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1.
Biochem Cell Biol ; 99(4): 499-507, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34357813

RESUMO

Adenylate kinases (AK) play a pivotal role in the regulation of cellular energy. The aim of our work was to achieve the overproduction and purification of AKs from two groups of bacteria and to determine, for the first time, the comprehensive biochemical and kinetic properties of adenylate kinase from Gram-negative Aquifex aeolicus (AKaq) and Gram-positive Geobacillus stearothermophilus (AKst). Therefore we determined KM and Vmax values, and the effects of temperature, pH, metal ions, donors of the phosphate groups and inhibitor Ap5A for both thermophilic AKs. The kinetic studies indicate that both AKs exhibit significantly higher affinity for substrates with the pyrophosphate group than for adenosine monophosphate. AK activation by Mg2+ and Mn2+ revealed that both ions are efficient in the synthesis of adenosine diphosphate and adenosine triphosphate; however, Mn2+ ions at 0.2-2.0 mmol/L concentration were more efficient in the activation of the ATP synthesis than Mg2+ ions. Our research demonstrates that zinc ions inhibit the activity of enzymes in both directions, while Ap5A at a concentration of 10 µmol/L and 50 µmol/L inhibited both enzymes with a different efficiency. Sigmoid-like kinetics were detected at high ATP concentrations not balanced by Mg2+, suggesting the allosteric effect of ATP for both bacterial AKs.


Assuntos
Trifosfato de Adenosina/metabolismo , Adenilato Quinase/metabolismo , Difosfatos/metabolismo , Geobacillus stearothermophilus/enzimologia , Zinco/metabolismo , Adenilato Quinase/química , Aquifex/enzimologia , Cinética
2.
Nucleic Acids Res ; 49(15): 8822-8835, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34352100

RESUMO

The catalytic subunit of SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) contains two active sites that catalyze nucleotidyl-monophosphate transfer (NMPylation). Mechanistic studies and drug discovery have focused on RNA synthesis by the highly conserved RdRp. The second active site, which resides in a Nidovirus RdRp-Associated Nucleotidyl transferase (NiRAN) domain, is poorly characterized, but both catalytic reactions are essential for viral replication. One study showed that NiRAN transfers NMP to the first residue of RNA-binding protein nsp9; another reported a structure of nsp9 containing two additional N-terminal residues bound to the NiRAN active site but observed NMP transfer to RNA instead. We show that SARS-CoV-2 RdRp NMPylates the native but not the extended nsp9. Substitutions of the invariant NiRAN residues abolish NMPylation, whereas substitution of a catalytic RdRp Asp residue does not. NMPylation can utilize diverse nucleotide triphosphates, including remdesivir triphosphate, is reversible in the presence of pyrophosphate, and is inhibited by nucleotide analogs and bisphosphonates, suggesting a path for rational design of NiRAN inhibitors. We reconcile these and existing findings using a new model in which nsp9 remodels both active sites to alternately support initiation of RNA synthesis by RdRp or subsequent capping of the product RNA by the NiRAN domain.


Assuntos
Nidovirales/enzimologia , Nucleotídeos/metabolismo , Domínios Proteicos , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismo , SARS-CoV-2/enzimologia , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Coenzimas/metabolismo , RNA-Polimerase RNA-Dependente de Coronavírus/metabolismo , Difosfatos/farmacologia , Difosfonatos/farmacologia , Guanosina Trifosfato/metabolismo , Manganês , Modelos Moleculares , Nidovirales/química , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Uridina Trifosfato/metabolismo
3.
Sensors (Basel) ; 21(16)2021 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-34450980

RESUMO

Copper (II) ions have been shown to greatly improve the chemical stability and peroxidase-like activity of gold nanoclusters (AuNCs). Since the affinity between Cu2+ and pyrophosphate (PPi) is higher than that between Cu2+ and AuNCs, the catalytic activity of AuNCs-Cu2+ decreases with the introduction of PPi. Based on this principle, a new colorimetric detection method of PPi with high sensitivity and selectivity was developed by using AuNCs-Cu2+ as a probe. Under optimized conditions, the detection limit of PPi was 0.49 nM with a linear range of 0.51 to 30,000 nM. The sensitivity of the method was three orders of magnitude higher than that of a fluorescence method using AuNCs-Cu2+ as the probe. Finally, the AuNCs-Cu2+ system was successfully applied to directly determine the concentration of PPi in human urine samples.


