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1.
Neurology ; 91(22): e2078-e2088, 2018 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-30413629

RESUMO

OBJECTIVE: To characterize the neurologic phenotypes associated with COL4A1/2 mutations and to seek genotype-phenotype correlation. METHODS: We analyzed clinical, EEG, and neuroimaging data of 44 new and 55 previously reported patients with COL4A1/COL4A2 mutations. RESULTS: Childhood-onset focal seizures, frequently complicated by status epilepticus and resistance to antiepileptic drugs, was the most common phenotype. EEG typically showed focal epileptiform discharges in the context of other abnormalities, including generalized sharp waves or slowing. In 46.4% of new patients with focal seizures, porencephalic cysts on brain MRI colocalized with the area of the focal epileptiform discharges. In patients with porencephalic cysts, brain MRI frequently also showed extensive white matter abnormalities, consistent with the finding of diffuse cerebral disturbance on EEG. Notably, we also identified a subgroup of patients with epilepsy as their main clinical feature, in which brain MRI showed nonspecific findings, in particular periventricular leukoencephalopathy and ventricular asymmetry. Analysis of 15 pedigrees suggested a worsening of the severity of clinical phenotype in succeeding generations, particularly when maternally inherited. Mutations associated with epilepsy were spread across COL4A1 and a clear genotype-phenotype correlation did not emerge. CONCLUSION: COL4A1/COL4A2 mutations typically cause a severe neurologic condition and a broader spectrum of milder phenotypes, in which epilepsy is the predominant feature. Early identification of patients carrying COL4A1/COL4A2 mutations may have important clinical consequences, while for research efforts, omission from large-scale epilepsy sequencing studies of individuals with abnormalities on brain MRI may generate misleading estimates of the genetic contribution to the epilepsies overall.

3.
Neurogenetics ; 19(1): 41-47, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29322350

RESUMO

Rett syndrome (RTT) is caused by mutations in methyl-CpG-binding protein 2 (MECP2), but defects in a handful of other genes (e.g., CDKL5, FOXG1, MEF2C) can lead to presentations that resemble, but do not completely mirror, classical RTT. In this study, we attempted to identify other monogenic disorders that share features with RTT. We performed a retrospective chart review on n = 319 patients who had undergone clinical whole exome sequencing (WES) for further etiological evaluation of neurodevelopmental diagnoses that remained unexplained despite extensive prior workup. From this group, we characterized those who (1) possessed features that were compatible with RTT based on clinical judgment, (2) subsequently underwent MECP2 sequencing and/or MECP2 deletion/duplication analysis with negative results, and (3) ultimately arrived at a diagnosis other than RTT with WES. n = 7 patients had clinical features overlapping RTT with negative MECP2 analysis but positive WES providing a diagnosis. These seven patients collectively possessed pathogenic variants in six different genes: two in KCNB1 and one each in FOXG1, IQSEC2, MEIS2, TCF4, and WDR45. n = 2 (both with KCNB1 variants) fulfilled criteria for atypical RTT. RTT-associated features included the following: loss of hand or language skills (n = 3; IQSEC2, KCNB1 x 2); disrupted sleep (n = 4; KNCB1, MEIS2, TCF4, WDR45); stereotyped hand movements (n = 5; FOXG1, KNCB1 x 2, MEIS2, TCF4); bruxism (n = 3; KCNB1 x 2; TCF4); and hypotonia (n = 7). Clinically based diagnoses can be misleading, evident by the increasing number of genetic conditions associated with features of RTT with negative MECP2 mutations.

