RESUMO
Bacteriophage T7 single-stranded DNA-binding protein (gp2.5) binds to and protects transiently exposed regions of single-stranded DNA (ssDNA) while dynamically interacting with other proteins of the replication complex. We directly visualize fluorescently labelled T7 gp2.5 binding to ssDNA at the single-molecule level. Upon binding, T7 gp2.5 reduces the contour length of ssDNA by stacking nucleotides in a force-dependent manner, suggesting T7 gp2.5 suppresses the formation of secondary structure. Next, we investigate the binding dynamics of T7 gp2.5 and a deletion mutant lacking 21 C-terminal residues (gp2.5-Δ21C) under various template tensions. Our results show that the base sequence of the DNA molecule, ssDNA conformation induced by template tension, and the acidic terminal domain from T7 gp2.5 significantly impact on the DNA binding parameters of T7 gp2.5. Moreover, we uncover a unique template-catalyzed recycling behaviour of T7 gp2.5, resulting in an apparent cooperative binding to ssDNA, facilitating efficient spatial redistribution of T7 gp2.5 during the synthesis of successive Okazaki fragments. Overall, our findings reveal an efficient binding mechanism that prevents the formation of secondary structures by enabling T7 gp2.5 to rapidly rebind to nearby exposed ssDNA regions, during lagging strand DNA synthesis.
Assuntos
Bacteriófago T7 , Proteínas Virais , Bacteriófago T7/genética , DNA/metabolismo , Replicação do DNA , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Conformação Molecular , Proteínas Virais/metabolismoRESUMO
Anti-cluster of differentiation (CD) 3 × α programmed death-ligand 1 (PD-L1) bispecific T-cell engager (BsTE)-bound T-cells (BsTE:T) are a promising new cancer treatment agent. However, the mechanisms of action of bispecific antibody-armed activated T-cells are poorly understood. Therefore, this study aimed to investigate the anti-tumor mechanism and efficacy of BsTE:T. The BsTE:T migration was assessed in vivo and in vitro using syngeneic and xenogeneic tumor models, flow cytometry, immunofluorescence staining, transwell migration assays, microfluidic chips, Exo View R100, western blotting, and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 technology. In murine B16 melanoma, MC38 colon cancer, and human multiple myeloma cells, BsTE:T exhibited superior tumor elimination relative to that of T-cells or BsTE alone. Moreover, BsTE:T migration into tumors was significantly enhanced owing to the presence of PD-L1 in tumor cells and secretion of PD-L1-containing exosomes. Furthermore, increased infiltration of CD44highCD62Llow effector memory CD8+ T-cells into tumors was closely associated with the anti-tumor effect of BsTE:T. Therefore, BsTE:T is an innovative potential anti-tumor therapy, and exosomal PD-L1 plays a crucial role both in vitro and in vivo in the anti-tumor activity of BsTE:T.
Assuntos
Anticorpos Biespecíficos , Antígeno B7-H1 , Exossomos , Animais , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/imunologia , Exossomos/metabolismo , Exossomos/imunologia , Camundongos , Antígeno B7-H1/metabolismo , Antígeno B7-H1/antagonistas & inibidores , Humanos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Camundongos Endogâmicos C57BL , Melanoma Experimental/imunologia , Melanoma Experimental/terapia , Complexo CD3/imunologia , Complexo CD3/metabolismo , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Linhagem Celular Tumoral , Feminino , Movimento Celular , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The B cell response to Salmonella typhimurium (STm) occurs massively at extrafollicular sites, without notable germinal centers (GCs). Little is known in terms of its specificity. To expand the knowledge of antigen targets, we screened plasmablast (PB)-derived monoclonal antibodies (mAbs) for Salmonella specificity, using ELISA, flow cytometry, and antigen microarray. Only a small fraction (0.5%-2%) of the response appeared to be Salmonella-specific. Yet, infection of mice with limited B cell receptor (BCR) repertoires impaired the response, suggesting that BCR specificity was important. We showed, using laser microdissection, that somatic hypermutation (SHM) occurred efficiently at extrafollicular sites leading to affinity maturation that in turn led to detectable STm Ag-binding. These results suggest a revised vision of how clonal selection and affinity maturation operate in response to Salmonella. Clonal selection initially is promiscuous, activating cells with virtually undetectable affinity, yet SHM and selection occur during the extrafollicular response yielding higher affinity, detectable antibodies.
