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1.
An Acad Bras Cienc ; 92(2): e20181165, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32556047

RESUMO

This study describes the histological characteristics and distribution of gastrointestinal tract endocrine cells (ECs) of Prochilodus lineatus (detritivorous fish) using immunohistochemical procedures. The digestive tract of P. lineatus was divided into seven portions: stomach (cardial and pyloric), pyloric caeca, and intestine (anterior, glandular, middle and posterior). A pool of specific antisera against cholecystokinin (CCK-8), -neuropeptide Y (NPY), -ghrelin (Ghre) and -leu-enkephalin (Leu-ENK) to identify ECs were used. According to the morphological characteristics of ECs, two different types were identified and classified as open or closed-type. The number of ECs varied throughout the gastrointestinal tract, though a high abundance was found in the anterior intestine and pyloric caeca. A large number of ECs immunoreactive to CCK-8 and NPY were recorded in the anterior, glandular and middle intestine. ECs immunopositive to Leu-ENK were distributed in the stomach and pyloric caeca. For Ghre, immunopositive ECs were restricted to the glandular intestine. The results of the present study indicate that P. lineatus presents an ECs distribution pattern with species-specific particularities. However, CCK showed a distribution similar to that of omnivores, which is possibly related to local signaling functions in order to achieve the correct digestion of the various organisms found in the detritus.


Assuntos
Caraciformes/classificação , Encefalina Leucina/análise , Trato Gastrointestinal/química , Grelina/análise , Neuropeptídeos/análise , Sincalida/análise , Animais , Imuno-Histoquímica
2.
Molecules ; 24(24)2019 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-31842282

RESUMO

As tool compounds to study cardiac ischemia, the endogenous δ-opioid receptors (δOR) agonist Leu5-enkephalin and the more metabolically stable synthetic peptide (d-Ala2, d-Leu5)-enkephalin are frequently employed. However, both peptides have similar pharmacological profiles that restrict detailed investigation of the cellular mechanism of the δOR's protective role during ischemic events. Thus, a need remains for δOR peptides with improved selectivity and unique signaling properties for investigating the specific roles for δOR signaling in cardiac ischemia. To this end, we explored substitution at the Phe4 position of Leu5-enkephalin for its ability to modulate receptor function and selectivity. Peptides were assessed for their affinity to bind to δORs and µ-opioid receptors (µORs) and potency to inhibit cAMP signaling and to recruit ß-arrestin 2. Additionally, peptide stability was measured in rat plasma. Substitution of the meta-position of Phe4 of Leu5-enkephalin provided high-affinity ligands with varying levels of selectivity and bias at both the δOR and µOR and improved peptide stability, while substitution with picoline derivatives produced lower-affinity ligands with G protein biases at both receptors. Overall, these favorable substitutions at the meta-position of Phe4 may be combined with other modifications to Leu5-enkephalin to deliver improved agonists with finely tuned potency, selectivity, bias and drug-like properties.


Assuntos
Encefalina Leucina/farmacologia , Receptores Opioides delta/metabolismo , Receptores Opioides mu/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Células CHO , Cricetulus , Encefalina Leucina/genética , Humanos , Fenilalanina , Receptores Opioides delta/agonistas , Receptores Opioides delta/genética , Receptores Opioides mu/agonistas , Receptores Opioides mu/genética , Transdução de Sinais/genética
3.
J Chem Theory Comput ; 15(11): 6456-6470, 2019 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-31553606

RESUMO

Accurate determinations of noncovalent interactions in biological systems are fundamental to rationalize the structure and to get insights into the functions and the dynamics of macromolecules. Here we propose a new tool for the efficient identification of noncovalent interactions in proteins. The noncovalent interaction (NCI) method, a well-established strategy to detect noncovalent interactions in chemical systems, is coupled with the libraries of extremely localized molecular orbitals (ELMOs), which allow instantaneous reconstruction of quantum mechanically rigorous electron distributions of polypeptides and proteins. Test calculations performed on different interactions show that the new NCI-ELMO strategy always outperforms the original NCI method based on the promolecular approximation, while it is in close agreement with original NCI analyses based on fully quantum mechanical calculations. The new technique allows for unraveling the network of interactions in biological systems and to rapidly monitor their evolutions with time, with possible consequences on the design of new drugs.


