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Eccema , Piel , Humanos , Eccema/microbiología , Femenino , Masculino , Piel/microbiología , Cohorte de Nacimiento , Lactante , Recién Nacido , Microbiota , Hong KongRESUMEN
Urban green waste and food waste are often used as bulking agents to prepare home compost in combination with animal manure in urban horticulture and community gardening. Although it is known that antimicrobial resistance genes (ARGs) persist in home compost, their origins have not been determined. In addition, the factors contributing to ARGs persistence remain unclear. In this study, we aim to (i) characterize the changes in the microbiome and antimicrobial resistome during the composting process of home compost using metagenomics shotgun sequencing, (ii) identify the source of the ARGs persisted in home compost using SourceTracker, and (iii) elucidate the collective effect of compost microbiome and environmental factors, including the physicochemical properties and antibiotics concentration of home compost, in contributing to ARG persistence using Procrustes analysis, co-occurrence network analysis, variation partitioning analysis, and structural equation modeling. SourceTracker analysis indicated that urban green waste bulking agent was the major source of the persisting ARGs in home compost instead of animal manure. Procrustes analysis and co-occurrence network analysis revealed a strong association between microbiome and antimicrobial resistome. Variation partitioning analysis and structural equation modeling suggested that physicochemical properties shaped the antimicrobial resistome directly and indirectly by influencing the microbiome. Our results indicated that the persistence of ARGs in home compost might be due to the succession of microbial species from the urban green waste bulking agent, and the physicochemical properties might have defined the compost environment to shape the microbiome in the compost, thus, in turn, the persisting antimicrobial resistome.
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Compostaje , Oxazolidinonas , Eliminación de Residuos , Animales , Antibacterianos/farmacología , Farmacorresistencia Bacteriana/genética , Estiércol/análisis , Alimentos , Genes BacterianosAsunto(s)
Fármacos Anti-VIH/farmacología , VIH-1/efectos de los fármacos , Piranocumarinas/farmacología , Fármacos Anti-VIH/química , Productos Biológicos/química , Productos Biológicos/farmacología , Farmacorresistencia Viral , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , VIH-1/genética , Humanos , Mutación , Piranocumarinas/químicaRESUMEN
AIMS/HYPOTHESIS: Most genetic variants identified for type 2 diabetes have been discovered in European populations. We performed genome-wide association studies (GWAS) in a Chinese population with the aim of identifying novel variants for type 2 diabetes in Asians. METHODS: We performed a meta-analysis of three GWAS comprising 684 patients with type 2 diabetes and 955 controls of Southern Han Chinese descent. We followed up the top signals in two independent Southern Han Chinese cohorts (totalling 10,383 cases and 6,974 controls), and performed in silico replication in multiple populations. RESULTS: We identified CDKN2A/B and four novel type 2 diabetes association signals with p < 1 × 10(-5) from the meta-analysis. Thirteen variants within these four loci were followed up in two independent Chinese cohorts, and rs10229583 at 7q32 was found to be associated with type 2 diabetes in a combined analysis of 11,067 cases and 7,929 controls (p meta = 2.6 × 10(-8); OR [95% CI] 1.18 [1.11, 1.25]). In silico replication revealed consistent associations across multiethnic groups, including five East Asian populations (p meta = 2.3 × 10(-10)) and a population of European descent (p = 8.6 × 10(-3)). The rs10229583 risk variant was associated with elevated fasting plasma glucose, impaired beta cell function in controls, and an earlier age at diagnosis for the cases. The novel variant lies within an islet-selective cluster of open regulatory elements. There was significant heterogeneity of effect between Han Chinese and individuals of European descent, Malaysians and Indians. CONCLUSIONS/INTERPRETATION: Our study identifies rs10229583 near PAX4 as a novel locus for type 2 diabetes in Chinese and other populations and provides new insights into the pathogenesis of type 2 diabetes.
