bluuebunny/crossref_metadata_2025 huggingface dataset:
MicroRNA‐203 regulates cell junction and adhesion proteins by modulating Zeb1/Vimentin axis in breast cancer cells after PARP1 inhibition.
Authors: SHUKLA NIDHI , Rajawat Jyotika , Mishra Durga Prasad
Published: June 2021

PARP inhibitors are currently in trend to treat various type of cancers and they have shown promising results in homologous recombination deficient breast and ovarian cancers like those bearing BRCA1/2 mutations. There are many reports describing about the failure of PARP inhibitors due to resistance or unknown factors in clinical trials. It is crucial to look into in‐depth molecular mechanisms behind the role of PARP in cancer pathogenesis and long term effect of PARP inhibition. MicroRNAs (miRNAs) are small non‐coding RNAs, acting as post‐transcriptional regulators of gene expression and known to regulate variety of cellular processes, including the DNA damage response and regulation of cell junction and adhesion proteins. However, little is known about miRNAs mediated regulation of sensitivity to PARP inhibitors and its pathological significance in context to regulation of cell junction and adhesion proteins. Aberrant miRNA regulation of junction proteins is one of the leading causes of cancer pathogenesis. Keeping these views in consideration, we aimed to identify novel miRNAs involved in the regulation of cell junction and adhesion proteins in breast cancer malignancy after PARP inhibition. To investigate this, we performed miRNA microarray analysis of PARP inhibitor treated MDA‐MB‐231 cells and found upregulation of tumor suppressor miR‐203 after PARP inhibition. PARP1 inhibition led to reduced expression of vimentin and Zeb1 while ZO‐1, E‐cadherin and RhoGDI were upregulated in breast cancer cells. To confirm the whether PARP inhibitory effects were mediated by miR‐203, cells were transfected with miR‐203 mimic and anti‐miR. We observed that miR‐203 mimic resulted in inhibition of junction proteins Zeb1 and vimentin, coinciding with PARP inhibition. We also found that PARP inhibition caused reduced cell growth and proliferation of breast cancer cells which correlated with the overexpression of miR‐203. Our findings indicates that upregulation of miR‐203 due to PARP inhibition is involved in regulating cell junction proteins vimentin and zeb1 as well as cytosketelal RhoGTPase and thus regulating cell migration, invasion and proliferation. Taken together, these observations raise the possibility that these miRNAs could be used as biomarkers to identify patients that may benefit from treatment with PARP inhibitors.

Support or Funding Information

JR acknowledge the support from Department of Biotechnology (DBT) for DBT Bio‐CARe fellowship. NS acknowledges the support by the senior research fellowship from the Indian Council of Medical Research (ICMR). This work was supported by the grants from Department of Biotechnology (GAP0187) to DP Mishra.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inhibition of endothelial SCUBE2, a novel VEGFR2 co‐receptor, suppresses tumor angiogenesis
Authors: Lin Yuh‐Charn , Liu Chun‐Yu , Kannagi Reiji , Yang Ruey‐Bing
Published: June 2021

Objective

Signal peptide‐CUB‐EGF domain containing protein 2 (SCUBE2), expressed on the endothelial cell (EC) surface, functions as a novel co‐receptor for vascular endothelial growth factor receptor 2 (VEGFR2) and enhances VEGF‐induced signaling in adult angiogenesis. However, whether SCUBE2 plays a role in pathological angiogenesis and whether anti‐SCUBE2 antibody is an effective strategy for blocking tumor angiogenesis remains unknown. The aim of this study was to investigate the pathological role and targeting therapy of SCUBE2 in tumor vasculature.

Approach and Results

Immunohistochemistry revealed that SCUBE2 is highly expressed in ECs of numerous carcinomas. Genetic EC knockout of SCUBE2 and pharmacological inhibition with the anti‐SCUBE2 monoclonal antibody (mAb) SP.B1 significantly reduced xenograft tumor growth, decreased tumor vascular density, increased apoptosis and decreased the proliferation of tumor cells. Mechanistic studies revealed that SP.B1 binds to SCUBE2 and induces its internalization for lysosomal degradation, thereby reducing its cell‐surface level and inhibiting the binding of and downstream signaling of VEGF, including VEGFR2 phosphorylation and AKT/mitogen‐activated protein kinase (MAPK) activation. Importantly, dual combination therapy with anti‐SCUBE2 mAb and anti‐VEGF antibody or chemotherapy was more effective than single‐agent therapy.

Conclusions

EC‐surface SCUBE2 is a VEGFR2 co‐receptor essential for pathological tumor angiogenesis, and anti‐SCUBE2 mAb acting as an internalization inducer may provide a potent combination therapy for tumor angiogenesis.

Support or Funding Information

This work was supported by grants from Academia Sinica and the Ministry of Science and Technology (MOST) of Taiwan (VTA 106‐T‐6‐2, MOST 106‐2320‐B‐001‐017‐MY3, MOST 106‐0210‐01‐15‐02, MOST 105‐0210‐01‐13‐01, MOST 104‐0210‐01‐09‐02, MOST 104‐2320‐B‐001‐011‐MY3) to R.‐B.Y.
Defensin γ‐Thionin from Capsicum chinense Induces Apoptosis in the Human Breast Cancer Cell Line MCF‐7 and Regulate Histone H3 Epigenetic Modifications
Authors: Arceo‐Martinez Maria Teresa , Guzmán‐Rodríguez Jaquelina , Palomera‐Sánchez Zoraya , Ochoa‐Zarzosa Alejandra , López‐Meza Joel Edmundo
Published: June 2021

Cellular homeostasis is regulated by genetic and epigenetic mechanisms controlling cell proliferation and differentiation; dysregulation in these functions has been associated to cancer. Breast cancer is considered the first worldwide cause of cancer death in women. Antimicrobial peptides (AMPs) have showed cytotoxic activity against diverse cancer cells through different mechanisms; however, only the effects of animal AMPs have been well characterized. Therefore, the aim of this work was to determine the cytotoxic effect of the plant defensin γ‐thionin on MCF‐7 breast cancer cells and to characterize its mechanism of action.

