Long-term Fate Mapping to Assess the Impact of Postnatal Isoflurane Exposure on Hippocampal Progenitor Cell Productivity

Abstract Background Exposure to isoflurane increases apoptosis among postnatally generated hippocampal dentate granule cells. These neurons play important roles in cognition and behavior, so their permanent loss could explain deficits after surgical procedures. Methods To determine whether developmental anesthesia exposure leads to persistent deficits in granule cell numbers, a genetic fate-mapping approach to label a cohort of postnatally generated granule cells in Gli1-CreERT2::GFP bitransgenic mice was utilized. Green fluorescent protein (GFP) expression was induced on postnatal day 7 (P7) to fate map progenitor cells, and mice were exposed to 6 h of 1.5% isoflurane or room air 2 weeks later (P21). Brain structure was assessed immediately after anesthesia exposure (n = 7 controls and 8 anesthesia-treated mice) or after a 60-day recovery (n = 8 controls and 8 anesthesia-treated mice). A final group of C57BL/6 mice was exposed to isoflurane at P21 and examined using neurogenesis and cell death markers after a 14-day recovery (n = 10 controls and 16 anesthesia-treated mice). Results Isoflurane significantly increased apoptosis immediately after exposure, leading to cell death among 11% of GFP-labeled cells. Sixty days after isoflurane exposure, the number of GFP-expressing granule cells in treated animals was indistinguishable from control animals. Rates of neurogenesis were equivalent among groups at both 2 weeks and 2 months after treatment. Conclusions These findings suggest that the dentate gyrus can restore normal neuron numbers after a single, developmental exposure to isoflurane. The authors’ results do not preclude the possibility that the affected population may exhibit more subtle structural or functional deficits. Nonetheless, the dentate appears to exhibit greater resiliency relative to nonneurogenic brain regions, which exhibit permanent neuron loss after isoflurane exposure.

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Asc Modulates the Function of NLRC4 in Response to Infection of Macrophages by Legionella pneumophila

mBio ◽

10.1128/mbio.00117-11 ◽

2011 ◽

Vol 2 (4) ◽

Cited By ~ 36

Author(s):

Christopher L. Case ◽

Craig R. Roy

Keyword(s):

Cell Death ◽

Legionella Pneumophila ◽

Fluorescent Protein ◽

Microbial Infection ◽

Content Type ◽

Caspase 1 ◽

Cell Death Pathway ◽

Dependent Cell ◽

Green Fluorescent ◽

In Cells

ABSTRACTNucleotide-binding domain, leucine-rich repeat containing proteins (NLRs) activate caspase-1 in response to a variety of bacterium-derived signals in macrophages. NLR-mediated activation of caspase-1 byLegionella pneumophilaoccurs through both an NLRC4/NAIP5-dependent pathway and a pathway requiring the adapter protein Asc. Both pathways are needed for maximal activation of caspase-1 and for the release of the cytokines interleukin-1β (IL-1β) and IL-18. Asc is not required for caspase-1-dependent pore formation and cell death induced upon infection of macrophages byL. pneumophila. Here, temporal and spatial localization of caspase-1-dependent processes was examined to better define the roles of Asc and NLRC4 during infection. Imaging studies revealed that caspase-1 localized to a single punctate structure in infected cells containing Asc but not in cells lacking this adapter. Both endogenous Asc and ectopically produced NLRC4 tagged with green fluorescent protein (GFP) were found to localize to caspase-1 puncta followingL. pneumophilainfection, suggesting that NLRC4 and Asc coordinate signaling through this complex during caspase-1 activation. Formation of caspase-1-containing puncta correlated with caspase-1 processing, suggesting a role for the Asc/NLRC4/caspase-1 complex in caspase-1 cleavage. In cells deficient for Asc, NLRC4 did not assemble into discrete puncta, and pyroptosis occurred at an accelerated rate. These data indicate that Asc mediates integration of NLR components into caspase-1 processing platforms and that recruitment of NLR components into an Asc complex can dampen pyroptotic responses. Thus, a negative feedback role of complexes containing Asc may be important for regulating caspase-1-mediated responses during microbial infection.IMPORTANCECaspase-1 is a protease activated during infection that is central to the regulation of several innate immune pathways. Studies examining the macromolecular complexes containing this protein, known as inflammasomes, have provided insight into the regulation of this protease. This work demonstrates that the intracellular bacteriumLegionella pneumophilainduces formation of complexes containing caspase-1 by multiple mechanisms and illustrates that an adapter molecule called Asc integrates signals from multiple independent upstream caspase-1 activators in order to assemble a spatially distinct complex in the macrophage. There were caspase-1-associated activities such as cytokine processing and secretion that were controlled by Asc. Importantly, this work uncovered a new role for Asc in dampening a caspase-1-dependent cell death pathway called pyroptosis. These findings suggest that Asc plays a central role in controlling a distinct subset of caspase-1-dependent activities by both assembling complexes that are important for cytokine processing and suppressing processes that mediate pyroptosis.

