RESUMO
Foreground detection is a classic video processing task, widely used in video surveillance and other fields, and is the basic step of many computer vision tasks. The scene in the real world is complex and changeable, and it is difficult for traditional unsupervised methods to accurately extract foreground targets. Based on deep learning theory, this paper proposes a foreground detection method based on the multiscale U-Net architecture with a fusion attention mechanism. The attention mechanism is introduced into the U-Net multiscale architecture through skip connections, causing the network model to pay more attention to the foreground objects, suppressing irrelevant background regions, and improving the learning ability of the model. We conducted experiments and evaluations on the CDnet-2014 dataset. The proposed model inputs a single RGB image and only utilizes spatial information, with an overall F-measure of 0.9785. The input of multiple images is fused, and the overall F-measure can reach 0.9830 by using spatiotemporal information. Especially in the Low Framerate category, the F-measure exceeds the current state-of-the-art methods. The experimental results demonstrate the effectiveness and superiority of our proposed method.
RESUMO
Protein phosphatase-2A (PP2A) is a heterotrimeric enzyme composed of a catalytic subunit, a regulatory subunit, and a structural subunit. In Candida albicans, Cdc55 and Rts1 have been identified as possible regulatory subunits of PP2A containing the catalytic subunit Pph21 and structural subunit Tpd3. The Tpd3-Pph21 phosphatase regulates cell morphogenesis and division. However, the functions of Cdc55 and Rts1 remain unclear. Here, we constructed cdc55Δ/Δ and rts1Δ/Δ mutants and found that they exhibit different defects in multiple phenotypes although both show similar hyperphosphorylation of the septin Sep7 and aberrant septin organization. Under yeast growth conditions, the cdc55Δ/Δ mutant grows slowly as pseudohyphae with some cells lacking the nucleus, while rts1Δ/Δ cells are round and enlarged and seem to undergo incomplete cell separation producing multinucleated cells. Strong chitin deposition occurs at the septum of cdc55Δ/Δ cells and on the surface of rts1Δ/Δ cells, which likely contributes to increased susceptibility to caspofungin. Also, cdc55Δ/Δ exhibits severe defects in hyphal and biofilm formation, while rts1Δ/Δ is partially defective. Both mutants show reduced virulence in mice, suggesting that PP2A-B subunits could serve as potential antifungal targets.
Assuntos
Candida albicans/crescimento & desenvolvimento , Candida albicans/patogenicidade , Candidíase/patologia , Domínio Catalítico/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Fúngicas/metabolismo , Proteína Fosfatase 2/metabolismo , Animais , Candida albicans/efeitos dos fármacos , Candida albicans/genética , Candidíase/microbiologia , Caspofungina/farmacologia , Proteínas de Ciclo Celular/genética , Divisão Celular/genética , Modelos Animais de Doenças , Farmacorresistência Fúngica/genética , Feminino , Proteínas Fúngicas/genética , Deleção de Genes , Rim/microbiologia , Rim/patologia , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Fenótipo , Fosforilação/genética , Proteína Fosfatase 2/genética , Virulência/genéticaRESUMO
ß-1,6-glucan is an important cell wall component of Candida albicans. Deleted mutants of the two ß-1,6-glucan synthase genes KRE6 and SKN1 are viable albeit with a range of defects including slow growth. It remains unclear whether ß-1,6-glucan synthesis is not required under culture conditions or compensatory mechanisms exist in C. albicans. Here, we report that depleting ß-1,6-glucan synthases leads to a significant increase in cell wall chitin levels through the posttranscriptional regulation of the chitin synthase Chs3 which maintains cell viability. And depleting ß-1,6-glucan synthases in chs3Δ/Δ cells results in cell death. The elevation of cell wall chitin is mediated by the activation of the PKC signaling pathway and an unknown pathway(s) involving Ca2+ -calcineurin. Also, kre6Δ/Δ skn1Δ/Δ cells are not more susceptible to caspofungin, the antifungal drug that inhibits ß-1,3-glucan synthases, suggesting that ß-1,3-glucan has no role in compensating ß-1,6-glucan synthesis. Given the vital importance of elevating chitin synthesis in the absence of ß-1,6-glucan synthesis in C. albicans, antifungal drugs targeting ß-1,6-glucan and chitin synthesis could be used in combination therapies.
