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1.
Toxicology ; 432: 152380, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31981723

RESUMO

Bisphenol AF (BPAF) is a derivative of bisphenol A (BPA) that is widely used in fluorinated polymers, fluorinated rubber, electronic equipment, plastic optical fibers, etc. Studies have shown that BPAF exposure is associated with a number of diseases; however, little is known about the effects of BPAF on cardiomyocytes. We investigated the impact of chronic exposure to BPAF on cardiomyocytes derived from embryonic stem cells (ESCs). The present study showed that chronic exposure to various concentrations of BPAF (0, 8, 200 and 1000 ng/ml) induces cardiomyocyte hypertrophy. The ratios of microfilaments to mitochondrial length and the ratio of microfilaments to cell nuclei and MYH7b levels indicate that BPAF exposure alters the morphology of the cells and mitochondria. Furthermore, BPAF exposure at concentrations from 8 to 1000 ng/ml results in an increase in G protein-coupled estrogen receptor (GPER) expression. Additionally, our results suggest that these effects of BPAF mediate cardiomyocyte hypertrophy apparently due to an increase in the production of reactive nitrogen species (RNS) via an increase in endothelial NO synthase (eNOS). These results imply that ESC-based myocardial differentiation can be an excellent cellular model to study BPAF-induced cardiotoxicity at the cellular and molecular levels.


Assuntos
Compostos Benzidrílicos/toxicidade , Células-Tronco Embrionárias/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Fenóis/toxicidade , Transdução de Sinais/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Animais , Calmodulina/biossíntese , Calmodulina/genética , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/ultraestrutura , Tamanho Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/ultraestrutura , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/ultraestrutura , Óxido Nítrico Sintase Tipo III/biossíntese , Óxido Nítrico Sintase Tipo III/genética , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/efeitos dos fármacos
2.
J Neurochem ; 151(6): 795-809, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31237956

RESUMO

Overactivation of N-methyl-d-aspartate glutamate receptors (NMDARs) after traumatic brain injury (TBI) contributes to excitotoxic cell death. The hyperactivation of NMDARs results in toxic levels of intracellular Ca2+ and in the activation of p53-mediated apoptosis pathway. Neuronal Ca2+ -dependent activator protein 1 (NCDAP1) was identified as an epileptogenic gene of unknown function in our laboratory. In this study, we investigated the expression and cellular localization of NCDAP1 in rat models of fluid percussion-induced TBI. NCDAP1 expression increased in the ipsilateral cortex and hippocampus adjacent to the lesion of the TBI rats compared with that in the sham-operated controls. In addition, NCDAP1 was co-expressed with neuronal marker (NeuN), and the results of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining suggest that NCDAP1 is involved in neuronal apoptosis that occurs after brain injury. In addition, the expression levels of p53, Bax, and active caspase-3 correlated with those of NCDAP1. To further investigate the function of NCDAP1, primary cultured neurons were employed to establish an apoptosis model. The expression of NCDAP1 was induced by NMDA-induced Ca2+ influx, and the knockdown of NCDAP1 by siRNA decreased apoptosis caused by treatment with NMDA. Silencing of NCDAP1 also reduced p53 expression, whereas the over-expression of NCDAP1 induced cell death and up-regulated the expression of p53. The inhibition of p53 with pifithrin alpha or siRNA counteracted the effects of NCDAP1. Based on our data, we suggest that NCDAP1 plays an important role in p53-mediated neuronal apoptosis following TBI.


Assuntos
Lesões Encefálicas Traumáticas/metabolismo , Calmodulina/biossíntese , Neurônios/metabolismo , Transdução de Sinais/fisiologia , Proteína Supressora de Tumor p53/biossíntese , Animais , Lesões Encefálicas Traumáticas/genética , Lesões Encefálicas Traumáticas/patologia , Calmodulina/genética , Morte Celular/fisiologia , Masculino , Neurônios/patologia , Ratos , Ratos Sprague-Dawley , Proteína Supressora de Tumor p53/genética
3.
J Am Heart Assoc ; 7(10)2018 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-29720499

