RESUMO
Ebola virus (EBOV) inclusion bodies (IBs) are cytoplasmic sites of nucleocapsid formation and RNA replication, housing key steps in the virus life cycle that warrant further investigation. During infection, IBs display dynamic properties regarding their size and location. The contents of IBs also must transition prior to further viral maturation, assembly, and release, implying additional steps in IB function. Interestingly, the expression of the viral nucleoprotein (NP) alone is sufficient for the generation of IBs, indicating that it plays an important role in IB formation during infection. In addition to NP, other components of the nucleocapsid localize to IBs, including VP35, VP24, VP30, and the RNA polymerase L. We previously defined and solved the crystal structure of the C-terminal domain of NP (NP-Ct), but its role in virus replication remained unclear. Here, we show that NP-Ct is necessary for IB formation when NP is expressed alone. Interestingly, we find that NP-Ct is also required for the production of infectious virus-like particles (VLPs), and that defective VLPs with NP-Ct deletions are significantly reduced in viral RNA content. Furthermore, coexpression of the nucleocapsid component VP35 overcomes deletion of NP-Ct in triggering IB formation, demonstrating a functional interaction between the two proteins. Of all the EBOV proteins, only VP35 is able to overcome the defect in IB formation caused by the deletion of NP-Ct. This effect is mediated by a novel protein-protein interaction between VP35 and NP that controls both regulation of IB formation and RNA replication itself and that is mediated by a newly identified functional domain of NP, the central domain.IMPORTANCE Inclusion bodies (IBs) are cytoplasmic sites of RNA synthesis for a variety of negative-sense RNA viruses, including Ebola virus. In addition to housing important steps in the viral life cycle, IBs protect new viral RNA from innate immune attack and contain specific host proteins whose function is under study. A key viral factor in Ebola virus IB formation is the nucleoprotein, NP, which also is important in RNA encapsidation and synthesis. In this study, we have identified two domains of NP that control inclusion body formation. One of these, the central domain (CD), interacts with viral protein VP35 to control both inclusion body formation and RNA synthesis. The other is the NP C-terminal domain (NP-Ct), whose function has not previously been reported. These findings contribute to a model in which NP and its interactions with VP35 link the establishment of IBs to the synthesis of viral RNA.
Assuntos
Ebolavirus/metabolismo , Corpos de Inclusão Viral/metabolismo , Nucleoproteínas/fisiologia , Linhagem Celular , Ebolavirus/patogenicidade , Genoma Viral/genética , Células HEK293 , Doença pelo Vírus Ebola/virologia , Humanos , Corpos de Inclusão/metabolismo , Nucleocapsídeo/metabolismo , Nucleocapsídeo/fisiologia , Proteínas do Nucleocapsídeo/metabolismo , Proteínas do Nucleocapsídeo/fisiologia , Nucleoproteínas/metabolismo , RNA/biossíntese , RNA Viral/genética , Fatores de Transcrição/metabolismo , Proteínas Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Proteínas Virais Reguladoras e Acessórias/fisiologia , Vírion/metabolismo , Replicação Viral/fisiologiaRESUMO
PURPOSE: Thrombin inhibits cadherin on vascular endothelial cells, rapidly and reversibly increasing endothelial permeability. The purpose of this study was to evaluate the feasibility of trans-arterial infusion with thrombin. MATERIAL AND METHODS: Ten rabbits with right thigh tumor were randomly divided into two groups: A thrombin group and a control group. In the thrombin group, a suspension of thrombin (300 IU), cisplatin (3 mg), lipiodol (0.3 ml) and iopamidol (0.3 ml) was infused into the right femoral artery. In the control group, a suspension of cisplatin, lipiodol and iopamidol was infused. Platinum concentrations in plasma were measured five and ten minutes after administration. Platinum concentrations were also measured in tumor specimens excised 30 minutes after infusion. RESULTS: At both five and ten minutes after infusion, platinum concentrations in plasma were significantly lower for the thrombin group than for the control group. Platinum concentration in tumor tissue was significantly higher for the thrombin group than for the control group. CONCLUSION: The present results suggest that transarterial infusion with thrombin may offer a number of pharmacological advantages.
