RESUMO
The quantity of snow crabs (Chionoecetes opilio) harvested in Korea is subject to seasonal restrictions; therefore, snow crabs are imported from Russia. Metabolites in snow crabs from two geographic origins were compared. The metabolites were subjected to metabolomic analysis to prevent fraudulent sales of marine products from a particular country. Capillary electrophoresis time-of-flight mass spectrometry was used. Seventy-seven target metabolites were identified using a mass spectral library. Through orthogonal partial least squares discriminant analysis, the top 25 biomarker candidates were evaluated based on p-values and fold changes. A total of 246 peaks (187 and 59 in the cation and anion modes, respectively) were identified. Among the biomarker candidates, 2-oxovaleric acid, asymmetric dimethylarginine, hypotaurine, and allo-threonine were selected as final biomarkers to unequivocally determine the geographic origin. Overall, metabolic analyses allowed us to differentiate snow crabs from different geographic origins. This method could also be extended of other marine products.
Assuntos
Biomarcadores , Braquiúros , Eletroforese Capilar , Metabolômica , Animais , Biomarcadores/análise , Biomarcadores/metabolismo , Braquiúros/química , Braquiúros/metabolismo , Braquiúros/classificação , Federação Russa , República da Coreia , Espectrometria de Massas , Análise Discriminante , Frutos do Mar/análiseRESUMO
The yellow goosefish is a benthic fish that belongs to the family Lophiidae and order Lophiiformes and is distributed in the Yellow and East China Seas. This study aimed to distinguish between yellow goosefish from different geographical origins by analyzing their metabolites. Capillary electrophoresis time-of-flight mass spectrometry was used to analyze metabolite profiles in the muscle tissues of yellow goosefish to distinguish between Korean and Chinese yellow goosefish. In total, 271 putative metabolites were extracted using 50% acetonitrile in water. Principal component analysis and orthogonal partial least squares discriminant analysis (OPLS-DA) were used to distinguish different geographical origins using the metabolite profiles obtained. The R2 and Q2 values of the OPLS-DA model were 0.856 and 0.695, respectively, indicating that the model was well-fitted and had good predictability. The heat map revealed that nucleic acid and amino compounds differed between the Korean and Chinese fish, and the variable importance in the projection scores obtained from OPLS-DA showed that there were geographical differences in the primary metabolites (5'-methylthioadenosine, adenosine, uridine 5-diphosphate, guanosine 5-diphosphate, urea, homocarnosine, O-acetylcarnitine, cycloleucine, cycloleucine S-adenosylmethionine, S-adenosylhomocysteine, ethanolamine, myo-inositol 1-phosphate), which were identified as potential candidate biomarkers.
Assuntos
Cicloleucina , Peixes , Músculos , Animais , Cicloleucina/análise , Eletroforese Capilar , Peixes/metabolismo , República da Coreia , China , Músculos/química , Músculos/metabolismoRESUMO
Because of the wide use of Fingolimod for the treatment of multiple sclerosis (MS) and its cardiovascular side effects such as bradycardia, second-generation sphingosine 1-phosphate receptor 1 (S1P1) agonist drugs for MS have been developed and approved by FDA. The issue of bradycardia is still present with the new drugs, however, which necessitates further exploration of S1P1 agonists with improved safety profiles for next-generation MS drugs. Herein, we report a tetrahydroisoquinoline or a benzo[c]azepine core-based S1P1 agonists such as 32 and 60 after systematic examination of hydrophilic groups and cores. We investigated the binding modes of our representative compounds and their molecular interactions with S1P1 employing recent S1P1 cryo-EM structures. Also, favorable ADME properties of our compounds were shown. Furthermore, in vivo efficacy of our compounds was clearly demonstrated with PLC and EAE studies. Also, the preliminary in vitro cardiovascular safety of our compound was verified with human iPSC-derived cardiomyocytes.