4.
JAMA Neurol ; 74(10): 1228-1236, 2017 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-28806457

RESUMO

Importance: Knowing the range of symptoms seen in patients with a missense or loss-of-function variant in KCNB1 and how these symptoms correlate with the type of variant will help clinicians with diagnosis and prognosis when treating new patients. Objectives: To investigate the clinical spectrum associated with KCNB1 variants and the genotype-phenotype correlations. Design, Setting, and Participants: This study summarized the clinical and genetic information of patients with a presumed pathogenic variant in KCNB1. Patients were identified in research projects or during clinical testing. Information on patients from previously published articles was collected and authors contacted if feasible. All patients were seen at a clinic at one of the participating institutes because of presumed genetic disorder. They were tested in a clinical setting or included in a research project. Main Outcomes and Measures: The genetic variant and its inheritance and information on the patient's symptoms and characteristics in a predefined format. All variants were identified with massive parallel sequencing and confirmed with Sanger sequencing in the patient. Absence of the variant in the parents could be confirmed with Sanger sequencing in all families except one. Results: Of 26 patients (10 female, 15 male, 1 unknown; mean age at inclusion, 9.8 years; age range, 2-32 years) with developmental delay, 20 (77%) carried a missense variant in the ion channel domain of KCNB1, with a concentration of variants in region S5 to S6. Three variants that led to premature stops were located in the C-terminal and 3 in the ion channel domain. Twenty-one of 25 patients (84%) had seizures, with 9 patients (36%) starting with epileptic spasms between 3 and 18 months of age. All patients had developmental delay, with 17 (65%) experiencing severe developmental delay; 14 (82%) with severe delay had behavioral problems. The developmental delay was milder in 4 of 6 patients with stop variants and in a patient with a variant in the S2 transmembrane element rather than the S4 to S6 region. Conclusions and Relevance: De novo KCNB1 missense variants in the ion channel domain and loss-of-function variants in this domain and the C-terminal likely cause neurodevelopmental disorders with or without seizures. Patients with presumed pathogenic variants in KCNB1 have a variable phenotype. However, the type and position of the variants in the protein are (imperfectly) correlated with the severity of the disorder.


Assuntos
Mutação de Sentido Incorreto/genética , Transtornos do Neurodesenvolvimento/genética , Transtornos do Neurodesenvolvimento/fisiopatologia , Canais de Potássio Shab/genética , Adolescente , Adulto , Encéfalo/diagnóstico por imagem , Criança , Pré-Escolar , Eletroencefalografia , Feminino , Estudo de Associação Genômica Ampla , Genótipo , Humanos , Imagem por Ressonância Magnética , Masculino , Transtornos do Neurodesenvolvimento/diagnóstico por imagem , Fenótipo , Adulto Jovem
5.
Am J Hum Genet ; 101(1): 65-74, 2017 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-28669405

RESUMO

KCNQ5 is a highly conserved gene encoding an important channel for neuronal function; it is widely expressed in the brain and generates M-type current. Exome sequencing identified de novo heterozygous missense mutations in four probands with intellectual disability, abnormal neurological findings, and treatment-resistant epilepsy (in two of four). Comprehensive analysis of this potassium channel for the four variants expressed in frog oocytes revealed shifts in the voltage dependence of activation, including altered activation and deactivation kinetics. Specifically, both loss-of-function and gain-of-function KCNQ5 mutations, associated with increased excitability and decreased repolarization reserve, lead to pathophysiology.


Assuntos
Epilepsia/genética , Predisposição Genética para Doença , Deficiência Intelectual/genética , Canais de Potássio KCNQ/genética , Mutação/genética , Eletroencefalografia , Humanos , Ativação do Canal Iônico , Canais de Potássio KCNQ/química , Proteínas Mutantes/química , Proteínas Mutantes/genética , Fenótipo , Alinhamento de Sequência
6.
J Chromatogr Sci ; 55(7): 706-711, 2017 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-28334842

RESUMO

Azadirachta indica has been used for its medicinal properties since time immemorial. Herbal medicines which are prepared using this medicinal tree are utilized to treat various diseases and disorders. No reports are available for marker-based standardization of these herbal medicines prepared from leaves of A. indica. Also existing HPLC methods for determination quercetin-3-O-ß-d-glucoside are time consuming. There is an obvious need for development of new HPLC method for quantification of quercetin-3-O-ß-d-glucoside which is fast enough to carry out analysis in stipulated time period. This article deals with the development, optimization and validation of fast HPLC-DAD method for the determination of quercetin-3-O-ß-d-glucoside in extracts containing A. indica for its successive application for marker-based standardization of herbal formulations containing A. indica. The retention time of quercetin-3-O-ß-d-glucoside was 11.213 min. The method was found to be linear in the range of 4.0-60 µg mL-1. Limit of detection and limit of quantitation of the proposed method were found to be 1.33 and 4.0 µg mL-1, respectively. The mean recoveries were found to be within 93.53-103.75%. The method can be used as quality control tool for routine analysis of herbal extracts and formulations containing A. indicia.