Assuntos
Modelos Moleculares , Proteínas/química , Teoria Quântica , Bases de Dados de Proteínas , Desenho de Fármacos , Encefalina Leucina/química , Encefalina Leucina/metabolismo , Ligação de Hidrogênio , Metais/química , Proteínas/metabolismo
4.
Bull Exp Biol Med ; 167(4): 428-431, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31502135

RESUMO

We studied the effects of neonatal administration of non-opioid leu-enkephalin analogue peptide NALE) on the morphological parameters of the liver and redox status of 60-dayold albino rats subjected to antenatal hypoxia. In animals subjected to antenatal hypoxia, a decrease body weight and reduction of the area of hepatocytes and total area of their nucleoli were observed; these changes were accompanied by activation of free-radical processes and impairment of antioxidant defense in the liver and blood serum. Intraperitoneal administration of NALE (100 µg/kg, daily) during postnatal days 2-6 normalized body weight, hepatocyte area, and total area of their nucleoli, significantly reduced the intensity of ROS generation, and improved antioxidant protection in the liver and blood serum in 60-day-old animals subjected to antenatal hypoxia.


Assuntos
Encefalina Leucina/análogos & derivados , Encefalina Leucina/uso terapêutico , Hipóxia/tratamento farmacológico , Animais , Peso Corporal/efeitos dos fármacos , Feminino , Hepatócitos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Masculino , Oxirredução/efeitos dos fármacos , Gravidez , Distribuição Aleatória , Ratos , Ratos Wistar
5.
J Coll Physicians Surg Pak ; 29(4): 341-344, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30925957

RESUMO

OBJECTIVE: To compare the influence of percutaneous transforaminal endoscopic discectomy (PTED) and traditional operation on the nervous system function and the serum leu-enkephalin (LEK), glial fibrillary acidic protein (GFAP) and prostaglandin E-2 (PGE-2) in patients with senile lumbar spinal stenosis. STUDY DESIGN: Experimental study. PLACE AND DURATION OF STUDY: Department of Orthopedics Two, Xinjiang Changji Hui Autonomous Prefecture People's Hospital, Xinjiang, China, from March 2017 to March 2018. METHODOLOGY: A total of 146 patients with senile lumbar spinal stenosis were randomly divided into control group and observation group, 73 in each group. Control group underwent traditional operation, while the observation group underwent PTED. General situation of operation, serum LEK, GFAP, PGE-2, American Spinal Injury Association (ASIA) score and Japanese Orthopaedic Association (JOA) score were compared. RESULTS: Intraoperative blood loss in observation group was less than that in control group (p<0.001). Both operation time and length of hospital stay in observation group were shorter than those in control group (both p<0.001). At 24 hours later after operation, both levels of serum LEK and ASIA score in observation group were higher than those in control group (p=0.006 and p<0.001, respectively), and levels of serum GFAP and PGE-2 and JOA score in observation group were all lower than those in control group (all p<0.001). CONCLUSION: Compared with traditional operation, PTED has the advantages of less intraoperative blood loss, shorter operation time and length of hospital stay, etc. Besides, PTED can effectively reduce serum LEK, BFGF and PGE-2 expression in patients; and dramatically improve their nervous system function and lumbar function.


Assuntos
Discotomia Percutânea/métodos , Endoscopia/métodos , Vértebras Lombares/cirurgia , Sistema Nervoso/fisiopatologia , Estenose Espinal/cirurgia , Adulto , Perda Sanguínea Cirúrgica , Encefalina Leucina/sangue , Feminino , Proteína Glial Fibrilar Ácida/sangue , Humanos , Tempo de Internação , Masculino , Pessoa de Meia-Idade , Fenômenos Fisiológicos do Sistema Nervoso , Duração da Cirurgia , Prostaglandinas E/sangue , Estudos Retrospectivos , Resultado do Tratamento
6.
Sci Adv ; 5(2): eaau5148, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30788432

RESUMO

The clinical use of endogenous neuropeptides has historically been limited due to pharmacokinetic issues, including plasma stability and blood-brain barrier permeability. In this study, we show that the rapidly metabolized Leu-enkephalin (LENK) neuropeptide may become pharmacologically efficient owing to a simple conjugation with the lipid squalene (SQ). The corresponding LENK-SQ bioconjugates were synthesized using different chemical linkers in order to modulate the LENK release after their formulation into nanoparticles. This new SQ-based nanoformulation prevented rapid plasma degradation of LENK and conferred on the released neuropeptide a notable antihyperalgesic effect that lasted longer than after treatment with morphine in a rat model of inflammation (Hargreaves test). The biodistribution study as well as the use of brain-permeant and -impermeant opioid receptor antagonists indicated that LENK-SQ NPs act through peripherally located opioid receptors. This study represents a novel nanomedicine approach, allowing the specific delivery of LENK neuropeptide into inflamed tissues for pain control.