Assuntos
Esclerose Múltipla , Tetra-Hidroisoquinolinas , Humanos , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/metabolismo , Receptores de Esfingosina-1-Fosfato , Bradicardia/induzido quimicamente , Receptores de Lisoesfingolipídeo/agonistas , Receptores de Lisoesfingolipídeo/metabolismo , Receptores de Lisoesfingolipídeo/uso terapêutico , Cloridrato de Fingolimode/farmacologia , Cloridrato de Fingolimode/uso terapêutico , Tetra-Hidroisoquinolinas/uso terapêutico , Esfingosina/metabolismoRESUMO
Red seabream (Pagrus major), a migratory fish, is characterized by high protein levels in the muscle. South Korean and Japanese red seabreams have a general distribution pattern; however, distinguishing them based on their geographical origin is difficult. In this study, we used capillary electrophoresis time-of-flight mass spectrometry (CE-TOF/MS) to analyze the red seabream muscle metabolome to investigate how can distinguish the origin of the fish. The metabolites were extracted using 50% acetonitrile in water. Chromatographic separation was successfully used to classify the metabolite profiles of Japanese and South Korean red seabream. Principal component analysis and hierarchical cluster analysis showed good ability to categorize the samples according to their origin. Amino acids showed the greatest quantitative difference in South Korean and Japanese muscle samples. Specifically, the L-alanine, L-glutamic acid, L-isoleucine, dimethylglycine, and L-valine levels in Japanese red seabream samples were significantly higher than those in South Korean samples. In contrast, the levels of trimethylamine N-oxide and inosine monophosphate in South Korean muscle samples were significantly higher than those in Japanese red muscle samples. The monitored metabolite profiles suggest that South Korean and Japanese red seabreams can be identified on the basis of amino acid levels.
Assuntos
Perciformes , Dourada , Aminoácidos/metabolismo , Animais , Eletroforese Capilar/métodos , Espectrometria de Massas/métodos , Metaboloma , Perciformes/metabolismo , Dourada/metabolismoRESUMO
Streptococcus mutans is a representative biofilm-forming bacterium that causes dental caries through glucosyltransferase (GTF) activity. Glucans are synthesized from sucrose by GTFs and provide binding sites for S. mutans to adhere tightly to the tooth enamel. Therefore, if a novel compound that interferes with GTF function is developed, biofilm formation control in S. mutans would be possible. We discovered that raffinose, an oligosaccharide from natural products, strongly inhibited biofilm formation, GTF-related gene expression, and glucan production. Furthermore, biofilm inhibition on saliva-coated hydroxyapatite discs through the reduction of bacterial adhesion indicated the applicability of raffinose in oral health. These effects of raffinose appear to be due to its ability to modulate GTF activity in S. mutans. Hence, raffinose may be considered an antibiofilm agent for use as a substance for oral supplies and dental materials to prevent dental caries. IMPORTANCE Dental caries is the most prevalent infectious disease and is expensive to manage. Dental biofilms can be eliminated via mechanical treatment or inhibited using antibiotics. However, bacteria that are not entirely removed or are resistant to antibiotics can still form biofilms. In this study, we found that raffinose inhibited biofilm formation by S. mutans, a causative agent of dental caries, possibly through binding to GtfC. Our findings support the notion that biofilm inhibition by raffinose can be exerted by interference with GTF function, compensating for the shortcomings of existing commercialized antibiofilm methods. Furthermore, raffinose is an ingredient derived from natural products and can be safely utilized in humans; it has no smell and tastes sweet. Therefore, raffinose, which can control S. mutans biofilm formation, has been suggested as a substance for oral supplies and dental materials to prevent dental caries.
