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1.
Curr Protoc ; 1(9): e236, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34491634

RESUMO

Human artificial chromosomes (HACs) are considered promising tools for gene delivery, functional analyses, and gene therapy. HACs have the potential to overcome many of the problems caused by the use of viral-based gene transfer systems, such as limited cloning capacity, lack of copy number control, and insertional mutagenesis during integration into host chromosomes. The recently developed alphoidtetO -HAC has an advantage over other HAC vectors because it can be easily eliminated from dividing cells by inactivation of its conditional kinetochore. This provides a unique control mechanism to study phenotypes induced by a gene or genes carried on the HAC. The alphoidtetO -HAC has a single gene acceptor loxP site that allows insertion of an individual gene of interest or a cluster of genes of up to several Mb in size in Chinese hamster ovary (CHO) hybrid cells. The HACs carrying chromosomal copies of genes can then be transferred from these donor CHO cells to different recipient cells of interest via microcell-mediated chromosome transfer (MMCT). Here, we describe a detailed protocol for loading a gene of interest into the alphoidtetO -HAC vector and for the subsequent transfer of the HAC to recipient cells using an improved MMCT protocol. The original MMCT protocol includes treatment of donor cells with colcemid to induce micronucleation, wherein the HAC becomes surrounded with a nuclear membrane. That step is followed by disarrangement of the actin cytoskeleton using cytochalasin B to help induce microcell formation. The updated MMCT protocol, described here, features the replacement of colcemid and cytochalasin B with TN16 + griseofulvin and latrunculin B, respectively, and the use of collagen/laminin surface coating to promote attachment of metaphase cells to plates during micronuclei induction. These modifications increase the efficiency of HAC transfer to recipient cells ten fold. The improved MMCT protocol has been successfully tested on several recipient cell lines, including human mesenchymal stem cells and mouse embryonic stem cells. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Insertion of a BAC containing a gene of interest into a single loxP loading site of alphoidtetO -HAC in hamster CHO cells Basic Protocol 2: Microcell-mediated chromosome transfer from donor hamster CHO cells to mammalian cells.


Assuntos
Cromossomos Artificiais Humanos , Animais , Células CHO , Cromossomos Artificiais Humanos/genética , Cricetinae , Cricetulus , Técnicas de Transferência de Genes , Genômica , Humanos , Camundongos
2.
Nat Commun ; 12(1): 5214, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34471131

RESUMO

Dyslipidemia and resulting lipotoxicity are pathologic signatures of metabolic syndrome and type 2 diabetes. Excess lipid causes cell dysfunction and induces cell death through pleiotropic mechanisms that link to oxidative stress. However, pathways that regulate the response to metabolic stress are not well understood. Herein, we show that disruption of the box H/ACA SNORA73 small nucleolar RNAs encoded within the small nucleolar RNA hosting gene 3 (Snhg3) causes resistance to lipid-induced cell death and general oxidative stress in cultured cells. This protection from metabolic stress is associated with broad reprogramming of oxidative metabolism that is dependent on the mammalian target of rapamycin signaling axis. Furthermore, we show that knockdown of SNORA73 in vivo protects against hepatic steatosis and lipid-induced oxidative stress and inflammation. Our findings demonstrate a role for SNORA73 in the regulation of metabolism and lipotoxicity.


Assuntos
Fígado Gorduroso/tratamento farmacológico , Fígado Gorduroso/metabolismo , Substâncias Protetoras/farmacologia , RNA Nucleolar Pequeno/metabolismo , Animais , Células CHO , Morte Celular/efeitos dos fármacos , Cricetulus , Diabetes Mellitus Tipo 2/metabolismo , Fígado Gorduroso/genética , Homeostase , Inflamação , Metabolismo dos Lipídeos , Lipídeos/farmacologia , Masculino , Síndrome Metabólica/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo/efeitos dos fármacos , RNA Longo não Codificante , RNA Nucleolar Pequeno/genética , Transdução de Sinais/efeitos dos fármacos
3.
Int J Mol Sci ; 22(16)2021 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-34445614