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Cromosomas Humanos Par 7 , Diabetes Mellitus Tipo 2/genética , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Proteínas de Homeodominio/genética , Factores de Transcripción Paired Box/genética , Adulto , Anciano , Pueblo Asiatico , China , Diabetes Mellitus Tipo 2/etnología , Femenino , Marcadores Genéticos , Variación Genética , Genotipo , Hong Kong , Humanos , Células Secretoras de Insulina/citología , Japón , Masculino , Persona de Mediana Edad , SingapurRESUMEN
While much effort has been made to characterize influenza A pdm09 virus (pH1N1), the flu that was responsible for the fourth influenza pandemic, there is a lack of study on the composition of bacteria that lead to secondary infection. In this study, we recruited pneumonia patients with and without pH1N1 infection and characterized their oropharyngeal microbiota by the unbiased high-throughput sequencing method. While there were no significant differences in common bacterial pneumonia-causative agents (Acinetobacter and Streptococcus species), previously unreported Pseudomonas species equipped with chemotaxis and flagellar assembly genes significantly increased (>20-fold) in the pH1N1-infected group. Bacillus and Ralstonia species that also increased significantly (5-10-fold) were also found to possess similar signaling and motility genes. In contrast, no such genes were found in oral commensal Prevotella, Veillonella and Neisseria species, which decreased significantly, or in either Acinetobacter or 10 out of 21 Streptococcus species, including Streptococcus pneumoniae. Our results support the notion that pH1N1 infection provides a niche for previously unnoticed potential respiratory pathogens that were able to access the lower respiratory tract with weakened immunity.
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Bacterias/genética , Bacterias/patogenicidad , Coinfección/microbiología , Subtipo H1N1 del Virus de la Influenza A/fisiología , Gripe Humana/microbiología , Neumonía Bacteriana/microbiología , Adolescente , Adulto , Anciano , Bacterias/clasificación , Bacterias/aislamiento & purificación , Quimiotaxis/genética , Quimiotaxis/fisiología , China/epidemiología , Coinfección/epidemiología , Coinfección/virología , Femenino , Humanos , Gripe Humana/epidemiología , Gripe Humana/virología , Masculino , Microbiota , Persona de Mediana Edad , Orofaringe/microbiología , Orofaringe/virología , Neumonía Bacteriana/epidemiología , Neumonía Bacteriana/virología , Pseudomonas/fisiología , Adulto JovenRESUMEN
A low level of CD4+ lymphocyte cells makes end-stage HIV/AIDS patients highly susceptible to microbial infections. We have adopted the next generation sequencing method to identify the spectrum of bacterial plasma and viral elements that might be present in these patients. The HIV/AIDS plasma microbiome was dominated by bacterial elements in the taxonomical order Pseudomonadales, while healthy people carried fewer bacterial DNA in the plasma. We have found that many of the bacterial elements in HIV/AIDS plasma are similar to those of the microbes found in the human gut, suggesting potential acquisition of microbial elements from the gut. The HIV/AIDS and normal plasma DNA virome shared some similarities in the presence of common ubiquitous eukaryotic viruses. The normal DNA virome was mainly composed of viruses from Anelloviridae. In contrast, the HIV/AIDS DNA virome contained a large proportion of bacteriophages, endogenous retroviruses and a non-human virus. In addition, several sequences, which might belong to novel bacteria or endogenous retroviruses, were identified. Taken together, the use of high-throughput sequencing technology in unveiling microbial metagenomics may facilitate future research in combating HIV/AIDS and its associated microbial complications.
