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1.
Biosci Biotechnol Biochem ; 85(12): 2434-2441, 2021 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-34506620

RESUMO

Rapeseed contains high levels of glucosinolates (GSLs), playing pivotal roles in defense against herbivores and pests. As their presence in rapeseed reduces the value of the meal for animal feeding, intensive efforts to reduce them produced low-seed GSL cultivars. However, there is no such variety suitable for the south part of Japan. Here, we tested the effects of cold oxygen plasma (oxygen CP) on seed germination and GSL and lipid content, in 3 rapeseed cultivars. According to the cultivars, oxygen CP slightly stimulated seed germination and modified the GSL levels, and decreased GSL levels in Kizakinonatane but increased those in Nanashikibu. In contrast, it negligibly affected the lipid content and composition in the 3 cultivars. Thus, oxygen CP modulated seed GSL levels without affecting seed viability and lipid content. Future optimization of this technique may help optimize rapeseed GSL content without plant breeding.


Assuntos
Glucosinolatos
2.
Plant Cell Physiol ; 61(6): 1095-1106, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-32255184

RESUMO

Glucosinolates (GSLs) are secondary metabolites that play important roles in plant defense and are suggested to act as storage compounds. Despite their important roles, metabolic dynamics of GSLs under various growth conditions remain poorly understood. To determine how light conditions influence the levels of different GSLs and their distribution in Arabidopsis leaves, we visualized the GSLs under different light conditions using matrix-assisted laser desorption/ionization mass spectrometry imaging. We observed the unique distribution patterns of each GSL in the inner regions of leaves and marked decreases under darkness, indicating light conditions influenced GSL metabolism. GSLs are hydrolyzed by a group of ß-glucosidase (BGLU) called myrosinase. Previous transcriptome data for GSL metabolism under light and dark conditions have revealed the highly induced expression of BGLU30, one of the putative myrosinases, which is also annotated as Dark INducible2, under darkness. Impairment of the darkness-induced GSL decrease in the disruption mutants of BGLU30, bglu30, indicated that BGLU30 mediated GSL hydrolysis under darkness. Based on the GSL profiles in the wild-type and bglu30 leaves under both conditions, short-chain GSLs were potentially preferable substrates for BGLU30. Our findings provide an effective way of visualizing GSL distribution in plants and highlighted the carbon storage GSL function.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Ciclo Celular/metabolismo , Glucosinolatos/metabolismo , Folhas de Planta/metabolismo , Celulases , Cisteína/metabolismo , Escuridão , Glutationa/metabolismo , Metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
3.
Plant Cell Physiol ; 61(4): 803-813, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32049325

RESUMO

Sulfur (S) is an essential element for plants, and S deficiency causes severe growth retardation. Although the catabolic process of glucosinolates (GSLs), the major S-containing metabolites specific to Brassicales including Arabidopsis, has been recognized as one of the S deficiency (-S) responses in plants, the physiological function of this metabolic process is not clear. Two ß-glucosidases (BGLUs), BGLU28 and BGLU30, are assumed to be responsible for this catabolic process as their transcript levels were highly upregulated by -S. To clarify the physiological function of BGLU28 and BGLU30 and their roles in GSL catabolism, we analyzed the accumulation of GSLs and other S-containing compounds in the single and double mutant lines of BGLU28 and BGLU30 and in wild-type plants under different S conditions. GSL levels were highly increased, while the levels of sulfate, cysteine, glutathione and protein were decreased in the double mutant line of BGLU28 and BGLU30 (bglu28/30) under -S. Furthermore, transcript level of Sulfate Transporter1;2, the main contributor of sulfate uptake from the environment, was increased in bglu28/30 mutants under -S. With these metabolic and transcriptional changes, bglu28/30 mutants displayed obvious growth retardation under -S. Overall, our results indicate that BGLU28 and BGLU30 are required for -S-induced GSL catabolism and contribute to sustained plant growth under -S by recycling sulfate to primary S metabolism.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Celulases/metabolismo , Glucosinolatos/metabolismo , Desenvolvimento Vegetal/genética , Enxofre/deficiência , Enxofre/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Cisteína/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica de Plantas , Glutationa/metabolismo , Sulfatos/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
4.
Anal Bioanal Chem ; 409(6): 1697-1706, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27933363