Assuntos
Analgésicos Opioides/farmacocinética , Barreira Hematoencefálica/metabolismo , Morfina/farmacocinética , Nanomedicina Teranóstica , Analgésicos Opioides/administração & dosagem , Analgésicos Opioides/química , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Encefalina Leucina/química , Encefalina Leucina/farmacocinética , Hiperalgesia/tratamento farmacológico , Masculino , Camundongos , Estrutura Molecular , Morfina/administração & dosagem , Morfina/química , Nanopartículas/química , Nanopartículas/ultraestrutura , Ratos , Esqualeno/química , Distribuição Tecidual
7.
Biochemistry ; 58(8): 1032-1037, 2019 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-30719916

RESUMO

This study reports a general method to calculate dihedral angles (φ and ψ) of a given amino acid sequence, focusing on potential energy and torque moment concepts. By defining these physical measures in relation to the chemical interactions that occur on each single amino acid residue within a peptide, we analyze the folding process as the result of main mechanical forces (MMFs) exerted in the specific amino acid chain of interest. As a proof of concept, Leu-enkephalin was initially used as a model peptide to carry out the theoretical study. Our data show agreement between calculated Leu-enkephalin backbone dihedral angles and the corresponding experimentally determined X-ray values. Hence, we used calcitonin to validate our MMF-based method on a larger peptide, i.e., 32 amino acid residues forming an α-helix. Through a similar approach (although simplified with regard to electrostatic interactions), the calculations for calcitonin also demonstrate a good agreement with experimental values. This study offers new opportunities to analyze peptides' amino acid sequences and to help in the prediction of how they must fold, assisting in the development of new computational techniques in the field.


Assuntos
Calcitonina/química , Encefalina Leucina/química , Fragmentos de Peptídeos/química , Dobramento de Proteína , Estresse Mecânico , Animais , Humanos , Modelos Moleculares , Conformação Molecular
8.
ACS Chem Neurosci ; 10(3): 1615-1626, 2019 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-30614675

RESUMO

Leu-enkephalin and d-Ala2-Leu-enkephalin were modified at their N- and C-termini with guanidyl and tetrazole groups. The resulting molecules were prepared in solution or by solid phase peptide synthesis. The affinity of the different analogues at mu (MOP) and delta opioid receptors (DOP) was then assessed by competitive binding in stably transfected DOP and MOP HEK293 cells. Inhibition of cAMP production and recruitment of ß-arrestin were also investigated. Finally, lipophilicity (logD7.4) and plasma stability of each compound were measured. Compared to the native ligands, we found that the replacement of the terminal carboxylate by a tetrazole slightly decreased both the affinity at mu and delta opioid receptors as well as the half-life. By contrast, replacing the ammonium at the N-terminus with a guanidyl significantly improved the affinity, the potency, as well as the lipophilicity and the stability of the resulting peptides. Replacing the glycine residue with a d-alanine in position 2 consistently improved the potency as well as the stability of the analogues. The best peptidomimetic of the whole series, guanidyl-Tyr-d-Ala-Gly-Phe-Leu-tetrazole, displayed sub-nanomolar affinity and an increased lipophilicity. Moreover, it proved to be stable in plasma for up to 24 h, suggesting that the modifications are protecting the compound against protease degradation.