Assuntos
Antineoplásicos/administração & dosagem , Sistemas de Liberação de Medicamentos , Platina/farmacocinética , Trombina/farmacologia , Experimentação Animal , Animais , Cisplatino/administração & dosagem , Cisplatino/farmacocinética , Meios de Contraste/administração & dosagem , Óleo Etiodado/administração & dosagem , Óleo Etiodado/farmacocinética , Estudos de Viabilidade , Artéria Femoral , Infusões Intra-Arteriais , Iopamidol/administração & dosagem , Iopamidol/farmacocinética , Neoplasias Hepáticas Experimentais/patologia , Masculino , Coelhos , Trombina/administração & dosagem , Fatores de TempoRESUMO
Although viruses subvert innate immune pathways for their replication, there is evidence they can also co-opt anti-viral responses for their benefit. The ubiquitous human pathogen, Herpes Simplex Virus-1 (HSV-1), encodes a protein (UL12.5) that induces the release of mitochondrial nucleic acid into the cytosol, which activates immune sensing pathways and reduces productive replication in non-neuronal cells. HSV-1 establishes latency in neurons and can reactivate to cause disease. We found that UL12.5 is required for HSV-1 reactivation in neurons and acts to directly promote viral lytic gene expression during initial exit from latency. Further, the direct activation of innate immune sensing pathways triggered HSV reactivation and compensated for a lack of UL12.5. Finally, we found that the induction of HSV-1 lytic genes during reactivation required intact RNA and DNA sensing pathways, demonstrating that HSV-1 can both respond to and active antiviral nucleic acid sensing pathways to reactivate from a latent infection.
RESUMO
The quality of rice koji greatly affects the quality of sake. To accurately evaluate the quality of rice koji, various approaches for the evaluation of rice koji are required. In this study, we directly and simultaneously visualized the distribution of polypeptides in rice koji using mass spectrometry imaging. We demonstrated four koji-specific polypeptides at m/z 4660, 6140, 8170, and 11,840 and one rice-derived polypeptide at m/z 5330. To identify the koji-specific polypeptides, extracts from rice koji were separated using tricine SDS-PAGE, and the band appeared to coincide with the polypeptide at m/z 11,840 was identified to be the N-terminal fragment of α-amylase. The polypeptide seemed to have no hydrolytic activity based on the primary structure of α-amylase. The polypeptide at m/z 11,840 seemed to coincide with the fragmented α-amylase was detected at the later stage of koji making (after 42 h). At the same period during koji making, the increasing rate of α-amylase activity decreased compared to that of glucoamylase activity, suggesting that α-amylase fragmentation possibly leads to the deceleration of the increase in α-amylase activity at the later stage of koji making. This is the first study to directly and simultaneously demonstrate the distribution of polypeptides in rice koji using mass spectrometry imaging and imply the relationship between α-amylase fragmentation and activity in rice koji.
Assuntos
Aspergillus oryzae , Oryza , Aspergillus oryzae/química , Espectrometria de Massas , Oryza/química , Peptídeos , alfa-Amilases/químicaRESUMO
p190RhoGAP-A (p190) is a GTPase-activating protein known to regulate actin cytoskeleton dynamics by decreasing RhoGTP levels through activation of Rho intrinsic GTPase activity. We have previously shown that p190 protein levels are cell cycle-regulated, decreasing in mitosis, and that this decrease is mediated by the ubiquitin-proteasome pathway. In addition, overexpression of p190 results in decreased RhoGTP levels at the cleavage furrow during cytokinesis, p190 and the RhoGEF Ect2 play opposing roles in cytokinesis, and sustained levels of p190 in mitosis are associated with cytokinesis failure, all findings that suggest but do not directly demonstrate that completion of cytokinesis is dependent on reduced levels of p190. Here we report, using an RNAi reconstitution approach with a degradation-resistant mutant, that decreased p190 levels are required for successful cytokinesis. We also show that the multinucleation phenotype is dependent on p190 RhoGAP activity, determine that the N-terminal GBDS1 region is necessary and sufficient for p190 mitotic ubiquitination and degradation, and identify four N-terminal residues as necessary for the degradation of p190 in mitosis. Our data indicate that in addition to activation of RhoGEF(s), reduction of RhoGAP (p190) is a critical mechanism by which increased RhoGTP levels are achieved in late mitosis, thereby ensuring proper cell division.