RESUMO

BACKGROUND: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a familial arrhythmogenic syndrome characterized by sudden death. There are several genetic forms of CPVT associated with mutations in genes encoding the cardiac ryanodine receptor (RyR2) and its auxiliary proteins including calsequestrin (CASQ2) and calmodulin (CaM). It has been suggested that impairment of the ability of RyR2 to stay closed (ie, refractory) during diastole may be a common mechanism for these diseases. Here, we explore the possibility of engineering CaM variants that normalize abbreviated RyR2 refractoriness for subsequent viral-mediated delivery to alleviate arrhythmias in non-CaM-related CPVT. METHODS AND RESULTS: To that end, we have designed a CaM protein (GSH-M37Q; dubbed as therapeutic CaM or T-CaM) that exhibited a slowed N-terminal Ca dissociation rate and prolonged RyR2 refractoriness in permeabilized myocytes derived from CPVT mice carrying the CASQ2 mutation R33Q. This T-CaM was introduced to the heart of R33Q mice through recombinant adeno-associated viral vector serotype 9. Eight weeks postinfection, we performed confocal microscopy to assess Ca handling and recorded surface ECGs to assess susceptibility to arrhythmias in vivo. During catecholamine stimulation with isoproterenol, T-CaM reduced isoproterenol-promoted diastolic Ca waves in isolated CPVT cardiomyocytes. Importantly, T-CaM exposure abolished ventricular tachycardia in CPVT mice challenged with catecholamines. CONCLUSIONS: Our results suggest that gene transfer of T-CaM by adeno-associated viral vector serotype 9 improves myocyte Ca handling and alleviates arrhythmias in a calsequestrin-associated CPVT model, thus supporting the potential of a CaM-based antiarrhythmic approach as a therapeutic avenue for genetically distinct forms of CPVT.


Assuntos
Calmodulina/genética , Técnicas de Transferência de Genes , Terapia Genética/métodos , Frequência Cardíaca , Taquicardia Ventricular/terapia , Animais , Sinalização do Cálcio , Calmodulina/biossíntese , Calsequestrina/deficiência , Calsequestrina/genética , Modelos Animais de Doenças , Predisposição Genética para Doença , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos Cardíacos/metabolismo , Fenótipo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Taquicardia Ventricular/genética , Taquicardia Ventricular/metabolismo , Taquicardia Ventricular/fisiopatologia
4.
ACS Synth Biol ; 7(2): 405-411, 2018 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-29370697

RESUMO

Single-molecule FRET (smFRET) is a powerful tool to investigate conformational changes of biological molecules. In general, smFRET studies require protein samples that are site-specifically double-labeled with a pair of donor and acceptor fluorophores. The common approaches to produce such samples cannot be applied when studying the synthesis and folding of the polypeptide chain on the ribosome. The best strategy is to incorporate two fluorescent amino acids cotranslationally using cell-free protein synthesis systems. Here, we demonstrate the cotranslational site-specific incorporation into a model protein of Atto633, a dye with excellent photophysical properties, suitable for single molecule spectroscopy, together with a second dye using a combination of the sense cysteine and the nonsense amber codon. In this work we show that cotranslational incorporation of good fluorophores into proteins is a viable strategy to produce suitable samples for smFRET studies.


Assuntos
Calmodulina , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Engenharia de Proteínas/métodos , Modificação Traducional de Proteínas , Coloração e Rotulagem/métodos , Calmodulina/biossíntese , Calmodulina/química , Calmodulina/genética , Escherichia coli , Humanos
5.
Artigo em Inglês | MEDLINE | ID: mdl-27899032

RESUMO

BACKGROUND: We aimed to gain new insights into the pathogenesis of sporadic ALS (sALS) through a comprehensive proteomic analysis. METHODS: Protein profiles of the anterior and posterior horn in post-mortem spinal cord samples of 10 ALS patients and 10 controls were analysed using 2D-differential gel electrophoresis. The identified protein spots with statistically significant level changes and a spot ratio >2.0 were analysed by LC-MS/MS. RESULTS: In the posterior horn only 3 proteins were differentially expressed. In the anterior horn, 16 proteins with increased levels and 2 proteins with decreased levels were identified in ALS compared to controls. The identified proteins were involved in mitochondrial metabolism, calcium homeostasis, protein metabolism, glutathione homeostasis, protein transport and snRNP assembly. The two proteins with decreased levels, ATP5D and calmodulin, were validated by Western blot and immunostaining. Immunohistochemical and immunofluorescent double staining of ATP5D and synaptophysin showed that the reduction of ATP5D was most pronounced at synapses. CONCLUSIONS: We speculate that mitochondrial dysfunction in synaptic clefts could play an important role in sALS pathogenesis. A similar approach revealed decreased calmodulin expression mainly in the neuronal body and dendrites of ALS patients. These findings contribute to a deeper understanding of the disease process underlying ALS.


