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1.
Sci Adv ; 10(7): eadi8847, 2024 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-38363840

RESUMO

Various control strategies are available for building fluorogenic probes to visualize biological events in terms of a fluorescence change. Here, we performed the time-dependent density functional theory (TD-DFT) computational analysis of the twisted intramolecular charge transfer (TICT) process in rhodamine dyes. On the basis of the results, we designed and synthesized a series of rhodamine dyes and established a fluorescence quenching strategy that we call steric repulsion-induced TICT (sr-TICT), in which the fluorescence quenching process is greatly accelerated by simple intramolecular twisting. As proof of concept of this design strategy, we used it to develop a fluorogenic probe, 2-Me PeER (pentyloxyethylrhodamine), for the N-dealkylation activity of CYP3A4. We applied 2-Me PeER for CYP3A4 activity-based fluorescence-activated cell sorting (FACS), providing access to homogeneous, highly functional human-induced pluripotent stem cell (hiPSC)-derived hepatocytes and intestinal epithelial cells. Our results suggest that sr-TICT represents a general fluorescence control method for fluorogenic probes.


Assuntos
Corantes , Citocromo P-450 CYP3A , Humanos , Fluorescência , Mercaptoetanol , Rodaminas
2.
J Org Chem ; 89(4): 2104-2126, 2024 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-37267444

RESUMO

This work describes the reactivity and properties of fluorinated derivatives (F-PD and F-PDO) of plasmodione (PD) and its metabolite, the plasmodione oxide (PDO). Introduction of a fluorine atom on the 2-methyl group markedly alters the redox properties of the 1,4-naphthoquinone electrophore, making the compound highly oxidizing and particularly photoreactive. A fruitful set of analytical methods (electrochemistry, absorption and emission spectrophotometry, and HRMS-ESI) have been used to highlight the products resulting from UV photoirradiation in the absence or presence of selected nucleophiles. With F-PDO and in the absence of nucleophile, photoreduction generates a highly reactive ortho-quinone methide (o-QM) capable of leading to the formation of a homodimer. In the presence of thiol nucleophiles such as ß-mercaptoethanol, which was used as a model, o-QMs are continuously regenerated in sequential photoredox reactions generating mono- or disulfanylation products as well as various unreported sulfanyl products. Besides, these photoreduced adducts derived from F-PDO are characterized by a bright yellowish emission due to an excited-state intramolecular proton transfer (ESIPT) process between the dihydronapthoquinone and benzoyl units. In order to evidence the possibility of an intramolecular coupling of the o-QM intermediate, a synthetic route to the corresponding anthrones is described. Tautomerization of the targeted anthrones occurs and affords highly fluorescent stable hydroxyl-anthraquinones. Although probable to explain the intense visible fluorescence emission also observed in tobacco BY-2 cells used as a cellular model, these coupling products have never been observed during the photochemical reactions performed in this study. Our data suggest that the observed ESIPT-induced fluorescence most likely corresponds to the generation of alkylated products through reduction species, as demonstrated with the ß-mercaptoethanol model. In conclusion, F-PDO thus acts as a novel (pro)-fluorescent probe for monitoring redox processes and protein alkylation in living cells.


Assuntos
Indolquinonas , Vitamina K 3/análogos & derivados , Mercaptoetanol , Indolquinonas/química , Antraquinonas
3.
Peptides ; 171: 171116, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37951356

RESUMO

Previously, we have investigated three C-terminal esterified endomorphin-2 (EM-2) analogs EM-2-Me, EM-2-Et and EM-2-Bu with methyl, ethyl and tert-butyl ester modifications, respectively. These analogs produced significant antinociception in acute pain at the spinal and supraspinal levels, with reduced tolerance and gastrointestinal side effects. The present study was undertaken to determine the analgesic effects and opioid mechanisms of these three analogs in the formalin pain test. Our results demonstrated that intracerebroventricular (i.c.v.) administration of 0.67-20 nmol EM-2 analogs EM-2-Me, EM-2-Et and EM-2-Bu produced dose-dependent antinociceptive effects in both phase Ⅰ and phase Ⅱ of formalin pain. EM-2-Me and EM-2-Bu displayed more potent antinociception than morphine. Especially, EM-2-Bu exhibited the highest antinociception in phase Ⅱ of formalin pain, with the ED50 value being 2.1 nmol. Naloxone (80 nmol, i.c.v.) completely antagonized the antinociceptive effects of EM-2-Me, EM-2-Et and EM-2-Bu (20 nmol, i.c.v.) in both phase I and phase Ⅱ of formalin pain, suggesting a central opioid mechanism. Nevertheless, the antinociception induced by EM-2-Me might be involved in the release of dynorphin A, which subsequently acted on κ- opioid receptor. EM-2-Bu produced the antinociception probably by the direct activation of both µ- and δ-opioid receptors. EM-2-Me, EM-2-Et and EM-2-Bu also produced significant analgesic effects after peripheral administration, and the central opioid receptors were involved. Furthermore, EM-2-Bu had no influence on the locomotor activity after i.c.v. injection. The present investigation demonstrated that C-terminal esterified modifications of EM-2 will be beneficial for developing novel therapeutics in formalin pain.


