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1.
Anal Chem ; 93(29): 10075-10083, 2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34270209

RESUMO

Metabolomics is a powerful and essential technology for profiling metabolic phenotypes and exploring metabolic reprogramming, which enables the identification of biomarkers and provides mechanistic insights into physiology and disease. However, its applications are still limited by the technical challenges particularly in its detection sensitivity for the analysis of biological samples with limited amount, necessitating the development of highly sensitive approaches. Here, we developed a highly sensitive liquid chromatography tandem mass spectrometry method based on a 3-nitrophenylhydrazine (3-NPH) derivatization strategy that simultaneously targets carbonyl, carboxyl, and phosphoryl groups for targeted metabolomic analysis (HSDccp-TM) in biological samples. By testing 130 endogenous metabolites including organic acids, amino acids, carbohydrates, nucleotides, carnitines, and vitamins, we showed that the derivatization strategy resulted in significantly improved detection sensitivity and chromatographic separation capability. Metabolic profiling of merely 60 oocytes and 5000 hematopoietic stem cells primarily isolated from mice demonstrated that this method enabled routine metabolomic analysis in trace amounts of biospecimens. Moreover, the derivatization strategy bypassed the tediousness of inferring the MS fragmentation patterns and simplified the complexity of monitoring ion pairs of metabolites, which greatly facilitated the metabolic flux analysis (MFA) for glycolysis, the tricarboxylic acid (TCA) cycle, and pentose phosphate pathway (PPP) in cell cultures. In summary, the novel 3-NPH derivatization-based method with high sensitivity, good chromatographic separation, and broad coverage showed great potential in promoting metabolomics and MFA, especially in trace amounts of biospecimens.


Assuntos
Metabolômica , Espectrometria de Massas em Tandem , Animais , Cromatografia Líquida , Camundongos , Fenil-Hidrazinas
2.
Talanta ; 233: 122522, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34215137

RESUMO

Aldehydes are toxic carbonyl compounds that are identified in various matrices surrounding us. For instance, aldehydes could be formed during the cooking and frying of foods which affects the food quality and safety. Derivatization is a must for the determination of aldehydes as they lack intrinsic chromophoric groups. 2,4-Dinitrophenyl hydrazine (DNPH) is the most used derivatizing reagent for aldehydes and the formed hydrazones could be determined by either HPLC-UV or LC-MS. However, UV detection is non-sensitive, and the MS equipment is expensive and not widely available. Thus, herein we report a smart chemiluminescence (CL) detection method for the DNPH aldehydes derivatives. These derivatives are supposed to possess photosensitization ability due to the presence of strong chromophoric structures; nitrobenzene and phenyl hydrazone. Upon their UV irradiation, singlet oxygen is found to be produced which then converts the DNPH-aldehyde derivative into hydroperoxide. Next, the hydroperoxide reacts with luminol in an alkaline medium producing a strong CL. An HPLC system with online UV irradiation and online reaction with luminol followed by CL detection was constructed and used for the determination of aldehydes after their derivatization with DNPH. The developed method showed excellent sensitivity with detection limits down to 1.5-18.5 nM. The achieved sensitivity is superior to that obtained by HPLC-UV and LC-MS detection methods for DNPH-aldehydes derivatives. Additionally, our approach is an chemiluminogenic where the DNPH reagent itself does not produce CL which is an excellent advantage. The method was applied successfully for the determination of aldehydes in canola oil samples using simple liquid-liquid extraction showing good recovery (87.0-106.0%), accuracy (87.2-106.6), and precision (RSD≤10.2%). After analysis of fresh and heated oil samples, it was demonstrated that heating of oil, even for short time, strongly elevated the level of their aldehydes' content. At last, it was found that the results of the analysis of aldehydes in oil samples using the proposed method perfectly matched those obtained by a reference LC-MS method.


Assuntos
Aldeídos , Luminol , Cromatografia Líquida de Alta Pressão , Hidrazinas , Luminescência , Fenil-Hidrazinas
3.
Phytomedicine ; 87: 153579, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33991865

RESUMO

BACKGROUND: Hydroxysafflor yellow A (HSYA) from the flower of Carthamus tinctorius (Safflower) has been reported to have various pharmacological effects. However, little is known about the bioactivities of other chemical constituents in Safflower and the relationship between enhancement of blood circulation and hepatoprotection by HSYA. PURPOSE: The present research was to evaluate the antithrombotic and hepatoprotective activities of HSYA and C, examine their mechanisms of actions, including influence on the excretion velocity of acetaminophen, and the relationship between the antithrombotic, hepatoprotective, and other bioactivities. METHODS: The hepatoprotective activities were examined by acetaminophen (APAP)-induced zebrafish toxicity and carbon tetrachloride (CCl4)-induced mouse liver injury. The concentrations of APAP in zebrafish and APAP that was excreted to the culture media were quantified by UHPLC-MS. The anti-thrombosis effect of HSYA and C were examined by the phenylhydrazine (PHZ)-induced zebrafish thrombosis. RESULTS: HSYA and HSYC showed robust protection on APAP-induced toxicity and PHZ-induced thrombosis. The hepatoprotective effects of HSYA and C were more potent than that of the positive control, acetylcysteine (61.7% and 58.0%, respectively, vs. 56.9% at 100 µM) and their antithrombosis effects were more robust than aspirin (95.1% and 86.2% vs. 52.7% at 100 µM). HSYA and C enhanced blood circulation, rescued APAP-treated zebrafish from morphological abnormalities, and mitigated APAP-induced toxicity in liver development in liver-specific RFP-expressing transgenic zebrafish. HSYC attenuated CCl4-induced mouse liver injury and regulated the levels of HIF-1α, iNOS, TNF-α, α-SMA, and NFκB in liver tissues. HSYA was also protective in a dual thrombotic and liver toxicity zebrafish model. By UHPLC-MS, HSYA accelerated the excretion of APAP. CONCLUSION: HSYA and C are the bioactive constituents of Safflower that are responsible for the herbal drug's traditional use in promoting blood circulation to remove blood stasis. Safflower and its chalcone constituents may protect from damage due to exogenous or disease-induced endogenous toxins by enhancing the excretion velocity of toxins.


