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1.
Int J Mol Sci ; 22(19)2021 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-34638532

RESUMO

Bone metastases from prostate cancer (PCa) result from a complex cross-talk between PCa cells and osteoblasts (OB). Thus, targeting this interplay has become an attractive strategy to interfere with PCa bone dissemination. The agents currently used in clinical trials have proved ineffective, boosting research to identify additional mechanisms that may be involved in this two-directional talk. Here, we investigated whether and how 5-hydro-5-methylimidazolone (MG-H1), a specific methylglyoxal (MG)-derived advanced glycation end product (AGE), was a novel player in the dialogue between PCa and OB to drive PCa bone metastases. Conditioned medium from osteotropic PC3 PCa cells, pre-treated or not with a specific MG scavenger, was administrated to human primary OB and cell morphology, mesenchymal trans-differentiation, pro-osteogenic determinants, PCa-specific molecules, and migration/invasion were studied by phase-contrast microscopy, real-time PCR, western blot and specific assays, respectively. We found that PC3 cells were able to release MG-H1 that, by binding to the receptor for AGEs (RAGE) on OB, reprogrammed them into a less-differentiate phenotype, endowed with some PCa-specific molecular features and malignant properties, in a mechanism involving reactive oxidative species (ROS) production and NF-kB pathway activation. These findings provide novel insights into the mechanisms of PCa osteoblastic metastases and foster in vivo research toward new therapeutic strategies interfering with PCa/OB cross-talk.


Assuntos
Neoplasias Ósseas/secundário , Desdiferenciação Celular/fisiologia , Imidazóis/metabolismo , Ornitina/análogos & derivados , Osteoblastos/citologia , Neoplasias da Próstata/patologia , Antígenos de Neoplasias/metabolismo , Osso e Ossos/patologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Meios de Cultivo Condicionados/farmacologia , Humanos , Masculino , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Ornitina/metabolismo , Células PC-3 , Próstata/patologia , Espécies Reativas de Oxigênio/metabolismo
2.
J Coll Physicians Surg Pak ; 31(11): 1354-1356, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34689497

RESUMO

Gyrate atrophy is a metabolic disorder characterised by typical progressive circular chorioretinal atrophy, myopia and early developmental cataract. The disease is caused by deficiency of ornithine aminotransferase (OAT) enzyme. Although OAT is expressed in most tissues of the body, but the main target of the disease appears to be the retina. A case is presented here of a 21-year woman, who came to our clinic with the complaint of decline in central vision for eight months. She had progressive poor night vision and was diagnosed with OAT deficiency five years ago. Her systemic history was unremarkable, except for femoral deep vein thrombosis (DVT) which occurred two years ago. Laboratory tests performed at that time had revealed elevated serum ornithine and low serum lysin levels. Optic coherence tomography (OCT) scans showed foveoschisis bilaterally. In summary, gyrate atrophy may present as macular involvement in the form of foveoschisis and may lead to impaired central vision. Key Words: Foveoschisis, Gyrate atrophy, Ornithine aminotransferase.


Assuntos
Atrofia Girata , Atrofia/patologia , Feminino , Angiofluoresceinografia , Atrofia Girata/complicações , Atrofia Girata/diagnóstico , Atrofia Girata/patologia , Humanos , Ornitina , Ornitina-Oxo-Ácido Transaminase/genética , Retina/patologia
3.
Sheng Wu Gong Cheng Xue Bao ; 37(8): 2903-2914, 2021 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-34472307

RESUMO

Ornithine decarboxylase (ODC) is a key enzyme in the biosynthetic pathway of polyamines and catalyzes the decarboxylation of ornithine to produce putrescine. Inhibition of ODC activity is a potential approach for the prevention and treatment of many diseases including cancer, as the expression levels and the activities of ODC in many abnormal cells and tumor cells are generally higher than those of normal cells. The discovery and evaluation of ODC inhibitors rely on the monitoring of the reaction processes catalyzed by ODC. There are several commonly used methods for analyzing the activity of ODC, such as measuring the yield of putrescine by high performance liquid chromatography, or quantifying the yield of isotope labelled carbon dioxide. However, the cumbersome operation and cost of these assays, as well as the difficulty to achieve high-throughput and real-time detection, hampered their applications. In this work, we optimized a real-time label-free method for analyzing the activity of ODC based on the macromolecule cucurbit[6]uril (CB6) and a fluorescent dye, DSMI (trans-4-[4-(dimethylamino) styryl]-1-methylpyridinium iodide). Finally, the optimized method was used to determine the activities of different ODC inhibitors with different inhibition mechanisms.