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Infecciones Oportunistas Relacionadas con el SIDA/diagnóstico , Bacterias/aislamiento & purificación , Infecciones por VIH/complicaciones , Plasma/microbiología , Plasma/virología , Virus/aislamiento & purificación , Infecciones Oportunistas Relacionadas con el SIDA/microbiología , Infecciones Oportunistas Relacionadas con el SIDA/virología , Adulto , Bacterias/clasificación , Bacterias/genética , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Masculino , Virus/clasificación , Virus/genética , Adulto JovenRESUMEN
c-Met represents an important emerging therapeutic target in cancer. In this study, we demonstrate the mechanism by which c-Met tyrosine kinase inhibition inhibits tumor growth in a highly invasive Asian-prevalent head and neck cancer, nasopharyngeal cancer (NPC). c-Met tyrosine kinase inhibitors (TKIs; AM7 and c-Met TKI tool compound SU11274) downregulated c-Met phosphorylation, resulting in marked inhibition of NPC cell growth and invasion. Strikingly, inhibition of c-Met resulted in significant downregulation of TP53-induced Glycolysis and Apoptosis Regulator (TIGAR) and subsequent depletion of intracellular NADPH. Importantly, overexpression of TIGAR ameliorated the effects of c-Met kinase inhibition, confirming the importance of TIGAR downregulation in the growth inhibitory activity of c-Met TKI. The effects of c-Met inhibition on TIGAR and NADPH levels were observed with two different c-Met TKIs (AM7 and SU11274) and with multiple cell lines. As NADPH provides a crucial reducing power required for cell survival and proliferation, our findings reveal a novel mechanistic action of c-Met TKI, which may represent a key effect of c-Met kinase inhibition. Our data provide the first evidence linking c-Met, TIGAR and NADPH regulation in human cancer cells suggesting that inhibition of a tyrosine kinase/TIGAR/NADPH cascade may have therapeutic applicability in human cancers.
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Indoles/farmacología , Péptidos y Proteínas de Señalización Intracelular/genética , NADP/biosíntesis , Neoplasias Nasofaríngeas/metabolismo , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Pirimidinonas/farmacología , Quinolinas/farmacología , Sulfonamidas/farmacología , Apoptosis , Proteínas Reguladoras de la Apoptosis , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular , Regulación hacia Abajo , Humanos , Neoplasias Nasofaríngeas/tratamiento farmacológico , Neoplasias Nasofaríngeas/patología , Monoéster Fosfórico Hidrolasas , Fosforilación , Proteínas Proto-Oncogénicas c-met/genética , Proteínas Proto-Oncogénicas c-met/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genéticaRESUMEN
The fibroblast growth factor 8b (FGF8b) oncogene is known to be primarily involved in the tumorigenesis and progression of hormone-related cancers. Its role in other epithelial cancers has not been investigated, except for esophageal cancer, in which FGF8b overexpression was mainly found in tumor biopsies of male patients. These observations were consistent with previous findings in these cancer types that the male sex-hormone androgen is responsible for FGF8b expression. Nasopharyngeal carcinoma (NPC) is a highly metastatic cancer of head and neck commonly found in Asia. It is etiologically associated with Epstein-Barr Virus (EBV) infection, inflammatory tumor microenvironment and relatively higher male predominance. Here, we reported for the first time that FGF8b is overexpressed in this EBV-associated non-hormone-related cancer of the head and neck, NPC. More importantly, overexpression of FGF8b mRNA and protein was detected in a large majority of NPC tumors from both male and female genders, in addition to multiple NPC cell lines. We hypothesized that FGF8b overexpression may contribute to NPC tumorigenesis. Using EBV-associated NPC cell lines, we demonstrated that specific knockdown of FGF8b by small interfering RNA inhibited cell proliferation, migration and invasion, whereas exogenous FGF8b stimulated these multiple phenotypes. Further mechanistic investigation revealed that in addition to NF-κB signaling (a major inflammatory signaling pathway known to be activated in NPC), an important EBV oncoprotein, the latent membrane protein 1 (LMP1), was found to be a direct inducer of FGF8b overexpression in NPC cells, whereas androgen (testosterone) has minimal effect on FGF8b expression in EBV-associated NPC cells. In summary, our study has identified LMP1 as the first viral oncogene capable of directly inducing FGF8b (an important cellular oncogene) expression in human cancer cells. This novel mechanism of viral-mediated FGF8 upregulation may implicate a new role of oncoviruses in human carcinogenesis.
