RESUMO
The transmembrane domain (TMD) of the syndecans, a family of transmembrane heparin sulfate proteoglycans, is involved in forming homo- and heterodimers and oligomers that transmit signaling events. Recently, we reported that the unique phenylalanine in TMD positively regulates intramolecular interactions of syndecan-2. Besides the unique phenylalanine, syndecan-2 contains a conserved phenylalanine (SDC2-Phe-169) that is present in all syndecan TMDs, but its function has not been determined. We therefore investigated the structural role of SDC2-Phe-169 in syndecan TMDs. Replacement of SDC2-Phe-169 by tyrosine (S2F169Y) did not affect SDS-resistant homodimer formation but significantly reduced SDS-resistant heterodimer formation between syndecan-2 and -4, suggesting that SDC2-Phe-169 is involved in the heterodimerization/oligomerization of syndecans. Similarly, in an in vitro binding assay, a syndecan-2 mutant (S2(F169Y)) showed a significantly reduced interaction with syndecan-4. FRET assays showed that heteromolecular interactions between syndecan-2 and -4 were reduced in HEK293T cells transfected with S2(F169Y) compared with syndecan-2. Moreover, S2(F169Y) reduced downstream reactions mediated by the heterodimerization of syndecan-2 and -4, including Rac activity, cell migration, membrane localization of PKCα, and focal adhesion formation. The conserved phenylalanine in syndecan-1 and -3 also showed heterodimeric interaction with syndecan-2 and -4. Taken together, these findings suggest that the conserved phenylalanine in the TMD of syndecans is crucial in regulating heteromeric interactions of syndecans.
Assuntos
Sequência Conservada , Fenilalanina/metabolismo , Multimerização Proteica , Sindecanas/química , Sindecanas/metabolismo , Sequência de Aminoácidos , Animais , Sobrevivência Celular/efeitos dos fármacos , Células HEK293 , Humanos , Dados de Sequência Molecular , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Ratos , Dodecilsulfato de Sódio/farmacologiaRESUMO
In this study, the roles of reactive oxygen species (ROS) and NF-κB on inflammation induction in lipopolysaccharide (LPS)-stimulated zebrafish embryos were evaluated using N-acetyl-l-cysteine (NAC) and pyrrolidine dithiocarbamate (PDTC), specific inhibitors of ROS and NF-κB, respectively. LPS-stimulated zebrafish embryos showed increasing production of NO and ROS and expression of iNOS and COX-2 protein, compared to a control group without LPS. However, NAC significantly inhibited production of NO and ROS and markedly suppressed expression of iNOS and COX-2 protein in LPS-stimulated zebrafish embryos. The mRNA expressions of NF-κB such as p65NF-κB and IκB-A were significantly increased after LPS stimulation, whereas PDTC attenuated mRNA expression of NF-κB. PDTC also inhibited production of NO and reduced expression of iNOS and COX-2 protein in LPS-stimulated zebrafish embryos. Taken together, these results indicated that LPS increases pro-inflammatory mediators in zebrafish embryos through ROS and NF-κB regulation.
Assuntos
Doenças dos Peixes/imunologia , Proteínas de Peixes/metabolismo , Mediadores da Inflamação/metabolismo , Inflamação/veterinária , NF-kappa B/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Peixe-Zebra , Acetilcisteína/metabolismo , Animais , Embrião não Mamífero , Regulação da Expressão Gênica , Inflamação/imunologia , Lipopolissacarídeos/administração & dosagem , Pirrolidinas/metabolismo , Tiocarbamatos/metabolismoRESUMO
Many multicomponent protein complexes mediating diverse cellular processes are assembled through scaffolds with specialized protein interaction modules. The multi-tRNA synthetase complex (MSC), consisting of nine different aminoacyl-tRNA synthetases and three non-enzymatic factors (AIMP1-3), serves as a hub for many signaling pathways in addition to its role in protein synthesis. However, the assembly process and structural arrangement of the MSC components are not well understood. Here we show the heterotetrameric complex structure of the glutathione transferase (GST) domains shared among the four MSC components, methionyl-tRNA synthetase (MRS), glutaminyl-prolyl-tRNA synthetase (EPRS), AIMP2 and AIMP3. The MRS-AIMP3 and EPRS-AIMP2 using interface 1 are bridged via interface 2 of AIMP3 and EPRS to generate a unique linear complex of MRS-AIMP3:EPRS-AIMP2 at the molar ratio of (1:1):(1:1). Interestingly, the affinity at interface 2 of AIMP3:EPRS can be varied depending on the occupancy of interface 1, suggesting the dynamic nature of the linear GST tetramer. The four components are optimally arranged for maximal accommodation of additional domains and proteins. These characteristics suggest the GST tetramer as a unique and dynamic structural platform from which the MSC components are assembled. Considering prevalence of the GST-like domains, this tetramer can also provide a tool for the communication of the MSC with other GST-containing cellular factors.