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Minocycline decreases CCR2-positive monocytes in the retina and ameliorates photoreceptor degeneration in a mouse model of retinitis pigmentosa

PLoS ONE ◽

10.1371/journal.pone.0239108 ◽

2021 ◽

Vol 16 (4) ◽

pp. e0239108

Author(s):

Ryo Terauchi ◽

Hideo Kohno ◽

Sumiko Watanabe ◽

Saburo Saito ◽

Akira Watanabe ◽

...

Keyword(s):

Cell Death ◽

Retinal Degeneration ◽

Photoreceptor Cell ◽

Fluorescent Protein ◽

Outer Nuclear Layer ◽

Inhibitory Effect ◽

Subretinal Space ◽

Photoreceptor Degeneration ◽

Retinal Inflammation ◽

Green Fluorescent

Retinal inflammation accelerates photoreceptor cell death caused by retinal degeneration. Minocycline, a semisynthetic broad-spectrum tetracycline antibiotic, has been previously reported to rescue photoreceptor cell death in retinal degeneration. We examined the effect of minocycline on retinal photoreceptor degeneration using c-mer proto-oncogene tyrosine kinase (Mertk)−/−Cx3cr1GFP/+Ccr2RFP/+ mice, which enabled the observation of CX3CR1-green fluorescent protein (GFP)- and CCR2-red fluorescent protein (RFP)-positive macrophages by fluorescence. Retinas of Mertk−/−Cx3cr1GFP/+Ccr2RFP/+ mice showed photoreceptor degeneration and accumulation of GFP- and RFP-positive macrophages in the outer retina and subretinal space at 6 weeks of age. Mertk−/−Cx3cr1GFP/+Ccr2RFP/+ mice were intraperitoneally administered minocycline. The number of CCR2-RFP positive cells significantly decreased after minocycline treatment. Furthermore, minocycline administration resulted in partial reversal of the thinning of the outer nuclear layer and decreased the number of apoptotic cells, as assessed by the TUNEL assay, in Mertk−/−Cx3cr1GFP/+Ccr2RFP/+ mice. In conclusion, we found that minocycline ameliorated photoreceptor cell death in an inherited photoreceptor degeneration model due to Mertk gene deficiency and has an inhibitory effect on CCR2 positive macrophages, which is likely to be a neuroprotective mechanism of minocycline.

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Microscopic Observation and Processing Validation of Fruit Sanitizing Treatments for the Enhanced Microbiological Safety of Fresh Orange Juice†

Journal of Food Protection ◽

10.4315/0362-028x-64.3.310 ◽

2001 ◽

Vol 64 (3) ◽

pp. 310-314 ◽

Cited By ~ 19

Author(s):

STEVEN PAO ◽

CRAIG L. DAVIS ◽

MICKEY E. PARISH

Keyword(s):

Fluorescent Protein ◽

Citrus Fruit ◽

Microbial Reduction ◽

Hot Water ◽

Near Surface ◽

E Coli ◽

Surface Areas ◽

Orange Fruit ◽

Green Fluorescent ◽

The Impact

Studies were conducted to evaluate the infiltration of dye and bacteria into the interior of orange fruit and the impact of possible infiltration on achieving a 5-log microbial reduction during fresh juice processing. Fresh orange fruit were treated at the stem end area with dye and either Salmonella Rubislaw or Escherichia coli strains expressing green fluorescent protein. Microscopic images showed that bacterial contaminants localized at the surface or near surface areas that may be sanitized by surface treatments. Dye infiltration was not a reliable indicator of bacterial penetration in citrus fruit. To quantify the reduction of bacterial contamination, orange fruit were inoculated with E. coli and processed with and without hot water treatments. Greater than 5-log reductions were achieved in juice extracted from fruit immersed in hot water for 1 or 2 min at 80°C, in comparison to the E. coli level detected in the control juice obtained by homogenization of inoculated fruit.

