A Mitochondrially Targeted Nitroxide JP4-039 Protects and Mitigates against Total Body Irradiation Induced Hematopoietic Syndrome

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4721-4721 ◽  
Author(s):  
Michael W Epperly ◽  
Joshua G Pierce ◽  
Tracy Dixon ◽  
Darcy Franicola ◽  
Suhua Nie ◽  
...  
Keyword(s):  
Total Body Irradiation ◽  
Fold Increase ◽  
Linear Quadratic ◽  
Mitochondrial Fractions ◽  
High Concentrations ◽  
Total Body ◽  
Induced Apoptosis ◽  
In Vivo Effects

Abstract Nitroxides are efficient scavengers of free radicals and have been proposed for use as radioprotective agents. To circumvent the need for high concentrations for in vivo effects, and to target drugs to the mitochondria to prevent ionizing irradiation-induced apoptosis, we tested a family of compounds based on combining a hemi-gramicidin linker to a nitroxide. One small molecule (JP4-039) produced a 32-fold increased mitochondrial nitroxide localization in 32D cl 3 cells in vitro after one hour incubation at 37°C compared to the unlinked nitroxide tempo at 10uM. The cells were lyzed by rapidly freezing then thawing, followed by centrifugation to yield the nuclear, cytoplasmic, and mitochondrial fractions. EPR was used to quantitate nitroxide in each JP4-039 or tempo treated cell fraction. There was a 32-fold increase in the nitroxide signal for JP4-039 accumulation in the mitochondria compared to the nitroxide tempo. Irradiation survival curves were performed by incubating 32D cl 3 murine hematopoietic progenitor cells in JP4-039 or tempo (1 μM) for one hour before irradiation or by addition immediately following irradiation. The cells were irradiated to doses ranging from 0 to 8 Gy, suspended in methylcellulose-containing media, incubated for seven days at 37°C and colonies of greater than 50 cells were scored. The data was analyzed using linear quadratic and single-hit, multi-target models. Incubation of 32D cl 3 cells in JP4-039 for one hour prior to irradiation resulted in a significant increase in Do to 2.25 ± 0.11 Gy compared to 1.19 ± 0.13 Gy for 32D cl 3 cells alone or 1.32 ± 0.09 Gy for 32D cl 3 cells incubated in tempo (p=0.0042 or 0.0073, respectively). Similar results were achieved giving JP4-039 after irradiation with a Do of 1.97 ± 0.01 Gy (p < 0.0001). To test effectiveness in vivo, C57BL/6NHsd female mice were injected intraperitoneally with JP4-039 or tempo (10 mg/kg) 10 minutes before (or four hours after) total body irradiation to the LD 75/30 dose of 9.75 Gy. Mice were monitored and those moribund from the hematopoietic syndrome were sacrificed. Mice injected with 10 mg/kg JP4-039 before irradiation had a significant increase in survival of 60% compared to 28% for control irradiated mice (p = 0.0005) or 44% for tempo treated mice (p = 0.2252). JP4-039 given after irradiation had a survival of 53% at 30 days after irradiation (p = 0.0474). Thus, JP4-039, a mitochondrial targeted nitroxide, is an effective radioprotector, and mitigator.

Lycorine Modulates the Expression of p21 Via a p53-Independent Pathway in HL-60 Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4297-4297
Author(s):  
Jing Liu ◽  
Shu-Ling Wang ◽  
Lin Fang ◽  
Mao Ye ◽  
Zhi-Wei Sun ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Fold Increase ◽  
Alternative Pathways ◽  
Cyclin Dependent Kinase Inhibitor ◽  
M Phase ◽  
Induced Apoptosis

Abstract Abstract 4297 Leukemia is one of the most life-threatening cancers today, and acute promyelogenous leukemia is a common type of leukemia. We have previously shown that lycorine, a natural alkaloid extract from Amaryllidaceae, exhibited anti-leukemia effects in vitro and in vivo. Lycorine treatment of HL-60 cell arrested cell cycle at G2/M phase and induced apoptosis. In the present study, we sought to explore the molecular mechanisms for the anti-leukemia action of lycorine. Gene chip analysis revealed that lycorine treatment of HL-60 cells induced more than 9 fold increase of p21, a cyclin-dependent kinase inhibitor, whose expression is mainly regulated by p53. Since HL-60 cells are p53 null, the above findings suggest that lycorine activates p21 expression through p53-independent pathway. To further explore the alternative pathways for the activation of p21 induced by lycorine, we examined the effect of lycorine on the expression of Rb, pRb, E2F, c-Myc and HDACs which have shown to regulate p21 expression. We show that expression of pRb (ser780) and c-Myc was down-regulated, Rb and E2F were up-regulated, while the expression of HDAC1 and HDAC3 was not changed. Together these findings suggest that lycorine exerts its anti-leukemia effect by activating p21 expression via pRb/E2F and c-Myc pathways. Disclosures: No relevant conflicts of interest to declare.