Assuntos
Citocinese/fisiologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Mitose/fisiologia , Proteínas Repressoras/metabolismo , Actinas/genética , Actinas/metabolismo , Citoesqueleto/genética , Citoesqueleto/metabolismo , Ativação Enzimática/fisiologia , Fatores de Troca do Nucleotídeo Guanina/genética , Células HeLa , Humanos , Estrutura Terciária de Proteína , Proteínas Repressoras/genética , Fatores de Troca de Nucleotídeo Guanina Rho , Ubiquitinação/fisiologiaRESUMO
Co-overexpression of the epidermal growth factor (EGF) receptor (EGFR) and c-Src frequently occurs in human tumors and is linked to enhanced tumor growth. In experimental systems this synergistic growth requires EGF-dependent association of c-Src with the EGFR and phosphorylation of Tyr-845 of the receptor by c-Src. A search for signaling mediators of Tyr(P)-845 revealed that mitochondrial cytochrome c oxidase subunit II (CoxII) binds EGFR in a Tyr(P)-845- and EGF-dependent manner. In cells this association involves translocation of EGFR to the mitochondria, but regulation of this process is ill-defined. The current study demonstrates that c-Src translocates to the mitochondria with similar kinetics as EGFR and that the catalytic activity of EGFR and c-Src as well as endocytosis and a mitochondrial localization signal are required for these events. CoxII can be phosphorylated by EGFR and c-Src, and EGF stimulation reduces Cox activity and cellular ATP, an event that is dependent in large part on EGFR localized to the mitochondria. These findings suggest EGFR plays a novel role in modulating mitochondrial function via its association with, and modification of CoxII.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Mitocôndrias/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Proteína Tirosina Quinase CSK , Linhagem Celular Tumoral , Complexo IV da Cadeia de Transporte de Elétrons/genética , Fator de Crescimento Epidérmico/genética , Receptores ErbB/genética , Humanos , Camundongos , Mitocôndrias/genética , Fosforilação/fisiologia , Transporte Proteico/fisiologia , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Quinases da Família srcRESUMO
The present study demonstrates different functional domains of a recently described centrosomal protein, Cep57 (centrosomal protein 57). Endogenous Cep57 protein and ectopic expression of full-length protein or the N-terminal coiled-coil domain localize to the centrosome internal to gamma-tubulin, suggesting that it is either on both centrioles or on a centromatrix component. The N-terminus can also multimerize with the N-terminus of other Cep57 molecules. The C-terminus contains a second coiled-coil domain that directly binds to MTs (microtubules). This domain both nucleates and bundles MTs in vitro. This activity was also seen in vivo, as overexpression of full-length Cep57 or the C-terminus generates nocodazole-resistant MT cables in cells. Based on the present findings, we propose that Cep57 serves as a link with its N-terminus anchored to the centriole or centromatrix and its C-terminus to MTs.