Assuntos
Azadirachta/química , Cromatografia Líquida de Alta Pressão/métodos , Extratos Vegetais/química , Quercetina/análogos & derivados , Limite de Detecção , Modelos Lineares , Folhas de Planta/química , Quercetina/análise , Reprodutibilidade dos Testes
7.
Cold Spring Harb Mol Case Stud ; 2(6): a001172, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27900362

RESUMO

Using whole-exome sequencing, we have identified novel de novo heterozygous pleckstrin homology domain-interacting protein (PHIP) variants that are predicted to be deleterious, including a frameshift deletion, in two unrelated patients with common clinical features of developmental delay, intellectual disability, anxiety, hypotonia, poor balance, obesity, and dysmorphic features. A nonsense mutation in PHIP has previously been associated with similar clinical features. Patients with microdeletions of 6q14.1, including PHIP, have a similar phenotype of developmental delay, intellectual disability, hypotonia, and obesity, suggesting that the phenotype of our patients is a result of loss-of-function mutations. PHIP produces multiple protein products, such as PHIP1 (also known as DCAF14), PHIP, and NDRP. PHIP1 is one of the multiple substrate receptors of the proteolytic CUL4-DDB1 ubiquitin ligase complex. CUL4B deficiency has been associated with intellectual disability, central obesity, muscle wasting, and dysmorphic features. The overlapping phenotype associated with CUL4B deficiency suggests that PHIP mutations cause disease through disruption of the ubiquitin ligase pathway.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/genética , Pré-Escolar , Deficiências do Desenvolvimento/genética , Exoma , Feminino , Mutação da Fase de Leitura , Heterozigoto , Humanos , Deficiência Intelectual/genética , Hipotonia Muscular/genética , Mutação , Obesidade/genética , Fenótipo , Domínios de Homologia à Plecstrina , Sequenciamento Completo do Exoma/métodos
8.
Hum Mutat ; 37(8): 786-93, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27120018

RESUMO

Retinoic acid (RA) signaling plays a key role in the development and function of several systems in mammals. We previously discovered that the de novo mutations c.1159C>T (p.Arg387Cys) and c.1159C>A (p.Arg387Ser) in the RA Receptor Beta (RARB) gene cause microphthalmia and diaphragmatic hernia. However, the natural history of affected subjects beyond the prenatal or neonatal period was unknown. Here, we describe nine additional subjects with microphthalmia who have de novo mutations in RARB, including the previously described p.Arg387Cys as well as the novel c.887G>C (p.Gly296Ala) and c.638T>C (p.Leu213Pro). Moreover, we review the information on four previously reported cases. All subjects who survived the neonatal period (n = 10) displayed severe global developmental delay with progressive motor impairment due to spasticity and/or dystonia (with or without chorea). The majority of subjects also showed Chiari type I malformation and severe feeding difficulties. We previously found that p.Arg387Cys and p.Arg387Ser induce a gain-of-function. We show here that the p.Gly296Ala and p.Leu213Pro RARB mutations further promote the RA ligand-induced transcriptional activity by twofold to threefold over the wild-type receptor, also indicating a gain-of-function mechanism. These observations suggest that precise regulation of RA signaling is required for brain development and/or function in humans.


Assuntos
Mutação com Ganho de Função , Deficiência Intelectual/genética , Transtornos dos Movimentos/genética , Receptores do Ácido Retinoico/genética , Adolescente , Criança , Pré-Escolar , Distúrbios Distônicos , Feminino , Humanos , Recém-Nascido , Masculino , Modelos Moleculares , Mutação de Sentido Incorreto , Conformação Proteica , Receptores do Ácido Retinoico/química , Ativação Transcricional
9.
Am J Med Genet A ; 170A(5): 1330-2, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26834045

RESUMO

We evaluated a 13-year-old East Pakistani male affected with microcephaly, apparent intellectual disability, hypotonia, and brisk reflexes without spasticity. His parents were first cousins. The patient also had a brother who was similarly affected and died at 10 years due to an accident. Previous SNP array testing showed a 1.63 Mb duplication at 16p13.11 of uncertain significance along with regions of homozygosity. Exome sequencing identified a known pathogenic homozygous alteration in DEAF1, c.676C>T (p.R226W), in this patient. The alteration had been reported in two individuals from a consanguineous Saudi Arabian family. Both individuals had microcephaly, intellectual disability, hypotonia, feeding difficulties, and poor growth. The patient reported here did not have evidence of white matter disease, as had been reported with prior patients. We conclude that this DEAF1 gene alteration caused this patient's symptoms and that white matter disease should not be considered a obligate feature of this syndrome.