Druggable Genome siRNA-Screening Identifies Glybenclamide as a Radioprotector against Total Body Irradiation

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 504-504
Author(s):  
Peter R McDonald ◽  
Jian Fei Jiang ◽  
Tracy Dixon ◽  
Darcy Franicola ◽  
Xichen Zhang ◽  
...  
Keyword(s):  
Total Body Irradiation ◽  
Survival Curve ◽  
New Drugs ◽  
Linear Quadratic ◽  
Cell Viability Assay ◽  
Sirna Library ◽  
Total Body ◽  
Druggable Genome ◽  
Sirna Screening

Abstract New drugs to protect or mitigate total body irradiation (TBI) damage are urgently required. To determine whether existing pharmacologic agents have this second function, we used siRNA to screen 5,520 druggable targets in the Silencer Druggable Genome siRNA library (Version 1.1, Ambion, Austin, TX). We sought to identify genes which if silenced protected against or mitigated radiation damage. T98G human glioblastoma cells were transfected with each target in the siRNA library in triplicate at a final concentration of 20nM/target in a one-gene, one-well format using DharmaFECT2 transfection reagent and 48 hours later were irradiated to 25 Gy. Viability was measured 72 hours later using the CellTiter-Blue cell viability assay. The siRNA screen was performed three times and a hit was scored as a gene, which when silenced, protected T98G cells by at least 15% in each of the triplicate screens. We identified 117 candidate protective genes. These were further analyzed using Ingenuity Pathways Analysis to identify potential radiation inhibitor signaling pathways and drug candidates. One pharmacologically attractive candidate was glybenclamide. Glybenclamide is a sulfonylurea currently used to treat diabetes that inhibits the ATP-binding cassette, sub-family A (ABC1) and selectively blocks ATP sensitive potassium channels. Glybenclamide (10 μM) was added to 32D cl 3 murine hematopoietic progenitor cells one hour before irradiation, or immediately following irradiation. Cells were then irradiated to doses ranging from 0 to 8 Gy, plated in methylcellulose, and incubated for seven days at 37° C in a humidified CO2 incubator at which time colonies of greater than 50 cells were counted. The data were analyzed using linear quadratic or single-hit, multi-target models. Glybenclamide pre-irradiation treated cells were protected from irradiation as demonstrated by an increased shoulder on the survival curve with a n of 34.9 ± 0.5 compared to untreated 32D cl 3 cells or cells treated with glybenclamide after irradiation with a n of 3.1 ± 1.3 or 2.5 ± 0.9 (p = 0.0018 or < 0.0001, respectively). To determine if glybenclamide was radioprotective in vivo, we dissolved the drug in 50% Cremophor EL and 50% ethanol and then diluted 1:4 in water. C57BL/6NHsd female mice were injected intraperitoneally with 5 mg/kg glybenclamide either 10 minutes before or 10 minutes after TBI to the LD50/30 dose of 9.5 Gy. Duplicate groups received sucrose supplement to counteract potential hypoglycemia. We found that 60% of mice injected with glybenclamide before irradiation survived for 35 days compared to 33% for the TBI alone group (p = 0.0361), while 44% of mice injected with glybenclamide following irradiation survived. There was no significant effect of sucrose supplement or difference between irradiated controls, mice injected with vehicle only or glybenclamide following irradiation. These results demonstrate the power of unbiased siRNA screening with a druggable genome and suggest that glybenclamide might be a clinically useful radioprotector.

