RESUMO
Certain ciliary transmembrane and membrane-tethered signaling proteins migrate from the ciliary tip to base via retrograde intraflagellar transport (IFT), essential for maintaining their ciliary dynamics to enable cells to sense and transduce extracellular stimuli inside the cell. During this process, the BBSome functions as an adaptor between retrograde IFT trains and these signaling protein cargoes. The Arf-like 13 (ARL13) small GTPase resembles ARL6/BBS3 in facilitating these signaling cargoes to couple with the BBSome at the ciliary tip prior to loading onto retrograde IFT trains for transporting towards the ciliary base, while the molecular basis for how this intricate coupling event happens remains elusive. Here, we report that Chlamydomonas ARL13 only in a GTP-bound form (ARL13GTP) anchors to the membrane for diffusing into cilia. Upon entering cilia, ARL13 undergoes GTPase cycle for shuttling between the ciliary membrane (ARL13GTP) and matrix (ARL13GDP). To achieve this goal, the ciliary membrane-anchored BBS3GTP binds the ciliary matrix-residing ARL13GDP to activate the latter as an ARL13 guanine nucleotide exchange factor. At the ciliary tip, ARL13GTP recruits the ciliary matrix-residing and post-remodeled BBSome as an ARL13 effector to anchor to the ciliary membrane. This makes the BBSome spatiotemporally become available for the ciliary membrane-tethered phospholipase D (PLD) to couple with. Afterward, ARL13GTP hydrolyzes GTP for releasing the PLD-laden BBSome to load onto retrograde IFT trains. According to this model, hedgehog signaling defects associated with ARL13b and BBS3 mutations in humans could be satisfactorily explained, providing us a mechanistic understanding behind BBSome-cargo coupling required for proper ciliary signaling.
Assuntos
Síndrome de Bardet-Biedl , Cílios , Humanos , Cílios/metabolismo , Transporte Proteico/genética , Síndrome de Bardet-Biedl/genética , Proteínas Hedgehog/metabolismo , Proteínas de Membrana/metabolismo , Guanosina Trifosfato/metabolismo , Flagelos/metabolismoRESUMO
Certain transmembrane and membrane-tethered signaling proteins export from cilia as BBSome cargoes via the outward BBSome transition zone (TZ) diffusion pathway, indispensable for maintaining their ciliary dynamics to enable cells to sense and transduce extracellular stimuli inside the cell. Murine Rab-like 2 (Rabl2) GTPase resembles Chlamydomonas Arf-like 3 (ARL3) GTPase in promoting outward TZ passage of the signaling protein cargo-laden BBSome. During this process, ARL3 binds to and recruits the retrograde IFT train-dissociated BBSome as its effector to diffuse through the TZ for ciliary retrieval, while how RABL2 and ARL3 cross talk in this event remains uncertain. Here, we report that Chlamydomonas RABL2 in a GTP-bound form (RABL2GTP) cycles through cilia via IFT as an IFT-B1 cargo, dissociates from retrograde IFT trains at a ciliary region right above the TZ, and converts to RABL2GDP for activating ARL3GDP as an ARL3 guanine nucleotide exchange factor. This confers ARL3GTP to detach from the ciliary membrane and become available for binding and recruiting the phospholipase D (PLD)-laden BBSome, autonomous of retrograde IFT association, to diffuse through the TZ for ciliary retrieval. Afterward, RABL2GDP exits cilia by being bound to the ARL3GTP/BBSome entity as a BBSome cargo. Our data identify ciliary signaling proteins exported from cilia via the RABL2-ARL3 cascade-mediated outward BBSome TZ diffusion pathway. According to this model, hedgehog signaling defect-induced Bardet-Biedl syndrome caused by RABL2 mutations in humans could be well explained in a mutation-specific manner, providing us with a mechanistic understanding behind the outward BBSome TZ passage required for proper ciliary signaling.
