RESUMO
Heavy alcohol consumption is the dominant risk factor for chronic pancreatitis (CP); however, treatment and prevention strategies for alcoholic chronic pancreatitis (ACP) remains limited. The present study demonstrates that ACP induction in C57BL/6 mice causes significant acinar cell injury, pancreatic stellate cell (PSC) activation, exocrine function insufficiency, and an increased fibroinflammatory response when compared with alcohol or CP alone. Although the withdrawal of alcohol during ACP recovery led to reversion of pancreatic damage, continued alcohol consumption with established ACP perpetuated pancreatic injury. In addition, phosphokinase array and Western blot analysis of ACP-induced mice pancreata revealed activation of the phosphatidylinositol 3 kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) and cyclic AMP response element binding protein (CREB) signaling pathways possibly orchestrating the fibroinflammatory program of ACP pathogenesis. Mice treated with urolithin A (Uro A, a gut-derived microbial metabolite) in the setting of ACP with continued alcohol intake (during the recovery period) showed suppression of AKT and P70S6K activation, and acinar damage was significantly reduced with a parallel reduction in pancreas-infiltrating macrophages and proinflammatory cytokine accumulation. These results collectively provide mechanistic insight into the impact of Uro A on attenuation of ACP severity through suppression of PI3K/AKT/mTOR signaling pathways and can be a useful therapeutic approach in patients with ACP with continuous alcohol intake.NEW & NOTEWORTHY Our novel findings presented here demonstrate the utility of Uro A as an effective therapeutic agent in attenuating alcoholic chronic pancreatitis (ACP) severity with alcohol continuation after established disease, through suppression of the PI3K/AKT/mTOR signaling pathway.
Assuntos
Pancreatite Alcoólica , Proteínas Proto-Oncogênicas c-akt , Camundongos , Animais , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/farmacologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Camundongos Endogâmicos C57BL , Serina-Treonina Quinases TOR/metabolismo , Transdução de Sinais , Pancreatite Alcoólica/patologia , Sirolimo/farmacologia , Citocinas/farmacologia , Consumo de Bebidas Alcoólicas , Mamíferos/metabolismoRESUMO
Environmental chemicals such as inorganic arsenic (iAs) significantly contribute to redox toxicity in the human body by enhancing oxidative stress. Imbalanced oxidative stress rapidly interferes with gut homeostasis and affects variety of cellular processes such as proliferation, apoptosis, and maintenance of intestinal barrier integrity. It has been shown that gut microbiota are essential to protect against iAs3+-induced toxicity. However, the effect of microbial metabolites on iAs3+-induced toxicity and loss of gut barrier integrity has not been investigated. The objectives of the study are to investigate impact of iAs on gut barrier function and determine benefits of gut microbial metabolite, urolithin A (UroA) against iAs3+-induced adversaries on gut epithelium. We have utilized both colon epithelial cells and in a human intestinal 3D organoid model system to investigate iAs3+-induced cell toxicity, oxidative stress, and gut barrier dysfunction in the presence or absence of UroA. Here, we report that treatment with UroA attenuated iAs3+-induced cell toxicity, apoptosis, and oxidative stress in colon epithelial cells. Moreover, our data suggest that UroA significantly reduces iAs3+-induced gut barrier permeability and inflammatory markers in both colon epithelial cells and in a human intestinal 3D organoid model system. Mechanistically, UroA protected against iAs3+-induced disruption of tight junctional proteins in intestinal epithelial cells through blockade of oxidative stress and markers of inflammation. Taken together, our studies for the first time suggest that microbial metabolites such as UroA can potentially be used to protect against environmental hazards by reducing intestinal oxidative stress and by enhancing gut barrier function.