Assuntos
Acetaminofen/toxicidade , Chalcona/análogos & derivados , Fibrinolíticos/farmacologia , Substâncias Protetoras/farmacologia , Quinonas/farmacologia , Acetaminofen/farmacocinética , Animais , Animais Geneticamente Modificados , Circulação Sanguínea/efeitos dos fármacos , Tetracloreto de Carbono/toxicidade , Carthamus tinctorius/química , Chalcona/isolamento & purificação , Chalcona/farmacologia , Chalconas/isolamento & purificação , Chalconas/farmacologia , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Doença Hepática Induzida por Substâncias e Drogas/patologia , Glicosídeos/isolamento & purificação , Glicosídeos/farmacologia , Hepatócitos/efeitos dos fármacos , Humanos , Masculino , Camundongos Endogâmicos ICR , Fenil-Hidrazinas/toxicidade , Substâncias Protetoras/química , Substâncias Protetoras/isolamento & purificação , Quinonas/isolamento & purificação , Trombose/induzido quimicamente , Trombose/tratamento farmacológico , Peixe-Zebra/genética
4.
Front Immunol ; 12: 680855, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34054870

RESUMO

Clearance of red blood cells and hemoproteins is a key metabolic function of macrophages during hemolytic disorders and following tissue injury. Through this archetypical phagocytic function, heme is detoxified and iron is recycled to support erythropoiesis. Reciprocal interaction of heme metabolism and inflammatory macrophage functions may modify disease outcomes in a broad range of clinical conditions. We hypothesized that acute hemolysis and heme induce acute anti-inflammatory signals in liver macrophages. Using a macrophage-driven model of sterile liver inflammation, we showed that phenylhydrazine (PHZ)-mediated acute erythrophagocytosis blocked the anti-CD40 antibody-induced pathway of macrophage activation. This process attenuated the inflammatory cytokine release syndrome and necrotizing hepatitis induced by anti-CD40 antibody treatment of mice. We further established that administration of heme-albumin complexes specifically delivered heme to liver macrophages and replicated the anti-inflammatory effect of hemolysis. The anti-inflammatory heme-signal was induced in macrophages by an increased intracellular concentration of the porphyrin independently of iron. Overall, our work suggests that induction of heme-signaling strongly suppresses inflammatory macrophage function, providing protection against sterile liver inflammation.


Assuntos
Anticorpos/imunologia , Antígenos CD40/antagonistas & inibidores , Antígenos CD40/imunologia , Hemólise/imunologia , Hepatite/etiologia , Albuminas/metabolismo , Animais , Anticorpos/efeitos adversos , Biópsia , Modelos Animais de Doenças , Suscetibilidade a Doenças , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Eritrócitos/patologia , Perfilação da Expressão Gênica , Heme/metabolismo , Hepatite/metabolismo , Hepatite/patologia , Ferro/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Fenil-Hidrazinas/efeitos adversos , Porfirinas/metabolismo , Ligação Proteica
5.
Bioorg Chem ; 112: 104957, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34020240

RESUMO

Members of the ectonucleoside triphosphate diphosphohydrolases (NTPDases) constitute the major family of enzymes responsible for the maintenance of extracellular levels of nucleotides and nucleosides by catalyzing the hydrolysis of nucleoside triphosphate (NTP) and nucleoside diphosphates (NDP) to nucleoside monophosphate (NMP). Although, NTPDase inhibitors can act as potential drug candidates for the treatment of various diseases, there is lack of potent as well as selective inhibitors of NTPDases. The current study describes the synthesis of a number of carboxamide derivatives that were tested on recombinant human (h) NTPDases. The most promising inhibitors were 2h (h-NTPDase1, IC50: 0.12 ± 0.03 µM), 2d (h-NTPDase2, IC50: 0.15 ± 0.01 µM) and 2a (h-NTPDase3, IC50: 0.30 ± 0.04 µM; h-NTPDase8, IC50: 0.16 ± 0.02 µM). Four compounds (2e, 2f, 2g and 2h) were associated with the selective inhibition of h-NTPDase1 while 2b was identified as a selective h-NTPDase3 inhibitor. Considering the importance of NTPDase3 in the regulation of insulin release, the NTPDase3 inhibitors were further investigated to elucidate their role in the insulin release. The obtained data suggested that compound 2a was actively participating in regulating the insulin release without producing any effect on NTPDase3 mRNA. Moreover, the most potent inhibitors were docked within the active site of respective enzyme and the observed interactions were in compliance with in vitro results. Hence, these compounds can be used as pharmacological tool to further investigate the role of NTPDase3 coupled to insulin release.


Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Indóis/farmacologia , Simulação de Acoplamento Molecular , Fenil-Hidrazinas/farmacologia , Adenosina Trifosfatases/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Indóis/síntese química , Indóis/química , Estrutura Molecular , Fenil-Hidrazinas/síntese química , Fenil-Hidrazinas/química , Relação Estrutura-Atividade
6.
J Biol Chem ; 296: 100397, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33571527

RESUMO

Since the discovery of the prolyl hydroxylases domain (PHD) proteins and their canonical hypoxia-inducible factor (HIF) substrate two decades ago, a number of in vitro hydroxylation (IVH) assays for PHD activity have been developed to measure the PHD-HIF interaction. However, most of these assays either require complex proteomics mass spectrometry methods that rely on the specific PHD-HIF interaction or require the handling of radioactive material, as seen in the most commonly used assay measuring [14C]O2 release from labeled [14C]α-ketoglutarate. Here, we report an alternative rapid, cost-effective assay in which the consumption of α-ketoglutarate is monitored by its derivatization with 2,4-dinitrophenylhydrazine (2,4-DNPH) followed by treatment with concentrated base. We extensively optimized this 2,4-DNPH α-ketoglutarate assay to maximize the signal-to-noise ratio and demonstrated that it is robust enough to obtain kinetic parameters of the well-characterized PHD2 isoform comparable with those in published literature. We further showed that it is also sensitive enough to detect and measure the IC50 values of pan-PHD inhibitors and several PHD2 inhibitors in clinical trials for chronic kidney disease (CKD)-induced anemia. Given the efficiency of this assay coupled with its multiwell format, the 2,4-DNPH α-KG assay may be adaptable to explore non-HIF substrates of PHDs and potentially to high-throughput assays.


Assuntos
Colorimetria/métodos , Prolina Dioxigenases do Fator Induzível por Hipóxia/análise , Ácidos Cetoglutáricos/análise , Fenil-Hidrazinas/química , Ensaios Enzimáticos/métodos , Humanos , Hidroxilação , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Ácidos Cetoglutáricos/química , Cinética , Especificidade por Substrato
7.
J Sep Sci ; 44(7): 1552-1563, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33475238

RESUMO

Danqi tablet composed of the dried roots of Salvia miltiorrhiza and Panax notoginseng is a well-known Chinese patent medicine commonly used for the treatment of cardio-cerebrovascular diseases such as coronary heart disease and myocardial ischemia. Numerous chemical constituents belonging to S. miltiorrhiza and P. notoginseng were detectable in Danqi tablet. Here, we established and validated a rapid and sensitive ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry method for simultaneous quantification of 23 components in Danqi tablet and then successfully applied to assay 12 batches of samples from ten manufacturers. Our results demonstrated that the contents of 23 components in 12 batches of Danqi tablets varied significantly and their quality indeed existed differently based on the principal component analysis. According to the quantitative data and the loading plot of principal component analysis, five abundant compounds in Danqi tablet were selected as characteristic chemical markers possibly responsible for the quality assessment. Among them, salvianolic acid B and ginsenoside Rg1 were further chosen to be combined at 2:5 ratio to evaluate the anti-thrombotic activity on phenylhydrazine-induced zebrafish heart thrombosis model. Expectedly, this component combination increased the heart red blood cells intensity compared with the model group and the median effective concentration was 123.4 µg/mL, suggestive of its well anti-thrombotic effect. This study contributed to the quantitative evaluation of Danqi tablet and indicated the combination of salvianolic acid B and ginsenoside Rg1 may be capable of reflecting the effect of Danqi tablet, thereby providing a reference for further investigations on the improvement of quality control and clinical application of Danqi tablet.


Assuntos
Medicamentos de Ervas Chinesas/análise , Animais , Bioensaio , Cromatografia Líquida de Alta Pressão , Medicamentos de Ervas Chinesas/uso terapêutico , Espectrometria de Massas , Fenil-Hidrazinas , Comprimidos/análise , Trombose/induzido quimicamente , Trombose/tratamento farmacológico , Peixe-Zebra
8.
J Agric Food Chem ; 69(4): 1405-1412, 2021 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-33470094

RESUMO

A rapid, high-throughput method for the quantitation of the 2-acetyl azaheterocycles, 2-acetyl-1-pyrroline, 2-acetyl-1,4,5,6-tetrahydropyridine, 2-acetylpyrazine, and 2-acetyl-2-thiazoline, in different food products, by liquid chromatography-tandem mass spectrometry (LC-MS/MS), was developed. The quick extraction by bead beater homogenization, fast derivatization by 3-nitrophenylhydrazine (40 °C, 2 h), and efficient LC separation make this method suitable for high-throughput analysis. As established in this study, the highly precise LC-MS/MS method applies to different food products or beverages without requiring further adjustment. The analysis was performed with sample amounts of 0.2-0.5 g, and limit of quantitation values of 0.6, 0.5, 0.6, and 1.0 µg/kg were obtained for 2-acetyl-1-pyrroline, 2-acetyl-1,4,5,6-tetrahydropyridine, 2-acetylpyrazine, and 2-acetyl-2-thiazoline, respectively. Thus, it was possible to quantitate the analytes in the range of their odor thresholds.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Aromatizantes/química , Odorantes/análise , Oryza/química , Espectrometria de Massas em Tandem/métodos , Fenil-Hidrazinas/análise , Pirazinas/análise , Pirróis/análise
9.
Artigo em Inglês | MEDLINE | ID: mdl-33035680