Assuntos
Analgésicos Opioides , Dor , Animais , Camundongos , Analgésicos Opioides/farmacologia , Esterificação , Mercaptoetanol , Dor/tratamento farmacológico , Receptores Opioides , Receptores Opioides kappa , Receptores Opioides mu/metabolismo , Relação Dose-Resposta a Droga
4.
Biochem Biophys Res Commun ; 690: 149295, 2024 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-38000295

RESUMO

BACKGROUND: Bmal1 (Brain and muscle arnt-like, or Arntl) is a bHLH/PAS domain transcription factor central to the transcription/translation feedback loop of the circadian clock. Mast cells are crucial for effector functions in allergic reaction and their activity follows a circadian rhythm. However, the functional roles of Bmal1 in mast cells remain to be determined. PURPOSE: This study aimed to elucidate the specific roles of Bmal1 in IgE-dependent mast cell degranulation. RESULTS: IgE-dependent degranulation was enhanced in bone marrow-derived mast cells (BMMCs) derived from Bmal1-deficient mice (Bmal1-KO mice) compared to that in BMMCs derived from wild-type mice (WT mice) in the absence of 2-Mercaptoethanol (2-ME) in culture. Mast cell-deficient KitW-sh mice reconstituted with Bmal1-KO BMMCs showed more robust passive cutaneous anaphylactic (PCA) reactions, an in vivo model of IgE-dependent mast cell degranulation, than KitW-sh mice reconstituted with WT BMMCs. In the absence of 2-ME in culture, the mRNA expression of the anti-oxidative genes NF-E2-related factor 2 (Nrf2), superoxide dismutase 2 (SOD2), and heme oxygenase-1 (HO-1) was lower and reactive oxygen species (ROS) generation was higher in Bmal1-KO BMMCs than in WT BMMCs at steady state. The IgE-dependent ROS generation and degranulation were enhanced in Bmal1-KO BMMCs compared to WT BMMCs in the absence of 2-ME in culture. The addition of 2-ME into the culture abrogated or weakened the differences in anti-oxidative gene expression, ROS generation, and IgE-dependent degranulation between WT and Bmal1-KO BMMCs. CONCLUSION: The current findings suggest that Bmal1 controls the expression of anti-oxidative genes in mast cells and Bmal1 deficiency enhanced IgE-dependent degranulation associated with promotion of ROS generation. Thus, Bmal1 may function as a key molecule that integrates redox homeostasis and effector functions in mast cells.


Assuntos
Fatores de Transcrição ARNTL , Mastócitos , Animais , Camundongos , Fatores de Transcrição ARNTL/genética , Fatores de Transcrição ARNTL/metabolismo , Degranulação Celular , Imunoglobulina E/metabolismo , Mastócitos/metabolismo , Mercaptoetanol/metabolismo , Camundongos Knockout , Espécies Reativas de Oxigênio/metabolismo
5.
Chembiochem ; 25(1): e202300595, 2024 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-37815851

RESUMO

Methanogenic and methanotrophic archaea play important roles in the global carbon cycle by interconverting CO2 and methane. To conserve energy from these metabolic pathways that happen close to the thermodynamic equilibrium, specific electron carriers have evolved to balance the redox potentials between key steps. Reduced ferredoxins required to activate CO2 are provided by energetical coupling to the reduction of the high-potential heterodisulfide (HDS) of coenzyme M (2-mercaptoethanesulfonate) and coenzyme B (7-mercaptoheptanoylthreonine phosphate). While the standard redox potential of this important HDS has been determined previously to be -143 mV (Tietze et al. 2003 DOI: 10.1002/cbic.200390053), we have measured thiol disulfide exchange kinetics and reassessed this value by equilibrating thiol-disulfide mixtures of coenzyme M, coenzyme B, and mercaptoethanol. We determined the redox potential of the HDS of coenzyme M and coenzyme B to be -16.4±1.7 mV relative to the reference thiol mercaptoethanol (E0 '=-264 mV). The resulting E0 ' values are -281 mV for the HDS, -271 mV for the homodisulfide of coenzyme M, and -270 mV for the homodisulfide of coenzyme B. We discuss the importance of these updated values for the physiology of methanogenic and methanotrophic archaea and their implications in terms of energy conservation.