RESUMO

The anorexigenic neuropeptide prolactin-releasing peptide (PrRP) is involved in the regulation of food intake and energy expenditure. Lipidization of PrRP stabilizes the peptide, facilitates central effect after peripheral administration and increases its affinity for its receptor, GPR10, and for the neuropeptide FF (NPFF) receptor NPFF-R2. The two most potent palmitoylated analogs with anorectic effects in mice, palm11-PrRP31 and palm-PrRP31, were studied in vitro to determine their agonist/antagonist properties and mechanism of action on GPR10, NPFF-R2 and other potential off-target receptors related to energy homeostasis. Palmitoylation of both PrRP31 analogs increased the binding properties of PrRP31 to anorexigenic receptors GPR10 and NPFF-R2 and resulted in a high affinity for another NPFF receptor, NPFF-R1. Moreover, in CHO-K1 cells expressing GPR10, NPFF-R2 or NPFF-R1, palm11-PrRP and palm-PrRP significantly increased the phosphorylation of extracellular signal-regulated kinase (ERK), protein kinase B (Akt) and cAMP-responsive element-binding protein (CREB). Palm11-PrRP31, unlike palm-PrRP31, did not activate either c-Jun N-terminal kinase (JNK), p38, c-Jun, c-Fos or CREB pathways in cells expressing NPFF-1R. Palm-PrRP31 also has higher binding affinities for off-target receptors, namely, the ghrelin, opioid (KOR, MOR, DOR and OPR-L1) and neuropeptide Y (Y1, Y2 and Y5) receptors. Palm11-PrRP31 exhibited fewer off-target activities; therefore, it has a higher potential to be used as an anti-obesity drug with anorectic effects.


Assuntos
Cálcio/metabolismo , Lipoilação , Hormônio Liberador de Prolactina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neuropeptídeos/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Humanos , Técnicas In Vitro , Hormônio Liberador de Prolactina/química , Hormônio Liberador de Prolactina/genética , Receptores Acoplados a Proteínas G/genética , Receptores de Neuropeptídeos/genética
4.
Int J Mol Sci ; 22(16)2021 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-34445224

RESUMO

The tightly localized noradrenergic neurons (NA) in the locus coeruleus (LC) are well recognized as essential for focused arousal and novelty-oriented responses, while many children with autism spectrum disorder (ASD) exhibit diminished attention, engagement and orienting to exogenous stimuli. This has led to the hypothesis that atypical LC activity may be involved in ASD. Oxytocin (OXT) neurons and receptors are known to play an important role in social behavior, pair bonding and cognitive processes and are under investigation as a potential treatment for ASD. However, little is known about the neurotransmission from hypothalamic paraventricular (PVN) OXT neurons to LC NA neurons. In this study, we test, in male and female rats, whether PVN OXT neurons excite LC neurons, whether oxytocin is released and involved in this neurotransmission, and whether activation of PVN OXT neurons alters novel object recognition. Using "oxytocin sniffer cells" (CHO cells that express the human oxytocin receptor and a Ca indicator) we show that there is release of OXT from hypothalamic PVN OXT fibers in the LC. Optogenetic excitation of PVN OXT fibers excites LC NA neurons by co-release of OXT and glutamate, and this neurotransmission is greater in males than females. In male, but not in female animals, chemogenetic activation of PVN OXT neurons increases attention to novel objects.


Assuntos
Atenção , Locus Cerúleo/metabolismo , Neurônios/metabolismo , Ocitocina/metabolismo , Núcleo Hipotalâmico Paraventricular/metabolismo , Caracteres Sexuais , Transmissão Sináptica , Animais , Células CHO , Cricetulus , Feminino , Humanos , Masculino , Ocitocina/genética , Ratos , Receptores de Ocitocina/genética , Receptores de Ocitocina/metabolismo
5.
Biomolecules ; 11(7)2021 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-34356625

RESUMO

Monoamine oxidases (MAOs) and muscarinic acetylcholine receptors (mAChRs) are considered important therapeutic targets for Parkinson's disease (PD). Lipophilic tanshinones are major phytoconstituents in the dried roots of Salvia miltiorrhiza that have demonstrated neuroprotective effects against dopaminergic neurotoxins and the inhibition of MAO-A. Since MAO-B inhibition is considered an effective therapeutic strategy for PD, we tested the inhibitory activities of three abundant tanshinone congeners against recombinant human MAO (hMAO) isoenzymes through in vitro experiments. In our study, tanshinone I (1) exhibited the highest potency against hMAO-A, followed by tanshinone IIA and cryptotanshinone, with an IC50 less than 10 µM. They also suppressed hMAO-B activity, with an IC50 below 25 µM. Although tanshinones are known to inhibit hMAO-A, their enzyme inhibition mechanism and binding sites have yet to be investigated. Enzyme kinetics and molecular docking studies have revealed the mode of inhibition and interactions of tanshinones during enzyme inhibition. Proteochemometric modeling predicted mAChRs as possible pharmacological targets of 1, and in vitro functional assays confirmed the selective M4 antagonist nature of 1 (56.1% ± 2.40% inhibition of control agonist response at 100 µM). These findings indicate that 1 is a potential therapeutic molecule for managing the motor dysfunction and depression associated with PD.