Assuntos
Arsênio , Arsenicais , Microbioma Gastrointestinal , Arsênio/metabolismo , Arsênio/toxicidade , Arsenicais/metabolismo , Colo , Cumarínicos/metabolismo , Cumarínicos/farmacologia , Humanos , Mucosa Intestinal/metabolismoRESUMO
The biological activities of chemokine (C-C motif) ligand 2 (CCL2) are mediated via C-C chemokine receptor-2 (CCR2). Increased CCL2 level is associated with metastasis of many cancers. In our study, we investigated the role of the CCL2/CCR2 axis in the development of spontaneous intestinal tumorigenesis using the ApcMin/+ mouse model. Ablation of CCR2 in ApcMin/+ mice significantly increased the overall survival and reduced intestinal tumor burden. Immune cell analysis showed that CCR2-/- ApcMin/+ mice exhibited significant reduction in the myeloid cell population and increased interferon γ (IFN-γ) producing T cells both in spleen and mesenteric lymph nodes compared to ApcMin/+ mice. The CCR2-/- ApcMin/+ tumors showed significantly reduced levels of interleukin (IL)-17 and IL-23 and increased IFN-γ and Granzyme B compared to ApcMin/+ tumors. Transfer of CCR2+/+ ApcMin/+ CD4+ T cells into Rag2-/- mice led to development of colitis phenotype with increased CD4+ T cells hyper proliferation and IL-17 production. In contrast, adoptive transfer of CCR2-/- ApcMin/+ CD4+ T cells into Rag2-/- mice failed to enhance colonic inflammation or IL-17 production. These results a suggest novel additional role for CCR2, where it regulates migration of IL-17 producing cells mediating tumor-promoting inflammation in addition to its role in migration of tumor associated macrophages.
RESUMO
Leukotriene B4 (LTB4) receptor (BLT)1 is expressed on variety of immune cells and has been implicated as a mediator of diverse inflammatory diseases. However, whether biological responses initiated via this receptor generate tumor-promoting inflammation or antitumor immunity remains unexplored. In this study, we investigated the role of BLT1 in antitumor immunity using syngeneic TC-1 cervical cancer model, and observed accelerated tumor growth and reduced survival in BLT1â»/â» mice compared with BLT1âº/⺠mice. Analysis of the tumor infiltrates by flow cytometry and confocal microscopy revealed a significant decrease in effector immune cells, most notably, CD8⺠T cells and NK cells in the tumors of the BLT1â»/â» mice. Gene expression profiling confirmed the dramatic decrease of IFN-γ, granzyme B, and IL-2 in tumors growing in BLT1â»/â» mice. Furthermore, depletion of CD8⺠T cells enhanced the tumor growth in BLT1âº/⺠but not in BLT1â»/â» mice. However, similar levels of Ag-dependent CD8⺠T cell-mediated killing activity were observed in spleens of BLT1âº/⺠and BLT1â»/â» mice. Adoptive transfer of CD8⺠T cells from tumor-bearing BLT1âº/⺠but not BLT1â»/â» mice significantly reduced tumor growth and increased the survival of Rag2â»/â» mice. Although the homeostatic proliferation and expression profiles of other chemokine receptors of adoptively transferred BLT1âº/⺠and BLT1â»/â» CD8⺠T cells appears to be similar, BLT1âº/⺠T lymphocytes entered the tumors in greater numbers. These results suggest that BLT1 expression on CD8⺠T cells plays an important role in their trafficking to tumors.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Quimiotaxia de Leucócito/imunologia , Receptores do Leucotrieno B4/biossíntese , Neoplasias do Colo do Útero/imunologia , Transferência Adotiva , Animais , Linfócitos T CD8-Positivos/metabolismo , Modelos Animais de Doenças , Feminino , Citometria de Fluxo , Imunofluorescência , Linfócitos do Interstício Tumoral , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Reação em Cadeia da Polimerase em Tempo Real , Neoplasias do Colo do Útero/metabolismoRESUMO
Clostridioides difficile is a Gram-positive, anaerobic, spore-forming bacterium responsible for antibiotic-associated pseudomembranous colitis. Clostridioides difficile infection (CDI) symptoms can range from diarrhea to life-threatening colon damage. Toxins produced by C. difficile (TcdA and TcdB) cause intestinal epithelial injury and lead to severe gut barrier dysfunction, stem cell damage, and impaired regeneration of the gut epithelium. Current treatment options for intestinal repair are limited. In this study, we demonstrate that treatment with the microbial metabolite urolithin A (UroA) attenuates CDI-induced adverse effects on the colon epithelium in a preclinical model of CDI-induced colitis. Moreover, our analysis suggests that UroA treatment protects against C. difficile-induced inflammation, disruption of gut barrier integrity, and intestinal tight junction proteins in the colon of CDI mice. Importantly, UroA treatment significantly reduced the expression and release of toxins from C. difficile without inducing bacterial cell death. These results indicate the direct regulatory effects of UroA on bacterial gene regulation. Overall, our findings reveal a novel aspect of UroA activity, as it appears to act at both the bacterial and host levels to protect against CDI-induced colitis pathogenesis. This research sheds light on a promising avenue for the development of novel treatments for C. difficile infection.IMPORTANCETherapy for Clostridioides difficile infections includes the use of antibiotics, immunosuppressors, and fecal microbiota transplantation. However, these treatments have several drawbacks, including the loss of colonization resistance, the promotion of autoimmune disorders, and the potential for unknown pathogens in donor samples. To date, the potential benefits of microbial metabolites in CDI-induced colitis have not been fully investigated. Here, we report for the first time that the microbial metabolite urolithin A has the potential to block toxin production from C. difficile and enhance gut barrier function to mitigate CDI-induced colitis.