Assuntos
Calcineurina/metabolismo , Cálcio/metabolismo , Candida albicans/crescimento & desenvolvimento , Candida albicans/metabolismo , Parede Celular/metabolismo , Quitina/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteína Quinase C/metabolismo , beta-Glucanas/metabolismo , Calcineurina/genética , Candida albicans/enzimologia , Candida albicans/genética , Parede Celular/genética , Quitina Sintase/genética , Quitina Sintase/metabolismo , Proteínas Fúngicas/genética , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Proteínas Quinases Ativadas por Mitógeno/genética , Proteína Quinase C/genética , Transdução de SinaisRESUMO
Phosphatidate phosphatases play essential roles in lipid metabolism by converting phosphatidic acid to diacylglycerol. Here, we have investigated the roles of a phosphatidate phosphatase, Pah1, in the fungal pathogen Candida albicans. Deleting PAH1 causes multiple phenotypes, especially severe hyphal defects, increased sensitivity to cell wall stress, and reduced virulence in mice. By qPCR, we detected a significant downregulation of hyphal-specific genes including two key hyphal-promoting genes UME6 and HGC1. Overexpression of UME6 in pah1Δ/Δ cells largely restored the hyphal growth, indicating that the reduced expression of UME6 is primarily responsible for the hyphal defects. We also detected decreased expression of three hyphal-promoting transcription factors EFG1, FLO8, and CPH1 in pah1 mutants, consistent with the reduced expression of UME6. Furthermore, the pah1Δ/Δ mutant exhibited increased sensitivity to cell wall stress. During systemic infection of mice, the mutant showed significantly impaired ability to colonize the kidney and to kill the host. Together, C. albicans PAH1 plays an important role in hyphal growth, adaptability to environmental stresses, and virulence. Thus, Pah1 could be targeted for the development of new antifungal drugs.
Assuntos
Candida albicans/crescimento & desenvolvimento , Candida albicans/patogenicidade , Proteínas Fúngicas/fisiologia , Hifas/crescimento & desenvolvimento , Fosfatidato Fosfatase/fisiologia , Animais , Candidíase/microbiologia , Feminino , Deleção de Genes , Camundongos Endogâmicos BALB C , Estresse Fisiológico , Fatores de Transcrição/metabolismo , VirulênciaRESUMO
ß-1,6-glucan is an important component of the fungal cell wall. The ß-1,6-glucan synthase gene KRE6 was thought to be essential in the fungal pathogen Candida albicans because it could not be deleted in previous efforts. Also, the role of its homolog SKN1 was unclear because its deletion caused no defects. Here, we report the construction and characterization of kre6Δ/Δ, skn1Δ/Δ and kre6Δ/Δ skn1Δ/Δ mutants in C. albicans. While deleting KRE6 or SKN1 had no obvious phenotypic consequence, deleting both caused slow growth, cell separation failure, cell wall abnormalities, diminished hyphal growth, poor biofilm formation and loss of virulence in mice. Furthermore, the GPI-linked cell surface proteins Hwp1 and the invasin Als3 or Ssa1 were not detected in kre6Δ/Δ skn1Δ/Δ mutant. In GMM medium, RT-qPCR and western blotting revealed a constitutive expression of KRE6 and growth conditions-associated activation of SKN1. Like many hypha-specific genes, SKN1 is repressed by Nrg1, but its activation does not involve the transcription factor Efg1. Dysregulation of SKN1 reduces C. albicans ability to damage epithelial and endothelial cells and attenuates its virulence. Given the vital role of ß-1,6-glucan synthesis in C. albicans physiology and virulence, Kre6 and Skn1 are worthy targets for developing antifungal agents.