Assuntos
Abietanos , Inibidores da Monoaminoxidase , Monoaminoxidase , Fenantrenos , Receptor Muscarínico M4 , Salvia miltiorrhiza/química , Abietanos/química , Abietanos/farmacologia , Animais , Células CHO , Cricetulus , Humanos , Monoaminoxidase/química , Monoaminoxidase/genética , Monoaminoxidase/metabolismo , Inibidores da Monoaminoxidase/química , Inibidores da Monoaminoxidase/farmacologia , Fenantrenos/química , Fenantrenos/farmacologia , Receptor Muscarínico M4/antagonistas & inibidores , Receptor Muscarínico M4/química , Receptor Muscarínico M4/genética , Receptor Muscarínico M4/metabolismo
6.
Int J Mol Sci ; 22(15)2021 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-34360706

RESUMO

For the treatment of severe COVID-19, supplementation with human plasma-purified α-1 antitrypsin (AAT) to patients is currently considered. AAT inhibits host proteases that facilitate viral entry and possesses broad anti-inflammatory and immunomodulatory activities. Researchers have demonstrated that an interaction between SARS-CoV-2 spike protein (S) and lipopolysaccharides (LPS) enhances pro-inflammatory responses in vitro and in vivo. Hence, we wanted to understand the potential anti-inflammatory activities of plasma-derived and recombinant AAT (recAAT) in a model of human total peripheral blood mononuclear cells (PBMCs) exposed to a combination of CHO expressed trimeric spike protein and LPS, ex vivo. We confirmed that cytokine production was enhanced in PBMCs within six hours when low levels of LPS were combined with purified spike proteins ("spike"). In the presence of 0.5 mg/mL recAAT, however, LPS/spike-induced TNF-α and IL-1ß mRNA expression and protein release were significantly inhibited (by about 46-50%) relative to LPS/spike alone. Although without statistical significance, recAAT also reduced production of IL-6 and IL-8. Notably, under the same experimental conditions, the plasma-derived AAT preparation Respreeza (used in native and oxidized forms) did not show significant effects. Our findings imply that an early pro-inflammatory activation of human PBMCs is better controlled by the recombinant version of AAT than the human plasma-derived AAT used here. Considering the increasing clinical interest in AAT therapy as useful to ameliorate the hyper-inflammation seen during COVID-19 infection, different AAT preparations require careful evaluation.


Assuntos
Anti-Inflamatórios/farmacologia , Leucócitos Mononucleares/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , alfa 1-Antitripsina/farmacologia , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/imunologia , Células CHO , COVID-19/terapia , Células Cultivadas , Cricetulus , Citocinas/metabolismo , Humanos , Inflamação/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/toxicidade , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , alfa 1-Antitripsina/química , alfa 1-Antitripsina/imunologia
7.
Molecules ; 26(16)2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34443436

RESUMO

The clinical success of PD-1/PD-L1 immune checkpoint targeting antibodies in cancer is followed by efforts to develop small molecule inhibitors with better penetration into solid tumors and more favorable pharmacokinetics. Here we report the crystal structure of a macrocyclic peptide inhibitor (peptide 104) in complex with PD-L1. Our structure shows no indication of an unusual bifurcated binding mode demonstrated earlier for another peptide of the same family (peptide 101). The binding mode relies on extensive hydrophobic interactions at the center of the binding surface and an electrostatic patch at the side. An interesting sulfur/π interaction supports the macrocycle-receptor binding. Overall, our results allow a better understanding of forces guiding macrocycle affinity for PD-L1, providing a rationale for future structure-based inhibitor design and rational optimization.