Assuntos
Toxinas Bacterianas , Clostridioides difficile , Infecções por Clostridium , Colite , Cumarínicos , Enterocolite Pseudomembranosa , Animais , Camundongos , Toxinas Bacterianas/genética , Enterotoxinas/genética , Clostridioides difficile/metabolismo , Proteínas de Bactérias/genética , Enterocolite Pseudomembranosa/tratamento farmacológico , Infecções por Clostridium/tratamento farmacológico , Colite/induzido quimicamenteRESUMO
Clostridioides difficile is a gram-positive, anaerobic, spore-forming bacterium that is responsible for antibiotic-associated pseudomembranous colitis. Clostridioides difficile infection (CDI) symptoms can range from diarrhea to life-threatening colon damage. Toxins produced by C. difficile (TcdA and TcdB) cause intestinal epithelial injury and lead to severe gut barrier dysfunction, stem cell damage, and impaired regeneration of the gut epithelium. Current treatment options for intestinal repair are limited. In this study, we demonstrate that treatment with the microbial metabolite urolithin A (UroA) attenuates CDI-induced adverse effects on the colon epithelium in a preclinical model of CDI-induced colitis. Moreover, our analysis suggests that UroA treatment protects against C. difficile-induced inflammation, disruption of gut barrier integrity, and intestinal tight junction proteins in the colon of CDI mice. Importantly, UroA treatment significantly reduced the expression and release of toxins from C. difficile, without inducing bacterial cell death. These results indicate the direct regulatory effects of UroA on bacterial gene regulation. Overall, our findings reveal a novel aspect of UroA activities, as it appears to act at both the bacterial and host levels to protect against CDI-induced colitis pathogenesis. This research sheds light on a promising avenue for the development of novel treatments for C. difficile infection.
RESUMO
Diabetic nephropathy (DN) remains the leading cause of vascular morbidity and mortality in diabetes patients. Despite the progress in understanding the diabetic disease process and advanced management of nephropathy, a number of patients still progress to end-stage renal disease (ESRD). The underlying mechanism still needs to be clarified. Gaseous signaling molecules, so-called gasotransmitters, such as nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H2S), have been shown to play an essential role in the development, progression, and ramification of DN depending on their availability and physiological actions. Although the studies on gasotransmitter regulations of DN are still emerging, the evidence revealed an aberrant level of gasotransmitters in patients with diabetes. In studies, different gasotransmitter donors have been implicated in ameliorating diabetic renal dysfunction. In this perspective, we summarized an overview of the recent advances in the physiological relevance of the gaseous molecules and their multifaceted interaction with other potential factors, such as extracellular matrix (ECM), in the severity modulation of DN. Moreover, the perspective of the present review highlights the possible therapeutic interventions of gasotransmitters in ameliorating this dreaded disease.