Assuntos
Archaea , Mesna , Mesna/metabolismo , Archaea/metabolismo , Compostos de Sulfidrila , Mercaptoetanol , Dissulfetos/metabolismo , Dióxido de Carbono/metabolismo , Elétrons , Transporte de Elétrons , Metano/metabolismo , Oxirredução
6.
Anim Biotechnol ; 34(9): 5180-5191, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37965764

RESUMO

Sperm mRNA transcriptional profiling can be used to evaluate the fertility of breeding bulls. The aim of the study was to compare the modified RNA isolation methods for higher RNA yield and quality from freshly ejaculated sperm of cattle and buffalo bulls. Ten fresh ejaculates from each Sahiwal (n = 10 bulls × 10 ejaculates) and Murrah bulls (n = 10 bulls x 10 ejaculates) were used for RNA isolation. From the recovered live sperm, total sperm RNA was isolated by conventional methods (TRIzol, Double TRIzol), membrane-based methods combined with TRIzol (RNeasy + TRIzol) with the addition of ß-mercaptoethanol (BME) and Kit (RNeasy mini) methods in fresh semen. Among different isolation methods; the membrane-based modified methods combined with TRIzol (RNeasy + TRIzol) with the addition of ß-mercaptoethanol (BME) resulted significantly (p < .05) higher total RNA quantity (300-340 ng/µL) and better purity in different concentrations of spermatozoa viz., 30-40 million, 70-80 million and 300-400 million sperm. The study concluded that the inclusion of BME to the combined membrane-based methods with somatic cell lysis buffer solution was best for constant increased yield and purity of RNA isolation from Sahiwal cattle and Murrah buffalo bull sperm.


Assuntos
Búfalos , Guanidinas , Fenóis , Preservação do Sêmen , Bovinos , Masculino , Animais , Búfalos/genética , Sêmen , RNA/genética , Mercaptoetanol/farmacologia , Espermatozoides , Preservação do Sêmen/veterinária , Motilidade dos Espermatozoides
7.
Cell Biochem Funct ; 41(7): 898-911, 2023 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-37649158

RESUMO

The prevalence of breast cancer (BC) continues to increase and is the leading cause of cancer deaths in many countries. Numerous in vitro and in vivo studies have demonstrated that 2-methoxyestradiol (2-ME) has antiproliferative and antiangiogenic effects in BC, thereby inhibiting tumour growth and metastasis. We compared the effect of 2-ME in early- and late-stage BC using a transgenic mouse model-FVB/N-Tg(MMTV-PyVT)-of spontaneously development of aggressive mammary carcinoma with lung metastasis. Mice received 100 mg/kg 2-ME treatment immediately when palpable mammary tumours were identified (early-stage BC; Experimental group 1) and 28 days after palpable mammary tumours were detected (late-stage BC; Experimental group 2). 2-ME was administered via oral gavage three times a week for 28 days after initiation of treatment, whereas control mice received the vehicle containing 10% dimethyl sulfoxide and 90% sunflower oil for the same duration as the treatment group. Mammary tumours were measured weekly over the 28 days and at termination, blood, mammary and lung tissue were collected for analysis. Mice with a tumour volume threshold of 4000 mm3 were killed before the treatment regime was completed. 2-ME treatment of early-stage BC led to lower levels of mammary tumour necrosis, whereas tumour mass and volume were increased. Additionally, necrotic lesions and anti-inflammatory CD163-expressing cells were more frequent in pulmonary metastatic tumours in this group. In contrast, 2-ME treatment of late-stage BC inhibited tumour growth over the 28-day period and resulted in increased CD3+ cell number and tumour necrosis. Furthermore, 2-ME treatment slowed down pulmonary metastasis but did not increase survival of late-stage BC mice. Besides late-stage tumour necrosis, none of the other results were statistically significant. This study demonstrates that 2-ME treatment has an antitumour effect on late-stage BC, however, with no increase in survival rate, whereas the treatment failed to demonstrate any benefit in early-stage BC.


Assuntos
Neoplasias Pulmonares , Neoplasias Mamárias Animais , Camundongos , Animais , 2-Metoxiestradiol/farmacologia , Mercaptoetanol , Camundongos Transgênicos , Neoplasias Pulmonares/tratamento farmacológico , Necrose
8.
Chemosphere ; 338: 139499, 2023 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-37467859

RESUMO

Nitrophenols (NPs) have significant impacts on human health, climate, and atmospheric chemistry. Despite numerous measurements of particulate NPs, still little is known about their gaseous atmospheric abundances, sources, and fate. Here, four gaseous NPs [2,4-dinitrophenol (2,4-DNP), 4-nitrophenol (4-NP), 2-nitrophenol (2-NP), and 2-Methyl-4-nitrophenol (2-Me-4-NP)] were continuously monitored during late Spring at an urban site in Houston, Texas. Among the four NPs, 4-NP showed the highest abundance, followed by 2-Me-4-NP, 2-NP, and 2,4-DNP with average concentrations of 1.07 ± 0.19 ppt, 0.47 ± 0.12 ppt, 0.41 ± 0.16 ppt, and 0.27 ± 0.09 ppt, respectively. The positive matrix factorization (PMF) model identified seven sources: industrial NPs, secondary formation, phenol sources, acetonitrile source, natural gas/crude oil, traffic, and petrochemical industries/oil refineries. A zero-dimensional photochemical box model was used to simulate the observed 2-NP and 2,4-DNP. A 50.0% and 70.0% jNO2 was found to be consistent with the measured 2-NP and 2,4-DNP. This yields a nitrous acid (HONO) production of 7.5 ± 2.5 ppt/h from 06:00 to 18:00 Central Standard Time (CST) from both NPs. An extrapolation including other known NPs suggests a maximum HONO formation of 13.8 ppt/h. The results of this study suggest that using PMF analysis supplemented by photochemical box model provides identification of the NPs sources and their atmospheric implication to HONO formation.