Assuntos
Linfócitos B/imunologia , Seleção Clonal Mediada por Antígeno/imunologia , Centro Germinativo/imunologia , Salmonella typhimurium/imunologia , Hipermutação Somática de Imunoglobulina/imunologia , Animais , Anticorpos Monoclonais/imunologia , Seleção Clonal Mediada por Antígeno/genética , Imunoglobulina G/biossíntese , Imunoglobulina G/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/imunologia , Infecções por Salmonella/imunologia , Infecções por Salmonella/microbiologia , Hipermutação Somática de Imunoglobulina/genética , Baço/citologia , Baço/imunologiaRESUMO
The T7 primase-helicase plays a pivotal role in the replication of T7 DNA. Using affinity isolation of peptide-nucleic acid crosslinks and mass spectrometry, we identify protein regions in the primase-helicase and T7 DNA polymerase that form contacts with the RNA primer and DNA template. The contacts between nucleic acids and the primase domain of the primase-helicase are centered in the RNA polymerase subdomain of the primase domain, in a cleft between the N-terminal subdomain and the topoisomerase-primase fold. We demonstrate that residues along a beta sheet in the N-terminal subdomain that contacts the RNA primer are essential for phage growth and primase activity in vitro. Surprisingly, we found mutations in the primase domain that had a dramatic effect on the helicase. Substitution of a residue conserved in other DnaG-like enzymes, R84A, abrogates both primase and helicase enzymatic activities of the T7 primase-helicase. Alterations in this residue also decrease binding of the primase-helicase to ssDNA. However, mass photometry measurements show that these mutations do not interfere with the ability of the protein to form the active hexamer.
Assuntos
Bacteriófago T7 , DNA Helicases , DNA Primase , DNA , Proteínas Virais , Sequência de Aminoácidos , Bacteriófago T7/enzimologia , DNA/metabolismo , DNA Helicases/química , DNA Helicases/metabolismo , DNA Primase/química , DNA Primase/genética , DNA Primase/metabolismo , Mutação , Proteínas Virais/química , Proteínas Virais/metabolismoRESUMO
T cell effector functions can be elicited by noncognate stimuli, but the mechanism and contribution of this pathway to the resolution of intracellular macrophage infections have not been defined. Here, we show that CD4(+) T helper 1 (Th1) cells could be rapidly stimulated by microbe-associated molecular patterns during active infection with Salmonella or Chlamydia. Further, maximal stimulation of Th1 cells by lipopolysaccharide (LPS) did not require T-cell-intrinsic expression of toll-like receptor 4 (TLR4), interleukin-1 receptor (IL-1R), or interferon-γ receptor (IFN-γR) but instead required IL-18R, IL-33R, and adaptor protein MyD88. Innate stimulation of Th1 cells also required host expression of TLR4 and inflammasome components that together increased serum concentrations of IL-18. Finally, the elimination of noncognate Th1 cell stimulation hindered the resolution of primary Salmonella infection. Thus, the in vivo bactericidal capacity of Th1 cells is regulated by the response to noncognate stimuli elicited by multiple innate immune receptors.