RESUMO

Information on spatiotemporal metabolic behavior is indispensable for a precise understanding of physiological changes and responses, including those of ripening processes and wounding stress, in fruit, but such information is still limited. Here, we visualized the spatial distribution of metabolites within tissue sections of tomato (Solanum lycopersicum L.) fruit using a matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) technique combined with a matrix sublimation/recrystallization method. This technique elucidated the unique distribution patterns of more than 30 metabolite-derived ions, including primary and secondary metabolites, simultaneously. To investigate spatiotemporal metabolic alterations during physiological changes at the whole-tissue level, MALDI-MSI was performed using the different ripening phenotypes of mature green and mature red tomato fruits. Although apparent alterations in the localization and intensity of many detected metabolites were not observed between the two tomatoes, the amounts of glutamate and adenosine monophosphate, umami compounds, increased in both mesocarp and locule regions during the ripening process. In contrast, malate, a sour compound, decreased in both regions. MALDI-MSI was also applied to evaluate more local metabolic responses to wounding stress. Accumulations of a glycoalkaloid, tomatine, and a low level of its glycosylated metabolite, esculeoside A, were found in the wound region where cell death had been induced. Their inverse levels were observed in non-wounded regions. Furthermore, the amounts of both compounds differed in the developmental stages. Thus, our MALDI-MSI technique increased the understanding of the physiological changes and responses of tomato fruit through the determination of spatiotemporally resolved metabolic alterations. Graphical abstract ᅟ.


Assuntos
Metaboloma , Metabolômica/métodos , Solanum lycopersicum/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Frutas/crescimento & desenvolvimento , Frutas/metabolismo , Frutas/fisiologia , Solanum lycopersicum/crescimento & desenvolvimento , Solanum lycopersicum/fisiologia
5.
Food Chem Toxicol ; 169: 113421, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36100043

RESUMO

Anticancer agents can cause various side effects, including tissue damages/inflammatory reactions. Drug-responsive biomarkers are essential for evaluating drug toxicity in disease processes. S100 calcium-binding proteins A8/A9 (S100A8/A9) are highly expressed in neutrophils and monocytes/macrophages accumulated at inflammatory sites and are known to be related to tissue damage/inflammation; however, their response to drug toxicity has not been reported. Herein, we investigated the effects of anticancer agents (doxorubicin, cisplatin, and docetaxel) on S100A8/A9 gene expression profiles in four representative tissues (heart, kidney, liver, and lung) in normal C57BL/6J mice. Both S100A8/A9 expression was transiently or time-dependently elevated in four tissues within 48 h after dosing of the three anticancer agents under toxicity-inducing conditions. S100A8/A9 patterns differed among agents and tissues. This result suggests that S100A8/A9 is useful for evaluating anticancer agent-induced tissue damage. Metabolomic analysis revealed that some metabolites showed temporal patterns similar to that of S100A8/A9 expression. The amounts of fumarate (doxorubicin-treated heart), tyrosine (cisplatin-treated kidney), acetylcarnosine (doxorubicin-treated liver), and 2-phosphoglycerate (docetaxel-treated lung) showed similar patterns to that of S100A8/A9 expression. Although these metabolites showed different behaviors between tissues and serum, they may be useful marker candidates for evaluating anticancer agent-induced tissue damage at an earlier stage after dosing.


Assuntos
Antineoplásicos , Biomarcadores Farmacológicos , Calgranulina A , Calgranulina B , Inflamação , Animais , Camundongos , Antineoplásicos/administração & dosagem , Antineoplásicos/toxicidade , Biomarcadores Farmacológicos/metabolismo , Calgranulina A/genética , Calgranulina A/metabolismo , Calgranulina B/genética , Calgranulina B/metabolismo , Cisplatino/administração & dosagem , Cisplatino/toxicidade , Docetaxel/administração & dosagem , Docetaxel/toxicidade , Doxorrubicina/administração & dosagem , Doxorrubicina/toxicidade , Fumaratos/análise , Inflamação/induzido quimicamente , Inflamação/metabolismo , Camundongos Endogâmicos C57BL , Tirosina/análise
6.
J Agric Food Chem ; 70(21): 6455-6466, 2022 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-35543229

RESUMO

There are numerous cultivars of tea (Camellia sinensis L.), but the differences in their anti-hyperglycemic-related effects are largely unknown. The inhibition of the dipeptidyl peptidase (DPP)-IV enzyme plays an essential role in controlling hyperglycemia in diabetes by blocking the degradation of incretin hormones, which is necessary for insulin secretion. In this study, we examined the DPP-IV inhibitory activity of leaf extracts from diverse Japanese green tea cultivars. The inhibitory rates differed among tea extracts. Metabolic profiling (MP), using liquid chromatography-mass spectrometry, of all cultivars revealed compositional differences among cultivars according to their DPP-IV inhibitory capacity. Epigallocatechin-3-O-(3-O-methyl)gallate, kaempferol-3-O-rutinoside, myricetin-3-O-glucoside/galactoside, and theogallin were newly identified as DPP-IV inhibitors. The bioactivity of a tea extract was potentiated by adding these ingredients in combination. Our results show that MP is a useful approach for evaluating the DPP-IV inhibitory potency of green tea and for determining bioactivity-related ingredients and combinations.