Assuntos
Candida albicans/enzimologia , Candida albicans/patogenicidade , Deleção de Genes , Glucosiltransferases/deficiência , Fatores de Virulência/deficiência , beta-Glucanas/metabolismo , Animais , Candida albicans/crescimento & desenvolvimento , Candida albicans/metabolismo , Candidíase/microbiologia , Candidíase/patologia , Modelos Animais de Doenças , Glucosiltransferases/metabolismo , Camundongos , Virulência , Fatores de Virulência/metabolismoRESUMO
Melanoma-associated antigen A1 (MAGEA1) and BORIS (also known as CTCFL) are members of the cancer testis antigen (CTA) family. Their functions and expression-regulation mechanisms are not fully understood. In this study, we reveal new functions and regulatory mechanisms of MAGEA1 and BORIS in breast cancer cells, which we investigated in parental and genetically manipulated breast cancer cells via gene overexpression or siRNA-mediated downregulation. We identified the interaction between MAGEA1 and CTCF, which is required for the binding of MAGEA1 to the BORIS promoter and is critical for the recruitment of DNMT3a. A protein complex containing MAGEA1, CTCF and DNMT3a was formed before or after conjunction with the BORIS promoter. The binding of this complex to the BORIS promoter accounts for the hypermethylation and repression of BORIS expression, which results in cell death in the breast cancer cell lines tested. Multiple approaches were employed, including co-immunoprecipitation, glutathione S-transferase pull-down assay, co-localization and cell death analyses using annexin V-FITC/propidium iodide double-staining and caspase 3 activation assays, chromatin immunoprecipitation and bisulfite sequencing PCR assays for methylation. Our results have implications for the development of strategies in CTA-based immune therapeutics.
Assuntos
Neoplasias da Mama/patologia , Metilação de DNA , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica , Antígenos Específicos de Melanoma/metabolismo , Neoplasias Ovarianas/patologia , Regiões Promotoras Genéticas , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Fator de Ligação a CCCTC/genética , Fator de Ligação a CCCTC/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA Metiltransferase 3A , Proteínas de Ligação a DNA/metabolismo , Epigênese Genética , Feminino , Humanos , Antígenos Específicos de Melanoma/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Células Tumorais CultivadasRESUMO
PP2A-like phosphatases share high homology with PP2A enzymes and are composed of a catalytic subunit and a regulatory subunit. In Candida albicans, the PP2A-like catalytic subunit SIT4 regulates cell growth, morphogenesis, and virulence. However, the functions of its regulatory subunits remain unclear. Here, by homology analysis and co-IP experiments, we identified two regulatory subunits of SIT4 in C. albicans, SAP155 (orf19.642) and SAP190 (orf19.5160). We constructed sit4Δ/Δ, sap155Δ/Δ, sap190Δ/Δ, and sap155Δ/Δ sap190Δ/Δ mutants and found that deleting SAP155 had no apparent phenotypic consequence, while deleting SAP190 caused slow growth, hypersensitivity to cell wall stress, abnormal morphogenesis in response to serum or genotoxic stress (HU and MMS), less damage to macrophages, and attenuated virulence in mice. However, deleting both SAP155 and SAP190 caused significantly stronger defects, which was similar to deleting SIT4. Together, our results suggest that SAP190 is required for the function of SIT4 and that SAP155 can partially compensate for the loss of SAP190 in C. albicans. Given the vital role of these regulatory subunits of SIT4 in C. albicans physiology and virulence, they could serve as potential antifungal targets.
RESUMO
Genotoxic stress causes DNA damage or stalled DNA replication and filamentous growth in the pathogenic fungus Candida albicans The DNA checkpoint kinase Rad53 critically regulates by phosphorylation effectors that execute the stress response. Rad53 itself is activated by phosphorylation and inactivated by dephosphorylation. Previous studies have suggested that the phosphatase Pph3 dephosphorylates Rad53. Here, we used mass spectrometry and mutagenesis to identify Pph3 dephosphorylation sites on Rad53 in C. albicans We found that serine residues 351, 461 and 477, which were dephosphorylated in wild-type cells during the recovery from DNA damage caused by methyl methanesulfonate (MMS), remained phosphorylated in pph3Δ/Δ cells. Phosphomimetic mutation of the three residues (rad53-3D) impaired Rad53 dephosphorylation, exit from cell cycle arrest, dephosphorylation of two Rad53 effectors Dun1 and Dbf4, and the filament-to-yeast growth transition during the recovery from MMS-induced DNA damage. The phenotypes observed in the rad53-3D mutant also occurred in the pph3Δ/Δ mutant. Together, our findings reveal a molecular mechanism by which Pph3 controls DNA damage response in C. albicans.