RESUMO
BACKGROUND: G-protein-coupled estrogen receptor (GPER/GPR30) was reported to bind 17ß-estradiol (E2), tamoxifen, and ICI 182,780 (fulvestrant) and promotes activation of epidermal growth factor receptor (EGFR)-mediated signaling in breast, endometrial and thyroid cancer cells. Although lung adenocarcinomas express estrogen receptors α and ß (ERα and ERß), the expression of GPER in lung cancer has not been investigated. The purpose of this study was to examine the expression of GPER in lung cancer. METHODS: The expression patterns of GPER in various lung cancer lines and lung tumors were investigated using standard quantitative real time PCR (at mRNA levels), Western blot and immunohistochemistry (IHC) methods (at protein levels). The expression of GPER was scored and the pairwise comparisons (cancer vs adjacent tissues as well as cancer vs normal lung tissues) were performed. RESULTS: Analysis by real-time PCR and Western blotting revealed a significantly higher expression of GPER at both mRNA and protein levels in human non small cell lung cancer cell (NSCLC) lines relative to immortalized normal lung bronchial epithelial cells (HBECs). The virally immortalized human small airway epithelial cell line HPL1D showed higher expression than HBECs and similar expression to NSCLC cells. Immunohistochemical analysis of tissue sections of murine lung adenomas as well as human lung adenocarcinomas, squamous cell carcinomas and non-small cell lung carcinomas showed consistently higher expression of GPER in the tumor relative to the surrounding non-tumor tissue. CONCLUSION: The results from this study demonstrate increased GPER expression in lung cancer cells and tumors compared to normal lung. Further evaluation of the function and regulation of GPER will be necessary to determine if GPER is a marker of lung cancer progression.
Assuntos
Adenocarcinoma/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas de Neoplasias/metabolismo , Receptores de Estrogênio/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Western Blotting , Linhagem Celular Tumoral , Humanos , Imuno-Histoquímica , Camundongos , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Background: Cytochrome P450 Family 1 Subfamily A Member 1 (CYP1A1) pathway, which is regulated by aryl hydrocarbon receptor (AhR) plays an important role in chemical carcinogenesis and xenobiotic metabolism. Recently, we demonstrated that the microbial metabolite Urolithin A (UroA) mitigates colitis through its gut barrier protective and anti-inflammatory activities in an AhR-dependent manner. Here, we explored role of CYP1A1 in UroA-mediated gut barrier and immune functions in regulation of inflammatory bowel disease (IBD). Methods: To determine the role of CYP1A1 in UroA-mediated protectives activities against colitis, we subjected C57BL/6 mice and Cyp1a1 -/- mice to dextran sodium sulphate (DSS)-induced acute colitis model. The phenotypes of the mice were characterized by determining loss of body weight, intestinal permeability, systemic and colonic inflammation. Further, we evaluated the impact of UroA on regulation of immune cell populations by flow cytometry and confocal imaging using both in vivo and ex vivo model systems. Results: UroA treatment mitigated DSS-induced acute colitis in the wildtype mice. However, UroA-failed to protect Cyp1a1 -/- mice against colitis, as evident from non-recovery of body weight loss, shortened colon lengths and colon weight/length ratios. Further, UroA failed to reduce DSS-induced inflammation, intestinal permeability and upregulate tight junction proteins in Cyp1a1 -/- mice. Interestingly, UroA induced the expansion of T-reg cells in a CYP1A1-dependent manner both in vivo and ex vivo models. Conclusion: Our results suggest that CYP1A1 expression is essential for UroA-mediated enhanced gut barrier functions and protective activities against colitis. We postulate that CYP1A1 plays critical and yet unknown functions beyond xenobiotic metabolism in the regulation of gut epithelial integrity and immune systems to maintain gut homeostasis in IBD pathogenesis.