The cytotoxic effect of γ‐thionin on MCF‐7 cells was assessed by trypan blue exclusion assays. This AMP was cytotoxic in a concentration‐dependent manner, with an IC50=117.29 μg/ml. Interestingly, viability of normal bovine mammary epithelial cells and peripheral blood mononuclear cells was unaffected. Additionally, this AMP induced apoptosis in MCF‐cells and did not affect the membrane integrity or cytosolic calcium. Further, γ‐thionin IC50 increased the expression of apoptosis related genes such as caspase 7, 8, 9, Apaf‐1 and cytochrome c. Transcriptional regulation has been linked to H3 epigenetic marks, for this reason, the global levels of H3 modifications were determined by western blotting. Data showed that γ‐thionin IC50 increases the mark of active transcription H3K9ac after 24 h of treatment. Intriguingly, the level of H3S10ph in cells treated with the defensin was higher at 12 h, which suggests an active proliferation mark. The mRNA expression of desacetylases HDAC1, HDAC2 and demethylase KDM4A was increased after 6 h of γ‐thionin treatment, while the expression of the kinase AURKB was induced at 12 h. Also, the expression of estrogen receptor gene (ESR1) was increased at 6 h and decreased at 24 h. Taken together, these results show that γ‐thionin induces apoptosis in MCF‐7 breast cancer cells and regulates changes in genetic expression, which could be associated with H3 epigenetic modifications. This is the first study of a plant AMP inducing cytotoxicity through epigenetic mechanisms in a hormone response breast cancer model.

Support or Funding Information

CIC‐UMSNH 14.5

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evidence of natural transcriptome regulation by cinnamon extract identified by changes in Akt1 mRNA levels of MCF‐7 breast cancer cells
Authors: Hill Matthew , Hall Seth , Almehmadi Seham , Lin Zachary , Aulthouse Amy , Kinder David , Stockert Amy
Published: June 2021

Research in recent years has focused heavily on different tumorigenesis mechanisms. One such mechanism is the anti‐apoptotic effects regulated by varying Akt isoforms. Akt1 signaling can result in nuclear exclusion and subsequent cytosolic degradation of key transcription factors. It is proposed that transcriptional regulation of Akt1 offers potential benefits for both preventative and adjunct therapy for cancer treatment. Many natural products including cinnamon contain high amounts of polyphenolic compounds that have been shown to regulate both signaling proteins as well as key transcription factors. Previous research in the lab has suggested that cinnamon extract treatment of MCF7 breast cancer cells leads to epigenetic changes, specifically methylation patterns. Preliminary experiments demonstrate potential methylation changes at the Akt1 gene that alter expression. Real‐time PCR was used to evaluate the expression levels in MCF7 breast cancer cells under cinnamon treatment conditions and vehicle. Duplicate cell sets were grown to allow separate isolation of both DNA and RNA. Cells were grown to 70–80% confluence and treated with either cinnamon extract or vehicle for 24 hours. Cells were collected at the 24 hour time point by trypsin release and stored at −80 degrees C until use. Analysis of mRNA levels of Akt1 suggest a change in Akt1 expression level that is proposed to result from changes in methylation. In order to better substantiate the potential change in Akt1 methylation, MethylLight ® was completed with Akt1 specific primers and results were compared to the observed mRNA levels. Further research will allow comparison of Akt1 location under these conditions as well as Akt1 activity.

Support or Funding Information

Departments of Pharmaceutical and Biomedical Sciences and Biological and Allied Health Sciences, Ohio Northern University

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Copper Binding Regulates Cellular Prion Protein Function
Authors: Nguyen Xuan T. A. , Tran Hoa T. , Cojoc Dan , Legname Giuseppe
Published: June 2021

The cellular prion protein (PrPC), mainly known for its role in neurodegenerative diseases, is involved in several physiological processes including neuritogenesis. By combining genomic approaches, cellular assays and focal stimulation technique, we have explored the molecular mechanism underlining PrPC function as a signaling molecule in neuritogenesis. Several recombinant prion protein (recPrP) mutants were obtained to treat primary hippocampal cultures in bulk or exposed near the hippocampal growth cones (GC) of single neurons in a local stimulation manner. While focal stimulation of GC with wild‐type recPrP induced neurite outgrowth and rapid GC turning towards the source, N‐terminal mutants fail to support this function.

In particular, the copper‐binding sites mutants present at the N‐terminus of PrPC are toxic to neurons indicating this region being crucial for the function of the protein. Mutants of recPrP including a key mutation for prion conversion H95Y (Giachin, Mai et al. 2015) or a GSS‐linked mutation abolish the function on neuritogenesis. Altogether, our findings indicate the functional regions for PrPC involved in neuritogenesis, suggest a potential link between loss‐of‐function of the protein and disease initiation.

