scholarly journals Heat-Induced Oxidation of the Nuclei and Cytosol

2021 ◽  
Vol 11 ◽  
Author(s):  
Richa Babbar ◽  
Barbara Karpinska ◽  
Anil Grover ◽  
Christine H. Foyer
Keyword(s):  
Fluorescent Protein ◽  
Plant Responses ◽  
Specific Information ◽  
Redox Potentials ◽  
Heat Treated ◽  
Ros Accumulation ◽  
Stomatal Guard Cells ◽  
Transcript Profiles ◽  
Green Fluorescent ◽  
Induced Changes

The concept that heat stress (HS) causes a large accumulation of reactive oxygen species (ROS) is widely accepted. However, the intracellular compartmentation of ROS accumulation has been poorly characterized. We therefore used redox-sensitive green fluorescent protein (roGFP2) to provide compartment-specific information on heat-induced redox changes of the nuclei and cytosol of Arabidopsis leaf epidermal and stomatal guard cells. We show that HS causes a large increase in the degree of oxidation of both compartments, causing large shifts in the glutathione redox potentials of the cells. Heat-induced increases in the levels of the marker transcripts, heat shock protein (HSP)101, and ascorbate peroxidase (APX)2 were maximal after 15 min of the onset of the heat treatment. RNAseq analysis of the transcript profiles of the control and heat-treated seedlings revealed large changes in transcripts encoding HSPs, mitochondrial proteins, transcription factors, and other nuclear localized components. We conclude that HS causes extensive oxidation of the nucleus as well as the cytosol. We propose that the heat-induced changes in the nuclear redox state are central to both genetic and epigenetic control of plant responses to HS.

scholarly journals Colonization of Arabidopsis thaliana with Salmonella enterica and Enterohemorrhagic Escherichia coli O157:H7 and Competition by Enterobacter asburiae

2003 ◽  
Vol 69 (8) ◽  
pp. 4915-4926 ◽  
Author(s):  
Michael B. Cooley ◽  
William G. Miller ◽  
Robert E. Mandrell
Keyword(s):  
Escherichia Coli ◽  
Arabidopsis Thaliana ◽  
Salmonella Enterica ◽  
Fluorescent Protein ◽  
Enterohemorrhagic Escherichia Coli ◽  
Plant Responses ◽  
Human Pathogens ◽  
E Coli ◽  
Green Fluorescent ◽  
Enterobacter Asburiae

ABSTRACT Enteric pathogens, such as Salmonella enterica and Escherichia coli O157:H7, have been shown to contaminate fresh produce. Under appropriate conditions, these bacteria will grow on and invade the plant tissue. We have developed Arabidopsis thaliana (thale cress) as a model system with the intention of studying plant responses to human pathogens. Under sterile conditions and at 100% humidity, S. enterica serovar Newport and E. coli O157:H7 grew to 109 CFU g−1 on A. thaliana roots and to 2 × 107 CFU g−1 on shoots. Furthermore, root inoculation led to contamination of the entire plant, indicating that the pathogens are capable of moving on or within the plant in the absence of competition. Inoculation with green fluorescent protein-labeled S. enterica and E. coli O157:H7 showed invasion of the roots at lateral root junctions. Movement was eliminated and invasion decreased when nonmotile mutants of S. enterica were used. Survival of S. enterica serovar Newport and E. coli O157:H7 on soil-grown plants declined as the plants matured, but both pathogens were detectable for at least 21 days. Survival of the pathogen was reduced in unautoclaved soil and amended soil, suggesting competition from indigenous epiphytes from the soil. Enterobacter asburiae was isolated from soil-grown A. thaliana and shown to be effective at suppressing epiphytic growth of both pathogens under gnotobiotic conditions. Seed and chaff harvested from contaminated plants were occasionally contaminated. The rate of recovery of S. enterica and E. coli O157:H7 from seed varied from undetectable to 19% of the seed pools tested, depending on the method of inoculation. Seed contamination by these pathogens was undetectable in the presence of the competitor, Enterobacter asburiae. Sampling of 74 pools of chaff indicated a strong correlation between contamination of the chaff and seed (P = 0.025). This suggested that contamination of the seed occurred directly from contaminated chaff or by invasion of the flower or silique. However, contaminated seeds were not sanitized by extensive washing and chlorine treatment, indicating that some of the bacteria reside in a protected niche on the seed surface or under the seed coat.