Assuntos
Inibidores da Ornitina Descarboxilase , Ornitina Descarboxilase , Hidrocarbonetos Aromáticos com Pontes , Imidazóis , Ornitina , Putrescina
4.
ACS Infect Dis ; 7(10): 2917-2929, 2021 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-34570483

RESUMO

Multifunctional antimicrobial peptides that combine the intrinsic microbicidal property of cationic polypeptide chains and additional antibacterial strategy hold promising applications for the treatment of infections caused by antibiotic-resistant bacteria, especially "superbugs". In the present study, star-shaped copolymers ZnPc-g-PLO with a zinc phthalocyanine (ZnPc) core and four poly(l-ornithine) (PLO) arms were designed, synthesized, and evaluated as dual-functional antimicrobial agents, that is, intrinsic membrane damage and photothermal ablation capacity. In an aqueous solution, amphiphilic ZnPc-g-PLO molecules self-assemble into nanosized polymeric micelles with an aggregated ZnPc core and star-shaped PLO periphery, where the ZnPc core exhibits appreciable aggregation-induced photothermal conversion efficiency. In the absence of laser irradiation, ZnPc-g-PLO micelles display potent and broad-spectrum antibacterial activities via physical bacterial membrane disruption as a result of the high cationic charge density of the star-shaped PLO. Upon laser irradiation, significant improvement in bactericidal potency was realized due to the efficacious photothermal sterilization from the ZnPc core. Notably, ZnPc-g-PLO micelles did not induce drug-resistance upon subinhibitory passages. In summary, dual-functional ZnPc-g-PLO copolymers can serve as promising antibacterial agents for the treatment of infectious diseases caused by antibiotic-resistant bacteria.


Assuntos
Anti-Infecciosos , Compostos Organometálicos , Indóis , Isoindóis , Ornitina , Compostos de Zinco
5.
Cells ; 10(8)2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34440845

RESUMO

Keratoconus (KC) is a common corneal ectatic disease that affects 1:500-1:2000 people worldwide and is associated with a progressive thinning of the corneal stroma that may lead to severe astigmatism and visual deficits. Riboflavin-mediated collagen crosslinking currently remains the only approved treatment to halt progressive corneal thinning associated with KC by improving the biomechanical properties of the stroma. Treatments designed to increase collagen deposition by resident corneal stromal keratocytes remain elusive. In this study, we evaluated the effects of arginine supplementation on steady-state levels of arginine and arginine-related metabolites (e.g., ornithine, proline, hydroxyproline, spermidine, and putrescine) and collagen protein expression by primary human corneal fibroblasts isolated from KC and non-KC (healthy) corneas and cultured in an established 3D in vitro model. We identified lower cytoplasmic arginine and spermidine levels in KC-derived constructs compared to healthy controls, which corresponded with overall higher gene expression of arginase. Arginine supplementation led to a robust increase in cytoplasmic arginine, ornithine, and spermidine levels in controls only and a significant increase in collagen type I secretion in KC-derived constructs. Further studies evaluating safety and efficacy of arginine supplementation are required to elucidate the potential therapeutic applications of modulating collagen deposition in the context of KC.


Assuntos
Arginina/farmacologia , Matriz Extracelular/metabolismo , Ceratocone/patologia , Regulação para Cima/efeitos dos fármacos , Arginase/metabolismo , Arginina/metabolismo , Arginina/uso terapêutico , Estudos de Casos e Controles , Técnicas de Cultura de Células , Células Cultivadas , Colágeno/metabolismo , Colágeno Tipo I/metabolismo , Córnea/citologia , Córnea/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Ceratocone/tratamento farmacológico , Ceratocone/metabolismo , Óxido Nítrico Sintase/metabolismo , Ornitina/metabolismo , Espermidina/metabolismo
6.
BMJ Case Rep ; 14(8)2021 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-34404670

RESUMO

A family of three siblings affected with gyrate atrophy of the choroid and retina is presented. Ultrawide field fundus imaging was used to monitor the progression of the disease objectively over 5 years.


Assuntos
Atrofia Girata , Adolescente , Atrofia/patologia , Criança , Corioide/diagnóstico por imagem , Corioide/patologia , Diagnóstico por Imagem , Feminino , Fundo de Olho , Atrofia Girata/complicações , Atrofia Girata/diagnóstico por imagem , Humanos , Masculino , Ornitina , Retina/diagnóstico por imagem , Retina/patologia
7.
Biomolecules ; 11(8)2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34439753

RESUMO

There is a pressing need for molecular targets and biomarkers in gastric cancer (GC). We aimed at identifying aberrations in L-arginine metabolism with therapeutic and diagnostic potential. Systemic metabolites were quantified using mass spectrometry in 293 individuals and enzymes' gene expression was quantified in 29 paired tumor-normal samples using qPCR and referred to cancer pathology and molecular landscape. Patients with cancer or benign disorders had reduced systemic arginine, citrulline, and ornithine and elevated symmetric dimethylarginine and dimethylamine. Citrulline and ornithine depletion was accentuated in metastasizing cancers. Metabolite diagnostic panel had 91% accuracy in detecting cancer and 70% accuracy in differentiating cancer from benign disorders. Gastric tumors had upregulated NOS2 and downregulated ASL, PRMT2, ORNT1, and DDAH1 expression. NOS2 upregulation was less and ASL downregulation was more pronounced in metastatic cancers. Tumor ASL and PRMT2 expression was inversely related to local advancement. Enzyme up- or downregulation was greater or significant solely in cardia subtype. Metabolic reprogramming in GC includes aberrant L-arginine metabolism, reflecting GC subtype and pathology, and is manifested by altered interplay of its intermediates and enzymes. Exploiting L-arginine metabolic pathways for diagnostic and therapeutic purposes is warranted. Functional studies on ASL, PRMT2, and ORNT1 in GC are needed.