Assuntos
Camellia sinensis , Inibidores da Dipeptidil Peptidase IV , Camellia sinensis/química , Dipeptidil Peptidase 4 , Inibidores da Dipeptidil Peptidase IV/química , Metabolômica/métodos , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Chá/química
7.
Mol Cancer Res ; 18(9): 1354-1366, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32467171

RESUMO

DNA replication stress (DRS) is a predominant cause of genome instability, a driver of tumorigenesis and malignant progression. Nucleoside analogue-type chemotherapeutic drugs introduce DNA damage and exacerbate DRS in tumor cells. However, the mechanisms underlying the antitumor effect of these drugs are not fully understood. Here, we show that the fluorinated thymidine analogue trifluridine (FTD), an active component of the chemotherapeutic drug trifluridine/tipiracil, delayed DNA synthesis by human replicative DNA polymerases by acting both as an inefficient deoxyribonucleotide triphosphate source (FTD triphosphate) and as an obstacle base (trifluorothymine) in the template DNA strand, which caused DRS. In cells, FTD decreased the thymidine triphosphate level in the dNTP pool and increased the FTD triphosphate level, resulting in the activation of DRS-induced cellular responses during S-phase. In addition, replication protein A-coated single-stranded DNA associated with FancD2 and accumulated after tumor cells completed S-phase. Finally, FTD activated the p53-p21 pathway and suppressed tumor cell growth by inducing cellular senescence via mitosis skipping. In contrast, tumor cells that lost wild-type p53 underwent apoptotic cell death via aberrant late mitosis with severely impaired separation of sister chromatids. These results demonstrate that DRS induced by a nucleoside analogue-type chemotherapeutic drug suppresses tumor growth irrespective of p53 status by directing tumor cell fate toward cellular senescence or apoptotic cell death according to p53 status. IMPLICATIONS: Chemotherapeutic drugs that increase DRS during S-phase but allow tumor cells to complete S-phase may have significant antitumor activity even when functional p53 is lost.


Assuntos
Antivirais/uso terapêutico , Replicação do DNA/efeitos dos fármacos , Trifluridina/uso terapêutico , Proteína Supressora de Tumor p53/genética , Animais , Antivirais/farmacologia , Humanos , Masculino , Camundongos , Camundongos Nus , Trifluridina/farmacologia
8.
Plants (Basel) ; 8(4)2019 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-30974830

RESUMO

Plants take up sulfur (S), an essential element for all organisms, as sulfate, which is mainly attributed to the function of SULTR1;2 in Arabidopsis. A disruption mutant of SULTR1;2, sel1-10, has been characterized with phenotypes similar to plants grown under sulfur deficiency (-S). Although the effects of -S on S metabolism were well investigated in seedlings, no studies have been performed on mature Arabidopsis plants. To study further the effects of -S on S metabolism, we analyzed the accumulation and distribution of S-containing compounds in different parts of mature sel1-10 and of the wild-type (WT) plants grown under long-day conditions. While the levels of sulfate, cysteine, and glutathione were almost similar between sel1-10 and WT, levels of glucosinolates (GSLs) differed between them depending on the parts of the plant. GSLs levels in the leaves and stems were generally lower in sel1-10 than those in WT. However, sel1-10 seeds maintained similar levels of aliphatic GSLs to those in WT plants. GSL accumulation in reproductive tissues is likely to be prioritized even when sulfate supply is limited in sel1-10 for its role in S storage and plant defense.