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.

ANG II and calmodulin/CaMKII regulate surface expression and functional activity of NBCe1 via separate means

2007 ◽  
Vol 293 (1) ◽  
pp. F68-F77 ◽  
Author(s):  
Clint Perry ◽  
Hong Le ◽  
Irina I. Grichtchenko
Keyword(s):  
Fluorescent Protein ◽  
Surface Expression ◽  
Inhibitory Effect ◽  
Additive Effect ◽  
Xenopus Laevis Oocytes ◽  
Ang Ii ◽  
Early Endosomes ◽  
Green Fluorescent ◽  
Induced Changes

We recently reported that ANG II inhibits NBCe1 current and surface expression in Xenopus laevis oocytes (Perry C, Blaine J, Le H, and Grichtchenko II. Am J Physiol Renal Physiol 290: F417–F427, 2006). Here, we investigated mechanisms of ANG II-induced changes in NBCe1 surface expression. We showed that the PKC inhibitor GF109203X blocks and EGTA reduces surface cotransporter loss in ANG II-treated oocytes, suggesting roles for PKC and Ca2+. Using the endosomal marker FM 4-64 and enhanced green fluorescent protein (EGFP)-tagged NBCe1, we showed that ANG II stimulates endocytosis of NBCe1. To eliminate the possibility that ANG II inhibits NBCe1 recycling, we demonstrated that the recycling inhibitor monensin decreases surface expression, accumulates NBCe1-EGFP in endosomes, and inhibits NBCe1 current. Monensin and ANG II applied together produce greater inhibition of NBCe1 current than either did alone. This additive effect of monensin and ANG II suggests that ANG II stimulates internalization of NBCe1. We used the calmodulin (CaM) antagonist W13, which controls recycling by blocking the exit of the endocytosed cargo from early endosomes, to determine the role of CaM in NBCe1 trafficking. We demonstrated that W13 decreases surface expression of NBCe1, accumulates NBCe1-EGFP in endosomal-like formations, and inhibits NBCe1 current. W13 and ANG II applied together produce greater inhibition of NBCe1 current than either does alone, while W13 and monensin applied together do not. The additive effect of ANG II and W13 and lack of additive effect of monensin and W13 suggest that CaM is not involved in ANG II stimulation of internalization but controls recycling of endocytosed NBCe1. The CaM-activated enzyme CaM kinase II (CaMKII) applied with ANG II also gives an additive inhibitory effect, suggesting a role for CaMKII in NBCe1 recycling.

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.

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

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.

Electric-Field-Induced Changes in Absorption and Fluorescence of the Green Fluorescent Protein Chromophore in a PMMA Film

2011 ◽  
Vol 115 (26) ◽  
pp. 8622-8626 ◽  
Author(s):  
Takakazu Nakabayashi ◽  
Kazuyuki Hino ◽  
Yuka Ohta ◽  
Sayuri Ito ◽  
Hirofumi Nakano ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Electric Field ◽  
Fluorescent Protein ◽  
Pmma Film ◽  
Green Fluorescent ◽  
Induced Changes

scholarly journals Synaptic correlates of associative fear memory in the lateral amygdala

2020 ◽  
Author(s):  
Dong Il Choi ◽  
Ji-il Kim ◽  
Jooyoung Kim ◽  
Hoonwon Lee ◽  
Ja Eun Choi ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Fear Memory ◽  
Neural Substrates ◽  
Lateral Amygdala ◽  
Spine Morphology ◽  
External Threats ◽  
Memory Extinction ◽  
Green Fluorescent ◽  
Induced Changes ◽  
Protein Reconstitution