RESUMO

Hematopoiesis, the complex developmental process that forms blood components and replenishes the blood system, involves multiple intracellular and extracellular mechanisms. We previously demonstrated that lysophosphatidic acid (LPA), a lipid growth factor, has opposing regulatory effects on erythrocyte differentiation through activation of LPA receptors 2 and 3; yet the mechanisms underlying this process remain unclear. In this study, LPA2 is observed that highly expressed in common myeloid progenitors (CMP) in murine myeloid cells, whereas the expression of LPA3 displaces in megakaryocyte-erythroid progenitors (MEP) of later stage of myeloid differentiation. Therefore, we hypothesized that the switching expression of LPA2 and LPA3 determine the hematic homeostasis of mammalian megakaryocytic-erythroid lineage. In vitro colony-forming unit assays of murine progenitors reveal that LPA2 agonist GRI reduces the erythroblast differentiation potential of CMP. In contrast, LPA3 agonist OMPT increases the production of erythrocytes from megakaryocyte-erythrocyte progenitor cells (MEP). In addition, treatment with GRI reduces the erythroid, CMP, and MEP populations in mice, indicating that LPA2 predominantly inhibits myeloid differentiation at an early stage. In contrast, activation of LPA3 increases the production of terminally differentiated erythroid cells through activation of erythropoietic transcriptional factor. We also demonstrate that the LPA3 signaling is essential for restoration of phenylhydrazine (PHZ)-induced acute hemolytic anemia in mice and correlates to erythropoiesis impairment of Hutchinson-Gilford progeria Symptom (HGPS) premature aging expressed K562 model. Our results reveal the distinct roles of LPA2 and LPA3 at different stages of hematopoiesis in vivo, providing potentiated therapeutic strategies of anemia treatment.


Assuntos
Anemia Hemolítica/genética , Células Eritroides/metabolismo , Eritropoese/genética , Células Mieloides/metabolismo , Receptores de Ácidos Lisofosfatídicos/genética , Células-Tronco/metabolismo , Anemia Hemolítica/induzido quimicamente , Anemia Hemolítica/tratamento farmacológico , Anemia Hemolítica/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Linhagem da Célula/genética , Modelos Animais de Doenças , Células Eritroides/citologia , Células Eritroides/efeitos dos fármacos , Eritropoese/efeitos dos fármacos , Regulação da Expressão Gênica , Humanos , Isoquinolinas/farmacologia , Células K562 , Lisofosfolipídeos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Células Mieloides/citologia , Células Mieloides/efeitos dos fármacos , Organotiofosfatos/farmacologia , Fenil-Hidrazinas/administração & dosagem , Ácidos Fosfatídicos/farmacologia , Receptores de Ácidos Lisofosfatídicos/agonistas , Receptores de Ácidos Lisofosfatídicos/metabolismo , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos
10.
Molecules ; 25(23)2020 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-33297566

RESUMO

In leathers, formaldehyde is currently analyzed according to EN ISO 17226-1 standard, by reversed phase liquid chromatography after off-line precolumn derivatization with 2,4 dinitrophenylhydrazine (DNPH) in strong acidic conditions. We first demonstrate that this standard is not adapted to leather retanned with resins likely to release formaldehyde by hydrolysis. Indeed, formaldehyde content may be largely overestimated due to concomitant resin hydrolysis (in harsh acidic conditions) that releases formaldehyde during the derivatization step and during the waiting time on autosampler before analysis. Therefore, we thoroughly studied the derivatization step in order to propose new derivatization conditions. Replacing orthophosphoric acid by less acidic buffer solutions is not enough to avoid hydrolysis. A derivatization without adding acid is realized by solubilizing DNPH in acetonitrile instead of orthophosphoric acid. These conditions lead to a complete derivatization of formaldehyde in 3 h at 50 °C (in a water bath) while avoiding the hydrolysis of co-extracted dicyandiamide and melamine resins. The as-obtained leather extracts are stable over time. Formaldehyde contents found with this method agree with the formaldehyde content measured immediately at the end of derivatization reaction in standard conditions or with formaldehyde content measured by a home-designed flow injection analysis with acetylacetone online derivatization and UV detection.


