Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 16 de 16
Filtrar
1.
Hum Mol Genet ; 24(13): 3638-50, 2015 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-25801283

RESUMO

Protein aggregate myopathies (PAMs) define muscle disorders characterized by protein accumulation in muscle fibres. We describe a new PAM in a patient with proximal muscle weakness and hypertrophic cardiomyopathy, whose muscle fibres contained inclusions containing myosin and myosin-associated proteins, and aberrant distribution of microtubules. These lesions appear as intact A- and M-bands lacking thin filaments and Z-discs. These features differ from inclusions in myosin storage myopathy (MSM), but are highly similar to those in mice deficient for the muscle-specific RING finger proteins MuRF1 and MuRF3. Sanger sequencing excluded mutations in the MSM-associated gene MYH7 but identified mutations in TRIM63 and TRIM54, encoding MuRF1 and MuRF3, respectively. No mutations in other potentially disease-causing genes were identified by Sanger and whole exome sequencing. Analysis of seven family members revealed that both mutations segregated in the family but only the homozygous TRIM63 null mutation in combination with the heterozygous TRIM54 mutation found in the proband caused the disease phenotype. Both MuRFs are microtubule-associated proteins localizing to sarcomeric M-bands and Z-discs. They are E3 ubiquitin ligases that play a role in degradation of sarcomeric proteins, stabilization of microtubules and myogenesis. Lack of ubiquitin and the 20S proteasome subunit in the inclusions found in the patient suggested impaired turnover of thick filament proteins. Disruption of microtubules in cultured myotubes was rescued by transient expression of wild-type MuRF1. The unique features of this novel myopathy point to defects in homeostasis of A-band proteins in combination with instability of microtubules as cause of the disease.


Assuntos
Proteínas Musculares/genética , Debilidade Muscular/genética , Mutação , Agregação Patológica de Proteínas/genética , Ubiquitina-Proteína Ligases/genética , Adulto , Idoso , Animais , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Células Musculares/metabolismo , Proteínas Musculares/metabolismo , Debilidade Muscular/metabolismo , Músculo Esquelético/metabolismo , Linhagem , Agregados Proteicos , Agregação Patológica de Proteínas/metabolismo , Espanha , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases/metabolismo
3.
Artigo em Inglês | MEDLINE | ID: mdl-22919407

RESUMO

Natural honey is well known for its therapeutic value and has been used in traditional medicine of different cultures throughout the world. The aim of this study was to investigate the anti-inflammatory effect of Malaysian Gelam honey in inflammation-induced rats. Paw edema was induced by a subplantar injection of 1% carrageenan into the rat right hind paw. Rats were treated with the nonsteroidal anti-inflammatory drug (NSAID) Indomethacin (10 mg/kg, p.o.) or Gelam honey at different doses (1 or 2 g/kg, p.o.). The increase in footpad thickness was considered to be edema, which was measured using a dial caliper. Plasma and paw tissue were collected to analyze the production of inflammatory mediators, such as NO, PGE(2), TNF-α, and IL-6, as well as iNOS and COX-2. The results showed that Gelam honey could reduce edema in a dose-dependent fashion in inflamed rat paws, decrease the production of NO, PGE(2), TNF-α, and IL-6 in plasma, and suppress the expression of iNOS, COX-2, TNF-α, and IL-6 in paw tissue. Oral pretreatment of Gelam honey at 2 g/kg of body weight at two time points (1 and 7 days) showed a significantly decreased production of proinflammatory cytokines, which was similar to the effect of the anti-inflammatory drug Indomethacin (NSAID), both in plasma and tissue. Thus, our results suggest that Gelam honey has anti-inflammatory effects by reducing the rat paw edema size and inhibiting the production of proinflammatory mediators. Gelam honey is potentially useful for treating inflammatory conditions.

