The effects of L-NAME on neuronal NOS and SOD1 expression in the DRG–spinal cord network of axotomised Thy 1.2 eGFP mice

2011 ◽  
Vol 7 (2-4) ◽  
pp. 129-141 ◽  
Author(s):  
Matthew J. G. Bradman ◽  
Richard Morris ◽  
Anne McArdle ◽  
Malcolm J. Jackson ◽  
Thimmasettappa Thippeswamy
Keyword(s):  
Spinal Cord ◽  
Fluorescent Protein ◽  
No Production ◽  
Dependent Manner ◽  
Differential Distribution ◽  
Copper Zinc Superoxide Dismutase ◽  
Spinal Network ◽  
Copper Zinc ◽  
Green Fluorescent ◽  
Induced Changes

Nitric oxide (NO) plays an important role in pathophysiology of the nervous system. Copper/zinc superoxide dismutase (SOD1) reacts with superoxide, which is also a substrate for NO, to provide antioxidative protection. NO production is greatly altered following nerve injury, therefore we hypothesised that SOD1 and NO may be involved in modulating axotomy responses in dorsal root ganglion (DRG)–spinal network. To investigate this interaction, adult Thy1.2 enhanced membrane-bound green fluorescent protein (eGFP) mice underwent sciatic nerve axotomy and received NG-nitro- <l-arginine methylester (L-NAME) or vehicle 7–9 days later. L4–L6 spinal cord and DRG were harvested for immunohistochemical analyses. Effect of injury was confirmed by axotomy markers; small proline-rich repeat protein 1A (SPRR1A) was restricted to ipsilateral neuropathology, while Thy1.2 eGFP revealed also contralateral crossover effects. L-NAME, but not axotomy, increased neuronal NO synthase (nNOS) and SOD1 immunoreactive neurons, with no colocalisation, in a lamina-dependent manner in the dorsal horn of the spinal cord. Axotomy and/or L-NAME had no effect on total nNOS+and SOD1+neurons in DRG. However, L-NAME altered SOD1 expression in subsets of axotomised DRG neurons. These findings provide evidence for differential distribution of SOD1 and its modulation by NO, which may interact to regulate axotomy-induced changes in DRG–spinal network.

scholarly journals Extracellular Vesicles-Encapsulated Yeast Prions and What They Can Tell Us about the Physical Nature of Propagons

2020 ◽  
Vol 22 (1) ◽  
pp. 90
Author(s):  
Mehdi Kabani
Keyword(s):  
Protein Quality ◽  
Fluorescent Protein ◽  
Physical Nature ◽  
Dependent Manner ◽  
Yeast Saccharomyces Cerevisiae ◽  
Yeast Prions ◽  
Daughter Cells ◽  
Cytoplasmic Proteins ◽  
Green Fluorescent ◽  
Functionally Diverse

The yeast Saccharomyces cerevisiae hosts an ensemble of protein-based heritable traits, most of which result from the conversion of structurally and functionally diverse cytoplasmic proteins into prion forms. Among these, [PSI+], [URE3] and [PIN+] are the most well-documented prions and arise from the assembly of Sup35p, Ure2p and Rnq1p, respectively, into insoluble fibrillar assemblies. Yeast prions propagate by molecular chaperone-mediated fragmentation of these aggregates, which generates small self-templating seeds, or propagons. The exact molecular nature of propagons and how they are faithfully transmitted from mother to daughter cells despite spatial protein quality control are not fully understood. In [PSI+] cells, Sup35p forms detergent-resistant assemblies detectable on agarose gels under semi-denaturant conditions and cytosolic fluorescent puncta when the protein is fused to green fluorescent protein (GFP); yet, these macroscopic manifestations of [PSI+] do not fully correlate with the infectivity measured during growth by the mean of protein infection assays. We also discovered that significant amounts of infectious Sup35p particles are exported via extracellular (EV) and periplasmic (PV) vesicles in a growth phase and glucose-dependent manner. In the present review, I discuss how these vesicles may be a source of actual propagons and a suitable vehicle for their transmission to the bud.