Assuntos
Aminoacil-tRNA Sintetases/química , Glutationa Transferase/química , Metionina tRNA Ligase/química , Proteínas Nucleares/química , Fatores de Alongamento de Peptídeos/química , Proteínas Supressoras de Tumor/química , Sequência de Aminoácidos , Animais , Células CHO , Cromatografia , Cricetinae , Cricetulus , Transferência Ressonante de Energia de Fluorescência , Humanos , Microscopia Eletrônica , Dados de Sequência Molecular , Complexos Multiproteicos , Conformação Proteica , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Transdução de SinaisRESUMO
In this study, we demonstrated that survivin downregulation with TRAIL expression greatly enhanced the cytotoxic death of pancreatic cancer cells after gemcitabine treatment. Using real-time RT-PCR, we analyzed five survivin shRNAs to identify the best target sequence for suppression of human survivin, with the goal of treating gemcitabine-resistant pancreatic cancer cells. Survivin shRNA 5, corresponding to target 5, showed the greatest reduction in survivin mRNA levels. Furthermore, combined treatment with survivin shRNA-expressing adenovirus with gemcitabine plus TRAIL decreased uncleaved PARP and increased consequent PARP cleavage, which was correlated with the greatest levels of survivin downregulation and cell death. These results indicate that survivin functions as a common mediator of gemcitabine- and TRAIL-induced cell death. Using a nude mouse model implanted with MiaPaCa-2 pancreatic cancer cells, we observed tumor regression induced by an oncolytic adenovirus expressing survivin shRNA and TRAIL plus gemcitabine. Together, our findings provide a strong rationale for treating pancreatic cancer patients with both gemcitabine and oncolytic adenovirus armed with survivin shRNA and TRAIL.
Assuntos
Antimetabólitos Antineoplásicos/uso terapêutico , Desoxicitidina/análogos & derivados , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas Inibidoras de Apoptose/genética , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos , Neoplasias Pancreáticas/terapia , Ligante Indutor de Apoptose Relacionado a TNF/genética , Adenoviridae , Animais , Apoptose , Linhagem Celular Tumoral , Terapia Combinada , Desoxicitidina/uso terapêutico , Regulação para Baixo , Feminino , Células HEK293 , Humanos , Camundongos , Camundongos Nus , Interferência de RNA , RNA Interferente Pequeno/genética , Survivina , Ensaios Antitumorais Modelo de Xenoenxerto , GencitabinaRESUMO
BACKGROUND: It has been reported that both chemical and physical surface patterns influence cellular behaviors, such as cell alignment and elongation. However, it still remains unclear how actin filament and microtubules (MTs) differentially respond to these patterns. RESULTS: We examined the effects of chemical and physical patterns on cell elongation and alignment by observing actin filament and MTs of retinal pigment epithelium-1(RPE-1) cells, which were cultured on either fibronectin (FN)-line pattern (line width and spacing: 1 µm) or FN-coated 1 µm gratings with two different depths (0.35 or 1 µm). On the surface with either FN-line pattern or micrograting structure, the cell aspect ratios were at least two times higher than those on the surface with no pattern. Cell elongation on the gratings depended on the depth of the gratings. Cell elongation and alignment on both FN-line pattern and 1 µm gratings with 0.35 µm depth were perturbed either by inhibition of actin polymerization or MT depletion, while cell elongation and alignment on 1 µm gratings with 1 µm depth were perturbed only by MT depletion. CONCLUSIONS: Our results suggest that the contribution of actin filaments and MTs to the elongation and alignment of epithelial cells on microgratings depends on the groove depth of these gratings.