Support or Funding Information

Fondo per gli Investimenti della Ricerca di Base (FIRB) program project (grant number RBAP11FRE9_001 to GL) from Ministero dell'Istruzione, dell'Università e della Ricerca(MIUR), Italy

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Spectral Sensitivity of Sensory Motor Integration of Gill Lateral Cell Cilia in the Bivalve Mollusc Crassostrea virginica
Authors: Buchanan Reniece , Jean‐Pierre Johanne , Catapane Edward J. , Carroll Margaret A.
Published: June 2021

Gill lateral cells of Crassostrea virginica are innervated by serotonin and dopamine nerves from their ganglia. Most bivalves have lateral cell cilia that respond to serotonin, which is cilio‐excitatory, and dopamine, which is cilio‐inhibitory. The motor aspects of gill lateral cell innervation have been well studied, but not the sensory side. Recently we found various sensory cues, including light, initiated a sensory‐motor integration response between mantle rim sensory tentacles and gill lateral cell cilia, via the visceral and cerebral ganglia. We also found that in animal preparations in which their shells removed, the cerebral ocelli (eyespots) responded to light causing lateral cell cilia to decrease beating rates. We hypothesize that light can penetrate oyster shell and stimulate cerebral ocelli to initiate a motor response of gill lateral cell cilia. In addition we hypothesize that if light does penetrate the shell, certain wavelengths will be more effective in stimulating sensory cells in ocelli and mantle rim tentacles. To test this we first measured light transmittance through shells using a Beckman DU800 spectrophotometer and found red light (650+ nm) produced the greatest degree of transmittance through the shell. Using animal preparations with intact gill to cerebral/visceral ganglia innervation we found that white light shown though the shell stimulated ocelli to initiate a motor response to slow down gill lateral cell cilia beating rates. Cilia beating was measured by stroboscopic microscopy and expressed as beats/sec ± sem. Then using different wavelengths of light (blue ‐ 405 nm, green ‐525 nm, amber ‐ 591 nm, red – 680 nm) we stimulated mantle rim sensory tentacles and cerebral ocelli with the shell in place. Under these conditions we found that only red light resulted in a sensory‐motor response that slowed lateral cell cilia beating rates. Cilia beating rates slowed from a basal rate of about 15 beats/sec to zero over a 20 minute period. These results were statistically significant and the other tested wavelengths had no significant effect on cilia beating rates. This study further demonstrates the integration of photosensory signals in the control of gill lateral cell cilia in the bivalve C. virginica, and adds new knowledge that demonstrates a spectral sensitivity of sensory cells in the cerebral ocelli and mantle rim tentacle involved with this sensory‐motor integration response.

Support or Funding Information

Supported by grant 690340047 of PSC‐CUNY, grant 2R25GM06003 of the Bridge Program of NIGMS and a Carnegie Foundation award.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Gene Expression in the Rat Thalamus following Chronic Neuropathic Pain Development: mRNA sequencing analysis
Authors: Clifford John L. , Kumar Raina , Srinivasan Seshamalini , Dmitrov George , Gautam Aarti , Walsh Andrianna , Workman Emily , Chavez Roger , Sosanya Natasha , Christy Robert J. , Hammamieh Rasha
Published: June 2021

Chronic pain afflicts roughly 44% of Service Members returning from Iraq and Afghanistan, and it is estimated that 100 million Americans suffer from this condition. The underlying molecular mechanisms of chronic pain development have remained elusive. The broad objective of this study is to identify molecular signals and indications of chronic pain in key brain regions during the onset and progression of neuropathic pain, in a rodent model. For this experiment, we collected tissue from five brain regions (thalamus, amygdala, cingulate cortex, insular cortex, and somatosensory cortex), as well as the L4–L6 spinal cord and L5 dorsal root ganglia, from rats subjected to the spared nerve injury (SNI) protocol, a model for chronic neuropathic pain. Tissue was collected at 1, 3, 7, 14 and 21 days post‐injury from each tissue and RNA extracted for mRNA sequencing (TruSeq total RNA protocol and Illumina HISCAN‐SQ platform). We present here the detailed mRNA sequencing workflow (pipeline) customized to provide the full complement of mRNA data (transcriptome) for multiple brain regions over the time course of the SNI protocol. In our initial analysis we focused on the assessment of the transcriptome of the thalamus, which is an important center of pain processing in the brain. We focused in particular on the 21 day time point, which is when the mechanical allodynia reaches its peak and therefore the most severe alterations in transcription are expected. In rats experiencing chronic pain relative to controls, we observed a reduction in the expression of several neurotransmitter receptors, such as CHRM4, CHRNB3, CHRNA5, CHRNB4, CNR1, ADORA2A, GPR85, BDKRB2, MC5R, DRD1, DRD2, and associated G proteins, such as GPR85, GNAT2, GNB3, GNG7, as well as the neurotransmitter TAC1. These findings suggest a global desensitization of neural signaling in the thalamus corresponding to chronic pain. Analysis of all time points and brain regions is underway and will provide a comprehensive picture of gene expression changes during the onset and progression of chronic pain.

Research was conducted in compliance with the Animal Welfare Act, and all other Federal requirements. The views expressed are those of the authors and do not constitute endorsement by the U.S. Army.