Assuntos
Candida albicans/efeitos dos fármacos , Quinase do Ponto de Checagem 2/genética , Reparo do DNA , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Metanossulfonato de Metila/farmacologia , Fosfoproteínas Fosfatases/genética , Candida albicans/genética , Candida albicans/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2/metabolismo , Dano ao DNA , Proteínas Fúngicas/metabolismo , Deleção de Genes , Fosfoproteínas Fosfatases/deficiência , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Serina/metabolismoRESUMO
The protein kinase Rad53 and its orthologs play a fundamental role in regulating the DNA damage checkpoint in eukaryotes. Rad53 is activated by phosphorylation in response to DNA damage and deactivated by dephosphorylation after the damage is repaired. However, the phosphatases involved in Rad53 deactivation are not entirely understood. In this study, by investigating the consequences of overexpressing SDS22, a gene encoding a regulatory subunit of the PP1 phosphatase Glc7, in the human fungal pathogen Candida albicans, we discovered that Sds22 plays an important role in Rad53 dephosphorylation and thus the deactivation of the DNA damage checkpoint. Sds22 cellular levels increase when cells are exposed to DNA damaging agents and decrease after removing the genotoxins. Depletion of Glc7 has similar phenotypes. We provide evidence that Sds2 acts through inhibitory physical association with Glc7. Our findings provide novel insights into the mechanisms for the control of DNA damage checkpoint. Furthermore, SDS22 overexpression reduces C. albicans virulence in a mouse model of systemic infection, suggesting potential targets for developing antifungal drugs.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Candida albicans/genética , Candidíase/genética , Dano ao DNA/genética , Proteínas Fúngicas/genética , Proteína Fosfatase 1/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas de Caenorhabditis elegans/biossíntese , Candida albicans/patogenicidade , Candidíase/microbiologia , Núcleo Celular/genética , Dano ao DNA/efeitos dos fármacos , Proteínas Fúngicas/biossíntese , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Humanos , Metanossulfonato de Metila/toxicidade , Fosforilação , Proteínas Serina-Treonina Quinases/biossínteseRESUMO
Septins are a component of the cytoskeleton and play important roles in diverse cellular processes including cell cycle control, cytokinesis and polarized growth. In fungi, septin organization, dynamics and function are regulated by phosphorylation, and several kinases responsible for the phosphorylation of several septins have been identified. However, little is known about the phosphatases that dephosphorylate septins. Here, we report the characterization of Tpd3, a structural subunit of the PP2A family of phosphatases, in the pathogenic fungus Candida albicans. We found that tpd3Δ/Δ cells are defective in hyphal growth and grow as pseudohyphae under yeast growth conditions with aberrant septin organization. Western blotting detected hyperphosphorylation of the septin Sep7 in cells lacking Tpd3. Tpd3 and Sep7 colocalize at the bud neck and can coimmunoprecipitate. Furthermore, we discovered similar defects in cells lacking Pph21, a catalytic subunit of the PP2A family, and its physical association with Tpd3. Importantly, purified Tpd3-Pph21 complexes can dephosphorylate Sep7 in vitro. Together, our findings strongly support the idea that the Tpd3-Pph21 complex dephosphorylates Sep7 and regulates morphogenesis and cytokinesis. The tpd3Δ/Δ mutant is greatly reduced in virulence in mice, providing a potential antifungal target.