Assuntos
Encefalina Leucina/análogos & derivados , Oligopeptídeos/farmacologia , Receptores Opioides delta/agonistas , Receptores Opioides mu/agonistas , Animais , Células HEK293 , Humanos , Peptídeos Opioides/efeitos dos fármacos , Receptores Opioides delta/metabolismo , Receptores Opioides mu/metabolismo
9.
Free Radic Biol Med ; 131: 126-132, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30502457

RESUMO

Fast Photochemical Oxidation of Protein (FPOP), based on a pulsed KrF laser (248 nm) for free-radical generation, is a biophysical method that utilizes hydroxyl radicals to footprint proteins in solution. FPOP has been recognized for structural proteomics investigations, including epitope mapping, protein-aggregation characterization, protein-folding monitoring, and binding-affinity determination. The distinct merits of the platform are: i) the use of a scavenger to control radical lifetime and allow fast ("snapshot") footprinting of solvent-accessible residues in a protein; ii) the employment of a flow system to enable single-shot irradiation of small plugs of the targeted sample; iii) the use of methionine and catalase after radical oxidation chemistry to prevent post-oxidation with residual oxidizing species; and iv) the utilization of mature mass spectrometry-based proteomic methods to afford detailed analysis. In addition to •OH, other reactive reagents (e.g., carbenes, iodide, sulfate radical anion, and trifluoromethyl radical) can be implemented on this platform to increase the versatility and scope. In this study, we further elaborate the use of FPOP platform to generate secondary radicals and establish a workflow to answer fundamental questions regarding the intrinsic selectivity and reactivity of radicals that are important in biology. Carbonate radical anion is the example we chose owing to its oxidative character and important putative pathogenic roles in inflammation. This systematic study with model proteins/peptides gives consistent results with a previous study that evaluated reactivity with free amino acids and shows that methionine and tryptophan are the most reactive residues with CO3-•. Other aromatic amino acids (i.e., tyrosine, histidine and phenylalanine) exhibit moderate reactivity, whereas, aliphatic amino acids are inert, unlike with •OH. The outcome demonstrates this approach to be appropriate for studying the fast reactions of radicals with proteins.


Assuntos
Angiotensina I/química , Bombesina/química , Bradicinina/química , Carbonatos/química , Encefalina Leucina/química , Sequência de Aminoácidos , Catalase/química , Radicais Livres , Peróxido de Hidrogênio/química , Cinética , Lasers de Excimer , Luz , Metano/análogos & derivados , Metano/química , Metionina/química , Oxirredução , Processos Fotoquímicos , Soluções , Triptofano/química
10.
Pharmacol Rep ; 71(1): 42-47, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30391790

RESUMO

BACKGROUND: Inflammatory bowel diseases (IBD) are a group of chronic and recurrent gastrointestinal disorders that are difficult to control. Recently, a new IBD therapy based on the targeting of the endogenous opioid system has been proposed. Consequently, due to the fact that endogenous enkephalins have an anti-inflammatory effect, we aimed at investigating the degradation of serum enkephalin (Met- and Leu-enkephalin) in patients with IBD. METHODS: Enkephalin degradation in serum of patients with IBD was characterized using mass spectrometry methods. Calculated half-life (T1/2) of enkephalins were compared and correlated with the disease type and gender of the patients. Additionally, statistical analysis was used to examine the dynamics of changes in terms of inhibition of enkephalins degradation within research groups. RESULTS: Our research indicates that the degree of enkephalins degradation depends on the gender of the patients. The difference is most evident for the rate of Met-enkephalin degradation between men (mean T1/2 = 13.61 min) and women (mean T1/2 = 21.84 min) with Crohn's disease (CD). CONCLUSIONS: The most significant alternation of enkephalins degradation in serum samples of IBD patients, compared to control group, were observed in both Crohn's disease and ulcerative colitis (UC) female patients. We suggest that the differences observed between the genders in IBD patients may be explained by regulation of enkephalinases activity by estradiol.


Assuntos
Colite Ulcerativa/sangue , Doença de Crohn/sangue , Encefalina Leucina/sangue , Encefalina Metionina/sangue , Adulto , Biomarcadores/sangue , Estudos de Casos e Controles , Cromatografia Líquida de Alta Pressão , Colite Ulcerativa/diagnóstico , Doença de Crohn/diagnóstico , Feminino , Meia-Vida , Humanos , Masculino , Estudos Prospectivos , Proteólise , Fatores Sexuais , Espectrometria de Massas por Ionização por Electrospray
11.
Amino Acids ; 51(2): 319-329, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30392096