Assuntos
Arginina/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Gástricas/metabolismo , Idoso , Argininossuccinato Liase/biossíntese , Diferenciação Celular , Citrulina/metabolismo , DNA Complementar/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/biossíntese , Masculino , Espectrometria de Massas , Metabolômica , Pessoa de Meia-Idade , Proteínas de Transporte da Membrana Mitocondrial/biossíntese , Metástase Neoplásica , Óxido Nítrico Sintase Tipo II , Ornitina/metabolismo , Reação em Cadeia da Polimerase , Proteína-Arginina N-Metiltransferases/biossíntese , Reprodutibilidade dos Testes , Neoplasias Gástricas/tratamento farmacológico , Transcriptoma
8.
Commun Biol ; 4(1): 927, 2021 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-34326456

RESUMO

Human Arginase 1 (hArg1) is a metalloenzyme that catalyzes the hydrolysis of L-arginine to L-ornithine and urea, and modulates T-cell-mediated immune response. Arginase-targeted therapies have been pursued across several disease areas including immunology, oncology, nervous system dysfunction, and cardiovascular dysfunction and diseases. Currently, all published hArg1 inhibitors are small molecules usually less than 350 Da in size. Here we report the cryo-electron microscopy structures of potent and inhibitory anti-hArg antibodies bound to hArg1 which form distinct macromolecular complexes that are greater than 650 kDa. With local resolutions of 3.5 Å or better we unambiguously mapped epitopes and paratopes for all five antibodies and determined that the antibodies act through orthosteric and allosteric mechanisms. These hArg1:antibody complexes present an alternative mechanism to inhibit hArg1 activity and highlight the ability to utilize antibodies as probes in the discovery and development of peptide and small molecule inhibitors for enzymes in general.


Assuntos
Arginase/genética , Arginase/metabolismo , Arginina/química , Sítios de Ligação , Microscopia Crioeletrônica , Ornitina/química , Ligação Proteica , Especificidade por Substrato
9.
Angew Chem Int Ed Engl ; 60(42): 22776-22782, 2021 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-34258835

RESUMO

Although ß-hairpins are widespread in proteins, there is no tool to coax any small peptide to adopt a ß-hairpin conformation, regardless of sequence. Here, we report that δ-linked γ(R)-methyl-ornithine (δ MeOrn) provides an improved ß-turn template for inducing a ß-hairpin conformation in peptides. We developed a synthesis of protected δ MeOrn as a building block suitable for use in Fmoc-based solid-phase peptide synthesis. The synthesis begins with l-leucine and affords gram quantities of the Nα -Boc-Nδ -Fmoc-γ(R)-methyl-ornithine building block. X-ray crystallography confirms that the δ MeOrn turn unit adopts a folded structure in a macrocyclic ß-hairpin peptide. CD and NMR spectroscopy allow comparison of the δ MeOrn turn template to the δ-linked ornithine (δ Orn) turn template that we previously introduced and to the popular d-Pro-Gly turn template. These studies show that the folding of the δ MeOrn turn template is substantially better than that of δ Orn and is comparable to d-Pro-Gly.


Assuntos
Peptídeos/química , Dicroísmo Circular , Cristalografia por Raios X , Ressonância Magnética Nuclear Biomolecular , Ornitina/química , Peptídeos/síntese química , Estrutura Secundária de Proteína , Técnicas de Síntese em Fase Sólida
10.
BMJ Case Rep ; 14(7)2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-34210698

RESUMO

Hyperornithinaemia-hyperammonaemia-homocitrullinuria (HHH) syndrome is a rare inherited metabolic disorder of the urea cycle. Few reports exist to guide practices during pregnancy and fetal delivery. Yet, with affected patients often surviving into reproductive age, appropriate management of the peripartum phase is essential to ensure positive maternal and fetal outcomes.Reassuringly, the vast majority of offspring of parturients with HHH syndrome have normal developmental outcomes; yet as seen here, fetal growth restriction does appear more frequently. Furthermore, in addition to the absent fetal corpus callosum observed in this case, other fetal cerebral abnormalities, including speech delay and intellectual impairment, have been recognised.Unregulated dietary intake is one proposed factor for the observed disruption in fetal growth and early cerebral development. These stipulations not only reinforce the importance of extensive planning and teamwork, but also demonstrate the importance of timely intervention by a metabolic dietician and dietary compliance in the early organogenesis stage of pregnancy.