Assuntos
Antígeno B7-H1/metabolismo , Proteínas de Checkpoint Imunológico/metabolismo , Compostos Macrocíclicos/química , Compostos Macrocíclicos/farmacologia , Peptídeos/química , Peptídeos/farmacologia , Receptor de Morte Celular Programada 1/metabolismo , Animais , Células CHO , Cricetulus , Humanos , Células Jurkat , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Ligação Proteica
8.
J Anim Sci ; 99(8)2021 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-34337647

RESUMO

Chinese hamster ovary cell constructs expressing either the ß 1-, ß 2- or ß 3-adrenergic receptor (AR) were used to determine whether a novel ß-AR modulator, lubabegron fumarate (LUB; Experior, Elanco Animal Health) might exert greater potency for a specific ß-AR subtype. EC50 values calculated based on cAMP accumulation in dose response curves indicate that LUB is highly selective for the ß 3-AR subtype, with an EC50 of 6 × 10-9 M, with no detectible agonistic activity at the ß 2-AR. We hypothesized that the accumulation of lipolytic markers would reflect the agonist activity at each of the ß-receptor subtypes of the specific ligand; additionally, there would be differences in receptor subtype expression in subcutaneous (s.c.) and intrmuscular (i.m.) adipose tissues. Total RNA was extracted from adipose tissue samples and relative mRNA levels for ß 1-, ß2-, and ß 3-AR were measured using real-time quantitative polymerase chain reaction. Fresh s.c. and i.m. adipose tissue explants were incubated with isoproterenol hydrochloride (ISO; ß-AR pan-agonist), dobutamine hydrochloride (DOB; specific ß 1-AA), salbutamol sulfate (SAL; specific ß 2-AA), ractopamine hydrochloride (RAC), zilpaterol hydrochloride (ZIL), BRL-37344 (specific ß 3-agonist), or LUB for 30 min following preincubation with theophylline (inhibitor of phosphodiesterase). Relative mRNA amounts for ß 1-, ß 2-, and ß 3-AR were greater (P < 0.05) in s.c. than in i.m. adipose tissue. The most abundant ß-AR mRNA in both adipose tissues was the ß 2-AR (P < 0.05), with the ß 1- and ß 3-AR subtypes being minimally expressed in i.m. adipose tissue. ISO, RH, and ZH stimulated the release of glycerol and nonesterified fatty acid (NEFA) from s.c. adipose tissue, but these ß-AR ligands did not alter concentrations of these lipolytic markers in i.m. adipose tissue. LUB did not affect glycerol or NEFA concentrations in s.c. or i.m. adipose tissue, but attenuated (P < 0.05) the accumulation of cAMP mediated by the ß 1- and ß 2-AR ligands DOB and SAL in s.c. adipose tissue. Collectively, these data indicate that bovine i.m. adipose tissue is less responsive than s.c. adipose tissue to ß-adrenergic ligands, especially those that are agonists at the ß 1- and ß3-receptor subtypes. The minimal mRNA expression of the ß 1- and ß 3 subtypes in i.m. adipose tissue likely limits the response potential to agonists for these ß-AR subtypes.


Assuntos
Agonistas Adrenérgicos beta , Receptores Adrenérgicos beta , Tecido Adiposo , Agonistas Adrenérgicos beta/farmacologia , Animais , Células CHO , Bovinos , Cricetinae , Cricetulus , Fumaratos , Receptores Adrenérgicos beta/genética
9.
Nat Commun ; 12(1): 4171, 2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34234116

RESUMO

Here we report the pharmacologic blockade of voltage-gated sodium ion channels (NaVs) by a synthetic saxitoxin derivative affixed to a photocleavable protecting group. We demonstrate that a functionalized saxitoxin (STX-eac) enables exquisite spatiotemporal control of NaVs to interrupt action potentials in dissociated neurons and nerve fiber bundles. The photo-uncaged inhibitor (STX-ea) is a nanomolar potent, reversible binder of NaVs. We use STX-eac to reveal differential susceptibility of myelinated and unmyelinated axons in the corpus callosum to NaV-dependent alterations in action potential propagation, with unmyelinated axons preferentially showing reduced action potential fidelity under conditions of partial NaV block. These results validate STX-eac as a high precision tool for robust photocontrol of neuronal excitability and action potential generation.