Support or Funding Information

This work was supported by funding from the United States Army Medical Research and Material Command.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Antimutator Activity of the Nudix Hydrolases from E. coli
Authors: Hynes Thomas William , O'Handley Suzanne Freeman
Published: June 2021

Enzymes of the Nudix Hydrolase superfamily are characterized by the ability to hydrolyze substrates containing nucleoside diphosphate linked to some moiety x, hence the acronym Nudix. Within the Nudix superfamily, MutT is an established antimutator. This functionality is a point of debate for some of the other members of the superfamily. Experiments to analyze antimutator activity were carried out on the 13 Nudix hydrolase E. coli knockouts. Overnight cultures of each E. coli knockout were grown and plated on LB‐agar containing Streptomycin or Nalidixic Acid, or diluted a million‐fold and plated on LB‐agar or LB‐agar containing Kanamycin (knockouts all contain Kanamycin resistance markers). After ~16 hours incubation at 37 °C, colonies were counted. The MutT knockout demonstrated the ability to gain antibiotic resistance to Streptomycin and Nalidixic Acid as observed by many viable colonies on all plates. The other Nudix hydrolase E. coli knockouts grew on LB‐agar and in the presence of Kanamycin but not in the presence of Streptomycin or Nalidixic Acid. These results indicate that MutT from E. coli is in fact an antimutator, as previously established, but the other Nudix hydrolases from E. coli are not antimutators. Complementation studies with Nudix hydrolases from M. tuberculosis will be carried out to determine which, if any, are antimutators comparable to MutT from E. coli.

Support or Funding Information

RIT College of Science FEAD grant (SOH)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Redox Regulation of FGF21 in an Obese “Stress‐less” Mouse Model
Authors: Roberts Aaron , Amos Debbie , Santanam Nalini
Published: June 2021

Background

Oxidative stress plays a key role in the metabolic syndrome including Type 2 Diabetes and Obesity. It is implicated that oxygen‐derived free radicals alter the function of metabolic tissues thus altering glucose and lipid homeostasis. Catalase is an antioxidant enzyme that helps to catabolize hydrogen peroxide generated by superoxide dismutation which subsequently reduces oxidative stress. The protein hormone Fibroblast Growth Factor 21 (FGF21) plays regulatory roles in lipid and glucose metabolism, and more recently, has been shown to be a novel regulator of oxidative stress giving it clinical significance in the context of Type 2 Diabetes and Obesity. FGF21 expression has been shown to increase under higher oxidative stress conditions.

Hypothesis

We hypothesized that excess catalase expression would deter oxidative stress mediated metabolic regulation in adipose tissue as exhibited by changes in FGF21 expression levels and insulin sensitivity.

Methods

To test this hypothesis, male control C57Bl6, Catalase transgenic (Cat‐tg) mice that expressed 3–4 fold excess catalase, as well as the newly engineered Bob‐Cat mice, which is a hybrid of Cat‐tg and the leptin resistant obese Ob‐Ob mice (study approved by MU IACUC), were fed normal chow (NC), 45% fish oil (OM3), or 45% high fat (HFD) diet for 8 weeks. Weekly body weights and food consumption were measured. ECHO‐MRI was used to analyze body fat and lean mass. HOMA‐IR was calculated based on fasting glucose and plasma insulin levels. Changes in mRNA expression of key genes regulating metabolic homeostasis (FGF‐21 and Nrf2) were measured in adipose tissue. ANOVA was used for statistical comparisons using GraphPad Prism.

Results

The HOMO‐IR data showed that the Cat‐tg mice became insulin resistant while on the HF diet, while the C57 and Bob‐Cat mice maintained their insulin sensitivity although fat mass increased on the high fat diet across all three genotypes. Gene expression data showed a significant increase in FGF21 and Nrf2 in the Bob‐Cat mice that were on the HF and OM3 diets. There was a negative correlation between Nrf2 and FGF21.

Conclusion

The results of the gene expression data and insulin homeostasis in the presence of excess antioxidants (catalase) show that proper redox balance is necessary for the signaling and function of FGF21. The correlation in FGF21 expression in the HF and OM3 diets of the Bob‐Cat and the maintenance of insulin sensitivity indicate that FGF21 acts to minimize the effects of poor nutritive conditions.

Support or Funding Information

Department of Biomedical Sciences, Joan C. Edwards School of Medicine, Marshall University, Huntington, WV

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Metabolic stress directs WNK kinases to aggresomes for pooling and degradation in the distal convoluted tubule of the kidney
Authors: Thomson Martin Nikolaus , Gallardo Catherina Cuevas , Bewarder Tim Michael , Ellison David , Mutig Kerim , Bachmann Sebastian
Published: June 2021

Objective

Renal salt retention due to excessive function of the renal NaCl cotransporter (NCC) along the distal convoluted tubule (DCT) plays a central role in the pathogenesis of hypertension. The Western type diet with high salt and low potassium content (HS/LK) has been shown to increase NCC activity by stimulating its phosphorylation via a kinase cascade comprising with‐no‐lysine kinases (WNKs) and a Ste20‐related kinase (SPAK). WNKs and SPAK have been shown to accumulate in perinuclear aggregates in response to potassium depletion, but the functional significance of these aggregates remained obscure. These structures may in part reflect a degradative pathway but may serve pooling functions as well.

Methods

Mice received HS/LK or control diets for 10 days. WNK1‐transfected DCT cells were exposed to vehicle, hyperosmotic stress, or potassium depletion. WNK1, WNK4, SPAK, phospho‐S383 SPAK, and various autophagy markers were studied by immunofluorescence and immunoblotting. Electron microscopy was applied for ultrastructural analysis.