Assuntos
Imunidade Inata/imunologia , Inflamassomos/metabolismo , Transdução de Sinais , Células Th1/imunologia , Receptores Toll-Like/metabolismo , Animais , Carga Bacteriana/imunologia , Antígenos CD4/imunologia , Chlamydia/fisiologia , Citometria de Fluxo , Interleucina-18/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase em Tempo Real , Salmonella/fisiologia , Receptor 4 Toll-Like/metabolismoRESUMO
The vast majority of intracellular protein targets are refractory toward small-molecule therapeutic engagement, and additional therapeutic modalities are needed to overcome this deficiency. Here, the identification and characterization of a natural product, WDB002, reveals a therapeutic modality that dramatically expands the currently accepted limits of druggability. WDB002, in complex with the FK506-binding protein (FKBP12), potently and selectively binds the human centrosomal protein 250 (CEP250), resulting in disruption of CEP250 function in cells. The recognition mode is unprecedented in that the targeted domain of CEP250 is a coiled coil and is topologically featureless, embodying both a structural motif and surface topology previously considered on the extreme limits of "undruggability" for an intracellular target. Structural studies reveal extensive protein-WDB002 and protein-protein contacts, with the latter being distinct from those seen in FKBP12 ternary complexes formed by FK506 and rapamycin. Outward-facing structural changes in a bound small molecule can thus reprogram FKBP12 to engage diverse, otherwise "undruggable" targets. The flat-targeting modality demonstrated here has the potential to expand the druggable target range of small-molecule therapeutics. As CEP250 was recently found to be an interaction partner with the Nsp13 protein of the SARS-CoV-2 virus that causes COVID-19 disease, it is possible that WDB002 or an analog may exert useful antiviral activity through its ability to form high-affinity ternary complexes containing CEP250 and FKBP12.
Assuntos
Actinobacteria/genética , Antivirais/farmacologia , Genoma Bacteriano , Macrolídeos/farmacologia , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Proteína 1A de Ligação a Tacrolimo/química , Proteína 1A de Ligação a Tacrolimo/metabolismo , Actinobacteria/metabolismo , Sequência de Aminoácidos , Antivirais/química , Antivirais/metabolismo , Autoantígenos/genética , Autoantígenos/metabolismo , Calcineurina/genética , Calcineurina/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Evolução Molecular , Células HEK293 , Humanos , Macrolídeos/química , Macrolídeos/metabolismo , Modelos Moleculares , Conformação Proteica , Homologia de Sequência , Sirolimo/química , Sirolimo/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
The volatile compounds and sensory profiles of 18 different types of distilled soju, chosen with regard to various raw materials and distillation methods (atmospheric vs. vacuum), were explored using headspace solid-phase microextraction (HS-SPME) with gas chromatography-mass spectrometry (GC-MS) and descriptive analysis. General chemical properties such as pH, total acidity (TA), total soluble solids (°Brix), and lactic acid concentration were also determined. A total of 56 volatile compounds, comprising 31 esters, 11 alcohols, 1 acid, 4 aldehydes, 3 ketones, and 6 miscellaneous compounds, were identified. From the principal component analysis (PCA) of the volatile data, samples made using atmospheric distillation such as MSO and PJU showed a clear difference from decompressed distillation samples. Based on the PCA of the sensory data, there was also a clear distinction between samples by their distillation method. To explore relationships among chemical, volatile, and sensory data sets, multiple factor analysis (MFA) was applied. Yeasty and earthy flavors showed a close relationship with 1-nonanol, octatonic acid, and longer-chain esters such as ethyl phenylacetate and ethyl tetradecanoate, and with chemical parameters such as TA, °Brix, and lactic acid.