9.
J Am Soc Mass Spectrom ; 30(8): 1512-1520, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31044355

RESUMO

Matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry imaging (MSI) is a powerful technique to visualize the distributions of biomolecules without any labeling. In MALDI-MSI experiments, the choice of matrix deposition method is important for acquiring favorable MSI data with high sensitivity and high reproducibility. Generally, manual or automated spray-coating and automated sublimation methods are used, but these methods have some drawbacks with respect to detection sensitivity, spatial resolution, and data reproducibility. Herein, we present an optimized matrix deposition method of sublimation coupled with recrystallization using 9-aminoacridine (9-AA) as a matrix capable of ionizing endogenous metabolites. The matrix recrystallization process after sublimation was optimized for the solvent concentration and reaction temperature for matrix-metabolite co-crystallization. This optimized method showed excellent reproducibility and spatial resolution compared to the automatic spray-coating method. Furthermore, the recrystallization step after sublimation remarkably improved the detectability of metabolites, including amino acids, nucleotide derivatives, and lipids, compared with the conventional sublimation method. To date, there have been no other reports of 9-AA-based sublimation combined with recrystallization. The present method provides an easy, sensitive, and reproducible matrix deposition method for MALDI-MSI of endogenous metabolites. Graphical Abstract.


Assuntos
Química Encefálica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Desenho de Equipamento , Masculino , Camundongos Endogâmicos C57BL , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/instrumentação
10.
Mol Cancer Res ; 16(10): 1483-1490, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29866926

RESUMO

Acquired resistance to therapeutic drugs is a serious problem for patients with cancer receiving systemic treatment. Experimentally, drug resistance is established in cell lines in vitro by repeated, continuous exposure to escalating concentrations of the drug; however, the precise mechanism underlying the acquired resistance is not always known. Here, it is demonstrated that the human colorectal cancer cell line DLD1 with acquired resistance to trifluridine (FTD), a key component of the novel, orally administered nucleoside analogue-type chemotherapeutic drug trifluridine/tipiracil, lacks functional thymidine kinase 1 (TK1) expression because of one nonsense mutation in the coding exon. Targeted disruption of the TK1 gene also conferred severe FTD resistance, indicating that the loss of TK1 protein expression is the primary cause of FTD resistance. Both FTD-resistant DLD1 cells and DLD1-TK1 -/- cells exhibited similar 5-fluorouracil (5-FU) sensitivity to that of the parental DLD1 line. The quantity of cellular pyrimidine nucleotides in these cells and the kinetics of thymidylate synthase ternary complex formation in 5-FU-treated cells is similar to DLD1 cells, indicating that 5-FU metabolism and cytotoxicity were unaffected. The current data provide molecular-based evidence that acquired resistance to FTD does not confer 5-FU resistance, implying that 5-FU-based chemotherapy would be effective even in tumors that become refractory to FTD during trifluridine/tipiracil treatment. Mol Cancer Res; 16(10); 1483-90. ©2018 AACR.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Timidina Quinase/genética , Animais , Antimetabólitos Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Éxons/genética , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Trifluridina/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Toxins (Basel) ; 10(1)2017 12 28.
Artigo em Inglês | MEDLINE | ID: mdl-29283413

RESUMO

Elevated circulating uremic toxins are associated with a variety of symptoms and organ dysfunction observed in patients with chronic kidney disease (CKD). Indoxyl sulfate (IS) and p-cresyl sulfate (PCS) are representative uremic toxins that exert various harmful effects. We recently showed that IS induces metabolic alteration in skeletal muscle and causes sarcopenia in mice. However, whether organ-specific accumulation of IS and PCS is associated with tissue dysfunction is still unclear. We investigated the accumulation of IS and PCS using liquid chromatography/tandem mass spectrometry in various tissues from mice with adenine-induced CKD. IS and PCS accumulated in all 15 organs analyzed, including kidney, skeletal muscle, and brain. We also visualized the tissue accumulation of IS and PCS with immunohistochemistry and mass spectrometry imaging techniques. The oral adsorbent AST-120 prevented some tissue accumulation of IS and PCS. In skeletal muscle, reduced accumulation following AST-120 treatment resulted in the amelioration of renal failure-associated muscle atrophy. We conclude that uremic toxins can accumulate in various organs and that AST-120 may be useful in treating or preventing organ dysfunction in CKD, possibly by reducing tissue accumulation of uremic toxins.