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ROMK1 (Kir1.1) Causes Apoptosis and Chronic Silencing of Hippocampal Neurons

Journal of Neurophysiology ◽

10.1152/jn.2000.84.2.1062 ◽

2000 ◽

Vol 84 (2) ◽

pp. 1062-1075 ◽

Cited By ~ 62

Author(s):

H. Nadeau ◽

S. McKinney ◽

D. J. Anderson ◽

H. A. Lester

Keyword(s):

Cell Death ◽

Hippocampal Neurons ◽

Fluorescent Protein ◽

Input Resistance ◽

Firing Frequency ◽

Compensatory Mechanisms ◽

Spike Shape ◽

Infected Cells ◽

Green Fluorescent ◽

Potential Threshold

Lentiviral vectors were constructed to express the weakly rectifying kidney K+ channel ROMK1 (Kir1.1), either fused to enhanced green fluorescent protein (EGFP) or as a bicistronic message (ROMK1-CITE-EGFP). The channel was stably expressed in cultured rat hippocampal neurons. Infected cells were maintained for 2–4 wk without decrease in expression level or evidence of viral toxicity, although 15.4 mM external KCl was required to prevent apoptosis of neurons expressing functional ROMK1. No other trophic agents tested could prevent cell death, which was probably caused by K+loss. This cell death did not occur in glia, which were able to support ROMK1 expression indefinitely. Functional ROMK1, quantified as the nonnative inward current at −144 mV in 5.4 mM external K+blockable by 500 μM Ba2+, ranged from 1 to 40 pA/pF. Infected neurons exhibited a Ba2+-induced depolarization of 7 ± 2 mV relative to matched EGFP-infected controls, as well as a 30% decrease in input resistance and a shift in action potential threshold of 2.6 ± 0.5 mV. This led to a shift in the relation between injected current and firing frequency, without changes in spike shape, size, or timing. This shift, which quantifies silencing as a function of ROMK1 expression, was predicted from Hodgkin-Huxley models. No cellular compensatory mechanisms in response to expression of ROMK1 were identified, making ROMK1 potentially useful for transgenic studies of silencing and neurodegeneration, although its lethality in normal K+ has implications for the use of K+ channels in gene therapy.

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Cell Death of Nicotiana benthamiana Is Induced by Secreted Protein NIS1 of Colletotrichum orbiculare and Is Suppressed by a Homologue of CgDN3

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-12-11-0316 ◽

2012 ◽

Vol 25 (5) ◽

pp. 625-636 ◽

Cited By ~ 33

Author(s):

Kae Yoshino ◽

Hiroki Irieda ◽

Fumie Sugimoto ◽

Hirofumi Yoshioka ◽

Tetsuro Okuno ◽

...

Keyword(s):

Cell Death ◽

Nicotiana Benthamiana ◽

Protein Gene ◽

Fluorescent Protein ◽

Expression System ◽

Functional Screening ◽

Secreted Protein ◽

Virus Induced Gene Silencing ◽

Colletotrichum Orbiculare ◽

Green Fluorescent

Colletotrichum orbiculare, the causal agent of cucumber anthracnose, infects Nicotiana benthamiana. Functional screening of C. orbiculare cDNAs in a virus vector-based plant expression system identified a novel secreted protein gene, NIS1, whose product induces cell death in N. benthamiana. Putative homologues of NIS1 are present in selected members of fungi belonging to class Sordariomycetes, Dothideomycetes, or Orbiliomycetes. Green fluorescent protein–based expression studies suggested that NIS1 is preferentially expressed in biotrophic invasive hyphae. NIS1 lacking signal peptide did not induce NIS1-triggered cell death (NCD), suggesting apoplastic recognition of NIS1. NCD was prevented by virus-induced gene silencing of SGT1 and HSP90, indicating the dependency of NCD on SGT1 and HSP90. Deletion of NIS1 had little effect on the virulence of C. orbiculare against N. benthamiana, suggesting possible suppression of NCD by C. orbiculare at the postinvasive stage. The CgDN3 gene of C. gloeosporioides was previously identified as a secreted protein gene involved in suppression of hypersensitive-like response in Stylosanthes guianensis. Notably, we found that NCD was suppressed by the expression of a CgDN3 homologue of C. orbiculare. Our findings indicate that C. orbiculare expresses NIS1 at the postinvasive stage and suggest that NCD could be repressed via other effectors, including the CgDN3 homologue.