Results

HS/LK diet induced accumulation of WNKs and SPAK in the form of perinuclear protein aggregates within DCT cells. These aggregates were rare in DCT of mice treated with control diet and virtually absent after administration of high potassium diet. The autophagy marker ATG5 was present in the aggregates and the 20S proteasome signal was intensified adjacently, whereas standard lysosomal markers showed no significant association with the punctate WNK/SPAK signal. Ultrastructural analysis of DCT in kidneys of mice treated with HS/LK revealed abundant aggresomes adjacent to rough endoplasmic reticulum and clathrin‐coated vesicles. Evaluation of cultured DCT cells transfected with WNK1 or KS‐WNK1 revealed formation of similar aggresomes in response to potassium depletion, whereas hyperosmotic stress induced formation of autophagosomes. Pretreatment of cells with proteasome inhibitor MG‐132 or autophagy/lysosome inhibitor bafilomycin A1 prior to potassium depletion substantially augmented the size of WNK1‐containing aggresomes.

Conclusion

Challenging of DCT epithelium induces the formation of perinuclear aggresomes which pool the components of the kinase cascade controlling NCC activity. From there, WNKs and SPAK may either be degraded via proteasomes or lysosomes, or reintroduced into the signal chain.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Methylenedioxypryovalerone (MDPV) Rapidly Increases Dopamine Transporter and Vesicular Monoamine Transporter‐2 Function
Authors: German Christopher L. , Magee Charlotte P. , Curtis Peter S. , Hanson Glen R. , Fleckenstein Annette E.
Published: June 2021

Methylenedioxypyrovalerone (MDPV) is a synthetic cathinone designer drug primarily abused for its psychostimulant properties. Rapid dysregulation of central monoamine neurotransmitter transport into presynaptic terminals and synaptic vesicles are principal mechanisms of psychostimulant action, and contribute to the development of persistent monoaminergic deficits. Still, the impact of in vivo MDPV administration on the function of central monoamine transporters is not fully elucidated. Consequently, this study examined the response of the striatal dopamine transporter (DAT) and vesicular monoamine transporter‐2 (VMAT2) to a single MDPV exposure in adult male rats. A single in vivo MDPV administration rapidly (within 1 h) and reversibly (effect diminishes after 6 h) increased both DAT and VMAT2 function when assessed ex vivo in striatal synaptosomes and non‐membrane‐associated (presumably cytoplasmic) synaptic vesicles, respectively. MDPV treatment did not increase VMAT2 immunoreactivity within the cytoplasmic synaptic vesicle fraction, suggesting increased VMAT2 activity is unlikely due to intracellular trafficking increasing the number of available cytoplasmic VMAT2‐associated synaptic vesicles. In contrast to other psychostimulants, most notably methamphetamine and methylphenidate, MDPV acutely increases both DAT and VMAT2 function, and increased VMAT2 function is likely independent of vesicular trafficking‐mediated mechanisms. Together, these data suggest that MDPV has a unique and undescribed mechanism of action on striatal dopaminergic neurons.

Support or Funding Information

This work was supported by National Institutes of Health grants DA039145 and DA031883.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The effects of lipid lowering drugs on neuroinflammation in metabolic syndrome mice
Authors: Hsieh Liang‐Po , Yeh Ching Hua
Published: June 2021

People get used to high‐calorie and refined dishes diet and lack of physical exercises with rapid modern lifestyles change. Those bad habits contribute to the increasing prevalence of metabolic syndrome. The peripheral chronic inflammatory responses are related to the adipose tissue accumulation and hyperlipidemia in the metabolic syndrome patients. Depression is an important comorbidity with metabolic syndrome. The inflammatory cytokines secreted by activated inflammatory cells in peripheral tissue would induce central neural inflammation. Chronic brain inflammation may be linked to depression. We hypothesize that depression occurrence in metabolic syndrome patients is due to the peripheral and central neural inflammation. We evaluated whether fenofibrate or Statin could improve depression‐like behavior in metabolic syndrome mice. The metabolic syndrome mice were induced by high‐fat‐diet. The depression‐like behaviors in metabolic syndrome mice were analyzed by tail suspension test, open field test and sucrose preference test. After 4‐week treatment of fenofibrtae and Statin, the depression‐like behavior in mice were analyzed and then sacrificed. The expressions of inflammatory cytokines in the brain of mice were detected by RT‐PCR. The activation of macrophage and microglia were analyzed using IHC stain. The results showed that body weight, TG, TC in serum and adipose tissue weight of mice were increased after high‐fat‐diet fed. That confirmed metabolic syndrome was induced successfully in the mice. In behavior analysis, fenofibrate decreased immobility times of tail suspension, increased number of line crossing of open field test, and increase sucrose intake of sucrose preference test indicating that PPARα activation may improve depression‐like behavior. The data showed that fenofibrate could improve depression level in metabolic syndrome mice.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Pharmacological Cathepsin C Inactivation Eliminates Proteinase 3, the Antigen in Autoimmune Vasculitis
Authors: seren seda , Dallet‐Choisy Sandrine , Marchand‐Adam Sylvain , Korkmaz Brice
Published: June 2021