Assuntos
Proteínas de Transporte/química , Centrossomo/química , Microtúbulos/química , Conformação Proteica , Estrutura Terciária de Proteína , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular , Centrossomo/metabolismo , Centrossomo/ultraestrutura , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Humanos , Camundongos , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Interferência de RNA , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
5-Fluoro-2'-deoxyuridine (FUdR), a potent anticancer agent, exerts its effects by inhibiting thymidylate synthase, an essential machinery for DNA synthesis in cell proliferation. Also, cell death is caused by FUdR, primarily due to an imbalance in the nucleotide pool resulting from this enzyme inhibition. We have investigated the cancer cell death induced by FUdR, focusing on its molecular mechanisms. Using mouse mammary tumor FM3A cell lines, the original clone F28-7 and its variant F28-7-A cells, we previously reported an interesting observation that FUdR induces a necrotic morphology in F28-7, but induces, in contrast, an apoptotic morphology in F28-7-A cells. In the present study, to understand the molecular mechanisms underlying these differential cell deaths, i.e., necrosis and apoptosis, we investigated the gene expression changes occurring in these processes. Using the cDNA microarray technology, we found 215 genes being expressed differentially in the necrosis and apoptosis. Further analysis revealed differences between these cell lines in terms of the expressions of both a cluster of heat shock protein (HSP)-related genes and a cluster of apoptosis-related genes. Notably, inhibition of HSP90 in F28-7 cells caused a shift from the FUdR-induced necrosis into apoptosis. These findings are expected to lead to a better understanding of this anticancer drug FUdR for its molecular mechanisms and also of the general biological issue, necrosis and apoptosis.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Apoptose/genética , Floxuridina/farmacologia , Perfilação da Expressão Gênica , Necrose/induzido quimicamente , Necrose/genética , Animais , Linhagem Celular Tumoral , Citocromos c/metabolismo , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Necrose/metabolismo , Análise de Sequência com Séries de OligonucleotídeosRESUMO
The vast majority of platforms for the detection of viral or bacterial antigens rely on immunoassays, typically ELISA or sandwich ELISA, that are contingent on the availability of suitable monoclonal antibodies (mAbs). This is a major bottleneck, since the generation and production of mAbs is time-consuming and expensive. Synthetic antibody fragments (sFabs) generated by phage-display selection offer an alternative with many advantages over Fabs obtained from natural antibodies using hybridoma technology. Unlike mAbs, sFabs are generated using phage display, allowing selection for binding to specific strains or for pan-specificity, for identification of structural epitopes or unique protein conformations and even for complexes. Further, they can easily be produced in Escherichia coli in large quantities and engineered for purposes of detection technologies and other applications. Here, the use of phage-display selection to generate a pan-specific Fab (MJ20), based on a Herceptin Fab scaffold, with the ability to bind selectively and with high affinity to the C-terminal domains of the nucleoproteins (NPs) from all five known strains of the Ebola virus is reported. The high-resolution crystal structure of the complex of MJ20 with the antigen from the Bundibugyo strain of the Ebola virus reveals the basis for pan-specificity and illustrates how the phage-display technology can be used to manufacture suitable Fabs for use in diagnostic or therapeutic applications.
Assuntos
Complexo Antígeno-Anticorpo/química , Ebolavirus/química , Fragmentos Fab das Imunoglobulinas/química , Nucleoproteínas/química , Técnicas de Visualização da Superfície Celular , Cristalografia por Raios X , Humanos , Fragmentos de Imunoglobulinas/química , Biblioteca de Peptídeos , Ligação Proteica , Domínios ProteicosRESUMO
Autonomously replicating sequence-binding factor 1 (ABF1) is a multifunctional, site-specific DNA binding protein that is essential for cell viability in Saccharomyces cerevisiae. ABF1 plays a direct role in transcriptional activation, stimulation of DNA replication, and gene silencing at the mating-type loci. Here we demonstrate that all three activities of ABF1 are conferred by the C terminus of the protein (amino acids [aa] 604 to 731). Furthermore, a detailed mutational analysis has revealed two important clusters of amino acid residues in the C terminus (C-terminal sequence 1 [CS1], aa 624 to 628; and CS2, aa 639 to 662). While both regions play a pivotal role in supporting cell viability, they make distinct contributions to ABF1 functions in various nuclear processes. CS1 specifically participates in transcriptional silencing and/or repression in a context-dependent manner, whereas CS2 is universally required for all three functions of ABF1. When tethered to specific regions of the genome, a 30-aa fragment that contains CS2 alone is sufficient for activation of transcription and chromosomal replication. In addition, CS2 is responsible for ABF1-mediated chromatin remodeling. Based on these results, we suggest that ABF1 may function as a chromatin-reorganizing factor to increase accessibility of the local chromatin structure, which in turn facilitates the action of additional factors to establish either an active or repressed chromatin state.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Cromatina/genética , Cromossomos Fúngicos/genética , Replicação do DNA , Proteínas de Ligação a DNA/química , Inativação Gênica , Dados de Sequência Molecular , Plasmídeos/genética , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/citologia , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/química , Ativação TranscricionalRESUMO
Autonomously replicating sequence binding factor 1 (ABF1) and repressor/activator protein 1 (RAP1) from budding yeast are multifunctional, site-specific DNA-binding proteins, with roles in gene activation and repression, replication, and telomere structure and function. Previously we have shown that RAP1 can prevent nucleosome positioning in the vicinity of its binding site and have provided evidence that this ability to create a local region of "open" chromatin contributes to RAP1 function at the HIS4 promoter by facilitating binding and activation by GCN4. Here we examine and directly compare to that of RAP1 the ability of ABF1 to create a region of open chromatin near its binding site and to contribute to activated transcription at the HIS4, ADE5,7, and HIS7 promoters. ABF1 behaves similarly to RAP1 in these assays, but it shows some subtle differences from RAP1 in the character of the open chromatin region near its binding site. Furthermore, although the two factors can similarly enhance activated transcription at the promoters tested, RAP1 binding is continuously required for this enhancement, but ABF1 binding is not. These results indicate that ABF1 and RAP1 achieve functional similarity in part via mechanistically distinct pathways.