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Factor 8 de Crecimiento de Fibroblastos/fisiología , Regulación Neoplásica de la Expresión Génica , Herpesvirus Humano 4/patogenicidad , Oncogenes , Carcinoma , Movimiento Celular , Proliferación Celular , Femenino , Factor 8 de Crecimiento de Fibroblastos/antagonistas & inhibidores , Factor 8 de Crecimiento de Fibroblastos/genética , Humanos , Masculino , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/fisiología , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/patología , Neoplasias Nasofaríngeas/virología , Invasividad Neoplásica , ARN Mensajero/análisis , ARN Interferente Pequeño/genética , Proteínas de la Matriz Viral/fisiologíaAsunto(s)
Apoptosis , Fragmentación del ADN , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/patogenicidad , Proteínas Virales/metabolismo , Animales , Chlorocebus aethiops , Cromatina/metabolismo , Efecto Citopatogénico Viral , ADN Complementario , Proteínas Fluorescentes Verdes , Humanos , Etiquetado Corte-Fin in Situ , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/crecimiento & desarrollo , Síndrome Respiratorio Agudo Grave/metabolismo , Coloración y Etiquetado , Células Vero , Proteínas del Envoltorio Viral , Proteínas Virales/fisiología , Proteínas ViroporinasRESUMEN
BACKGROUND AND PURPOSE: Statins (3-hydroxy-3-methyl-glutaryl coenzyme A (HMG CoA) reductase inhibitors) have been demonstrated to reduce cardiovascular mortality. It is unclear how the expression level of HMG CoA reductase in cardiovascular tissues compares with that in cells derived from the liver. We hypothesized that this enzyme exists in different cardiovascular tissues, and simvastatin modulates the vascular iberiotoxin-sensitive Ca2+-activated K(+) (BK(Ca)) channels. EXPERIMENTAL APPROACHES: Expression of HMG CoA reductase in different cardiovascular preparations was measured. Effects of simvastatin on BK(Ca) channel gatings of porcine coronary artery smooth muscle cells were evaluated. KEY RESULTS: Western immunoblots revealed the biochemical existence of HMG CoA reductase in human cardiovascular tissues and porcine coronary artery. In porcine coronary artery smooth muscle cells, extracellular simvastatin (1, 3 and 10 microM) (hydrophobic), but not simvastatin Na+ (hydrophilic), inhibited the BK(Ca) channels with a minimal recovery upon washout. Isopimaric acid (10 microM)-mediated enhancement of the BK(Ca) amplitude was reversed by external simvastatin. Simvastatin Na+ (10 microM, applied internally), markedly attenuated isopimaric acid (10 microM)-induced enhancement of the BK(Ca) amplitude. Reduced glutathione (5 mM; in the pipette solution) abolished simvastatin -elicited inhibition. Mevalonolactone (500 microM) and geranylgeranyl pyrophosphate (20 microM) only prevented simvastatin (1 and 3 microM)-induced responses. simvastatin (10 microM ) caused a rottlerin (1 microM)-sensitive (cycloheximide (10 microM)-insensitive) increase of PKC-delta protein expression. CONCLUSIONS AND IMPLICATIONS: Our results demonstrated the biochemical presence of HMG CoA reductase in different cardiovascular tissues, and that simvastatin inhibited the BK(Ca) channels of the arterial smooth muscle cells through multiple intracellular pathways.