Membrane‐bound proteinase 3 (PR3) is the main target antigen of anti‐neutrophil cytoplasmic autoantibodies (ANCA) in granulomatosis with polyangiitis (GPA), a systemic small‐vessel vasculitis. Binding of ANCA to membrane PR3 triggers neutrophil activation with the secretion of enzymatically active PR3 and additional granule proteins, thereby contributing to vascular damage. PR3 and related neutrophil serine proteases (NSP) are activated from proforms by the lysosomal cysteine protease cathepsin C (CatC) during neutrophil maturation. We hypothesized that pharmacological inhibition of CatC provides an effective measure to reduce intracellular and membrane‐bound PR3 and has therefore implications as a novel therapeutic approach in GPA. We first studied PR3 in neutrophils from 21 patients with Papillon‐Lefèvre syndrome (PLS), a genetic form of CatC deficiency. PLS neutrophil lysates showed a largely reduced, but still detectable (0.5–4%) PR3 activity when compared to healthy control cells. The PR3 membrane expression on activated PLS neutrophils was diminished by approximately 80% as shown by flow cytometry. We then explored the effect of a potent cell permeable nitrile CatC inhibitor on PR3 expression in normal neutrophils using a CD34+ hematopoietic stem cell model. Human CD34+ hematopoietic stem cells were treated with a CatC inhibitor during neutrophil differentiation over 10 days. We observed complete abrogation of membrane PR3, cellular PR3 protein and proteolytic PR3 activity whereas neutrophil differentiation was not compromised. Our data suggest that pharmacological CatC inhibition could provide an attractive novel treatment strategy for PR3‐ANCA associated autoimmune vasculitis that should be explored further.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Role of Cytoplasmic Deacetylase, SIRT2, in Stabilizing Lung Endothelial Barrier Function
Authors: Yang Xiaoyan , Yang Deying , Chen Zhang , Komarova Yulia
Published: June 2021

Lung microvascular endothelial functional and structural integrity is essential for maintenance of tissue‐fluid homeostasis and normal function of the respiratory system. Disruption of the pulmonary endothelial barrier leads to pulmonary edema, acute lung injury, and respiratory failure. Adherens junction (AJ) proteins, actin cytoskeleton, and microtubules are major components required for maintenance of barrier integrity. Post‐translational protein modifications of AJ constituents, such as phosphorylation, nitration, and nitrosylation, have been well known to regulate endothelial barrier function. More recently, lysine acetylation/deacetylation has emerged as an important and physiologically significant posttranslational protein modification in physiological and pathological conditions. However, its role in endothelial barrier function remains largely unknown. Sirtuins (SIRTs) are NAD+‐dependent deacetylases, and critical regulators of energy metabolism and oxidative stress response in different cell types. We studied the function of SIRT2, the only cytosolic sirtuin family member, in regulating pulmonary endothelial barrier function during inflammatory response. We showed that SIRT2 is the most abundant sirtuin in human lung endothelial cells. Importantly, SIRT2 protein level is significantly increased in lung endothelial cells upon challenging mice with a sublethal dose of lipopolysaccharides, a gram‐negative bacteria endotoxin which causes systemic inflammatory responses resulting in acute lung injury. Increase in SIRT2 protein level was also observed in human lung microvascular endothelial cells (HLMVEC) upon challenge with permeability‐increasing agents such as human α‐thrombin and TNF‐α, further emphasizing the potentially important role of SIRT2 in regulating adaptation of endothelial cells to inflammatory responses. To determine the role of SIRT2 activity in the maintenance of lung endothelial barrier function, we knocked down SIRT2 in HLMVECs and assessed changes in endothelial permeability upon challenge with α‐thrombin. We found that SIRT2 knock down significantly augmented α‐thrombin‐induced permeability increases as compared to control siRNA‐treated cells, suggesting that SIRT2 activity may play a critical role in regulating endothelial barrier integrity. Furthermore, treatment of endothelial cells with AGK2, a SIRT2‐selective inhibitor, significantly decreased assembly of endothelial junctions due to increased accumulation of VE‐cadherin in the cytoplasm. Our data suggest that SIRT2 plays a critical role in the maintenance of endothelial barrier function under basal conditions and upon inflammatory response.

Support or Funding Information

Xiaoyan Yang : T32HL007829‐23, AHA 17SDG33410608.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Effect of Beetroot on Vascular Contractile Response in Ovariectomized Rats
Authors: Deng Arlinda , Nguyen Huy , Longo Amanda B. , Ward Wendy , MacPherson Rebecca EK. , Di Wang Hui
Published: June 2021

Hypertension in women is known to peak dramatically following menopause, potentially attributable to the decreasing levels of sex hormones such as estrogen. Estrogen has regulating effects on nitric oxide (NO) bioavailability, a molecule which plays an important role in regulating blood pressure by dilating arteries. However, in post‐menopausal women estrogen therapy causes many adverse events, and an alternate treatment option is required. Beetroot (BR) is a plant that contains dietary nitrates that can increase blood NO levels. In this study, we tested the hypothesis that ingestion of BR will alter vascular tone and control blood pressure in an ovariectomized rodent model.

Twelve‐week old female ovariectomized (OVX) Sprague‐Dawley rats were divided to receive BR juice (containing 18.75 mg nitrates), nitroglycerin (NG) (0.2 mg/kg), estradiol replacement (1.5 mg) or no treatment for 12 weeks (n=4 per group). Isolated aortic rings were mounted in tissue baths and isometric contractile force was measured. Cumulative concentration‐response curves to phenylephrine (PE) (10−8 M to 10−5 M) and potassium chloride (KCL 120 mM) were generated to examine contractile function. Superoxide was measured by using chemiluminescence.