RESUMO

Quercetin and resveratrol are polyphenolic compounds, members of the flavonoid and the stilbene family, respectively, both medicinally important as dietary anticancer and antioxidant agents. They are present in a variety of foods-including fruits, vegetables, tea, wine, as well as other dietary supplements-and are responsible for various health benefits. Different quercetin and resveratrol esters of Leu/Met-enkephalin and tetrapeptide Leu-Ser-Lys-Leu (LSKL) were synthesized as model systems for monitoring the influence of the peptides on biological activity of resveratrol and quercetin. General formula of the main peptidyl-quercetin derivatives is 2-[3-(aa)n-4-hydroxyphenyl]-3,5,7-tri-hydroxy-4H-1-benzopyran-4-on, and the general formula of the main peptidyl-resveratrol derivatives is (E)-5-[4-(aa)n)styryl]benzene-1,3-diol. The antioxidant and anticancer activities of prepared compounds were investigated. Significant anticancer activity was obtained for the LSKL-based both quercetin and resveratrol derivatives. All prepared compounds exhibit antioxidant activity, in particular quercetin derivative containing Met-enkephalin.


Assuntos
Antineoplásicos/farmacologia , Antioxidantes/farmacologia , Neoplasias/dietoterapia , Quercetina/análogos & derivados , Quercetina/farmacologia , Resveratrol/análogos & derivados , Resveratrol/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/uso terapêutico , Antioxidantes/síntese química , Antioxidantes/uso terapêutico , Proliferação de Células/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Suplementos Nutricionais , Encefalina Leucina/química , Encefalina Metionina/química , Ésteres/síntese química , Células HCT116 , Humanos , Células MCF-7 , Peptídeos/química , Compostos Fitoquímicos/síntese química , Quercetina/síntese química , Quercetina/uso terapêutico , Resveratrol/síntese química , Resveratrol/uso terapêutico , Solubilidade , Fator de Crescimento Transformador beta/metabolismo
12.
Eur Neuropsychopharmacol ; 29(3): 450-456, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30591345

RESUMO

The impact that ß-arrestin proteins have on G protein-coupled receptor trafficking, signaling and physiological behavior has gained much appreciation over the past decade. A number of studies have attributed the side effects associated with the use of naturally occurring and synthetic opioids, such as respiratory depression and constipation, to excessive recruitment of ß-arrestin. These findings have led to the development of biased opioid small molecule agonists that do not recruit ß-arrestin, activating only the canonical G protein pathway. Similar G protein-biased small molecule opioids have been found to occur in nature, particularly within kratom, and opioids within salvia have served as a template for the synthesis of other G protein-biased opioids. Here, we present the first report of naturally occurring peptides that selectively activate G protein signaling pathways at δ opioid receptors, but with minimal ß-arrestin recruitment. Specifically, we find that rubiscolin peptides, which are produced as cleavage products of the plant protein rubisco, bind to and activate G protein signaling at δ opioid receptors. However, unlike the naturally occurring δ opioid peptides leu-enkephalin and deltorphin II, the rubiscolin peptides only very weakly recruit ß-arrestin 2 and have undetectable recruitment of ß-arrestin 1 at the δ opioid receptor.


Assuntos
Receptores Opioides delta/química , Receptores Opioides delta/metabolismo , Ribulose-Bifosfato Carboxilase/metabolismo , Animais , Células CHO , Cricetulus , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Encefalina Leucina/farmacologia , Modelos Moleculares , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Ensaio Radioligante , Receptores Opioides delta/genética , Ribulose-Bifosfato Carboxilase/síntese química , Ribulose-Bifosfato Carboxilase/química , Ribulose-Bifosfato Carboxilase/farmacologia , Transfecção , beta-Arrestina 2/genética , beta-Arrestina 2/metabolismo
13.
Phys Chem Chem Phys ; 20(38): 24894-24901, 2018 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-30234204

RESUMO

The intrinsic structure of an opioid peptide [Ala2, Leu5]-leucine enkephalin (ALE) has been investigated using first-principles based vibrational self-consistent field (VSCF) theory and cold ion spectroscopy. IR-UV double resonance spectroscopy revealed the presence of only one highly abundant conformer of the singly protonated ALE, isolated and cryogenically cooled in the gas phase. High-level quantum mechanical calculations of electronic structures in conjunction with a systematic conformational search allowed for finding a few low-energy candidate structures. In order to identify the observed structure, we computed vibrational spectra of the candidate structures and employed the theory at the semi-empirically scaled harmonic level and at the first-principles based anharmonic VSCF levels. The best match between the calculated "anharmonic" and the measured spectra appeared, indeed, for the most stable candidate. An average of two spectra calculated with different quantum mechanical potentials is proposed for the best match with experiment. The match thus validates the calculated intrinsic structure of ALE and demonstrates the predictive power of first-principles theory for solving structures of such large molecules.