Assuntos
Ouro , Nanopartículas Metálicas , Colorimetria , Cobre , Difosfatos , Corantes Fluorescentes , Humanos , Limite de Detecção , Peroxidase , Peroxidases , Espectrometria de Fluorescência
4.
Nucleic Acids Res ; 49(14): 8324-8338, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34302475

RESUMO

Bacterial replication is a fast and accurate process, with the bulk of genome duplication being catalyzed by the α subunit of DNA polymerase III within the bacterial replisome. Structural and biochemical studies have elucidated the overall properties of these polymerases, including how they interact with other components of the replisome, but have only begun to define the enzymatic mechanism of nucleotide incorporation. Using transient-state methods, we have determined the kinetic mechanism of accurate replication by PolC, the replicative polymerase from the Gram-positive pathogen Staphylococcus aureus. Remarkably, PolC can recognize the presence of the next correct nucleotide prior to completing the addition of the current nucleotide. By modulating the rate of pyrophosphate byproduct release, PolC can tune the speed of DNA synthesis in response to the concentration of the next incoming nucleotide. The kinetic mechanism described here would allow PolC to perform high fidelity replication in response to diverse cellular environments.


Assuntos
Proteínas de Bactérias/genética , Replicação do DNA/genética , DNA Polimerase Dirigida por DNA/genética , Infecções Estafilocócicas/genética , Staphylococcus aureus/genética , Difosfatos/metabolismo , Humanos , Cinética , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/patogenicidade
5.
Spectrochim Acta A Mol Biomol Spectrosc ; 263: 120169, 2021 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-34273894

RESUMO

A simple Schiff-base fluorescent chemosensor (1) was synthesized by the reaction of 3-amino-pyrazine-2-carbohydrazide and 7-diethylamino-3-formylcoumarin; the sensor 1 displayed a notable green emission at 524 nm in DMSO and an aggregation-induced ratiometric emission (AIRE) at 555 nm in an almost buffered aqueous media (0.5% DMSO content). The AIRE of 1 was quenched following binding to Zn2+ ions, while the fluorescence emission in the far-red region was evidently enhanced at 628 nm. Notably, the ratiometric signal output could be utilized to specifically distinguish Zn2+ among various metal ions. Moreover, the 1-Zn2+ complex was effectively employed as a fluorescent ratiometric chemosensor for pyrophosphate (PPi) detection. The detection limit was 3.52 µM and 2.45 µM for Zn2+ and PPi, respectively. The binding mechanism was evaluated by 1H NMR, ESI-MS, single-crystal X-ray diffraction, TEM, time-resolved fluorescence spectrophotometry, and density functional theory studies. Overall, owing to its sensitive fluorescence behavior, cell imaging studies demonstrated that this sensor is capable of sensing Zn2+ and PPi in living cells.


Assuntos
Difosfatos , Corantes Fluorescentes , Espectrometria de Fluorescência , Água , Zinco
6.
J Periodontal Res ; 56(5): 982-990, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34142719