The aorta rings in the no treatment group developed highest contraction force in response to PE compared to aortic rings from the NG or estradiol‐treated groups (P<0.01). The BR‐treated group had a significantly reduced contractile force compared to no treatment, which was closer to NG and estradiol group levels (P<0.05). BR treatment did not reduce vascular contractions in response to KCL (P>0.05) but significantly reduced superoxide production (P<0.05).

These experiments suggest that dietary beetroot would be able to reduce oxidative stress and vascular contraction in response to PE, possibly through the production of nitric oxide.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Extracellular ATP promotes protein synthesis in skeletal muscle through activation of the Akt‐mTOR signaling pathway
Authors: Jiménez Camilo Morales , Calderón Manuel Arias , Hernández Nadia , Jaimovich Enrique , Radic Sonja Buvinic
Published: June 2021

Objective

Extracellular nucleotides are released from different cells surrounding skeletal muscle, like motoneuron, endothelial cells, fibroblasts and bone cells during exercise. Even the muscle fibers themselves release ATP during membrane depolarization and contraction. Could this ATP‐enriched environment modulate protein synthesys in skeletal muscle? In this study, we assessed if extracellular ATP leads to protein synthesis in adult muscle through activation of Akt‐mTOR signaling pathway.

Methods

Flexor Digitorium Brevis (FDB) muscles from 6–7 weeks‐old BalbC mice were dissected. Isolated muscles were stimulated with exogenous ATP in a time‐ and concentration‐dependent manner (3–20 min, 0.1 – 100 μM). The relative protein levels of p‐Akt(Ser473), p‐mTOR( Ser2448), p‐p70S6K(Thr389) and p‐4E‐BP1(Thr37/46) were evaluated by immunoblot. Normalization with the non‐phosphorylated protein and GAPDH was performed. The protein synthesis was estimated by the nonradioactive SUrface SEnsing of Translation (SUnSET) technique.

Results

100 μM ATP increased p‐Akt(Ser473) levels by 3‐fold in 10–15 min. Akt phosphorylation was concentration‐dependent, with a bell‐shaped curve that peaked in 3 μM ATP. Phosphorylation of Akt signalling‐related proteins (mTOR, p70S6K, and 4E‐BP1) and protein synthesis levels were also increased after 10–20 min with 3 μM ATP and UTP. Preincubation for 30 min with a general P2Y/P2X receptors antagonist (100 μM suramin), PI3‐kinase inhibitor (50 μM LY294002) or mTOR inhibitor (100 nM Rapamycin) abolished the effect of ATP on the protein synthesis.

Conclusion

These findings suggest that extracellular ATP promotes protein synthesis in skeletal muscle through the activation of P2YRs and the Akt‐mTOR signaling pathway. Considering that effect on protein synthesis was mimicked by UTP (P2Y2R/P2Y4R agonist), and that P2Y2R is the mostly expressed nucleotide receptor in FDB muscle (as we previously described), a relevant role of P2Y2R in controlling protein synthesis in skeletal muscles is proposed. This proposed pathway could have a fine‐tuned role in the regulation of muscle mass during the integrated musculoskeletal activity.

Support or Funding Information

Funded by Fondecyt‐1151353(SB)‐1151293(EJ)‐CONICYT‐PCHA‐63140009(CM)‐21151035(MA‐C).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cerebral Hyperperfusion and Metabolic Regulation in Response to Hypoxia: Do ATP‐Sensitive Potassium Channels Play a Role?
Authors: Alves Marcos Paulo Rocha , Campos Monique Opuszcka , Mattos João Dario , Mansur Daniel Elias , Rocha Helena , Secher Niels Henry , Nóbrega Antonio Claudio , Fernandes Igor Alexandre
Published: June 2021

ATP‐sensitive K+ channels (K+ATP) mediate hypoxia‐induced cerebral vasodilatation and hyperperfusion in animals, a compensatory mechanism that maintains the oxidative metabolic stability of the brain. We then tested whether K+ATP blockade attenuates the increase in cerebral blood flow (CBF) in humans and leads to a shift towards brain non‐oxidative metabolism during isocapnic hypoxia (IH). Seven men (25 ± 4 yrs.) were exposed to 5‐min of normoxia and IH (10% O2) before (BG) and 3 h after Glibenclamide oral administration (AG – 5 mg). Invasive mean arterial pressure (MAP), radial artery and right jugular venous blood saturation, oxygen (PO2) and carbon dioxide (PCO2) partial pressure, glucose and lactate levels, and internal carotid (ICA) and vertebral (VA) artery blood flow (BF, Doppler ultrasound) were quantified over the last 30 s. In another protocol, 5 male subjects (24 ± 4 yrs.) underwent the same procedures before (BPL) and 3 h after placebo ingestion (APL – 5 mg of starch). CBF ((ICA BF + VA BF)×2 (mL/min)), cerebral metabolic rate of O2 (CMRO2), oxygen‐glucose (OGI) and oxygen‐carbohydrate (OCI) indexes were determined in each trial. IH provoked similar arterial desaturation (ΔBG −18.3 ± 6.0 vs. ΔAG −17.9 ± 3.5%, p = 0.784) and PaO2 reduction (ΔBG −80.6 ± 4.8 vs. ΔAG −77.6 ± 9.0 mmHg, p = 0.351). MAP and PaCO2 remained unchanged under both conditions. Smaller increases in ICA (ΔBG +111 ± 94 vs. ΔAG +66 ± 68 mL/min, p = 0.021) and VA BF (ΔBG +44 ± 38 vs. ΔAG +26 ± 39 mL/min, p=0.035) were observed under K+ATP blockade. IH‐induced increase in CBF (ΔBG +310 ± 215 vs. ΔAG +184 ± 150 mL/min, p = 0.017) was attenuated and followed by a smaller increase in CMRO2 (ΔBG +18.9 ± 14.8 vs. ΔAG +4.5 ± 12.2 mmol/100 g/min, p = 0.015). There was no IH‐induced shift to cerebral non‐oxidative metabolism as OGI and OCI did not change. CBF and metabolic responses to IH did not differ between BPL and APL. Therefore, our findings indicate that K+ATP play a role in the human cerebral hyperemia and maintenance of the brain metabolic stability during isocapnic hypoxia.