Assuntos
Encefalina Leucina/química , Espectrofotometria Infravermelho/métodos , Modelos Moleculares , Conformação Proteica , Reprodutibilidade dos Testes
14.
Anat Histol Embryol ; 47(6): 517-526, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30105873

RESUMO

The gastrointestinal (GI) tract is innervated by nerve processes derived from the intramural enteric neurons and neurons localized outside the digestive tract. This study analysed the neurochemical characterization of nerves in the wall of the porcine oesophagus using single immunofluorescence technique. Immunoreactivity to vesicular acetylcholine transporter (VAChT), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), somatostatin (SOM), galanin (GAL), neuronal isoform of nitric oxide synthase (nNOS), substance P (SP), leucine enkephalin (LENK), calcitonin gene-related peptide (CGRP) or dopamine beta-hydroxylase (DBH) was investigated in intramuscular and intramucosal nerves of the cervical, thoracic and abdominal oesophagus. The results indicate that all of the substances studied were present in the oesophageal nerves. The density of particular populations of fibres depended on the segment of the oesophagus. The most numerous were fibres immunoreactive to VIP in the longitudinal and circular muscle layers of the abdominal oesophagus: The number of these fibres amounted to 16.4 ± 0.8 and 18.1 ± 3.1, respectively. In turn, the least numerous were CGRP-positive fibres, which were present only in the circular muscle layer of the cervical oesophagus and mucosal layer of the abdominal oesophagus in the number of 0.3 ± 0. The obtained results show that nerves in the porcine oesophageal wall are very diverse in their neurochemical coding, and differences between particular parts of the oesophagus suggest that organization of the innervation clearly depends on the fragment of this organ.


Assuntos
Sistema Nervoso Entérico/química , Esôfago/inervação , Imunofluorescência/veterinária , Fibras Nervosas/química , Neuropeptídeos/análise , Animais , Peptídeo Relacionado com Gene de Calcitonina/análise , Dopamina beta-Hidroxilase/análise , Encefalina Leucina/análise , Feminino , Galanina/análise , Neuropeptídeo Y/análise , Óxido Nítrico Sintase Tipo I/análise , Somatostatina/análise , Substância P/análise , Suínos , Peptídeo Intestinal Vasoativo/análise , Proteínas Vesiculares de Transporte de Acetilcolina/análise
15.
J Diabetes Res ; 2018: 4735659, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30140706

RESUMO

One of the most frequently reported disorders associated with diabetes is gastrointestinal (GI) disturbance. Although pathogenesis of these complications is multifactorial, the complicity of the enteric nervous system (ENS) in this respect has significant importance. Therefore, this paper analysed changes in substance P- (SP-), calcitonin gene-related peptide- (CGRP-), and leu5-enkephalin- (L-ENK-) like immunoreactivity (LI) in enteric stomach neurons caused by chemically induced diabetes in a porcine model. Using double immunofluorescent labelling, it was found that acute hyperglycaemia led to significant changes in the chemical coding of stomach enteric neurons. Generally, the response to artificially inducted diabetes depended on the "kind" of enteric plexus as well as the stomach region studied. A clear increase in the percentage of neurons immunoreactive to SP and CGRP was visible in the myenteric plexus (MP) in the antrum, corpus, and pylorus as well as in the submucosal plexus (SmP) in the corpus. For L-ENK, an increase in the number of L-ENK-LI neurons was observed in the MP of the antrum and SmP in the corpus, while in the MP of the corpus and pylorus, a decrease in the percentage of L-ENK-LI neurons was noted.