Assuntos
Técnicas Biossensoriais/métodos , Formaldeído/análise , Pele/química , Animais , Cromatografia Líquida de Alta Pressão/métodos , Formaldeído/química , Fenil-Hidrazinas/análise , Fenil-Hidrazinas/química
11.
Bioorg Med Chem Lett ; 30(21): 127519, 2020 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-32860979

RESUMO

Aiming to discover novel high-efficient antifungal leads that possess an innovative action mechanism, twenty-three carboxylated pyrroline-2-one derivatives, bearing a phenylhydrazine moiety, were rationally designed and firstly prepared in this letter. The in vitro bioassays showed that most of the compounds possessed excellent antifungal effects with the EC50 values of less than 1 µg/mL against the phytopathogenic fungi Fusarium graminearum (Fg), Botrytis cinerea (Bc), Rhizoctonia solani (Rs) and Colletotrichum capsici (Cc). The further bioassays showed that the compound 6u showed the comparable in vivo control effect with carbendazim against fusarium head blight and rice sheath blight. The 3D-QSAR model revealed the pivotal effects of a bulky electron-donating group at the 1-position of pyrrole ring, a bulky electron-withdrawing group at the 4-position of phenyl ring and a small alkyl at the carbonate group on the anti-Rs activities of target compounds. The abnormal mycelial morphology and delayed spore germination were observed in the treatments of compound 6u. Given the excellent and broad-spectrum antifungal effects the target compounds have, we unfeignedly anticipated that the above finding could motivate the discovery of high-efficient antifungal leads, which might possess an innovative action mechanism against phytopathogenic fungi.


Assuntos
Antifúngicos/farmacologia , Desenho de Fármacos , Fenil-Hidrazinas/farmacologia , Pirróis/farmacologia , Relação Quantitativa Estrutura-Atividade , Antifúngicos/síntese química , Antifúngicos/química , Botrytis/efeitos dos fármacos , Colletotrichum/efeitos dos fármacos , Relação Dose-Resposta a Droga , Fusarium/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Fenil-Hidrazinas/química , Pirróis/síntese química , Pirróis/química , Rhizoctonia/efeitos dos fármacos
12.
Front Immunol ; 11: 1772, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32849636

RESUMO

Intravascular hemolysis of any cause can induce acute kidney injury (AKI). Hemolysis-derived product heme activates the innate immune complement system and contributes to renal damage. Therefore, we explored the role of the master complement regulator Factor H (FH) in the kidney's resistance to hemolysis-mediated AKI. Acute systemic hemolysis was induced in mice lacking liver expression of FH (hepatoFH-/-, ~20% residual FH) and in WT controls, by phenylhydrazine injection. The impaired complement regulation in hepatoFH-/- mice resulted in a delayed but aggravated phenotype of hemolysis-related kidney injuries. Plasma urea as well as markers for tubular (NGAL, Kim-1) and vascular aggression peaked at day 1 in WT mice and normalized at day 2, while they increased more in hepatoFH-/- compared to the WT and still persisted at day 4. These were accompanied by exacerbated tubular dilatation and the appearance of tubular casts in the kidneys of hemolytic hepatoFH-/- mice. Complement activation in hemolytic mice occurred in the circulation and C3b/iC3b was deposited in glomeruli in both strains. Both genotypes presented with positive staining of FH in the glomeruli, but hepatoFH-/- mice had reduced staining in the tubular compartment. Despite the clear phenotype of tubular injury, no complement activation was detected in the tubulointerstitium of the phenylhydrazin-injected mice irrespective of the genotype. Nevertheless, phenylhydrazin triggered overexpression of C5aR1 in tubules, predominantly in hepatoFH-/- mice. Moreover, C5b-9 was deposited only in the glomeruli of the hemolytic hepatoFH-/- mice. Therefore, we hypothesize that C5a, generated in the glomeruli, could be filtered into the tubulointerstitium to activate C5aR1 expressed by tubular cells injured by hemolysis-derived products and will aggravate the tissue injury. Plasma-derived FH is critical for the tubular protection, since pre-treatment of the hemolytic hepatoFH-/- mice with purified FH attenuated the tubular injury. Worsening of acute tubular necrosis in the hepatoFH-/- mice was trigger-dependent, as it was also observed in LPS-induced septic AKI model but not in chemotherapy-induced AKI upon cisplatin injection. In conclusion, plasma FH plays a key role in protecting the kidneys, especially the tubules, against hemolysis-mediated injury. Thus, FH-based molecules might be explored as promising therapeutic agents in a context of AKI.


Assuntos
Ativação do Complemento , Fator H do Complemento/metabolismo , Hemólise , Hepatócitos/metabolismo , Glomérulos Renais/metabolismo , Necrose Tubular Aguda/prevenção & controle , Túbulos Renais/metabolismo , Animais , Complemento C5a/genética , Complemento C5a/metabolismo , Fator H do Complemento/genética , Modelos Animais de Doenças , Regulação da Expressão Gênica , Glomérulos Renais/patologia , Necrose Tubular Aguda/sangue , Necrose Tubular Aguda/induzido quimicamente , Necrose Tubular Aguda/patologia , Túbulos Renais/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenil-Hidrazinas , Receptor da Anafilatoxina C5a/genética , Receptor da Anafilatoxina C5a/metabolismo , Transdução de Sinais
13.
Eur J Med Chem ; 206: 112676, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32858418

