RESUMO
While the existence and functional role of class C G-protein-coupled receptors (GPCR) dimers is well established, there is still a lack of consensus regarding class A and B GPCR multimerization. This lack of consensus is largely due to the inherent challenges of demonstrating the presence of multimeric receptor complexes in a physiologically relevant cellular context. The C-X-C motif chemokine receptor 4 (CXCR4) is a class A GPCR that is a promising target of anticancer therapy. Here, we investigated the potential of CXCR4 to form multimeric complexes with other GPCRs and characterized the relative size of the complexes in a live-cell environment. Using a bimolecular fluorescence complementation (BiFC) assay, we identified the ß2 adrenergic receptor (ß2AR) as an interaction partner. To investigate the molecular scale details of CXCR4-ß2AR interactions, we used a time-resolved fluorescence spectroscopy method called pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). PIE-FCCS can resolve membrane protein density, diffusion, and multimerization state in live cells at physiological expression levels. We probed CXCR4 and ß2AR homo- and heteromultimerization in model cell lines and found that CXCR4 assembles into multimeric complexes larger than dimers in MDA-MB-231 human breast cancer cells and in HCC4006 human lung cancer cells. We also found that ß2AR associates with CXCR4 multimers in MDA-MB-231 and HCC4006 cells to a higher degree than in COS-7 and CHO cells and in a ligand-dependent manner. These results suggest that CXCR4-ß2AR heteromers are present in human cancer cells and that GPCR multimerization is significantly affected by the plasma membrane environment.
Assuntos
Neoplasias , Receptores Adrenérgicos beta 2 , Receptores CXCR4 , Transdução de Sinais , Animais , Cricetinae , Humanos , Células CHO , Cricetulus , Proteínas de Membrana/metabolismo , Neoplasias/metabolismo , Receptores CXCR4/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Multimerização ProteicaRESUMO
BACKGROUND: G protein-coupled receptor heteromerization is believed to exert dynamic regulatory impact on signal transduction. CXC chemokine receptor 4 (CXCR4) and its ligand CXCL12, both of which are overexpressed in many cancers, play a pivotal role in metastasis. Likewise, lysophosphatidic acid receptor 1 (LPA1) is implicated in cancer cell proliferation and migration. In our preliminary study, we identified LPA1 as a prospective CXCR4 interactor. In the present study, we investigated in detail the formation of the CXCR4-LPA1 heteromer and characterized the unique molecular features and function of this heteromer. METHODS: We employed bimolecular fluorescence complementation, bioluminescence resonance energy transfer, and proximity ligation assays to demonstrate heteromerization between CXCR4 and LPA1. To elucidate the distinctive molecular characteristics and functional implications of the CXCR4-LPA1 heteromer, we performed various assays, including cAMP, BRET for G protein activation, ß-arrestin recruitment, ligand binding, and transwell migration assays. RESULTS: We observed that CXCR4 forms heteromers with LPA1 in recombinant HEK293A cells and the human breast cancer cell line MDA-MB-231. Coexpression of LPA1 with CXCR4 reduced CXCL12-mediated cAMP inhibition, ERK activation, Gαi/o activation, and ß-arrestin recruitment, while CXCL12 binding to CXCR4 remained unaffected. In contrast, CXCR4 had no impact on LPA1-mediated signaling. The addition of lysophosphatidic acid (LPA) further hindered CXCL12-induced Gαi/o recruitment to CXCR4. LPA or alkyl-OMPT inhibited CXCL12-induced migration in various cancer cells that endogenously express both CXCR4 and LPA1. Conversely, CXCL12-induced calcium signaling and migration were increased in LPAR1 knockout cells, and LPA1-selective antagonists enhanced CXCL12-induced Gαi/o signaling and cell migration in the parental MDA-MB-231 cells but not in LPA1-deficient cells. Ultimately, complete inhibition of cell migration toward CXCL12 and alkyl-OMPT was only achieved in the presence of both CXCR4 and LPA1 antagonists. CONCLUSIONS: The presence and impact of CXCR4-LPA1 heteromers on CXCL12-induced signaling and cell migration have been evidenced across various cell lines. This discovery provides crucial insights into a valuable regulatory mechanism of CXCR4 through heteromerization. Moreover, our findings propose a therapeutic potential in combined CXCR4 and LPA1 inhibitors for cancer and inflammatory diseases associated with these receptors, simultaneously raising concerns about the use of LPA1 antagonists alone for such conditions. Video Abstract.
