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Since interleukin-8 (IL-8/CXCL8) and its receptor, CXCR1 and CXCR2, were known in the early 1990s, biological pathways related to these proteins were proven to have high clinical value in cancer and inï¬ammatory/autoimmune conditions treatment. Recently, IL-8 has been identified as biomarker for severe COVID-19 patients and COVID-19 prognosis. Boyles et al. (mAbs 12 (2020), pp. 1831880) have published a high-resolution X-ray crystal structure of the LY3041658 Fab in a complex human CXCL8. They described the ability to bind to IL-8 and the blocking of IL-8/its receptors interaction by the LY3041658 monoclonal antibody. Therefore, the study has been designed to identify potential small molecules inhibiting interleukin-8 by targeting LY3041658/IL-8 complex structure using an in silico approach. A structurebased pharmacophore and molecular docking models of the protein active site cavity were generated to identify possible candidates, followed by virtual screening with the ZINC database. ADME analysis of hit compounds was also conducted. Molecular dynamics simulations were then performed to survey the behaviour and stability of the ligand-protein complexes. Furthermore, the MM/PBSA technique has been utilized to evaluate the free binding energy. The final data confirmed that one newly obtained compound, ZINC21882765, may serve as the best potential inhibitor for IL-8.
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Tratamento Farmacológico da COVID-19 , Interleucina-8 , Humanos , Simulação de Acoplamento Molecular , Relação Quantitativa Estrutura-Atividade , Simulação de Dinâmica Molecular , LigantesRESUMO
Cells within cartilaginous tissues are mechanosensitive and thus require mechanical loading for regulation of tissue homeostasis and metabolism. Mechanical loading plays critical roles in cell differentiation, proliferation, biosynthesis, and homeostasis. Inflammation is an important event occurring during multiple processes, such as aging, injury, and disease. Inflammation has significant effects on biological processes as well as mechanical function of cells and tissues. These effects are highly dependent on cell/tissue type, timing, and magnitude. In this review, we summarize key findings pertaining to effects of inflammation on multiscale mechanical properties at subcellular, cellular, and tissue level in cartilaginous tissues, including alterations in mechanotransduction and mechanosensitivity. The emphasis is on articular cartilage and the intervertebral disc, which are impacted by inflammatory insults during degenerative conditions such as osteoarthritis, joint pain, and back pain. To recapitulate the pro-inflammatory cascades that occur in vivo, different inflammatory stimuli have been used for in vitro and in situ studies, including tumor necrosis factor (TNF), various interleukins (IL), and lipopolysaccharide (LPS). Therefore, this review will focus on the effects of these stimuli because they are the best studied pro-inflammatory cytokines in cartilaginous tissues. Understanding the current state of the field of inflammation and cell/tissue biomechanics may potentially identify future directions for novel and translational therapeutics with multiscale biomechanical considerations.
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BACKGROUND AND PURPOSE: Recent evidence suggested that urotensin II (UII) and its paralog peptide UII-related peptide (URP) might exert common but also divergent physiological actions. Unfortunately, none of the existing antagonists were designed to discriminate specific UII- or URP-associated actions, and our understanding, on how these two endogenous peptides can trigger different, but also common responses, is limited. EXPERIMENTAL APPROACH: Ex vivo rat and monkey aortic ring contraction as well as dissociation kinetics studies using transfected CHO cells expressing the human urotensin (UT) receptors were used in this study. KEY RESULTS: Ex vivo rat and monkey aortic ring contraction studies revealed the propensity of [Pep(4)]URP to decrease the maximal response of human UII (hUII) without any significant change in potency, whereas no effect was noticeable on the URP-induced vasoconstriction. Dissociation experiments demonstrated the ability of [Pep(4)]URP to increase the dissociation rate of hUII, but not URP. Surprisingly, URP, an equipotent UII paralog, was also able to accelerate the dissociation rate of membrane-bound (125)I-hUII, whereas hUII had no noticeable effect on URP dissociation kinetics. Further experiments suggested that an interaction between the glutamic residue at position 1 of hUII and the UT receptor seems to be critical to induce conformational changes associated with agonistic activation. Finally, we demonstrated that the N-terminal domain of the rat UII isoform was able to act as a specific antagonist of the URP-associated actions. CONCLUSION: Such compounds, that is [Pep(4)]URP and rUII(1-7), should prove to be useful as new pharmacological tools to decipher the specific role of UII and URP in vitro but also in vivo.