AbstractSuccessful adaptation to the environment requires accurate responding to external threats by recalling specific memories. However, elucidating underlying neural substrates of associative fear memory was limited due to the difficulties in direct examination of extinction-induced changes of specific synapses that encode an auditory fear memory. Using dual-eGRASP (enhanced Green Fluorescent Protein Reconstitution Across Synaptic Partners), we found that synapses between engram cells or synaptic engram showed a significantly larger spine morphology at auditory cortex (AC) to lateral amygdala (LA) projections after auditory fear conditioning. Fear extinction reversed the enhanced synaptic engram spines while re-conditioning with the same tone and shock restored the size of the synaptic engram. Taken together, we suggest that the synaptic engram may represent a different state of fear memory.One Sentence SummaryAssociative fear memory enlarged the spine morphology of synapses between engram neurons in the amygdala, which was diminished by memory extinction and restored by re-conditioning, suggesting that connections between engram cells represent a different state of fear memory.

scholarly journals Cellular Leptin Resistance Impairs the Leptin-Mediated Suppression of Neuropeptide Y Secretion in Hypothalamic Neurons

Endocrinology ◽  
2011 ◽  
Vol 152 (11) ◽  
pp. 4138-4147 ◽  
Author(s):  
Sandeep S. Dhillon ◽  
Sean A. McFadden ◽  
Jennifer A. Chalmers ◽  
Maria-Luisa Centeno ◽  
Ginah L. Kim ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Neuropeptide Y ◽  
Energy Homeostasis ◽  
Fluorescent Protein ◽  
Primary Cultures ◽  
Leptin Resistance ◽  
Cellular Processes ◽  
Compound C ◽  
Green Fluorescent ◽  
Induced Changes

Evidence shows that neuropeptide Y (NPY) neurons are involved in mediating the anorexigenic action of leptin via neuronal circuits in the hypothalamus. However, studies have produced limited data on the cellular processes involved and whether hypothalamic NPY neurons are susceptible to cellular leptin resistance. To investigate the direct regulation of NPY secretion by leptin, we used novel NPY-synthesizing, immortalized mHypoA-NPY/green fluorescent protein and mHypoA-59 hypothalamic cell lines derived from adult hypothalamic primary cultures. We report that leptin treatment significantly suppressed NPY secretion in the cells by approximately 20%. We found a decrease in c-fos expression upon leptin exposure, indicating deactivation or hyperpolarization of the neurons. Protein analysis indicated that leptin inhibits AMP-activated protein kinase (AMPK) activity and activates acetyl-coenzyme A carboxylase in NPY neurons, supporting the hypothesis of an AMPK-dependent mechanism. Inhibiting both AMPK with Compound C or phosphatidylinositol 3 kinase (PI3K) with 2-(4-morpholinyl)-8-phenyl-1(4H)-1-benzopyran-4-one hydrochloride prevented the leptin-mediated decrease in NPY secretion, indicating both AMPK- and PI3K-mediated mechanisms. Further, NPY secretion was stimulated by 30% by the AMPK activator, aminoimidazole carboxamide ribonucleotide. Importantly, prolonged leptin exposure in the mHypoA-NPY/green fluorescent protein cells prevented leptin-induced changes in AMPK phosphorylation and suppression of NPY secretion, indicating that NPY neurons are susceptible to leptin resistance. Our studies indicate that AMPK and PI3K pathways are involved in leptin action in NPY neurons and that leptin resistance blocks the feedback response likely required to maintain energy homeostasis.

scholarly journals Influence of a Fungus-Feeding Nematode on Growth and Biocontrol Efficacy of Trichoderma harzianum

2001 ◽  
Vol 91 (3) ◽  
pp. 301-306 ◽  
Author(s):  
Yeoung-Seuk Bae ◽  
Guy R. Knudsen
Keyword(s):  
Trichoderma Harzianum ◽  
Fluorescent Protein ◽  
Epifluorescence Microscopy ◽  
Field Soil ◽  
Untreated Soil ◽  
Trichoderma Sp ◽  
Heat Treated ◽  
C 30 ◽  
Green Fluorescent ◽  
Pellet Formulation