Assuntos
2,4-Dinitrofenol , Gases , Humanos , Mercaptoetanol , Nitrofenóis
9.
J Pharm Sci ; 112(9): 2552-2560, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37482124

RESUMO

Leiomyomas, the most common benign neoplasms of the female reproductive tract, currently have limited medical treatment options. Drugs targeting estrogen/progesterone signaling are used, but side effects and limited efficacy in many cases are major limitation of their clinical use. Previous studies from our laboratory and others demonstrated that 2-methoxyestradiol (2-ME) is promising treatment for uterine fibroids. However, its poor bioavailability and rapid degradation hinder its development for clinical use. The objective of this study is to evaluate the in vivo effect of biodegradable and biocompatible 2-ME-loaded polymeric nanoparticles in a patient-derived leiomyoma xenograft mouse model. PEGylated poly(lactide-co-glycolide) (PEG-PLGA) nanoparticles loaded with 2-ME were prepared by nanoprecipitation. Female 6-week age immunodeficient NOG (NOD/Shi-scid/IL-2Rγnull) mice were used. Estrogen-progesterone pellets were implanted subcutaneously. Five days later, patient-derived human fibroid tumors were xenografted bilaterally subcutaneously. Engrafted mice were treated with 2-ME-loaded or blank (control) PEGylated nanoparticles. Nanoparticles were injected intraperitoneally and after 28 days of treatment, tumor volume was measured by caliper following hair removal, and tumors were removed and weighed. Up to 99.1% encapsulation efficiency was achieved, and the in vitro release profile showed minimal burst release, thus confirming the high encapsulation efficiency. In vivo administration of the 2-ME-loaded nanoparticles led to 51% growth inhibition of xenografted tumors compared to controls (P < 0.01). Thus, 2-ME-loaded nanoparticles may represent a novel approach for the treatment of uterine fibroids.


Assuntos
Leiomioma , Nanopartículas , Humanos , Camundongos , Feminino , Animais , 2-Metoxiestradiol/uso terapêutico , Progesterona , Xenoenxertos , Mercaptoetanol/uso terapêutico , Camundongos Endogâmicos NOD , Leiomioma/tratamento farmacológico , Leiomioma/patologia , Polímeros , Polietilenoglicóis , Estrogênios
10.
Redox Biol ; 63: 102758, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37245287

RESUMO

Ferroptosis is an iron dependent form of cell death, that is triggered by the discoordination of iron, lipids, and thiols. Its unique signature that distinguishes it from other forms of cell death is the formation and accumulation of lipid hydroperoxides, particularly oxidized forms of polyunsaturated phosphatidylethanolamines (PEs), which drives cell death. These readily undergo iron-catalyzed secondary free radical reactions leading to truncated products which retain the signature PE headgroup and which can readily react with nucleophilic moieties in proteins via their truncated electrophilic acyl chains. Using a redox lipidomics approach, we have identified oxidatively-truncated PE species (trPEox) in enzymatic and non-enzymatic model systems. Further, using a model peptide we demonstrate adduct formation with Cys as the preferred nucleophilic residue and PE(26:2) +2 oxygens, as one of the most reactive truncated PE-electrophiles produced. In cells stimulated to undergo ferroptosis we identified PE-truncated species with sn-2 truncations ranging from 5 to 9 carbons. Taking advantage of the free PE headgroup, we have developed a new technology using the lantibiotic duramycin, to enrich and identify the PE-lipoxidated proteins. Our results indicate that several dozens of proteins for each cell type, are PE-lipoxidated in HT-22, MLE, and H9c2 cells and M2 macrophages after they were induced to undergo ferroptosis. Pretreatment of cells with the strong nucleophile, 2-mercaptoethanol, prevented the formation of PE-lipoxidated proteins and blocked ferroptotic death. Finally, our docking simulations showed that the truncated PE species bound at least as good to several of the lantibiotic-identified proteins, as compared to the non-truncated parent molecule, stearoyl-arachidonoyl PE (SAPE), indicating that these oxidatively-truncated species favor/promote the formation of PEox-protein adducts. The identification of PEox-protein adducts during ferroptosis suggests that they are participants in the ferroptotic process preventable by 2-mercaptoethanol and may contribute to a point of no return in the ferroptotic death process.