Assuntos
Diabetes Mellitus Experimental/metabolismo , Sistema Nervoso Entérico/metabolismo , Imunofluorescência , Neurônios/metabolismo , Neuropeptídeos/metabolismo , Estômago/inervação , Animais , Peptídeo Relacionado com Gene de Calcitonina/imunologia , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/imunologia , Encefalina Leucina/imunologia , Encefalina Leucina/metabolismo , Sistema Nervoso Entérico/imunologia , Feminino , Neurônios/imunologia , Neuropeptídeos/imunologia , Estreptozocina , Substância P/imunologia , Substância P/metabolismo , Sus scrofa
16.
Anal Biochem ; 559: 24-29, 2018 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-29981318

RESUMO

The aim of this work was to assess the influence of preanalytical variables on the stability of two endogenous opioid peptides (Methionine-Enkephalin and Leucine-Enkephalin) in human plasma. For this purpose, first a sensitive LC-MS/MS analytical method was developed and validated for the simultaneous quantitative analysis of these two peptides. The methodology consisted of a simple protein precipitation step followed by UPLC separation and MRM quantitative analysis using a stable isotope labelled Methionine-Enkephalin as internal standard. The method with a limit of quantitation of 10 pg/mL showed good reproducibility with excellent accuracy and precision, and was linear up to 2000 pg/mL. An extensive evaluation of the pre-analytical stability of these peptides in human blood was carried out to ensure an adequate sample collection procedure to obtain reliable results in the analysis of clinical samples.


Assuntos
Encefalina Leucina/sangue , Encefalina Metionina/sangue , Cromatografia Líquida de Alta Pressão , Encefalina Leucina/química , Encefalina Metionina/química , Humanos , Espectrometria de Massas , Estrutura Molecular
17.
Rev Sci Instrum ; 89(4): 043104, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29716322

RESUMO

In the present paper, we describe a new home-built crossed-beam apparatus devoted to ion-induced ionization and fragmentation of isolated biologically relevant molecular systems. The biomolecular ions are produced by an electrospray ionization source, mass-over-charge selected, accumulated in a 3D ion trap, and then guided to the extraction region of an orthogonal time-of-flight mass spectrometer. Here, the target molecular ions interact with a keV atomic ion beam produced by an electron cyclotron resonance ion source. Cationic products from the collision are detected on a position sensitive detector and analyzed by time-of-flight mass spectrometry. A detailed description of the operation of the setup is given, and early results from irradiation of a protonated pentapeptide (leucine-enkephalin) by a 7 keV He+ ion beam are presented as a proof-of-principle.


Assuntos
Espectrometria de Massas por Ionização por Electrospray/instrumentação , Espectrometria de Massas em Tandem/instrumentação , Elétrons , Encefalina Leucina/química , Desenho de Equipamento , Gases/química , Hélio/química , Íons/química , Cinética , Estudo de Prova de Conceito
18.
BMC Vet Res ; 14(1): 169, 2018 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-29793486

RESUMO

BACKGROUND: The enteric nervous system (ENS), located in the intestinal wall and characterized by considerable independence from the central nervous system, consists of millions of cells. Enteric neurons control the majority of functions of the gastrointestinal tract using a wide range of substances, which are neuromediators and/or neuromodulators. One of them is leucine-enkephalin (leuENK), which belongs to the endogenous opioid family. It is known that opioids in the gastrointestinal tract have various functions, including visceral pain conduction, intestinal motility and secretion and immune processes, but many aspects of distribution and function of leuENK in the ENS, especially during pathological states, remain unknown. RESULTS: During this experiment, the distribution of leuENK - like immunoreactive (leuENK-LI) nervous structures using the immunofluorescence technique were studied in the porcine colon in physiological conditions, during chemically-induced inflammation and after axotomy. The study included the circular muscle layer, myenteric (MP), outer submucous (OSP) and inner submucous plexus (ISP) and the mucosal layer. In control animals, the number of leuENK-LI neurons amounted to 4.86 ± 0.17%, 2.86 ± 0.28% and 1.07 ± 0.08% in the MP, OSP and ISP, respectively. Generally, both pathological stimuli caused an increase in the number of detected leuENK-LI cells, but the intensity of the observed changes depended on the factor studied and part of the ENS. The percentage of leuENK-LI perikarya amounted to 11.48 ± 0.96%, 8.71 ± 0.13% and 9.40 ± 0.76% during colitis, and 6.90 ± 0.52% 8.46 ± 12% and 4.48 ± 0.44% after axotomy in MP, OSP and ISP, respectively. Both processes also resulted in an increase in the number of leuENK-LI nerves in the circular muscle layer, whereas changes were less visible in the mucosa during inflammation and axotomy did not change the number of leuENK-LI mucosal fibers. CONCLUSIONS: LeuENK in the ENS takes part in intestinal regulatory processes not only in physiological conditions, but also under pathological factors. The observed changes are probably connected with the participation of leuENK in sensory and motor innervation and the neuroprotective effects of this substance. Differences in the number of leuENK-LI neurons during inflammation and after axotomy may suggest that the exact functions of leuENK probably depend on the type of pathological factor acting on the intestine.


