intron 4, immediately following the DSP coding region. the use of this polyadenylation signal deletes the DPP coding region and places a TGA translation termination signal as the fourth codon following the exon 4-encoded segment. The DSPP cDNAs contain open reading frames of 593 and 600 codons. Northern blots hybridized to radiolabeled DSP probes showed bands at 1.4, 2.5, 4.4, and 4.8 kb. Cloning and characterization of reverse transcriptase polymerase chain reaction products confirmed the existence of mRNA encoding pDSP386, pDSPP593, and pDSPP600in vivo, but also suggested that DNA sequence redundancies in the DSPP coding region make it prone to cloning artifacts.
Assuntos
Dentina/química , Dentinogênese/genética , Precursores de Proteínas/química , Precursores de Proteínas/genética , Sequência de Aminoácidos , Animais , Northern Blotting , Clonagem Molecular , DNA Complementar/análise , Proteínas da Matriz Extracelular , Humanos , Camundongos , Dados de Sequência Molecular , Fosfoproteínas/biossíntese , Fosfoproteínas/química , Fosfoproteínas/genética , Poliadenilação , Precursores de Proteínas/biossíntese , RNA Mensageiro/análise , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sialoglicoproteínas/biossíntese , Sialoglicoproteínas/química , Sialoglicoproteínas/genética , SuínosRESUMO
Mucopolysaccharidosis type IVA (Morquio A syndrome, MPS IVA) is a rare, autosomal recessive disorder with a prevalence of 1 in 170,000 live births. It is caused by a deficiency of N-acetylgalactosamine 6-sulfatase (GALNS), a lysosomal hydrolase encoded by a gene on human chromosome 16q24.3. Mucopolysaccharidosis type IVA is the only known MPS that is associated with structural defects in dental enamel. GALNS cleaves the sulfate group from N-acetylgalactosamine 6-sulfate and galactose 6-sulfate, which are specifically found in keratan sulfate and chondroitin 6-sulfate. A pathologic absence of GALNS activity results in the accumulation of these glycosaminoaglycans in the urine and in the lysosomes of tissues that turn them over. There is currently no animal model for MPS IVA. To learn more about how a GALNS deficit could lead to enamel defects, we have cloned and characterized a full-length pig GALNS cDNA. GALNS mRNA was localized in developing teeth by in situ hybridization, Northern blot, and reverse-transcription polymerase chain reaction analyses, while GALNS substrates were localized using immunohistochemistry. We report that secretory ameloblasts were positive for GALNS mRNA, as well as for keratan sulfate and chondroitin 6-sulfate. We conclude that enamel defects associated with the loss of GALNS activity in persons with MPS IVA are likely to result from the pathological accumulation of keratan sulfate and chondroitin 6-sulfate in the lysosomes of secretory stage ameloblasts.
Assuntos
Condroitina Sulfatases/genética , DNA Complementar/genética , Expressão Gênica , Odontogênese/fisiologia , Suínos/crescimento & desenvolvimento , Suínos/genética , Sequência de Aminoácidos/genética , Animais , Sequência de Bases/genética , Sulfatos de Condroitina/metabolismo , Imuno-Histoquímica , Sulfato de Queratano/metabolismo , Dados de Sequência Molecular , RNA Mensageiro/metabolismo , Dente/fisiologiaRESUMO
Kallikrein-4 (KLK4) is a serine proteinase believed to be important in the normal development of dental enamel. We isolated native KLK4 from developing pig enamel and expressed four recombinant forms. Pig KLK4 was expressed in bacteria with and without the propeptide, and in two eukaryotic systems. Recombinant pig KLK4 was secreted as a zymogen by '293' cells and purified. The proKLK4 was activated in vitro by thermolysin and recombinant pig enamelysin, but not by native KLK4. These results were confirmed using a fluorescent peptide analog of the KLK4 propeptide-enzyme junction. Native KLK4 appears as a doublet at 37 kDa and 34 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Removal of N-linked oligosaccharides by digestion with deglycosidase-F reduced the doublet to a single band at approximately 28 kDa, demonstrating that the active enzyme is glycosylated, and that the 37 kDa and 34 kDa forms differ only in their number of glycosylations. Deglycosylation was also associated with a loss of proteolytic activity. We digested recombinant pig amelogenin with native KLK4 and characterized the cleavage products by N-terminal sequencing and mass spectrometry. Eleven cleavage sites in the amelogenin protein were identified, demonstrating that KLK4 degrades amelogenin and is likely to participate in the degradation of enamel proteins in vivo.