Interleukin-1 (IL-1) Inhibits Growth of Cytomegalovirus in Human Marrow Stromal Cells: Inhibition Is Reversed Upon Removal of IL-1

Blood ◽  
1999 ◽  
Vol 94 (2) ◽  
pp. 572-578 ◽  
Author(s):  
Mineo Iwata ◽  
Jeff Vieira ◽  
Michael Byrne ◽  
Heidi Horton ◽  
Beverly Torok-Storb
Keyword(s):  
Stromal Cells ◽  
Neutralizing Antibodies ◽  
Fluorescent Protein ◽  
Interleukin 1 ◽  
Elongation Factor ◽  
Marrow Stromal Cells ◽  
Type I ◽  
Untreated Culture ◽  
Green Fluorescent ◽  
Induced Changes

Abstract A Toledo strain cytomegalovirus (CMV) containing the gene for green fluorescent protein (GFP) under the control of elongation factor-1 promoter was used to study infection of human marrow stromal cells. Two stromal cell lines were used: HS-5, which secretes copious amounts of known cytokines and interleukins; and HS-27a, which does not secrete these activities. CMV growth and spread was monitored by counting green plaques and quantitating GFP intensity. Initial studies indicated that, whereas HS-5 and 27a have similar susceptibilities to infection, as evidenced by the same number of GFP+ cells at day 2, HS-5 appears more resistant to growth and spread of CMV. Furthermore, conditioned media from HS-5 (HS-5 CM) inhibited CMV plaque formation in HS-27a, suggesting that factors secreted by HS-5 are responsible for limiting CMV growth. Neutralizing antibodies against interleukin-1 (IL-1) and IL-1β completely blocked the ability of HS-5 CM to limit viral growth, suggesting that IL-1, which is known to be present in HS-5 CM, is responsible for this effect. When exogenous IL-1β was added to CMV-infected HS-27a, both the number of plaques and the intensity of GFP was significantly reduced in IL-1–treated HS-27a compared with untreated HS-27a (the number of plaques by day 18 was 20 ± 3 v 151 ± 12/well, respectively; GFP intensity was 535 ± 165 v 6,516 ± 652/well, respectively, in 4 separate experiments). At day 21, when IL-1β–treated, CMV-infected cultures were passaged and then cultured in the absence of IL-1β, CMV growth progressed with the kinetics of the original untreated culture, indicating that the IL-1β effect is reversible. Because HS-27a expresses the type I IL-1 receptor, we speculate that the antiviral effects are mediated through IL-1–induced changes in cellular gene expression. DNA chip analysis of mRNA from IL-1β–treated and nontreated HS-27a cells has identified some candidate molecules.

scholarly journals The sustainability of interactions between the orexin-1 receptor and β-arrestin-2 is defined by a single C-terminal cluster of hydroxy amino acids and modulates the kinetics of ERK MAPK regulation

2005 ◽  
Vol 387 (3) ◽  
pp. 573-584 ◽  
Author(s):  
Sandra MILASTA ◽  
Nicholas A. EVANS ◽  
Laura ORMISTON ◽  
Shelagh WILSON ◽  
Robert J. LEFKOWITZ ◽  
...  
Keyword(s):  
Amino Acids ◽  
Fluorescent Protein ◽  
Weak Interactions ◽  
Dependent Manner ◽  
Wild Type ◽  
Erk Mapk ◽  
Hydroxy Amino ◽  
C Terminus ◽  
Green Fluorescent ◽  
Kinetics Of

