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1.
Sci Rep ; 10(1): 2936, 2020 02 19.
Artículo en Inglés | MEDLINE | ID: mdl-32076074

RESUMEN

To visualize protein-protein interactions in Candida albicans with the bimolecular fluorescence complementation (BiFC) approach, we created a Tet-on system with the plasmids pWTN1 and pWTN2. Both plasmids bear a hygromycin B-resistant marker (CaHygB) that is compatible with the original Tet-on plasmid pNIM1, which carries a nourseothricin-resistant marker (CaSAT1). By using GFPmut2 and mCherry as reporters, we found that the two complementary Tet-on plasmids act synergistically in C. albicans with doxycycline in a dose-dependent manner and that expression of the fusion proteins, CaCdc11-GFPmut2 and mCherry-CaCdc10, derived from this system, is septum targeted. Furthermore, to allow detection of protein-protein interactions with the reassembly of a split fluorescent protein, we incorporated mCherry into our system. We generated pWTN1-RN and pNIM1-RC, which express the N-terminus (amino acids 1-159) and C-terminus (amino acids 160-237) of mCherry, respectively. To verify BiFC with mCherry, we created the pWTN1-CDC42-RN (or pWTN1-RN-CDC42) and pNIM1-RC-RDI1 plasmids. C. albicans cells containing these plasmids treated with doxycycline co-expressed the N- and C-terminal fragments of mCherry either N-terminally or C-terminally fused with CaCdc42 and CaRdi1, respectively, and the CaCdc42-CaRdi1 interaction reconstituted a functional form of mCherry. The establishment of this Tet-on-based BiFC system in C. albicans should facilitate the exploration of protein-protein interactions under a variety of conditions.


Asunto(s)
Bioensayo/métodos , Candida albicans/metabolismo , Proteínas Fúngicas/metabolismo , Mapeo de Interacción de Proteínas , Tetraciclina/farmacología , Candida albicans/efectos de los fármacos , Candida albicans/genética , Doxiciclina/farmacología , Fluorescencia , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Marcadores Genéticos , Higromicina B/farmacología , Proteínas Luminiscentes/metabolismo , Unión Proteica/efectos de los fármacos , Septinas/metabolismo
2.
Mol Plant Microbe Interact ; 32(3): 286-295, 2019 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-30133338

RESUMEN

We identified a protein spot showing downregulation in the presence of Cryphonectria hypovirus 1 and tannic acid supplementation as a septin subunit with the highest homology to the Aspergillus nidulans aspA gene, an ortholog of the Saccharomyces cerevisiae Cdc11 gene. To analyze the functional role of this septin component (CpSep1), we constructed its null mutant and obtained a total of eight CpSep1-null mutants from 137 transformants. All CpSep1-null mutants showed retarded growth, with fewer aerial mycelia and intense pigmentation on plates of potato dextrose agar supplemented with L-methionine and biotin. When the marginal hyphae were examined, hyperbranching was observed in contrast to the wild type. The inhibition of colonial growth was partially recovered when the CpSep1-null mutants were cultured in the presence of the osmostabilizing sorbitol. Conidia production of the CpSep1-null mutants was significantly increased by at least 10-fold more. Interestingly, the conidial morphology of the CpSep1-null mutants changed to circular in contrast to the typical rod-shaped spores of the wild type, indicating a role of septin in the spore morphology of Cryphonectria parasitica. However, no differences in the germination process were observed. Virulence assays using excised chestnut bark, stromal pustule formation on chestnut stems, and apple inoculation indicated that the CpSep1 gene is important in pathogenicity.


Asunto(s)
Ascomicetos , Virus ARN , Septinas , Ascomicetos/genética , Ascomicetos/patogenicidad , Ascomicetos/virología , Regulación hacia Abajo , Mutación , Virus ARN/metabolismo , Septinas/genética , Esporas Fúngicas/genética , Virulencia/genética
3.
FASEB J ; 31(8): 3622-3635, 2017 08.
Artículo en Inglés | MEDLINE | ID: mdl-28432198

RESUMEN

Periconception maternal folic acid (vitamin B9) supplementation can reduce the prevalence of neural tube defects (NTDs), although just how folates benefit the developing embryo and promote closing of the neural tube and other morphologic processes during development remains unknown. Folate contributes to a 1-carbon metabolism, which is essential for purine biosynthesis and methionine recycling and affects methylation of DNA, histones, and nonhistone proteins. Herein, we used animal models and cultured mammalian cells to demonstrate that disruption of the methylation pathway mediated by folate compromises normal neural tube closure (NTC) and ciliogenesis. We demonstrate that the embryos with NTD failed to adequately methylate septin2, a key regulator of cilium structure and function. We report that methylation of septin2 affected its GTP binding activity and formation of the septin2-6-7 complex. We propose that folic acid promotes normal NTC in some embryos by regulating the methylation of septin2, which is critical for normal cilium formation during early embryonic development.-Toriyama, M., Toriyama, M., Wallingford, J. B., Finnell, R. H. Folate-dependent methylation of septins governs ciliogenesis during neural tube closure.