Assuntos
Sinalização do Cálcio , Quimiocina CXCL12 , Receptores CXCR4 , Receptores de Ácidos Lisofosfatídicos , Humanos , Movimento Celular , Ligantes , Estudos ProspectivosRESUMO
G protein-coupled receptors (GPCRs) are the largest family of cell-surface receptors and are involved in a variety of pathological conditions including cancer and cardiovascular, metabolic, neurological, and autoimmune diseases. GPCRs are being intensively investigated as targets for therapeutic intervention, and the ß-arrestin recruitment assay has become a popular tool for analyzing GPCR activation. Here, we report a high-throughput method for cloning GPCR cDNAs into adenoviral bimolecular fluorescence complementation (BiFC) vectors and performing the ß-arrestin BiFC assay in cells transduced with recombinant adenoviruses. An analysis of the activation of somatostatin receptor 2 (SSTR2) with the adenovirus-based ß-arrestin BiFC assay showed that the assay is suitable for quantifying SSTR2 activation in response to specific agonists or antagonists. Furthermore, the adenovirus-based ß-arrestin BiFC assay was able to detect the activation of a broad range of GPCRs. Collectively, our data indicate that the adenovirus-based ß-arrestin BiFC assay can serve as a simple and universal platform for studying GPCR activation and thus will be useful for high-throughput screening of drugs that target GPCRs.
Assuntos
Adenoviridae/genética , Arrestinas/metabolismo , Técnicas Biossensoriais/métodos , Receptores Acoplados a Proteínas G/metabolismo , Clonagem Molecular , DNA Complementar/genética , Fluorescência , Células HEK293 , Ensaios de Triagem em Larga Escala/métodos , Humanos , Receptores Acoplados a Proteínas G/genética , beta-ArrestinasRESUMO
C-X-C chemokine receptor 4 (CXCR4) is widely overexpressed in various types of cancer and is involved in several cancer phenotypes including tumor growth, survival, and metastasis. The roles of histamine and histamine receptor H1 (HRH1) in cancer pathogenesis remain controversial. Here, we show that HRH1 is widely expressed in various cancer cell lines and cancer tissues and that coexpression of CXCR4 and HRH1 is associated with poor prognosis in breast cancer. Using bimolecular fluorescence complementation and bioluminescence resonance energy transfer donor saturation assays, we demonstrate that CXCR4 and HRH1 can assemble into a heteromeric complex. Simultaneous activation of CXCR4 and HRH1 synergistically increases calcium flux in MDA-MB-231 cells that endogenously express CXCR4 and HRH1 but not in cells deficient in CXCR4 or HRH1. Costimulation of CXCR4 and HRH1 also significantly enhances CXCL12-induced MDA-MB-231 cell migration, while histamine alone does not induce cell migration. Synergistic effects on calcium flux and cell migration are inhibited by the Gαi inhibitor pertussis toxin and the Gαq inhibitor YM254890, suggesting that the Gαi and Gαq pathways are involved in the synergy. Enhanced calcium signaling and cell migration are also observed in NCI-H23 and HeLa cells, which coexpress CXCR4 and HRH1. Taken together, our findings demonstrate an interplay between CXCR4 and HRH1, and suggest the possibility of the CXCR4-HRH1 heteromer as a potential therapeutic target for anticancer therapy.
