bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cholinergic regulation of epithelial Na+ channel (ENaC) in alveolar type 2 cells.
Authors: Takemura Yoshizumi , Helms My N. , Self Julie , Ramosevac Semra , Eaton Douglas C. 
Published: June 2021

We and others have shown that epithelial Na+ channels (ENaC) in alveolar type 2 (AT2) cells are activated by β2 agonists, steroid hormones, elevated oxygen tension and by dopamine. Acetylcholine (Ach) receptors in bronchial smooth muscle are well known therapeutic targets in certain respiratory diseases, but there have been few reports on whether cholinergic agonists alter sodium transport in airway epithelial cells; therefore we investigated how cholinergic receptors regulate ENaC activity in primary cultures of rat AT2 cells and found that muscarinic agonists such as carbachol and oxotremorine activate ENaC. Rat AT2 cells were isolated by elastase treatment and were placed in primary culture. Cell attached‐patch clamp recording were used to investigate ENaC activities measured as the number of channels per patch times the open probability, NPo.Results of western blotting suggested M1 and M2 receptors were present in rat AT2 cells, but nicotinic receptors were not. Carbachol and oxotremorine increased NPo of ENaC in a dose‐dependent manner but nicotine did not. Atropine almost completely blocked ENaC activation by carbachol or oxotremorine.Our results showed that ENaC can be activated in rat AT2 cells by muscarinic agonists acting through M1 and M2 cholinergic receptors.
Heat shock protein 72 stabilizes the mitochondrial membrane potential in the anoxia tolerant turtle (Trachemys scripta) neuron
Authors: Milton Sarah Louise , Kesaraju Shailaja 
Published: June 2021

This study was undertaken to determine if Heat Shock Protein 70 (Hsp72), which doubles in the anoxia tolerant freshwater turtle brain, provides protection against cell death. The turtle survives both extended anoxia and reoxygenation, through such protective responses as alterations in heat shock proteins and increases in adenosine and GABA, and by suppression of reactive oxygen species (ROS). Neuronal cell cultures were transfected with specific siRNA against Hsp72 and apoptosis investigated by Western blot. ROS production was measured by Amplex Red, and the mitochondrial membrane potential (MMP) by JC‐1. Hsp72 in vitro increased 2X over basal by 16h. SiRNA treatment knocked down Hsp72 by 60%. Cell death doubled in anoxic and A/R cells; the Bcl‐2/Bax ratio decreased from 1.2 in controls to 0.8 in knockdown cultures. Cyt C and caspase‐3 levels did not change, although AIF increased. H2O2 release though increased 6‐7‐fold upon reoxygenation compared to controls, and preliminary evidence suggests an organelle‐associated localization for Hsp72 which could be mitochondrial. The MMP does not recover in knockdown cells upon reoxygenation following anoxic depolarization, suggesting that in the anoxia tolerant neuron, Hsp72 does not prevent cell death by directly inhibiting the intrinsic apoptotic pathway but instead stabilizes the mitochondrial membrane itself.Research supported by an NIH R‐15 grant to SLM.
Preliminary studies of myosin isoforms in the masseter muscle of mice fed a soft diet
Authors: Druzinsky Robert E. , Adamczyk Agnieszka , Hennessy Sara 
Published: June 2021

Plasticity in myosin isoform expression resulting from increases and decreases in functional muscle activities is well known. Expression of myosin isoforms changes according to a rigid format (Schiaffino and Reggiani, 1996; Reader et al., 2001):I/alpha <‐‐‐‐> IIa <‐‐‐‐> IIx <‐‐‐‐> IIb. Adult mice were fed a soft diet (standard mouse diet in powdered form) for ten days as a model of disuse atrophy of the jaw closing muscles. Masseter muscles were dissected free, blotted dry, and snap frozen in isopentane cooled in liquid nitrogen. Ten micron sections were cut on a cryostat and stained using monoclonal antibodies to myosin heavy chain isoforms (BA‐G5, 10F5, 6H1, 2F7, SC‐71, A4.840). No fibers positive for A4.840 (Type I) were found in either control or experimental masseters. Although the masseter is composed entirely of type II fibers, some fibers are labeled by BA‐G5 (alpha). Preliminary results suggest that BA‐G5 expression is increased in the masseter muscles of animals fed the soft diet. Thus, the masseter shifts toward the type I phenotype without actually producing a true type I isoform. This result is in accord with Kitagawa et al. (2004) who found increased numbers of type IIa fibers in the masseter of rabbits fed a soft diet.Supported by a Governors State University Research Grant and NIH P20 MD001816.
TP508, a thrombin‐derived peptide, modulates TLR‐4 dependent cytokine expression in differentiated THP‐1 cells
Authors: Rousseau Emma , Smoke Chris , Zepp Jarod , Dinarello Charles A. , Sheller Michael R. 
Published: June 2021