Asunto(s)
Cilios/fisiología , Embrión de Mamíferos/metabolismo , Embrión no Mamífero/metabolismo , Ácido Fólico/metabolismo , Tubo Neural/fisiología , Septinas/metabolismo , Animales , Dactinomicina/análogos & derivados , Desarrollo Embrionario/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Células HEK293 , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Humanos , Metilación , Ratones , Defectos del Tubo Neural/etiología , Plásmidos , Transducción de Señal , Xenopus/embriología
4.
J Med Food ; 20(3): 279-287, 2017 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-28256936

RESUMEN

Gelidium amansii is an edible and economically important red alga consumed in South Eastern Asia. In previous studies, we reported that the ethanol extracts of G. amansii (GAE) has promising modulatory activity with respect to the morphological and functional maturation of hippocampal neurons in culture. In this study, we show that the chloroform (CHCl3) subfraction of GAE and the ethyl acetate (EtOAc) fraction dose-dependently promoted neurite outgrowth, and their effects were comparable with that of GAE. We further assessed in cultured cortical neurons, proteins differentially expressed in the presence/absence of the GAE, CHCl3 subfraction, and the EtOAc fraction by 2D-PAGE and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Proteomic data revealed that a number of proteins responsible for multiple cellular and biochemical functions vital for neuronal development and maturation were significantly upregulated in neurons treated with the GAE, CHCl3 subfraction, and the EtOAc fraction. Of the identified proteins, profilin 2a, septin 7, cdc42, protein phosphatase 2A, DA11, eukaryotic translation initiation factor 5A-1, and γ-enolase are known to play important roles in neuritogenesis and dendritic arborization. Immunofluorescence data demonstrate that GAE-treated hippocampal neurons showed greater intensity ratios in the expressions of the septin 7 and cdc42 compared to vehicle control, validating their proteomic profiles. Together these results suggest that the GAE/CHCl3 subfraction and EtOAc fraction promote neurite development by up or downregulating several key proteins.


Asunto(s)
Neuronas/efectos de los fármacos , Neuronas/metabolismo , Extractos Vegetales/farmacología , Rhodophyta/química , Animales , Células Cultivadas , Hipocampo/química , Hipocampo/citología , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Neurogénesis/efectos de los fármacos , Neuronas/química , Neuronas/citología , Proteómica , Ratas , Ratas Sprague-Dawley , Septinas/genética , Septinas/metabolismo , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/metabolismo
5.
J Anim Sci ; 94(8): 3169-3184, 2016 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-27695782

RESUMEN

The objective of this study was to investigate the effect of a high dietary Se supplementation on the whole transcriptome of sheep. A custom sheep whole-transcriptome microarray, with more than 23,000 unique transcripts, was designed and then used to profile the global gene expression of sheep after feeding a high dietary supplementation of organic Se. Lactating crossbred ewes ( = 10; 3 to 4 yr of age and 55 to 65 kg BW) at late lactation (100 ± 8 d in milk) were acclimated to indoor individual pen feeding of a basal control diet (0.40 mg Se/d, sodium selenite) for 4 wk. Sheep were then kept on a diet with an extra (high) supplementation of organic Se (1.45 mg Se/d as Sel-Plex; Alltech Biotechnology Pty Ltd, Dandenong, Victoria, Australia) for 40 d. Whole blood was collected at 2 time points (last day of the acclimatization period [T0] and after 40 d of the organic Se supplementation [T40]), and then total RNA was isolated and labeled for the subsequent microarray analysis. Significance Analysis of Microarrays, using the -statistic, of the microarray data (T40 versus T0) evidenced the up- and downregulation of 942 and 244 transcripts (false discovery rate < 0.05), respectively. Seven genes showed the same trend of expression (up- or downregulation) when tested by quantitative real-time PCR (qPCR) in a cross-validation step. The microarray showed significant upregulation of the following selenoproteins at T40: selenium binding protein 1 (SELENBP1), selenoprotein W1 (SEPW1), glutathione peroxidase 3 (GPX3), and septin 8 (SEPT8). And the expression trends for SEPW1 and SEPT8 were validated using qPCR. Functional annotation of the differentially expressed genes showed the enrichment of several immune system-related biological processes (lymphocyte activation, cytokine binding, leukocyte activation, T cell differentiation, and B cell activation) and pathways (cytokine and interleukin signaling). Moreover, Gene Set Enrichment Analysis evidenced the enrichment of B and T cell receptors signaling pathways, with an enrichment score of 0.63 and 0.59, respectively. Overall, from a global gene expression (whole-transcriptome) point of view, short-term supplementation of a high dietary organic Se to Se-nondeficient sheep results in a transcriptomic signature that mainly reflects an induced immune system and a modulation of transcription effect. Also, the present study provides a custom whole-transcriptome microarray platform that can be used in further global gene expression studies in the ovine species.