Assuntos
Microextração em Fase Sólida , Compostos Orgânicos Voláteis , Ésteres/análise , Cromatografia Gasosa-Espectrometria de Massas/métodos , Ácido Láctico/análise , República da Coreia , Microextração em Fase Sólida/métodos , Compostos Orgânicos Voláteis/análiseRESUMO
Bacteriophage T7 encodes its own DNA polymerase, the product of gene 5 (gp5). In isolation, gp5 is a DNA polymerase of low processivity. However, gp5 becomes highly processive upon formation of a complex with Escherichia coli thioredoxin, the product of the trxA gene. Expression of a gp5 variant in which aspartate residues in the metal-binding site of the polymerase domain were replaced by alanine is highly toxic to E. coli cells. This toxicity depends on the presence of a functional E. coli trxA allele and T7 RNA polymerase-driven expression but is independent of the exonuclease activity of gp5. In vitro, the purified gp5 variant is devoid of any detectable polymerase activity and inhibited DNA synthesis by the replisomes of E. coli and T7 in the presence of thioredoxin by forming a stable complex with DNA that prevents replication. On the other hand, the highly homologous Klenow fragment of DNA polymerase I containing an engineered gp5 thioredoxin-binding domain did not exhibit toxicity. We conclude that gp5 alleles encoding inactive polymerases, in combination with thioredoxin, could be useful as a shutoff mechanism in the design of a bacterial cell-growth system.
Assuntos
Bacteriófago T7 , Replicação do DNA , DNA Viral , DNA Polimerase Dirigida por DNA , Proteínas de Escherichia coli , Escherichia coli , Tiorredoxinas , Bacteriófago T7/enzimologia , Bacteriófago T7/genética , DNA Viral/biossíntese , DNA Viral/química , DNA Viral/genética , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/virologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Domínios Proteicos , Tiorredoxinas/química , Tiorredoxinas/genética , Tiorredoxinas/metabolismoRESUMO
PURPOSE: To evaluate the effect of the location of the femoral tunnel on 3-dimensional (3D) computed tomography (CT) upon the postoperative tunnel widening after anterior cruciate ligament (ACL) reconstructions. METHODS: Inclusion criteria were patients who underwent hamstring ACL reconstructions using an adjustable-loop cortical suspension device, underwent 3D CT at the day after surgery, and were followed for a minimum of 2 years after surgery. Exclusion criteria were patients with combined ligament injury and reinjury after reconstruction. Using 3D CT, the center of the femoral tunnel aperture was located on a standardized grid system. The center of the ACL footprint was defined from the literature. The femoral tunnel location was classified as anatomic if it located within 2 standard deviations of the center position. If it was outside the 2 standard deviations, the tunnel was classified as nonanatomic. The patients were divided into either anatomic or nonanatomic groups. Femoral tunnel angles on both sagittal and coronal planes were measured. Both femoral and tibial tunnels measured on anteroposterior and lateral radiographs at immediate postoperative day and at 2 years after surgery. Postoperative knee stability and patient-reported outcomes were evaluated. RESULTS: There were 37 patients in anatomical group and 52 patients in nonanatomical group among enrolled 87 patients. There were no differences in demographics between the 2 groups. There were no differences in the femoral tunnel angles and postoperative tunnel widening between the 2 groups. A higher position correlated to the femoral tunnel widening at 2 years postoperatively. Postoperative knee stability and patient-reported outcomes showed no statistically significant differences between the 2 groups. CONCLUSIONS: There was no significant difference in postoperative tunnel widening or clinical outcomes between anatomic and nonanatomic femoral tunnel location after hamstring ACL reconstructions. A higher position correlated to the femoral tunnel widening at 2 years postoperatively. LEVEL OF EVIDENCE: Level III, Retrospective comparative study.