Assuntos
Deficiência Intelectual/genética , Microcefalia/genética , Hipotonia Muscular/genética , Proteínas Nucleares/genética , Adolescente , Duplicação Cromossômica , Exoma , Humanos , Deficiência Intelectual/fisiopatologia , Leucoencefalopatias/genética , Leucoencefalopatias/patologia , Masculino , Microcefalia/fisiopatologia , Hipotonia Muscular/fisiopatologia , Mutação , Paquistão , Linhagem , Análise de Sequência de DNA
10.
J Child Neurol ; 30(14): 1871-6, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26303410

RESUMO

Tuberous sclerosis complex is a multisystem, chronic genetic condition characterized by systemic growth of benign tumors and often accompanied by epilepsy, autism spectrum disorders, and intellectual disability. Nonetheless, the neurodevelopmental phenotype of these patients is not often detailed. The authors describe 3 individuals with tuberous sclerosis complex who share common characteristics that can help to identify a distinct profile of autism spectrum disorder. These findings include typical cognitive development, expressive and pragmatic language deficits, and anxiety. The authors also describe features specific to tuberous sclerosis complex that require consideration before diagnosing an autism spectrum disorder. Identifying distinct profiles of autism spectrum disorder in tuberous sclerosis complex can help optimize treatment across the life span.


Assuntos
Transtorno do Espectro Autista/diagnóstico , Transtorno do Espectro Autista/terapia , Encéfalo/patologia , Esclerose Tuberosa/diagnóstico , Esclerose Tuberosa/terapia , Adolescente , Transtorno do Espectro Autista/patologia , Transtorno do Espectro Autista/fisiopatologia , Criança , Diagnóstico Diferencial , Humanos , Imagem por Ressonância Magnética , Masculino , Fenótipo , Esclerose Tuberosa/patologia , Esclerose Tuberosa/fisiopatologia
11.
PLoS One ; 9(2): e88556, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24551118

RESUMO

Prostate cancer (PCa) cells undergoing neuroendocrine differentiation (NED) are clinically relevant to the development of relapsed castration-resistant PCa. Increasing evidences show that autophagy involves in the development of neuroendocrine (NE) tumors, including PCa. To clarify the effect of autophagy on NED, androgen-sensitive PCa LNCaP cells were examined. Treatment of LNCaP cells with IL-6 resulted in an induction of autophagy. In the absence of androgen, IL-6 caused an even stronger activation of autophagy. Similar result was identified in NED induction. Inhibition of autophagy with chloroquine (CQ) markedly decreased NED. This observation was confirmed by beclin1 and Atg5 silencing experiments. Further supporting the role of autophagy in NED, we found that LC3 was up-regulated in PCa tissue that had relapsed after androgen-deprivation therapy when compared with their primary tumor counterpart. LC3 staining in relapsed PCa tissue showed punctate pattern similar to the staining of chromogranin A (CgA), a marker for NED cells. Moreover, autophagy inhibition induced the apoptosis of IL-6 induced NE differentiated PCa cells. Consistently, inhibition of autophagy by knockdown of beclin1 or Atg5 sensitized NE differentiated LNCaP cells to etoposide, a chemotherapy drug. To identify the mechanisms, phosphorylation of IL-6 downstream targets was analyzed. An increase in phospho-AMPK and a decrease in phospho-mTOR were found, which implies that IL-6 regulates autophagy through the AMPK/mTOR pathway. Most important to this study is the discovery of REST, a neuronal gene-specific transcriptional repressor that is involved in autophagy activation. REST was down-regulated in IL-6 treatment. Knockdown experiments suggest that REST is critical to NED and autophagy activation by IL-6. Together, our studies imply that autophagy is involved in PCa progression and plays a cytoprotective role when NED is induced in PCa cells by IL-6 treatment. These results reveal the potential of targeting autophagy as part of a combined therapeutic regime for NE tumors.