RESUMO

We report here an extensive structure-activity relationship study of balsalazide, which was previously identified in a high-throughput screening as an inhibitor of Sirt5. To get a closer understanding why this compound is able to inhibit Sirt5, we initially performed docking experiments comparing the binding mode of a succinylated peptide as the natural substrate and balsalazide with Sirt5 in the presence of NAD+. Based on the evidence gathered here, we designed and synthesized 13 analogues of balsalazide, in which single functional groups were either deleted or slightly altered to investigate which of them are mandatory for high inhibitory activity. Our study confirms that balsalazide with all its given functional groups is an inhibitor of Sirt5 in the low micromolar concentration range and structural modifications presented in this study did not increase potency. While changes on the N-aroyl-ß-alanine side chain eliminated potency, the introduction of a truncated salicylic acid part minimally altered potency. Calculations of the associated reaction paths showed that the inhibition potency is very likely dominated by the stability of the inhibitor-enzyme complex and not the type of inhibition (covalent vs. non-covalent). Further in-vitro characterization in a trypsin coupled assay determined that the tested inhibitors showed no competition towards NAD+ or the synthetic substrate analogue ZKsA. In addition, investigations for subtype selectivity revealed that balsalazide is a subtype-selective Sirt5 inhibitor, and our initial SAR and docking studies pave the way for further optimization.


Assuntos
Inibidores de Histona Desacetilases/química , Inibidores de Histona Desacetilases/farmacologia , Mesalamina/química , Mesalamina/farmacologia , Simulação de Acoplamento Molecular , Fenil-Hidrazinas/química , Fenil-Hidrazinas/farmacologia , Sirtuínas/antagonistas & inibidores , Desenho de Fármacos , Inibidores de Histona Desacetilases/metabolismo , Mesalamina/metabolismo , Fenil-Hidrazinas/metabolismo , Conformação Proteica , Ácido Salicílico/química , Sirtuínas/química , Sirtuínas/metabolismo , Relação Estrutura-Atividade
14.
J Inorg Biochem ; 210: 111172, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32659518

RESUMO

Anemia is highly prevalent in people with chronic kidney disease (CKD), and CKD patients always have lower plasma but higher erythrocyte Zn levels than healthy people. To date, no satisfactory mechanism has explained these Zn metabolism abnormalities. We collected blood samples from patients on hemodialysis, 5/6 nephrectomized rats and phenylhydrazine (PHZ)-induced anemic mice and rats and compared them with their normal counterparts. We found that all the anemic animals had significantly decreased plasma Zn levels but elevated erythrocyte Zn levels. We also found that in anemic mice, new red blood cells (reticulocytes) had a ~7-fold higher Zn concentration than mature erythrocytes. When excess Zn was supplied to the rats, there was a ~1.2-fold increase in the Zn level in the rat bones. When Zn was depleted in the rats, the bones lost the greatest amount of Zn in the body (a 45% decrease). We prepared Zn-depleted rats and rendered these rats anemic by treating them with PHZ, and we compared them with normal rats. We found that in PHZ-induced anemia, rats released ~16% of Zn from their bones. Rat bones not only act as a 'reservoir' to adjust the excess or deficient Zn levels but also release Zn in anemia, and the released Zn stimulates erythropoiesis in the bone marrow. In anemia, Zn is redistributed from the plasma (causing the plasma Zn level to decreases) and bones to the bone marrow to produce reticulocytes (causing erythrocyte Zn level elevation).


Assuntos
Anemia/fisiopatologia , Osso e Ossos/metabolismo , Eritrócitos/metabolismo , Plasma/metabolismo , Zinco/metabolismo , Adulto , Idoso , Anemia/sangue , Anemia/induzido quimicamente , Animais , Contagem de Eritrócitos , Eritropoese/fisiologia , Feminino , Humanos , Masculino , Camundongos Endogâmicos ICR , Pessoa de Meia-Idade , Fenil-Hidrazinas , Ratos , Ratos Sprague-Dawley , Zinco/deficiência , Zinco/farmacologia
15.
Life Sci ; 253: 117732, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32360570

RESUMO

AIMS: Recently, the zebrafish has gained attention as an innovative experimental model to decipher molecular and cellular mechanisms involved in cardiovascular development and diseases. Nevertheless, the use of zebrafish models has been challenged because the transparency of these fish, which allows for accurate cardiac evaluation, disappears in adulthood. In this study, the epicardial outline method was performed to investigate the feasibility of echocardiography in assessing cardiac function in pathological adult zebrafish. MATERIALS AND METHODS: We attempted to estimate heart failure in adult zebrafish treated with three distinct regulators of cardiac function: phenylhydrazine hydrochloride (PHZ), doxorubicin (DOX), and ethanol. B-mode and Doppler images were evaluated at frequencies of up to 50 MHz and 40 MHz, respectively. The correlation between alterations in cardiac function, haemoglobin concentration, and myocardial histopathology were assessed. KEY FINDINGS: Cardiac output (CO) in PHZ-treated zebrafish was significantly higher than that in control zebrafish (151 ± 67 vs. 84 ± 37 µl/min, P = 0.004), whereas ejection fraction (EF) was lower (36.3 ± 10.9 vs. 50.9 ± 8.7%, P < 0.001), indicating typical high output heart failure derived from anaemia. Additionally, ventricular dysfunction in DOX-treated zebrafish was characterised by low CO (57 ± 38 µl/min) and EF (28.8 ± 10.4%), accompanied by an enlarged ventricle in diastole and systole, representing low output heart failure. For ethanol-treated zebrafish, EF was markedly reduced (39.6 ± 7.2%) indicating a dilated heart, while CO remained unchanged (90 ± 40 µl/min). SIGNIFICANCE: The epicardial outline method is an effective way of using echocardiography to assess cardiac dysfunction in pathological adult zebrafish, unlocking a major bottleneck in this research field with limited cardiac functional assays.