Assuntos
Cílios , Proteínas Hedgehog , Humanos , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Cílios/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas Hedgehog/metabolismo , Proteínas de Membrana/metabolismo , Transporte Proteico/genética , Proteínas rab de Ligação ao GTP/metabolismo , ChlamydomonasRESUMO
Many G protein-coupled receptors and other signaling proteins localize to the ciliary membrane for regulating diverse cellular processes. The BBSome composed of multiple Bardet-Biedl syndrome (BBS) proteins is an intraflagellar transport (IFT) cargo adaptor essential for sorting signaling proteins in and/or out of cilia via IFT. Leucine zipper transcription factor-like 1 (LZTFL1) protein mediates ciliary signaling by controlling BBSome ciliary content, reflecting how LZTFL1 mutations could cause BBS. However, the mechanistic mechanism underlying this process remains elusive thus far. Here, we show that LZTFL1 maintains BBSome ciliary dynamics by finely controlling BBSome recruitment to the basal body and its reassembly at the ciliary tip simultaneously in Chlamydomonas reinhardtii LZTFL1 directs BBSome recruitment to the basal body via promoting basal body targeting of Arf-like 6 GTPase BBS3, thus deciding the BBSome amount available for loading onto anterograde IFT trains for entering cilia. Meanwhile, LZTFL1 stabilizes the IFT25/27 component of the IFT-B1 subcomplex in the cell body so as to control its presence and amount at the basal body for entering cilia. Since IFT25/27 promotes BBSome reassembly at the ciliary tip for loading onto retrograde IFT trains, LZTFL1 thus also directs BBSome removal out of cilia. Therefore, LZTFL1 dysfunction deprives the BBSome of ciliary presence and generates Chlamydomonas cells defective in phototaxis. In summary, our data propose that LZTFL1 maintains BBSome dynamics in cilia by such a dual-mode system, providing insights into how LZTFL1 mediates ciliary signaling through maintaining BBSome ciliary dynamics and the pathogenetic mechanism of the BBS disorder as well.
Assuntos
Chlamydomonas reinhardtii/fisiologia , Cílios/fisiologia , Fototaxia , Fatores de Transcrição/fisiologia , Síndrome de Bardet-Biedl , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ligação Proteica , Transdução de SinaisRESUMO
Certain ciliary transmembrane and membrane-associated signaling proteins export from cilia as intraflagellar transport (IFT) cargoes in a BBSome-dependent manner. Upon reaching the ciliary tip via anterograde IFT, the BBSome disassembles before being reassembled to form an intact entity for cargo phospholipase D (PLD) coupling. During this BBSome remodeling process, Chlamydomonas Rab-like 4 GTPase IFT27, by binding its partner IFT25 to form the heterodimeric IFT25/27, is indispensable for BBSome reassembly. Here, we show that IFT27 binds IFT25 in an IFT27 nucleotide-independent manner. IFT25/27 and the IFT subcomplexes IFT-A and -B are irrelevant for maintaining the stability of one another. GTP-loading onto IFT27 enhances the IFT25/27 affinity for binding to the IFT-B subcomplex core IFT-B1 entity in cytoplasm, while GDP-bound IFT27 does not prevent IFT25/27 from entering and cycling through cilia by integrating into IFT-B1. Upon at the ciliary tip, IFT25/27 cycles on and off IFT-B1 and this process is irrelevant with the nucleotide state of IFT27. During BBSome remodeling at the ciliary tip, IFT25/27 promotes BBSome reassembly independent of IFT27 nucleotide state, making postremodeled BBSomes available for PLD to interact with. Thus, IFT25/27 facilitates BBSome-dependent PLD export from cilia via controlling availability of intact BBSomes at the ciliary tip, while IFT27 nucleotide state does not participate in this regulatory event.