Assuntos
Potenciais de Ação/efeitos dos fármacos , Canal de Sódio Disparado por Voltagem NAV1.2/metabolismo , Saxitoxina/farmacologia , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Animais , Axônios/efeitos dos fármacos , Axônios/metabolismo , Células CHO , Células Cultivadas , Corpo Caloso/citologia , Corpo Caloso/efeitos dos fármacos , Corpo Caloso/metabolismo , Cricetulus , Embrião de Mamíferos , Feminino , Hipocampo/citologia , Masculino , Camundongos , Canal de Sódio Disparado por Voltagem NAV1.2/genética , Técnicas de Patch-Clamp , Cultura Primária de Células , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saxitoxina/análogos & derivados , Saxitoxina/efeitos da radiação , Análise de Célula Única , Análise Espaço-Temporal , Raios Ultravioleta , Bloqueadores do Canal de Sódio Disparado por Voltagem/efeitos da radiação
10.
Int J Mol Sci ; 22(12)2021 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-34207579

RESUMO

Biomanufacturing processes may be optimized by storing cell culture media at room temperature, but this is currently limited by their instability and change in color upon long-term storage. This study demonstrates that one of the critical contributing factors toward media browning is tryptophan. LC-MS technology was utilized to identify tryptophan degradation products, which are likely formed primarily from oxidation reactions. Several of the identified compounds were shown to contribute significantly to color in solutions but also to exhibit toxicity against CHO cells. A cell-culture-compatible antioxidant, a-ketoglutaric acid, was found to be an efficient cell culture media additive for stabilizing components against degradation, inhibiting the browning of media formulations, and decreasing ammonia production, thus providing a viable method for developing room-temperature stable cell culture media.


Assuntos
Meios de Cultura/química , Triptofano/metabolismo , Animais , Células CHO , Cricetulus , Oxirredução , Triptofano/análise
11.
Int J Mol Sci ; 22(12)2021 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-34207662

RESUMO

p62/Sequestosome-1 (p62) is a multifunctional adaptor protein and is also a constant component of disease-associated protein aggregates, including Mallory-Denk bodies (MDBs), in steatohepatitis and hepatocellular carcinoma. We investigated the interaction of the two human p62 isoforms, p62-H1 (full-length isoform) and p62-H2 (partly devoid of PB1 domain), with keratins 8 and 18, the major components of MDBs. In human liver, p62-H2 is expressed two-fold higher compared to p62-H1 at the mRNA level and is present in slightly but not significantly higher concentrations at the protein level. Co-transfection studies in CHO-K1 cells, PLC/PRF/5 cells as well as p62- total-knockout and wild-type mouse fibroblasts revealed marked differences in the cytoplasmic distribution and aggregation behavior of the two p62 isoforms. Transfection-induced overexpression of p62-H2 generated large cytoplasmic aggregates in PLC/PRF/5 and CHO-K1 cells that mostly co-localized with transfected keratins resembling MDBs or (transfection without keratins) intracytoplasmic hyaline bodies. In fibroblasts, however, transfected p62-H2 was predominantly diffusely distributed in the cytoplasm. Aggregation of p62-H2 and p62ΔSH2 as well as the interaction with K8 (but not with K18) involves acquisition of cross-ß-sheet conformation as revealed by staining with luminescent conjugated oligothiophenes. These results indicate the importance of considering p62 isoforms in protein aggregation disease.


Assuntos
Queratinas/metabolismo , Agregados Proteicos , Proteína Sequestossoma-1/metabolismo , Animais , Células CHO , Cricetulus , Humanos , Queratinas/genética , Camundongos , Camundongos Knockout , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteína Sequestossoma-1/genética
12.
Life Sci ; 282: 119824, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34265361

RESUMO

AIM: Berberine (BBR) is an alkaloid extracted from Coptidis Rhizoma, also known as Huang-Lian. Huang-Lian has been used extensively in traditional Chinese medicine for the treatment of various diseases, including diabetes and dementia. Because Alzheimer's disease (AD) is a complex disease that involves various pathophysiological changes, the diverse neuroprotective effects of BBR may be useful for improving the brain's energy state at an early stage of the disease. MAIN METHODS: We performed extracellular flux and 1H NMR-based metabolic profiling analyses to investigate the effects of BBR on metabolic processes in these cells. Pioglitazone (PIO), a peroxisome proliferator-activated receptor-γ (PPARγ) agonist has been studied extensively for the treatment of AD. We explored the combination dosing effects of BBR and PIO in vitro, then leveraged computational methods to explain the experimental finding. KEY FINDINGS: BBR demonstrates potential in modulating the mitochondrial bioenergetics and attenuating dysfunction of the primary energy and glutathione metabolism pathways in an AD cell model. It also suppresses basal respiration and reduces the production of pro-inflammatory cytokines in activated microglial cells. Both experimental and computational observations indicate that BBR and PIO have comparable binding affinities to the PPARγ protein, suggesting both drugs may have some overlapping effects for AD. SIGNIFICANCE: BBR exerts beneficial effects on disrupted metabolic processes in amyloidogenic cells and activated microglial cells, which are important for preventing or delaying early-stage disease progression. The choice of BBR or PIO for AD treatment depends on their respective pharmacokinetic profiles, delivery, efficacy and safety, and warrants further study.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/metabolismo , Berberina/farmacologia , Microglia/metabolismo , Mitocôndrias/metabolismo , Modelos Biológicos , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Células CHO , Cricetulus , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Inflamação/patologia , Camundongos , Microglia/patologia , Mitocôndrias/patologia
13.
Nat Immunol ; 22(9): 1093-1106, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34282331