Assuntos
Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional , Oxirredutases do Álcool , Aminoidrolases/genética , Aminoidrolases/metabolismo , Sítios de Ligação , Proteínas de Ligação a DNA/genética , Ativação Enzimática , Nucleossomos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Pirofosfatases , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Complexo Shelterina , Proteínas de Ligação a Telômeros/genética , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genéticaRESUMO
Detoxification is essential for the fungal growth in the drug stress environments, and the multidrug transporters play an important role in this process. Here a cerulenin transporter gene (MpMdt, AB206476) was identified from Monascus pilosus. MpMdt mRNA contains 1951 bp and encodes a protein of 559 amino acid residues with 11 trans-membrane domains; and there is no difference in the sequence of MpMdt mRNA between the wild type M. pilosus IFO4520 and its cerulenin resistant mutant MK-1. Up-expression of MpMdt renders the cerulenin resistance of the mutant MK-1. Over-expression of MpMdt could also increase the cerulenin tolerance in the transgenic M. pilosus IFO4520. These results suggested that MpMdt is able to efflux-transport the anti-fungal antibiotic cerulenin and increase the cerulenin resistance of M. pilosus.
Assuntos
Proteínas de Transporte/genética , Cerulenina/metabolismo , Resistência a Medicamentos/genética , Monascus/genética , Sequência de Bases , Proteínas de Transporte/metabolismo , Primers do DNA , Etiquetas de Sequências ExpressasRESUMO
Epidural spinal myelolipoma was diagnosed in a 13-year-old, male Siberian husky that was referred for evaluation of progressive pelvic limb paresis and urinary incontinence. An epidural mass was detected by magnetic resonance imaging and computed tomography. The mass was removed and identified histopathologically as an epidural myelolipoma. Pelvic limb paresis improved after surgery, but urinary retention associated with neurological bladder dysfunction persisted.
Assuntos
Neoplasias das Glândulas Suprarrenais/veterinária , Doenças do Cão/cirurgia , Neoplasias Epidurais/veterinária , Mielolipoma/veterinária , Paresia/veterinária , Neoplasias das Glândulas Suprarrenais/complicações , Neoplasias das Glândulas Suprarrenais/diagnóstico , Neoplasias das Glândulas Suprarrenais/cirurgia , Animais , Doenças do Cão/diagnóstico , Doenças do Cão/etiologia , Doenças do Cão/patologia , Cães , Neoplasias Epidurais/complicações , Neoplasias Epidurais/diagnóstico , Neoplasias Epidurais/cirurgia , Laminectomia/veterinária , Imageamento por Ressonância Magnética/veterinária , Masculino , Mielolipoma/complicações , Mielolipoma/diagnóstico , Mielolipoma/cirurgia , Paresia/etiologia , Tomografia Computadorizada por Raios X/veterinária , Resultado do Tratamento , Bexiga Urinaria Neurogênica/etiologia , Bexiga Urinaria Neurogênica/veterinária , Incontinência Urinária/etiologia , Incontinência Urinária/veterinária , Retenção Urinária/etiologia , Retenção Urinária/veterináriaRESUMO
The construction of an experimental system that can mimic koji making in the manufacturing setting of a sake brewery is initially required for the quantitative evaluation of mycelia grown on/in koji pellets (haze formation). Koji making with rice was investigated with a solid-state fermentation (SSF) system using a non-airflow box (NAB), which produced uniform conditions in the culture substrate with high reproducibility and allowed for the control of favorable conditions in the substrate during culture. The SSF system using NAB accurately reproduced koji making in a manufacturing setting. To evaluate haze formation during koji making, surfaces and cross sections of koji pellets obtained from koji making tests were observed using a digital microscope. Image analysis was used to distinguish between haze and non-haze sections of koji pellets, enabling the evaluation of haze formation in a batch by measuring the haze rate of a specific number of koji pellets. This method allowed us to obtain continuous and quantitative data on the time course of haze formation. Moreover, drying koji during the late stage of koji making was revealed to cause further penetration of mycelia into koji pellets (internal haze). The koji making test with the SSF system using NAB and quantitative evaluation of haze formation in a batch by image analysis is a useful method for understanding the relations between haze formation and koji making conditions.