Assuntos
Reconstrução do Ligamento Cruzado Anterior/métodos , Ligamento Cruzado Anterior/cirurgia , Fêmur/cirurgia , Tíbia/cirurgia , Adolescente , Adulto , Ligamento Cruzado Anterior/anatomia & histologia , Lesões do Ligamento Cruzado Anterior/cirurgia , Feminino , Fêmur/anatomia & histologia , Músculos Isquiossurais/anatomia & histologia , Músculos Isquiossurais/cirurgia , Humanos , Processamento de Imagem Assistida por Computador/métodos , Imageamento Tridimensional , Articulação do Joelho/anatomia & histologia , Articulação do Joelho/cirurgia , Masculino , Medidas de Resultados Relatados pelo Paciente , Período Pós-Operatório , Radiografia , Estudos Retrospectivos , Tíbia/anatomia & histologia , Tomografia Computadorizada por Raios X , Adulto JovemRESUMO
The Ras proteins are aberrantly activated in a wide range of human cancers, often endowing tumors with aggressive properties and resistance to therapy. Decades of effort to develop direct Ras inhibitors for clinical use have thus far failed, largely because of a lack of adequate small-molecule-binding pockets on the Ras surface. Here, we report the discovery of Ras-binding miniproteins from a naïve library and their evolution to afford versions with midpicomolar affinity to Ras. A series of biochemical experiments indicated that these miniproteins bind to the Ras effector domain as dimers, and high-resolution crystal structures revealed that these miniprotein dimers bind Ras in an unprecedented mode in which the Ras effector domain is remodeled to expose an extended pocket that connects two isolated pockets previously found to engage small-molecule ligands. We also report a Ras point mutant that stabilizes the protein in the open conformation trapped by these miniproteins. These findings provide new tools for studying Ras structure and function and present opportunities for the development of both miniprotein and small-molecule inhibitors that directly target the Ras proteins.
Assuntos
Proteínas/metabolismo , Proteínas/farmacologia , Proteínas ras/química , Proteínas ras/metabolismo , Sequência de Aminoácidos , Bases de Dados de Proteínas , Descoberta de Drogas , Modelos Moleculares , Mutação , Ligação Proteica , Domínios Proteicos/efeitos dos fármacos , Multimerização Proteica , Estrutura Quaternária de Proteína , Proteínas/química , Proteínas/genéticaRESUMO
The development of a subunit Salmonella vaccine has been hindered by the absence of detailed information about antigenic targets of protective Salmonella-specific T and B cells. Recent studies have identified SseB as a modestly protective Ag in susceptible C57BL/6 mice, but the mechanism of protective immunity remains undefined. In this article, we report that simply combining Salmonella SseB with flagellin substantially enhances protective immunity, allowing immunized C57BL/6 mice to survive for up to 30 d following challenge with virulent bacteria. Surprisingly, the enhancing effect of flagellin did not require flagellin Ag targeting during secondary responses or recognition of flagellin by TLR5. Although coimmunization with flagellin did not affect SseB-specific Ab responses, it modestly boosted CD4 responses. In addition, protective immunity was effectively transferred in circulation to parabionts of immunized mice, demonstrating that tissue-resident memory is not required for vaccine-induced protection. Finally, protective immunity required host expression of IFN-γR but was independent of induced NO synthase expression. Taken together, these data indicate that Salmonella flagellin has unique adjuvant properties that improve SseB-mediated protective immunity provided by circulating memory.
Assuntos
Proteínas de Bactérias/imunologia , Flagelina/imunologia , Memória Imunológica , Chaperonas Moleculares/imunologia , Salmonelose Animal/prevenção & controle , Vacinas contra Salmonella/imunologia , Adjuvantes Imunológicos , Animais , Anticorpos Antibacterianos/sangue , Linfócitos T CD4-Positivos/imunologia , Feminino , Imunização , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , Receptores de Interferon/genética , Receptores de Interferon/imunologia , Vacinas contra Salmonella/administração & dosagem , Salmonella typhimurium/imunologia , Receptor 5 Toll-Like/imunologia , Receptor de Interferon gamaRESUMO
DNA replication occurs semidiscontinuously due to the antiparallel DNA strands and polarity of enzymatic DNA synthesis. Although the leading strand is synthesized continuously, the lagging strand is synthesized in small segments designated Okazaki fragments. Lagging-strand synthesis is a complex event requiring repeated cycles of RNA primer synthesis, transfer to the lagging-strand polymerase, and extension effected by cooperation between DNA primase and the lagging-strand polymerase. We examined events controlling Okazaki fragment initiation using the bacteriophage T7 replication system. Primer utilization by T7 DNA polymerase is slower than primer formation. Slow primer release from DNA primase allows the polymerase to engage the complex and is followed by a slow primer handoff step. The T7 single-stranded DNA binding protein increases primer formation and extension efficiency but promotes limited rounds of primer extension. We present a model describing Okazaki fragment initiation, the regulation of fragment length, and their implications for coordinated leading- and lagging-strand DNA synthesis.