Assuntos
Hiperamonemia , Distúrbios Congênitos do Ciclo da Ureia , Citrulina , Feminino , Humanos , Hiperamonemia/diagnóstico , Hiperamonemia/terapia , Ornitina/deficiência , Gravidez , Distúrbios Congênitos do Ciclo da Ureia/complicações , Distúrbios Congênitos do Ciclo da Ureia/diagnóstico , Distúrbios Congênitos do Ciclo da Ureia/genética
11.
Molecules ; 26(11)2021 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-34205146

RESUMO

The objective of the present study was to investigate the proximate composition, antiradical properties and hepatoprotective activity of three species of shellfish, Corbicula japonica, Spisula sachalinensis, and Anadara broughtonii, from the coastal areas of Far East Russia. Biologically active peptides such as taurine (3.74 g/100 g protein) and ornithine (2.12 g/100 g protein) have been found in the tissues of A. broughtonii. C. japonica contains a high amount of ornithine (5.57 g/100 g protein) and taurine (0.85 g/100 g protein). The maximum DPPH and ABTS radical scavenging activity (36.0 µg ascorbic acid/g protein and 0.68 µmol/Trolox equiv/g protein, respectively) was determined for the tissue of C. japonica. The protein and peptide molecular weight distribution of the shellfish tissue water extracts was investigated using HPLC. It was found that the amount of low molecular weight proteins and peptides were significantly and positively correlated with radical scavenging activity (Pearson's correlation coefficient = 0.96), while the amount of high molecular weight proteins negatively correlated with radical scavenging activity (Pearson's correlation coefficient = -0.86). Hepatoprotective activity, measured by the survival rate of HepG2 hepatocytes after cotreatment with t-BHP, was detected for C. japonica. The highest protection (95.3 ± 2.4%) was achieved by the cold water extract of C. japonica at the concentration of 200 mg/mL. Moreover, oral administration of hot water extract of C. japonica to rats before the treatment with CCl4 exhibited a markedly protective effect by lowering serum levels of ALT and AST, inhibiting the changes in biochemical parameters of functional state of rat liver, including MDA, SOD, GSH and GST.


Assuntos
Antioxidantes/farmacologia , Arcidae/química , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Corbicula/química , Hepatócitos/citologia , Frutos do Mar/análise , Spisula/química , terc-Butil Hidroperóxido/efeitos adversos , Administração Oral , Animais , Antioxidantes/química , Tetracloreto de Carbono/efeitos adversos , Sobrevivência Celular , Cromatografia Líquida de Alta Pressão , Modelos Animais de Doenças , Células Hep G2 , Hepatócitos/efeitos dos fármacos , Humanos , Masculino , Peso Molecular , Ornitina/isolamento & purificação , Ratos , Federação Russa , Frutos do Mar/classificação , Taurina/isolamento & purificação
12.
Int J Food Microbiol ; 352: 109281, 2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34126526

RESUMO

Food-borne pathogenic bacteria are dispersed throughout the entire chain of the food industry. However, many food preservatives are limited by poor biocompatibility such as cumulative poisoning. The antimicrobial peptide is increasingly regarded as a promising preservative in food research due to its high bioactivity and low cytotoxicity. In this study, thirteen peptides were designed, synthesized, and screened for application as food preservatives. One of them, termed zp65, whose sequence is GIOAOIIIOIOO-NH2, demonstrated potent bactericidal effect against common Gram-negative strains including enterohemorrhagic Escherichia coli, Salmonella, and Citrobacter freundii. Encouragingly, zp65 showed negligible cytotoxicity to both mammalian cells and Galleria mellonella larvae. Peptide zp65 was prone to form α-helix structure in amphiphilic environments, facilitating its affinity with bacterial membrane. Furthermore, the proteolytic stability of zp65 was much higher than its derivatives consisting of totally natural amino acids. Isothermal titration calorimetry indicated that zp65 has a strong binding affinity to lipopolysaccharide with Kd = 1.3 µM, suggesting its possible action target on the bacterial envelope. Mechanistic studies revealed that this peptide also influenced the membrane potential of E.coli O157:H7 (O157) in a dose-dependent manner. Surprisingly, peptide zp65 did not induce disruption of membrane permeability even at a higher concentration of 4-fold minimal inhibitory concentration. By employing confocal microscopy, peptide zp65 labeled by fluorescein isothiocyanate mainly aggregated on the bacterial membrane. These results suggested that the bactericidal mode of action of zp65 is likely attributed to depolarization of the cell membrane. The minced lean beef experiment indicated that the maximum reduction of O157 reached 1.46 log colony-forming unit (CFU) per gram on day 1 after zp65 treatment at the dosage of 40 µg/g. Compared with the untreated cooked beef sample, the CFU of the zp65-treated group remained at a much lower level after 10-day storage. Subsequently, treatment with zp65 at concentrations above 32 µM also significantly reduced O157 viable counts in fresh tomato juice. And the zp65 treatment could rescue about 40% of Galleria mellonella larvae injected with O157-contaminated tomato juice. The peptide zp65 exhibits great potential and deserves further study as a candidate for food preservative.