4.
BMC Musculoskelet Disord ; 13: 262, 2012 Dec 29.
Artigo em Inglês | MEDLINE | ID: mdl-23273262

RESUMO

BACKGROUND: The formation of contractile myofibrils requires the stepwise onset of expression of muscle specific proteins. It is likely that elucidation of the expression patterns of muscle-specific sarcomeric proteins is important to understand muscle disorders originating from defects in contractile sarcomeric proteins. METHODS: We investigated the expression profile of a panel of sarcomeric components with a focus on proteins associated with a group of congenital disorders. The analyses were performed in cultured human skeletal muscle cells during myoblast proliferation and myotube development. RESULTS: Our culture technique resulted in the development of striated myotubes and the expression of adult isoforms of the sarcomeric proteins, such as fast TnI, fast TnT, adult fast and slow MyHC isoforms and predominantly skeletal muscle rather than cardiac actin. Many proteins involved in muscle diseases, such as beta tropomyosin, slow TnI, slow MyBPC and cardiac TnI were readily detected in the initial stages of muscle cell differentiation, suggesting the possibility of an early role for these proteins as constituent of the developing contractile apparatus during myofibrillogenesis. This suggests that in disease conditions the mechanisms of pathogenesis for each of the mutated sarcomeric proteins might be reflected by altered expression patterns, and disturbed assembly of cytoskeletal, myofibrillar structures and muscle development. CONCLUSIONS: In conclusion, we here confirm that cell cultures of human skeletal muscle are an appropriate tool to study developmental stages of myofibrillogenesis. The expression of several disease-associated proteins indicates that they might be a useful model system for studying the pathogenesis of muscle diseases caused by defects in specific sarcomeric constituents.


Assuntos
Diferenciação Celular , Perfilação da Expressão Gênica , Desenvolvimento Muscular , Fibras Musculares de Contração Rápida/metabolismo , Fibras Musculares de Contração Lenta/metabolismo , Proteínas Musculares/metabolismo , Doenças Musculares/metabolismo , Actinas/metabolismo , Diferenciação Celular/genética , Proliferação de Células , Células Cultivadas , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Humanos , Desenvolvimento Muscular/genética , Fibras Musculares de Contração Rápida/patologia , Fibras Musculares de Contração Lenta/patologia , Proteínas Musculares/genética , Doenças Musculares/genética , Doenças Musculares/patologia , Cadeias Pesadas de Miosina/metabolismo , Isoformas de Proteínas , Sarcômeros/metabolismo , Sarcômeros/patologia , Fatores de Tempo , Tropomiosina/metabolismo , Troponina I/metabolismo , Troponina T/metabolismo
5.
Open Access Rheumatol ; 14: 113-121, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35756976

RESUMO

Purpose: To assess the clinical impact of rheumatoid factor (RF) and anti-cyclic citrullinated peptide antibody (ACPA)'s seropositivity on treatment response in patients with rheumatoid arthritis (RA) treated with etanercept. Patients and Methods: A retrospective analysis of patients with RA registered in Baghdad Teaching Hospital Registry from May 2012 to August 2019 was conducted. Patients aged ≥18 years, meeting the ACR/EULAR 2010 criteria for RA, being treated with etanercept, and followed up at ≥1 year after etanercept initiation were included; patients who received any other biologics for RA were excluded. Patients were classified as seropositive (RF- and ACPA-positive), seronegative (RF- and ACPA-negative), RF-positive, RF-negative, ACPA-positive, and ACPA-negative. The primary outcomes included Clinical Disease Activity Index (CDAI) and Disease Activity Score 28 (DAS28) which were measured at one year after treatment initiation. Results: At baseline, a total of 1318 (88.3%) patients were seropositive; 1122 (75.2%) and 1054 (70.6%) patients were RF- and ACPA-positive, respectively. Baseline mean CDAI scores were significantly (P = 0.001) higher among seropositive patients compared with seronegative patients. The baseline mean DAS28 score was also significantly higher in ACPA-positive group compared with the ACPA-negative group (P = 0.021). At baseline, the number of patients who had high CDAI scores was significantly higher among the seropositive, RF-positive, and ACPA-positive groups (P = 0.001, P = 0.001, and P = 0.002, respectively). After one year of treatment with etanercept, among seropositive versus seronegative and ACPA-positive versus ACPA-negative groups, there was a significant improvement in terms of the mean CDAI score (P = 0.004 and P = 0.017, respectively) and CDAI response (P = 0.011 and P = 0.048, respectively). At one year, the proportion of patients among the seropositive versus seronegative group who reached remission were 566 (42.9%) versus 78 (44.6%) and 642 (47.3%) versus 83 (47.4%), for CDAI and DAS28 response, respectively. Conclusion: The results imply that seropositivity and ACPA-positivity may influence the treatment response in patients with RA, who were treated with etanercept.