Assuntos
Carbono/uso terapêutico , Cresóis/metabolismo , Indicã/metabolismo , Falência Renal Crônica/tratamento farmacológico , Óxidos/uso terapêutico , Ésteres do Ácido Sulfúrico/metabolismo , Toxinas Biológicas/metabolismo , Administração Oral , Adsorção , Animais , Cromatografia Líquida , Falência Renal Crônica/diagnóstico por imagem , Falência Renal Crônica/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Espectrometria de Massas em Tandem , Uremia
12.
Sci Rep ; 6: 36618, 2016 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-27830716

RESUMO

Sarcopenia is associated with increased morbidity and mortality in chronic kidney disease (CKD). Pathogenic mechanism of skeletal muscle loss in CKD, which is defined as uremic sarcopenia, remains unclear. We found that causative pathological mechanism of uremic sarcopenia is metabolic alterations by uremic toxin indoxyl sulfate. Imaging mass spectrometry revealed indoxyl sulfate accumulated in muscle tissue of a mouse model of CKD. Comprehensive metabolomics revealed that indoxyl sulfate induces metabolic alterations such as upregulation of glycolysis, including pentose phosphate pathway acceleration as antioxidative stress response, via nuclear factor (erythroid-2-related factor)-2. The altered metabolic flow to excess antioxidative response resulted in downregulation of TCA cycle and its effected mitochondrial dysfunction and ATP shortage in muscle cells. In clinical research, a significant inverse association between plasma indoxyl sulfate and skeletal muscle mass in CKD patients was observed. Our results indicate that indoxyl sulfate is a pathogenic factor for sarcopenia in CKD.


Assuntos
Indicã/metabolismo , Músculo Esquelético/metabolismo , Insuficiência Renal Crônica/metabolismo , Sarcopenia/metabolismo , Uremia/metabolismo , Animais , Modelos Animais de Doenças , Masculino , Camundongos , Músculo Esquelético/patologia , Insuficiência Renal Crônica/patologia , Sarcopenia/patologia , Uremia/patologia
13.
Mol Cancer Ther ; 14(10): 2332-42, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26208523

RESUMO

Platinum-based chemotherapeutic drugs are widely used as components of combination chemotherapy in the treatment of cancer. One such drug, oxaliplatin, exerts a synergistic effect against advanced colorectal cancer in combination with 5-fluorouracil (5-FU) and leucovorin. In the p53-proficient colorectal cancer cell line HCT116, oxaliplatin represses the expression of deoxyuridine triphosphatase (dUTPase), a ubiquitous pyrophosphatase that catalyzes the hydrolysis of dUTP to dUMP and inhibits dUTP-mediated cytotoxicity. However, the underlying mechanism of this activity has not been completely elucidated, and it remains unclear whether factors other than downregulation of dUTPase contribute to the synergistic effect of 5-FU and oxaliplatin. In this study, we found that oxaliplatin and dachplatin, platinum-based drugs containing the 1,2-diaminocyclohexane (DACH) carrier ligand, repressed the expression of nuclear isoform of dUTPase (DUT-N), whereas cisplatin and carboplatin did not. Oxaliplatin induced early p53 accumulation, upregulation of primary miR-34a transcript expression, and subsequent downregulation of E2F3 and E2F1. Nutlin-3a, which activates p53 nongenotoxically, had similar effects. Introduction of miR-34a mimic also repressed E2F1 and DUT-N expression, indicating that this miRNA plays a causative role. In addition to DUT-N, oxaliplatin repressed, in a p53-dependent manner, the expression of genes encoding enzymes involved in thymidylate biosynthesis. Consequently, oxaliplatin significantly decreased the level of dTTP in the dNTP pool in a p53-dependent manner. These data indicate that the DACH carrier ligand in oxaliplatin triggers signaling via the p53-miR-34a-E2F axis, leading to transcriptional regulation that ultimately results in accumulation of dUTP and reduced dTTP biosynthesis, potentially enhancing 5-FU cytotoxicity.


Assuntos
Inativação Gênica/efeitos dos fármacos , Compostos Organoplatínicos/farmacologia , Timidina Monofosfato/biossíntese , Transcrição Gênica/efeitos dos fármacos , Proteína Supressora de Tumor p53/fisiologia , Vias Biossintéticas , Replicação do DNA , Regulação para Baixo , Sinergismo Farmacológico , Fator de Transcrição E2F1/genética , Fator de Transcrição E2F1/metabolismo , Fator de Transcrição E2F3/genética , Fator de Transcrição E2F3/metabolismo , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HCT116 , Humanos , Imidazóis/farmacologia , MicroRNAs/genética , MicroRNAs/metabolismo , Compostos Organoplatínicos/química , Oxaliplatina , Piperazinas/farmacologia , Pirofosfatases/genética , Pirofosfatases/metabolismo , Fator de Transcrição Sp1/metabolismo
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