RESUMO

BACKGROUND AND OBJECTIVE: Periodontal ligament stem cells (PDLSCs) are the primary cell source for the regeneration and remodeling of periodontal ligament (PDL). It is crucial to prevent PDLSCs from mineralization when using the PDLSCs for PDL regeneration. At present, little is known about how to inhibit PDLSC mineralization. This study investigates the effects of pyrophosphate (PPi) on inhibiting PDLSC osteogenic differentiation and mineralization as well as the underlying mechanism. MATERIALS AND METHODS: Human PDLSCs were cultured in an osteogenic differentiation medium with different PPi concentrations (0, 10, or 100 µM). The effects of PPi on osteogenic differentiation were assessed by ALP activity and the expressions of osteogenic related proteins (OPN, RUNX2, OSX, and DMP1). The mineralization formation was detected by alizarin red staining. The activation of MAPK signaling pathways (ERK1/2, JNK, and p38) was determined by western blotting and pathway blockade assays. The gene expressions of PPi's regulators (Ank, Enpp1, and Alpl) were assessed by real-time PCR. RESULTS: Both low and high concentrations (10 µM and 100 µM) of PPi inhibited the mineralization of PDLSCs. The addition of PPi (10 µM or 100 µM) decreased the ALP activity of the PDLSCs to approximately two-thirds of the control group on day 3. PPi reduced the expressions of RUNX2, OSX, and DMP1 on days 7, 14, and 21, while it increased the expression of OPN at the three time points. PPi enhanced the phosphorylation of MAPK pathways, and the application of corresponding MAPK pathway inhibitors reversed the osteogenic inhibition effects of PPi. CONCLUSION: PPi inhibits the osteogenic differentiation and mineralization of PDLSCs in vitro through activating ERK1/2, JNK, and p38 signaling pathways.


Assuntos
Osteogênese , Ligamento Periodontal , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Difosfatos , Humanos , Sistema de Sinalização das MAP Quinases
7.
Biosensors (Basel) ; 11(6)2021 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-34070918

RESUMO

In this paper, we propose a simple colorimetric method for the sensitive and selective detection of alkaline phosphatase (ALP) activity based on the turn off/turn on oxidase mimic activity of His@AuNCs. His@AuNCs/graphene oxide hybrids (His@AuNCs/GO) were easily obtained using the self-assembly method with poly (diallyldimethylammonium chloride) (PDDA)-coated GO and showed high oxidase-like activity compared with His@AuNCs. We found that the pyrophosphate ion (P2O74-, PPi) could effectively inhibit the oxidase mimic activity of His@AuNCs/GO, and the hydrolysis of PPi by ALP restored the inhibited activity of His@AuNCs/GO, enabling them to efficiently catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) to generate the blue oxidized product oxTMB. The intensity of the color showed a linear dependency with the ALP activity. ALP was detected in the linear range of 0-40 mU/mL with a low detection limit (LOD) of 0.26 mU/mL (S/N = 3). The proposed method is fast, easy, and can be applied to monitor the ALP activity in serum samples accurately and effectively, which suggests its practicability and reliability in the detection of ALP activity in clinical practice.


Assuntos
Fosfatase Alcalina/análise , Nanopartículas Metálicas/química , Benzidinas , Catálise , Colorimetria , Difosfatos , Ouro , Humanos , Hidrólise , Oxirredução , Oxirredutases , Reprodutibilidade dos Testes
8.
Semin Arthritis Rheum ; 51(3): 655-660, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33941385

RESUMO

OBJECTIVE: To explore the lived experience of people with calcium pyrophosphate deposition (CPPD) disease and the impact of this condition on their daily lives. METHODS: Patients with CPPD and their caregivers were invited to take part in a one-to-one (patient only) or paired (patient and caregiver) semi-structured interview. Interviews covered patients' diagnosis and treatment experiences, and the impact of CPPD on their daily lives. Transcribed interviews were analysed using inductive thematic analysis. RESULTS: 28 patient interviews, six of which included a caregiver, were conducted across five countries. Acute CPP crystal arthritis flares resulted in temporary but profound disability for most patients, disrupting their ability to go about day-to-day activities, and they sought immediate medical attention. CPPD+OA and chronic CPP crystal inflammatory arthritis presented patients with longer term limitations in daily lives. Patients and their caregivers described these disruptions and limitations, which included a reduced ability or inability to complete household and self-care tasks, exercise, socialise, work and drive. They also described how arthritis pain and resulting limitations adversely impacted upon patients' psychological wellbeing. Delays in referral to specialists and diagnostic uncertainty were described by many. Lack of appropriate treatment or access to treatments only upon worsening of symptoms impacted upon the length of time some patients spent in pain and with functional limitations. CONCLUSION: This study is the first to demonstrate the wide-ranging impact of CPPD, and highlights the need for improved diagnosis, physician training, as well as greater emphasis upon finding targeted therapies to specifically treat CPPD.