Assuntos
Escherichia coli O157/efeitos dos fármacos , Microbiologia de Alimentos , Conservantes de Alimentos/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Proteínas Citotóxicas Formadoras de Poros/farmacologia , Animais , Antibacterianos/farmacologia , Bovinos , Contagem de Colônia Microbiana , Larva/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Mariposas/efeitos dos fármacos , Ornitina/química , Carne Vermelha/microbiologia
13.
Molecules ; 26(9)2021 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-34067107

RESUMO

Increased blood glucose in diabetic individuals results in the formation of advanced glycation end products (AGEs), causing various adverse effects on kidney cells, thereby leading to diabetic nephropathy (DN). In this study, the antiglycative potential of Swertiamarin (SM) isolated from the methanolic extract of E. littorale was explored. The effect of SM on protein glycation was studied by incubating bovine serum albumin with fructose at 60 °C in the presence and absence of different concentrations of swertiamarin for 24 h. For comparative analysis, metformin was also used at similar concentrations as SM. Further, to understand the role of SM in preventing DN, in vitro studies using NRK-52E cells were done by treating cells with methylglyoxal (MG) in the presence and absence of SM. SM showed better antiglycative potential as compared to metformin. In addition, SM could prevent the MG mediated pathogenesis in DN by reducing levels of argpyrimidine, oxidative stress and epithelial mesenchymal transition in kidney cells. SM also downregulated the expression of interleukin-6, tumor necrosis factor-α and interleukin-1ß. This study, for the first time, reports the antiglycative potential of SM and also provides novel insights into the molecular mechanisms by which SM prevents toxicity of MG on rat kidney cells.


Assuntos
Células Epiteliais/patologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Glucosídeos Iridoides/farmacologia , Rim/patologia , Estresse Oxidativo/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Pironas/farmacologia , Animais , Bovinos , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Células Epiteliais/metabolismo , Fluorescência , Frutose , Produtos Finais de Glicação Avançada/metabolismo , Glicosilação/efeitos dos fármacos , Inflamação/patologia , Glucosídeos Iridoides/química , Glucosídeos Iridoides/isolamento & purificação , Ligantes , Malondialdeído/metabolismo , Espectrometria de Massas , Ornitina/análogos & derivados , Ornitina/química , Ornitina/farmacologia , Carbonilação Proteica/efeitos dos fármacos , Pirimidinas/química , Pirimidinas/farmacologia , Pironas/química , Pironas/isolamento & purificação , Aldeído Pirúvico , Ratos , Espécies Reativas de Oxigênio/metabolismo , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Soroalbumina Bovina/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier
14.
Anal Bioanal Chem ; 413(17): 4531-4543, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34050775

RESUMO

We adapted a radioligand receptor binding assay for measuring insulin levels in unknown samples. The assay enables rapid and accurate determination of insulin concentrations in experimental samples, such as from insulin-secreting cells. The principle of the method is based on the binding competition of insulin in a measured sample with a radiolabeled insulin for insulin receptor (IR) in IM-9 cells. Both key components, radiolabeled insulin and IM-9 cells, are commercially available. The IR binding assay was used to determine unknown amounts of insulin secreted by MIN6 ß cell line after stimulation with glucose, arginine, ornithine, dopamine, and serotonin. The experimental data obtained by the IR binding assay were compared to the results determined by RIA kits and both methods showed a very good agreement of results. We observed the stimulation of glucose-induced insulin secretion from MIN6 cells by arginine, weaker stimulation by ornithine, but inhibitory effects of dopamine. Serotonin effects were either stimulatory or inhibitory, depending on the concentration of serotonin used. The results will require further investigation. The study also clearly revealed advantages of the IR binding assay that allows the measuring of a higher throughput of measured samples, with a broader range of concentrations than in the case of RIA kits. The IR binding assay can provide an alternative to standard RIA and ELISA assays for the determination of insulin levels in experimental samples and can be especially useful in scientific laboratories studying insulin production and secretion by ß cells and searching for new modulators of insulin secretion.