6.
J Med Genet ; 47(8): 575-7, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19858127

RESUMO

BACKGROUND: Myosin binding protein C (MyBPC) is essential for the structure of the sarcomeres in striated muscle. There is one cardiac specific isoform and two skeletal muscle specific isoforms. Mutations in MYBPC3 encoding the cardiac isoform cause cardiomyopathy. METHODS AND RESULTS: We have identified an infant with fatal cardiomyopathy due to a homozygous mutation, p.R943X, in MYBPC3. The patient also had an unexpected skeletal myopathy. The patient expressed the cardiac specific MyBPC isoform in skeletal muscle at transcript and protein levels. Numerous muscle fibres expressing the mutant cardiac isoform showed structural abnormalities with disorganisation of sarcomeres and depletion of myosin thick filaments. CONCLUSIONS: The surprising identification of a skeletal myopathy in this patient was due to aberrant expression of mutant cardiac MyBPC in skeletal muscle.


Assuntos
Cardiomiopatias/genética , Proteínas de Transporte/genética , Predisposição Genética para Doença , Doenças Musculares/genética , Mutação/genética , Miocárdio/metabolismo , Miocárdio/patologia , Sequência de Bases , Análise Mutacional de DNA , Evolução Fatal , Feminino , Humanos , Lactente , Dados de Sequência Molecular , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Músculo Esquelético/ultraestrutura , Doenças Musculares/patologia
7.
Molecules ; 16(8): 6378-95, 2011 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-21796076

RESUMO

Two types of monofloral Malaysian honey (Gelam and Nenas) were analyzed to determine their antioxidant activities and total phenolic and flavonoid contents, with and without gamma irradiation. Our results showed that both types of honey can scavenge free radicals and exhibit high antioxidant-reducing power; however, Gelam honey exhibited higher antioxidant activity (p < 0.05) than Nenas honey, which is in good correlation (r = 0.9899) with its phenolic contents. Interestingly, we also noted that both irradiated honeys have higher antioxidant activities and total phenolic and flavonoid contents compared to nonirradiated honeys by Folin-Ciocalteu and UV-spectrophotometry methods, respectively. However, HPLC analysis for phenolic compounds showed insignificant increase between irradiated and nonirradiated honeys. The phenolic compounds such as: caffeic acid, chlorogenic acid, ellagic acid, p- coumaric acid, quercetin and hesperetin as indicated by HPLC method were found to be higher in Gelam honey versus Nenas honey. In conclusion, irradiation of honey causes enhanced antioxidant activities and flavonoid compounds.


Assuntos
Antioxidantes/farmacologia , Compostos de Bifenilo/antagonistas & inibidores , Irradiação de Alimentos , Gastroenteropatias/dietoterapia , Mel/efeitos da radiação , Picratos/antagonistas & inibidores , Compostos de Bifenilo/metabolismo , Ácidos Cafeicos/farmacologia , Ácido Clorogênico/farmacologia , Cromatografia Líquida de Alta Pressão , Ácidos Cumáricos/farmacologia , Ácido Elágico/farmacologia , Raios gama , Gastroenteropatias/fisiopatologia , Hesperidina/farmacologia , Mel/análise , Humanos , Malásia , Oxirredução/efeitos da radiação , Picratos/metabolismo , Propionatos , Quercetina/farmacologia , Espectrofotometria
8.
Curr Cancer Drug Targets ; 18(8): 807-815, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29141549