Assuntos
Ferroptose , Humanos , Mercaptoetanol , Oxirredução , Morte Celular , Ferro/metabolismo , Peroxidação de Lipídeos
11.
IEEE Trans Biomed Eng ; 70(6): 1891-1901, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-37015385

RESUMO

OBJECTIVE: Multicellular tumor spheroids (MCTs) are indispensable models for evaluating drug efficacy for precision cancer therapeutic strategies as well as for repurposing FDA-approved drugs for ovarian cancer. However, current imaging techniques cannot provide effective monitoring of pathological responses due to shallow penetration and experimentally operative destruction. We plan to utilize a noninvasive optical imaging tool to achieve in vivo longitudinal monitoring of the growth of MCTs and therapeutic responses to repurpose three FDA-approved drugs for ovarian cancer therapy. METHODS: A swept-source optical coherence tomography (SS-OCT) system was used to monitor the volume growth of MCTs over 11 days. Three inhibitors of 2-Methoxyestradiol (2-ME), AZD1208, and R-Ketorolac (R-keto) with concentrations of 1, 10, and 25 µM were employed to treat ovarian MCTs on day 5. Three-dimensional (3D), intrinsic optical attenuation contrast, and degree of uniformity were applied to analyze the therapeutic effect of these inhibitors on ovarian MCTs. RESULTS: We found that 2-ME, AZD1208, and R-keto with concentration of 10 and 25 µM significantly inhibited the volume growth of ovarian MCTs. There was no effect to necrotic tissues from all concentrations of 2-ME, AZD1208, and R-keto inhibitors from our OCT results. 2-ME and AZD1208 inhibited the growth of high uniformity tissues within MCTs and higher concentrations provided more significant inhibitory effects. CONCLUSION: Our results indicated that OCT was capable and reliable to monitor the therapeutic effect of inhibitors to ovarian MCTs and it can be used for the rapid characterization of novel therapeutics for ovarian cancers in the future.


Assuntos
Reposicionamento de Medicamentos , Neoplasias Ovarianas , Humanos , Feminino , Tomografia de Coerência Óptica/métodos , Mercaptoetanol/uso terapêutico , Neoplasias Ovarianas/diagnóstico por imagem , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia
12.
Int J Cardiol ; 379: 136-142, 2023 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-36907447

RESUMO

AIMS: Intracardiac dynamics during atrial fibrillation(AF) complicated by heart failure(HF) are not fully understood. The aim of this study was to evaluate the impact of intracardiac dynamics assessed by echo-vector flow mapping on AF complicated by HF. METHODS AND RESULTS: We enrolled 76 AF patients receiving sinus restoration therapy and assessed energy loss(EL) by echo-vector flow mapping during both AF rhythm and sinus rhythm. Patients were divided into 2 groups according to serum NT-proBNP level: high NT-proBNP group(≥1800 pg/ml during AF rhythm: N = 19), and low NT-proBNP group(N = 57). Outcome measures were average ELs/stroke volume(SV) in left ventricle(LV) and left atrium(LA). Average EL/SVs during AF rhythm in the LV and LA were significantly larger in the high NT-proBNP group than the low NT-proBNP group(54.2 mE/m·L versus 41.2 mE/m·L, P = 0.02; 3.2 mE/m·L versus 1.9 mE/m·L, P = 0.01). The significantly larger EL/SV in the high NT-proBNP group was recorded for maximum EL/SVs. In patients with high NT-proBNP, large vortex formation with extreme EL was detected in the LV and LA during the diastolic phase. After sinus restoration, the average decrease of EL/SV in the LV and LA was larger in the high NT-proBNP group(-21.4 mE/m·L versus +2.6 mE/m·L, P = 0.04; -1.6 mE/m·L versus -0.3 mE/m·L, P = 0.02). Average EL/SV during sinus rhythm did not significantly differ between the high and low NT-proBNP groups in the LV and LA. CONCLUSIONS: High EL during AF rhythm as intracardiac energy inefficiency was associated with high serum NT-proBNP levels and improved after sinus restoration.


Assuntos
Fibrilação Atrial , Insuficiência Cardíaca , Humanos , Fibrilação Atrial/diagnóstico por imagem , Fibrilação Atrial/complicações , Mercaptoetanol , Biomarcadores , Insuficiência Cardíaca/diagnóstico por imagem , Peptídeo Natriurético Encefálico , Átrios do Coração , Fragmentos de Peptídeos
13.
Protein Expr Purif ; 201: 106185, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36195295

RESUMO

In-gel hydrolysis of para-nitrophenyl phosphate (p-NPP) to yellow colored para-nitrophenol was used to locate precisely the K. pneumoniae alkaline phosphatase (Kp-ALKP) on 7% native PAGE. Subsequent removal of the yellow-stained band and electroelution yielded a 54 kDa, Kp-ALKP with Km, Vmax and kcat values of (0.7 ± 0.02) mM, (80 ± 4.5) µmol min-1 and (39.2 ± 2.2) × 104 s-1 respectively for p-NPP. Kp-ALKP was optimally active at 70 °C and pH 7.2 that was activated by Mg2+, Ca2+, Co2+ and inhibited by EDTA, PO4, Pb2+, Cu2+ and Hg2+. The enzyme was trypsin resistant and retained 75% activity in presence of 10 mM PO4 and 65% activity at 3 mM Hg2+ showing it's PO43- irrepressibility and Hg2+-tolerance. Molecular dynamics simulation revealed increased structural stability of Kp-ALKP at 70 °C that accounts for it's optimal temperature. Zymography revealed that both DTT and ß-mercaptoethanol induced activity loss accompanied by mobility retardation of Kp-ALKP on 7% native PAGE. These results and in Silico analysis shows that both DTT and ßME reduce the C308-C358 disulfide bond, leading to an open conformation of the enzyme. However, Hg2+ had negligible effect on the in-gel mobility of Kp-ALKP indicating it's plausible non-covalent interaction with surface-accessible amino-acids without significant conformational change. For the first time our study reveals the zymography as an easy, inexpensive and convenient tool for quick purification, characterization and conformational analysis of K. pneumoniae alkaline phosphatase.