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Músculo Liso Vascular/efectos de los fármacos , Péptidos/farmacología , Canales de Potasio Calcio-Activados/antagonistas & inhibidores , Simvastatina/farmacología , Adulto , Anciano , Animales , Western Blotting , Caveolina 1/biosíntesis , Línea Celular , Línea Celular Tumoral , Vasos Coronarios/citología , Vasos Coronarios/efectos de los fármacos , Vasos Coronarios/fisiología , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Femenino , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Imidazoles/farmacología , Técnicas In Vitro , Masculino , Potenciales de la Membrana/efectos de los fármacos , Persona de Mediana Edad , Músculo Liso Vascular/citología , Músculo Liso Vascular/fisiología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/fisiología , Ésteres del Forbol/farmacología , Canales de Potasio Calcio-Activados/metabolismo , Canales de Potasio Calcio-Activados/fisiología , Proteína Quinasa C-delta/metabolismo , Piridinas/farmacología , Simvastatina/química , PorcinosRESUMEN
Traditional Chinese medicine (TCM) has been used for prevention and treatment of severe acute respiratory syndrome (SARS) in Hong Kong during the outbreak in spring 2003. We investigated the immunomodulating effects of an innovative TCM regimen derived from two herbal formulas (Sang Ju Yin and Yu Ping Feng San) for treating febrile diseases. Thirty-seven healthy volunteers were given the oral TCM regimen daily for 14 days. Peripheral venous blood samples were taken on days 0, 15 and 29 for hematology, biochemistry and immunology tests, including the measurement of blood lymphocyte subsets and plasma T-helper lymphocyte types 1 and 2 cytokines and receptor. After 3 months, 23 of the volunteers participated in a control study without TCM treatment for the same time course of blood tests. Two volunteers withdrew on day 2, due to headache and dizziness. All others remained well without any side effects. No participants showed significant changes in their blood test results, except that the T-lymphocyte CD4/CD8 ratio increased significantly from 1.31 +/- 0.50 (mean +/- SD) on day 0 to 1.41 +/- 0.63 on day 15 (p < 0.02), and reduced to 1.32 +/- 0.47 on day 29 (p < 0.05). In the control study, there were no changes in the CD4/CD8 ratio. The transient increase in CD4/CD8 ratio was likely due to the TCM intake. We postulate that the administration of the innovative TCM may have beneficial immunomodulatory effects for preventing viral infections including SARS.
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Relación CD4-CD8 , Medicamentos Herbarios Chinos/uso terapéutico , Medicina Tradicional China , Adulto , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Severe acute respiratory syndrome (SARS) is a global health concern. In Hong Kong, two major outbreaks, one hospital based and the other in the Amoy Gardens apartments, were identified. The frequency of diarrhoea, admission to intensive care, and mortality differed significantly between the two outbreaks. We did genomic sequencing for viral isolates from five Amoy Gardens patients. The virus sequence was identical in four of these five patients. The sequence data from one hospital case and the four identical community cases had only three nucleotide differences. Alterations in the SARS coronavirus genome are unlikely to have caused the distinctive clinical features of the Amoy Gardens patients, and these results highlight the importance of non-viral genomic factors in this outbreak.
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Brotes de Enfermedades/estadística & datos numéricos , Genoma Viral , Síndrome Respiratorio Agudo Grave/virología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genética , Secuencia de Bases/genética , Infección Hospitalaria/virología , Hong Kong/epidemiología , Humanos , Síndrome Respiratorio Agudo Grave/diagnóstico , Síndrome Respiratorio Agudo Grave/epidemiologíaRESUMEN
Elfin (previously named CLIM1) is a protein that possesses an N-terminal PDZ domain and a C-terminal LIM domain. It belongs to the family of Enigma proteins. Enigma proteins are a family of cytoplasmic proteins that contain an N-terminal PDZ domain and a series of C-terminal LIM domains. By virtue of these two protein interacting domains, Enigma proteins are capable of protein-protein interactions. It has been proposed that Enigma proteins may act as adapters between kinases and the cytoskeleton. We have previously shown that Elfin is most abundantly expressed in the heart and it colocalizes with alpha-actinin 2 at the Z-disks of the myocardium. In this report, Elfin was shown to localize at the actin stress fibers of myoblasts, as revealed by green fluorescent protein (GFP) tagging. In situ hybridization and immunostaining showed that Elfin expression begins at an early stage in mouse development and is present throughout the developing heart. Taken together, our experimental results suggest that Elfin may play an important role in myofibrillogenesis and heart development.