Assuntos
Neoplasias , Receptores CXCR4 , Humanos , Receptores CXCR4/metabolismo , Histamina/farmacologia , Histamina/metabolismo , Sinalização do Cálcio , Células HeLa , Cálcio/metabolismo , Quimiocina CXCL12/metabolismo , Movimento Celular , Receptores Histamínicos H1/metabolismo , Linhagem Celular Tumoral , Neoplasias/genéticaRESUMO
Autologous Stem Cell Transplant (ASCT) is increasingly used to treat hematological malignancies. A key requisite for ASCT is mobilization of hematopoietic stem cells into peripheral blood, where they are collected by apheresis and stored for later transplantation. However, success is often hindered by poor mobilization due to factors including prior treatments. The combination of G-CSF and GPC-100, a small molecule antagonist of CXCR4, showed potential in a multiple myeloma clinical trial for sufficient and rapid collection of CD34+ stem cells, compared to the historical results from the standards of care, G-CSF alone or G-CSF with plerixafor, also a CXCR4 antagonist. In the present study, we show that GPC-100 has high affinity towards the chemokine receptor CXCR4, and it potently inhibits ß-arrestin recruitment, calcium flux and cell migration mediated by its ligand CXCL12. Proximity Ligation Assay revealed that in native cell systems with endogenous receptor expression, CXCR4 co-localizes with the beta-2 adrenergic receptor (ß2AR). Co-treatment with CXCL12 and the ß2AR agonist epinephrine synergistically increases ß-arrestin recruitment to CXCR4 and calcium flux. This increase is blocked by the co-treatment with GPC-100 and propranolol, a non-selective beta-adrenergic blocker, indicating a functional synergy. In mice, GPC-100 mobilized more white blood cells into peripheral blood compared to plerixafor. GPC-100 induced mobilization was further amplified by propranolol pretreatment and was comparable to mobilization by G-CSF. Addition of propranolol to the G-CSF and GPC-100 combination resulted in greater stem cell mobilization than the G-CSF and plerixafor combination. Together, our studies suggest that the combination of GPC-100 and propranolol is a novel strategy for stem cell mobilization and support the current clinical trial in multiple myeloma registered as NCT05561751 at www.clinicaltrials.gov.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Compostos Heterocíclicos , Mieloma Múltiplo , Animais , Camundongos , Mobilização de Células-Tronco Hematopoéticas/métodos , Mieloma Múltiplo/tratamento farmacológico , Propranolol/uso terapêutico , Cálcio/metabolismo , Compostos Heterocíclicos/uso terapêutico , Células-Tronco Hematopoéticas/metabolismo , Receptores CXCR4/metabolismo , Fator Estimulador de Colônias de Granulócitos/farmacologia , beta-Arrestinas/metabolismo , Benzilaminas/metabolismoRESUMO
We have previously reported anti-obesity effects of Lysimachia foenum-graecum in high-fat diet (HFD)-induced obesity model. Here we isolated a triterpene saponin foenumoside B as an active component of L. foenum-graecum. Foenumoside B blocked the differentiation of 3T3-L1 preadipocytes in a dose-dependent manner with an IC50 of 0.2 µg/ml in adipogenesis assay and suppressed the induction of PPARγ, the master regulator of adipogenesis. Foenumoside B induced the activation of AMP-activated protein kinase (AMPK), and modulated the expression of genes involved in lipid metabolism towards lipid breakdown in differentiated adipocytes. In mouse model, oral administration of foenumoside B (10mg/kg/day for 6 weeks) reduced HFD-induced body weight gain significantly without affecting food intake. Treatment of foenumoside B suppressed lipid accumulation in white adipose tissues and the liver, and lowered blood levels of glucose, triglycerides, ALT, and AST in HFD-induced obese mice. Consistent with the in vitro results, foenumoside B activated AMPK signaling, suppressed the expression of lipogenic genes, and enhanced the expression of lipolytic genes in vivo. Foenumoside B also blocked HFD-induced proinflammatory cytokine production in adipose tissue, suggesting its protective role against insulin resistance. Taken together, these findings demonstrate that foenumoside B represents the anti-obesity effects of L. foenum-graecum, and suggest therapeutic potential of foenumoside B in obesity and obesity-related metabolic diseases.
Assuntos
Adipócitos/efeitos dos fármacos , Adipogenia/efeitos dos fármacos , Fármacos Antiobesidade/uso terapêutico , Dieta Hiperlipídica/efeitos adversos , Obesidade/tratamento farmacológico , Primulaceae/química , Saponinas/uso terapêutico , Células 3T3-L1 , Quinases Proteína-Quinases Ativadas por AMP , Adipócitos/citologia , Animais , Fármacos Antiobesidade/farmacologia , Ativação Enzimática , Expressão Gênica/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Metabolismo dos Lipídeos/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/etiologia , Proteínas Quinases/biossíntese , Saponinas/farmacologiaRESUMO
We developed one-step sequence- and ligation-independent cloning (SLIC) as a simple, cost-effective, time-saving, and versatile cloning method. Highly efficient and directional cloning can be achieved by direct bacterial transformation 2.5 min after mixing any linearized vector, an insert(s) prepared by PCR, and T4 DNA polymerase in a tube at room temperature.