Assuntos
Chlamydomonas , Cílios , Nucleotídeos , Fosfolipase D , Proteínas rab de Ligação ao GTP , Cílios/química , Cílios/metabolismo , Flagelos/química , Flagelos/metabolismo , Fosfolipase D/metabolismo , Transporte Proteico , Transdução de Sinais , Chlamydomonas/citologia , Chlamydomonas/enzimologia , Chlamydomonas/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Guanosina Trifosfato/metabolismo , Guanosina Difosfato/metabolismoRESUMO
Bardet-Biedl syndrome (BBS) is a ciliopathy caused by defects in the assembly or distribution of the BBSome, a conserved protein complex. The BBSome cycles via intraflagellar transport (IFT) through cilia to transport signaling proteins. How the BBSome is recruited to the basal body for binding to IFT trains for ciliary entry remains unknown. Here, we show that the Rab-like 5 GTPase IFT22 regulates basal body targeting of the BBSome in Chlamydomonas reinhardtii Our functional, biochemical and single particle in vivo imaging assays show that IFT22 is an active GTPase with low intrinsic GTPase activity. IFT22 is part of the IFT-B1 subcomplex but is not required for ciliary assembly. Independent of its association to IFT-B1, IFT22 binds and stabilizes the Arf-like 6 GTPase BBS3, a BBS protein that is not part of the BBSome. IFT22/BBS3 associates with the BBSome through an interaction between BBS3 and the BBSome. When both IFT22 and BBS3 are in their guanosine triphosphate (GTP)-bound states they recruit the BBSome to the basal body for coupling with the IFT-B1 subcomplex. The GTP-bound BBS3 likely remains to be associated with the BBSome upon ciliary entry. In contrast, IFT22 is not required for the transport of BBSomes in cilia, indicating that the BBSome is transferred from IFT22 to the IFT trains at the ciliary base. In summary, our data propose that nucleotide-dependent recruitment of the BBSome to the basal body by IFT22 regulates BBSome entry into cilia.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Corpos Basais/metabolismo , Chlamydomonas reinhardtii/metabolismo , Flagelos/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Fatores de Ribosilação do ADP/genética , Síndrome de Bardet-Biedl/genética , Síndrome de Bardet-Biedl/metabolismo , Chlamydomonas reinhardtii/genética , Cílios/genética , Cílios/metabolismo , Flagelos/genética , GTP Fosfo-Hidrolases/genética , Humanos , Ligação Proteica , Transporte ProteicoRESUMO
Food safety become a hot issue currently with globalization of food trade and food supply chains. Chemical pollution, microbial contamination and adulteration in food have attracted more attention worldwide. Contamination with antibiotics, estrogens and heavy metals in water environment and soil environment have also turn into an enormous threat to food safety. Traditional small-scale, long-term detection technologies have been unable to meet the current needs. In the monitoring process, rapid, convenient, accurate analysis and detection technologies have become the future development trend. We critically synthesizing the current knowledge of various rapid detection technology, and briefly touched upon the problem which still exist in research process. The review showed that the application of novel materials promotes the development of rapid detection technology, high-throughput and portability would be popular study directions in the future. Of course, the ultimate aim of the research is how to industrialization these technologies and apply to the market.
Assuntos
Inocuidade dos Alimentos , Metais Pesados , Abastecimento de Alimentos , Solo , TecnologiaRESUMO
The roles of cellular orientation during trabecular and ventricular wall morphogenesis are unknown, and so are the underlying mechanisms that regulate cellular orientation. Myocardial-specific Numb and Numblike double-knockout (MDKO) hearts display a variety of defects, including in cellular orientation, patterns of mitotic spindle orientation, trabeculation, and ventricular compaction. Furthermore, Numb- and Numblike-null cardiomyocytes exhibit cellular behaviors distinct from those of control cells during trabecular morphogenesis based on single-cell lineage tracing. We investigated how Numb regulates cellular orientation and behaviors and determined that N-cadherin levels and membrane localization are reduced in MDKO hearts. To determine how Numb regulates N-cadherin membrane localization, we generated an mCherry:Numb knockin line and found that Numb localized to diverse endocytic organelles but mainly to the recycling endosome. Consistent with this localization, cardiomyocytes in MDKO did not display defects in N-cadherin internalization but rather in postendocytic recycling to the plasma membrane. Furthermore, N-cadherin overexpression via a mosaic model partially rescued the defects in cellular orientation and trabeculation of MDKO hearts. Our study unravels a phenomenon that cardiomyocytes display spatiotemporal cellular orientation during ventricular wall morphogenesis, and its disruption leads to abnormal trabecular and ventricular wall morphogenesis. Furthermore, we established a mechanism by which Numb modulates cellular orientation and consequently trabecular and ventricular wall morphogenesis by regulating N-cadherin recycling to the plasma membrane.