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Regulación del Desarrollo de la Expresión Génica , Corazón/embriología , Miocardio/metabolismo , Factores de Transcripción/biosíntesis , Factores de Transcripción/química , Actinina/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Citoplasma/metabolismo , Citoesqueleto/metabolismo , ADN Complementario/metabolismo , Proteínas Fluorescentes Verdes , Hibridación in Situ , Proteínas con Dominio LIM , Proteínas Luminiscentes/metabolismo , Ratones , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , ARN Mensajero/metabolismo , Homología de Secuencia de Aminoácido , Fibras de Estrés/metabolismo , Factores de Tiempo , Distribución TisularRESUMEN
Yeast Apc11p together with Rbx1 and Roc2/SAG define a new class of RING-H2 fingers in a superfamily of E3 ubiquitin ligases. The human homolog of Apc11p, ANAPC11 was identified during a large-scale partial sequencing of a human liver cancer cDNA library and partial characterization was performed. This 514 bp full-length cDNA has a predicted open reading frame (ORF) encoding 84 amino acids. The ORF codes for ANAPC11, the human anaphase promoting complex subunit 11 (yeast APC11 homolog), which possesses a RING-H2 finger motif and exhibits sequence similarity to subunits of E3 ubiquitin ligase complexes. In Northern blot hybridization with poly(A) RNA of various human tissues using radio-labelled ANAPC11 cDNA probe, we found strong signals detected in skeletal muscle and heart; moderate signals detected in brain, kidney, and liver; and detectable but low signals in colon, thymus, spleen, small intestine, placenta, lung, and peripheral blood leukocyte. The ANAPC11 gene is located at the human chromosome 17q25. ANAPC11 is distributed diffusely in the cytoplasm and nucleus with discrete accumulation in granular structures in all the cell lines (AML 12, HepG2, and C2C12) transfected. Expression level of ANAPC11 is found higher in certain types of cancer determined in the RNA dot blot experiment.
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Carcinoma Hepatocelular/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Ligasas/genética , Neoplasias Hepáticas/metabolismo , Proteínas de Neoplasias , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Complejos de Ubiquitina-Proteína Ligasa , Ciclosoma-Complejo Promotor de la Anafase , Animales , Subunidad Apc11 del Ciclosoma-Complejo Promotor de la Anafase , Secuencia de Bases/genética , Biomarcadores de Tumor/metabolismo , Encéfalo/metabolismo , Carcinoma Hepatocelular/genética , Mapeo Cromosómico/métodos , Clonación Molecular , ADN Complementario/análisis , ADN de Neoplasias/análisis , Proteínas Fúngicas/genética , Humanos , Células Híbridas , Riñón/metabolismo , Leucemia/metabolismo , Hígado/metabolismo , Neoplasias Hepáticas/genética , Datos de Secuencia Molecular , Músculo Esquelético/metabolismo , Miocardio/metabolismo , Complejo Represivo Polycomb 1 , Fracciones Subcelulares/metabolismo , Distribución Tisular/fisiología , Células Tumorales Cultivadas/metabolismo , Ubiquitina-Proteína Ligasas , Levaduras/genéticaRESUMEN
We have cloned and characterized another alternatively spliced isoform of the human four-and-a-half LIM domain protein 1 (FHL1), designated FHL1C. FHL1C contains a single zinc finger and two tandem repeats of LIM domains at the N-terminus followed by a putative RBP-J binding region at the C-terminus. FHL1C shares the same N-terminal two-and-a-half LIM domains with FHL1 but different C-terminal protein sequences. Due to the absence of the exon 4 in FHL1C, there is a frame-shift in the 3' coding region. Sequence analysis indicated that FHL1C is the human homolog of murine KyoT2. The Northern blot and RT-PCR results revealed that FHL1 is widely expressed in human tissues, including skeletal muscle and heart at a high level, albeit as a relatively low abundance transcript in brain, placenta, lung, liver, kidney, pancreas, and testis. In contrast, FHL1C is specifically expressed in testis, skeletal muscle, and heart at a relatively low level compared with FHL1. The expression of FHL1C transcripts was also seen in aorta, left atrium, left, and right ventricles of human heart at low level. Immunoblot analysis using affinity-purified anti-FHL1C antipeptide antibodies confirmed a 20 kDa protein of FHL1C in human skeletal muscle and heart. Unlike FHL1B, which is another FHL1 isoform recently reported by our group and localized predominantly in the nucleus [Lee et al., 1999], FHL1C is localized both in the nucleus and cytoplasm of mammalian cell.