Assuntos
Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , ATPases Translocadoras de Prótons/genética , Medula Espinal/patologia , Sinapses/patologia , Cálcio/metabolismo , Calmodulina/biossíntese , Calmodulina/genética , Regulação para Baixo/genética , Eletroforese em Gel Bidimensional , Feminino , Perfilação da Expressão Gênica , Homeostase/genética , Humanos , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Mitocôndrias/patologia , ATPases Mitocondriais Próton-Translocadoras , Proteínas/metabolismo , Proteômica , Medula Espinal/química
6.
PLoS One ; 11(4): e0152837, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27046189

RESUMO

Polycystic ovary syndrome (PCOS) is a heterogenetic disorder in women that is characterized by arrested follicular growth and anovulatory infertility. The altered protein expression levels in the ovarian tissues reflect the molecular defects in folliculogenesis. To identify aberrant protein expression in PCOS, we analyzed protein expression profiles in the ovarian tissues of patients with PCOS. We identified a total of 18 protein spots that were differentially expressed in PCOS compared with healthy ovarian samples. A total of 13 proteins were upregulated and 5 proteins were downregulated. The expression levels of heat shock protein 90B1 (HSP90B1) and calcium signaling activator calmodulin 1 (CALM1) were increased by at least two-fold. The expression levels of HSP90B1 and CALM1 were positively associated with ovarian cell survival and negatively associated with caspase-3 activation and apoptosis. Knock-down of HSP90B1 with siRNA attenuated ovarian cell survival and increased apoptosis. In contrast, ovarian cell survival was improved and cell apoptosis was decreased in cells over-expressing HSP90B1. These results demonstrated the pivotal role of HSP90B1 in the proliferation and survival of ovarian cells, suggesting a critical role for HSP90B1 in the pathogenesis of PCOS. We also observed a downregulation of anti-inflammatory activity-related annexin A6 (ANXA6) and tropomyosin 2 (TPM2) compared with the normal controls, which could affect cell division and folliculogenesis in PCOS. This is the first study to identify novel altered gene expression in the ovarian tissues of patients with PCOS. These findings may have significant implications for future diagnostic and treatment strategies for PCOS using molecular interventions.


Assuntos
Regulação da Expressão Gênica , Glicoproteínas de Membrana/biossíntese , Ovário/metabolismo , Síndrome do Ovário Policístico/metabolismo , Adulto , Apoptose , Calmodulina/biossíntese , Caspase 3/metabolismo , Linhagem Celular , Ativação Enzimática , Feminino , Humanos , Ovário/patologia , Síndrome do Ovário Policístico/patologia , Tropomiosina/biossíntese
7.
Oncotarget ; 5(8): 2318-29, 2014 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-24810858

RESUMO

Long non-coding RNAs (lncRNAs) play key roles in the progression and metastasis of some carcinomas. We previously showed that the expression of lncRNA H19 (H19) was higher in gastric cancer (GC) tissues than that in paired noncanerous tissues. However, the underlying mechanisms remain unclear. In this study, H19/miR-675 knockdown models in the MKN45 cell line and ectopic expression models in the SGC7901 cell line were established, and a co-expression network of H19 was generated to identify target genes by RIP and DLR. The results showed that overexpression of H19 promoted the features of GC including proliferation, migration, invasion and metastasis. An H19 co-expression network identified ISM1 as a binding protein of H19, and its expression was positively correlated with that of H19. CALN1 was identified as a target gene of miR-675 and its expression was negatively correlated with that of miR-675. H19 and MiR-675 function in a similar manner. However, H19 RNA actively binds to ISM1 and miR-675 targets CALN1. These differences suggest that H19 plays other roles besides encoding miR-675 in GC. Our results suggest that the effect of H19 in GC is mediated by the direct upregulation of ISM1 and the indirect suppression of CALN1 expression via miR-675.


Assuntos
Regulação Neoplásica da Expressão Gênica/genética , Invasividade Neoplásica/genética , RNA Longo não Codificante/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Idoso , Animais , Western Blotting , Calmodulina/biossíntese , Calmodulina/genética , Carcinogênese/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Feminino , Técnicas de Silenciamento de Genes , Xenoenxertos , Humanos , Imunoprecipitação , Estimativa de Kaplan-Meier , Masculino , Camundongos , Camundongos Nus , MicroRNAs/genética , Pessoa de Meia-Idade , Prognóstico , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Gástricas/mortalidade , Regulação para Cima
8.
J Cell Biochem ; 115(2): 303-12, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23996571

RESUMO

Human osteoclasts were differentiated using receptor activator of NFκB ligand (RANKL) and macrophage colony stimulating factor (M-CSF) from colony forming unit-granulocyte macrophage (CFU-GM) precursors of the myeloid lineage grown from umbilical cord blood. Gene expression profiling using quantitative polymerase chain reaction (Q-PCR) showed more than 1,000-fold induction of chemokine MCP-1 within 24 h of RANKL treatment. MCP-1 mRNA content exceeds that of other assayed chemokines (CCL1, 3, 4, and 5) at all time points up to day 14 of treatment. MCP-1 induction preceded peak induction of calcium signaling activator calmodulin 1 (CALM1) and transcription factors JUN and FOS, which were at 3 days. Key osteoclast related transcription factors NFATc1 and NFATc2 showed peak induction at 7 days, while marker genes for osteoclast function cathepsin K and tartrate resistance acid phosphatase (TRAP) were maximally induced at 14 days, corresponding with mature osteoclast function. To test whether the early and substantial peak in MCP-1 expression is part of human osteoclast differentiation events, a dominant negative inhibitor of MCP-1 (7ND) was added simultaneously with RANKL and M-CSF, resulting in blockade of CALM1, JUN and NFATc2 induction and strong inhibition of human osteoclast differentiation. These data show that a cascade of gene expression leading to osteoclast differentiation depends on intact early MCP-1 induction and signaling in human osteoclasts.


Assuntos
Diferenciação Celular/genética , Quimiocina CCL2/metabolismo , Osteoclastos/citologia , Transdução de Sinais/genética , Calmodulina/biossíntese , Células Cultivadas , Quimiocina CCL2/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Ligante RANK/genética , Cordão Umbilical/citologia
9.
Neurochem Int ; 63(3): 180-94, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23796501

RESUMO

Alpha-synuclein (α-Syn) is the principal protein component of Lewy bodies, a pathological hallmark of Parkinson's disease (PD). This protein may regulate protein phosphatase 2A (PP2A) activity, although the molecular mechanisms for α-Syn-mediated regulation of PP2A and the potential neuroprotective actions of PP2A against PD-associated pathology remain largely unexplored. We found that α-Syn gene overexpression in SK-N-SH cells and primary neurons led to PP2A/C phosphorylation at Y307, a known target of Src kinase, and consequent phosphatase inhibition. In addition, phospho-activated Src (p-Y416 Src, pSrc) was higher in SK-N-SH cells and primary neurons overexpressing α-Syn. Thus, α-Syn may promote Src activation and PP2A inactivation, leading to hyperphosphorylation of proteins. Immunoprecipitation revealed higher calmodulin/Src complex formation in α-Syn-overexpressing cells and α-Syn transgenic mice. A TUNEL apoptosis assay and an MTT cell viability assay demonstrated that the PP2A activator C2-ceramide protected neurons against α-Syn-induced cell injury. Buffering the Ca(2+) elevations induced by α-Syn overexpression ameliorated the cytotoxicity of α-Syn. Our findings define a potential molecular mechanism for α-Syn-mediated regulation of PP2A through formation of the calmodulin/Src complex, activation of Src, and Src-mediated phospho-inhibition of PP2A. Overexpression of α-Syn may lead to neurodegeneration in PD in part by suppressing the endogenous neuroprotective activity of PP2A.


Assuntos
Calmodulina/biossíntese , Proteína Fosfatase 2/metabolismo , alfa-Sinucleína/metabolismo , Quinases da Família src/biossíntese , Animais , Sequência de Bases , Linhagem Celular Tumoral , Primers do DNA , Humanos , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Transgênicos , Fosforilação , Proteína Fosfatase 2/antagonistas & inibidores , Ratos , Ratos Sprague-Dawley
10.
Biochem Biophys Res Commun ; 425(4): 750-4, 2012 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-22884799

RESUMO

The existence of extracellular calmodulin (CaM) has had a long and controversial history. CaM is a ubiquitous calcium-binding protein that has been found in every eukaryotic cell system. Calcium-free apo-CaM and Ca(2+)/CaM exert their effects by binding to and regulating the activity of CaM-binding proteins (CaMBPs). Most of the research done to date on CaM and its CaMBPs has focused on their intracellular functions. The presence of extracellular CaM is well established in a number of plants where it functions in proliferation, cell wall regeneration, gene regulation and germination. While CaM has been detected extracellularly in several animal species, including frog, rat, rabbit and human, its extracellular localization and functions are less well established. In contrast the study of extracellular CaM in eukaryotic microbes remains to be done. Here we show that CaM is constitutively expressed and secreted throughout asexual development in Dictyostelium where the presence of extracellular CaM dose-dependently inhibits cell proliferation but increases cAMP mediated chemotaxis. During development, extracellular CaM localizes within the slime sheath where it coexists with at least one CaMBP, the matricellular CaM-binding protein CyrA. Coupled with previous research, this work provides direct evidence for the existence of extracellular CaM in the Dictyostelium and provides insight into its functions in this model amoebozoan.


Assuntos
Calmodulina/metabolismo , Quimiotaxia/fisiologia , AMP Cíclico/fisiologia , Dictyostelium/crescimento & desenvolvimento , Calmodulina/biossíntese , Calmodulina/farmacologia , Proliferação de Células , Quimiotaxia/efeitos dos fármacos , AMP Cíclico/farmacologia , Dictyostelium/efeitos dos fármacos , Dictyostelium/fisiologia
11.
Exp Parasitol ; 123(4): 326-33, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19703447

RESUMO

We have cloned and expressed calmodulin (CaM) from Trypanosoma cruzi, for the first time, to obtain large amounts of protein. CaM is a very well conserved protein throughout evolution, sharing 100% amino acid sequence identity between different vertebrates and 99% between trypanosomatids. However, there is 89% amino acid sequence identity between T. cruzi and vertebrate CaMs. The results demonstrate significant differences between calmodulin from T. cruzi and mammals. First, a polyclonal antibody developed in an egg-yolk system to the T. cruzi CaM recognizes the autologous CaM but not the CaM from rat. Second, it undergoes a larger increase in the alpha-helix content upon binding with Ca(2+), when compared to CaM from vertebrates. Finally, two classic CaM antagonists, calmidazolium and trifluoperazine, capable of inhibiting the action of CaM in mammals when assayed on the plasma membrane Ca(2+) pump, showed a significant loss of activity when assayed upon stimulation with the T. cruzi CaM.


Assuntos
Calmodulina/biossíntese , Trypanosoma cruzi/metabolismo , Animais , Anticorpos Antiprotozoários/biossíntese , ATPases Transportadoras de Cálcio/sangue , Calmodulina/química , Calmodulina/genética , Calmodulina/imunologia , Galinhas , Dicroísmo Circular , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Ensaio de Imunoadsorção Enzimática , Membrana Eritrocítica/enzimologia , Feminino , Regulação da Expressão Gênica , Humanos , Imunoglobulinas/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Alinhamento de Sequência , Análise de Sequência de DNA , Trypanosoma cruzi/química , Trypanosoma cruzi/genética
12.
Mol Cells ; 27(4): 475-80, 2009 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-19390829

RESUMO

The transcription of soybean (Glycine max) calmodulin isoform-4 (GmCaM-4) is dramatically induced within 0.5 h of exposure to pathogen or NaCl. Core cis-acting elements that regulate the expression of the GmCaM-4 gene in response to pathogen and salt stress were previously identified, between -1,207 and -1,128 bp, and between -858 and -728 bp, in the GmCaM-4 promoter. Here, we characterized the properties of the DNA-binding complexes that form at the two core cis-acting elements of the GmCaM-4 promoter in pathogen-treated nuclear extracts. We generated GUS reporter constructs harboring various deletions of approximately 1.3-kb GmCaM-4 promoter, and analyzed GUS expression in tobacco plants transformed with these constructs. The GUS expression analysis suggested that the two previously identified core regions are involved in inducing GmCaM-4 expression in the heterologous system. Finally, a transient expression assay of Arabidopsis protoplasts showed that the GmCaM-4 promoter produced greater levels of GUS activity than did the CaMV35S promoter after pathogen or NaCl treatments, suggesting that the GmCaM-4 promoter may be useful in the production of conditional gene expression systems.


Assuntos
Calmodulina/genética , Glycine max/genética , Nicotiana/genética , Sítios de Ligação , Calmodulina/biossíntese , Proteínas de Ligação a DNA , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/química , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Isoformas de Proteínas , Análise de Sequência de DNA , Glycine max/metabolismo , Estresse Fisiológico , Nicotiana/metabolismo , Ativação Transcricional
13.
J Prosthodont ; 18(1): 11-6, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19166543

RESUMO

PURPOSE: The purpose of this study was to test whether calmodulin-like protein (CLP) is expressed in normal human oral mucosal cells and if downregulation of CLP occurs in malignant transformation. MATERIALS AND METHODS: Oral mucosal tissue was taken from three individuals in a double-blind manner. The samples were cut, measured, and homogenized. Total RNA was extracted and reverse transcribed. Each cDNA sample was subjected to polymerase chain reaction (PCR). PCR fragments were purified, cloned, and sequenced to verify the presence of CLP. Three oral mucosal tissue samples with biopsy-confirmed squamous cell carcinoma were obtained. These samples demonstrated regions of normal epithelial cells as well as invasive squamous cell carcinoma. One normal breast epithelial sample was also obtained for positive control. Sections were stained with an affinity-purified CLP antibody and counterstained with a diluted hematoxylin. Two observers evaluated the specimens for expression of CLP. Staining patterns and intensity were noted in normal oral mucosa, comparing them to the normal breast epithelium sample. Staining patterns and intensity were then observed in squamous tumor cells, comparing them to the patterns of benign squamous mucosa. RESULTS: CLP coding sequences were positively identified from the normal oral mucosal tissue samples by reverse transcription and polymerase chain reaction (RT-PCR) with 100% identity to the published CLP sequence (accession #M58026). In the three oral mucosa tissue samples with known squamous cell carcinoma, expression of CLP was readily detected in areas of normal oral mucosa, while a notable downregulation of CLP expression occurred in areas of malignant transformation. The staining intensity was equivalent to the staining seen in the benign breast epithelium used as a control. In the areas of squamous cell carcinoma, a decrease in CLP immunoreactivity occurred. There was a sharp contrast in staining quality and clarity between benign and malignant tissue. In the majority of the carcinoma regions, a complete lack of immunoreactivity was noted. CONCLUSIONS: The RNA for human CLP is found in normal oral mucosa. CLP expression is seen in normal oral mucosa with a downregulation of CLP expression in malignant transformation.