Assuntos
Fosfatase Alcalina , Mercúrio , Fosfatase Alcalina/genética , Dissulfetos , Ácido Edético , Concentração de Íons de Hidrogênio , Cinética , Chumbo , Mercaptoetanol , Nitrofenóis , Fosfatos , Tripsina
14.
Food Chem ; 409: 135281, 2023 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-36586251

RESUMO

The effects of transglutaminase (TGase, 1.0 unit/mL) with heat (95 °C, 5 min), 2-mercaptoethanol (2-ME, 0.83 %), and l-cysteine (l-Cys, 50 mM) pretreatment on the cross-linking of ovalbumin (OVA) and ovotransferrin (OVT) were investigated. SDS-PAGE revealed that although the polymerization of OVA and OVT did not occur after 3 h of incubation at 40 °C with TGase, OVA polymerized into high molecular weight polymers following TGase with 2-ME and heat pretreatment after 3 h of incubation. The surface hydrophobicity and reactive sulfhydryl (SH) groups of OVA samples significantly increased from 4065.7 ± 136.7 and 89.3 ± 1.2 SH groups (µmol/g) to 31483.6 ± 342.7 and 119.5 ± 3.7 SH groups (µmol/g), respectively. Similar results were obtained for OVT with TGase and l-Cys pretreatment and a 3-h incubation at 40 °C. The use of TGase, a reducing agent, and/or heat pretreatment can be used for the polymerization of OVA and OVT.


Assuntos
Substâncias Redutoras , Transglutaminases , Ovalbumina , Transglutaminases/metabolismo , Conalbumina , Temperatura Alta , Mercaptoetanol
15.
Molecules ; 27(19)2022 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-36234729

RESUMO

2,4,6-Trichloro-1,3,5-triazine (cyanuric chloride) is an excellent coupling reagent for the preparation of highly structured multifunctional molecules. Three component systems based on porphyrin, cyanuric chloride and carborane clusters were prepared by a one-pot stepwise amination of cyanuric chloride with 5-(4-aminophenyl)-10,15,20-triphenylporphyrin, followed by replacement of the remaining chlorine atoms with carborane S- or N-nucleophiles. Some variants of 1,3,5-triazine derivatives containing porphyrin, carborane and residues of biologically active compounds such as maleimide, glycine methyl ester as well as thioglycolic acid, mercaptoethanol and hexafluoroisopropanol were also prepared. A careful control of the reaction temperature during the substitution reactions will allow the synthesis of desired compounds in a good to high yields. The structures of synthesized compounds were determined with UV-vis, IR, 1H NMR, 11B NMR, MALDI-TOF or LC-MS spectroscopic data. The dark and photocytotoxicity as well as intracellular localization and photoinduced cell death for compounds 8, 9, 17, 18 and 24 were evaluated.


Assuntos
Boranos , Porfirinas , Cloro , Espectroscopia de Ressonância Magnética , Maleimidas , Mercaptoetanol , Estrutura Molecular , Porfirinas/química , Triazinas/química
16.
BMC Biol ; 20(1): 228, 2022 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-36209095

RESUMO

BACKGROUND: One-carbon metabolism, which includes the folate and methionine cycles, involves the transfer of methyl groups which are then utilised as a part of multiple physiological processes including redox defence. During the methionine cycle, the vitamin B12-dependent enzyme methionine synthetase converts homocysteine to methionine. The enzyme S-adenosylmethionine (SAM) synthetase then uses methionine in the production of the reactive methyl carrier SAM. SAM-binding methyltransferases then utilise SAM as a cofactor to methylate proteins, small molecules, lipids, and nucleic acids. RESULTS: We describe a novel SAM methyltransferase, RIPS-1, which was the single gene identified from forward genetic screens in Caenorhabditis elegans looking for resistance to lethal concentrations of the thiol-reducing agent dithiothreitol (DTT). As well as RIPS-1 mutation, we show that in wild-type worms, DTT toxicity can be overcome by modulating vitamin B12 levels, either by using growth media and/or bacterial food that provide higher levels of vitamin B12 or by vitamin B12 supplementation. We show that active methionine synthetase is required for vitamin B12-mediated DTT resistance in wild types but is not required for resistance resulting from RIPS-1 mutation and that susceptibility to DTT is partially suppressed by methionine supplementation. A targeted RNAi modifier screen identified the mitochondrial enzyme methylmalonyl-CoA epimerase as a strong genetic enhancer of DTT resistance in a RIPS-1 mutant. We show that RIPS-1 is expressed in the intestinal and hypodermal tissues of the nematode and that treating with DTT, ß-mercaptoethanol, or hydrogen sulfide induces RIPS-1 expression. We demonstrate that RIPS-1 expression is controlled by the hypoxia-inducible factor pathway and that homologues of RIPS-1 are found in a small subset of eukaryotes and bacteria, many of which can adapt to fluctuations in environmental oxygen levels. CONCLUSIONS: This work highlights the central importance of dietary vitamin B12 in normal metabolic processes in C. elegans, defines a new role for this vitamin in countering reductive stress, and identifies RIPS-1 as a novel methyltransferase in the methionine cycle.