Assuntos
Colite , Doenças Inflamatórias Intestinais , Animais , Colite/patologia , Cumarínicos , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Sulfato de Dextrana/efeitos adversos , Modelos Animais de Doenças , Inflamação , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Proteínas de Junções Íntimas/metabolismo , Xenobióticos/efeitos adversosRESUMO
The survival rate of colorectal cancer patients is adversely affected by the selection of tumors resistant to conventional anti-cancer drugs such as 5-fluorouracil (5FU). Although there is mounting evidence that commensal gut microbiota is essential for effective colon cancer treatment, the detailed molecular mechanisms and the role of gut microbial metabolites remain elusive. The goal of this study is to decipher the impact and mechanisms of gut microbial metabolite, urolithin A (UroA) and its structural analogue, UAS03 on reversal of 5FU-resistant (5FUR) colon cancers. Methods: We have utilized the SW480 and HCT-116 parental (5FU-sensitive) and 5FUR colon cancer cells to examine the chemosensitization effects of UroA or UAS03 by using both in vitro and in vivo models. The effects of mono (UroA/UAS03/5FU) and combinatorial therapy (UroA/UAS03 + 5FU) on cell proliferation, apoptosis, cell migration and invasion, regulation of epithelial mesenchymal transition (EMT) mediators, expression and activities of drug transporters, and their regulatory transcription factors were examined using molecular, cellular, immunological and flowcytometric methods. Further, the anti-tumor effects of mono/combination therapy (UroA or UAS03 or 5FU or UroA/UAS03 + 5FU) were examined using pre-clinical models of 5FUR-tumor xenografts in NRGS mice and azoxymethane (AOM)-dextran sodium sulfate (DSS)-induced colon tumors. Results: Our data showed that UroA or UAS03 in combination with 5FU significantly inhibited cell viability, proliferation, invasiveness as well as induced apoptosis of the 5FUR colon cancer cells compared to mono treatments. Mechanistically, UroA or UAS03 chemosensitized the 5FUR cancer cells by downregulating the expression and activities of drug transporters (MDR1, BCRP, MRP2 and MRP7) leading to a decrease in the efflux of 5FU. Further, our data suggested the UroA or UAS03 chemosensitized 5FUR cancer cells to 5FU treatment through regulating FOXO3-FOXM1 axis. Oral treatment with UroA or UAS03 in combination with low dose i.p. 5FU significantly reduced the growth of 5FUR-tumor xenografts in NRGS mice. Further, combination therapy significantly abrogated colonic tumors in AOM-DSS-induced colon tumors in mice. Conclusions: In summary, gut microbial metabolite UroA and its structural analogue UAS03 chemosensitized the 5FUR colon cancers for effective 5FU chemotherapy. This study provided the novel characteristics of gut microbial metabolites to have significant translational implications in drug-resistant cancer therapeutics.
Assuntos
Neoplasias do Colo , Resistencia a Medicamentos Antineoplásicos , Fluoruracila , Proteína Forkhead Box M1 , Proteína Forkhead Box O3 , Microbioma Gastrointestinal , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Animais , Antimetabólitos Antineoplásicos/metabolismo , Azoximetano , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/metabolismo , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Cumarínicos/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Resistencia a Medicamentos Antineoplásicos/fisiologia , Fluoruracila/metabolismo , Fluoruracila/farmacologia , Fluoruracila/uso terapêutico , Proteína Forkhead Box M1/genética , Proteína Forkhead Box M1/metabolismo , Proteína Forkhead Box O3/genética , Proteína Forkhead Box O3/metabolismo , Microbioma Gastrointestinal/genética , Microbioma Gastrointestinal/fisiologia , Humanos , Camundongos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismoRESUMO
The human gastrointestinal tract (GI) harbors a diverse population of microbial life that continually shapes host pathophysiological responses. Despite readily available abundant metagenomic data, the functional dynamics of gut microbiota remain to be explored in various health and disease conditions. Microbiota generate a variety of metabolites from dietary products that influence host health and pathophysiological functions. Since gut microbial metabolites are produced in close proximity to gut epithelium, presumably they have significant impact on gut barrier function and immune responses. The goal of this review is to discuss recent advances on gut microbial metabolites in the regulation of intestinal barrier function. While the mechanisms of action of these metabolites are only beginning to emerge, they mainly point to a small group of shared pathways that control gut barrier functions. Amidst expanding technology and broadening knowledge, exploitation of beneficial microbiota and their metabolites to restore pathophysiological balance will likely prove to be an extremely useful remedial tool.