RESUMO

Neutrophils display distinct gene expression patters depending on their developmental stage, activation state and tissue microenvironment. To determine the transcription factor networks that shape these responses in a mouse model, we integrated transcriptional and chromatin analyses of neutrophils during acute inflammation. We showed active chromatin remodeling at two transition stages: bone marrow-to-blood and blood-to-tissue. Analysis of differentially accessible regions revealed distinct sets of putative transcription factors associated with control of neutrophil inflammatory responses. Using ex vivo and in vivo approaches, we confirmed that RUNX1 and KLF6 modulate neutrophil maturation, whereas RELB, IRF5 and JUNB drive neutrophil effector responses and RFX2 and RELB promote survival. Interfering with neutrophil activation by targeting one of these factors, JUNB, reduced pathological inflammation in a mouse model of myocardial infarction. Therefore, our study represents a blueprint for transcriptional control of neutrophil responses in acute inflammation and opens possibilities for stage-specific therapeutic modulation of neutrophil function in disease.


Assuntos
Montagem e Desmontagem da Cromatina/genética , Inflamação/imunologia , Neutrófilos/imunologia , Ativação Transcricional/genética , Animais , Células CHO , Linhagem Celular , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Cricetulus , Feminino , Fatores Reguladores de Interferon/metabolismo , Fator 6 Semelhante a Kruppel/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/imunologia , Infarto do Miocárdio/patologia , Fatores de Transcrição de Fator Regulador X/metabolismo , Fator de Transcrição RelB/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética/genética
14.
Molecules ; 26(14)2021 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-34299490

RESUMO

In this study, we designed, synthesized and evaluated, in vitro, novel chalcone analogs containing dialkylamino pharmacophores in the cervical cancer cell line, OV2008. The compound, DML6 was selective and significantly decreased the proliferation of OV2008 and HeLa cells in sub-micromolar concentrations, compared to prostate, lung, colon, breast or human embryonic kidney cell line (HEK293). DML6, at 5 µM, arrested the OV2008 cells in the G2 phase. Furthermore, DML6, at 5 µM, increased the levels of reactive oxygen species and induced a collapse in the mitochondrial membrane potential, compared to OV2008 cells incubated with a vehicle. DML6, at 5 µM, induced intrinsic apoptosis by significantly (1) increasing the levels of the pro-apoptotic proteins, Bak and Bax, and (2) decreasing the levels of l the anti-apoptotic protein, Bcl-2, compared to cell incubated with a vehicle. Furthermore, DML6, at 5 and 20 µM, induced the cleavage of caspase-9, followed by subsequent cleavage of the executioner caspases, caspase-3 and caspase-7, which produced OV2008 cell death. Overall, our data suggest that DML6 is an apoptosis-inducing compound that should undergo further evaluation as a potential treatment for cervical cancer.


Assuntos
Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Chalconas/farmacologia , Mitose/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Neoplasias do Colo do Útero/tratamento farmacológico , Células A549 , Animais , Células CHO , Linhagem Celular , Linhagem Celular Tumoral , Cricetulus , Feminino , Células HEK293 , Células HeLa , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Neoplasias do Colo do Útero/metabolismo
15.
Molecules ; 26(14)2021 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-34299620