Asunto(s)
Análisis por Matrices de Proteínas/veterinaria , Ovinos/genética , Selenito de Sodio/farmacología , Transcriptoma , Animales , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Glutatión Peroxidasa/genética , Glutatión Peroxidasa/metabolismo , Lactancia , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Selenoproteínas/genética , Selenoproteínas/metabolismo , Septinas/genética , Septinas/metabolismo , Ovinos/fisiología , Selenito de Sodio/administración & dosificación
6.
Int J Hyperthermia ; 32(6): 648-56, 2016 09.
Artículo en Inglés | MEDLINE | ID: mdl-27269053

RESUMEN

PURPOSE: Modulated electro-hyperthermia (mEHT) has been shown to be effective against various types of human tumours, including hepatocellular carcinoma (HCC). Here we aimed to investigate the molecular mechanism underlying the cytotoxic effects of mEHT to HCC cells. MATERIALS AND METHODS: Human liver cancer cell lines, Huh7 and HepG2, were treated with mEHT (42 °C/60 min) three times at 2-day intervals. Growth inhibition and apoptotic induction were evaluated using MTS, microscopic analysis, a clonogenic assay, annexin V/PI staining and a ccK18 ELISA. Global changes in gene expression were examined using RNA sequencing to obtain insights into molecular changes in response to mEHT. For in vivo evaluation of mEHT we used HepG2 HCC xenografts grown in nude mice. RESULTS: mEHT suppressed HCC cell proliferation and long-term colony formation through induction of apoptosis. The growth inhibitory effects are induced through a subset of molecular changes. Notably the expression level of septin 4 (SEPT4) (involved in pro-apoptotic activity and growth suppression) was up-regulated, whereas a key regulator of invasiveness G-Protein coupled receptor 64 (GPR64) was repressed. Subsequent Western blotting confirmed that the common increase in tumour suppressor SEPT4 in both Huh7 and HepG2 cells is accompanied by the restoration of cyclin-dependent kinase (CDK) inhibitor p21 and decrease in pro-caspase 7 and pro-caspase 3, thereby accelerating apoptotic signalling in HCC cells. Additionally, mEHT significantly inhibited the growth of human HCC xenografts in nude mice. CONCLUSIONS: These findings suggest that apoptotic cell death induced by mEHT is mediated by the up-regulation of tumour suppressor SEPT4 in human HCC cells.


Asunto(s)
Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/terapia , Hipertermia Inducida , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/terapia , Septinas/metabolismo , Animales , Apoptosis , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Proliferación Celular , Humanos , Neoplasias Hepáticas/patología , Ratones Desnudos , Carga Tumoral , Regulación hacia Arriba
7.
Hear Res ; 289(1-2): 40-51, 2012 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-22575789

RESUMEN

Septins are a family of GTP binding proteins that are well conserved in eukaryotic species except plants. Septins contribute to the lateral compartmentalization of membranes, cortical rigidity, and the regulation of membrane trafficking by associating with membrane lipids, actin, and microtubules. The organ of Corti in the cochlea has pivotal roles in auditory perception and includes two kinds of highly polarized cells, hair and supporting cells, both of which are rich in actin and microtubules. To identify the roles of septins in the cochlea, we analyzed the localization of three septin proteins, septin 4 (SEPT4), septin 5 (SEPT5), and septin 7 (SEPT7) that are abundantly expressed in brain tissues, and also examined auditory functions of Sept4 and Sept5 null mice. SEPT4, SEPT5, and SEPT7 were expressed in inner and outer pillar cells and Deiters' cells but the distribution patterns of each protein in Deiters' cells were different. SEPT4 and SEPT7 were expressed in the phalangeal process where SEPT5 was not detected. In addition to these cells SEPT5 and SEPT7 were co-localized with presynaptic vesicles of efferent nerve terminals. Only SEPT7 was expressed in the cochlea at embryonic stages. Although expression patterns of septin proteins suggested their important roles in the function of the cochlea, both Sept4 and Sept5 null mice had similar auditory functions to their wild type littermates. Immunohistochemical analysis of Sept4 null mice showed that compensatory expression of SEPT5 in the phalangeal process of Deiters' cells may have caused functional compensation of hearing ability in Sept4 null mice.