RESUMO

BACKGROUND: Citrus bioactive compounds, as active anticancer agents, have been under focus by several studies worldwide. However, the underlying genes responsible for the anticancer potential have not been sufficiently highlighted. OBJECTIVES: The current study investigated the gene expression profile of hepatocellular carcinoma, HepG2, cells after treatment with Limonene. METHODS: The concentration that killed 50% of HepG2 cells was used to elucidate the genetic mechanisms of limonene anticancer activity. The apoptotic induction was detected by flow cytometry and confocal fluorescence microscope. Two of the pro-apoptotic events, caspase-3 activation and phosphatidylserine translocation were manifested by confocal fluorescence microscopy. Highthroughput real-time PCR was used to profile 1023 cancer-related genes in 16 different gene families related to the cancer development. RESULTS: In comparison to untreated cells, limonene increased the percentage of apoptotic cells up to 89.61%, by flow cytometry, and 48.2% by fluorescence microscopy. There was a significant limonene- driven differential gene expression of HepG2 cells in 15 different gene families. Limonene was shown to significantly (>2log) up-regulate and down-regulate 14 and 59 genes, respectively. The affected gene families, from the most to the least affected, were apoptosis induction, signal transduction, cancer genes augmentation, alteration in kinases expression, inflammation, DNA damage repair, and cell cycle proteins. CONCLUSION: The current study reveals that limonene could be a promising, cheap, and effective anticancer compound. The broad spectrum of limonene anticancer activity is interesting for anticancer drug development. Further research is needed to confirm the current findings and to examine the anticancer potential of limonene along with underlying mechanisms on different cell lines.


Assuntos
Antineoplásicos/uso terapêutico , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Genes Neoplásicos/efeitos dos fármacos , Limoneno/uso terapêutico , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Descoberta de Drogas/métodos , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica , Células Hep G2 , Humanos , Modelos Lineares , Microscopia Confocal , Oncogenes/efeitos dos fármacos , Extratos Vegetais/uso terapêutico , Reação em Cadeia da Polimerase em Tempo Real , Transcriptoma , Resultado do Tratamento
9.
Nucleic Acids Res ; 31(3): e9, 2003 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-12560512

RESUMO

The potential of expression analysis using cDNA microarrays to address complex problems in a wide variety of biological contexts is now being realised. A limiting factor in such analyses is often the amount of RNA required, usually tens of micrograms. To address this problem researchers have turned to methods of improving detection sensitivity, either through increasing fluorescent signal output per mRNA molecule or increasing the amount of target available for labelling by use of an amplification procedure. We present a novel DNA-based method in which an oligonucleotide is incorporated into the 3' end of cDNA during second-strand cDNA synthesis. This sequence provides an annealing site for a single complementary heel primer that directs Taq DNA polymerase amplification of cDNA following multiple cycles of denaturation, annealing and extension. The utility of this technique for transcriptome-wide screening of relative expression levels was compared to two alternative methodologies for production of labelled cDNA target, namely incorporation of fluorescent nucleotides by reverse transcriptase or the Klenow fragment. Labelled targets from two distinct mouse tissues, adult liver and kidney, were compared by hybridisation to a set of cDNA microarrays containing 6500 mouse cDNA probes. Here we demonstrate, through a dilution series of cDNA derived from 10 micro g of total RNA, that it is possible to produce datasets comparable to those produced with unamplified targets with the equivalent of 30 ng of total RNA. The utility of this technique for microarray analysis in cases where sample is limited is discussed.