Assuntos
Clonagem Molecular/métodos , Genômica/métodos , Transformação Bacteriana/genética , Sequência de Bases , DNA Polimerase Dirigida por DNA/metabolismo , Vetores Genéticos/genética , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Proteínas Virais/metabolismoRESUMO
Activating Transcription Factor 3 (ATF3), a member of the ATF/CREB family, is induced rapidly by various stresses. Its induction mechanism and role in response to changes in cellular redox status, however, have not been elucidated. Here, we found that NF-E2-related factor 2 (Nrf2), a transcription factor known to bind to antioxidant response element (ARE) in promoters, transcriptionally upregulated ATF3 expression in astrocytes. Treatment with Nrf2 activators and oxidants provoked ATF3 induction in astrocytes, whereas its expression was reduced in Nrf2-depleted cells. We further demonstrated that the consensus ARE in the ATF3 promoter is critical for Nrf2-mediation by promoter analyses using an ATF3 promoter-driven luciferase construct and a chromatin immunoprecipitation assay. In addition, we found that Nrf2-dependent ATF3 induction contributed to the antioxidative and cytoprotective functions of Nrf2 in astrocytes. Taken together, our findings suggest that ATF3 is a new target for Nrf2 and has a cytoprotective function in astrocytes.
Assuntos
Fator 3 Ativador da Transcrição/genética , Astrócitos/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Fator 3 Ativador da Transcrição/biossíntese , Animais , Astrócitos/efeitos dos fármacos , Sítios de Ligação , Células Cultivadas , Citoproteção , Humanos , Camundongos , Oxidantes/farmacologia , Elementos de RespostaRESUMO
A crude comparison of medical costs between people with disabilities (PWD) and without disabilities (PWoD) shows a much higher expenditure among PWD and such results have been a cause for further stigmatization. This study aims to empirically analyze whether the medical costs for PWD are actually high when characteristics related to medical costs are adjusted. Ten percent of the total population was randomly selected from the Korean National Health Insurance (NHI) Database in 2016. A crude comparative analysis was performed to calculate the medical cost of PWD and PWoD. A subsequent multiple regression analysis was conducted to adjust factors affecting the medical costs such as socioeconomic status, disease, and health behavior-related characteristics. The medical cost for PWD was 3.6 times higher than that for PWoD by crude comparison. However, after multiple regression analysis, margin of difference decreased to 1.5 times although the cost for PWD remained higher. Substantial decrease in higher medical costs for PWD after multiple analyses compared to crude analysis implies that additional adjustment using variables such as disease severity, not available in the NHI database, may predict a further reduction in differences. Thus, it is difficult to determine that the medical expenditure for PWD is excessive.
Assuntos
Pessoas com Deficiência , Gastos em Saúde , Programas Nacionais de Saúde , Humanos , República da Coreia , Classe SocialRESUMO
A comprehensive Dialectical Behavior Therapy (DBT) program was created within a VA Health Care System for patients with recent psychiatric hospitalization, suicidality and/or significant emotion dysregulation. The program was notable for being one of a relatively small number of comprehensive DBT programs in the VA system, and for including patients with psychosis and psychotic disorder, with a majority of patients (58%) having a documented history of psychosis or endorsing psychotic symptoms in assessments. We describe the process of creating this program at a VA medical center and present preliminary program evaluation data. All patients completed assessments of suicidality (C-SSRS), emotion dysregulation (DERS), skills use and dysfunctional coping (DBT-WCCL), borderline symptomatology (BSL-23), and depression (PHQ-9) at program entry and subsequently every 6-8 weeks through program completion. Suicide attempts and hospitalizations were also tracked. Twelve patients completed multiple (up to six) assessment timepoints, allowing for evaluation of change during treatment. Patients demonstrated improvements on most measures and no hospitalizations or suicide attempts during active treatment, and the subsample with psychosis showed average improvements on every outcome measure. Eleven of 12 patients completed a full six-month rotation.
