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1.
Biomolecules ; 12(8)2022 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-36008953

RESUMO

A peripheral nerve injury results in disruption of the fiber that usually protects axons from the surrounding environment. Severed axons from the proximal nerve stump are capable of regenerating, but axons are exposed to a completely new environment. Regeneration recruits cells that produce and deposit key molecules, including growth factor proteins and fibrils in the extracellular matrix (ECM), thus changing the chemical and geometrical environment. The regenerating axons thus surf on a newly remodeled micro-landscape. Strategies to enhance and control axonal regeneration and growth after injury often involve mimicking the extrinsic cues that are found in the natural nerve environment. Indeed, nano- and micropatterned substrates have been generated as tools to guide axons along a defined path. The mechanical cues of the substrate are used as guides to orient growth or change the direction of growth in response to impediments or cell surface topography. However, exactly how axons respond to biophysical information and the dynamics of axonal movement are still poorly understood. Here we use anisotropic, groove-patterned substrate topography to direct and enhance sensory axonal growth of whole mouse dorsal root ganglia (DRG) transplanted ex vivo. Our results show significantly enhanced and directed growth of the DRG sensory fibers on the hemi-3D topographic substrates compared to a 0 nm pitch, flat control surface. By assessing the dynamics of axonal movement in time-lapse microscopy, we found that the enhancement was not due to increases in the speed of axonal growth, but to the efficiency of growth direction, ensuring axons minimize movement in undesired directions. Finally, the directionality of growth was reproduced on topographic patterns fabricated as fully 3D substrates, potentially opening new translational avenues of development incorporating these specific topographic feature sizes in implantable conduits in vivo.


Assuntos
Gânglios Espinais , Regeneração Nervosa , Animais , Axônios/metabolismo , Células Cultivadas , Gânglios Espinais/metabolismo , Camundongos , Crescimento Neuronal
2.
Int J Mol Sci ; 21(5)2020 Feb 29.
Artigo em Inglês | MEDLINE | ID: mdl-32121406

RESUMO

Human cytomegalovirus (HCMV) infections are wide-spread among the general population with manifestations ranging from asymptomatic to severe developmental disabilities in newborns and life-threatening illnesses in individuals with a compromised immune system. Nearly all current drugs suffer from one or more limitations, which emphasizes the critical need to develop new approaches and new molecules. We reasoned that a 'poly-pharmacy' approach relying on simultaneous binding to multiple receptors involved in HCMV entry into host cells could pave the way to a more effective therapeutic outcome. This work presents the study of a synthetic, small molecule displaying pleiotropicity of interactions as a competitive antagonist of viral or cell surface receptors including heparan sulfate proteoglycans and heparan sulfate-binding proteins, which play important roles in HCMV entry and spread. Sulfated pentagalloylglucoside (SPGG), a functional mimetic of heparan sulfate, inhibits HCMV entry into human foreskin fibroblasts and neuroepithelioma cells with high potency. At the same time, SPGG exhibits no toxicity at levels as high as 50-fold more than its inhibition potency. Interestingly, cell-ELISA assays showed downregulation in HCMV immediate-early gene 1 and 2 (IE 1&2) expression in presence of SPGG further supporting inhibition of viral entry. Finally, HCMV foci were observed to decrease significantly in the presence of SPGG suggesting impact on viral spread too. Overall, this work offers the first evidence that pleiotropicity, such as demonstrated by SPGG, may offer a new poly-therapeutic approach toward effective inhibition of HCMV.


Assuntos
Infecções por Citomegalovirus/tratamento farmacológico , Citomegalovirus/efeitos dos fármacos , Glucosídeos/farmacologia , Proteoglicanas de Heparan Sulfato/genética , Ésteres do Ácido Sulfúrico/farmacologia , Células Cultivadas , Citomegalovirus/genética , Citomegalovirus/patogenicidade , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/virologia , Fibroblastos/efeitos dos fármacos , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Recém-Nascido , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
3.
Biometals ; 32(6): 951-964, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31754889

RESUMO

The understanding of cellular Cd2+ accumulation and toxicity is hampered by a lack of fluorescent indicators selective for intracellular free Cd2+ ([Cd2+]i). In this study, we used depolarized MIN6 mouse pancreatic beta cells as a model for evaluating [Cd2+]i detection with commercially available fluorescent probes, most of which have been traditionally used to visualize [Ca2+]i and [Zn2+]i. We trialed a panel of 12 probes including fura-2, FluoZin-3, Leadmium Green, Rhod-5N, indo-1, Fluo-5N, and others. We found that the [Zn2+]i probe FluoZin-3 and the traditional [Ca2+]i probe fura-2 responded most consistently and robustly to [Cd2+]i accumulation mediated by voltage-gated calcium channels. While selective detection of [Cd2+]i by fura-2 required the omission of Ca2+ from extracellular buffers, FluoZin-3 responded to [Cd2+]i similarly in the presence or absence of extracellular Ca2+. Furthermore, we showed that FluoZin-3 and fura-2 can be used together for simultaneous monitoring of [Ca2+]i and [Cd2+]i in the same cells. None of the other fluorophores tested were effective [Cd2+]i detectors in this model.