Assuntos
Calcinose , Condrocalcinose , Pirofosfato de Cálcio , Cuidadores , Difosfatos , Humanos
9.
PLoS Pathog ; 17(5): e1009532, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33984072

RESUMO

Bacteria inhabit diverse environmental niches and consequently must modulate their metabolism to adapt to stress. The nucleotide second messengers guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) (collectively referred to as (p)ppGpp) are essential for survival during nutrient starvation. (p)ppGpp is synthesized by the RelA-SpoT homologue (RSH) protein family and coordinates the control of cellular metabolism through its combined effect on over 50 proteins. While the role of (p)ppGpp has largely been associated with nutrient limitation, recent studies have shown that (p)ppGpp and related nucleotides have a previously underappreciated effect on different aspects of bacterial physiology, such as maintaining cellular homeostasis and regulating bacterial interactions with a host, other bacteria, or phages. (p)ppGpp produced by pathogenic bacteria facilitates the evasion of host defenses such as reactive nitrogen intermediates, acidic pH, and the complement system. Additionally, (p)ppGpp and pyrophosphorylated derivatives of canonical adenosine nucleotides called (p)ppApp are emerging as effectors of bacterial toxin proteins. Here, we review the RSH protein family with a focus on its unconventional roles during host infection and bacterial competition.


Assuntos
Bactérias/metabolismo , Infecções Bacterianas/microbiologia , Fenômenos Fisiológicos Bacterianos , Proteínas de Bactérias/metabolismo , Difosfatos/metabolismo , Nucleotídeos/metabolismo , Estresse Fisiológico , Animais , Infecções Bacterianas/metabolismo , Infecções Bacterianas/patologia , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Humanos , Fosforilação
10.
Meat Sci ; 180: 108557, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34052695

RESUMO

A dynamic model was developed to predict growth of Clostridium perfringens in cooked ground pork supplemented with salt (0-3% wt/wt) and sodium pyrophosphate (0-0.3% wt/wt) under varying temperatures. C. perfringens (NCTC 8238, NCTC 8239, and NCTC 10240) spores were heat shocked, cooled, and inoculated into ground pork. Isothermal bacterial growth was quantified with variable salt and phosphate concentrations at temperatures ranging from 15 to 51 °C. The primary Baranyi model was fitted to all C. perfringens growth profiles and gave a satisfactory fit (R2 ≥ 0.85). A quadratic polynomial secondary model was developed (P < 0.0001) to predict the maximum specific growth rate as a function of temperature, salt, and phosphate concentrations (R2 = 0.93). A dynamic model was developed and validated using growth data retrieved from 7 published studies. Thirty three out of 44 predictions were within the acceptable prediction zone (-0.5 ≤ prediction error ≤ 1.0). The developed predictive model can be used to minimize the risk of C. perfringens in pork products supplemented with additives during cooling.


Assuntos
Clostridium perfringens/crescimento & desenvolvimento , Produtos da Carne/microbiologia , Modelos Biológicos , Temperatura , Animais , Culinária , Difosfatos , Manipulação de Alimentos/métodos , Microbiologia de Alimentos , Cloreto de Sódio , Esporos Bacterianos/crescimento & desenvolvimento , Suínos
11.
Spectrochim Acta A Mol Biomol Spectrosc ; 257: 119792, 2021 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-33887510

RESUMO

Highly sensitive fluorescence detection of pyrophosphate ion (PPi) is in urgent demand but remains a great obstacle, ascribing to scarcity of high-performance materials with promising optical property and high affinity. Herein, we report the design and fabrication of a coumarin-based organic dye (DCCH-TPD) containing both hydrazide group and terpyridine moiety for PPi biosensing through Cu2+-induced photo-electron transfer (PET) effect and target analyte-switched competitive coordination reaction. Individual DCCH-TPD was found to be highly emissive, and displayed a turn-off response toward Cu2+ due to formation of Cu2+@DCCH-TPD and PET effect. The recognition of Cu2+@DCCH-TPD by PPi leads to generation of Cu2+@PPi complex, which greatly reduces the amount of Cu2+ coordinated with DCCH-TPD, subsequently decreasing PET effect. Significantly enhanced fluorescence is recorded and the fluorescence intensity is closely relied on PPi concentration. Thus, highly sensitive detection of PPi is achieved, and the detection limit was calculated to be 0.075 µM. Furthermore, the proposed sensor presented good selectivity, and excellent practical ability for application in arthritic fluid.