Assuntos
Citoesqueleto de Actina/ultraestrutura , Forma Celular/efeitos dos fármacos , Células Epiteliais/ultraestrutura , Microtúbulos/ultraestrutura , Citoesqueleto de Actina/efeitos dos fármacos , Linhagem Celular , Forma Celular/fisiologia , Citocalasina D/farmacologia , Células Epiteliais/efeitos dos fármacos , Fibronectinas/química , Fibronectinas/farmacologia , Humanos , Microtúbulos/efeitos dos fármacos , Nocodazol/farmacologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/ultraestrutura , Propriedades de SuperfícieRESUMO
Harmful algal blooms (HABs) can lead to substantial socio-economic losses and extensive damage to aquatic ecosystems, drinking water sources and human health. Common algicidal techniques, including ozonation, ultrasonic treatment and dispersion of algae-killing chemicals, are unsatisfactory both economically and ecologically. This study therefore presents a novel alternative strategy for the efficient control of deleterious algae via the use of host-specific virus-like particles (VLPs) combined with chemically synthesized algicidal compounds. The capsid protein of HcRNAV34, a single-stranded RNA virus that infects the toxic dinoflagellate, Heterocapsa circularisquama, was expressed in and purified from Escherichia coli and then self-assembled into VLPsâ in vitro. Next, the algicidal compound, thiazolidinedione 49 (TD49), was encapsidated into HcRNAV34 VLPs for specific delivery to H. circularisquama. Consequently, HcRNAV34 VLPs demonstrated the same host selectivity as naturally occurring HcRNAV34 virions, while TD49-encapsidated VLPs showed a more potent target-specific algicidal effect than TD49 alone. These results indicate that target-specific VLPs for the delivery of cytotoxic compounds to nuisance algae might provide a safe, environmentally friendly approach for the management of HABs in aquatic ecosystems.
Assuntos
Dinoflagellida/efeitos dos fármacos , Dinoflagellida/virologia , Sistemas de Liberação de Medicamentos/métodos , Proliferação Nociva de Algas/efeitos dos fármacos , Vírus de RNA/fisiologia , Tiazolidinedionas/farmacologia , Sistemas de Liberação de Medicamentos/instrumentação , Ecossistema , Vírus de RNA/genéticaRESUMO
Thiazolidinedione (TD) derivatives have been found to have an algicidal effect on harmful algal bloom microalgae. In this study, 75 TD derivatives were synthesized and analyzed for algicidal activity. Among these synthetic TDs, 18 TD derivatives showed specific algicidal activity on two strains belonging to the classes Raphidophyceae (Chattonella marina and Heterosigma akashiwo) and Dinophyceae (Cochlodinium polykrikoides). Two strains belonging to Bacillariophyceae (Navicula pelliculosa and Phaeodactylum EPV), one strain belonging to Dinophyceae (Amphidinium sp.), and a Eustigmatophycean microalga (Nannochloropsis oculata) showed less sensitivity to the TD derivatives than the other two phyla. The most reactive TD derivative, compound 2 (TD118), was selected and tested for morphological and physiological changes. TD118 effectively damaged the cell membrane of C. marina, H. akashiwo and C. polykrikoides. The O2 evolution and photosystem II efficiency (F(v)/F(m)) of C. marina, H. akashiwo and C. polykrikoides were also severely reduced by TD118 treatment. Amphidinium sp., N. pelliculosa, Phaeodactylum EPV and N. oculata showed less reduction of O2 evolution and the F(v)/F(m) by TD118. These results imply that the species-specific TD structure relationship may be due to structural and/or physiological differences among microalgal species.