Assuntos
Autofagia/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Interleucina-6/farmacologia , Células Neuroendócrinas/patologia , Neoplasias da Próstata/patologia , Transdução de Sinais/efeitos dos fármacos , Adenilato Quinase/metabolismo , Androgênios/farmacologia , Proteínas Reguladoras de Apoptose/metabolismo , Autofagia/genética , Proteína Beclina-1 , Diferenciação Celular/genética , Linhagem Celular Tumoral , Cloroquina/farmacologia , Citoproteção/efeitos dos fármacos , Citoproteção/genética , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Masculino , Proteínas de Membrana/metabolismo , Modelos Biológicos , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Células Neuroendócrinas/efeitos dos fármacos , Células Neuroendócrinas/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Serina-Treonina Quinases TOR/metabolismo
12.
J Thorac Oncol ; 9(1): 18-25, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24346090

RESUMO

INTRODUCTION: The objective of this study was to identify and characterize echinoderm microtubule-associated protein-like 4 anaplastic lymphoma kinase fusion (EML4-ALK+) cancers by variant-specific, quantitative reverse transcription polymerase chain reaction (RT-PCR) assays in a large cohort of North American non-small-cell lung cancer (NSCLC) patients. METHODS: We developed a panel of single and multiplex RT-PCR assays suitable for rapid and accurate detection of the eight most common EML4-ALK+ variants and ALK gene expression in archival formalin-fixed, paraffin-embedded NSCLC specimens. EGFR and KRAS genotyping and thymidylate synthase RNA level by RT-PCR assays were available in a subset of patients. RESULTS: Between December 2009 and September 2012, 7344 NSCLC specimens were tested. An EML4-ALK+ transcript was detected in 200 cases (2.7%), including 109 V1 (54.5%), 20 V2 (10.0%), 68 V3 (34.0%), and three V5a (1.5%) variants. Median age was 54.5 years (range, 23-89), and 104 patients (52.0%) were women. The great majority (n=188, 94.0%) of EML4-ALK+ NSCLC tumors had adenocarcinoma histology. ALK expression level varied significantly among different EML4-ALK+ variants and individual tumors. Only one case each of concurrent EGFR or KRAS mutation was detected. The median thymidylate synthase RNA level from 85 EML4-ALK+ cancers was significantly lower compared with that of EML4-ALK-negative lung adenocarcinomas (2.02 versus 3.29, respectively, p<0.001). CONCLUSIONS: This panel of variant-specific, quantitative RT-PCR assays detects common EML4-ALK+ variants as well as ALK gene expression level in archival formalin-fixed paraffin-embedded NSCLC specimens. These RT-PCR assays may be useful as an adjunct to the standard fluorescence in situ hybridization assay to better understand biologic variability and response patterns to anaplastic lymphoma kinase inhibitors.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/genética , Proteínas de Fusão Oncogênica/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Quinase do Linfoma Anaplásico , Receptores ErbB/genética , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Inclusão em Parafina , Receptores Proteína Tirosina Quinases/genética
13.
Liver Int ; 34(1): 110-7, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24028323

RESUMO

BACKGROUND & AIMS: Non-alcoholic steatohepatitis (NASH) in children is a significant public health concern. Oxidative stress is an important component in the pathophysiology of NASH. Several enzymatic antioxidant mechanisms protect the liver from oxidative injury. Examination of the expression of these enzymes in NASH livers may provide insight on the roles for these antioxidant mechanisms in the pathophysiology of NASH. METHODS: The mRNA expression of catalase, glutathione peroxidase 1 (GPX1), glutathione reductase (GSR), paraoxonase 1 (PON1) and other reactive oxygen species-related genes was evaluated by microarray and quantitative real-time PCR analyses. The PON1 protein levels were evaluated in liver and serum by Western blot analyses. Serum enzymatic activities of GPX, GSR and PON1 (paraoxonase and arylesterase activities) were examined. RESULTS: NASH livers exhibited elevated mRNA expression of catalase and PON1, but not GPX1 or GSR. No difference in serum GPX or GSR activity was detected between NASH patients and controls. Elevated expression of PON1 mRNA and protein was detected in NASH livers, but serum PON1 protein and activities were not elevated. CONCLUSIONS: Elevated expression of catalase and PON1 suggests protective roles for these antioxidants in NASH livers. Given the importance of oxidative stress in the pathophysiology of NASH, future studies focusing on these enzymes could identify important targets for therapeutic or preventive interventions for NASH patients.