Assuntos
Permeabilidade da Membrana Celular , Microbioma Gastrointestinal , Mucosa Intestinal/microbiologia , Mucosa Intestinal/fisiologia , Metaboloma , Animais , HumanosRESUMO
Urolithins are gut microbial metabolites derived from ellagitannins (ET) and ellagic acid (EA), and shown to exhibit anticancer, anti-inflammatory, anti-microbial, anti-glycative and anti-oxidant activities. Similarly, the parent molecules, ET and EA are reported for their neuroprotection and antidepressant activities. Due to the poor bioavailability of ET and EA, the in vivo functional activities cannot be attributed exclusively to these compounds. Elevated monoamine oxidase (MAO) activities are responsible for the inactivation of monoamine neurotransmitters in neurological disorders, such as depression and Parkinson's disease. In this study, we examined the inhibitory effects of urolithins (A, B and C) and EA on MAO activity using recombinant human MAO-A and MAO-B enzymes. Urolithin B was found to be a better MAO-A enzyme inhibitor among the tested urolithins and EA with an IC50 value of 0.88 µM, and displaying a mixed mode of inhibition. However, all tested compounds exhibited higher IC50 (>100 µM) for MAO-B enzyme.
RESUMO
The gut microbiota can be altered by dietary interventions to prevent and treat various diseases. However, the mechanisms by which food products modulate commensals remain largely unknown. We demonstrate that plant-derived exosome-like nanoparticles (ELNs) are taken up by the gut microbiota and contain RNAs that alter microbiome composition and host physiology. Ginger ELNs (GELNs) are preferentially taken up by Lactobacillaceae in a GELN lipid-dependent manner and contain microRNAs that target various genes in Lactobacillus rhamnosus (LGG). Among these, GELN mdo-miR7267-3p-mediated targeting of the LGG monooxygenase ycnE yields increased indole-3-carboxaldehyde (I3A). GELN-RNAs or I3A, a ligand for aryl hydrocarbon receptor, are sufficient to induce production of IL-22, which is linked to barrier function improvement. These functions of GELN-RNAs can ameliorate mouse colitis via IL-22-dependent mechanisms. These findings reveal how plant products and their effects on the microbiome may be used to target specific host processes to alleviate disease.
Assuntos
Complexo Multienzimático de Ribonucleases do Exossomo/farmacologia , Microbioma Gastrointestinal/efeitos dos fármacos , Intestinos/microbiologia , Intestinos/fisiologia , MicroRNAs/farmacologia , Plantas/química , Animais , Proteínas de Bactérias , Colite/terapia , Modelos Animais de Doenças , Suscetibilidade a Doenças , Feminino , Alimentos , Microbioma Gastrointestinal/genética , Vida Livre de Germes , Interações Hospedeiro-Patógeno , Imunidade nas Mucosas , Indóis/metabolismo , Interleucinas/metabolismo , Lacticaseibacillus rhamnosus/efeitos dos fármacos , Lacticaseibacillus rhamnosus/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Ribossômico 16S/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Serina Endopeptidases , Triptofano/metabolismo , Interleucina 22RESUMO
The composition of the upper respiratory tract microbiome may play an important role in the development of lower respiratory tract infections. Here, we characterised the microbiome of the nasopharynx and oropharynx of hospitalised patients with community-acquired pneumonia (CAP) with unknown aetiology in an attempt to obtain insight into the aetiology of CAP. A random sample of 10 patients hospitalised with CAP previously enrolled in a separate clinical trial (ClinicalTrials.gov registry, Study ID: NCT01248715) in which a complete microbiological workup was not able to define an aetiology were analysed in this pilot study. This larger trial (n = 1,221) enrolled patients from 9 adult hospitals in Louisville, Kentucky, USA. Nasopharyngeal and oropharyngeal swabs were obtained for metagenomic analysis. Polymerase chain reaction (PCR) for Streptococcus pneumoniae was performed in all patients. One patient had a distinct nasophararyngeal microbiome consisting largely of Haemophilus influenzae. This was the only patient with a negative PCR for S. pneumoniae in both nasophararyngeal and oropharyngeal specimens. Overall, substantial differences were found between nasophararyngeal and oropharyngeal microbiomes. The upper respiratory tract microbiome of only one patient suggested H. influenzae as a probable aetiology of CAP. Although this was a pilot study of only 10 patients, the presence of S. pneumoniae in the upper respiratory tract of the other 9 patients warrants further investigation.