Assuntos
Candida albicans/metabolismo , Proteína Fosfatase 2/metabolismo , Septinas/metabolismo , Animais , Candida albicans/enzimologia , Candida albicans/crescimento & desenvolvimento , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Citocinese/fisiologia , Citoesqueleto/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Hifas/crescimento & desenvolvimento , Camundongos , Camundongos Endogâmicos BALB C , Fosforilação , Proteína Fosfatase 2/genética , Septinas/genética , VirulênciaRESUMO
We previously produced an anti-idiotypic monoclonal antibody, 6B11, which mimics ovarian cancer antigen CA166-9 and induces cellular and humoral immunity. Here, to enhance the immunogenicity of 6B11, we constructed the 6B11ScFv-mIL-12 fusion protein (FP), by fusing single-chain fragment of 6B11 variable region (6B11ScFv) with mouse interleukin-12 (mIL-12), which was expressed in eukaryotic 293EBNA cells transfected with pSBI vectors. A binding activity assay showed 6B11ScFv-mIL-12 to have activities of both 6B11 and mIL-12-it specifically bound both ovarian monoclonal antibody COC166-9 and rabbit anti-mouse IL-12 antibody. The immune activity assay showed 6B11ScFv-mIL-12 to promote proliferation of lymphocytes stimulated by phytohemagglutinin, increase the absolute numbers and percentages of CD3(-)/CD56(+) natural killer cells and CD3(+)/CD56(+) natural killer T cells among peripheral lymphocytes, and increase interferon-γ. The FP was specifically cytotoxic to the CA166-9(+) ovarian cancer cell lines HOC1A and SKOV3 and inhibited growth of ID8 subcutaneous tumors in C57BL/6J mice. This study provides an experimental basis for clinical use of 6B11ScFv-mIL-12 in ovarian cancer therapy. To our knowledge, this is the first report of a fusion protein from an anti-idiotypic antibody and IL-12.
Assuntos
Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Monoclonais/uso terapêutico , Antígenos de Neoplasias/imunologia , Interleucina-12/imunologia , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/terapia , Proteínas Recombinantes de Fusão/administração & dosagem , Animais , Anticorpos Antineoplásicos/imunologia , Vacinas Anticâncer/uso terapêutico , Feminino , Humanos , Interferon gama/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Ovarianas/metabolismo , Coelhos , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Linfócitos T/imunologia , Células Tumorais CultivadasRESUMO
PURPOSE: Nuclear apoptosis-inducing factor 1 (NAIF1) could induce apoptosis in gastric cancer cells. Previously, we have reported that the expression of NAIF1 protein is down-regulated in gastric cancer tissues compared with the adjacent normal tissues. However, the role of NAIF1 in gastric cancer cells is not fully understood. METHODS: The effects of NAIF1 on cell viability were evaluated by MTT and colony formation assays. The ability of cellular migration and invasion were analyzed by transwell assays. The expression levels of targeted proteins were determined by western blot. The relative RNA expression levels were analyzed using quantitative polymerase chain reaction assays. Xenograft experiment was employed to determine the anti-tumor ability of NAIF1 in vivo. RESULTS: The study demonstrates that transient transfection of NAIF1 in gastric cancer cells BGC823 and MKN45 could inhibit the cell proliferation, migration, and invasion of the two gastric cancer cell lines. The tumor size is smaller in NAIF1-overexpressed MKN45 cell xenograft mice than in unexpressed group. Further in-depth analysis reveals that NAIF1 reduces the expression of MMP2 as well as MMP9, and inhibits the activation of FAK, all of which are key molecules involved in regulating cell migration and invasion. In addition, NAIF1 inhibits the expression of c-Jun N-terminal kinase (JNK) by accelerating its degradation through ubiquitin-proteasome pathway. Meanwhile, NAIF1 reduces the mRNA and protein expression of ERK1/2. CONCLUSIONS: Our study revealed that NAIF1 plays a role in regulating cellular migration and invasion through the MAPK pathways. It could be a therapeutic target for gastric cancer.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteínas Nucleares/metabolismo , Transdução de Sinais/efeitos dos fármacos , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Actinas/metabolismo , Animais , Proteínas Reguladoras de Apoptose/farmacologia , Western Blotting , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Progressão da Doença , Quinase 1 de Adesão Focal/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Invasividade Neoplásica , Proteínas Nucleares/farmacologia , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Neoplasias Gástricas/enzimologia , Ubiquitina/metabolismoRESUMO