RESUMO

Type 2 diabetes mellitus (T2DM) is linked to insulin resistance and a loss of insulin sensitivity, leading to millions of deaths worldwide each year. T2DM is caused by reduced uptake of glucose facilitated by glucose transporter 4 (GLUT4) in muscle and adipose tissue due to decreased intracellular translocation of GLUT4-containing vesicles to the plasma membrane. To treat T2DM, novel medications are required. Through a fluorescence microscopy-based high-content screen, we tested more than 600 plant extracts for their potential to induce GLUT4 translocation in the absence of insulin. The primary screen in CHO-K1 cells resulted in 30 positive hits, which were further investigated in HeLa and 3T3-L1 cells. In addition, full plasma membrane insertion was examined by immunostaining of the first extracellular loop of GLUT4. The application of appropriate inhibitors identified PI3 kinase as the most important signal transduction target relevant for GLUT4 translocation. Finally, from the most effective hits in vitro, four extracts effectively reduced blood glucose levels in chicken embryos (in ovo), indicating their applicability as antidiabetic pharmaceuticals or nutraceuticals.


Assuntos
Glicemia/efeitos dos fármacos , Glucose/metabolismo , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Extratos Vegetais/farmacologia , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Animais , Células CHO , Linhagem Celular , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Cricetulus , Diabetes Mellitus Tipo 2 , Transportador de Glucose Tipo 4/metabolismo , Células HeLa , Humanos , Resistência à Insulina/fisiologia , Camundongos , Transporte Proteico/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
16.
Genes (Basel) ; 12(6)2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34205689

RESUMO

Accumulation of α-Synuclein (αSyn) in nigral dopaminergic neurons is commonly seen in patients with Parkinson's disease (PD). We recently reported that transduction of intracellular single-chain intrabody targeting the 53-87 amino acid residues of human αSyn by recombinant adeno associated viral vector (AAV-NAC32) downregulated αSyn protein in SH-SY5Y cells and rat brain. This study characterizes the behavioral phenotype and dopaminergic protection in animals receiving AAV-NAC32. Our results show that adult DAT-Cre rats selectively overexpress αSyn in nigra dopaminergic neurons after local administration of AAV-DIO-αSyn. These animals develop PD-like phenotype, including bradykinesia and loss of tyrosine hydroxylase (TH) immunoreactivity in substantia nigra pars compacta dorsal tier (SNcd). An injection of AAV-NAC32 to nigra produces a selective antibody against αSyn and normalizes the behavior. AAV-NAC32 significantly increases TH, while reduces αSyn immunoreactivity in SNcd. Altogether, our data suggest that an AAV-mediated gene transfer of NAC32 antibody effectively antagonizes αSyn-mediated dopaminergic degeneration in nigra, which may be a promising therapeutic candidate for synucleinopathy or PD.


Assuntos
Anticorpos/uso terapêutico , Imunoterapia/métodos , Locomoção , Doença de Parkinson/terapia , alfa-Sinucleína/imunologia , Animais , Anticorpos/imunologia , Células CHO , Cricetinae , Cricetulus , Dependovirus/genética , Neurônios Dopaminérgicos/metabolismo , Vetores Genéticos/genética , Masculino , Doença de Parkinson/genética , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Ratos , Ratos Long-Evans , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , alfa-Sinucleína/química , alfa-Sinucleína/genética
17.
Molecules ; 26(14)2021 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-34299587

RESUMO

26RFa is a neuropeptide that activates the rhodopsin-like G protein-coupled receptor QRFPR/GPR103. This peptidergic system is involved in the regulation of a wide array of physiological processes including feeding behavior and glucose homeostasis. Herein, the pharmacological profile of a homogenous library of QRFPR-targeting peptide derivatives was investigated in vitro on human QRFPR-transfected cells with the aim to provide possible insights into the structural determinants of the Phe residues to govern receptor activation. Our work advocates to include in next generations of 26RFa(20-26)-based QRFPR agonists effective substitutions for each Phe unit, i.e., replacement of the Phe22 residue by a constrained 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid moiety, and substitution of both Phe24 and Phe26 by their para-chloro counterpart. Taken as a whole, this study emphasizes that optimized modifications in the C-terminal part of 26RFa are mandatory to design selective and potent peptide agonists for human QRFPR.