Assuntos
Ecocardiografia/métodos , Cardiopatias/diagnóstico por imagem , Insuficiência Cardíaca/diagnóstico por imagem , Pericárdio/diagnóstico por imagem , Animais , Débito Cardíaco/fisiologia , Modelos Animais de Doenças , Doxorrubicina/toxicidade , Etanol/toxicidade , Estudos de Viabilidade , Feminino , Cardiopatias/fisiopatologia , Insuficiência Cardíaca/fisiopatologia , Fenil-Hidrazinas/toxicidade , Disfunção Ventricular/diagnóstico por imagem , Disfunção Ventricular/fisiopatologia , Peixe-Zebra
16.
Drug Des Devel Ther ; 14: 1593-1607, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32425505

RESUMO

Purpose: Continuous efforts into the discovery and development of new antimalarials are required to face the emerging resistance of the parasite to available treatments. Thus, new effective drugs, ideally able to inhibit the Plasmodium life-cycle stages that cause the disease as well as those responsible for its transmission, are needed. Eight compounds from the Medicines for Malaria Venture (MMV) Malaria Box, potentially interfering with the parasite polyamine biosynthesis were selected and assessed in vitro for activity against malaria transmissible stages, namely mature gametocytes and early sporogonic stages. Methods: Compound activity against asexual blood stages of chloroquine-sensitive 3D7 and chloroquine-resistant W2 strains of Plasmodium falciparum was tested measuring the parasite lactate dehydrogenase activity. The gametocytocidal effect was determined against the P. falciparum 3D7elo1-pfs16-CBG99 strain with a luminescent method. The murine P. berghei CTRP.GFP strain was employed to assess compounds activities against early sporogonic stage development in an in vitro assay simulating mosquito midgut conditions. Results: Among the eight tested molecules, MMV000642, MMV000662 and MMV006429, containing a 1,2,3,4-tetrahydroisoquinoline-4-carboxamide chemical skeleton substituted at N-2, C-3 and C-4, displayed multi-stage activity. Activity against asexual blood stages of both strains was confirmed with values of IC50 (50% inhibitory concentration) in the range of 0.07-0.13 µM. They were also active against mature stage V gametocytes with IC50 values below 5 µM (range: 3.43-4.42 µM). These molecules exhibited moderate effects on early sporogonic stage development, displaying IC50 values between 20 and 40 µM. Conclusion: Given the multi-stage, transmission-blocking profiles of MMV000642, MMV000662, MMV006429, and their chemical characteristics, these compounds can be considered worthy for further optimisation toward a TCP5 or TCP6 target product profile proposed by MMV for transmission-blocking antimalarials.


Assuntos
Antimaláricos/farmacologia , Malária/tratamento farmacológico , Fenil-Hidrazinas/farmacologia , Plasmodium berghei/efeitos dos fármacos , Plasmodium falciparum/efeitos dos fármacos , Animais , Antimaláricos/administração & dosagem , Relação Dose-Resposta a Droga , Injeções Intraperitoneais , Malária/transmissão , Camundongos , Camundongos Endogâmicos BALB C , Estrutura Molecular , Testes de Sensibilidade Parasitária , Fenil-Hidrazinas/administração & dosagem , Relação Estrutura-Atividade
17.
Int J Mol Sci ; 21(10)2020 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-32443620

RESUMO

Starting from dansyl-chloride, in reaction with 1,1-diphenylhydrazine and methoxyamine, two new fluorescent derivatives 1 and 2 were obtained and characterized by NMR, IR, UV-Vis, HR-MS, and fluorescence spectroscopy. The single-crystal X-ray structure was obtained for compound 2. Both compounds generate free radicals by oxidation, as demonstrated by ESR spectroscopy. Compound 1 generates the corresponding hydrazyl-persistent free radical, evidenced directly by ESR spectroscopy, while compound 2 generates in the first instance the methoxyaminyl short-lived free radical, which decomposes rapidly with the formation of the methoxy radical, evidenced by the ESR spin-trapping technique. By oxidation of compounds 1 and 2, their fluorescence is quenched.