AIM: To study microchimerism's role and function after microtransplantation and identify novel genetic markers for microchimerism detection. METHODS: Analyzing microchimerisms from patients microtransplanted to determine the presence of GSTT1, GSTM1, SRY and other genetic markers by real-time PCR. RESULTS: Microchimerism could be detected for a short time after microtransplantation simultaneously with hematopoietic recovery. In conclusion, microchimerism might accelerate hematopoietic recovery and GSTT1 and GSTM1 genes could be used as genetic markers to differentiate donor cells. DISCUSSION: Microchimerism could exist for a short time after microtransplantation and appears to function in hematopoietic recovery. According to published reports, cytokines secreted from microchimerisms could be detected in recipients and exhibit some function on the host. Therefore, cytokines secreted from donor cells are hypothesized to accelerate hematopoietic recovery. The evidence to prove a longer existence for microchimerism is insufficient and needs supports by additional experiments; however, we cannot deny its existence just because of the limited sensitivity of methods.
Assuntos
Quimerismo , Leucemia Mieloide Aguda/terapia , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Transplante/métodos , Idoso , Sequência de Bases , Primers do DNA , Marcadores Genéticos , Glutationa Transferase/genética , Doença Enxerto-Hospedeiro , Humanos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Successful pathogens must be able to swiftly respond to and repair DNA damages inflicted by the host defence. The replication protein A (RPA) complex plays multiple roles in DNA damage response and is regulated by phosphorylation. However, the regulators of RPA phosphorylation remain unclear. Here, we investigated Rfa2 phosphorylation in the pathogenic fungus Candida albicans. Rfa2, a RFA subunit, is phosphorylated when DNA replication is inhibited by hydroxyurea and dephosphorylated during the recovery. By screening a phosphatase mutant library, we found that Pph3 associates with different regulatory subunits to differentially control Rfa2 dephosphorylation in stressed and unstressed cells. Site-directed mutagenesis revealed T11, S18, S29, and S30 being critical for Rfa2 phosphorylation in response to genotoxic insult. We obtained evidence that the genome integrity checkpoint kinase Mec1 and the cyclin-dependent kinase Clb2-Cdc28 mediate Rfa2 phosphorylation. Although cells expressing either a phosphomimetic or a non-phosphorylatable version of Rfa2 had defects, the latter exhibited greater sensitivity to genotoxic challenge, failure to repair DNA damages and to deactivate Rad53-mediated checkpoint pathways in a dosage-dependent manner. These mutants were also less virulent in mice. Our results provide important new insights into the regulatory mechanism and biological significance of Rfa2 phosphorylation in C. albicans.
Assuntos
Candida albicans/genética , Candidíase/microbiologia , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/metabolismo , Motivos de Aminoácidos , Animais , Candida albicans/química , Candida albicans/metabolismo , Candida albicans/patogenicidade , Replicação do DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Fosforilação , Proteína de Replicação A/genética , Proteína de Replicação A/metabolismo , VirulênciaRESUMO
The microchimerism is a status of the microcell or DNA of an individual in another one with genetic differences. Taking an overall view about the discovery and research of the microchimerism, it was found that although the study of the microchimerism emphasizes the formation, origin, distribution, type, relationship to disease and several other aspects, the objects of the study are always the microchimerism that obtained naturally. As it is known to all, the microchimerism can also be produced in some clinical treatment, such as in the transplant and transfusion, but compared with the microchimerism gained naturally, obviously, the study for the iatrogenic microchimerism formed in the treatment is not elaborate enough. The curative effect of micro transplantation, a new technique for leukemia treatment, is obvious, but its mechanism is unclear, whether that is related to microchimerism still needs further research. This review summarizes the study history and perspective of the microchimerism so as to provide some ideas for studying the action mechanism of microchimerism in micro transplantation.