Assuntos
Arildialquilfosfatase/sangue , Fígado Gorduroso/enzimologia , Fígado/enzimologia , Estresse Oxidativo , Adolescente , Fatores Etários , Arildialquilfosfatase/genética , Biomarcadores/sangue , Western Blotting , Catalase/sangue , Catalase/genética , Criança , Pré-Escolar , Estudos Transversais , Fígado Gorduroso/sangue , Fígado Gorduroso/genética , Feminino , Perfilação da Expressão Gênica/métodos , Glutationa Peroxidase/sangue , Glutationa Peroxidase/genética , Glutationa Redutase/sangue , Glutationa Redutase/genética , Humanos , Masculino , Hepatopatia Gordurosa não Alcoólica , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/sangue , Reação em Cadeia da Polimerase em Tempo Real , Regulação para Cima , Adulto Jovem
14.
PLoS One ; 7(4): e35143, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22496902

RESUMO

UNLABELLED: The exact pathophysiology of non-alcoholic steatohepatitis (NASH) is not known. Previous studies suggest that dietary advanced glycation end products (AGEs) can cause oxidative stress in liver. We aim to study the effects of dietary AGEs on liver health and their possible role in the pathogenesis of NASH. METHODS: Two groups of mice were fed the same diet except the AGE content varied. One group was fed a high AGE diet and the second group was fed a regular AGE diet. Liver histology, alanine aminotransferase, aspartate aminotransferase, fasting glucose, fasting insulin, insulin resistance and glucose tolerance were assessed. RESULTS: Histology revealed that neutrophil infiltration occurred in the livers of the high AGE group at week 26; steatosis did not accompany liver inflammation. At week 39 livers from both groups exhibited macro- or micro-steatosis, yet no inflammation was detected. Higher insulin levels were detected in the regular AGE group at week 26 (P = 0.034), compared to the high AGE group. At week 39, the regular AGE group showed higher levels of alanine aminotransferase (P<0.01) and aspartate aminotransferase (P = 0.02) than those of the high AGE group. CONCLUSIONS: We demonstrate that a high AGE diet can cause liver inflammation in the absence of steatosis. Our results show that dietary AGEs could play a role in initiating liver inflammation contributing to the disease progression of NASH. Our observation that the inflammation caused by high AGE alone did not persist suggests interesting future directions to investigate how AGEs contribute to pro-oxidative and anti-oxidative pathways in the liver.


Assuntos
Dieta/efeitos adversos , Fígado Gorduroso/induzido quimicamente , Produtos Finais de Glicação Avançada/administração & dosagem , Fígado/efeitos dos fármacos , Alanina Transaminase/análise , Animais , Aspartato Aminotransferases/análise , Jejum/sangue , Fígado Gorduroso/enzimologia , Fígado Gorduroso/patologia , Teste de Tolerância a Glucose , Insulina/sangue , Resistência à Insulina , Fígado/citologia , Fígado/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Infiltração de Neutrófilos/efeitos dos fármacos
16.
Indian J Pharm Sci ; 73(1): 79-84, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22131627

RESUMO

A simple isocratic reversed phase high performance liquid chromatography was used to separate three impurities present in the sample of 8-chlorotheophylline. LC-MS was used for the characterization of impurities. Based on mass spectral data, the structures of these impurities were characterized as 3,7-dihydro-1,3-dimethyl-1H-purine-2,6-dione (impurity I), 3,7-dihydro-1,3,7-trimethyl-1H-purine-2,6-dione (impurity II) and isomer of 8-chloro-1,3-dimethyl-2,6(3H,1H)-purinedione (impurity III).

18.
Genes Cancer ; 1(1): 40-9, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20811583

RESUMO

There is overwhelming evidence that tyrosine kinases play an important role in cancer development. As a prototype of targeted therapy, tyrosine kinase inhibitors are now successfully applied to cancer treatment. However, as single agents, tyrosine kinase inhibitors have not achieved satisfactory results in the treatment of prostate cancer, principally due to their inability to efficiently kill tumor cells. The authors' laboratory has been interested in the role of the Src complex in prostate cancer progression, including the induction of androgen independence and metastasis. Previously, the authors reported that Src inhibitors such as saracatinib and PP2 caused G1 growth arrest and diminished invasiveness in prostate cancer cells but rarely apoptosis. Here, they have shown that Src family kinase (SFK) inhibitors can induce a high level of autophagy, which protects treated cells from undergoing apoptosis. Src siRNA knockdown experiments confirmed that autophagy was indeed caused by the lack of Src activity. The SFK inhibitor-induced autophagy is accompanied by the inhibition of the PI3K (type I)/Akt/mTOR signaling pathway. To test whether autophagy blockade could lead to enhanced cell death, pharmacological inhibitors (3-methyladenine and chloroquine) and a genetic inhibitor (siRNA targeting Atg7) were used in combination with SFK inhibitors. The results showed that autophagy inhibition effectively enhanced cell killing induced by SFK inhibitors. Importantly, the authors showed that a combination of saracatinib with chloroquine in mice significantly reduced prostate cancer (PC3) xenograft growth compared with the control group. Taken together, these data suggest that (1) autophagy serves a protective role in SFK inhibitor-mediated cell killing, and (2) clinically acceptable autophagy modulators may be used beneficially as adjunctive therapeutic agents for SFK inhibitors.