Assuntos
Compostos de Dansil/química , Radicais Livres/síntese química , Hidroxilaminas/química , Fenil-Hidrazinas/química , Espectroscopia de Ressonância de Spin Eletrônica , Detecção de Spin
18.
Anticancer Drugs ; 31(1): 35-43, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31490285

RESUMO

Triple-negative breast cancer subtype is the most aggressive type of breast cancer due to the lack of specific therapeutic targets, having limited treatment options, low survival prognosis and high recurrence rates. In this work, we describe the effects of a semisynthetic derivative of [6]-gingerol (6G) called SSi6, produced by the addition of a 2,4-dinitrophenylhydrazine reagent on several aspects of triple-negative breast cancer biology. Human breast cancer cell lines MDA-MB-231 and MCF-10A were used in the experiments. MTT assays were used to detect cell viability. Cell cycle and apoptosis assay were analyzed using flow cytometer Accuri C6 and analysis of proteins as retinoblastoma Rb and kinases Cdk4/6 were analyzed by western blotting. SSi6 induced cytotoxic effects on triple-negative breast cancer cells, with higher selectivity when compared to the non-tumor MCF-10A cells. In addition, SSi6 inhibited migration and invasion of triple-negative breast cancer cells and was able to arrest cell cycle at the G1-phase, mainly by decreasing Cdk4/6-Rb axis levels. Therefore, SSi6 provoked the induction of apoptosis in triple-negative breast cancer cells. SSi6 was more efficient in producing these effects, compared to the original 6G natural product. This study may contribute to a better understanding of the effects of natural and semisynthetic products on the in-vitro metastatic processes in the MDA-MB-231 triple-negative breast cancer cell line. Additional, it can be useful to understand the effects of chemical modifications on already effective natural compounds aiming at the improvement of their bioactive properties, such as in the increase of the cytotoxic selectivity against tumor cells, compared to non-tumor ones.


Assuntos
Catecóis/farmacologia , Álcoois Graxos/farmacologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Apoptose/efeitos dos fármacos , Catecóis/química , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Álcoois Graxos/química , Feminino , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Humanos , Invasividade Neoplásica , Fenil-Hidrazinas/química , Neoplasias de Mama Triplo Negativas/patologia
19.
J Occup Health ; 62(1): e12105, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31883172

RESUMO

OBJECTIVES: The purpose of this research was to develop and validate an analytical method for rapid determination of the exposure of workers to ortho-phthalaldehyde (OPA) at the ceiling threshold concentration. METHODS: A 2,4-dinitrophenylhydrazine (DNPH)-silica cartridge was chosen as a sampler. OPA collected by the DNPH-silica cartridge was subsequently extracted with 5 mL of acetonitrile. A 50-µL aliquot of phosphoric acid/acetonitrile solution (2%, v/v) was added to 950 µL of the extraction solution and allowed to stand for 30 minutes at room temperature. This solution was then analyzed by high-performance liquid chromatography tandem mass spectrometry. The basic characteristics of the proposed method, such as recovery, repeatability, limit of quantification, and storage stability of the samples, were examined. RESULTS: The overall recoveries of OPA from OPA-spiked DNPH-silica cartridges were 93.6%-100.1% with relative standard deviations, representing the repeatability, of 1.5%-10.8%. The limit of quantification was 0.165 ng/sample. The recovery of OPA from DNPH-silica cartridges after 5 days of storage in a refrigerator exceeded 95%. CONCLUSIONS: The proposed method enabled the determination of the OPA concentration corresponding to the Threshold Limit Value-Ceiling of 0.1 ppb recommended by the American Conference of Governmental Industrial Hygienists, with a minimum sampling time of 18 seconds (corresponding to a sampling volume of 300 mL at 25°C and 1 atm). Thus, this method will be useful for estimating worker exposures to OPA.


Assuntos
Poluentes Ocupacionais do Ar/análise , Monitoramento Ambiental/métodos , Exposição Ocupacional/análise , Níveis Máximos Permitidos , o-Ftalaldeído/análise , Cromatografia Líquida de Alta Pressão , Humanos , Espectrometria de Massas , Estrutura Molecular , Fenil-Hidrazinas , Dióxido de Silício
20.
Exp Hematol ; 78: 21-34.e3, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31562902

RESUMO

Mouse models are widely used to study human erythropoiesis in vivo. One important caveat using mouse models is that mice often develop significant extramedullary erythropoiesis with anemia, which could mask important phenotypes. To overcome this drawback in mice, here we established in vitro and in vivo rat models for the studies of stress erythropoiesis. Using flow cytometry-based assays, we can monitor terminal erythropoiesis in rats during fetal and adult erythropoiesis under steady state and stress conditions. We used this system to test rat erythropoiesis under phenylhydrazine (PHZ)-induced hemolytic stress. In contrast to mice, rats did not have an increased proportion of early-stage erythroid precursors during terminal differentiation in the spleen or bone marrow. This could be explained by the abundant bone marrow spaces in rats that allow sufficient erythroid proliferation under stress. Consistently, the extent of splenomegaly in rats after PHZ treatment was significantly lower than that in mice. The level of BMP4, which was significantly increased in mouse spleen after PHZ treatment, remained unchanged in rat spleen. We further demonstrated that the bone marrow c-Kit positive progenitor population underwent a phenotype shift and became more CD71 positive and erythroid skewed with the expression of maturing erythroid markers under stress in rats and humans. In contrast, the phenotype shift to an erythroid-skewed progenitor population in mice occurred mainly in the spleen. Our study establishes rat in vitro and in vivo erythropoiesis models that are more appropriate and superior for the study of human stress erythropoiesis than mouse models.


Assuntos
Células Eritroides/metabolismo , Eritropoese/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Modelos Biológicos , Fenil-Hidrazinas/farmacologia , Estresse Fisiológico/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Eritroides/patologia , Humanos , Camundongos , Ratos
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