Assuntos
Cádmio/análise , Corantes Fluorescentes/análise , Fura-2/análise , Células Secretoras de Insulina/química , Células Secretoras de Insulina/metabolismo , Compostos Policíclicos/análise , Animais , Cádmio/metabolismo , Linhagem Celular , Corantes Fluorescentes/química , Fura-2/química , Espectrometria de Massas , Camundongos , Microscopia de Fluorescência , Compostos Policíclicos/química
4.
Mol Cell Endocrinol ; 478: 1-9, 2018 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-29959979

RESUMO

A previous body of work in bovine and rodent models shows that cholinergic agonists modulate the secretion of steroid hormones from the adrenal cortex. In this study we used live-cell Ca2+ imaging to investigate cholinergic activity in the HAC15 human adrenocortical carcinoma cell line. The cholinergic agonists carbachol and acetylcholine triggered heterogeneous Ca2+ oscillations that were strongly inhibited by antagonists with high affinity for the M3 muscarinic receptor subtype, while preferential block of M1 or M2 receptors was less effective. Acute exposure to carbachol and acetylcholine modestly elevated aldosterone secretion in HAC15 cells, and this effect was also diminished by M3 inhibition. HAC15 cells expressed relatively high levels of mRNA for M3 and M2 receptors, while M1 and M5 mRNA were much lower. In conclusion, our data extend previous findings in non-human systems to implicate the M3 receptor as the dominant muscarinic receptor in the human adrenal cortex.


Assuntos
Córtex Suprarrenal/citologia , Aldosterona/biossíntese , Sinalização do Cálcio , Receptor Muscarínico M3/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular , Fluorescência , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor Muscarínico M3/antagonistas & inibidores , Receptor Muscarínico M3/genética
5.
J Vis Exp ; (133)2018 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-29578527

RESUMO

This protocol describes an ex vivo model of mouse-derived dorsal root ganglia (DRG) explant and in vitro DRG-derived co-culture of dissociated sensory neurons and glial satellite cells. These are useful and versatile models to investigate a variety of biological responses associated with physiological and pathological conditions of the peripheral nervous system (PNS) ranging from neuron-glial interaction, neuroplasticity, neuroinflammation, and viral infection. The usage of DRG explant is scientifically advantageous compared to simplistic single cells models for multiple reasons. For instance, as an organotypic culture, the DRG explant allows ex vivo transfer of an entire neuronal network including the extracellular microenvironment that play a significant role in all the neuronal and glial functions. Further, DRG explants can also be maintained ex vivo for several days and the culture conditions can be perturbed as desired. In addition, the harvested DRG can be further dissociated into an in vitro co-culture of primary sensory neurons and satellite glial cells to investigate neuronal-glial interaction, neuritogenesis, axonal cone interaction with the extracellular microenvironment, and more general, any aspect associated with the neuronal metabolism. Therefore, the DRG-explant system offers a great deal of flexibility to study a wide array of events related to biological, physiological, and pathological conditions in a cost-effective manner.


Assuntos
Plasticidade Neuronal/imunologia , Células Receptoras Sensoriais/imunologia , Viroses/imunologia , Animais , Células Cultivadas , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Camundongos
6.
J Bacteriol ; 199(18)2017 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-28416708

RESUMO

Maintenance of cellular phosphate homeostasis is essential for cellular life. The PhoU protein has emerged as a key regulator of this process in bacteria, and it is suggested to modulate phosphate import by PstSCAB and control activation of the phosphate limitation response by the PhoR-PhoB two-component system. However, a proper understanding of PhoU has remained elusive due to numerous complications of mutating phoU, including loss of viability and the genetic instability of the mutants. Here, we developed two sets of strains of Sinorhizobium meliloti that overcame these limitations and allowed a more detailed and comprehensive analysis of the biological and molecular activities of PhoU. The data showed that phoU cannot be deleted in the presence of phosphate unless PstSCAB is inactivated also. However, phoU deletions were readily recovered in phosphate-free media, and characterization of these mutants revealed that addition of phosphate to the environment resulted in toxic levels of PstSCAB-mediated phosphate accumulation. Phosphate uptake experiments indicated that PhoU significantly decreased the PstSCAB transport rate specifically in phosphate-replete cells but not in phosphate-starved cells and that PhoU could rapidly respond to elevated environmental phosphate concentrations and decrease the PstSCAB transport rate. Site-directed mutagenesis results suggested that the ability of PhoU to respond to phosphate levels was independent of the conformation of the PstSCAB transporter. Additionally, PhoU-PhoU and PhoU-PhoR interactions were detected using a bacterial two-hybrid screen. We propose that PhoU modulates PstSCAB and PhoR-PhoB in response to local, internal fluctuations in phosphate concentrations resulting from PstSCAB-mediated phosphate import.IMPORTANCE Correct maintenance of cellular phosphate homeostasis is critical in all kingdoms of life and in bacteria involves the PhoU protein. This work provides novel insights into the role of the Sinorhizobium meliloti PhoU protein, which plays a key role in rapid adaptation to elevated phosphate concentrations. It is shown that PhoU rapidly responds to elevated phosphate levels by significantly decreasing the phosphate transport of PstSCAB, thereby preventing phosphate toxicity and cell death. Additionally, a new model for phosphate sensing in bacterial species which involves the PhoR-PhoB two-component system is presented. This work provides new insights into the bacterial response to changing environmental conditions and into regulation of the phosphate limitation response that influences numerous bacterial processes, including antibiotic production and virulence.