Assuntos
Quimerismo , Quimeras de Transplante , DNA/genética , HumanosRESUMO
Nuclear apoptosis-inducing factor 1 (NAIF1) was previously reported to induce apoptosis. Moreover, the expression of NAIF1 was significantly down-regulated in human gastric cancer tissues compared to adjacent normal tissues. However, the mechanism by which the NAIF1 gene induces apoptosis is not fully understood. Our results show that NAIF1 was minimally expressed in all the tested gastric cancer cell lines. Our data also demonstrates that NAIF1 is localized in the nuclei of cells as detected by monitoring the green fluorescence of NAIF1-GFP fusion protein using fluorescent confocal microscopy. Next, a comparative proteomic approach was used to identify the differential expression of proteins between gastric cancer cell lines MKN45/NAIF1 (-) and MKN45/NAIF1 (+). We found five proteins (proteasome 26S subunit 2, proteasome 26S subunit 13, NADH dehydrogenase Fe-S protein 1, chaperonin containing TCP1 subunit 3 and thioredoxin reductase 1) that were up-regulated and three proteins (ribonuclease inhibitor 1, 14-3-3 protein epsilon isoform and apolipoprotein A-I binding protein) that were down-regulated in the MKN45 cells overexpressing NAIF1. We also discovered that NAIF1 could induce cell cycle arrest at G1/S phase by altering the expression of cell cycle proteins cyclinD1, cdc2 and p21. The differentially expressed proteins identified here are related to various cellular programs involving cell cycle, apoptosis, and signal transduction regulation and suggest that NAIF1 may be a tumor suppressor in gastric cancer. Our research provides evidence that elucidates the role of how NAIF1 functions in gastric cancer.
Assuntos
Proteínas Reguladoras de Apoptose/genética , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Proteínas Nucleares/genética , Proteoma/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Pontos de Checagem do Ciclo Celular/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Metaboloma , Proteínas Nucleares/metabolismo , Proteoma/metabolismo , Proteômica , Neoplasias Gástricas/patologia , Transfecção , Células Tumorais Cultivadas , Regulação para CimaRESUMO
Traditionally, cell line generation requires several months and involves screening of over several hundred cell clones for high productivity before dozens are selected as candidate cell lines. Here, we have designed a new strategy for the generation of stable and high-expression cell lines by combining homologous recombination (HR) and fluorescence-activated cell sorting (FACS). High expression was indicated by the expression of secreted green fluorescent protein (SEGFP). Parental cell lines with the highest expression of SEGFP were then selected by FACS and identified by stability analysis. Consequently, HR vectors were constructed using the cassette for SEGFP as the HR region. After transfecting the HR vector, the cells with negative SEGFP expression were enriched by FACS. The complete exchange between SEGFP and target gene (TNFR-Fc) cassettes was demonstrated by DNA analysis. Compared with the traditional method, by integrating the cassette containing the gene of interest into the pre-selected site, the highest producing cells secreted a more than 8-fold higher titer of target protein. Hence, this new strategy can be applied to isolated stable cell lines with desirable expression of any gene of interest. The stable cell lines can rapidly produce proteins for researching protein structure and function and are even applicable in drug discovery.