The orexin-1 receptor interacts with β-arrestin-2 in an agonist-dependent manner. In HEK-293T cells, these two proteins became co-internalized into acidic endosomes. Truncations from the C-terminal tail did not prevent agonist-induced internalization of the orexin-1 receptor or alter the pathway of internalization, although such mutants failed to interact with β-arrestin-2 in a sustained manner or produce its co-internalization. Mutation of a cluster of three threonine and one serine residue at the extreme C-terminus of the receptor greatly reduced interaction and abolished co-internalization of β-arrestin-2–GFP (green fluorescent protein). Despite the weak interactions of this C-terminally mutated form of the receptor with β-arrestin-2, studies in wild-type and β-arrestin-deficient mouse embryo fibroblasts confirmed that agonist-induced internalization of this mutant required expression of a β-arrestin. Although without effect on agonist-mediated elevation of intracellular Ca2+ levels, the C-terminally mutated form of the orexin-1 receptor was unable to sustain phosphorylation of the MAPKs (mitogen-activated protein kinases) ERK1 and ERK2 (extracellular-signal-regulated kinases 1 and 2) to the same extent as the wild-type receptor. These studies indicate that a single cluster of hydroxy amino acids within the C-terminal seven amino acids of the orexin-1 receptor determine the sustainability of interaction with β-arrestin-2, and indicate an important role of β-arrestin scaffolding in defining the kinetics of orexin-1 receptor-mediated ERK MAPK activation.

scholarly journals Activation of Neurokinin 3 Receptors Stimulates GnRH Release in a Location-Dependent but Kisspeptin-Independent Manner in Adult Mice

Endocrinology ◽  
2013 ◽  
Vol 154 (11) ◽  
pp. 3984-3989 ◽  
Author(s):  
Garrett T. Gaskins ◽  
Katarzyna M. Glanowska ◽  
Suzanne M. Moenter
Keyword(s):  
Green Fluorescent Protein ◽  
Carbon Fiber ◽  
Fluorescent Protein ◽  
Hypogonadotropic Hypogonadism ◽  
Brain Slices ◽  
Dependent Manner ◽  
Gnrh Secretion ◽  
Gnrh Release ◽  
Carbon Fiber Microelectrodes ◽  
Green Fluorescent

GnRH neurons form the final common pathway for the central control of reproduction. GnRH release occurs from terminals in the external layer of the median eminence (ME) for neuroendocrine control of the pituitary, and near GnRH-GnRH fiber appositions within the preoptic area (POA). Whether or not control of GnRH secretion by neuromodulators is different in these 2 areas is unknown. Mutations in neurokinin B (NKB) or the neurokinin-3 receptor (NK3R) are linked to hypogonadotropic hypogonadism in humans, suggesting that NKB may regulate GnRH secretion. Using fast scan cyclic voltammetry through carbon-fiber microelectrodes, we examined real-time GnRH release in response to the NK3R agonist senktide in the ME and POA. Coronal brain slices were acutely prepared from adult gonad-intact GnRH-green fluorescent protein male mice, and carbon-fiber microelectrodes were placed either within green fluorescent protein-positive terminal fields of the ME or near GnRH-GnRH fiber appositions in the POA. Senktide induced GnRH release consistently in the ME but not the POA, indicating that GnRH release is differentially regulated by NKB in a location-dependent manner. Senktide also induced GnRH secretion in the ME of kisspeptin-knockout (Kiss1 knockout) mice. Interestingly, release amplitude was lower compared with wild-type mice. These data indicate regulation of GnRH release by NK3R agonists is site specific and suggest that kisspeptin is not a required mediator between NK3R activation and GnRH secretion in the ME. This information will be useful for informing future models of afferent regulation of GnRH release.

scholarly journals Simvastatin Efficiently Reduces Levels of Alzheimer’s Amyloid Beta in Yeast

2019 ◽  
Vol 20 (14) ◽  
pp. 3531 ◽  
Author(s):  
Sudip Dhakal ◽  
Mishal Subhan ◽  
Joshua M. Fraser ◽  
Kenneth Gardiner ◽  
Ian Macreadie
Keyword(s):  
Fusion Protein ◽  
Amyloid Beta ◽  
Large Scale ◽  
Fluorescent Protein ◽  
Model Organism ◽  
Dependent Manner ◽  
Green Fluorescence ◽  
Convenient Means ◽  
Ergosterol Synthesis ◽  
Green Fluorescent