Assuntos
Transtorno da Personalidade Borderline , Terapia do Comportamento Dialético , Veteranos , Terapia Comportamental , Transtorno da Personalidade Borderline/psicologia , Transtorno da Personalidade Borderline/terapia , Atenção à Saúde , Humanos , Avaliação de Programas e Projetos de Saúde , Tentativa de Suicídio/prevenção & controle , Tentativa de Suicídio/psicologia , Resultado do TratamentoRESUMO
Polo-like kinase 1 (Plk1) is central to cell division. Here, we report that Plk1 is critical for mitosis in the embryonic development of zebrafish. Using a combination of several cell biology tools, including single-cell live imaging applied to whole embryos, we show that Plk1 is essential for progression into mitosis during embryonic development. Plk1 morphant cells displayed mitotic infidelity, such as abnormal centrosomes, irregular spindle assembly, hypercondensed chromosomes, and a failure of chromosome arm separation. Consequently, depletion of Plk1 resulted in mitotic arrest and finally death by 6days post-fertilization. In comparison, Plk2 or Plk3 morphant embryos did not display any significant abnormalities. Treatment of embryos with the Plk1 inhibitor, BI 2536, caused a block in mitosis, which was more severe when used to treat plk1 morphants. Finally, using an assay to rescue the Plk1 morphant phenotype, we found that the kinase domain and PBD domains are both necessary for Plk1 function in zebrafish development. Our studies demonstrate that Plk1 is required for embryonic proliferation because its activity is crucial for mitotic integrity. Furthermore, our study suggests that zebrafish will be an efficient and economical in vivo system for the validation of anti-mitotic drugs.
Assuntos
Proteínas de Ciclo Celular/genética , Embrião não Mamífero/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Proteínas de Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Apoptose , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células , Segregação de Cromossomos/efeitos dos fármacos , Embrião não Mamífero/citologia , Embrião não Mamífero/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Histonas/genética , Histonas/metabolismo , Hibridização In Situ , Cinética , Microscopia de Vídeo/métodos , Mitose/efeitos dos fármacos , Dados de Sequência Molecular , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , Pteridinas/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/metabolismo , Quinase 1 Polo-LikeRESUMO
Fibroblast growth factor 11 (FGF11) is a member of the intracellular fibroblast growth factor superfamily. Here, we identified FGF11 as a novel mediator of adipogenesis. During 3T3-L1 adipocyte differentiation, the expression of FGF11 decreased at the mitotic clonal expansion stage and increased at the terminal differentiation stage. FGF11 knockdown reduced the expression of peroxisome proliferator-activated receptor gamma (PPARγ), a master regulator of adipogenesis, resulting in the inhibition of adipocyte differentiation. Treatment with the PPARγ agonist rosiglitazone restored the inhibition of adipogenesis caused by FGF11 knockdown. We also report that the expression of the PPARγ regulators CCAAT/enhancer-binding protein α, sterol regulatory element-binding protein 1, KLF9, KLF2, GATA binding factor 2, and GATA binding factor 3 was influenced by FGF11. These results suggest that FGF11 indirectly controls the expression of PPARγ through modifying the expression of multiple PPARγ regulators, thereby mediating adipogenesis.
Assuntos
Adipogenia/genética , Fatores de Crescimento de Fibroblastos/genética , PPAR gama/genética , Células 3T3-L1 , Animais , Fatores de Crescimento de Fibroblastos/metabolismo , Camundongos , PPAR gama/metabolismoRESUMO
The blood-brain barrier (BBB) is essential for maintaining brain homeostasis and protecting the brain from toxic substances. Breakdown of this barrier results in severe brain pathologies, whereas impermeability of the BBB is a major obstacle for drug delivery to the brain. Despite its importance, our understanding of the maturation and modulation of the BBB is limited. Zebrafish (Danio rerio) has emerged as a useful model organism for studying vertebrate development and disease mechanisms, as well as for preclinical drug screening. However, the nature of the BBB has not yet been examined in teleost fish. In this paper, we report that with the exception of the circumventricular organs, the cerebral microvessels in zebrafish are impermeable to sulfo-NHS-biotin and horseradish peroxidase (HRP). Brain endothelial cells show immunoreactivity to Claudin-5 and Zonula Occludens-1 (ZO-1), implying the presence of tight junctions in these cells. The expression of Claudin-5 and ZO-1 was detected in cerebral microvessels from 3 days post-fertilization (dpf), concomitant with maturation of the BBB, as determined by restricted permeability to HRP and various fluorescent tracers. Real-time analysis of fluorescent tracer leakage in embryonic zebrafish suggests that they may be used as an in vivo model for BBB breakdown. Taken together, our results show that the endothelial tight junction-based BBB of zebrafish is similar to that of higher vertebrates and thus, zebrafish may be an excellent genetic and experimental model organism for studying development and maintenance of the BBB.