Asunto(s)
Vías Auditivas/metabolismo , Cóclea/metabolismo , Células Ciliadas Auditivas/metabolismo , Células Laberínticas de Soporte/metabolismo , Septinas/metabolismo , Estimulación Acústica , Animales , Umbral Auditivo , Cóclea/embriología , Potenciales Evocados Auditivos del Tronco Encefálico , Regulación de la Expresión Génica , Genotipo , Inmunohistoquímica , Ratones , Ratones Endogámicos ICR , Ratones Noqueados , Fenotipo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Septinas/deficiencia , Septinas/genética
8.
Schizophr Res ; 96(1-3): 257-66, 2007 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-17644312

RESUMEN

Schizophrenic brains exhibit various neuro-pathological changes in size, volume and structure as compared to normal brains. These structural abnormalities could be the result of apoptotic cell death. ARTS/Sept4 protein plays an important role in induction and promotion of apoptosis. Though ARTS is highly expressed in the healthy human brain, most of tested schizophrenic brain samples showed no expression of ARTS protein. Specifically, using Western blot analysis with monoclonal anti-ARTS antibody we found that only 1 out of 14 schizophrenic samples (7%) showed a strong ARTS signal as compared to 10 out of 15 (66.6%) found in the normal controls group. Furthermore, using immunohistochemistry assay only 33.3% (5 of 15) (SE+/-12.5) of the schizophrenic patients samples showed any ARTS immunoreactivity as compared to (13 of 15) 87% (SE+/-9) of bipolar, (11 of 14) 78% (SE+/-11.3) of major depression and (10 of 14) 71% (SE+/-12.5) of normal controls. A four-fold reduction in apoptosis rate was measured in these schizophrenic samples as compared to average apoptosis rate found in all other samples. These data support the linkage between loss of ARTS expression and the loss of sensitivity towards apoptosis. Interestingly, levels of ARTS were significantly lower in male schizophrenic patients as compared to female schizophrenic patients, and males of all other control groups. We propose that ARTS may play an important role in the pathogenesis of schizophrenia and could be used as a marker for this disease.


Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Lóbulo Frontal/metabolismo , Lóbulo Frontal/patología , GTP Fosfohidrolasas/metabolismo , Esquizofrenia/metabolismo , Esquizofrenia/patología , Apoptosis , Autopsia , Biomarcadores/metabolismo , Supervivencia Celular , Humanos , Etiquetado Corte-Fin in Situ , Cambios Post Mortem , Valores de Referencia , Septinas
9.
Neuron ; 53(4): 519-33, 2007 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-17296554

RESUMEN

In Parkinson disease (PD), alpha-synuclein aggregates called Lewy bodies often involve and sequester Septin4 (Sept4), a polymerizing scaffold protein. However, the pathophysiological significance of this phenomenon is unclear. Here, we show the physiological association of Sept4 with alpha-synuclein, the dopamine transporter, and other presynaptic proteins in dopaminergic neurons; mice lacking Sept4 exhibit diminished dopaminergic neurotransmission due to scarcity of these presynaptic proteins. These data demonstrate an important role for septin scaffolds in the brain. In transgenic mice that express human alpha-synuclein(A53T) (a mutant protein responsible for familial PD), loss of Sept4 significantly enhances neuropathology and locomotor deterioration. In this PD model, insoluble deposits of Ser129-phosphorylated alpha-synuclein(A53T) are negatively correlated with the dosage of Sept4. In vitro, direct association with Sept4 protects alpha-synuclein against self-aggregation and Ser129 phosphorylation. Taken together, these data show that Sept4 may be involved in PD as a dual susceptibility factor, as its insufficiency can diminish dopaminergic neurotransmission and enhance alpha-synuclein neurotoxicity.