Assuntos
Sulfeto de Hidrogênio , Ácidos Nucleicos , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/genética , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/metabolismo , Animais , Caenorhabditis elegans/metabolismo , Carbono/metabolismo , Ditiotreitol/metabolismo , Ácido Fólico/metabolismo , Homocisteína/metabolismo , Sulfeto de Hidrogênio/metabolismo , Ligases/metabolismo , Lipídeos , Mercaptoetanol/metabolismo , Metionina/metabolismo , Metiltransferases/genética , Metiltransferases/metabolismo , Oxigênio/metabolismo , Substâncias Redutoras/metabolismo , S-Adenosilmetionina/metabolismo , Compostos de Sulfidrila/metabolismo , Vitamina B 12/metabolismo , Vitamina B 12/farmacologia , Vitaminas/metabolismo
17.
Toxins (Basel) ; 14(9)2022 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-36136547

RESUMO

Lecithin-dependent thermolabile hemolysin (LDH) is a virulence factor excreted by Vibrio parahaemolyticus, a marine bacterium that causes important losses in shrimp farming. In this study, the function of LDH was investigated through its inhibition by metal ions (Mg2+, Ca2+, Mn2+, Co2+, Ni2+ and Cu2+) and chemical modification reagents: ß-mercaptoethanol (ßME), phenylmethylsulfonyl fluoride (PMSF) and diethyl pyrocarbonate (DEPC). LDH was expressed in the Escherichia coli strain BL-21, purified under denaturing conditions, and the enzymatic activity was evaluated. Cu2+, Ni2+, Co2+ and Ca2+ at 1 mmol/L inhibited the LDH esterase activity by 20−95%, while Mg2+ and Mn2+ slightly increased its activity. Additionally, PMSF and DEPC at 1 mmol/L inhibited the enzymatic activity by 40% and 80%, respectively. Dose-response analysis showed that DEPC was the best-evaluated inhibitor (IC50 = 0.082 mmol/L), followed by Cu2+ > Co2+ > Ni2+ and PMSF (IC50 = 0.146−1.5 mmol/L). Multiple sequence alignment of LDH of V. parahaemolyticus against other Vibrio species showed that LDH has well-conserved GDSL and SGNH motifs, characteristic of the hydrolase/esterase superfamily. Additionally, the homology model showed that the conserved catalytic triad His-Ser-Asp was in the LDH active site. Our results showed that the enzymatic activity of LDH from V. parahaemolyticus was modulated by metal ions and chemical modification, which could be related to the interaction with catalytic amino acid residues such as Ser153 and/or His 393.


Assuntos
Proteínas Hemolisinas , Vibrio parahaemolyticus , Aminoácidos , Dietil Pirocarbonato , Escherichia coli/metabolismo , Esterases , Proteínas Hemolisinas/metabolismo , Hidrolases , Indicadores e Reagentes , Íons , Lecitinas , Mercaptoetanol , Fluoreto de Fenilmetilsulfonil , Vibrio parahaemolyticus/metabolismo , Fatores de Virulência
18.
Pharmacol Res Perspect ; 10(5): e00995, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36065843

RESUMO

Aldosterone exerts some of its effects not by binding to mineralocorticoid receptors, but rather by acting via G protein-coupled estrogen receptors (GPER). To determine if aldosterone binds directly to GPER, we studied the ability of aldosterone to compete for the binding of [3 H] 2-methoxyestradiol ([3 H] 2-ME), a high potency GPER-selective agonist. We used GPER gene transfer to engineer Sf9-cultured insect cells to express GPER. We chose insect cells to avoid interactions with any intrinsic mammalian receptors for aldosterone. [3 H] 2-ME binding was saturable and reversible to a high-affinity population of receptors with Kd  = 3.7 nM and Bmax  = 2.2 pmol/mg. Consistent with agonist binding to G Protein-coupled receptors, [3 H] 2-ME high-affinity state binding was reduced in the presence of the hydrolysis-resistant GTP analog, GppNHp. [3 H] 2-ME binding was competed for by the GPER agonist G1, the GPER antagonist G15, estradiol (E2), as well as aldosterone (Aldo). The order of potency for competing for [3 H] 2-ME binding, namely 2ME > Aldo > E2 ≥ G1, paralleled the orders of potency for inhibition of cell proliferation and inhibition of ERK phosphorylation by ligands acting at GPER. These data confirm the ability of aldosterone to interact with the GPER, consistent with the interpretation that aldosterone likely mediates its GPER-dependent effects by direct binding to the GPER. SIGNIFICANCE STATEMENT: Despite the growing evidence for aldosterone's actions via G protein-coupled estrogen receptors (GPER), there remains significant skepticism that aldosterone can directly interact with GPER. The current studies are the first to demonstrate directly that aldosterone indeed is capable of binding to the GPER and thus likely mediates its GPER-dependent effects by direct binding to the receptor.