Assuntos
Produtos Biológicos , Cárie Dentária , Antibacterianos/farmacologia , Biofilmes , Cárie Dentária/prevenção & controle , Materiais Dentários/metabolismo , Materiais Dentários/farmacologia , Glucanos , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Humanos , Rafinose/metabolismo , Rafinose/farmacologia , Streptococcus mutans/metabolismoRESUMO
Country-of-origin violations have occurred in which some merchants have fraudulently sold cheap Japanese yellowtail (Seriola quinqueradiata) by presenting them as domestic Korean products. There are many methods for determining the origins of marine organisms, such as molecular genetic methods and isotope analysis. However, this study aimed to develop a method for determining the origins of aquatic products using metabolite analysis technology. Ten yellowtail each from Korea and Japan were analyzed by capillary electrophoresis-time of flight/mass spectrometry (CETOF/MS). Hierarchical cluster analysis (HCA) and principal component analysis (PCA) results showed highly differing aspects between the Korean and Japanese samples. In the tricarboxylic acid (TCA) cycle, citric, malic, oxaloglutaric, and fumaric acids exhibited significant differences between Korean and Japanese yellowtail. Sixteen of the twenty essential amino acids analyzed as metabolites also differed significantly. All amino acids were involved in protein digestion, absorption, and metabolism. All 16 amino acid contents were higher in Japanese yellowtail than in Korean yellowtail, except for glutamine. The fasting period was found to be the biggest factor contributing to the difference in amino acid contents, in addition to environmental factors (including feeding habits). These significant differences indicated that metabolomics could be used to determine geographical origin.
RESUMO
Bacterial biofilm formation causes serious problems in various fields of medical, clinical, and industrial settings. Antibiotics and biocide treatments are typical methods used to remove bacterial biofilms, but biofilms are difficult to remove effectively from surfaces due to their increased resistance. An alternative approach to treatment with antimicrobial agents is using biofilm inhibitors that regulate biofilm development without inhibiting bacterial growth. In the present study, we found that linoleic acid (LA), a plant unsaturated fatty acid, inhibits biofilm formation under static and continuous conditions without inhibiting the growth of Pseudomonas aeruginosa. LA also influenced the bacterial motility, extracellular polymeric substance production, and biofilm dispersion by decreasing the intracellular cyclic diguanylate concentration through increased phosphodiesterase activity. Furthermore, quantitative gene expression analysis demonstrated that LA induced the expression of genes associated with diffusible signaling factor-mediated quorum sensing that can inhibit or induce the dispersion of P. aeruginosa biofilms. These results suggest that LA is functionally and structurally similar to a P. aeruginosa diffusible signaling factor (cis-2-decenoic acid) and, in turn, act as an agonist molecule in biofilm dispersion.
Assuntos
Biofilmes/efeitos dos fármacos , Ácidos Graxos Monoinsaturados/metabolismo , Ácido Linoleico/farmacologia , Pseudomonas aeruginosa/fisiologia , Percepção de Quorum/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimentoRESUMO
Development of highly effective, safe, and fast-acting anti-depressants is urgently required for the treatment of major depressive disorder. It has been suggested that targeting 5-HT2A and 5-HT2C in addition to inhibition of serotonin reuptake may be beneficial in generating anti-depressant agents with better pharmacology and less adverse effects. We have developed phthalazinone-based compounds that potently bind to 5-HT2A, 5-HT2C, and the serotonin transporter. The representative compounds 11j and 11l displayed strong binding affinities against these targets, and showed favorable toxicity profiles as determined by hERG binding and CYP inhibition assays. Furthermore, these compounds presented promising anti-depressant effects comparable to fluoxetine and also synergistic effects with fluoxetine in forced swimming test, which implicates these compounds can be developed to help the treatment of major depressive disorder.