Assuntos
Engenharia Celular/métodos , Citometria de Fluxo , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Recombinação Homóloga , Imunoglobulina G/genética , Receptores do Fator de Necrose Tumoral/genética , Animais , Células CHO , Células Clonais , Cricetulus , Eletroporação , Etanercepte , Efeito Fundador , Vetores Genéticos , Proteínas de Fluorescência Verde/metabolismo , Imunoglobulina G/biossíntese , Receptores do Fator de Necrose Tumoral/biossíntese , Seleção Genética , Transfecção , TransgenesRESUMO
BACKGROUND: Ovarian cancer G protein coupled receptor 1 (OGR1) mediates inhibitory effects on cell migration in human prostate and ovarian cancer cells. However, the mechanisms and signaling pathways that mediate these inhibitory effects are essentially unknown. METHODS: MCF7 cell line was chosen as a model system to study the mechanisms by which OGR1 regulates cell migration, since it expresses very low levels of endogenous OGR1. Cell migratory activities were assessed using both wound healing and transwell migration assays. The signaling pathways involved were studied using pharmacological inhibitors and genetic forms of the relevant genes, as well as small G protein pull-down activity assays. The expression levels of various signaling molecules were analyzed by Western blot and quantitative PCR analysis. RESULTS: Over-expression of OGR1 in MCF7 cells substantially enhanced activation of Rho and inhibition of Rac1, resulting in inhibition of cell migration. In addition, expression of the Gα12/13 specific regulator of G protein signaling (RGS) domain of p115RhoGEF, but not treatment with pertussis toxin (PTX, a Gαi specific inhibitor), could abrogate OGR1-dependent Rho activation, Rac1 inactivation, and inhibition of migration in MCF7 cells. The bioactive lipids tested had no effect on OGR1 function in cell migration. CONCLUSION: Our data suggest, for the first time, that OGR1 inhibits cell migration through a Gα12/13 -Rho-Rac1 signaling pathway in MCF7 cells. This pathway was not significantly affected by bioactive lipids and all the assays were conducted at constant pH, suggesting a constitutive activity of OGR1. This is the first clear delineation of an OGR1-mediated cell signaling pathway involved in migration.
RESUMO
CCDC134 (coiled coil domain containing 134), a novel secretory protein, acts as an inhibitor of Erk1/2 and JNK/SAPK pathways. However, the role of CCDC134 in cancer development is still lacking. In this study, we found that CCDC134 expression significantly reduced in gastric cancer tissues compared with normal tissues (P < 0.001) and lesion tissues (P < 0.001). But no statistically significant difference was observed between normal and lesion tissues (P = 0.842). In vitro transient transfection of CCDC134-specific siRNA significantly promoted the migration and invasion of both the normal gastric epithelial cell line GES-1 and gastric cancer cell line AGS cells. Further analysis revealed that the attenuated expression of CCDC134 promoted the activation of Erk1/2 and JNK/SAPK, but had no effect on p38. The activation of Erk1/2 and JNK/SAPK was required for CCDC134-mediated migration and invasion. Besides, CCDC134-RNAi could induce the expression of MMP-2 and MMP-9, which are key molecules involved in regulating cell migration and invasion. Therefore, CCDC134 may be a candidate biomarker for malignant transformation. It plays a role in regulation of cell migration and invasion, and could be a therapeutic target of gastric cancer.
Assuntos
Movimento Celular , Sistema de Sinalização das MAP Quinases , Proteínas Nucleares/genética , Interferência de RNA , Neoplasias Gástricas/metabolismo , Linhagem Celular , Regulação para Baixo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , MAP Quinase Quinase 4/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Proteínas de Membrana , Invasividade Neoplásica , Proteínas Nucleares/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , RNA Interferente Pequeno , Neoplasias Gástricas/patologia , Análise Serial de Tecidos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Rfa2 is a ssDNA (single-stranded DNA)-binding protein that plays an important role in DNA replication, recombination and repair. Rfa2 is regulated by phosphorylation, which alters its protein-protein interaction and protein-DNA interaction. In the present study, we found that the Pph3-Psy2 phosphatase complex is responsible for Rfa2 dephosphorylation both during normal G1-phase and under DNA replication stress in Candida albicans. Phosphorylated Rfa2 extracted from pph3Δ or psy2Δ G1 cells exhibited diminished binding affinity to dsDNA (double-stranded DNA) but not to ssDNA. We also discovered that Cdc28 (cell division cycle 28) and Mec1 are responsible for Rfa2 phosphorylation in G1-phase and under DNA replication stress respectively. Moreover, MS revealed that the domain of Rfa2 that was phosphorylated in G1-phase differed from that phosphorylated under the stress conditions. The results of the present study imply that differential phosphorylation plays a crucial role in RPA (replication protein A) regulation.