Assuntos
Barreira Hematoencefálica/crescimento & desenvolvimento , Barreira Hematoencefálica/metabolismo , Animais , Animais Geneticamente Modificados , Transporte Biológico/fisiologia , Biotina/metabolismo , Permeabilidade Capilar , Embrião não Mamífero , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Peroxidase do Rábano Silvestre/metabolismo , Proteínas de Membrana , Proteína Proto-Oncogênica c-fli-1/biossíntese , Proteína Proto-Oncogênica c-fli-1/genética , Junções Íntimas/fisiologia , Peixe-ZebraRESUMO
Fibroblast growth factor 11 (FGF11) is an intracellular FGF. Although induction of FGF11 by hypoxia has been observed in several cell types, the molecular function of FGF11 is not clearly understood yet. Here, we investigated the role of FGF11 under hypoxia. We identified hypoxia-inducible factor-1α (HIF-1α) as an interacting protein of FGF11 using immunoprecipitation and mass spectrometry. FGF11 knockdown decreased HIF-1α protein, while FGF11 overexpression increased it, without affecting HIF-1α mRNA. Protein stability test and ubiquitination assay showed that FGF11 increased HIF-1α stability by acting upstream of proteasomal degradation. Altogether, these results suggest a cross-regulation between HIF-1α and FGF11, through which hypoxia-induced FGF11 reinforces hypoxia responses by enhancing the stability of HIF-1α.
Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Hipóxia Celular , Linhagem Celular Tumoral , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Ligação Proteica , Estabilidade Proteica , Proteólise , Transcrição GênicaRESUMO
Previously, we developed a one-step sequence- and ligation-independent cloning (SLIC) method that is simple, fast, and cost-effective. However, although one-step SLIC generally works well, its cloning efficiency is occasionally poor, potentially due to formation of stable secondary structures within the single-stranded DNA (ssDNA) region generated by T4 DNA polymerase during the 2.5 min treatment at room temperature. To overcome this problem, we developed a modified thermo-regulated one-step SLIC approach by testing shorter T4 DNA polymerase treatment durations (5 s-2.5 min) over a wide range of temperatures (25-75°C). The highest cloning efficiency resulted when inserts with homology lengths <20 bases were treated with T4 DNA polymerase for 30 s at 50°C. This briefer T4 polymerase treatment at a higher temperature helps increase cloning efficiency for inserts with strong secondary structures at their ends, increasing the utility of one-step SLIC for the cloning of short fragments.
Assuntos
Clonagem Molecular/métodos , DNA Polimerase Dirigida por DNA/química , Análise de Sequência de DNA , Proteínas Virais/química , DNA Recombinante/química , DNA Recombinante/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Vetores Genéticos/química , Vetores Genéticos/genética , Temperatura , Fatores de TempoRESUMO
Cetacean body structure and physiology exhibit dramatic adaptations to their aquatic environment. Fibroblast growth factors (FGFs) are a family of essential factors that regulate animal development and physiology; however, their role in cetacean evolution is not clearly understood. Here, we sequenced the fin whale genome and analysed FGFs from 8 cetaceans. FGF22, a hair follicle-enriched gene, exhibited pseudogenization, indicating that the function of this gene is no longer necessary in cetaceans that have lost most of their body hair. An evolutionary analysis revealed signatures of positive selection for FGF3 and FGF11, genes related to ear and tooth development and hypoxia, respectively. We found a D203G substitution in cetacean FGF9, which was predicted to affect FGF9 homodimerization, suggesting that this gene plays a role in the acquisition of rigid flippers for efficient manoeuvring. Cetaceans utilize low bone density as a buoyancy control mechanism, but the underlying genes are not known. We found that the expression of FGF23, a gene associated with reduced bone density, is greatly increased in the cetacean liver under hypoxic conditions, thus implicating FGF23 in low bone density in cetaceans. Altogether, our results provide novel insights into the roles of FGFs in cetacean adaptation to the aquatic environment.