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Analyzing Defects in the Caenorhabditis elegans Nervous System Using Organismal and Cell Biological Approaches

Cell Biology Education ◽

10.1187/cbe.01-08-0001 ◽

2002 ◽

Vol 1 (1) ◽

pp. 18-25 ◽

Cited By ~ 4

Author(s):

Megan Guziewicz ◽

Toni Vitullo ◽

Bethany Simmons ◽

Rebecca Eustance Kohn

Keyword(s):

Nervous System ◽

Caenorhabditis Elegans ◽

Synaptic Vesicle ◽

Cell Biology ◽

Fluorescent Protein ◽

Vesicle Transport ◽

Laboratory Exercise ◽

Cellular Analysis ◽

Green Fluorescent ◽

The Impact

The goal of this laboratory exercise is to increase student understanding of the impact of nervous system function at both the organismal and cellular levels. This inquiry-based exercise is designed for an undergraduate course examining principles of cell biology. After observing the movement of Caenorhabditis elegans with defects in their nervous system, students examine the structure of the nervous system to categorize the type of defect. They distinguish between defects in synaptic vesicle transport and defects in synaptic vesicle fusion with membranes. The synaptic vesicles are tagged with green fluorescent protein (GFP), simplifying cellular analysis. The expected outcome of this experiment is that students will better understand the concepts of vesicle transport, neurotransmitter release, GFP, and the relation between the nervous system and behavior.

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A C-Terminal Domain Targets the Pseudomonas aeruginosa Cytotoxin ExoU to the Plasma Membrane of Host Cells

Infection and Immunity ◽

10.1128/iai.74.5.2552-2561.2006 ◽

2006 ◽

Vol 74 (5) ◽

pp. 2552-2561 ◽

Cited By ~ 35

Author(s):

Shira D. P. Rabin ◽

Jeffrey L. Veesenmeyer ◽

Kathryn T. Bieging ◽

Alan R. Hauser

Keyword(s):

Pseudomonas Aeruginosa ◽

Cell Death ◽

Plasma Membrane ◽

Hela Cells ◽

Type Iii Secretion ◽

Fluorescent Protein ◽

Host Cells ◽

Type Iii ◽

C Terminus ◽

Green Fluorescent

ABSTRACT ExoU, a phospholipase injected into host cells by the type III secretion system of Pseudomonas aeruginosa, leads to rapid cytolytic cell death. Although the importance of ExoU in infection is well established, the mechanism by which this toxin kills host cells is less clear. To gain insight into how ExoU causes cell death, we examined its subcellular localization following transfection or type III secretion/translocation into HeLa cells. Although rapid cell lysis precluded visualization of wild-type ExoU by fluorescence microscopy, catalytically inactive toxin was readily detected at the periphery of HeLa cells. Biochemical analysis confirmed that ExoU was targeted to the membrane fraction of transfected cells. Visualization of ExoU peptides fused with green fluorescent protein indicated that the domain responsible for this targeting was in the C terminus of ExoU, between residues 550 and 687. Localization to the plasma membrane occurred within 1 h of expression, which is consistent with the kinetics of cytotoxicity. Together, these results indicate that a domain between residues 550 and 687 of ExoU targets this toxin to the plasma membrane, a process that may be important in cytotoxicity.

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Transgenic Mice Expressing Green Fluorescent Protein under the Control of the Corticotropin-Releasing Hormone Promoter

Endocrinology ◽

10.1210/en.2009-0881 ◽

2009 ◽

Vol 150 (12) ◽

pp. 5626-5632 ◽

Cited By ~ 38

Author(s):

Tamar Alon ◽

Ligang Zhou ◽

Cristian A. Pérez ◽

Alastair S. Garfield ◽

Jeffrey M. Friedman ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Fluorescent Protein ◽

Artificial Chromosome ◽

Brain Regions ◽

Ingestive Behavior ◽

Physiological Processes ◽

Functional Relevance ◽

Green Fluorescent ◽

And Function ◽

The Brain

Abstract CRH is widely expressed in the brain and is of broad functional relevance to a number of physiological processes, including stress response, parturition, immune response, and ingestive behavior. To delineate further the organization of the central CRH network, we generated mice expressing green fluorescent protein (GFP) under the control of the CRH promoter, using bacterial artificial chromosome technology. Here we validate CRH-GFP transgene expression within specific brain regions and confirm the distribution of central GFP-producing cells to faithfully recapitulate that of CRH-expressing cells. Furthermore, we confirm the functional integrity of a population of GFP-producing cells by demonstrating their apposite responsiveness to nutritional status. We anticipate that this transgenic model will lend itself as a highly tractable tool for the investigation of CRH expression and function in discrete brain regions.

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