Assuntos
Caderinas/metabolismo , Ventrículos do Coração/embriologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Miócitos Cardíacos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Organogênese , Animais , Caderinas/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Miócitos Cardíacos/citologia , Proteínas do Tecido Nervoso/genéticaRESUMO
The epicardium contributes to multiple cardiac lineages and is essential for cardiac development and regeneration. However, the mechanism of epicardium formation is unclear. This study aimed to establish the cellular and molecular mechanisms underlying the dissociation of pro-epicardial cells (PECs) from the pro-epicardium (PE) and their subsequent translocation to the heart to form the epicardium. We used lineage tracing, conditional deletion, mosaic analysis and ligand stimulation in mice to determine that both villous protrusions and floating cysts contribute to PEC translocation to myocardium in a CDC42-dependent manner. We resolved a controversy by demonstrating that physical contact of the PE with the myocardium constitutes a third mechanism for PEC translocation to myocardium, and observed a fourth mechanism in which PECs migrate along the surface of the inflow tract to reach the ventricles. Epicardial-specific Cdc42 deletion disrupted epicardium formation, and Cdc42 null PECs proliferated less, lost polarity and failed to form villous protrusions and floating cysts. FGF signaling promotes epicardium formation in vivo, and biochemical studies demonstrated that CDC42 is involved in the trafficking of FGF receptors to the cell membrane to regulate epicardium formation.
Assuntos
Membrana Celular/metabolismo , Pericárdio/citologia , Pericárdio/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Animais , Polaridade Celular , Proliferação de Células , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Espaço Intracelular/metabolismo , Camundongos Knockout , Modelos Biológicos , Miocárdio/citologia , Miocárdio/metabolismo , Fosforilação , Transporte Proteico , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Peste des petits ruminants (PPR) is a highly contagious and fatal disease of small ruminants, particularly sheep and goats. This disease leads to high morbidity and mortality of small ruminants, thus resulting in devastating economic loss to the livestock industry globally. The severe disease impact has prompted the Food and Agriculture Organization of the United Nations (FAO) and the World Organization for Animal Health (OIE) to develop a global strategy for the control and eradication of PPR by 2030. Over the past decades, the control of PPR is mainly achieved through vaccinating the animals with live-attenuated vaccines, e.g., rinderpest vaccines. As a closely related disease to PPR of large ruminants, rinderpest was eradicated in 2011 and its vaccines subsequently got banned in order to keep rinderpest-free zones. Consequently, it is desirable to develop homologous PPR vaccines to control the disease. The present review summarizes the objectives of PPR control and eradication by focusing on the homologous PPR vaccines.
RESUMO
Hispidalin is a novel antimicrobial peptide isolated from the seeds of Benincasa hispida and is reported to have broad antimicrobial activity against various bacterial and fungal pathogens. To produce significant amounts of Hispidalin, a recombinant Hispidalin with an N-terminal 6â¯×â¯His tag and an enterokinase sequence, for the first time, was successfully expressed in Escherichia coli or Pichia pastoris cell factory. Results showed that the E. coli-derived recombinant Hispidalin did not show any antimicrobial activity against all the tested strains, whereas the P. pastoris-derived recombinant Hispidalin (rHispidalin) showed a broad antibacterial spectrum against five pathogenic bacteria of both Gram-negative and Gram-positive. rHispidalin also has bactericidal activity and completely killed all of the Staphylococcus aureus within 40â¯min. Additionally, rHispidalin showed a broad range of thermostability and pH stability, and a hemolytic activity of less than 2% even at a concentration of 300⯵g/ml; it was resistant to trypsin and proteinase K, but was moderately sensitive to pepsin and papain. Moreover, rHispidalin effectively permeabilized the cytoplasmic membrane and disrupted the morphology of targeted bacterial cells. After an initial optimization was performed, the amount of rHispidalin accumulation could reach as high as 98.6⯵g/ml. These results indicate that Hispidalin could be produced on a large scale by P. pastoris and has a great potential to be utilized as a new antibacterial agent for further development.