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Proteínas de Drosophila , Proteínas de Homeodominio/genética , Proteínas Musculares , Músculo Esquelético/metabolismo , Miocardio/metabolismo , Testículo/metabolismo , Empalme Alternativo , Animales , Secuencia de Bases/genética , Northern Blotting/métodos , Encéfalo/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Exones/genética , Mutación del Sistema de Lectura/genética , Proteínas de Homeodominio/química , Proteínas de Homeodominio/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteínas con Dominio LIM , Masculino , Ratones , Datos de Secuencia Molecular , Isoformas de Proteínas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/instrumentación , Homología de Secuencia , Fracciones Subcelulares/química , Fracciones Subcelulares/metabolismo , Secuencias Repetidas en Tándem/genética , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Dedos de Zinc/fisiologíaRESUMEN
LIM domain proteins are found to be important regulators in cell growth, cell fate determination, cell differentiation and remodeling of the cell cytoskeleton. Human Four-and-a-half LIM-only protein 3 (FHL3) is a type of LIM-only protein that contains four tandemly repeated LIM motifs with an N-terminal single zinc finger (half LIM motif). FHL3 expresses predominantly in human skeletal muscle. In this report, FHL3 was shown to be a novel interacting partner of FHL2 using the yeast two-hybrid assay. Furthermore, site-directed mutagenesis of FHL3 indicated that the LIM2 of FHL3 is the essential LIM domain for interaction with FHL2. Green fluorescent protein (GFP) was used to tag FHL3 in order to study its distribution during myogenesis. Our result shows that FHL3 was localized in the focal adhesions and nucleus of the cells. FHL3 mainly stayed in the focal adhesion during myogenesis. Moreover, using site-directed mutagenesis, the LIM1 of FHL3 was identified as an essential LIM domain for its subcellular localization. Mutants of GFP have given rise to a novel technique, two-fusion fluorescence resonance energy transfer (FRET), in the determination of protein-protein interaction at particular subcellular locations of eukaryotic cells. To determine whether FHL2 and FHL3 can interact with one another and to locate the site of this interaction in a single intact mammalian cell, we fused FHL2 and FHL3 to different mutants of GFP and studied their interactions using FRET. BFP/GFP fusion constructs were cotransfected into muscle myoblast C2C12 to verify the colocalization and subcellular localization of FRET. We found that FHL2 and FHL3 were colocalized in the mitochondria of the C2C12 cells and FRET was observed by using an epi-fluorescent microscope equipped with an FRET specific filter set.
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Proteínas de Homeodominio/metabolismo , Proteínas Musculares , Factores de Transcripción , Secuencia de Bases , Línea Celular , Núcleo Celular/metabolismo , Cartilla de ADN , Proteínas Fluorescentes Verdes , Proteínas de Homeodominio/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteínas con Dominio LIM , Proteínas con Homeodominio LIM , Proteínas Luminiscentes/metabolismo , Mutagénesis Sitio-Dirigida , Unión Proteica , Espectrometría de Fluorescencia , Técnicas del Sistema de Dos HíbridosRESUMEN
LIM domain proteins are found to be important regulators in cell growth, cell fate determination, cell differentiation, and remodeling of the cell cytoskeleton. Human Four-and-a-half LIM-only protein 2 (FHL2) is expressed predominantly in human heart and is only slightly expressed in skeletal muscle. Since FHL2 is an abundant protein in human heart, it may play an important role in the regulation of cell differentiation and myofibrillogenesis of heart at defined subcellular compartment. Therefore, we hypothesized that FHL2 act as a multi-functional protein by the specific arrangement of the LIM domains of FHL2 and that one of the LIM domains of FHL2 can function as an anchor and localizes it into a specific subcellular compartment in a cell type specific manner to regulate myofibrillogenesis. From our results, we observed that FHL2 is localized at the focal adhesions of the C2C12, H9C2 myoblast as well as a nonmyogenic cell line, HepG2 cells. Colocalization of vinculin-CFP and FHL2-GFP at focal adhesions was also observed in cell lines. Site-directed mutagenesis, in turn, suggested that the second LIM domain-LIM2 is essential for its specific localization to focal adhesions. Moreover, FHL2 was observed along with F-actin and focal adhesion of C2C12 and H9C2 myotubes. Finally, we believe that FHL2 moves from focal adhesions and then stays at the Z-discs of terminally differentiated heart muscle.