Assuntos
Antidepressivos/química , Azóis/química , Receptor 5-HT2A de Serotonina/química , Receptor 5-HT2C de Serotonina/química , Proteínas da Membrana Plasmática de Transporte de Serotonina/química , Animais , Antidepressivos/metabolismo , Antidepressivos/farmacologia , Comportamento Animal/efeitos dos fármacos , Desenho de Fármacos , Fluoxetina/química , Fluoxetina/farmacologia , Humanos , Concentração Inibidora 50 , Camundongos , Receptor 5-HT2A de Serotonina/metabolismo , Receptor 5-HT2C de Serotonina/metabolismo , Antagonistas do Receptor 5-HT2 de Serotonina/química , Antagonistas do Receptor 5-HT2 de Serotonina/metabolismo , Antagonistas do Receptor 5-HT2 de Serotonina/farmacologia , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Inibidores Seletivos de Recaptação de Serotonina/química , Inibidores Seletivos de Recaptação de Serotonina/metabolismo , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Relação Estrutura-AtividadeRESUMO
In this study, a quorum-quenching (QQ) bacterium named HEMM-1 was isolated at a membrane bioreactor (MBR) plant. HEMM-1 has diplococcal morphology and 99% sequence identity to Enterococcus species. The HEMM-1 cell-free supernatant (CFS) showed higher QQ activities than the CFS of other QQ bacteria, mostly by degrading N-acyl homoserine lactones (AHLs) with short acyl chains. Instrumental analyses revealed that HEMM-1 CFS degraded AHLs via lactonase activity. Under static, flow, and shear conditions, the HEMM-1 CFS was effective in reducing bacterial and activated-sludge biofilms formed on membrane surfaces. In conclusion, the HEMM-1 isolate is a QQ bacterium applicable to the control of biofouling in MBRs via inhibition of biofilm formation on membrane surfaces.
Assuntos
Incrustação Biológica , Reatores Biológicos , Percepção de Quorum , Acil-Butirolactonas , Bactérias , BiofilmesRESUMO
Pseudomonas aeruginosa is a causative agent of chronic infections in immunocompromised patients. Disruption of quorum sensing circuits is an attractive strategy for treating diseases associated with P. aeruginosa infection. In this study, we designed and synthesized a series of gingerol analogs targeting LasR, a master regulator of quorum sensing networks in P. aeruginosa. Structure-activity relationship studies showed that a hydrogen-bonding interaction in the head section, stereochemistry and rotational rigidity in the middle section, and optimal alkyl chain length in the tail section are important factors for the enhancement of LasR-binding affinity and for the inhibition of biofilm formation. The most potent compound 41, an analog of (R)-8-gingerol with restricted rotation, showed stronger LasR-binding affinity and inhibition of biofilm formation than the known LasR antagonist (S)-6-gingerol. This new LasR antagonist can be used as an early lead compound for the development of anti-biofilm agents to treat P. aeruginosa infections.
Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Catecóis/farmacologia , Álcoois Graxos/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Antibacterianos/química , Proteínas de Bactérias/metabolismo , Catecóis/química , Álcoois Graxos/química , Humanos , Simulação de Acoplamento Molecular , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/fisiologia , Percepção de Quorum/efeitos dos fármacos , Transativadores/metabolismoRESUMO
p53 is an important tumor-suppressor protein deactivation of which by mdm2 results in cancers. A SUMO-specific proteaseâ 4 (SUSP4) was shown to rescue p53 from mdm2-mediated deactivation, but the mechanism is unknown. The discovery by NMR spectroscopy of a "p53 rescue motif" in SUSP4 that disrupts p53-mdm2 binding is presented. This 29-residue motif is pre-populated with two transient helices connected by a hydrophobic linker. The helix at the C-terminus binds to the well-known p53-binding pocket in mdm2 whereas the N-terminal helix serves as an affinity enhancer. The hydrophobic linker binds to a previously unidentified hydrophobic crevice in mdm2. Overall, SUSP4 appears to use two synergizing modules, the p53 rescue motif described here and a globular-structured SUMO-binding catalytic domain, to stabilize p53. A p53 rescue motif peptide exhibits an anti-tumor activity in cancer cell lines expressing wild-type p53. A pre-structures motif in the intrinsically disordered proteins is thus important for target recognition.
Assuntos
Cisteína Endopeptidases/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Domínio Catalítico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cisteína Endopeptidases/química , Humanos , Simulação de Dinâmica Molecular , Mutagênese , Peptídeos/farmacologia , Ligação Proteica , Proteínas Proto-Oncogênicas c-mdm2/química , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/genéticaRESUMO
Biofilm formation on biotic or abiotic surfaces has unwanted consequences in medical, clinical, and industrial settings. Treatments with antibiotics or biocides are often ineffective in eradicating biofilms. Promising alternatives to conventional agents are biofilm-inhibiting compounds regulating biofilm development without toxicity to growth. Here, we screened a biofilm inhibitor, raffinose, derived from ginger. Raffinose, a galactotrisaccharide, showed efficient biofilm inhibition of Pseudomonas aeruginosa without impairing its growth. Raffinose also affected various phenotypes such as colony morphology, matrix formation, and swarming motility. Binding of raffinose to a carbohydrate-binding protein called LecA was the cause of biofilm inhibition and altered phenotypes. Furthermore, raffinose reduced the concentration of the second messenger, cyclic diguanylate (c-di-GMP), by increased activity of a c-di-GMP specific phosphodiesterase. The ability of raffinose to inhibit P. aeruginosa biofilm formation and its molecular mechanism opens new possibilities for pharmacological and industrial applications.
Assuntos
Adesinas Bacterianas/metabolismo , Antibacterianos/metabolismo , Biofilmes/efeitos dos fármacos , GMP Cíclico/análogos & derivados , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/fisiologia , Rafinose/metabolismo , GMP Cíclico/metabolismo , Zingiber officinale/química , Rafinose/isolamento & purificaçãoRESUMO
The eIF4E-binding protein 1 (4EBP1) has long been known to be completely unstructured without any secondary structures, which contributed significantly to the proposal of the induced fit mechanism for target binding of intrinsically disordered proteins. We show here that 4EBP1 is not completely unstructured, but contains a pre-structured helix.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Intrinsicamente Desordenadas/química , Fosfoproteínas/química , Proteínas de Ciclo Celular , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Estrutura Secundária de ProteínaRESUMO
The hepatitis B virus x protein (HBX) is expressed in HBV-infected liver cells and can interact with a wide range of cellular proteins. In order to understand such promiscuous behavior of HBX we expressed a truncated mini-HBX protein (named Tr-HBX) (residues 18-142) with 5 Cys â Ser mutations and characterized its structural features using circular dichroism (CD) spectropolarimetry, NMR spectroscopy as well as bioinformatics tools for predicting disorder in intrinsically unstructured proteins (IUPs). The secondary structural content of Tr-HBX from CD data suggests that Tr-HBX is only partially folded. The protein disorder prediction by IUPred reveals that the unstructured region encompasses its N-terminal ~30 residues of Tr-HBX. A two-dimensional (1)H-(15)N HSQC NMR spectrum exhibits fewer number of resonances than expected, suggesting that Tr-HBX is a hybrid type IUP where its folded C-terminal half coexists with a disordered N-terminal region. Many IUPs are known to be capable of having promiscuous interactions with a multitude of target proteins. Therefore the intrinsically disordered nature of Tr-HBX revealed in this study provides a partial structural basis for the promiscuous structure-function behavior of HBX.
Assuntos
Vírus da Hepatite B/metabolismo , Transativadores/química , Transativadores/metabolismo , Sequência de Aminoácidos , Dicroísmo Circular/métodos , Vírus da Hepatite B/genética , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Estrutura Secundária de Proteína , Desdobramento de Proteína , Transativadores/genética , Transativadores/isolamento & purificação , Proteínas Virais Reguladoras e AcessóriasRESUMO
Intrinsically unfolded proteins (IUPs) do not obey the golden rule of structural biology, 3D structure = function, as they manifest their inherent functions without resorting to three-dimensional structures. Absence of a compact globular topology in these proteins strongly implies that their ligand recognition processes should involve factors other than spatially well-defined binding pockets. Heteronuclear multidimensional (HetMulD) NMR spectroscopy assisted with a stable isotope labeling technology is a powerful tool for quantitatively investigating detailed structural features in IUPs. In particular, it allows us to delineate the presence and locations of pre-structured motifs (PreSMos) on a per-residue basis. PreSMos are the transient local structural elements that presage target-bound conformations and act as specificity determinants for IUP recognition by target proteins. Here, we present a brief chronicle of HetMulD NMR studies on IUPs carried out over the past two decades along with a discussion on the functional significance of PreSMos in IUPs.