Assuntos
Difosfatos , Líquido Sinovial , Cobre , Cumarínicos , Fluorescência , Corantes Fluorescentes
12.
Int J Mol Sci ; 22(6)2021 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-33799449

RESUMO

(1) Background: Tissue non-specific alkaline phosphatase (TNAP) is suspected to induce atherosclerosis plaque calcification. TNAP, during physiological mineralization, hydrolyzes the mineralization inhibitor inorganic pyrophosphate (PPi). Since atherosclerosis plaques are characterized by the presence of necrotic cells that probably release supraphysiological concentrations of ATP, we explored whether this extracellular adenosine triphosphate (ATP) is hydrolyzed into the mineralization inhibitor PPi or the mineralization stimulator inorganic phosphate (Pi), and whether TNAP is involved. (2) Methods: Murine aortic smooth muscle cell line (MOVAS cells) were transdifferentiated into chondrocyte-like cells in calcifying medium, containing ascorbic acid and ß-glycerophosphate. ATP hydrolysis rates were determined in extracellular medium extracted from MOVAS cultures during their transdifferentiation, using 31P-NMR and IR spectroscopy. (3) Results: ATP and PPi hydrolysis by MOVAS cells increased during transdifferentiation. ATP hydrolysis was sequential, yielding adenosine diphosphate (ADP), adenosine monophosphate (AMP), and adenosine without any detectable PPi. The addition of levamisole partially inhibited ATP hydrolysis, indicating that TNAP and other types of ectonucleoside triphoshatediphosphohydrolases contributed to ATP hydrolysis. (4) Conclusions: Our findings suggest that high ATP levels released by cells in proximity to vascular smooth muscle cells (VSMCs) in atherosclerosis plaques generate Pi and not PPi, which may exacerbate plaque calcification.


Assuntos
Aterosclerose/genética , Transdiferenciação Celular/genética , Difosfatos/metabolismo , Calcificação Vascular/genética , Trifosfato de Adenosina , Fosfatase Alcalina/genética , Animais , Aorta/citologia , Aorta/metabolismo , Ácido Ascórbico/farmacologia , Aterosclerose/metabolismo , Aterosclerose/patologia , Condrócitos/metabolismo , Condrócitos/patologia , Glicerofosfatos/genética , Glicerofosfatos/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Camundongos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Fosfatos/metabolismo , Calcificação Vascular/metabolismo , Calcificação Vascular/patologia
13.
Molecules ; 26(8)2021 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-33919593

RESUMO

Inorganic pyrophosphatase (PPase) is a ubiquitous enzyme that converts pyrophosphate (PPi) to phosphate and, in this way, controls numerous biosynthetic reactions that produce PPi as a byproduct. PPase activity is generally assayed by measuring the product of the hydrolysis reaction, phosphate. This reaction is reversible, allowing PPi synthesis measurements and making PPase an excellent model enzyme for the study of phosphoanhydride bond formation. Here we summarize our long-time experience in measuring PPase activity and overview three types of the assay that are found most useful for (a) low-substrate continuous monitoring of PPi hydrolysis, (b) continuous and fixed-time measurements of PPi synthesis, and (c) high-throughput procedure for screening purposes. The assays are based on the color reactions between phosphomolybdic acid and triphenylmethane dyes or use a coupled ATP sulfurylase/luciferase enzyme assay. We also provide procedures to estimate initial velocity from the product formation curve and calculate the assay medium's composition, whose components are involved in multiple equilibria.