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Aorta Torácica/efeitos dos fármacos , Hormônios Peptídicos/antagonistas & inibidores , Hormônios Peptídicos/farmacologia , Receptores Acoplados a Proteínas G/metabolismo , Urotensinas/antagonistas & inibidores , Animais , Células CHO , Cricetinae , Cricetulus , Humanos , Técnicas In Vitro , Peptídeos e Proteínas de Sinalização Intracelular , Macaca fascicularis , Masculino , Hormônios Peptídicos/química , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas G/genética , Transfecção , Urotensinas/farmacologia , Vasoconstrição/efeitos dos fármacosRESUMO
OBJECTIVE: To assess the activation grade of intestinal eosinophils in patients with eosinophilic gastroenteritis (EOG), ulcerative colitis (UC), Crohn's disease (CD), and controls by immunohistochemistry. METHODS: Cecal biopsies were collected from healthy controls and from patients with EOG, CD, UC, and other noninflammatory GI diseases. Immunohistochemistry was performed in sequential sections stained with monoclonal antibodies directed against eosinophil cationic protein (ECP) or eosinophil protein X (EPX) stored in eosinophil granules (EG1) and that secreted by activated eosinophils (EG2). The ratio of EG1 to EG2-positive eosinophils expressed as percentage of lamina propria cells was calculated. ECP and EPX were measured in serum and feces. RESULTS: The percentage of EG1 and EG2-positive lamina propria cells was elevated in EOG and slightly, but not significantly, in UC. The ratio of EG1 to EG2-positive cells was decreased in CD, UC, and other patients as compared to healthy controls. Particularly low EG1 to EG2 ratios were found in EOG. Correspondingly, fecal and serum levels of ECP and EPX, respectively, were highest in patients with EOG. The EG1 to EG2 ratio was negatively correlated with fecal ECP and EPX levels. At sites of actively inflamed mucosa, the EG1 to EG2 ratio was lower than in noninflamed tissue. CONCLUSIONS: Our data strongly suggest that the EG1 to EG2 ratio may be a marker of tissue eosinophil activation. Low ratios (<1) indicate eosinophil activation, whereas ratios > or =1 are found in healthy controls. Furthermore, we show that EOG is characterized by a pronounced intestinal eosinophil accumulation and activation, whereas in CD and UC, eosinophils seem to be activated but their number is not or only slightly elevated compared to controls.
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Proteínas Sanguíneas/análise , Colite Ulcerativa/imunologia , Doença de Crohn/imunologia , Eosinofilia/imunologia , Gastroenterite/imunologia , Ativação de Neutrófilo/imunologia , Ribonucleases , Adulto , Idoso , Biópsia , Doenças do Ceco/imunologia , Doenças do Ceco/patologia , Ceco/imunologia , Ceco/patologia , Colite Ulcerativa/patologia , Doença de Crohn/patologia , Proteínas Granulares de Eosinófilos , Neurotoxina Derivada de Eosinófilo , Eosinofilia/patologia , Fezes/química , Feminino , Gastroenterite/patologia , Humanos , Técnicas Imunoenzimáticas , Mediadores da Inflamação/análise , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Masculino , Pessoa de Meia-IdadeRESUMO
The prenylated protein carboxyl methyltransferase (PPMT) catalyzes the posttranslational methylation of isoprenylated C-terminal cysteine residues found in many signaling proteins such as the small monomeric G proteins and the gamma subunits of heterotrimeric G proteins. Here we report that both membrane-bound PPMT from rat kidney and the recombinant bacterially expressed form of the enzyme required divalent cations for catalytic activity. Unlike EDTA and EGTA, the metal chelator 1,10-phenanthroline strongly inhibited the PPMT activity of kidney intracellular membranes in a dose- and time-dependent manner. 1,10-Phenanthroline was found to inhibit the methylation of the prenylcysteine analog N-acetyl-S-all-trans-geranylgeranyl-l-cysteine, a synthetic substrate for PPMT, with an IC(50) of 2.2 mM. Gel electrophoretic analysis demonstrated that 1,10-phenanthroline almost totally abolished the labeling of methylated proteins in kidney intracellular membranes. Immunoblotting analysis showed that one of the two major peaks of (3)H-methylated proteins in intracellular membranes comigrated with the small G proteins Ras, Cdc42, RhoA, and Rab1. In addition, the methylation of immunoprecipitated Ras and RhoA from kidney intracellular membranes was strongly inhibited when 1,10-phenanthroline was present. Treatment of kidney intracellular membranes with 1,10-phenanthroline increased the proteolytic degradation of PPMT by exogenous trypsin, compared to untreated membranes. We conclude from these data that metal ions are essential for the activity and the stabilization of PPMT. The finding that PPMT is a metalloenzyme may provide new insights into the functions played by this methyltransferase in signal transduction processes.