In vitro and in vivo effects of increased concentrations of free fatty acids on free thyroxin measurements as determined by five assays

1990 ◽  
Vol 36 (4) ◽  
pp. 611-613 ◽  
Author(s):  
R Sapin ◽  
J L Schlienger ◽  
F Grunenberger ◽  
F Gasser ◽  
J Chambron
Keyword(s):  
Fatty Acids ◽  
Free Fatty Acids ◽  
The Other ◽  
In Vitro Experiments ◽  
Fat Emulsion ◽  
Before And After ◽  
High Concentrations ◽  
In Vivo Effects

Abstract To compare in vitro and in vivo effects of increased concentrations of free fatty acids (FFA) on free thyroxin (FT4) values, we measured FT4 in three pooled sera supplemented with oleate and in serum from 18 euthyroid patients before and after an infusion of fat emulsion (Intralipid). We used five FT4 RIA kits: two two-step methods [Gammacoat, Baxter (GC); Ria-gnost, Behring (RG)], two analog RIAs [Amerlex-M, Amersham (AM); Coat-Ria, BioMérieux (CR)], and one kit with labeled antibodies [Amerlex-MAB*, Amersham (AA)]. In vitro, at the maximum oleate addition of 5 mmol/L, FT4 increased when measured by the GC and RG kits, decreased by the AM kit, and showed no significant change by the CR and AA kits. In vivo, post-Intralipid, FFA concentrations rose significantly and the FT4 changes agreed with the results of the in vitro experiments, except for the RG kit, for which FT4 increased in only nine patients. We conclude that in vitro oleate addition is useful to predict the in vivo effect of increased FFA on FT4 values; moreover, in serum from euthyroid subjects with high concentrations of FFA, FT4 analyzed with the CR or AA kits should better agree with normal results for thyrotropin than FT4 values measured with the other kits.

Mass-Spectrometric Characterization of Phospholipids and Their Hydroperoxide Derivatives In Vivo: Effects of Total Body Irradiation

Lipidomics ◽  
2009 ◽  
pp. 153-183 ◽  
Author(s):  
Yulia Y. Tyurina ◽  
Vladimir A. Tyurin ◽  
Valentina I. Kapralova ◽  
Andrew A. Amoscato ◽  
Michael W. Epperly ◽  
...  
Keyword(s):  
Total Body Irradiation ◽  
Mass Spectrometric ◽  
Total Body ◽  
In Vivo Effects

scholarly journals Enterococcus faecalis exploits the human fibrinolytic system to drive excess collagenolysis: implications in gut healing and identification of druggable targets

2020 ◽  
Vol 318 (1) ◽  
pp. G1-G9 ◽  
Author(s):  
Richard A. Jacobson ◽  
Kiedo Wienholts ◽  
Ashley J. Williamson ◽  
Sara Gaines ◽  
Sanjiv Hyoju ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Healing Process ◽  
Abdominal Sepsis ◽  
Infectious Complications ◽  
Fibrinolytic System ◽  
Clinical Safety ◽  
High Concentrations ◽  
Clinically Significant

Perforations, anastomotic leak, and subsequent intra-abdominal sepsis are among the most common and feared complications of invasive interventions in the colon and remaining intestinal tract. During physiological healing, tissue protease activity is finely orchestrated to maintain the strength and integrity of the submucosa collagen layer in the wound. We (Shogan, BD et al. Sci Trans Med 7: 286ra68, 2015.) have previously demonstrated in both mice and humans that the commensal microbe Enterococcus faecalis selectively colonizes wounded colonic tissues and disrupts the healing process by amplifying collagenolytic matrix-metalloprotease activity toward excessive degradation. Here, we demonstrate for the first time, to our knowledge, a novel collagenolytic virulence mechanism by which E. faecalis is able to bind and locally activate the human fibrinolytic protease plasminogen (PLG), a protein present in high concentrations in healing colonic tissue. E. faecalis-mediated PLG activation leads to supraphysiological collagen degradation; in this study, we demonstrate this concept both in vitro and in vivo. This pathoadaptive response can be mitigated with the PLG inhibitor tranexamic acid (TXA) in a fashion that prevents clinically significant complications in validated murine models of both E. faecalis- and Pseudomonas aeruginosa-mediated colonic perforation. TXA has a proven clinical safety record and is Food and Drug Administration approved for topical application in invasive procedures, albeit for the prevention of bleeding rather than infection. As such, the novel pharmacological effect described in this study may be translatable to clinical trials for the prevention of infectious complications in colonic healing. NEW & NOTEWORTHY This paper presents a novel mechanism for virulence in a commensal gut microbe that exploits the human fibrinolytic system and its principle protease, plasminogen. This mechanism is targetable by safe and effective nonantibiotic small molecules for the prevention of infectious complications in the healing gut.