A large-scale epidemiology study on statins previously showed that simvastatin was unique among statins in reducing the incidence of dementia. Since amyloid beta (Aβ42) is the protein that is most associated with Alzheimer’s disease, this study has focused on how simvastatin influences the turnover of native Aβ42 and Aβ42 fused with green fluorescent protein (GFP), in the simplest eukaryotic model organism, Saccharomyces cerevisiae. Previous studies have established that yeast constitutively producing Aβ42 fused to GFP offer a convenient means of analyzing yeast cellular responses to Aβ42. Young cells clear the GFP fusion protein and do not have green fluorescence while the older population of cells retains the fusion protein and exhibits green fluorescence, offering a fast and convenient means of studying factors that affect Aβ42 turnover. In this study the proportion of cells having GFP fused to Aβ after exposure to simvastatin, atorvastatin and lovastatin was analyzed by flow cytometry. Simvastatin effectively reduced levels of the cellular Aβ42 protein in a dose-dependent manner. Simvastatin promoted the greatest reduction as compared to the other two statins. A comparison with fluconazole, which targets that same pathway of ergosterol synthesis, suggests that effects on ergosterol synthesis do not account for the reduced amounts of Aβ42 fused to GFP. The levels of native Aβ42 following treated with simvastatin were also examined using a more laborious approach, quantitative MALDI TOF mass spectrometry. Simvastatin efficiently reduced levels of native Aβ42 from the population. This work indicates a novel action of simvastatin in reducing levels of Aβ42 providing new insights into how simvastatin exerts its neuroprotective role. We hypothesize that this reduction may be due to protein clearance.

NR2B-siRNA Mediated by Hydroxyapatite Nanoparticles Relieves for Malin-Induced Pain of Mice

2011 ◽  
Vol 343-344 ◽  
pp. 926-932 ◽  
Author(s):  
Yong Hong Gu ◽  
Xue Bin Yan ◽  
Dong Huang ◽  
Rui Han ◽  
Li Xiang Wu
Keyword(s):  
Spinal Cord ◽  
Immunohistochemical Staining ◽  
Formalin Test ◽  
Fluorescent Protein ◽  
Intrathecal Injection ◽  
Nociceptive Response ◽  
Hydroxyapatite Nanoparticles ◽  
Green Fluorescent ◽  
Tonic Phase

To observe the effect of NR2B-siRNA mediated by hydroxyapatite nanoparticles (HA) on formalin-induced inflammatory pain of mice and the expression of NR2B in spinal cord. To preliminarily investigate the feasibility of HA as siRNA carrier to transfer NR2B-siRNA in vivo. The sequence-specific NR2B-siRNA of mice was designed and synthesized initially. Using HA as a siRNA carrier, green fluorescent protein(GFP)-siRNA as the control, 4 ug of NR2B-siRNA was administered into subarachnoid space of mice via conscious injection. On 7th day after intrathecal injection, formalin test was observed for 1 hour in each group, followed by dissection of lumbar segments of spinal cords immediately for use in immunohistochemical staining of NR2B. The results show that NR2B-siRNA not only significantly abolish the nociceptive response of mice in the tonic phase induced by formalin, but also decrease the amount of cells expressing NR2B protein in spinal cord, while GFP-siRNA mediated by HA don’t produce the same effects, which demonstrates that HA is capable of effectively transfering NR2B-siRNA via intrathecal injection, furthermore, HA/NR2B-siRNA complex can significantly reduce formalin-induced pain of mice, and specificly inhibit NR2B expression in spinal cord of mice.

scholarly journals Modulation of Virulence by Two Acidified Nitrite-Responsive Loci of Salmonella enterica Serovar Typhimurium