RESUMO
The crystal structure of human liver cytosolic recombinant serine hydroxymethyltransferase (hcSHMT) suggested that Ser53 and Arg 263 could participate in the reaction catalyzed by SHMT. The mutation of Arg262 (corresponding to Arg263 in hcSHMT) to "A" in sheep liver cytosolic SHMT (scSHMT) resulted in a 5-fold increase in Km for L-Ser and a 5-fold decrease in kcat compared to scSHMT. Further, in R262A SHMT-glycine complex, the peak at 343 nm (geminal diamine) was more pronounced, compared to wild-type enzyme. Stopped-flow studies showed that the rate constant for the formation of glycine-geminal diamine for R262A SHMT was also decreased. The rate of reaction, concentration of spectral intermediates, fluorescence excitation maximum of glycine geminal diamine and interaction with methoxyamine were altered in R262A SHMT. Although Arg263 in hcSHMT is located outside the PLP binding pocket, it positions Tyr73 for interaction with PLP, by forked H-bonding with the carbonyl groups of main chain residues, Asn71 and Lys72 of the other subunit of the tight dimer. Mutation of Arg262 to Ala and the consequent alteration in orientation of PLP leads to decreased catalytic efficiency. Ser53 (in hcSHMT) is in hydrogen bonding distance to one of the carboxylate oxygens of the amino acid substrate, which also interacts with Tyr83 and Arg402. Replacement of Ser53 with Cys (using 'O' software program) in the structure of hcSHMT resulted in disruption of these interactions, whereas replacement with Ala (S53A) only weakened the substrate interactions. There was a 10-fold increase in Km and 20-fold decrease in catalytic activity efficiency for S52C SHMT, whereas S52A SHMT retained 20% of the activity without change in Km for serine. These results suggest that S52 affects substrate binding and catalysis.
Assuntos
Arginina/metabolismo , Citosol/enzimologia , Glicina Hidroximetiltransferase/metabolismo , Fígado/citologia , Mutagênese Sítio-Dirigida , Serina/metabolismo , Ovinos , Animais , Biocatálise , Estabilidade Enzimática , Glicina Hidroximetiltransferase/química , Glicina Hidroximetiltransferase/genética , Modelos Moleculares , Mutação , Ligação Proteica , Fosfato de Piridoxal/metabolismo , TemperaturaRESUMO
Serine hydroxymethyltransferase (SHMT), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, catalyses the transfer of the hydroxymethyl group from serine to tetrahydrofolate to yield glycine and N (5), N (10)-methylenetetrahydrofolate. An analysis of the known SHMT sequences indicated that several amino acid residues were conserved. In this paper, we report the identification of the amino acid residues essential for maintaining the oligomeric structure of sheep liver cytosolic recombinant SHMT (scSHMT) through intra- and inter-subunit interactions and by stabilizing the binding of PLP at the active site. The mutation of Lys-71, Arg-80 and Asp-89, the residues involved in intra-subunit ionic interactions, disturbed the oligomeric structure and caused a loss of catalytic activity. Mutation of Trp-110 to Phe was without effect, while its mutation to Ala resulted in the enzyme being present in the insoluble fraction. These results suggested that Trp-110 located in a cluster of hydrophobic residues was essential for proper folding of the enzyme. Arg-98 and His-304, residues involved in the inter-subunit interactions, were essential for maintaining the tetrameric structure. Mutation of Tyr-72, Asp-227 and His-356 at the active site which interact with PLP resulted in the loss of PLP, and hence loss of tetrameric structure. Mutation of Cys-203, located away from the active site, weakened PLP binding indirectly. The results demonstrate that in addition to residues involved in inter-subunit interactions, those involved in PLP binding and intra-subunit interactions also affect the oligomeric structure of scSHMT.