Assuntos
Difosfatos/metabolismo , Pirofosfatase Inorgânica/isolamento & purificação , Fosfatos/metabolismo , Ensaios Enzimáticos/métodos , Humanos , Hidrólise , Pirofosfatase Inorgânica/química , Luciferases/química , Fosfatos/química
14.
Int J Mol Sci ; 22(9)2021 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-33925341

RESUMO

Pathological (ectopic) mineralization of soft tissues occurs during aging, in several common conditions such as diabetes, hypercholesterolemia, and renal failure and in certain genetic disorders. Pseudoxanthoma elasticum (PXE), a multi-organ disease affecting dermal, ocular, and cardiovascular tissues, is a model for ectopic mineralization disorders. ABCC6 dysfunction is the primary cause of PXE, but also some cases of generalized arterial calcification of infancy (GACI). ABCC6 deficiency in mice underlies an inducible dystrophic cardiac calcification phenotype (DCC). These calcification diseases are part of a spectrum of mineralization disorders that also includes Calcification of Joints and Arteries (CALJA). Since the identification of ABCC6 as the "PXE gene" and the development of several animal models (mice, rat, and zebrafish), there has been significant progress in our understanding of the molecular genetics, the clinical phenotypes, and pathogenesis of these diseases, which share similarities with more common conditions with abnormal calcification. ABCC6 facilitates the cellular efflux of ATP, which is rapidly converted into inorganic pyrophosphate (PPi) and adenosine by the ectonucleotidases NPP1 and CD73 (NT5E). PPi is a potent endogenous inhibitor of calcification, whereas adenosine indirectly contributes to calcification inhibition by suppressing the synthesis of tissue non-specific alkaline phosphatase (TNAP). At present, therapies only exist to alleviate symptoms for both PXE and GACI; however, extensive studies have resulted in several novel approaches to treating PXE and GACI. This review seeks to summarize the role of ABCC6 in ectopic calcification in PXE and other calcification disorders, and discuss therapeutic strategies targeting various proteins in the pathway (ABCC6, NPP1, and TNAP) and direct inhibition of calcification via supplementation by various compounds.


Assuntos
Calcificação Fisiológica/genética , Calcificação Fisiológica/fisiologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , 5'-Nucleotidase/genética , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Calcinose , Difosfatos/metabolismo , Proteínas Ligadas por GPI/genética , Humanos , Artropatias , Camundongos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Pseudoxantoma Elástico/genética , Pseudoxantoma Elástico/fisiopatologia , Pirofosfatases/genética , Pirofosfatases/metabolismo , Ratos , Calcificação Vascular , Doenças Vasculares
15.
Wiad Lek ; 74(1): 43-47, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33851585

RESUMO

OBJECTIVE: The aim: Of our study was to measure the mRNA expression of the investigated odontogenesis factors in mandible tissue of mouse embryos (17th day of pregnancy) gestated by females, kept on a E450 rich diet since 30 days before fertilization to gestation. PATIENTS AND METHODS: Materials and methods: The effect of food supplements was studied in «Overload phosphates model¼. Experiments were carried out on white nonlinear outbred mice with mass 25-28g (n=40). The females from the control group were fed with standard rodent food, whereas the experimental females were fed with pyrophosphate-enriched food. The materials, used for the molecular genetic study, were the lower jaws of 17-days old mouse embryos (E-17). RESULTS: Results: The investigated BMP2 and osteocalcin genes are expressed at approximately the same level. Pyrophosphate-rich diet does not alter BMP2 gene expression, but it significantly increases the expression of osteocalcin. CONCLUSION: Conclusions: The present study is the first one to describe the impact of the pyrophosphate-rich diet on mRNA expression of key osteogenesis regulators - osteocalcin and BMP2.


Assuntos
Difosfatos , Osteoblastos , Animais , Proteína Morfogenética Óssea 2 , Proteínas Morfogenéticas Ósseas/metabolismo , Dieta , Feminino , Mandíbula , Camundongos , Osteoblastos/metabolismo , Osteocalcina/genética
16.
ACS Chem Biol ; 16(5): 794-799, 2021 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-33877806

RESUMO

Most kinases utilize ATP as a phosphate donor and phosphorylate a wide range of phosphate acceptors. An alternative phosphate donor is inorganic pyrophosphate (PPi), which costs only 1/1000 of ATP. To develop a method to engineer PPi-dependent kinases, we herein aimed to alter the product of PPi-dependent myo-inositol kinase from d-myo-inositol 1-phosphate to d-myo-inositol 3-phosphate. For this purpose, we introduced the myo-inositol recognition residues of the ATP-dependent myo-inositol-3-kinase into the PPi-dependent myo-inositol-1-kinase. This replacement was expected to change the 3D arrangements of myo-inositol in the active site and bring the hydroxyl group at the 3C position close to the catalytic residue. LC-MS and NMR analyses proved that the engineered enzyme successfully produced myo-inositol 3-phosphate from PPi and myo-inositol.