Assuntos
Bacteriófago T7/fisiologia , Replicação do DNA/fisiologia , DNA Viral/biossíntese , DNA Polimerase Dirigida por DNA/metabolismo , Escherichia coli/virologia , Modelos Biológicos , DNA/genética , DNA/metabolismo , DNA Viral/genética , DNA Polimerase Dirigida por DNA/genética , Escherichia coli/genética , Escherichia coli/metabolismoRESUMO
Bacteriophage T7 uses the thioredoxin of its host, Escherichia coli, to enhance the processivity of its DNA polymerase, a requirement for the growth of phage T7. The evolutionarily conserved structure and high degree of homology of amino acid sequence of the thioredoxin family imply that homologues from other organisms might also interact with T7 DNA polymerase to support the phage growth. Despite the structural resemblance, human thioredoxin, whose X-ray crystallographic structure overlaps with that of the E. coli protein, cannot support T7 phage growth. It does not form a complex with T7 DNA polymerase as determined by surface plasmon resonance and thus does not increase the processivity. Homologous scanning analysis using this nonfunctional homologue reveals that the 60 N-terminal and the 12 C-terminal amino acid residues of E. coli thioredoxin can be substituted for its human counterpart without significantly affecting phage growth. Comparison of chimeric thioredoxins, followed by site-directed mutagenesis, identifies leucine 95 as a critical element. This residue may contribute to hydrophobic interactions with the thioredoxin-binding loop of the polymerase; levels of DNA binding and thus nucleotide polymerization are significantly decreased in the absence of this residue. The results suggest that the specific interactions at the interface of thioredoxin and DNA polymerase, rather than the overall structure, are important in the interactions that promote high processivity.
Assuntos
Bacteriófago T7/enzimologia , DNA Polimerase Dirigida por DNA/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Tiorredoxinas/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Estrutura Secundária de Proteína , Ressonância de Plasmônio de SuperfícieRESUMO
The formation of a replication loop on the lagging strand facilitates coordinated synthesis of the leading- and lagging-DNA strands and provides a mechanism for recycling of the lagging-strand DNA polymerase. As an Okazaki fragment is completed, the loop is released, and a new loop is formed as the synthesis of a new Okazaki fragment is initiated. Loop release requires the dissociation of the complex formed by the interactions among helicase, DNA polymerase, and DNA. The completion of the Okazaki fragment may result in either a nick or a single-stranded DNA region. In the replication system of bacteriophage T7, the dissociation of the polymerase from either DNA region is faster than that observed for the dissociation of the helicase from DNA polymerase, implying that the replication loop is released more likely through the dissociation of the lagging-strand DNA from polymerase, retaining the polymerase at replication fork. Both dissociation of DNA polymerase from DNA and that of helicase from a DNA polymerase · DNA complex are much faster at a nick DNA region than the release from a ssDNA region. These results suggest that the replication loop is released as a result of the nick formed when the lagging-strand DNA polymerase encounters the previously synthesized Okazaki fragment, releasing lagging-strand DNA and retaining DNA polymerase at the replication fork for the synthesis of next Okazaki fragment.