Assuntos
Antifúngicos/farmacologia , Proliferação Nociva de Algas/efeitos dos fármacos , Tiazolidinedionas/farmacologia , Antifúngicos/química , Microalgas/classificação , Microalgas/efeitos dos fármacos , Relação Estrutura-Atividade , Tiazolidinedionas/químicaRESUMO
Vitiligo is an autoimmune skin disease caused by cutaneous melanocyte loss. Although phototherapy and T cell suppression therapy have been widely used to induce epidermal re-pigmentation, full pigmentation recovery is rarely achieved due to our poor understanding of the cellular and molecular mechanisms governing this process. Here, we identify unique melanocyte stem cell (McSC) epidermal migration rates between male and female mice, which is due to sexually dimorphic cutaneous inflammatory responses generated by ultra-violet B exposure. Using genetically engineered mouse models, and unbiased bulk and single-cell mRNA sequencing approaches, we determine that manipulating the inflammatory response through cyclooxygenase and its downstream prostaglandin product regulates McSC proliferation and epidermal migration in response to UVB exposure. Furthermore, we demonstrate that a combinational therapy that manipulates both macrophages and T cells (or innate and adaptive immunity) significantly promotes epidermal melanocyte re-population. With these findings, we propose a novel therapeutic strategy for repigmentation in patients with depigmentation conditions such as vitiligo.
Assuntos
Vitiligo , Humanos , Masculino , Feminino , Animais , Camundongos , Vitiligo/terapia , Caracteres Sexuais , Pele , Melanócitos , Células-Tronco , Imunoglobulinas , Pigmentação da PeleRESUMO
Lithium manganese oxide-based adsorbents have been developed for the recovery of lithium from seawater. To maximize the recovery efficiency, it is important to prevent microfouling of lithium adsorbents by marine bacteria. To identify the marine bacteria that cause biofouling against the lithium adsorbents, lithium adsorbents were installed into a non-coated frame or a frame coated with an antifouling agent soaked in seawater. Microorganisms from the surface of lithium adsorbents were collected for 30 days at 10-day intervals, cultured in marine broth, isolated, and identified by 16S rDNA sequencing. Pseudoalteromonas and Vibrio were constituted to 35.6 and 28.8 % of total isolates, respectively, and were predominant in the non-coated frame, whereas Vibrio was poorly isolated (2.3 %) from the antifouling agent-coated frame. In this study, antifouling strategy for maximum lithium recovery efficiency in the marine area takes account of Pseudoalteromonas and Vibrio.
Assuntos
Incrustação Biológica/prevenção & controle , Lítio/química , Manganês/química , Óxidos/química , Microbiologia da Água , Adsorção , DNA Bacteriano/genética , DNA Ribossômico/genética , Pseudoalteromonas/isolamento & purificação , RNA Ribossômico 16S/genética , Água do Mar/microbiologia , Vibrio/isolamento & purificaçãoRESUMO
Two-photon excited fluorescence microscopy is a widely-employed imaging technique that enables the noninvasive study of biological specimens in three dimensions with sub-micrometer resolution. Here, we report an assessment of a gain-managed nonlinear (GMN) fiber amplifier for multiphoton microscopy. This recently-developed source delivers 58-nJ and 33-fs pulses at 31-MHz repetition rate. We show that the GMN amplifier enables high-quality deep-tissue imaging, and furthermore that the broad spectral bandwidth of the GMN amplifier can be exploited for superior spectral resolution when imaging multiple distinct fluorophores.
RESUMO
A decreased production of interferon gamma (IFNG) has been observed in acute schizophrenia. In order to explore the possible relationship between IFNG and schizophrenia, we attempted to analyze the differentially expressed proteins in the brains of interferon-gamma knockout (Ifng-KO) mice. Five upregulated and five downregulated proteins were identified with 2D gels and MALDI-TOF/TOF MS analyses in Ifng-KO mouse brain. Of the identified proteins, we focused on creatine kinase brain (CKB) and triose phosphate isomerase 1 (TPI1). Consistent with the proteomic data, reverse transcriptase-mediated PCR, immunoblotting, and immunohistochemistry analyses confirmed that the levels of gene expressions of Ckb and Tpi1 were downregulated and upregulated, respectively. When we analyzed the genetic polymorphisms of the single nucleotide polymorphisms (SNPs) of their human orthologous genes in a Korean population, the promoter SNPs of CKB and TPI1 were weakly associated with schizophrenia. In addition, IFNG polymorphisms were associated with schizophrenia. These results suggest that IFNG and proteins affected by IFNG may play a role in the pathogenesis of schizophrenia.