Assuntos
Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana Transportadoras/metabolismo , Fosfatos/metabolismo , Sinorhizobium meliloti/genética , Sinorhizobium meliloti/metabolismo , Fatores de Transcrição/metabolismo , Deleção de Genes , Proteínas de Membrana Transportadoras/genética , Mutagênese Sítio-Dirigida , Mapeamento de Interação de Proteínas , Fatores de Transcrição/genética , Técnicas do Sistema de Duplo-Híbrido
7.
J Neurovirol ; 23(3): 483-491, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28326469

RESUMO

The molecular mechanism of herpes simplex virus (HSV) entry and the associated inflammatory response in the nervous system remain poorly understood. Using mouse-derived ex vivo dorsal root ganglia (DRG) explant model and single cell neurons (SCNs), in this study, we provided a visual evidence for the expression of heparan sulfate (HS) and 3-O-sulfated heparan sulfate (3-OS HS) followed by their interactions with HSV-1 glycoprotein B (gB) and glycoprotein D (gD) during cell entry. Upon heparanase treatment of DRG-derived SCN, a significant inhibition of HSV-1 entry was observed suggesting the involvement of HS role during viral entry. Finally, a cytokine array profile generated during HSV-1 infection in DRG explant indicated an enhanced expression of chemokines (LIX, TIMP-2, and M-CSF)-known regulators of HS. Taken together, these results highlight the significance of HS during HSV-1 entry in DRG explant. Further investigation is needed to understand which isoforms of 3-O-sulfotransferase (3-OST)-generated HS contributed during HSV-1 infection and associated cell damage.


Assuntos
Gânglios Espinais/metabolismo , Heparitina Sulfato/metabolismo , Herpesvirus Humano 1/genética , Interações Hospedeiro-Patógeno , Neurônios/metabolismo , Animais , Biomarcadores/metabolismo , Quimiocina CXCL5/genética , Quimiocina CXCL5/metabolismo , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/virologia , Expressão Gênica , Glucuronidase/farmacologia , Herpesvirus Humano 1/efeitos dos fármacos , Herpesvirus Humano 1/crescimento & desenvolvimento , Herpesvirus Humano 1/metabolismo , Hidrólise , Fator Estimulador de Colônias de Macrófagos/genética , Fator Estimulador de Colônias de Macrófagos/metabolismo , Camundongos , Neurônios/efeitos dos fármacos , Neurônios/virologia , Cultura Primária de Células , Análise de Célula Única/métodos , Técnicas de Cultura de Tecidos , Inibidor Tecidual de Metaloproteinase-2/genética , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Internalização do Vírus/efeitos dos fármacos
8.
Biometals ; 29(4): 625-35, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27260023

RESUMO

Leadmium Green is a commercially available, small molecule, fluorescent probe advertised as a detector of free intracellular cadmium (Cd(2+)) and lead (Pb(2+)). Leadmium Green has been used in various paradigms, such as tracking Cd(2+) sequestration in plant cells, heavy metal export in protozoa, and Pb(2+) absorption by vascular endothelial cells. However very little information is available regarding its affinity and selectivity for Cd(2+), Pb(2+), and other metals. We evaluated the in vitro selectivity of Leadmium Green using spectrofluorimetry. Consistent with manufacturer's claims, Leadmium Green was sensitive to Cd(2+) (KD ~600 nM) and also Pb(2+) (KD ~9.0 nM) in a concentration-dependent manner, and furthermore proved insensitive to Ca(2+), Co(2+), Mn(2+) and Ni(2+). Leadmium Green also responded to Zn(2+) with a KD of ~82 nM. Using fluorescence microscopy, we evaluated Leadmium Green in live mouse hippocampal HT22 cells. We demonstrated that Leadmium Green detected ionophore-mediated acute elevations of Cd(2+) or Zn(2+) in a concentration-dependent manner. However, the maximum fluorescence produced by ionophore-delivered Zn(2+) was much less than that produced by Cd(2+). When tested in a model of oxidant-induced liberation of endogenous Zn(2+), Leadmium Green responded weakly. We conclude that Leadmium Green is an effective probe for monitoring intracellular Cd(2+), particularly in models where Cd(2+) accumulates rapidly, and when concomitant fluctuations of intracellular Zn(2+) are minimal.


Assuntos
Cádmio/análise , Fluorescência , Corantes Fluorescentes/análise , Corantes Fluorescentes/química , Animais , Células Cultivadas , Camundongos , Microscopia Confocal , Espectrometria de Fluorescência , Zinco/análise
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