Assuntos
Primers do DNA , DNA Complementar/síntese química , Perfilação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase/métodos , Animais , DNA Polimerase I/metabolismo , Feminino , Camundongos , DNA Polimerase Dirigida por RNA/metabolismo , Reprodutibilidade dos Testes , Taq Polimerase/metabolismo
10.
Nat Struct Mol Biol ; 23(3): 248-55, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26828964

RESUMO

To fully understand the transport mechanism of Na(+)/H(+) exchangers, it is necessary to clearly establish the global rearrangements required to facilitate ion translocation. Currently, two different transport models have been proposed. Some reports have suggested that structural isomerization is achieved through large elevator-like rearrangements similar to those seen in the structurally unrelated sodium-coupled glutamate-transporter homolog GltPh. Others have proposed that only small domain movements are required for ion exchange, and a conventional rocking-bundle model has been proposed instead. Here, to resolve these differences, we report atomic-resolution structures of the same Na(+)/H(+) antiporter (NapA from Thermus thermophilus) in both outward- and inward-facing conformations. These data combined with cross-linking, molecular dynamics simulations and isothermal calorimetry suggest that Na(+)/H(+) antiporters provide alternating access to the ion-binding site by using elevator-like structural transitions.


Assuntos
Trocadores de Sódio-Hidrogênio/química , Trocadores de Sódio-Hidrogênio/metabolismo , Thermus thermophilus/enzimologia , Calorimetria , Cristalografia por Raios X , Modelos Moleculares , Simulação de Dinâmica Molecular , Conformação Proteica
11.
Nat Protoc ; 11(8): 1554-71, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27466713

RESUMO

The thermostability of an integral membrane protein (MP) in detergent solution is a key parameter that dictates the likelihood of obtaining well-diffracting crystals that are suitable for structure determination. However, many mammalian MPs are too unstable for crystallization. We developed a thermostabilization strategy based on systematic mutagenesis coupled to a radioligand-binding thermostability assay that can be applied to receptors, ion channels and transporters. It takes ∼6-12 months to thermostabilize a G-protein-coupled receptor (GPCR) containing 300 amino acid (aa) residues. The resulting thermostabilized MPs are more easily crystallized and result in high-quality structures. This methodology has facilitated structure-based drug design applied to GPCRs because it is possible to determine multiple structures of the thermostabilized receptors bound to low-affinity ligands. Protocols and advice are given on how to develop thermostability assays for MPs and how to combine mutations to make an optimally stable mutant suitable for structural studies. The steps in the procedure include the generation of ∼300 site-directed mutants by Ala/Leu scanning mutagenesis, the expression of each mutant in mammalian cells by transient transfection and the identification of thermostable mutants using a thermostability assay that is based on binding of an (125)I-labeled radioligand to the unpurified, detergent-solubilized MP. Individual thermostabilizing point mutations are then combined to make an optimally stable MP that is suitable for structural biology and other biophysical studies.


Assuntos
Proteínas de Membrana/química , Proteínas de Membrana/genética , Mutagênese , Temperatura , Sequência de Aminoácidos , Detergentes/química , Modelos Moleculares , Mutação , Conformação Proteica , Estabilidade Proteica , Solubilidade
12.
PLoS One ; 10(11): e0142094, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26544689

RESUMO

OBJECTIVE: An essential role for embryonic MyHC in foetal development has been found from its association with distal arthrogryposis syndromes, a heterogeneous group of disorders characterised by congenital contractions. The latter probably result from severe myopathy during foetal development. Lack of embryonic muscle biopsy material and suitable animal models has hindered study of the pathomechanisms linking mutations in MYH3 to prenatal myopathy. METHODS AND RESULTS: We determined the pathomechanisms of developmental myopathy caused by recurrent p.Thr178Ile MYH3 heterozygosity, using patient-derived skeletal muscle cells in culture as an experimental disease model to emulate early embryonic development. These cultured cells were processed for discrimination and quantitative analysis of mutant and wild-type MYH3 alleles and MyHC transcripts, real-time RT-qPCR, sequence analysis, immunofluorescence microscopy, immunoblot, and proteomic assessments. Involvement of the ubiquitin proteasome system was investigated in patients with p.Thr178Ile mutations in MYH3 and MYH2. We found equal overall expression of mutant and wild-type MyHC mRNAs and proteins. Compared to the controls, however, expression of embryonic MyHC transcripts and proteins was reduced whereas expression of myosin-specific E3 ubiquitin ligase (MuRF1) was increased. We also found delayed myofibrillogenesis and atrophic myotubes but structured sarcomeres. CONCLUSION: In conclusion, this study suggests that developmental p.Thr178Ile MYH3 myopathy is associated with a combined pathomechanism of insufficient dosage of functional embryonic MyHC and production of mutant protein.