Assuntos
Bacteriófago T7/enzimologia , DNA Primase/metabolismo , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , DNA/metabolismo , Modelos Moleculares , Complexos Multienzimáticos/metabolismo , Bacteriófago T7/química , Bacteriófago T7/metabolismo , Sítios de Ligação , DNA/biossíntese , DNA/química , DNA Primase/química , DNA Primase/genética , DNA Bacteriano/química , DNA Bacteriano/metabolismo , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , DNA Viral/química , DNA Viral/metabolismo , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/genética , Escherichia coli/metabolismo , Escherichia coli/virologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Deleção de Genes , Cinética , Complexos Multienzimáticos/química , Conformação de Ácido Nucleico , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismoRESUMO
We investigated the effects of Sarcodon aspratus, Agaricus bisporus, and Lentinula edodes aqueous extracts on the tenderization of bovine longissimus dorsi muscle. Meat quality and muscle protein degradation were examined as well. Beef chunks were marinated in distilled water (control), 5% S. aspratus (SA), 5% A. bisporus (AB), or 5% L. edodes (LE) extracts. SA was shown to have a higher enzymatic activity (p < 0.001) and water-holding capacity than LE (p < 0.01). SA and AB extracts exhibited lower shear force values compared with the control (p < 0.05). SA, AB, and LE showed superior muscle proteolytic effects compared with the control. SA demonstrated the ability to degrade myosin heavy chains and actin, which was not observed after AB and LE extract treatments. This suggests that SA extract may affect tenderization. Taken together, our results show that aqueous extract of S. aspratus affects the tenderness of the bovine longissimus dorsi muscle.
Assuntos
Basidiomycota/química , Carne , Proteínas Musculares/química , Músculo Esquelético/química , Agaricus/química , Animais , Bovinos , Cor , Eletroforese em Gel de Poliacrilamida , Enzimas/química , Enzimas/metabolismo , Qualidade dos Alimentos , Calefação , Humanos , Concentração de Íons de Hidrogênio , Proteínas Musculares/análise , Proteínas Musculares/metabolismo , Cogumelos Shiitake/química , PaladarRESUMO
This study analyzed the dietary pattern of Southeast Asian workers (Vietnamese, Thais, Cambodians and Myanmar) living in South Korea in order to recognize the dietary changes after they moved to South Korea. Questionnaires were completed by 251 Southeast Asian workers living in South Korea. Using a self-administered questionnaire, we assessed the diets before and after living in the hometown and in South Korea. Significant changes observed in the Southeast Asian workers were decreased in consumption frequency of fresh fruits, cooked vegetables, rice noodles, green tea and glutinous rice, and increase in consumption of Kimchi, seaweed, milk, coffee and pizza. These changes were attributed to rapid dietary acculturation. The frequencies of eating homemade food were significantly decreased after they came to Korea except for Thais. Thais showed the highest frequencies of eating homemade food daily among others. 28.2% of respondents said their health condition had deteriorated after living in South Korea due to difficulties to adapt Korean food, increased frequencies of eating instant food, and lacking exercises. By providing understanding of the dietary patterns of Southeast Asian workers, these results can be used for preliminary data to develop a program for their Korean food adaptation.
Assuntos
Aculturação , Dieta Saudável , Exercício Físico , Preferências Alimentares , Estilo de Vida Saudável , Sobrepeso/prevenção & controle , Cooperação do Paciente , Adulto , Sudeste Asiático/etnologia , Culinária , Dieta Saudável/etnologia , Emigrantes e Imigrantes , Fast Foods/efeitos adversos , Feminino , Preferências Alimentares/etnologia , Humanos , Masculino , Indústria Manufatureira , Pessoa de Meia-Idade , Inquéritos Nutricionais , Sobrepeso/etnologia , Sobrepeso/etiologia , Cooperação do Paciente/etnologia , República da Coreia , Comportamento Sedentário/etnologia , Migrantes , Recursos Humanos , Adulto JovemRESUMO