Assuntos
Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos/farmacologia , Pichia/genética , Antibacterianos/química , Antibacterianos/metabolismo , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/metabolismo , Bactérias/efeitos dos fármacos , Bactérias/crescimento & desenvolvimento , Cucurbitaceae/química , Cucurbitaceae/genética , Cucurbitaceae/metabolismo , Estabilidade de Medicamentos , Expressão Gênica , Concentração de Íons de Hidrogênio , Testes de Sensibilidade Microbiana , Pichia/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologiaRESUMO
BACKGROUND: In the present study, we investigated the role of ornithine decarboxylase (ODC) in the methyl jasmonate (MeJA)-regulated postharvest quality maintenance of Agaricus bisporus (J. E. Kange) Imbach button mushrooms by pretreating mushrooms with a specific irreversible inhibitor called α-difluoromethylornithine (DFMO) before exposure to MeJA vapor. RESULTS: Mushrooms were treated with 0 or 100 µmol L-1 MeJA or a combination of 120 µmol L-1 DFMO and 100 µmol L-1 MeJA, respectively, before storage at 4 °C for 21 days. Treatment with MeJA alone induced the increase in ODC activity whereas this effect was greatly suppressed by pretreatment with DFMO. α-Difluoromethylornithine strongly attenuated the effect of MeJA on decreasing cap opening, slowing the decline rate of soluble protein and total sugar, and accumulating total phenolics and flavonoids. α-Difluoromethylornithine pretreatment also counteracted the ability of MeJA to inhibit polyphenol oxidase and lipoxygenase activities, and malondialdehyde production, and to stimulate superoxide dismutase and catalase activities. It also largely downregulated MeJA-induced accumulation of free putrescine (Put). CONCLUSION: These results reveal that ODC is involved in MeJA-regulated postharvest quality retention of button mushrooms, and this involvement is likely to be associated with Put levels. © 2018 Society of Chemical Industry.
Assuntos
Acetatos/farmacologia , Agaricus/química , Agaricus/efeitos dos fármacos , Ciclopentanos/farmacologia , Proteínas Fúngicas/metabolismo , Ornitina Descarboxilase/metabolismo , Oxilipinas/farmacologia , Agaricus/enzimologia , Agaricus/crescimento & desenvolvimento , Catecol Oxidase/metabolismo , Flavonoides/análise , Flavonoides/metabolismo , Malondialdeído/metabolismo , Fenóis/análise , Fenóis/metabolismo , Putrescina/análise , Putrescina/metabolismo , Controle de Qualidade , Superóxido Dismutase/metabolismoRESUMO
VpDef is a novel defensin isolated from the clam Venerupis philippinarum. Previously it was expressed in Escherichia coli; however, the E. coli-derived recombinant VpDef did not show effective antimicrobial activity against Staphyloccocus aureus or the Gram-negative bacteria tested. As such, the goal of this study was to design, express, and purify a recombinant VpDef (rVpDef) in Pichia pastoris and to determine its antibacterial potency and stability. A 6.9â¯KDa rVpDef was successfully expressed as a secreted peptide in P. pastoris, and the amount of rVpDef accumulation was shown to reach as high as approximate 60⯵g per 1â¯ml of culture medium only after an initial optimization was performed. The purified rVpDef demonstrated a broad antibacterial spectrum and was active against six typical common bacteria, both Gram-positive and Gram-negative. A minimal inhibition concentration of as low as 50⯵g/ml was observed for rVpDef against the growth of E. coli O157 (ATCC 35150). Moreover, rVpDef was tolerant to temperature shock and proteinase digestion and maintained a high stability over a relatively broad pH range. In addition, rVpDef had a low hemolytic activity against rabbit erythrocytes. Taken together, this study demonstrated that rVpDef could be produced in a large-scale manner in P. pastoris and has a good antibacterial activity and suitable stability. This is the first report on heterologous expression of a biologically active VpDef in P. pastoris, supporting its use for both research and application purposes.
Assuntos
Bivalves/metabolismo , Defensinas/metabolismo , Peptídeos/metabolismo , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Animais , Antibacterianos/farmacologia , Sequência de Bases , Bivalves/genética , Defensinas/genética , Defensinas/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/crescimento & desenvolvimento , Bactérias Gram-Positivas/efeitos dos fármacos , Bactérias Gram-Positivas/genética , Hemólise/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Peptídeos/farmacologia , Pichia/genética , Estabilidade Proteica , Coelhos , Proteínas Recombinantes/farmacologia , TemperaturaRESUMO
The antimicrobial peptide PaDef was isolated from Mexican avocado fruit and was reported to inhibit the growth of Escherichia coli and Staphylococcus aureus in 2013. In this study, an N-terminal 6 × His tagged recombinant PaDef (rPaDef) with a molecular weight of 7.5 KDa, for the first time, was expressed as a secreted peptide in Pichia pastoris. The optimal culture condition for rPaDef expression was determined to be incubation with 1.5% methanol for 72 h at 28 °C under pH 6.0. Under this condition, the amount of the rPaDef accumulation reached as high as 79.6 µg per 1 ml of culture medium. Once the rPaDef peptide was purified to reach a 95.7% purity using one-step nickel affinity chromatography, its strong and concentration-dependent antimicrobial activity was detected to be against a broad-spectrum of bacteria of both Gram-negative and Gram-positive. The growth of these bacterial pathogens was almost completely inhibited when the rPaDef peptide was at a concentration of as low as 90 µg/ml. In summary, our data showed that rPaDef derived from Mexican avocado fruit can be expressed and secreted efficiently when P. pastoris was used as a cell factory. This is the first report on heterologous expression of PaDef in P. pastoris and the approach described holds great promise for antibacterial drug development.