Asunto(s)
Proteínas del Citoesqueleto/metabolismo , GTP Fosfohidrolasas/metabolismo , Enfermedad de Parkinson/metabolismo , Terminales Presinápticos/metabolismo , alfa-Sinucleína/metabolismo , Estimulación Acústica/métodos , Anciano , Anciano de 80 o más Años , Animales , Encéfalo/patología , Proteínas del Citoesqueleto/genética , Modelos Animales de Enfermedad , Dopamina/metabolismo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Femenino , GTP Fosfohidrolasas/genética , Humanos , Masculino , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Mutación , Inhibición Neural/fisiología , Neuronas/metabolismo , Enfermedad de Parkinson/patología , Enfermedad de Parkinson/fisiopatología , Enfermedad de Parkinson/terapia , Reflejo de Sobresalto/fisiología , Septinas , Serina/metabolismo , Sinaptofisina/metabolismo , alfa-Sinucleína/genética
10.
Gene Expr ; 11(5-6): 271-8, 2004.
Artículo en Inglés | MEDLINE | ID: mdl-15200239

RESUMEN

Septin 3 is a novel member of the septin subfamily of GTPase domain proteins. Human septin 3 was originally cloned during a screening of genes expressed in human teratocarcinoma cells induced to differentiate with retinoic acid. Alternative splicing of the septin 3 gene transcript produces two isoforms, A and B, in the human brain, though their regional expression and physiological function remain to be determined. The purpose of the present study was to identify the expression patterns of human septin 3 isoforms in normal human brain and a human neuroblastoma cell line, SH-SY5Y, after retinoic acid-induced differentiation. The expression and distribution patterns of septin 3 isoforms A and B were similar and resembled that of another septin, CDCrel-1. Septin 3A and 3B were expressed in normal human brain in a region-specific manner, with the highest level in the temporal cortex and hippocampus and the lowest level in the brainstem regions. Prominent immunoreactivity was observed diffusely in the neocortices in association with neuropils and punctate structures suggestive of synaptic junctions. Immunoprecipitation studies revealed that septin 3A, 3B, and CDCrel-1 form a complex in the frontal cortex of human brain. These findings, taken together, suggest that septin 3A and 3B, along with CDCrel-1, play some fundamental role(s) in synaptogenesis and neuronal development.


Asunto(s)
Encéfalo/metabolismo , GTP Fosfohidrolasas/metabolismo , Anticuerpos/inmunología , Química Encefálica , Proteínas de Ciclo Celular/análisis , Diferenciación Celular , Línea Celular , Lóbulo Frontal/química , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/inmunología , Expresión Génica , Humanos , Inmunoquímica , Inmunoprecipitación , Unión Proteica , Isoformas de Proteínas/análisis , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , ARN Mensajero/análisis , Septinas , Tretinoina/farmacología , Regulación hacia Arriba
11.
Cancer Res ; 62(2): 333-7, 2002 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-11809673

RESUMEN

t(X;11) is a recurrent translocation in pediatric acute myeloid leukemia (AML). We showed that the MLL gene on 11q23 was fused to the SEPTIN6 gene on Xq24, a human homologue to mouse Septin6, in three de novo infant AML with complex chromosomal abnormalities involving 11q23 and Xq22-24. SEPTIN6 consisted of at least 12 exons and was predicted to encode at least two types of proteins by alternative splicing. Expression of approximately 2.3-, 3.1-, and 4.6-kb SEPTIN6 transcripts was simultaneously detected in fetal lung, liver, and brain, in all of the adult tissues except brain, and in acute lymphoblastic leukemia and AML cell lines. However, the expression of an approximately 2.7-kb transcript was detected alone in fetal heart and adult brain. The SEPTIN6 protein is homologous to septin family members including CDCREL1 and AF17q25/MSF, which generate fusion products with MLL. The MLL-SEPTIN6 fusion proteins contain almost the entire septin protein, similar to MLL-CDCREL1 and MLL-AF17q25/MSF. Notably, all three of the patients were diagnosed with M1 or M2. Combined present results and literatures suggest that AML with the MLL-SEPTIN6 fusion gene is a subset of infant AML, which differentiate into the myeloid lineage, although AML with other MLL fusion genes is capable of differentiating into the myelomonocytic or monocytic lineage.


Asunto(s)
Cromosomas Humanos Par 11 , Proteínas de Unión al ADN/genética , Proteínas de Unión al GTP/genética , Leucemia Mieloide Aguda/genética , Proto-Oncogenes , Factores de Transcripción , Translocación Genética , Cromosoma X , Adulto , Animales , Fusión Artificial Génica , Clonación Molecular , Proteínas del Citoesqueleto , ADN Complementario/genética , ADN de Neoplasias/genética , Femenino , Proteínas de Unión al GTP/biosíntesis , Expresión Génica , N-Metiltransferasa de Histona-Lisina , Humanos , Lactante , Leucemia Mieloide Aguda/metabolismo , Masculino , Ratones , Proteína de la Leucemia Mieloide-Linfoide , Septinas
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