Pharmacology of 5-HT1A Receptors: Critical Aspects

CNS Spectrums ◽  
1998 ◽  
Vol 3 (10) ◽  
pp. 17-38 ◽  
Author(s):  
Franco Borsini
Keyword(s):  
Receptor Subtypes ◽  
Laboratory Conditions ◽  
In Vivo Effects ◽  
Critical Aspects

AbstractMyriad difficulties exist in analyzing the pharmacology of the serotonin 1A (5-HT1A) receptor. The receptor may demonstrate a different activity depending on the tissue or species used for analysis, the agent used, laboratory conditions, and differences between in vitro and in vivo effects of compounds. Affinity for 5-HT receptors also varies widely, presenting difficulties in drawing definitive conclusions on affinity values for various compounds. At least two possibilities exist to explain the diversity of pharmacology of 5-HT receptors. First, it is possible that different 5-HT1A receptor subtypes exist. Second, the 5-HT1A receptors may play a far more complex role than previously believed.

scholarly journals Collagen-Based Electrospun Materials for Tissue Engineering: A Systematic Review

2021 ◽  
Vol 8 (3) ◽  
pp. 39
Author(s):  
Britani N. Blackstone ◽  
Summer C. Gallentine ◽  
Heather M. Powell
Keyword(s):  
Systematic Review ◽  
Tissue Engineering ◽  
Collagen Type I ◽  
The Body ◽  
Pool Data ◽  
Type I ◽  
High Concentrations ◽  
Increased Strength

Collagen is a key component of the extracellular matrix (ECM) in organs and tissues throughout the body and is used for many tissue engineering applications. Electrospinning of collagen can produce scaffolds in a wide variety of shapes, fiber diameters and porosities to match that of the native ECM. This systematic review aims to pool data from available manuscripts on electrospun collagen and tissue engineering to provide insight into the connection between source material, solvent, crosslinking method and functional outcomes. D-banding was most often observed in electrospun collagen formed using collagen type I isolated from calfskin, often isolated within the laboratory, with short solution solubilization times. All physical and chemical methods of crosslinking utilized imparted resistance to degradation and increased strength. Cytotoxicity was observed at high concentrations of crosslinking agents and when abbreviated rinsing protocols were utilized. Collagen and collagen-based scaffolds were capable of forming engineered tissues in vitro and in vivo with high similarity to the native structures.

scholarly journals Exosomal Vimentin from Adipocyte Progenitors Protects Fibroblasts against Osmotic Stress and Inhibits Apoptosis to Enhance Wound Healing

2021 ◽  
Vol 22 (9) ◽  
pp. 4678
Author(s):  
Sepideh Parvanian ◽  
Hualian Zha ◽  
Dandan Su ◽  
Lifang Xi ◽  
Yaming Jiu ◽  
...  
Keyword(s):  
Wound Healing ◽  
Osmotic Stress ◽  
Mechanical Stress ◽  
Impaired Wound Healing ◽  
In Vivo Experiments ◽  
Adipocyte Progenitors ◽  
Osmotic Balance ◽  
Induced Apoptosis

Mechanical stress following injury regulates the quality and speed of wound healing. Improper mechanotransduction can lead to impaired wound healing and scar formation. Vimentin intermediate filaments control fibroblasts’ response to mechanical stress and lack of vimentin makes cells significantly vulnerable to environmental stress. We previously reported the involvement of exosomal vimentin in mediating wound healing. Here we performed in vitro and in vivo experiments to explore the effect of wide-type and vimentin knockout exosomes in accelerating wound healing under osmotic stress condition. Our results showed that osmotic stress increases the size and enhances the release of exosomes. Furthermore, our findings revealed that exosomal vimentin enhances wound healing by protecting fibroblasts against osmotic stress and inhibiting stress-induced apoptosis. These data suggest that exosomes could be considered either as a stress modifier to restore the osmotic balance or as a conveyer of stress to induce osmotic stress-driven conditions.

scholarly journals iPLA2β Contributes to ER Stress-Induced Apoptosis during Myocardial Ischemia/Reperfusion Injury