Assuntos
Creatina Quinase Forma BB/metabolismo , Interferon gama/metabolismo , Esquizofrenia/genética , Esquizofrenia/metabolismo , Triose-Fosfato Isomerase/metabolismo , Animais , Encéfalo/metabolismo , Estudos de Casos e Controles , Creatina Quinase Forma BB/genética , Regulação para Baixo , Feminino , Humanos , Interferon gama/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , Proteoma/análise , Proteômica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , República da Coreia , Genética Reversa , Triose-Fosfato Isomerase/genética , Regulação para CimaRESUMO
Two-photon fluorescence microscopy is a nonlinear imaging modality frequently used in deep-tissue imaging applications. A tunable-wavelength multicolor short-pulse source is usually required to excite fluorophores with a wide range of excitation wavelengths. This need is most typically met by solid-state lasers, which are bulky, expensive, and complicated systems. Here, we demonstrate a compact, robust fiber system that generates naturally synchronized femtosecond pulses at 1050 nm and 1200 nm by using a combination of gain-managed and Raman amplification. We image the brain of a mouse and view the blood vessels, neurons, and other cell-like structures using simultaneous degenerate and nondegenerate excitation.
RESUMO
To construct a highly sensitive detection system for endocrine disruptors, we have compared the activity of promoters with the ALK1, ICL1, RPS7 and TEF1 for heterologous gene in Yarrowia lipolytica. The promoters were introduced into the upstream of lacZ or hERalpha reporter gene, respectively, and the activity was evaluated by beta-galactosidase assay by lacZ or western blot analysis by hERalpha. The expression analysis revealed that the ALK1 and ICL1 promoter were induced by n-decane and by EtOH, respectively. The constitutive promoter of RPS7 and TEF1 showed mostly high level of expression in the presence of glucose and glycerol, respectively. Particularly, the TEF1 promoter showed the highest beta-galactosidase activity and a significant signal by western blotting with the anti-estrogen receptor compared with the other promoters. Moreover, the detection system was constructed with promoters were linked to the upstream of expression vector for hERalpha gene transformed into the Y. lipolytica with a chromosome-integrated lacZ reporter gene under the control of estrogen response elements (EREs). It was indicated that a combination of pTEF1p-hERalpha and CXAU1-2XERE was the most effective system for the E2-dependent induction of the beta-galactosidase activity. This system showed the highest beta-galactosidase activity at 10-6 M E2 and the activity could be detected at even the concentration of 10-10 M E2. As the result, we constructed a strongly sensitive detection system with Y. lipolitica to evaluate recognized/suspected ED chemicals, such as natural/synthetic hormones, pesticides, and commercial chemicals. The results demonstrate the utility, sensitivity and reproducibility of the system for characterizing environmental estrogens.
Assuntos
Alcanos/análise , Técnicas Biossensoriais/métodos , Disruptores Endócrinos/análise , Engenharia Genética , Yarrowia/genética , Yarrowia/metabolismo , Alcanos/metabolismo , Disruptores Endócrinos/metabolismo , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Regulação Fúngica da Expressão Gênica , Genes Reporter , Humanos , Regiões Promotoras Genéticas , Transformação GenéticaRESUMO
Intravital microscopy is a powerful technique to observe dynamic processes with single-cell resolution in live animals. No intravital window has been developed for imaging the colon due to its anatomic location and motility, although the colon is a key organ where the majority of microbiota reside and common diseases such as inflammatory bowel disease, functional gastrointestinal disorders, and colon cancer occur. Here we describe an intravital murine colonic window with a stabilizing ferromagnetic scaffold for chronic imaging, minimizing motion artifacts while maximizing long-term survival by preventing colonic obstruction. Using this setup, we image fluorescently-labeled stem cells, bacteria, and immune cells in live animal colons. Furthermore, we image nerve activity via calcium imaging in real time to demonstrate that electrical sacral nerve stimulation can activate colonic enteric neurons. The simple implantable apparatus enables visualization of live processes in the colon, which will open the window to a broad range of studies.