Assuntos
Aldosterona , Receptores de Estrogênio , Aldosterona/metabolismo , Animais , Estrogênios , Proteínas de Ligação ao GTP/metabolismo , Mamíferos/metabolismo , Mercaptoetanol , Receptores Acoplados a Proteínas G/metabolismo
19.
ACS Appl Mater Interfaces ; 14(38): 42976-42987, 2022 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-36103264

RESUMO

Local stimuli differentiate monocytes into M2-like macrophages that mechanistically drive the pathologies in cancer and age-related macular degeneration (AMD). A photo-controlled nanodrug that halts macrophage polarization through Rho-associated kinase (ROCK) inhibition was developed. A small-molecule ROCK inhibitor, fasudil, was conjugated to a photo-responsive group and a short poly(ethylene glycol) (PEG) chain. This resulted in the novel amphiphilic prodrug, PEG-2-(4'-(di(prop-2-yn-1-yl)amino)-4-nitro-[1,1'-biphenyl]-yl)propan-1-ol (PANBP)-Fasudil, that spontaneously formed micelles. Ultraviolet (UV) irradiation of PEG-PANBP-Fasudil nanoparticles rapidly released fasudil. For visualization of linker degradation, a reporter nanoprobe was synthesized, in which 2-Me-4-OMe TokyoGreen (TG), a fluorophore that does not fluoresce in conjugation, was incorporated. Irradiation of nanoprobe-laden monocytes activated the reporter fluorophore. Cytokine stimulation differentiated monocytes into macrophages, while UV irradiation prevented polarization of PEG-PANBP-Fasudil nanoparticle-laden monocytes. Nanoarchitectonics-based design opens new possibilities for intracellular drug delivery and precise spatiotemporal immune cell modulation toward the development of new therapies.


Assuntos
Pró-Fármacos , Quinases Associadas a rho , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , Citocinas/metabolismo , Liberação Controlada de Fármacos , Mercaptoetanol , Micelas , Polietilenoglicóis/metabolismo
20.
Eur J Pharmacol ; 933: 175276, 2022 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-36130639

RESUMO

Psoriasis is characterized by hyperproliferative keratinocytes, dilated capillaries and leukocyte infiltration. 2-Methoxyestradiol (2-ME) has shown significant inhibition on proliferation, angiogenesis and inflammation. To evaluate the anti-psoriatic potential of 2-ME, psoriasis-like dermatitis was induced by topical application of imiquimod (IMQ) on the dorsal skin of C57BL/6 mice for seven consecutive days, followed by treatment of vehicle or 2-ME ointment from Day 4 on. The psoriasis area and severity index (PASI) was assessed daily. On Day 8, skin histology and spleen index were assessed. The effects of 2-ME on the proliferation, apoptosis, cell cycle, vascular endothelial growth factor A (VEGFA), and Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathways of HaCaT cells stimulated by interleukin-17 (IL-17A) were detected, together with its effect on the proliferation, tube formation and VEGF receptor expression of human umbilical vein endothelial cells (HUVECs). We found that topical 2-ME treatment significantly improved IMQ-induced psoriasis-like dermatitis and decreased the PASI scores, the activation of STAT3 in the skin (P < 0.05), and the spleen index in mice (P < 0.01). In vitro, 2-ME inhibited the proliferation of HaCaT cells by inducing apoptosis and G2/M phase arrest (P < 0.01). Moreover, 2-ME suppressed IL-17A-induced VEGFA (2.5 µM: P < 0.05; 5 µM: P < 0.01) and phosphorylation of STAT3 by blocking p-JAK1 in HaCaT cells and prevented tube formation (P < 0.01) and proliferation by targeting VEGF receptors 1 (VEGFR1) and 2 (VEGFR2) in HUVECs. We conclude that 2-ME alleviated psoriasis in vivo and in vitro by inhibiting JAK1/STAT3 pathway and was a promising therapeutic agent for psoriasis.


Assuntos
Dermatite , Psoríase , 2-Metoxiestradiol/farmacologia , 2-Metoxiestradiol/uso terapêutico , Animais , Proliferação de Células , Dermatite/patologia , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Humanos , Imiquimode/efeitos adversos , Interleucina-17/metabolismo , Janus Quinase 1 , Queratinócitos , Mercaptoetanol/metabolismo , Mercaptoetanol/farmacologia , Mercaptoetanol/uso terapêutico , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Pomadas/efeitos adversos , Psoríase/induzido quimicamente , Psoríase/tratamento farmacológico , Psoríase/patologia , Fator de Transcrição STAT3 , Pele , Fator A de Crescimento do Endotélio Vascular/metabolismo
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