Assuntos
Secreção de Insulina , Insulina/análise , Insulina/metabolismo , Animais , Arginina/metabolismo , Linhagem Celular , Dopamina/metabolismo , Glucose/metabolismo , Humanos , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Camundongos , Ornitina/metabolismo , Radioimunoensaio/métodos , Ensaio Radioligante/métodos , Ratos , Ratos Wistar , Serotonina/metabolismo
15.
Molecules ; 26(8)2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33921162

RESUMO

Derivatization of amino acids by 2 M HCl/CH3OH (60 min, 80 °C) followed by derivatization of the intermediate methyl esters with pentafluoropropionic anhydride (PFPA) in ethyl acetate (30 min, 65 °C) is a useful two-step derivatization procedure (procedure A) for their quantitative measurement in biological samples by gas chromatography-mass spectrometry (GC-MS) as methyl ester pentafluoropropionic (PFP) derivatives, (Me)m-(PFP)n. This procedure allows in situ preparation of trideutero-methyl esters PFP derivatives, (d3Me)m-(PFP)n, from synthetic amino acids and 2 M HCl/CD3OD for use as internal standards. However, procedure A converts citrulline (Cit) to ornithine (Orn) and homocitrulline (hCit) to lysine (Lys) due to the instability of their carbamide groups under the acidic conditions of the esterification step. In the present study, we investigated whether reversing the order of the two-step derivatization may allow discrimination and simultaneous analysis of these amino acids. Pentafluoropropionylation (30 min, 65 °C) and subsequent methyl esterification (30 min, 80 °C), i.e., procedure B, of Cit resulted in the formation of six open and cyclic reaction products. The most abundant product is likely to be N5-Carboxy-Orn. The second most abundant product was confirmed to be Orn. The most abundant reaction product of hCit was confirmed to be Lys, with the minor reaction product likely being N6-Carboxy-Lys. Mechanisms are proposed for the formation of the reaction products of Cit and hCit via procedure B. It is assumed that at the first derivatization step, amino acids form (N,O)-PFP derivatives including mixed anhydrides. At the second derivatization step, the Cit-(PFP)4 and hCit-(PFP)4 are esterified on their C1-Carboxylic groups and on their activated Nureido groups. Procedure B also allows in situ preparation of (d3Me)m-(PFP)n from synthetic amino acids for use as internal standards. It is demonstrated that the derivatization procedure B enables discrimination between Cit and Orn, and between hCit and Lys. The utility of procedure B to measure simultaneously these amino acids in biological samples such as plasma and urine remains to be demonstrated. Further work is required to optimize the derivatization conditions of procedure B for biological amino acids.


Assuntos
Citrulina/análogos & derivados , Citrulina/química , Cromatografia Gasosa-Espectrometria de Massas/métodos , Aminoácidos/química , Fluorcarbonetos/química , Lisina/química , Ornitina/química
16.
Chem Asian J ; 16(11): 1382-1387, 2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-33886165

RESUMO

The incorporation of non-proteinogenic amino acids (NPAAs) enriches the structural diversity of nonribosomal peptides. Recently, four NPAA-containing cyclic hexapeptides, longicatenamides A-D, were isolated using a combined-culture strategy. Based on in silico analysis, we discovered their putative biosynthetic gene cluster (lon) and proposed a possible biosynthetic mechanism. Surprisingly, the lon22 gene encodes an atypical arginine dihydrolase, which can also catalyze the hydrolysis of citrulline to ornithine. Phylogenetic analysis showed that Lon22-like proteins form a novel clade that is separated from other guanidine-modifying enzymes. After rational design, the catalytic efficiencies of a Lon22 Y80F mutant for arginine and citrulline substrates were 2.31- and 4.70-fold that of the wild-type (WT), respectively. In addition, characterization of the Lon20-A4 adenylation domain suggested that it can incorporate both ornithine and lysine into the final products.


Assuntos
Hidrolases/metabolismo , Peptídeos Cíclicos/biossíntese , Aminoácidos/metabolismo , Sítios de Ligação , Citrulina/metabolismo , Hidrolases/classificação , Hidrolases/genética , Cinética , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Ornitina/metabolismo , Filogenia , Especificidade por Substrato
17.
mBio ; 12(2)2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33758091