Assuntos
Colo/diagnóstico por imagem , Microscopia Intravital/métodos , Imagem Óptica/métodos , Animais , Movimento Celular , Colo/microbiologia , Corantes Fluorescentes/química , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco/química , Células-Tronco/citologiaRESUMO
The DevS histidine kinase of Mycobacterium smegmatis contains tandem GAF domains (GAF-A and GAF-B) in its N-terminal sensory domain. The heme iron of DevS is in the ferrous state when purified and is resistant to autooxidation from a ferrous to a ferric state in the presence of O(2). The redox property of the heme and the results of sequence comparison analysis indicate that DevS of M. smegmatis is more closely related to DosT of Mycobacterium tuberculosis than DevS of M. tuberculosis. The binding of O(2) to the deoxyferrous heme led to a decrease in the autokinase activity of DevS, whereas NO binding did not. The regulation of DevS autokinase activity in response to O(2) and NO was not observed in the DevS derivatives lacking its heme, indicating that the ligand-binding state of the heme plays an important role in the regulation of DevS kinase activity. The redox state of the quinone/quinol pool of the respiratory electron transport chain appears not to be implicated in the regulation of DevS activity. Neither cyclic GMP (cGMP) nor cAMP affected DevS autokinase activity, excluding the possibility that the cyclic nucleotides serve as the effector molecules to modulate DevS kinase activity. The three-dimensional structure of the putative GAF-B domain revealed that it has a GAF folding structure without cyclic nucleotide binding capacity.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Mycobacterium smegmatis/fisiologia , Óxido Nítrico/metabolismo , Oxigênio/metabolismo , Protamina Quinase/química , Protamina Quinase/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Cristalografia por Raios X , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Compostos Ferrosos/metabolismo , Heme/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Fosforilação , Estrutura Terciária de Proteína , Alinhamento de Sequência , Ubiquinona/metabolismo , Vitamina K 2/metabolismoRESUMO
A methane-oxidizing bacterium was isolated from the effluent of manure and its molecular and biochemical properties were characterized. The isolate was aerobic, Gram-negative, and non-motile. The organism had a type I intracytoplasmic membrane structure and granular inclusion bodies. The outer cell wall surface (S-layers) was tightly packed with cup-shaped structures. Colonies were light yellow on nitrate mineral salt agar medium. In addition, the organism was catalase and oxidase positive. The isolate used the ribulose monophosphate (RuMP) pathway for carbon assimilation, and was able to utilize methane and methanol as a sole carbon and energy source, however, it could not utilize any other organic compounds that were tested. The cells grew well in a mixture of methane and air (methane:air=1:1, v/v) in a compulsory circulation diffusion system, and when grown under those conditions, the optimum pH was approximately 7.0 and the optimal temperature was 30 degrees. In addition, the specific growth rate and generation time were 0.13 per h and 5.43 h, respectively, when grown under the optimum conditions. The major ubiquinone was Q-8, and the G+C mol% of the DNA was 55.3. Phylogenetic analyses based on the 16S rRNA gene sequence comparisons showed that this bacterium belongs to a group of type I methanotrophs, and that it is most closely related to Methylomicrobium, with a sequence similarity of 99%. Therefore, the isolate was named Methylomicrobium sp. HG-1.
Assuntos
Metano/metabolismo , Methylococcaceae/classificação , Methylococcaceae/isolamento & purificação , Proteínas de Bactérias/genética , Composição de Bases , DNA Bacteriano/genética , DNA Ribossômico/genética , Ácidos Graxos , Esterco/microbiologia , Methylococcaceae/fisiologia , Methylococcaceae/ultraestrutura , Dados de Sequência Molecular , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
In this study, a volumetric scale-up of this system was designed and built on a field pilot-scale (total digester volume 10 m(3)), with the results from the field pilot-scale experiments compared with those from the bench-scale (total digester volume 0.4 m(3)) process prior to scale-up. The reduction rate of total chemical oxygen demand (tCOD) and the maximum methane content produced in the biogas from the bench-scale system were 90.6% and 72%; whereas those from the field pilot-scale system were 90.1% and 68%, respectively. The estimated methane yields were 282 and 254 l CH(4)/kg tCOD(degraded) in bench and field pilot-scale fermentation systems, respectively. These results indicate that the three stage fermentation system developed in this study can be applied as a commercial process for the disposal of food waste in view of process stability.