The highly mutagenic A:oxoG (8-oxoguanine) base pair in DNA most frequently arises by aberrant replication of the primary oxidative lesion C:oxoG. This lesion is particularly insidious because neither of its constituent nucleobases faithfully transmit genetic information from the original C:G base pair. Repair of A:oxoG is initiated by adenine DNA glycosylase, which catalyzes hydrolytic cleavage of the aberrant A nucleobase from the DNA backbone. These enzymes, MutY in bacteria and MUTYH in humans, scrupulously avoid processing of C:oxoG because cleavage of the C residue in C:oxoG would actually promote mutagenic conversion to A:oxoG. Here we analyze the structural basis for rejection of C:oxoG by MutY, using a synthetic crystallography approach to capture the enzyme in the process of inspecting the C:oxoG anti-substrate, with which it ordinarily binds only fleetingly. We find that MutY uses two distinct strategies to avoid presentation of C to the enzyme active site. Firstly, MutY possesses an exo-site that serves as a decoy for C, and secondly, repulsive forces with a key active site residue prevent stable insertion of C into the nucleobase recognition pocket within the enzyme active site.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , DNA Glicosilases/química , DNA Glicosilases/metabolismo , Reparo do DNA/fisiologia , Geobacillus stearothermophilus/enzimologia , Proteínas de Bactérias/genética , Domínio Catalítico , Cristalografia por Raios X , Dano ao DNA , DNA Glicosilases/genética , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Geobacillus stearothermophilus/genética , Guanina/análogos & derivados , Guanina/química , Guanina/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Conformação de Ácido Nucleico , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
Toll-like receptors (TLRs) can recognize microbial patterns and utilize adaptor molecules, such as-MyD88 or (TRIF TIR-domain-containing adapter-inducing interferon-ß), to initiate downstream signaling that ultimately affects the initiation of adaptive immunity. In addition to this inflammatory role, TLR5 expression on dendritic cells can favor antigen presentation of flagellin peptides and thus increase the sensitivity of flagellin-specific T-cell responses in vitro and in vivo. Here, we examined the role of alternative signaling pathways that might regulate flagellin antigen presentation in addition to MyD88. These studies suggest a requirement for spleen tyrosine kinase, a noncanonical TLR-signaling adaptor molecule, and its downstream molecule CARD9 in regulating the sensitivity of flagellin-specific CD4(+) T-cell responses in vitro and in vivo. Thus, a previously unappreciated signaling pathway plays an important role in regulating the dominance of flagellin-specific T-cell responses.
Assuntos
Proteínas Adaptadoras de Sinalização CARD/genética , Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/imunologia , Flagelina/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Tirosina Quinases/genética , Transdução de Sinais/imunologia , Imunidade Adaptativa , Animais , Apresentação de Antígeno , Proteínas Adaptadoras de Sinalização CARD/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Comunicação Celular , Proliferação de Células , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Flagelina/imunologia , Regulação da Expressão Gênica , Imunidade Inata , Interleucina-2/genética , Interleucina-2/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Lisossomos/imunologia , Lisossomos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/deficiência , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/imunologia , Fagossomos/imunologia , Fagossomos/metabolismo , Proteínas Tirosina Quinases/imunologia , Receptores de Interleucina-2/genética , Receptores de Interleucina-2/imunologia , Quinase Syk , Receptor 5 Toll-Like/genética , Receptor 5 Toll-Like/imunologiaRESUMO
CD4(+) T cells and B cells are both essential for acquired immunity to Salmonella infection. It is well established that Salmonella inhibit host CD4(+) T-cell responses, but a corresponding inhibitory effect on B cells is less well defined. Here, we utilize an Ag tetramer and pull-down enrichment strategy to directly visualize OVA-specific B cells in mice, as they respond to infection with Salmonella-OVA. Surprisingly, OVA-specific B-cell expansion and germinal center formation was not detected until bacteria were cleared from the host. Furthermore, Salmonella infection also actively inhibited both B- and T-cell responses to the same coinjected Ag but this did not require the presence of iNOS. The Salmonella Pathogenicity Island 2 (SPI2) locus has been shown to be responsible for inhibition of Salmonella-specific CD4(+) T-cell responses, and an examination of SPI2-deficient bacteria demonstrated a recovery in B-cell expansion in infected mice. Together, these data suggest that Salmonella can simultaneously inhibit host B- and T-cell responses using SPI2-dependent mechanisms.