Assuntos
Dessecação , Fermentação , Oryza/microbiologia , Bebidas Alcoólicas/microbiologia , Aspergillus oryzae/metabolismo , Manipulação de Alimentos , Micélio/metabolismo , Reprodutibilidade dos TestesRESUMO
The general transcription factor TFIID, composed of the TATA box-binding protein (TBP) and 14 TBP-associated factors (TAFs), is important for both basal and regulated transcription by RNA polymerase II. Although it is well known that the TAF N-terminal domain (TAND) at the amino-terminus of the TAF1 protein binds to TBP and thereby inhibits TBP function in vitro, the physiological role of this domain remains obscure. In our previous study, we screened for mutations that cause lethality when co-expressed with the TAF1 gene lacking TAND (taf1-DeltaTAND) and identified two DeltaTAND synthetic lethal (nsl) mutations as those in the SPT15 gene encoding TBP. In this study we isolated another nsl mutation in the same screen and identified it to be a mutation in the histone fold domain (HFD) of the TAF12 gene. Several other HFD mutations of this gene also exhibit nsl phenotypes, and all of them are more or less impaired in transcriptional activation in vivo. Interestingly, a set of genes affected in the taf1-DeltaTAND mutant is similarly affected in the taf12 HFD mutants but not in the nsl mutants of TBP. Therefore, we discovered that the nsl mutations of these two genes cause lethality in the taf1-DeltaTAND mutant by different mechanisms.
Assuntos
Proteínas de Ciclo Celular/genética , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Fatores Associados à Proteína de Ligação a TATA/genética , Proteína de Ligação a TATA-Box/genética , Fator de Transcrição TFIID/genética , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Bases , Sítios de Ligação/genética , Divisão Celular/genética , Genes Letais/genética , Histonas/química , Mutação , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Ligação Proteica , Dobramento de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Proteína de Ligação a TATA-Box/análogos & derivados , Transativadores/genética , Transativadores/metabolismoRESUMO
Nasunin, delphinidin-3-(p-coumaroylrutinoside)-5-glucoside, an antioxidant anthocyanin isolated from eggplant peels, was demonstrated as an angiogenesis inhibitor. Nasunin at higher 10 microM suppressed microvessel outgrowth in an ex vivo angiogenesis assay using a rat aortic ring. The effect of nasunin was examined in various in vitro angiogenesis models using human umbilical vein endothelial cells (HUVECs). Nasunin suppressed HUVEC proliferation in a dose-dependent manner (50-200 microM); however, it had no significant effect on HUVEC chemotaxis in a Boyden chamber assay and HUVEC tube formation on a reconstituted basement membrane. These results imply that nasunin with both antioxidant and antiangiogenic activities might be useful to prevent angiogenesis-related diseases.
Assuntos
Inibidores da Angiogênese/farmacologia , Antocianinas/farmacologia , Solanum melongena/química , Animais , Antocianinas/isolamento & purificação , Antioxidantes , Aorta/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Humanos , Masculino , Neovascularização Fisiológica/efeitos dos fármacos , Ratos , Ratos Wistar , Veias Umbilicais/efeitos dos fármacosRESUMO
The influence of drying the culture substrate during solid-state fermentation on enzyme production was investigated using a non-airflow box. The drying caused a significant increase in enzyme production, while the mycelium content decreased slightly. This suggests that changes in the water content in the substrate during culture affect enzyme production in fungi.