Assuntos
Colite/veterinária , Colo Descendente/metabolismo , Encefalina Leucina/metabolismo , Doenças dos Suínos/metabolismo , Animais , Axotomia/veterinária , Colite/metabolismo , Colite/fisiopatologia , Colo Descendente/inervação , Colo Descendente/fisiologia , Encefalina Leucina/fisiologia , Feminino , Imunofluorescência/veterinária , Suínos , Doenças dos Suínos/fisiopatologia
19.
ACS Chem Neurosci ; 9(7): 1735-1742, 2018 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-29648788

RESUMO

Opioid peptides are key regulators in cellular and intercellular physiological responses, and could be therapeutically useful for modulating several pathological conditions. Unfortunately, the use of peptide-based agonists to target centrally located opioid receptors is limited by poor physicochemical (PC), distribution, metabolic, and pharmacokinetic (DMPK) properties that restrict penetration across the blood-brain barrier via passive diffusion. To address these problems, the present paper exploits fluorinated peptidomimetics to simultaneously modify PC and DMPK properties, thus facilitating entry into the central nervous system. As an initial example, the present paper exploited the Tyr1-ψ[( Z)CF═CH]-Gly2 peptidomimetic to improve PC druglike characteristics (computational), plasma and microsomal degradation, and systemic and CNS distribution of Leu-enkephalin (Tyr-Gly-Gly-Phe-Leu). Thus, the fluoroalkene replacement transformed an instable in vitro tool compound into a stable and centrally distributed in vivo probe. In contrast, the Tyr1-ψ[CF3CH2-NH]-Gly2 peptidomimetic decreased stability by accelerating proteolysis at the Gly3-Phe4 position.


Assuntos
Encefalina Leucina/farmacocinética , Peptidomiméticos/química , Peptidomiméticos/farmacocinética , Animais , Transporte Biológico , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Estabilidade de Medicamentos , Encefalina Leucina/química , Encefalina Leucina/metabolismo , Feminino , Humanos , Camundongos Endogâmicos BALB C , Microssomos/efeitos dos fármacos , Microssomos/metabolismo , Estrutura Molecular , Peptidomiméticos/metabolismo , Ratos Sprague-Dawley , Solubilidade
20.
Biointerphases ; 13(3): 03B403, 2018 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-29351722

RESUMO

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) is one of the most powerful methods to analyze biomolecules in biological tissues and cells because it provides detailed chemical structure information and chemical images with a high spatial resolution. However, in terms of quantitative analysis, there are issues such as matrix effects that often cause secondary ion intensity changes regardless of the actual concentration in a sample. For instance, the intensity of secondary ions related to peptides is generally suppressed when lipids coexist. Since the evaluation of biomolecules is crucial to understand biological phenomena, it is required to analyze peptides or lipids without matrix effects. Therefore, the mechanism of matrix effects regarding peptides and lipids in TOF-SIMS was investigated in this study. Leu-enkephalin (YGGFL, molecular weight of 555.3 Da) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC, C44H84NO8P, molecular weight 785.6 Da) were employed to prepare model samples. Model samples contain different weight ratios of these two molecules. The intensity of secondary ions related to the peptide or the lipid was compared with control samples containing pure leu-enkephalin or DOPC. As a result, it is indicated that the intensity of DOPC related secondary ions is strongly enhanced by coexisting leu-enkephalin, while the intensity of leu-enkephalin related secondary ions is suppressed by coexisting DOPC especially in a low concentration range of the peptide.


Assuntos
Encefalina Leucina/análise , Fosfatidilcolinas/análise , Espectrometria de Massa de Íon Secundário/métodos
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