RESUMO

Among prokaryotes, cyanobacteria have an exclusive position as they perform oxygenic photosynthesis. Cyanobacteria substantially differ from other bacteria in further aspects, e.g., they evolved a plethora of unique regulatory mechanisms to control primary metabolism. This is exemplified by the regulation of glutamine synthetase (GS) via small proteins termed inactivating factors (IFs). Here, we reveal another small protein, encoded by the ssr0692 gene in the model strain Synechocystis sp. PCC 6803, that regulates flux into the ornithine-ammonia cycle (OAC), the key hub of cyanobacterial nitrogen stockpiling and remobilization. This regulation is achieved by the interaction with the central carbon/nitrogen control protein PII, which commonly controls entry into the OAC by activating the key enzyme of arginine synthesis, N-acetyl-l-glutamate kinase (NAGK). In particular, the Ssr0692 protein competes with NAGK for PII binding and thereby prevents NAGK activation, which in turn lowers arginine synthesis. Accordingly, we termed it P II-interacting regulator of arginine synthesis (PirA). Similar to the GS IFs, PirA accumulates in response to ammonium upshift due to relief from repression by the global nitrogen control transcription factor NtcA. Consistent with this, the deletion of pirA affects the balance of metabolite pools of the OAC in response to ammonium shocks. Moreover, the PirA-PII interaction requires ADP and is prevented by PII mutations affecting the T-loop conformation, the major protein interaction surface of this signal processing protein. Thus, we propose that PirA is an integrator determining flux into N storage compounds not only depending on the N availability but also the energy state of the cell.IMPORTANCE Cyanobacteria contribute a significant portion to the annual oxygen yield and play important roles in biogeochemical cycles, e.g., as major primary producers. Due to their photosynthetic lifestyle, cyanobacteria also arouse interest as hosts for the sustainable production of fuel components and high-value chemicals. However, their broad application as microbial cell factories is hampered by limited knowledge about the regulation of metabolic fluxes in these organisms. Our research identified a novel regulatory protein that controls nitrogen flux, in particular arginine synthesis. Besides its role as a proteinogenic amino acid, arginine is a precursor for the cyanobacterial storage compound cyanophycin, which is of potential interest to biotechnology. Therefore, the obtained results will not only enhance our understanding of flux control in these organisms but also help to provide a scientific basis for targeted metabolic engineering and, hence, the design of photosynthesis-driven biotechnological applications.


Assuntos
Amônia/metabolismo , Ornitina/metabolismo , Synechocystis/genética , Synechocystis/metabolismo , Arginina/biossíntese , Arginina/metabolismo , Nitrogênio/metabolismo , Proteínas PII Reguladoras de Nitrogênio/genética , Proteínas PII Reguladoras de Nitrogênio/metabolismo , Transdução de Sinais
18.
Carcinogenesis ; 42(5): 705-713, 2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-33780524

RESUMO

Advanced glycation end-products (AGEs) are a heterogeneous group of compounds formed by the non-enzymatic reaction between amino acids and reducing sugars, or dicarbonyls as intermediate compounds. Experimental studies suggest that AGEs may promote colorectal cancer, but prospective epidemiologic studies are inconclusive. We conducted a case-control study nested within a large European cohort. Plasma concentrations of three protein-bound AGEs-Nε-(carboxy-methyl)lysine (CML), Nε-(carboxy-ethyl)lysine (CEL) and Nδ-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine (MG-H1)-were measured by ultra-performance liquid chromatography-tandem mass spectrometry in baseline samples collected from 1378 incident primary colorectal cancer cases and 1378 matched controls. Multivariable-adjusted odds ratios (ORs) and 95% confidence intervals (CIs) were computed using conditional logistic regression for colorectal cancer risk associated with CML, CEL, MG-H1, total AGEs, and [CEL+MG-H1: CML] and [CEL:MG-H1] ratios. Inverse colorectal cancer risk associations were observed for CML (OR comparing highest to lowest quintile, ORQ5 versus Q1 = 0.40, 95% CI: 0.27-0.59), MG-H1 (ORQ5 versus Q1 = 0.73, 95% CI: 0.53-1.00) and total AGEs (OR Q5 versus Q1 = 0.52, 95% CI: 0.37-0.73), whereas no association was observed for CEL. A higher [CEL+MG-H1: CML] ratio was associated with colorectal cancer risk (ORQ5 versus Q1 = 1.91, 95% CI: 1.31-2.79). The associations observed did not differ by sex, or by tumour anatomical sub-site. Although individual AGEs concentrations appear to be inversely associated with colorectal cancer risk, a higher ratio of methylglyoxal-derived AGEs versus those derived from glyoxal (calculated by [CEL+MG-H1: CML] ratio) showed a strong positive risk association. Further insight on the metabolism of AGEs and their dicarbonyls precursors, and their roles in colorectal cancer development is needed.


Assuntos
Neoplasias Colorretais/genética , Produtos Finais de Glicação Avançada/genética , Lisina/análogos & derivados , Ornitina/análogos & derivados , Adulto , Idoso , Cromatografia Líquida , Estudos de Coortes , Neoplasias Colorretais/sangue , Neoplasias Colorretais/patologia , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Produtos Finais de Glicação Avançada/sangue , Humanos , Imidazóis/sangue , Lisina/sangue , Lisina/genética , Masculino , Pessoa de Meia-Idade , Razão de Chances , Ornitina/sangue , Ornitina/genética , Espectrometria de Massas em Tandem
19.
Sci Rep ; 11(1): 6389, 2021 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-33737637

RESUMO

There are three major folate uptake systems in human tissues and tumors, including the reduced folate carrier (RFC), folate receptors (FRs) and proton-coupled folate transporter (PCFT). We studied the functional interrelationships among these systems for the novel tumor-targeted antifolates AGF94 (transported by PCFT and FRs but not RFC) and AGF102 (selective for FRs) versus the classic antifolates pemetrexed, methotrexate and PT523 (variously transported by FRs, PCFT and RFC). We engineered HeLa cell models to express FRα or RFC under control of a tetracycline-inducible promoter with or without constitutive PCFT. We showed that cellular accumulations of extracellular folates were determined by the type and levels of the major folate transporters, with PCFT and RFC prevailing over FRα, depending on expression levels and pH. Based on patterns of cell proliferation in the presence of the inhibitors, we established transport redundancy for RFC and PCFT in pemetrexed uptake, and for PCFT and FRα in AGF94 uptake; uptake by PCFT predominated for pemetrexed and FRα for AGF94. For methotrexate and PT523, uptake by RFC predominated even in the presence of PCFT or FRα. For both classic (methotrexate, PT523) and FRα-targeted (AGF102) antifolates, anti-proliferative activities were antagonized by PCFT, likely due to its robust activity in mediating folate accumulation. Collectively, our findings describe a previously unrecognized interplay among the major folate transport systems that depends on transporter levels and extracellular pH, and that determines their contributions to the uptake and anti-tumor efficacies of targeted and untargeted antifolates.


Assuntos
Receptor 1 de Folato/genética , Ácido Fólico/metabolismo , Neoplasias/tratamento farmacológico , Transportador de Folato Acoplado a Próton/genética , Proteína Carregadora de Folato Reduzido/genética , Transporte Biológico/genética , Proliferação de Células/efeitos dos fármacos , Receptor 1 de Folato/metabolismo , Ácido Fólico/genética , Antagonistas do Ácido Fólico/farmacologia , Células HeLa , Humanos , Metotrexato/farmacologia , Neoplasias/genética , Neoplasias/metabolismo , Ornitina/análogos & derivados , Ornitina/farmacologia , Pemetrexede/farmacologia , Transportador de Folato Acoplado a Próton/metabolismo , Pterinas/farmacologia , Proteína Carregadora de Folato Reduzido/metabolismo
20.
PLoS One ; 16(3): e0248385, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33784308

RESUMO

N-hydroxylating flavin-dependent monooxygenases (FMOs) are involved in the biosynthesis of hydroxamate siderophores, playing a key role in microbial virulence. Herein, we report the first structural and kinetic characterization of a novel alkyl diamine N-hydroxylase DesB from Streptomyces sviceus (SsDesB). This enzyme catalyzes the first committed step in the biosynthesis of desferrioxamine B, a clinical drug used to treat iron overload disorders. X-ray crystal structures of the SsDesB holoenzyme with FAD and the ternary complex with bound NADP+ were solved at 2.86 Å and 2.37 Å resolution, respectively, providing a structural view of the active site environment. SsDesB crystallized as a tetramer and the structure of the individual protomers closely resembles the structures of homologous N-hydroxylating FMOs from Erwinia amylovora (DfoA), Pseudomonas aeruginosa (PvdA), and Aspergillus fumigatus (SidA). Using NADPH oxidation, oxygen consumption, and product formation assays, kinetic parameters were determined for various substrates with SsDesB. SsDesB exhibited typical saturation kinetics with substrate inhibition at high concentrations of NAD(P)H as well as cadaverine. The apparent kcat values for NADPH in steady-state NADPH oxidation and oxygen consumption assays were 0.28 ± 0.01 s-1 and 0.24 ± 0.01 s-1, respectively. However, in product formation assays used to measure the rate of N-hydroxylation, the apparent kcat for NADPH (0.034 ± 0.008 s-1) was almost 10-fold lower under saturating FAD and cadaverine concentrations, reflecting an uncoupled reaction, and the apparent NADPH KM was 33 ± 24 µM. Under saturating FAD and NADPH concentrations, the apparent kcat and KM for cadaverine in Csaky assays were 0.048 ± 0.004 s-1 and 19 ± 9 µM, respectively. SsDesB also N-hydroxylated putrescine, spermidine, and L-lysine substrates but not alkyl (di)amines that were branched or had fewer than four methylene units in an alkyl chain. These data demonstrate that SsDesB has wider substrate scope compared to other well-studied ornithine and lysine N-hydroxylases, making it an amenable biocatalyst for the production of desferrioxamine B, derivatives, and other N-substituted products.


Assuntos
Proteínas de Bactérias/metabolismo , Cadaverina/metabolismo , Desferroxamina/metabolismo , Oxigenases de Função Mista/biossíntese , Sideróforos/biossíntese , Streptomyces/enzimologia , Biocatálise , Domínio Catalítico , Dinitrocresóis/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Flavinas/metabolismo , Holoenzimas/metabolismo , Hidroxilação , Cinética , Oxigenases de Função Mista/química , Oxigenases de Função Mista/metabolismo , NADP/metabolismo , Ornitina/metabolismo , Oxirredução
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