2003 ◽  
Vol 71 (6) ◽  
pp. 3196-3205 ◽  
Author(s):  
Charles C. Kim ◽  
Denise Monack ◽  
Stanley Falkow
Keyword(s):  
Salmonella Enterica ◽  
Fluorescent Protein ◽  
Dependent Manner ◽  
Wild Type ◽  
Independent Manner ◽  
Inducible Promoters ◽  
Serovar Typhimurium ◽  
Bacterial Genes ◽  
Green Fluorescent

ABSTRACT Two acidified nitrite-inducible genes of Salmonella enterica serovar Typhimurium were identified with a green fluorescent protein-based promoter-trap screen. The nitrite-inducible promoters were located upstream of loci that we designated nipAB and nipC, which correspond to hcp-hcr (hybrid cluster protein) of Escherichia coli and norA of Alcaligenes eutrophus, respectively. Maximal induction of the promoters by nitrite was dependent on pH. The nipAB promoter was regulated by oxygen in an Fnr-dependent manner. The nipC promoter was also regulated by oxygen but in an Fnr-independent manner. The promoters were upregulated in activated RAW264.7 macrophage-like cells, which produce NO via the inducible nitric oxide synthase (iNOS), and the induction was inhibited by aminoguanidine, an inhibitor of iNOS. Although the nipAB and nipC mutants displayed no defects under a variety of in vitro conditions or in tissue culture infections, they exhibited lower oral 50% lethal doses (LD50s) than did the wild type in C57BL/6J mouse infections. The lower LD50s reflected an unexpected increased ability of small inoculating doses of the mutant bacteria to cause lethal infection 2 to 3 weeks after challenge, compared to a similar challenge dose of wild-type bacteria. We conclude that these genes are regulated by physiological nitrogen oxides and that the absence of these bacterial genes in some way diminishes the ability of mice to clear a low dose infection.

Measurement of proteases using chemiluminescence-resonance-energy-transfer chimaeras between green fluorescent protein and aequorin

2001 ◽  
Vol 357 (3) ◽  
pp. 687-697 ◽  
Author(s):  
Jonathan P. WAUD ◽  
Alexandra BERMÚDEZ FAJARDO ◽  
Thankiah SUDHAHARAN ◽  
Andrew R. TRIMBY ◽  
Jinny JEFFERY ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Green Fluorescent Protein ◽  
Caspase 3 ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Dependent Manner ◽  
Cell Extracts ◽  
Donor And Acceptor ◽  
Green Fluorescent

Homogeneous assays, without a separation step, are essential for measuring chemical events in live cells and for drug discovery screens, and are desirable for making measurements in cell extracts or clinical samples. Here we demonstrate the principle of chemiluminescence resonance energy transfer (CRET) as a homogeneous assay system, using two proteases as models, one extracellular (α-thrombin) and the other intracellular (caspase-3). Chimaeras were engineered with aequorin as the chemiluminescent energy donor and green fluorescent protein (GFP) or enhanced GFP as the energy acceptors, with a protease linker (6 or 18 amino acid residues) recognition site between the donor and acceptor. Flash chemiluminescent spectra (20–60 s) showed that the spectra of chimaeras matched GFP, being similar to that of luminous jellyfish, justifying their designation as ‘Rainbow’ proteins. Addition of the protease shifted the emission spectrum to that of aequorin in a time- and dose-dependent manner. Separation of the proteolysed fragments showed that the ratio of green to blue light matched the extent of proteolysis. The caspase-3 Rainbow protein was able to provide information on the specificity of caspases in vitro and in vivo. It was also able to monitor caspase-3 activation in cells provoked into apoptosis by staurosporine (1 or 2μM). CRET can also monitor GFP fluor formation. The signal-to-noise ratio of our Rainbow proteins is superior to that of fluorescence resonance energy transfer, providing a potential platform for measuring agents that interact with the reactive site between the donor and acceptor.

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