Assuntos
Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Doenças Musculares/embriologia , Doenças Musculares/genética , Proteínas Mutantes/genética , Cadeias Pesadas de Miosina/genética , Diferenciação Celular/genética , Humanos , Lactente , Desenvolvimento Muscular/genética , Fibras Musculares Esqueléticas/patologia , Proteínas Musculares/metabolismo , Doenças Musculares/patologia , Doenças Musculares/fisiopatologia , Isoformas de Proteínas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sarcômeros/metabolismo , Transcrição Gênica , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases/metabolismo
13.
PLoS One ; 8(8): e72365, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24015236

RESUMO

The activation of nuclear factor kappa B (NF-κB) plays a major role in the pathogenesis of a number of inflammatory diseases. In this study, we investigated the anti-inflammatory mechanism of Gelam honey in inflammation induced rats via NF-κB signalling pathway. Rats paw edema was induced by subplantar injection of 1% carrageenan into the right hind paw. Rats were pre-treated with Gelam honey at different doses (1 or 2 g/kg, p.o.) and NSAID Indomethacin (10 mg/kg, p.o.), in two time points (1 and 7 days). Our results showed that Gelam honey at both concentrations suppressed the gene expressions of NF-κB (p65 & p50) and IκBα in inflamed rats paw tissues. In addition, Gelam honey inhibited the nuclear translocation and activation of NF-κB and decreased the cytosolic degradation of IκBα dose dependently in inflamed rats paw tissues. The immunohistochemical expressions of pro-inflammatory mediators COX-2 and TNF-α were also decreased in inflamed rats paw tissues when treated with Gelam honey. The results of our findings suggest that Gelam honey exhibits its inhibitory effects by attenuating NF-κB translocation to the nucleus and inhibiting IκBα degradation, with subsequent decrease of inflammatory mediators COX-2 and TNF-α.


Assuntos
Anti-Inflamatórios/farmacologia , Mel , Fator de Transcrição RelA/metabolismo , Animais , Anti-Inflamatórios/administração & dosagem , Carragenina , Núcleo Celular/metabolismo , Ciclo-Oxigenase 2/metabolismo , Pé/patologia , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Masculino , Inibidor de NF-kappaB alfa , Subunidade p50 de NF-kappa B/genética , Subunidade p50 de NF-kappa B/metabolismo , Transporte Proteico , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Fator de Transcrição RelA/genética , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
14.
PLoS One ; 8(9): e72396, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24039757

RESUMO

Mutations in TPM2 result in a variety of myopathies characterised by variable clinical and morphological features. We used human and mouse cultured cells to study the effects of ß-TM mutants. The mutants induced a range of phenotypes in human myoblasts, which generally changed upon differentiation to myotubes. Human myotubes transfected with the E41K-ß-TM(EGFP) mutant showed perinuclear aggregates. The G53ins-ß-TM(EGFP) mutant tended to accumulate in myoblasts but was incorporated into filamentous structures of myotubes. The K49del-ß-TM(EGFP) and E122K-ß-TM(EGFP) mutants induced the formation of rod-like structures in human cells. The N202K-ß-TM(EGFP) mutant failed to integrate into thin filaments and formed accumulations in myotubes. The accumulation of mutant ß-TM(EGFP) in the perinuclear and peripheral areas of the cells was the striking feature in C2C12. We demonstrated that human tissue culture is a suitable system for studying the early stages of altered myofibrilogenesis and morphological changes linked to myopathy-related ß-TM mutants. In addition, the histopathological phenotype associated with expression of the various mutant proteins depends on the cell type and varies with the maturation of the muscle cell. Further, the phenotype is a combinatorial effect of the specific amino acid change and the temporal expression of the mutant protein.