Assuntos
Calmodulina/biossíntese , Carcinoma de Células Escamosas/metabolismo , Transformação Celular Neoplásica/metabolismo , Mucosa Bucal/metabolismo , Neoplasias Bucais/metabolismo , Calmodulina/genética , Transformação Celular Neoplásica/genética , Método Duplo-Cego , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa
14.
Angew Chem Int Ed Engl ; 48(9): 1633-5, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19156778

RESUMO

Ignoring the STOP sign: A pyrrolysine analogue bearing a terminal alkyne was site-specifically incorporated into recombinant calmodulin (CaM) through a UAG codon. The resulting protein was labeled with an azide-containing dye using a copper(I)-catalyzed click reaction. Subsequent application of an orthogonal cysteine tagging method yielded a CaM labeled with two distinct fluorophores that enabled its study by FRET spectroscopy.


Assuntos
Calmodulina/química , Lisina/análogos & derivados , Proteínas Recombinantes/química , Alcinos/química , Azidas/síntese química , Azidas/química , Calmodulina/biossíntese , Calmodulina/genética , Catálise , Códon de Terminação/genética , Cobre/química , Transferência Ressonante de Energia de Fluorescência , Lisina/síntese química , Lisina/química
15.
J Biomol NMR ; 42(4): 271-7, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18937031

RESUMO

We established a novel strategy for preparing uniformly stable isotope-labeled proteins by using suspension-cultured plant cells and an inducible virus vector encoding the research target. By using this new method, we demonstrated the expression of three proteins, namely, Escherichia coli dihydrofolate reductase (DHFR), chicken calmodulin (CaM), and porcine protein kinase C-dependent protein phosphatase-1 inhibitor with a molecular mass of 17-kDa (CPI-17). In addition, we successfully expressed bovine pancreatic trypsin inhibitor (BPTI), which contains three pairs of disulfide bonds, as the soluble form. In the most efficient case, as little as 50 ml culture yielded 3-4 mg (15)N-labeled protein suitable for NMR experiments. The (1)H-(15)N HSQC spectra of all of these proteins clearly indicated that their structures were identical to those of their counterparts reported previously. Thus, the present results suggest that our novel protocol is a potential method for NMR sample preparation.


Assuntos
Marcação por Isótopo/métodos , Nicotiana/metabolismo , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Agrobacterium tumefaciens/metabolismo , Aprotinina/biossíntese , Aprotinina/química , Calmodulina/biossíntese , Calmodulina/química , Células Cultivadas , Dissulfetos/metabolismo , Vetores Genéticos , Fosfoproteínas Fosfatases/biossíntese , Fosfoproteínas Fosfatases/química , Tetra-Hidrofolato Desidrogenase/biossíntese , Tetra-Hidrofolato Desidrogenase/química , Transfecção
16.
J Cell Biochem ; 103(2): 471-8, 2008 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-17546603

RESUMO

Tyroserleutide (YSL) is a tripeptide compound that has exhibited inhibitory effects on hepatocellular carcinoma in our previous research. The mechanism of this antitumor activity involves the second messenger, Ca(2+). Ca(2+) influences cell function through the Ca(2+)/calmodulin (CaM) pathway, and abnormality of the Ca(2+)/CaM system correlates closely with the occurrence of tumors. In addition, CaM associates with phosphatidylinositol 3 kinase (PI3K), thereby enhancing the activity of PI3K, which promotes cell proliferation. In order to investigate its anti-tumor mechanism, we studied the effects of YSL on CaM protein expression and mRNA level, PI3K activity, PI3K regulatory subunit p85 protein expression and mRNA level, and the mRNA level of PI3K catalytic subunits p110alpha and p110gamma in human hepatocellular carcinoma BEL-7402 xenograft tumors in nude mice. Our results showed that YSL decreased the mRNA level and protein expression of CaM, inhibited the activity of PI3K, and reduced the mRNA level and protein expression of the PI3K regulatory subunit p85 and mRNA level of PI3K catalytic subunits p110alpha and p110gamma. Accordingly, it is suggestive that the anti-tumor effects of YSL may be mediated by down regulation of CaM and PI3K subunits p85 and p110, influencing the signal transduction pathway in the tumor cells and perhaps overcoming the dysfunctional PI3K activity in tumors.