Support or Funding Information

CNPq, CAPES, FAPERJ, FINEP

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Postnatal Development of Hypoxic and Hypercapnic Ventilatory Responses in Coturnix Quail
Authors: Pratt Ashley E. , Song Monata , Bavis Ryan W.
Published: June 2021

Newborn mammals generally exhibit a biphasic hypoxic ventilatory response (HVR) in which the initial increase in ventilation is followed by a progressive decline toward (or below) baseline; the magnitude of the secondary decline diminishes with age. Considerably less is known about postnatal development of the HVR in birds, although a recent study did not detect a biphasic HVR in nestling red‐winged blackbirds (Dzialowski et al., Am J Physiol 311:R1105–R1112, 2016). We used head‐body plethysmography to characterize the ventilatory responses of Coturnix quail chicks to 15‐min challenges of 11% O2 or 4% CO2 at 0–1, 3–4, and 6–7 days of age. The magnitude of the HVR increased with postnatal age (21±4, 35±3, and 42±4 % increase from baseline, respectively; mean±SEM, P<0.01). The HVR was biphasic at P0–1 and (to a lesser extent) at P3–4, but not at P6–7. In contrast, the magnitude of the hypercapnic ventilatory response decreased with age (76±8, 54±4, 31±3 % increase from baseline, respectively; P<0.001). The hypercapnic ventilatory response was characterized by a relatively sustained increase in ventilation in all age groups, suggesting that the biphasic ventilatory response is specific to hypoxia. Additional experiments assessed hypoxic hypometabolism, and decreased metabolic rate does not appear to explain the biphasic HVR in the youngest quail chicks. We conclude that newborn quail exhibit a biphasic HVR similar to that observed in newborn mammals.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Contributions of TRPA1, TRPV1 and ROS to Neuronal Activation Following Mitochondrial Modulation with Antimycin A
Authors: Stanford Katherine , Barannikov Ivan , Taylor‐Clark Thomas
Published: June 2021

Mitochondrial dysfunction is linked to inflammatory nociceptive disorders which involve sensory nerve hyperexcitability. Inflammatory signaling pathways including interleukin‐1beta and tumor necrosis factor alpha induce mitochondrial dysfunction, resulting in mitochondrial reactive oxygen species (ROS) production, mitochondrial depolarization, and calcium release which influence cellular signaling. However, it is not known how these mitochondrial signaling events may influence nociception. Transient receptor potential channels (TRP) Ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1) are selectively expressed in nociceptive subtypes of vagal, trigeminal and dorsal root afferents and play critical roles in inflammation‐associated nociception. However, the mechanism by which TRPA1 and TRPV1 contribute to nociception downstream of mitochondrial dysfunction is unknown. Our lab has previously demonstrated that Antimycin A, a mitochondrial electron transport chain complex III inhibitor, activates bronchopulmonary C‐fibers through the gating of TRPA1 and TRPV1. Here we evaluated the contributions of TRPA1, TRPV1 and ROS to neuronal activation during treatment with Antimycin A using live cell imaging of dissociated vagal mouse neurons and transfected HEK293 cells. We used calcium imaging (FURA‐2AM) to determine the activation of these non‐selective cation channels. Antimycin A selectively activated a portion of nociceptive wild‐type neurons while failing to activate non‐nociceptive neurons. With TRPV1 inhibition (iodoresiniferatoxin) or knockout the magnitude of calcium flux in responding neurons was unchanged while the percentage on neurons responding was decreased. Conversely, with TRPA1 inhibition (A967079) or knockout, the magnitude of calcium flux in responding neurons was decreased while the percentage of neurons responding was unchanged. Using both the TRPA1 and TRPV1 inhibitors together or a combination of knockout and inhibitor, responses to Antimycin A were abolished. In both TRPA1 and TRPV1 transfected HEK293 cells, a substantial portion of cells exhibited a significant increase in cytosolic calcium. To evaluate the contribution of ROS to the mitochondrial dysfunction‐evoked TRP channel activation, we scavenged ROS with dithiothreitol (DTT), a reducing agent. DTT diminished the calcium response in TRPA1 but not TRPV1 transfected HEK293 cells. In vagal neurons, MnTMPyP (cell permeable superoxide dismutase/catalase mimetic) and Tempol (superoxide dismutase mimetic) largely abolished the response to Antimycin A in TRPA1 knockout neurons. These data suggest that modulation of mitochondrial function activates nociceptive neurons via TRPA1 through a ROS‐mediated and TRPV1 through a ROS‐independent mechanism.

Support or Funding Information

National Heart Lung and Blood Institute

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.