Assuntos
Peptídeos Catiônicos Antimicrobianos , Escherichia coli/crescimento & desenvolvimento , Persea/genética , Pichia/metabolismo , Proteínas de Plantas , Staphylococcus aureus/crescimento & desenvolvimento , Peptídeos Catiônicos Antimicrobianos/biossíntese , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos/farmacologia , Persea/química , Pichia/genética , Proteínas de Plantas/biossíntese , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/farmacologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologiaRESUMO
Chlamydomonas reinhardtii offers a great promise for large-scale production of multiple recombinant proteins of pharmaceutical and industrial interest. However, the nuclear-encoding transgenes usually are expressed at a low level, which severely hampers the use of this alga in molecular farming. In this study, the promoter of the endogenous intraflagellar transport 25 (IFT25) gene of C. reinhardtii was tested for its ability to drive the expression of green fluorescent protein (GFP), which functions as a readout for target gene expression. IFT25 promoter (IFT25P) alone was not able to drive GFP expression to a detectable level. IFT25P, however, can drive robust IFT25-GFP fusion protein expression when the intron-containing IFT25 gene was inserted between IFT25P and GFP cDNA. When an extended version of foot-and-mouth virus 2A protease (2AE) sequence was further inserted between the intron-containing IFT25 gene and the GFP cDNA, discrete GFP protein was observed to release from the IFT25-2AE-GFP polyprotein via 2A self-cleaving with a cleavage efficacy of approximately 99%. The monomer GFP was accumulated to a level of as high as 0.68% of total soluble proteins. To test whether the newly developed bicistronic IFT25P-IFT25-2AE expression system can be used to overexpress heterologous proteins of different origins and sizes, we inserted codon-optimized cDNAs encoding a Trichoderma reesei xylanase1 (25 kDa) and a Lachnospiraceae bacterium ND2006 type V CRISPR-Cas protein LbCpf1 (147 kDa) to the vector and found that the production of xylanase1 and LbCpf1 was as high as 0.69 and 0.49% of total soluble protein. Our result showed that IFT25P-IFT25-2AE system is more efficient to drive nuclear gene expression in C. reinhardtii than other conventionally used promoters, thus representing a novel efficient recombinant protein expression tool and has the potential to be scaled for commercial production of nuclear-encoded recombinant proteins of different sizes and origins in C. reinhardtii.
Assuntos
Chlamydomonas reinhardtii/genética , Expressão Gênica , Transgenes , Proteínas Virais/genética , Reatores Biológicos , Códon , DNA Complementar , Proteínas de Fluorescência Verde/genética , Proteínas Luminescentes/genética , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/biossínteseRESUMO
The cytotoxic activity of camptothecin derivatives is so high that these compounds need to be further modified before their successful application as anti-cancer agents clinically. In this study, we reported the synthesis and biological evaluation of a novel camptothecin derivative called compound 2-47. The changes in structure did not reduce its activity to inhibit DNA topoisomerase I. Compound 2-47 induced apoptosis of many tumor cells including leukemia cells K562, Jurkat, HL-60, breast cancer cell BT-549, colon cancer cell HT-29 and liver cancer cell HepG2 with a half maximal inhibitory concentration (IC50) of 2- to 3-fold lower than HCPT as a control. In particular, 2-47 inhibited the proliferation of Jurkat cells with an IC50 of as low as 40 nM. By making use of Jurkat cell as a model, following treatment of Jurkat cells, compound 2-47 activated caspase-3 and PARP, resulting in a decreased Bcl-2/Bax ratio. These data showed that compound 2-47 induces Jurkat cell death through the mitochondrial apoptotic pathway. In addition, compound 2-47 showed a decreased cytotoxic activity against normal cells and an improved solubility in low-polar solvent. For example, compound 2-47 solutes in CHCl3 130-fold higher than HCPT. Taken together, our data demonstrated that camptothecin derivative 2-47 notably inhibits the tumor cell proliferation through mitochondrial-mediated apoptosis in vitro.