A fungivorous nematode, Aphelenchoides sp., was isolated from field soil by baiting with mycelium of the biocontrol fungus Trichoderma harzianum ThzID1, and subsequently was maintained on agar cultures of the fungus. Interactions between the nematode and the green fluorescent protein-producing transformant, T. harzianum ThzID1-M3, were investigated in both heat-treated (80°C, 30 min) and untreated field soil. ThzID1-M3 was identified in soil by epifluorescence microscopy. When ThzID1-M3 was added to soil as an alginate pellet formulation, addition of the nematode (10 per gram of soil) significantly reduced radial growth and recoverable populations of the fungus, and the effect was greater in heat-treated soil than in untreated soil. Addition of ThzID1-M3 to soil pretreated with the nematode (10 per gram of soil) stimulated nematode population growth for approximately 10 to 20 days, whereas nematode populations decreased in the absence of added Trichoderma sp. When sclerotia of Sclerotinia sclerotiorum were added to soil (10 per 200 g of soil) with ThzID1-M3 (40 pellets per 200 g of soil), addition of Aphe-lenchoides sp. (2,000 per 200 g of soil) reduced the number of sclerotia colonized by ThzID1-M3. These results suggest that fungivorous nematodes may be a significant biotic constraint on activity of biocontrol fungi in the field.

scholarly journals CaHDZ27, a Homeodomain-Leucine Zipper I Protein, Positively Regulates the Resistance to Ralstonia solanacearum Infection in Pepper

2017 ◽  
Vol 30 (12) ◽  
pp. 960-973 ◽  
Author(s):  
Shaoliang Mou ◽  
Zhiqin Liu ◽  
Feng Gao ◽  
Sheng Yang ◽  
Meixia Su ◽  
...  
Keyword(s):  
Ralstonia Solanacearum ◽  
Leucine Zipper ◽  
Fluorescent Protein ◽  
Plant Immunity ◽  
Bimolecular Fluorescence Complementation ◽  
Marker Genes ◽  
Plant Responses ◽  
Dependent Manner ◽  
Virus Induced Gene Silencing ◽  
Green Fluorescent

Homeodomain-leucine zipper class I (HD-Zip I) transcription factors have been functionally characterized in plant responses to abiotic stresses, but their roles in plant immunity are poorly understood. Here, a HD-Zip I gene, CaHZ27, was isolated from pepper (Capsicum annum) and characterized for its role in pepper immunity. Quantitative real-time polymerase chain reaction showed that CaHDZ27 was transcriptionally induced by Ralstonia solanacearum inoculation and exogenous application of methyl jasmonate, salicylic acid, or ethephon. The CaHDZ27-green fluorescent protein fused protein was targeted exclusively to the nucleus. Chromatin immunoprecipitation demonstrated that CaHDZ27 bound to the 9-bp pseudopalindromic element (CAATAATTG) and triggered β-glucuronidase expression in a CAATAATTG-dependent manner. Virus-induced gene silencing of CaHDZ27 significantly attenuated the resistance of pepper plants against R. solanacearum and downregulated defense-related marker genes, including CaHIR1, CaACO1, CaPR1, CaPR4, CaPO2, and CaBPR1. By contrast, transient overexpression of CaHDZ27 triggered strong cell death mediated by the hypersensitive response and upregulated the tested immunity-associated marker genes. Ectopic CaHDZ27 expression in tobacco enhances its resistance against R. solanacearum. These results collectively suggest that CaHDZ27 functions as a positive regulator in pepper resistance against R. solanacearum. Bimolecular fluorescence complementation and coimmunoprecipitation assays indicate that CaHDZ27 monomers bind with each other, and this binding is enhanced significantly by R. solanacearum inoculation. We speculate that homodimerization of CaHZ27 might play a role in pepper response to R. solanacearum, further direct evidence is required to confirm it.

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