Assuntos
Substituição de Aminoácidos , Neuropeptídeos , Receptores Acoplados a Proteínas G/agonistas , Animais , Células CHO , Cricetulus , Humanos , Neuropeptídeos/química , Neuropeptídeos/genética , Neuropeptídeos/farmacologia , Fenilalanina/química , Fenilalanina/genética , Receptores Acoplados a Proteínas G/metabolismo , Relação Estrutura-Atividade
18.
Nat Commun ; 12(1): 4540, 2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34315875

RESUMO

The mTORC1 node plays a major role in autophagy modulation. We report a role of the ubiquitous Gαq subunit, a known transducer of plasma membrane G protein-coupled receptors signaling, as a core modulator of mTORC1 and autophagy. Cells lacking Gαq/11 display higher basal autophagy, enhanced autophagy induction upon different types of nutrient stress along with a decreased mTORC1 activation status. They are also unable to reactivate mTORC1 and thus inactivate ongoing autophagy upon nutrient recovery. Conversely, stimulation of Gαq/11 promotes sustained mTORC1 pathway activation and reversion of autophagy promoted by serum or amino acids removal. Gαq is present in autophagic compartments and lysosomes and is part of the mTORC1 multi-molecular complex, contributing to its assembly and activation via its nutrient status-sensitive interaction with p62, which displays features of a Gαq effector. Gαq emerges as a central regulator of the autophagy machinery required to maintain cellular homeostasis upon nutrient fluctuations.


Assuntos
Autofagia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Transdução de Sinais , Animais , Células CHO , Cricetulus , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Células HEK293 , Humanos , Lisossomos/metabolismo , Masculino , Camundongos , Modelos Biológicos , Fenótipo , Ligação Proteica , Domínios Proteicos , Ratos Wistar , Proteína Regulatória Associada a mTOR/metabolismo , Proteína Sequestossoma-1/metabolismo
19.
Molecules ; 26(13)2021 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-34202630

RESUMO

Serotonin is a neurotransmitter that plays a crucial role in the regulation of several behavioral and cognitive functions by binding to a number of different serotonin receptors present on the cell surface. We report here the synthesis and characterization of several novel fluorescent analogs of serotonin in which the fluorescent NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl) group is covalently attached to serotonin. The fluorescent ligands compete with the serotonin1A receptor specific radiolabeled agonist for binding to the receptor. Interestingly, these fluorescent ligands display a high environmental sensitivity of their fluorescence. Importantly, the human serotonin1A receptor stably expressed in CHO-K1 cells could be specifically labeled with one of the fluorescent ligands with minimal nonspecific labeling. Interestingly, we show by spectral imaging that the NBD-labeled ligand exhibits a red edge excitation shift (REES) of 29 nm when bound to the receptor, implying that it is localized in a restricted microenvironment. Taken together, our results show that NBD-labeled serotonin analogs offer an attractive fluorescent approach for elucidating the molecular environment of the serotonin binding site in serotonin receptors. In view of the multiple roles played by the serotonergic systems in the central and peripheral nervous systems, these fluorescent ligands would be useful in future studies involving serotonin receptors.


Assuntos
Azóis/química , Membrana Celular/química , Corantes Fluorescentes/química , Nitrobenzenos/química , Receptor 5-HT1A de Serotonina/química , Animais , Células CHO , Cricetulus , Humanos , Ligantes
20.
Molecules ; 26(13)2021 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-34206465

RESUMO

(1) Background: Two first-in-class racemic dopamine D1 receptor (D1R) positive allosteric modulator (PAM) chemotypes (1 and 2) were identified from a high-throughput screen. In particular, due to its selectivity for the D1R and reported lack of intrinsic activity, compound 2 shows promise as a starting point toward the development of small molecule allosteric modulators to ameliorate the cognitive deficits associated with some neuropsychiatric disease states; (2) Methods: Herein, we describe the enantioenrichment of optical isomers of 2 using chiral auxiliaries derived from (R)- and (S)-3-hydroxy-4,4-dimethyldihydrofuran-2(3H)-one (d- and l-pantolactone, respectively); (3) Results: We confirm both the racemate and enantiomers of 2 are active and selective for the D1R, but that the respective stereoisomers show a significant difference in their affinity and magnitude of positive allosteric cooperativity with dopamine; (4) Conclusions: These data warrant further investigation of asymmetric syntheses of optically pure analogues of 2 for the development of D1R PAMs with superior allosteric properties.


Assuntos
Dopamina , Receptores de Dopamina D1 , Regulação Alostérica , Animais , Células CHO , Cricetulus , Dopamina/análogos & derivados , Dopamina/química , Dopamina/farmacologia , Receptores de Dopamina D1/química , Receptores de Dopamina D1/metabolismo
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