Assuntos
Aspergillus oryzae/enzimologia , Dessecação , Enzimas/biossíntese , Fermentação , Enzimas/análise , Enzimas/metabolismo , Micélio/química , Micélio/metabolismo , Água/análise , Água/metabolismoRESUMO
Solid-state fermentation (SSF) has become an attractive alternative to submerged fermentation (SMF) for the production of enzymes, organic acids, and secondary metabolites, while there are many problems during the culture of SSF. We recently created a SSF system using a non-airflow box (NAB) in order to resolve the problems, which enabled the uniform culture in the whole substrate and high yield of many enzymes. In this paper, further characterization of SSF using the NAB was carried out to obtain other advantages. The NAB culture under the fixed environmental condition exhibited a rapid increase in enzyme production at earlier phase during the culture compared with conventional SSF. Total mycelial growth also exhibited the same trend as enzyme production. Thus, the increase in the rate of the enzyme production was thought to mainly be attributed to that of the growth. To support it, it was suggested that the NAB culture resulted in most optimal water activity for the growth just at the log phase. In addition, the NAB culture was able to achieve high reproducibility of enzyme production, derived from uniform condition of the substrate during the culture. The results indicate that the NAB culture has many benefits for SSF.
Assuntos
Reatores Biológicos/microbiologia , Enzimas/biossíntese , Fermentação , Micélio/crescimento & desenvolvimento , Micélio/metabolismo , Movimentos do Ar , Aspergillus oryzae/crescimento & desenvolvimento , Aspergillus oryzae/isolamento & purificação , Aspergillus oryzae/metabolismo , Umidade , Imersão , Micélio/isolamento & purificação , Reprodutibilidade dos Testes , Temperatura , Fatores de Tempo , Água/metabolismoRESUMO
Solid-state fermentation (SSF) has attracted a lot of interest for carrying out high-level protein production in filamentous fungi. However, it has problems such as the fermentation heat generated during the culture in addition to the reduced mobility of substances. These conditions lead to a nonuniform state in the culture substrate and result in low reproducibility. We constructed a non-airflow box (NAB) with a moisture permeable fluoropolymer membrane, thereby making it possible to control and maintain uniform and optimal conditions in the substrate. For the NAB culture in Aspergillus oryzae, temperature and water content on/in the whole substrate were more consistent than for a traditional tray box (TB) culture. Total weight after the culture remained constant and dry conditions could be achieved during the culture. These data demonstrate the possibility of growing a uniform culture of the whole substrate for SSF. The NAB is advantageous because it allows for the control of exact temperature and water content in the substrate during the culture by allowing vapor with latent heat to dissipate out of the box. In addition, several enzymes in the NAB culture exhibited higher production levels than in the TB culture. We believe that culturing in the constructed NAB could become a standard technique for commercial SSF.
Assuntos
Aspergillus oryzae/enzimologia , Reatores Biológicos/microbiologia , Enzimas/biossíntese , Fermentação , Microbiologia Industrial/métodos , Proteínas Fúngicas/biossíntese , Temperatura Alta , Concentração de Íons de Hidrogênio , Microbiologia Industrial/instrumentação , Temperatura , Água/metabolismoRESUMO
Two alternatively spliced mRNAs (d- and l-MpLaeA) of a methyltransferase gene (MpLaeA) were identified from Monascus pilosus IFO4520 and its mutant MK-1. Alternative splicing of the MpLaeA pre-mRNA occurred in the 5'-untranslated region (5'-UTR). The alternative splicing patterns of MpLaeA were regulated by the fungal growth stage and the principal nutrients: that is, the short l-MpLaeA mRNA was a constitutive transcript at all growth stages and different carbon or nitrogen sources, but the glutamate and NaNO(3) as main nitrogen source could up-regulate the long d-MpLaeA mRNA form. The long spliced 5'-UTR of d-MpLaeA blocked GFP expression in Escherichia coli , suggesting that d-MpLaeA mRNA was an ineffective spliced mRNA. Down-regulation of MpLaeA by transgenic antisense d-MpLaeA cDNA resulted in decreasing synthesis of monacolin K in M. pilosus. This suggested that the alternative splicing of MpLaeA mRNA might regulate the synthesis of monacolin K.