Assuntos
Adaptação Fisiológica , Evolução Molecular , Fatores de Crescimento de Fibroblastos/genética , Baleia Comum/genética , Baleia Comum/fisiologia , Animais , Genoma , Filogenia , Seleção GenéticaRESUMO
Low oxygen or hypoxia can be observed in the central region of solid tumors. Hypoxia is a strong stimulus for new blood vessel formation or angiogenesis, which is essential for tumor growth and progression. Fibroblast growth factor 11 (FGF11) is an intracellular non-secretory FGF whose function has not yet been fully characterized. In the present study, we demonstrated that FGF11 expression is upregulated under hypoxic conditions in human umbilical vein endothelial cells (HUVECs). FGF11 overexpression stimulated capillary-like tube formation, yet did not affect cell migration. Notably, FGF11 markedly increased the levels of tight junction proteins including occludin, zonula occludens-1 (ZO-1) and claudin-5 in HUVECs. The FGF11 promoter contains hypoxia response elements (HREs), and hypoxia-inducible factor-1 (HIF-1) binds to HREs to activate hypoxia-related genes. We demonstrated that hypoxia or HIF-1 expression under normoxic conditions increased the luciferase activity driven by the FGF11 promoter. However, deletion of the HREs from the FGF11 promoter rendered reporter gene activity unresponsive to hypoxia or HIF-1. Taken together, we propose that FGF11 may be involved in the stabilization of capillary-like tube structures associated with angiogenesis and may act as a modulator of hypoxia-induced pathological processes such as tumorigenesis.
Assuntos
Células Endoteliais/citologia , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Sítios de Ligação , Hipóxia Celular , Movimento Celular , Claudina-5/metabolismo , Células Endoteliais/metabolismo , Fatores de Crescimento de Fibroblastos/química , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Regiões Promotoras Genéticas , Regulação para Cima , Proteína da Zônula de Oclusão-1/metabolismoRESUMO
The shift from terrestrial to aquatic life by whales was a substantial evolutionary event. Here we report the whole-genome sequencing and de novo assembly of the minke whale genome, as well as the whole-genome sequences of three minke whales, a fin whale, a bottlenose dolphin and a finless porpoise. Our comparative genomic analysis identified an expansion in the whale lineage of gene families associated with stress-responsive proteins and anaerobic metabolism, whereas gene families related to body hair and sensory receptors were contracted. Our analysis also identified whale-specific mutations in genes encoding antioxidants and enzymes controlling blood pressure and salt concentration. Overall the whale-genome sequences exhibited distinct features that are associated with the physiological and morphological changes needed for life in an aquatic environment, marked by resistance to physiological stresses caused by a lack of oxygen, increased amounts of reactive oxygen species and high salt levels.
Assuntos
Adaptação Fisiológica/genética , Genoma , Baleia Anã/genética , Animais , Pressão Sanguínea/genética , Glutationa/metabolismo , Haptoglobinas/genética , Masculino , Baleia Anã/metabolismo , Família Multigênica , Mutação , Oceano Pacífico , Filogenia , Densidade Demográfica , Tolerância ao Sal , Estresse FisiológicoRESUMO
The need for efficient high-throughput gene delivery system for mammalian cells is rapidly increasing with the growing request for functional genomics studies and drug discoveries in various physiologically relevant systems. However, plasmid-based gene delivery has limitations in transfection efficiency and available cell types. Viral vectors have great advantages over plasmid-based vectors, but construction of recombinant viruses remains to be a big hurdle for high-throughput applications. Here we demonstrate a rapid and simple high-throughput system for constructing recombinant adenoviruses which have been used as efficient gene delivery tools in mammalian systems in vitro and in vivo. By combining Gateway-based site-specific recombination with Terminal protein-coupled adenovirus vector, the adenovirus high-throughput system (AdHTS) generates multiple recombinant adenoviruses in 96-well plates simultaneously without the need for additional cloning or recombination in bacteria or mammalian cells. The AdHTS allows rapid and robust cloning and expression of genes in mammalian cells by removing shuttle vector construction, bacterial transformation, or selection and by minimizing effort in plaque isolation. By shortening the time required to convert whole cDNA library into desired viral vector constructs, the AdHTS would greatly facilitate functional genomics and proteomics studies in various mammalian systems.