Assuntos
Fibras Musculares Esqueléticas/metabolismo , Doenças Musculares/genética , Tropomiosina/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Células Cultivadas , Citoesqueleto/metabolismo , Expressão Gênica , Estudos de Associação Genética , Proteínas de Fluorescência Verde/biossíntese , Humanos , Camundongos , Fibras Musculares Esqueléticas/patologia , Doenças Musculares/patologia , Mioblastos/metabolismo , Mioblastos/patologia , Fenótipo , Sarcômeros/metabolismo , Proteína Sequestossoma-1 , Técnicas de Cultura de Tecidos , Tropomiosina/metabolismo
15.
J Mol Biol ; 425(12): 2198-207, 2013 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-23706649

RESUMO

Structure determination of mammalian integral membrane proteins is challenging due to their instability upon detergent solubilisation and purification. Recent successes in the structure determination of G-protein-coupled receptors (GPCRs) resulted from the development of GPCR-specific protein engineering strategies. One of these, conformational thermostabilisation, could in theory facilitate structure determination of other membrane proteins by improving their tolerance to detergents and locking them in a specific conformation. We have therefore used this approach on the cocaine-sensitive rat serotonin transporter (SERT). Out of a panel of 554 point mutants throughout SERT, 10 were found to improve its thermostability. The most stabilising mutations were combined to make the thermostabilised mutants SAH6 (L99A+G278A+A505L) and SAH7 (L405A+P499A+A505L) that were more stable than SERT by 18°C and 16°C, respectively. Inhibitor binding assays showed that both of the thermostabilised SERT mutants bound [(125)I]RTI55 (ß-CIT) with affinity similar to that of the wild-type transporter, although cocaine bound with increased affinity (17- to 56-fold) whilst ibogaine, imipramine and paroxetine all bound with lower affinity (up to 90-fold). Neither SAH6 nor SAH7 was capable of transporting [(3)H]serotonin into HEK293 cell lines stably expressing the mutants, although serotonin bound to them with an apparent Ki of 155µM or 82µM, respectively. These data combined suggest that SAH6 and SAH7 are thermostabilised in a specific cocaine-bound conformation, making them promising candidates for crystallisation. Conformational thermostabilisation is thus equally applicable to membrane proteins that are transporters in addition to those that are GPCRs.


Assuntos
Cocaína/metabolismo , Estabilidade Proteica , Proteínas da Membrana Plasmática de Transporte de Serotonina/química , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Substituição de Aminoácidos , Animais , Linhagem Celular , Humanos , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Ligação Proteica , Ratos , Temperatura
16.
Asian Pac J Cancer Prev ; 13(4): 1605-10, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22799375

RESUMO

Gelam and Nenas monofloral honeys were investigated in this study for their chemopreventive effects against HT 29 colon cancer cells. MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H- tetrazolim) assays showed more effective inhibition of colon cancer cells proliferation by Gelam honey with IC50 values of 39.0 mg/ml and 85.5 mg/ml respectively after 24 hours of treatment. Alkali comet assays revealed both honeys increased DNA damage significantly in a dose dependent manner. In addition, annexin V-FITC/PI flow cytometry demonstrated that at IC50 concentrations and above, both Gelam and Nenas honeys induced apoptosis significantlyat values higher than for necrosis (p<0.05). Measurement of prostaglandin E2 (PGE2) confirmed that Gelam and Nenas honeys reduced its production in H2O2 inflammation-induced colon cancer cells. In conclusion, our study indicated and confirmed that both Gelam and Nenas honeys are capable of suppressing the growth of HT 29 colon cancer cells by inducing apoptosis and suppressing inflammation.


Assuntos
Anticarcinógenos/farmacologia , Mel , Inflamação/prevenção & controle , Análise de Variância , Anticarcinógenos/uso terapêutico , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Ensaio Cometa , Dano ao DNA/efeitos dos fármacos , Dinoprostona/metabolismo , Células HT29 , Humanos , Peróxido de Hidrogênio , Inflamação/induzido quimicamente , Inflamação/metabolismo , Concentração Inibidora 50
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA