RESUMO
Metabolites in human biofluids reflect individual physiological states influenced by various factors. Using liquid chromatography-mass spectrometry (LC-MS), we conducted non-targeted, non-invasive metabolomics using saliva of 27 healthy volunteers in Okinawa, comprising 13 young (30 ± 3 year) and 14 elderly (76 ± 4 year) subjects. Few studies have comprehensively identified age-dependent changes in salivary metabolites. Among 99 salivary metabolites, 21 were statistically age-related. All of the latter decline in abundance with advancing age, except ATP, which increased 1.96-fold in the elderly, possibly due to reduced ATP consumption. Fourteen age-linked and highly correlated compounds function in a metabolic network involving the pentose-phosphate pathway, glycolysis/gluconeogenesis, amino acids, and purines/pyrimidines nucleobases. The remaining seven less strongly correlated metabolites, include ATP, anti-oxidation-related glutathione disulfide, muscle-related acetyl-carnosine, N-methyl-histidine, creatinine, RNA-related dimethyl-xanthine and N-methyl-adenosine. In addition, glutamate and N-methyl-histidine are related to taste, so their decline suggests that the elderly lose some ability to taste. Reduced redox metabolism and muscle activity are suggested by changes in glutathione and acetyl-carnosine. These age-linked salivary metabolites together illuminate a metabolic network that reflects a decline of oral functions during human aging.
Assuntos
Metaboloma , Metabolômica , Saliva/metabolismo , Adolescente , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Biomarcadores/metabolismo , Cromatografia Líquida , Biologia Computacional/métodos , Feminino , Humanos , Masculino , Metabolômica/métodos , Pessoa de Meia-Idade , Espectrometria de Massas em Tandem , Adulto JovemRESUMO
Dementia is caused by factors that damage neurons. We quantified small molecular markers in whole blood of dementia patients, using nontargeted liquid chromatography-mass spectroscopy (LC-MS). Thirty-three metabolites, classified into five groups (A to E), differed significantly in dementia patients, compared with healthy elderly subjects. Seven A metabolites present in plasma, including quinolinic acid, kynurenine, and indoxyl-sulfate, increased. Possibly they act as neurotoxins in the central nervous system (CNS). The remaining 26 compounds (B to E) decreased, possibly causing a loss of support or protection of the brain in dementia. Six B metabolites, normally enriched in red blood cells (RBCs), all contain trimethylated ammonium moieties. These metabolites include ergothioneine and structurally related compounds that have scarcely been investigated as dementia markers, validating the examination of RBC metabolites. Ergothioneine, a potent antioxidant, is significantly decreased in various cognition-related disorders, such as mild cognitive impairment and frailty. C compounds also include some oxidoreductants and are normally abundant in RBCs (NADP+, glutathione, adenosine triphosphate, pantothenate, S-adenosyl-methionine, and gluconate). Their decreased levels in dementia patients may also contribute to depressed brain function. Twelve D metabolites contains plasma compounds, such as amino acids, glycerophosphocholine, dodecanoyl-carnitine, and 2-hydroxybutyrate, which normally protect the brain, but their diminution in dementia may reduce that protection. Seven D compounds have been identified previously as dementia markers. B to E compounds may be critical to maintain the CNS by acting directly or indirectly. How RBC metabolites act in the CNS and why they diminish significantly in dementia remain to be determined.
Assuntos
Envelhecimento , Biomarcadores/sangue , Encéfalo/metabolismo , Demência/patologia , Metaboloma , Idoso , Estudos de Casos e Controles , Demência/sangue , Humanos , Testes Neuropsicológicos , Transdução de SinaisRESUMO
S-adenosylmethionine is an important compound, because it serves as the methyl donor in most methyl transfer reactions, including methylation of proteins, nucleic acids, and lipids. However, cellular defects in the genetic disruption of S-adenosylmethionine synthesis are not well understood. Here, we report the isolation and characterization of temperature-sensitive mutants of fission yeast S-adenosylmethionine synthetase (Sam1). Levels of S-adenosylmethionine and methylated histone H3 were greatly diminished in sam1 mutants. sam1 mutants stopped proliferating in vegetative culture and arrested specifically in G2 phase without cell elongation. Furthermore, sam1 mutants lost viability during nitrogen starvation-induced G0 phase quiescence. After release from the G0 state, sam1 mutants could neither increase in cell size nor re-initiate DNA replication in the rich medium. Sam1 is thus required for cell growth and proliferation, and maintenance of and exit from quiescence. sam1 mutants lead to broad cellular and drug response defects, as expected, since S. pombe contains more than 90 S-adenosylmethionine-dependent methyltransferases.
RESUMO
Rapamycin inhibits TOR (target of rapamycin) kinase, and is being used clinically to treat various diseases ranging from cancers to fibrodysplasia ossificans progressiva. To understand rapamycin mechanisms of action more comprehensively, 1014 temperature-sensitive (ts) fission yeast (Schizosaccharomyces pombe) mutants were screened in order to isolate strains in which the ts phenotype was rescued by rapamycin. Rapamycin-rescued 45 strains, among which 12 genes responsible for temperature sensitivity were identified. These genes are involved in stress-activated protein kinase (SAPK) signalling, chromatin regulation, vesicle transport, and CoA- and mevalonate-related lipid metabolism. Subsequent metabolome analyses revealed that rapamycin upregulated stress-responsive metabolites, while it downregulated purine biosynthesis intermediates and nucleotide derivatives. Rapamycin alleviated abnormalities in cell growth and cell division caused by sty1 mutants (Δsty1) of SAPK. Notably, in Δsty1, rapamycin reduced greater than 75% of overproduced metabolites (greater than 2× WT), like purine biosynthesis intermediates and nucleotide derivatives, to WT levels. This suggests that these compounds may be the points at which the SAPK/TOR balance regulates continuous cell proliferation. Rapamycin might be therapeutically useful for specific defects of these gene functions.
Assuntos
Metabolismo dos Lipídeos/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/genética , Mutação , Schizosaccharomyces/crescimento & desenvolvimento , Sirolimo/farmacologia , Transporte Biológico/efeitos dos fármacos , Cromatina/metabolismo , Coenzima A/biossíntese , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Metaboloma , Ácido Mevalônico/metabolismo , Schizosaccharomyces/genética , Transdução de Sinais , TemperaturaAssuntos
Ensaios de Seleção de Medicamentos Antitumorais/métodos , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Antibióticos Antineoplásicos/farmacologia , Western Blotting , Linhagem Celular Tumoral , Fermentação , Humanos , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/patologia , Peptídeos Cíclicos/farmacologia , Inibidores da Síntese de Proteínas/farmacologia , Proteínas Proto-Oncogênicas B-raf/efeitos dos fármacosRESUMO
According to the chemical genetic approach, small molecules that bind directly to proteins are used to analyze protein function, thereby enabling the elucidation of complex mechanisms in mammal cells. Thus, it is very important to identify the molecular targets of compounds that induce a unique phenotype in a target cell. Phoslactomycin A (PLMA) is known to be a potent inhibitor of protein Ser/Thr phosphatase 2A (PP2A); however, the inhibitory mechanism of PP2A by PLMA has not yet been elucidated. Here, we demonstrated that PLMA directly binds to the PP2A catalytic subunit (PP2Ac) in cells by using biotinylated PLMA, and the PLMA-binding site was identified as the Cys-269 residue of PP2Ac. Moreover, we revealed that the Cys-269 contributes to the potent inhibition of PP2Ac activity by PLMA. These results suggest that PLMA is a PP2A-selective inhibitor and is therefore expected to be useful for future investigation of PP2A function in cells.
Assuntos
Domínio Catalítico , Cisteína/metabolismo , Inibidores Enzimáticos/farmacologia , Lactonas/farmacologia , Compostos Organofosforados/farmacologia , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/química , Sequência de Aminoácidos , Sítios de Ligação , Biotinilação , Linhagem Celular , Cisteína/genética , Inibidores Enzimáticos/química , Humanos , Lactonas/química , Dados de Sequência Molecular , Estrutura Molecular , Compostos Organofosforados/química , Fosfoproteínas Fosfatases/classificação , Fosfoproteínas Fosfatases/metabolismo , Proteína Fosfatase 2 , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Pironas , Alinhamento de SequênciaRESUMO
Heparanase is an endo-beta-D-glucuronidase that degrades heparan sulfate glycosaminoglycans in the extracellular matrix and the basement membrane and is well known to be involved in tumor cell invasion and angiogenesis. We have focused on heparanase as a target for antitumor agents, especially antimetastatic agents. (R)-3-hexadecanoyl-5-hydroxymethyltetronic acid (RK-682) was found to display an inhibitory activity against heparanase in our screening of natural sources. Because RK-682 has been reported to show inhibitory activities against several enzymes, we have tried to develop selective heparanase inhibitors using the method of rational drug design. Based on the structure of the heparanase/RK-682 complex, we speculated that selective inhibitory activity against heparanase could be acquired by arylalkylation, namely, by benzylation of the 4-position of RK-682. Among the rationally designed 4-alkyl-RK-682 derivatives, 4-benzyl-RK-682 has been found to possess a selective inhibitory activity for heparanase (IC50 for heparanase, 17 micromol/L; IC50 for other enzymes, >100 micromol/L). 4-Benzyl-RK-682 also inhibited the invasion and migration of human fibrosarcoma HT1080 cells (IC50 for invasion, 1.5 micromol/L; IC50 for migration, 3.0 micromol/L). On the other hand, RK-682 had no inhibitory effect on the invasion and migration of HT1080 cells at doses of up to 100 micromol/L.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Movimento Celular/efeitos dos fármacos , Glucuronidase/antagonistas & inibidores , Metástase Neoplásica , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/farmacologia , Desenho de Fármacos , Humanos , Ligantes , Invasividade Neoplásica , Relação Estrutura-AtividadeRESUMO
Heparan sulfate glycosaminoglycans (HSGAGs) are involved in tumor cell growth, adhesion, invasion, and migration, due to their interactions with various proteins. In this study, novel HSGAG-mimetic compounds (KI compounds) were designed and synthesized. As a result of cell-based assays, KI-105 was found to exert potent inhibitory activities against migration and invasion of human fibrosarcoma HT1080 cells. The present results indicate that a novel invasion/migration inhibitor, KI-105, can increase the adherence of HT1080 cells. It was conceivable that this cellular effect was caused by an increase in the amount of cell-surface HSGAGs and focal adhesions. Although further investigations are needed to decipher the molecular mechanism of KI-105, it is suggested that heparanase and Cdc42 are involved in its biological effects.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Materiais Biomiméticos/química , Materiais Biomiméticos/farmacologia , Desenho de Fármacos , Heparitina Sulfato/química , Heparitina Sulfato/farmacologia , Antineoplásicos/síntese química , Materiais Biomiméticos/síntese química , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Glucuronidase/antagonistas & inibidores , Glucuronidase/metabolismo , Humanos , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Fenótipo , Relação Estrutura-AtividadeRESUMO
A biological evaluation of the antiadhesive activity of novel heparan sulfate glycosaminoglycans mimetic compounds (KI-compounds) is described. In an adhesion assay, KI-111 [2-(4-fluoro-3-nitrobenzoyl)benzoic acetic anhydride] was found to exert potent inhibitory activities against the adhesion of human fibrosarcoma HT1080 cells and HeLa cells to fibronectin. Cell growth, migration, and invasion of HT1080 cells were also inhibited by KI-111 at almost equal concentrations.
Assuntos
Antineoplásicos/síntese química , Heparitina Sulfato/análogos & derivados , Heparitina Sulfato/farmacologia , Invasividade Neoplásica/prevenção & controle , Antineoplásicos/farmacologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células , Relação Dose-Resposta a Droga , Desenho de Fármacos , Fibronectinas/metabolismo , Humanos , Concentração Inibidora 50 , Mimetismo Molecular , Relação Estrutura-AtividadeRESUMO
In this paper we describe the establishment of an efficient visual method for screening heparanase inhibitors, and we present the results of screening 10,000 microbial culture broths. Heparanase-overexpressing stable clones of the human hepatocellular carcinoma HepG2 cells were established and used as an enzyme source. Digestion of heparan sulfate (HS) was detected using novel HS-containing tablets or SDS-polyacrylamide gel electrophoresis. This method was able to find suramin, a known heparanase inhibitor, from a library of typical enzyme inhibitors. By screening 10,000 culture broths of microorganisms (actinomycetes, fungi, and bacteria) an actinomycete strain, RK99-A234, was found to have heparanase inhibitory activity. RK-682 was identified in the fermentation broth as a heparanase inhibitor, IC50 = 17 microM.