Assuntos
Apoptose/efeitos dos fármacos , Camptotecina/análogos & derivados , Camptotecina/administração & dosagem , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/patologia , Camptotecina/toxicidade , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Desenho de Fármacos , Humanos , Solubilidade , Resultado do TratamentoRESUMO
Mytichitin-A is an antimicrobial peptide isolated from the serum of Mytilus coruscus and is reported to inhibit bacterial growth as tested on several Gram-positive bacteria. To produce large quantity of Mytichitin-A to further investigate its biological activity, nucleotide sequence encoding a recombinant 6 × His-Mytichitin-A (rMytichitin-A) peptide was synthesized and inserted into the inducible yeast expression vector pPICZαA. With the availability of such an expression vector called pPICZαA-Mytichitin-A, we transformed Pichia pastoris GS115 cells with a SacI-linearized pPICZαA-Mytichitin-A by electroporation. Transgenic strains secreting rMytichitin-A with a molecular weight of approximate 10 KDa as expected were obtained. The optimal culture condition for rMytichitin-A expression was determined to be 1.0% methanol induction, 96 h incubation at 28 °C and the amount of rMytichitin-A reached 45.5 µg/ml. The percentage of rMytichitin-A was estimated to be 73.6% of the total protein. After rMytichitin-A was purified using nickel ions affinity chromatography, approximate 9.1 mg pure rMytichitin-A was obtained from 500 ml of cell culture medium with 97.8% purity. More importantly, both the culture supernatant and purified rMytichitin-A inhibited the growth of Gram-positive bacteria, especially Staphylococcus aureus and Bacillus subtilis with a minimum inhibition concentration of as low as 31 and 48 µg/ml, respectively. Differently from the native protein, however, the rMytichitin-A is not active against Gram-negative bacteria. Taken together, this is the first report on the heterologous expression of Mytichitin-A in P. pastoris. Our study showed that P. pastoris is an effective expression system for producing large quantities of biologically active Mytichitin-A for both research and application purposes.
Assuntos
Peptídeos Catiônicos Antimicrobianos , Bacillus subtilis/crescimento & desenvolvimento , Bactérias Gram-Negativas/crescimento & desenvolvimento , Mytilus/genética , Pichia/metabolismo , Staphylococcus aureus/crescimento & desenvolvimento , Animais , Peptídeos Catiônicos Antimicrobianos/biossíntese , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos/farmacologia , Mytilus/metabolismo , Pichia/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacocinéticaRESUMO
Influenza A virus (IAV) is characterized by eight single-stranded, negative sense RNA segments, which allows for gene reassortment among different IAV subtypes when they co-infect a single host cell simultaneously. Genetic reassortment is an important way to favor the evolution of influenza virus. Novel reassortant virus may pose a pandemic among humans. In history, three human pandemic influenza viruses were caused by genetic reassortment between avian, human and swine influenza viruses. Since 2009, pandemic (H1N1) 2009 (pdm/09 H1N1) influenza virus composed of two swine influenza virus genes highlighted the genetic reassortment again. Due to wide host species and high transmission of the pdm/09 H1N1 influenza virus, many different avian, human or swine influenza virus subtypes may reassert with it to generate novel reassortant viruses, which may result in a next pandemic among humans. So, it is necessary to understand the potential threat of current reassortant viruses between the pdm/09 H1N1 and other influenza viruses to public health. This study summarized the status of the reassortant viruses between the pdm/09 H1N1 and other influenza viruses of different species origins in natural and experimental conditions. The aim of this summarization is to facilitate us to further understand the potential threats of novel reassortant influenza viruses to public health and to make effective prevention and control strategies for these pathogens.