Assuntos
Antineoplásicos/farmacologia , Calmodulina/biossíntese , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias Hepáticas Experimentais/tratamento farmacológico , Proteínas de Neoplasias/biossíntese , Oligopeptídeos/farmacologia , Fosfatidilinositol 3-Quinases/biossíntese , Animais , Antineoplásicos/uso terapêutico , Sinalização do Cálcio/efeitos dos fármacos , Calmodulina/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral/transplante , Classe I de Fosfatidilinositol 3-Quinases , Indução Enzimática/efeitos dos fármacos , Feminino , Humanos , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas Experimentais/metabolismo , Camundongos , Camundongos Nus , Proteínas de Neoplasias/genética , Oligopeptídeos/uso terapêutico , Fosfatidilinositol 3-Quinases/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , Organismos Livres de Patógenos Específicos , Ensaios Antitumorais Modelo de Xenoenxerto
17.
Artif Organs ; 31(4): 274-7, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17437495

RESUMO

Sustained myocardial hypertrophy is associated with an increased risk of sudden death and progression to heart failure. Multiple signal pathways are involved in cardiac hypertrophy and understanding their interaction may point to new therapeutic targets. In this work, we tested the hypothesis that adenovirus-mediated calmodulin (CaM) antisense expression will reduce the intracellular availability of CaM and inhibit the hypertrophic response. Three recombinant adenoviruses were constructed: AdASCaM, containing the AntiSense sequence of CaM and the enhanced green fluorescent protein (GFP) coding sequence; AdCaM, containing the coding sequence of CaM and the GFP sequence; and the AdGFP, containing the GFP coding sequence. Neonatal rat ventricular cardiomyocytes were infected with AdASCaM, AdCaM, or AdGFP and stimulated with phenylephrine (PE, 50 microM) or angiotensin II (AngII, 10 microM) for 48 h and cell surface area measured with planimetry. After PE treatment, the surface areas of cardiomyocytes infected with AdASCaM or AdGFP were 411 +/- 174.3 micro(2) and 832.6 +/- 372.3 micro(2), respectively (P < 0.01). After AngII treatment, the surface areas of cardiomyocytes infected with AdASCaM or AdGFP were 441.5 +/- 149.2 micro(2) and 726 +/- 328.3 micro(2), respectively (P < 0.01). Adenoviral expression of the CaM antisense (AdASCaM) significantly inhibited PE or AngII-induced cardiomyocyte hypertrophy. Cardiomyocytes infected with the AdCaM showed increased area when compared with those infected with the AdGFP. These results suggest that adenovirus-mediated changes in CaM expression may alter hypertrophy in cardiac myocytes.


Assuntos
Adenoviridae/genética , Calmodulina/biossíntese , Vetores Genéticos , Miócitos Cardíacos/patologia , Oligonucleotídeos Antissenso/farmacologia , Angiotensina II/farmacologia , Animais , Animais Recém-Nascidos , Calmodulina/genética , Crescimento Celular/efeitos dos fármacos , Células Cultivadas , Ventrículos do Coração/citologia , Miócitos Cardíacos/metabolismo , Fenilefrina/farmacologia , Ratos , Vasoconstritores/farmacologia
18.
Plant Cell Physiol ; 48(3): 414-23, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17251204

RESUMO

Thirteen tobacco calmodulin (CaM) genes fall into three distinct amino acid homology types. Wound-inducible type I isoforms NtCaM1 and 2 were moderately induced by tobacco mosaic virus (TMV)-mediated hypersensitive reaction, and the type III isoform NtCaM13 was highly induced, while the type II isoforms NtCaM3-NtCaM12 showed little response. Type I and III knockdown tobacco lines were generated using inverted repeat sequences from NtCaM1 and 13, respectively, to evaluate the contribution of pathogen-induced calmodulins (CaMs) to disease resistance. After specific reduction of type I and III CaM gene expression was confirmed in both transgenic lines, we analyzed the response to TMV infection, and found that TMV susceptibility was slightly enhanced in type III CaM knockdown lines compared with the control line. Resistance to a compatible strain of the bacterial pathogen Ralstonia solanacearum, and fungal pathogens Rhizoctonia solani and Pythium aphanidermatum was significantly lower in type III but not in type I CaM knockdown plants. Expression of jasmonic acid (JA)- and/or ethylene-inducible basic PR genes was not affected in these lines, suggesting that type III CaM isoforms are probably involved in basal defense against necrotrophic pathogens in a manner that is independent of JA and ethylene signaling.