Assuntos
Reatores Biológicos , Biotecnologia/métodos , Fermentação , Metano/química , Eliminação de Resíduos Líquidos/métodos , Anaerobiose , Biodegradação Ambiental , Fontes de Energia Bioelétrica , Conservação dos Recursos Naturais , Alimentos , Gases , Oxigênio/química , Projetos Piloto , Esgotos , Fatores de TempoRESUMO
The generation of virus-mimetic nanoparticles has received much attention in developing a new vaccine for overcoming the limitations of current vaccines. Thus, a method, encompassing most viral features for their size, hydrophobic domain and antigen display, would represent a meaningful direction for the vaccine development. In the present study, a polymer-templated protein nanoball with direction oriented hemagglutinin1 on its surface (H1-NB) was prepared as a new influenza vaccine, exhibiting most of the viral features. Moreover, the concentrations of antigen on the particle surface were controlled, and its effect on immunogenicity was estimated by in vivo studies. Finally, H1-NB efficiently promoted H1-specific immune activation and cross-protective activities, which consequently prevented H1N1 infections in mice.
Assuntos
Hemaglutininas Virais/metabolismo , Hemaglutininas/química , Vacinas contra Influenza/química , Nanopartículas/química , Polímeros/química , Animais , Materiais Biomiméticos/química , Materiais Biomiméticos/metabolismo , Células Dendríticas/fisiologia , Hemaglutininas/metabolismo , Humanos , Vírus da Influenza A Subtipo H1N1/imunologia , Vacinas contra Influenza/imunologia , Vacinas contra Influenza/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Nanopartículas/metabolismo , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Tamanho da Partícula , Baço/citologiaRESUMO
The DevSR (DosSR) two-component system, which is a major regulatory system involved in oxygen sensing in mycobacteria, plays an important role in hypoxic induction of many genes in mycobacteria. We demonstrated that overexpression of the kinase domain of Mycobacterium tuberculosis (Mtb) PknB inhibited transcriptional activity of the DevR response regulator in Mycobacterium smegmatis and that this inhibitory effect was exerted through phosphorylation of DevR on Thr180 within its DNA-binding domain. Moreover, the purified kinase domain of Mtb PknB significantly phosphorylated RegX3, NarL, KdpE, TrcR, DosR, and MtrA response regulators of Mtb that contain the Thr residues corresponding to Thr180 of DevR in their DNA-binding domains, implying that transcriptional activities of these response regulators might also be inhibited when the kinase domain of PknB is overexpressed.
Assuntos
Hipóxia Celular/genética , Mycobacterium smegmatis/genética , Mycobacterium tuberculosis/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA , Regulação Bacteriana da Expressão Gênica/genética , Humanos , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/patogenicidade , Oxigênio/química , Oxigênio/metabolismo , Fosforilação , Protamina Quinase/genética , Protamina Quinase/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Tuberculose/genética , Tuberculose/microbiologiaRESUMO
In the current study, we report the first example of polydiacetylenes (PDAs), where our PDA-based system acts as both a sensing probe and killer for bacteria. The contact of imidazolium and imidazole-derived PDA with various bacterial strains including MRSA (methicillin-resistant Staphylococcus aureus) and ESBL-EC (extended-spectrum ß-lactamase-producing Escherichia coli) results in a distinct blue-to-red colorimetric change of the solution as well as a rapid disruption of the bacterial membrane, which is demonstrated by transmission electron microscopy and confocal microscopy. Zeta potential analysis supports that antibacterial activity of the PDA solution originates from an electrostatic interaction between the negatively charged bacterial cell surface and the positively charged polymers. These results suggest that the PDA has a great potential to carry out the dual roles of a probe and killer for bacteria.