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1446
Author(s):  
Tingting Jin ◽  
Jun Lin ◽  
Yingchao Gong ◽  
Xukun Bi ◽  
Shasha Hu ◽  
...  
Keyword(s):  
Cell Death ◽  
Heart Disease ◽  
Myocardial Ischemia ◽  
Ischemic Heart Disease ◽  
Er Stress ◽  
Ischemia Reperfusion ◽  
Myocardial Ischemia Reperfusion ◽  
Induced Apoptosis

Both calcium-independent phospholipase A2 beta (iPLA2β) and endoplasmic reticulum (ER) stress regulate important pathophysiological processes including inflammation, calcium homeostasis and apoptosis. However, their roles in ischemic heart disease are poorly understood. Here, we show that the expression of iPLA2β is increased during myocardial ischemia/reperfusion (I/R) injury, concomitant with the induction of ER stress and the upregulation of cell death. We further show that the levels of iPLA2β in serum collected from acute myocardial infarction (AMI) patients and in samples collected from both in vivo and in vitro I/R injury models are significantly elevated. Further, iPLA2β knockout mice and siRNA mediated iPLA2β knockdown are employed to evaluate the ER stress and cell apoptosis during I/R injury. Additionally, cell surface protein biotinylation and immunofluorescence assays are used to trace and locate iPLA2β. Our data demonstrate the increase of iPLA2β augments ER stress and enhances cardiomyocyte apoptosis during I/R injury in vitro and in vivo. Inhibition of iPLA2β ameliorates ER stress and decreases cell death. Mechanistically, iPLA2β promotes ER stress and apoptosis by translocating to ER upon myocardial I/R injury. Together, our study suggests iPLA2β contributes to ER stress-induced apoptosis during myocardial I/R injury, which may serve as a potential therapeutic target against ischemic heart disease.

Application of the EYTEX™ System to the Evaluation of Cosmetic Products and their Ingredients

1992 ◽  
Vol 20 (1) ◽  
pp. 146-163
Author(s):  
Francis H. Kruszewski ◽  
Laura H. Hearn ◽  
Kyle T. Smith ◽  
Janice J. Teal ◽  
Virginia C. Gordon ◽  
...  
Keyword(s):  
Double Blind ◽  
Eye Irritation ◽  
Ocular Irritation ◽  
Rabbit Eye ◽  
Wide Range ◽  
High Concentrations ◽  
Alkaline Membrane ◽  
Positive Agreement

465 cosmetic product formulations and raw ingredients were evaluated with the EYTEX™ system to determine the potential of this in vitro alternative for identifying eye irritation potential. The EYTEX™ system is a non-animal, biochemical procedure developed by Ropak Laboratories, Irvine, CA, that was designed to approximate the Draize rabbit eye irritation assay for the evaluation of ocular irritation. Avon Products Inc. provided all the test samples, which included over 30 different product types and represented a wide range of eye irritancy. All the EYTEX™ protocols available at the time of this study were used. Samples were evaluated double-blind with both the membrane partition assay (MPA) and the rapid membrane assay (RMA). When appropriate, the standard assay (STD) and the alkaline membrane assay (AMA) were used, as well as specific, documented protocol modifications. EYTEX™ results were correlated with rabbit eye irritation data which was obtained from the historical records of Avon Products Inc. A positive agreement of EYTEX™ results with the in vivo assay was demonstrated by an overall concordance of 80%. The assay error was 20%, of which 18% was due to an overestimation of sample irritancy (false positives) and 2% was attributed to underestimation (false negatives). Overestimation error in this study was due in part to the inability of the protocols to accurately classify test samples with very low irritation potential. Underestimation of sample irritancy was generally associated with ethoxylated materials and high concentrations of specific types of surfactants. 100% sensitivity and 85% predictability were described by the data, indicating the efficiency of EYTEX™ in identifying known irritants. A specificity rate of 39% showed the EYTEX™ assay to be weak in discerning non-irritants. However, the EYTEX™ protocols used in this study were not designed to identify non-irritants. A compatibility rate of 99% proved the effectiveness of the EYTEX™ assay in accommodating a diversity of product types. The EYTEX™ system protocols, when used appropriately, can provide a conservative means of assessing the irritant potential of most cosmetic formulations and their ingredients.

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