Assuntos
Calmodulina/biossíntese , Nicotiana/metabolismo , Nicotiana/microbiologia , Proteínas de Plantas/biossíntese , Sequência de Bases , Calmodulina/genética , Primers do DNA/genética , DNA de Plantas/genética , Inativação Gênica , Genes de Plantas , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Pythium/patogenicidade , Ralstonia solanacearum/patogenicidade , Rhizoctonia/patogenicidade , Nicotiana/genética , Vírus do Mosaico do Tabaco/patogenicidade
19.
Zhonghua Yi Xue Za Zhi ; 86(29): 2017-20, 2006 Aug 08.
Artigo em Chinês | MEDLINE | ID: mdl-17064542

RESUMO

OBJECTIVE: To investigate the expression of calmodulin in the articular process of vertebrae of adolescent idiopathic scoliosis (AIS) patients and congenital scoliosis (CS) patients and its possible effects on the pathogenesis of AIS. METHODS: Nine AIS patients aged 13 - 17, and 10 patients of CS, aged 11 - 18, underwent orthopedic surgery. Specimens of the inferior articular process from the apical vertebrae and upper end vertebrae discarded during operation were collected from the AIS patients and specimens of the. Inferior articular process from the upper end vertebrae wee collected from the CS patients. Immunohistochemistry was used to detect the protein expression of calmodulin, and in situ hybridization was adopted to examine the mRNA expression of calmodulin. RESULTS: Calmodulin were expressed in the bone cells. There was no difference between the expression of calmodulin at the concave side and convex side, or between the apical vertebrae and end vertebrae. The expression of calmodulin of the AIS group was significant lower than that of the CS group (P < 0.05). The result of immunohistochemistry and that of in situ hybridization corresponded with each other. CONCLUSION: The expression of calmodulin in the articular process of vertebrae of AIS patients is low, suggesting that it plays a role in the development and progression of AIS.


Assuntos
Calmodulina/biossíntese , Escoliose/metabolismo , Articulação Zigapofisária/metabolismo , Adolescente , Criança , Humanos , RNA Mensageiro/biossíntese , Escoliose/congênito , Escoliose/patologia
20.
Zhonghua Yi Xue Za Zhi ; 84(16): 1358-61, 2004 Aug 17.
Artigo em Chinês | MEDLINE | ID: mdl-15387945

RESUMO

OBJECTIVE: The aim of this study was to identify whether one side of the apex of the scoliotic curve showed greater muscular abnormalities than the other. This investigation sought to compare directly the expression and localization of Calmodulin (CaM) and nNOS of the thoracic spinal musculature on convex side with those on the concave side in the scoliosis patients attending for spinal surgery. METHODS: The patient group comprised 8 females and 2 males, scheduled for spinal surgery, in whom the apex of the scoliotic curve arose between T6 and T11. Muscle biopsies were taken bilaterally during surgery from the superficial multifidus muscle at the apex of the curve between the 6th and 11th thoracic vertebral levels. Part of the tissue was fixed in Formalin and stained with hematoxylin and eosin; the remaining tissue was snap frozen and processed for immunohistochemistry and western blot. The following antibodies were used for the immunohistochemistry at dilutions optimized in preliminary experiments. Goat polyclonal anti-human-CaM and mouse monoclonal anti-human-nNOS were used as primary antibodies. CaM and nNOS were detected by immunohistochemical method and Western-blot. Western blotting was done with antibodies to CaM. RESULTS: Compared with control muscle and the muscle on the concave side of the scoliotic curve, A significant decrease in CaM and nNOS was found on the convex side of paraspinal muscle from idiopathic scoliosis patients, Western blotting with an CaM antibody and nNOS antibody shows that there are more CaM and nNOS protein in the concave side of paraspinal muscle in these patients with adolescent idiopathic scoliosis than in the convex side. CONCLUSION: This asymmetry of CaM and nNOS in the paraspinal muscle may play a role in the pathogenensis of IS.


Assuntos
Calmodulina/biossíntese , Músculo Esquelético/metabolismo , Óxido Nítrico Sintase/biossíntese , Escoliose/metabolismo , Adolescente , Adulto , Calmodulina/genética , Criança , Feminino , Humanos , Masculino , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/patologia , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo I , Escoliose/patologia , Vértebras Torácicas
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