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Proteínas de Ligação ao GTP/metabolismo , Proteínas Metiltransferases/metabolismo , Animais , Cátions Bivalentes , Membrana Celular/enzimologia , Quelantes/farmacologia , Ácido Edético/farmacologia , Ácido Egtázico/farmacologia , Inibidores Enzimáticos/farmacologia , Immunoblotting , Rim/enzimologia , Masculino , Metilação , Fenantrolinas/farmacologia , Proteínas Metiltransferases/antagonistas & inibidores , Processamento de Proteína Pós-Traducional , Ratos , Ratos Sprague-Dawley , Proteínas RecombinantesRESUMO
It has been postulated that, in the adult visual cortex, visual inputs modulate levels of mRNAs coding for neurotransmitter receptors in an activity-dependent manner. To investigate this possibility, we performed a monocular enucleation in adult rabbits and, 15 days later, collected their left and right visual cortices. Levels of mRNAs coding for voltage-activated sodium channels, and for receptors for kainate/alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), N-methyl-D-aspartate (NMDA), gamma-aminobutyric acid (GABA), and glycine were semiquantitatively estimated in the visual cortices ipsilateral and contralateral to the lesion by the Xenopus oocyte/voltage-clamp expression system. This technique also allowed us to study some of the pharmacological and physiological properties of the channels and receptors expressed in the oocytes. In cells injected with mRNA from left or right cortices of monocularly enucleated and control animals, the amplitudes of currents elicited by kainate or AMPA, which reflect the abundance of mRNAs coding for kainate and AMPA receptors, were similar. There was no difference in the sensitivity to kainate and in the voltage dependence of the kainate response. Responses mediated by NMDA, GABA, and glycine were unaffected by monocular enucleation. Sodium channel peak currents, activation, steady-state inactivation, and sensitivity to tetrodotoxin also remained unchanged after the enucleation. Our data show that mRNAs for major neurotransmitter receptors and ion channels in the adult rabbit visual cortex are not obviously modified by monocular deafferentiation. Thus, our results do not support the idea of a widespread dynamic modulation of mRNAs coding for receptors and ion channels by visual activity in the rabbit visual system.
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Ativação do Canal Iônico , Receptores de Aminoácido/biossíntese , Canais de Sódio/biossíntese , Visão Monocular/fisiologia , Córtex Visual/metabolismo , Vias Aferentes/cirurgia , Animais , Relação Dose-Resposta a Droga , Eletrofisiologia/métodos , Enucleação Ocular , Masculino , Oócitos , RNA Mensageiro/isolamento & purificação , Coelhos , Receptores de Aminoácido/genética , Bloqueadores dos Canais de Sódio , Canais de Sódio/genética , Tetrodotoxina/farmacologia , XenopusRESUMO
Two alternatively spliced terminal deoxynucleotidyl transferase transcripts, TdTS and TdTL which code respectively for proteins of 509 and 529 amino acids have been previously identified in the mouse thymus. Here we show that the same two transcripts are also present in B lineage cells from bone marrow. In addition we demonstrate that the corresponding 20 amino acid insertion found near the carboxy-terminal end of TdTL significantly alters the function of the enzyme. In contrast to TdTS, TdTL does not catalyse N region insertions at the recombination junction of a V(D)J site-specific recombination substrate. In an attempt to explain the lack of N region insertions we have characterized the different parameters which distinguish the two isoforms of TdT. Examination of transfected cell extracts revealed a reduced capacity of TdTL to add nucleotides to the 3' end of DNA, consistent with a lower terminal transferase activity. Furthermore, the half-life of the TdTL protein in these cells is 2-fold shorter than that of TdTS. Finally, despite the fact that TdTL has the same nuclear localization signal as TdTS, the cellular localization of the two isoforms was strikingly different. In contrast to nuclear TdTS, TdTL was found exclusively in the cytoplasm. All these characteristics could contribute to the functional difference between the two isoforms of TdT. However, the subcellular localization of TdTL on its own can account for its inability to add N regions.
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Processamento Alternativo , Medula Óssea/enzimologia , DNA Nucleotidilexotransferase/metabolismo , Isoenzimas/metabolismo , Timo/enzimologia , Animais , Linfócitos B/enzimologia , Sequência de Bases , Linhagem Celular , Chlorocebus aethiops , DNA Nucleotidilexotransferase/biossíntese , Primers do DNA , Células-Tronco Hematopoéticas/enzimologia , Isoenzimas/biossíntese , Cinética , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , Proteínas Recombinantes/metabolismo , Frações Subcelulares/enzimologia , TransfecçãoRESUMO
The mouse heavy chain immunoglobulin promoter VH441 can lead in vitro to bidirectional transcription, due to a symmetrical organization of immunoglobulin heavy chain promoters with two TATA-like sequences bracketing the upstream promoter element ATGCAAAT (the so called octamer). We demonstrate here that divergent transcription also occurs in vivo in mature B cells from a myeloma which expresses the VH441 gene and even from the spleen of BALB/c mice. The level of VH441 divergent transcript increases in the spleen of BALB/c mice after immunisation by beta-(1,6)-galactan, showing that it is expressed in B cells which actively transcribe the VH441 gene. The divergent transcript has been characterized: its major transcription start site was mapped within 33 base pairs from the divergent TATA-like region, it is unspliced and not polyadenylated. In the light of these results, the functions of the divergent transcript and the bidirectional promoter are discussed.