Assuntos
Difosfatos/química , Monoéster Fosfórico Hidrolases/química , Thermotoga maritima/enzimologia , Domínio Catalítico , Cristalização , Fosfatos de Inositol/química , Cinética , Espectroscopia de Ressonância Magnética , Proteínas Mutantes/química , Mutação , Fosforilação , Conformação Proteica , Espectrometria de Massas em Tandem
17.
Biochemistry ; 60(10): 791-801, 2021 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-33656855

RESUMO

S-Adenosyl-l-methionine (AdoMet) is synthesized by the MAT2A isozyme of methionine adenosyltransferase in most human tissues and in cancers. Its contribution to epigenetic control has made it a target for anticancer intervention. A recent kinetic isotope effect analysis of MAT2A demonstrated a loose nucleophilic transition state. Here we show that MAT2A has a sequential mechanism with a rate-limiting step of formation of AdoMet, followed by rapid hydrolysis of the ß-γ bond of triphosphate, and rapid release of phosphate and pyrophosphate. MAT2A catalyzes the slow hydrolysis of both ATP and triphosphate in the absence of other reactants. Positional isotope exchange occurs with 18O as the 5'-oxygen of ATP. Loss of the triphosphate is sufficiently reversible to permit rotation and recombination of the α-phosphoryl group of ATP. Adenosine (α-ß or ß-γ)-imido triphosphates are slow substrates, and the respective imido triphosphates are inhibitors. The hydrolytically stable (α-ß, ß-γ)-diimido triphosphate (PNPNP) is a nanomolar inhibitor. The MAT2A protein structure is highly stabilized against denaturation by binding of PNPNP. A crystal structure of MAT2A with 5'-methylthioadenosine and PNPNP shows the ligands arranged appropriately in the ATP binding site. Two magnesium ions chelate the α- and γ-phosphoryl groups of PNPNP. The ß-phosphoryl oxygen is in contact with an essential potassium ion. Imidophosphate derivatives provide contact models for the design of catalytic site ligands for MAT2A.


Assuntos
Trifosfato de Adenosina/metabolismo , Difosfatos/metabolismo , Inibidores Enzimáticos/farmacologia , Metionina Adenosiltransferase/antagonistas & inibidores , Metionina Adenosiltransferase/metabolismo , Polifosfatos/metabolismo , S-Adenosilmetionina/farmacologia , Sítios de Ligação , Humanos , Hidrólise , Cinética , Conformação Proteica
18.
BMC Oral Health ; 21(1): 113, 2021 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-33706750

RESUMO

OBJECTIVE: To assess the effects for controlling extrinsic tooth stain of a whitening toothpaste containing 10% high cleaning silica, 0.5% sodium phytate and 0.5% sodium pyrophosphate, in comparison with a negative control toothpaste. METHODS: A total of 86 adults who met with the inclusion and exclusion criteria were invited to take part in the study. They were distributed into test and control groups randomly. At baseline, 4 weeks and 8 weeks, the same examiner provided the clinical examinations, including evaluations of oral soft and hard tissues and measurements of tooth stain of the anterior teeth using the Lobene Stain Index. Adverse events and any changes in general health conditions of the patients were monitored. RESULTS: When the study was completed, comparisons between patients in test and control groups yielded statistically significant differences in Lobene stain adjusted mean area score [0.83 (0.05) vs. 1.13 (0.05)], Lobene stain adjusted mean intensity score [0.99 (0.06) vs. 1.32 (0.06)] and Lobene stain adjusted mean composite score [1.45 (0.13) vs. 2.50 (0.13)] (All, P < 0.001). Patients in the test group exhibited reductions of 26.55%, 25% and 42%, respectively in Lobene stain area, intensity and composite scores, relative to patients in the control group. Comparisons within groups showed that all three Lobene scores at 8 weeks in both groups were lower than those at baseline (All, P < 0.001). CONCLUSION: This study demonstrates that 8-week use of a toothpaste containing 10% high cleaning silica, 0.5% sodium phytate and 0.5% sodium pyrophosphate can effectively reduce extrinsic tooth stain. Trial registration NCT04238429 (before enrollment of the first participant). Data register: March 4, 2018.