Assuntos
Evolução Molecular , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Vírus Reordenados/genética , Vírus Reordenados/isolamento & purificação , Animais , Humanos , Vírus da Influenza A Subtipo H1N1/classificação , Influenza Humana/epidemiologia , Vírus Reordenados/classificaçãoRESUMO
Our previous study successfully identified that the novel isatin derivative (E)-methyl 3-(1-(4-methoxybenzyl)-2,3-dioxoindolin-5-yl) acrylate (HKL 2H) acts as an anticancer agent at an inhibitory concentration (IC50) level of 3nM. In this study, the molecular mechanism how HKL 2H induces cytotoxic activity in the human chronic myelogenous leukemia K562 cells was investigated. Flow cytometric analysis showed that the cells were arrested in the G2/M phase and accumulated subsequently in the sub-G1 phase in the presence of HKL 2H. HKL 2H treatment down-regulated the expressions of CDK1 and cyclin B but up-regulated the level of phosphorylated CDK1. Annexin-V staining and the classic DNA ladder studies showed that HKL 2H induced the apoptosis of K562 cells. Our study further showed that HKL 2H treatment caused the dissipation of mitochondrial membrane potential, activated caspase-3 and lowered the Bcl-2/Bax ratio in K562 cells, suggesting that the HKL 2H-causing programmed cell death of K562 cells was caused via the mitochondrial apoptotic pathway. Taken together, our data demonstrated that HKL 2H, a 5-(2-carboxyethenyl) isatin derivative, notably induces G2/M cell cycle arrest and mitochondrial-mediated apoptosis in K562 cells, indicating that this compound could be a promising anticancer candidate for further investigation.
Assuntos
Acrilatos/farmacologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Fase G2/efeitos dos fármacos , Isatina/análogos & derivados , Humanos , Isatina/farmacologia , Células K562 , Mitocôndrias/efeitos dos fármacosRESUMO
As one of the major microbes in the soy sauce fermentation, Candida versatilis enriches the flavor and improves the quality of soy sauce. In this study, a combination of five different GC-MS and LC-MS-based metabolome analytical approaches was used to analyze the intracellular, extracellular and whole metabolites of C. versatilis. Our results found out that a total of 132, 244 and 267 different metabolites were detectable from the intracellular, extracellular and whole part, respectively. When exposed to 0. 9 and 18 % salt, respectively, 114, 123 and 129 different intracellular metabolites, 184, 200 and 178 extracellular metabolites and 177, 188 and 186 whole metabolites were detected, respectively. Our data showed that salt enhances the metabolic capacity of C. versatilis, especially its amino acid and enhances the synthesis and secretion of some metabolites of C. versatilis, especially the aldehydes and phenols, such as vanillin, guaiacol and 5-hydroxymethylfurfural. Our data also showed that special attention has to be paid to the generation of biogenic amines when C. versatilis was treated with salt.
Assuntos
Candida/metabolismo , Metaboloma , Cloreto de Sódio/metabolismo , Estresse Fisiológico , Benzaldeídos/metabolismo , Aminas Biogênicas/biossíntese , Furaldeído/análogos & derivados , Furaldeído/metabolismo , Guaiacol/metabolismo , Análise de Componente Principal , Tolerância ao SalRESUMO
BACKGROUND: This study aimed to enhance and improve the quality and safety of soy sauce. In the present work, the change of biogenic amines, such as histamine, tyramine, cadaverine, spermidine, was examined by the treatment of Candida versatilis and Zygosaccharomyces rouxii, and the influence of salt-tolerant yeast on biogenic amines was analysed during the whole fermentation process. RESULTS: The results showed that the content of biogenic amines was elevated after yeast treatment and the content of biogenic amines was influenced by using yeast. The dominating biogenic amine in soy sauce was tyramine. At the end of fermentation, the concentrations of biogenic amines produced by Zygosaccharomyces rouxii and Candida versatilis in the soy mash were 122.71 mg kg(-1) and 69.96 mg kg(-1) . CONCLUSIONS: The changes of biogenic amines in high-salt liquid soy mash during fermentation process indicated that a variety of biogenic amines were increased in the fermentation ageing period, which may be due to amino acid decarboxylation to form biogenic amines by yeast decarboxylase. The fermentation period of soy sauce should be longer than 5 months because biogenic amines began to decline after this time period.