19.
Cancer Res ; 66(21): 10449-59, 2006 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-17079466

RESUMO

The inappropriate activation of androgen receptor (AR) by nonsteroids is considered a potential mechanism in the emergence of hormone-refractory prostate tumors, but little is known about the properties of these "pseudoactivated" AR. Here, we present the first comprehensive analysis closely examining the properties of AR activated by the neuropeptide bombesin that distinguish it from androgen-activated AR. We show that bombesin-activated AR (a) is required for bombesin-induced growth of LNCaP cells, (b) has a transcriptional profile overlapping with, but not identical to, androgen-activated AR, (c) activates prostate-specific antigen by preferentially binding to its proximal promoter, and (d) assembles a distinct coactivator complex. Significantly, we found that Src kinase is critical for bombesin-induced AR-mediated activity and is required for translocation and transactivation of AR. Additionally, we identify c-Myc, a Src target gene, to be activated by bombesin and a potential coactivator of AR-mediated activity specific to bombesin-induced signaling. Because Src kinase is often activated by other nonsteroids, such as other neuropeptides, growth factors, chemokines, and cytokines, our findings have general applicability and provide rationale for investigating the efficacy of the Src kinase pathway as a target for the prevention of relapsed prostate cancers.


Assuntos
Bombesina/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Receptores Androgênicos/efeitos dos fármacos , Quinases da Família src/fisiologia , Bombesina/uso terapêutico , Proliferação de Células/efeitos dos fármacos , Peptídeo Liberador de Gastrina/farmacologia , Humanos , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , Antígeno Prostático Específico/genética , Neoplasias da Próstata/patologia , Transporte Proteico/efeitos dos fármacos , Receptores Androgênicos/fisiologia , Transdução de Sinais
20.
Cancer Res ; 66(17): 8439-47, 2006 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-16951154

RESUMO

Androgen receptor (AR) is a ligand-induced transcriptional factor, which plays an important role in the normal development of prostate as well as in the progression of prostate cancer. Numerous coactivators, which associate with AR and function to remodel chromatin and recruit RNA polymerase II to enhance the transcriptional potential of AR, have been identified. Among these coactivators, few are protein kinases. In this study, we describe the characterization of a novel protein kinase, male germ cell-associated kinase (MAK), which serves as a coactivator of AR. We present evidence, which indicates that (a) MAK physically associates with AR (MAK and AR are found to be coprecipitated from cell extracts, colocalized in nucleus, and corecruited to prostate-specific antigen promoter in LNCaP as well as in transfected cells); (b) MAK is able to enhance AR transactivation potential in an androgen- and kinase-dependent manner in several prostate cancer cells and synergize with ACTR/steroid receptor coactivator-3 coactivator; (c) small hairpin RNA (shRNA) knocks down MAK expression resulting in the reduction of AR transactivation ability; (d) MAK-shRNA or kinase-dead mutant, when introduced into LNCaP cells, reduces the growth of the cells; and (e) microarray analysis of LNCaP cells carrying kinase-dead MAK mutant showed a significant impediment of AR signaling, indicating that endogenous MAK plays a general role in AR function in prostate cancer cells and likely to be a general coactivator of AR in prostate tissues. The highly restricted expression of this kinase makes it a potentially useful target for intervention of androgen independence.


Assuntos
Androgênios/fisiologia , Neoplasias da Próstata/genética , Proteínas Serina-Treonina Quinases/metabolismo , Linhagem Celular Tumoral , Cromatina/genética , Cromatina/patologia , Genes Reporter , Humanos , Cinética , Masculino , Próstata/patologia , Próstata/fisiologia , Próstata/fisiopatologia , Neoplasias da Próstata/patologia , Neoplasias da Próstata/fisiopatologia , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética
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