Assuntos
Dentifrícios , Clareamento Dental , Descoloração de Dente , Adulto , Difosfatos , Humanos , Ácido Fítico/uso terapêutico , Dióxido de Silício , Fluoreto de Sódio/uso terapêutico , Descoloração de Dente/tratamento farmacológico , Cremes Dentais/uso terapêutico , Resultado do Tratamento
19.
Int J Mol Sci ; 22(4)2021 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-33671604

RESUMO

Phosphatases are hydrolytic enzymes that cleave the phosphoester bond of numerous substrates containing phosphorylated residues. The typical classification divides them into acid or alkaline depending on the pH at which they have optimal activity. The histidine phosphatase (HP) superfamily is a large group of functionally diverse enzymes characterized by having an active-site His residue that becomes phosphorylated during catalysis. HP enzymes are relevant biomolecules due to their current and potential application in medicine and biotechnology. Entamoeba histolytica, the causative agent of human amoebiasis, contains a gene (EHI_146950) that encodes a putative secretory acid phosphatase (EhHAPp49), exhibiting sequence similarity to histidine acid phosphatase (HAP)/phytase enzymes, i.e., branch-2 of HP superfamily. To assess whether it has the potential as a biocatalyst in removing phosphate groups from natural substrates, we studied the EhHAPp49 structural and functional features using a computational-experimental approach. Although the combined outcome of computational analyses confirmed its structural similarity with HP branch-2 proteins, the experimental results showed that the recombinant enzyme (rEhHAPp49) has negligible HAP/phytase activity. Nonetheless, results from supplementary activity evaluations revealed that rEhHAPp49 exhibits Mg2+-dependent alkaline pyrophosphatase activity. To our knowledge, this study represents the first computational-experimental characterization of EhHAPp49, which offers further insights into the structure-function relationship and the basis for future research.


Assuntos
Entamoeba histolytica/enzimologia , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/metabolismo , Relação Estrutura-Atividade , 6-Fitase/metabolismo , Sítios de Ligação , Domínio Catalítico , Difosfatos/metabolismo , Entamoeba histolytica/genética , Humanos , Simulação de Acoplamento Molecular , Monoéster Fosfórico Hidrolases/genética , Conformação Proteica , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
20.
Bioresour Technol ; 329: 124922, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33713899

RESUMO

In China, more than 3.5 million tons of Camellia oleifera discarded shells are produced every year. This work first prepared phosphorus-containing biochar (PBC) from C. oleifera shells and was successfully applied to the efficient removal of tetracycline (TC) from solutions. The prepared PBC exhibits superior TC adsorption capacity of 451.5 mg/g, and TC uptake rapidly reached 315.5 mg/g at the first 5 min (C0 = 50 mg/L). Furthermore, PBC also shows excellent applicability to the broad range pH value (1-9) and superior selective removal in the presence of various high concentration coexisting ions (1 mM). Mechanisms underlying TC adsorption were also put forward, and analysis suggested that pyrophosphate-like surface functional groups (C-O-P bond) played a critical role in this process. Notably, treating pharmaceutical wastewater with PBC can efficiently reduce chemical oxygen demand (COD) and total organic carbon (TOC) concentration below the discharge standard of China (GB21904-2008).


Assuntos
Camellia , Poluentes Químicos da Água , Adsorção , Carvão Vegetal , China , Difosfatos , Cinética , Fósforo , Tetraciclina/análise , Poluentes Químicos da Água/análise
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