Accelerated wound healing by TP508, a thrombin‐derived synthetic peptide, may involve inflammatory mediators. The mechanism of action for TP508 is hypothesized to be integrin binding at its RGD site. THP‐1 monocytes were differentiated to an adherent macrophage phenotype with 50 ng/mL phorbol 12‐myristate 13‐acetate (PMA). Cells were pretreated with TP508 for 30 minutes, followed by lipopolysaccharide (LPS) for 3.5 hours. TNFα, IL‐8 and IL‐1 β were analyzed via ELISA. TP508 decreased expression of TNFα and IL‐8 and increased IL‐1 β expression independently of LPS. When heparin‐sulfate was used to stimulate TLR4 receptors, TP508 again reduced TNFα expression.Given the peptide sequence of TP508, direct blocking of the TLR4 receptor is improbable, so the TP508 RGD site was explored for potential interaction with TLR4‐dependent cytokine stimulation. Variants of TP508 were used to test whether the RGD‐integrin binding was responsible for the reduction in cytokine expression in these cells. An αV β3 integrin blocking antibody was as effective in reducing LPS‐stimulated cytokine expression as the TLR4 activity‐blocking antibody. Treatment of cells with αV β3 integrin‐binding peptide GRGDSP also significantly reduced TNFα expression in response to LPS. This preliminary evidence suggests that TP508 may modulate cytokine expression through an integrin dependent mechanism.
The interplay between microRNA‐21 and endothelin receptor B (EDNRB) in gastric cancer
Authors: David Stefan , Olaru Alexandru V , Paun Bogdan C , Jin Zhe , Meltzer Stephen J. , Selaru Florin M 
Published: June 2021

BackgroundMicroRNAs (miRs) are short noncoding RNA species that bind to mRNAs reducing the expression of corresponding proteins. Endothelins (ET 1‐3) are involved in vasoconstriction, angiogenesis, mitogenesis and apoptosis acting through the endothelin receptors EDNRA and EDNRB.Methods7 normal gastric (N) and 3 gastric cancer (GC) tissues were used for microRNA arrays. Global miR profiles were analyzed by Significance Analysis of Microarrays. MiR results were confirmed by qRT‐PCR on additional 72 gastric tissues. TargetScan identified EDNRB as a putative miR‐21 target. The EDNRB expression was evaluated by qRT‐PCR on all 82 gastric tissues. MKN28 GC cells were transfected with miR‐21 inhibitor (mir21i) and EDNRB level was assessed by western blotting.ResultsThe array data showed a 6‐fold miR‐21 upregulation in GC vs. N. qRT‐PCR confirmed the miR‐21 upregulation GC (p=0.011). qRT‐PCR also showed that EDNRB expression was 1.9 times lower in GC (p=0.015) vs. N, suggesting that EDNRB may function as a tumor suppressor, potentially downregulated by miR‐21. Confirming this hypothesis, miR‐21 inhibitor (miR21i) enhanced EDNRB protein in the MKN28 cells (Fig.1).Conclusionour data confirm miR‐21 overexpression in a large cohort of GC and indicate, for the first time, that EDNRB is a miR‐21 target in GC. Our findings suggest that miR‐21 plays an etiologic role in GC partly through EDNRB regulation. Lastly, EDNRB may deserve further study as a tumor suppressor in GC.
Apigenin protects HT22 murine hippocampal neuronal cells against ER stress‐induced apoptosis
Authors: Choi A Young , Kang Insug 
Published: June 2021

Endoplasmic reticulum (ER)‐stress is known to induce neuronal cell death and is involved in neurodegenerative diseases including Alzheimer's diseases, Parkinson diseases, and cerebral ischemia. Apigenin, a bioactive flavonoid, is shown to have anti‐tumorigenic and anti‐inflammatory properties in various cell types. In the present study, we investigated the effect of apigenin on ER stress‐induced apoptosis and action mechanism involved in murine HT22 hippocampal neuronal cells. Apigenin blocked apoptotic cell death of HT22 cells induced by ER stress inducers, thapsigargin (TG) and brefeldin A (BFA). We showed that apigenin reduced the expression levels of unfolded protein response (UPR) proteins including C/EBP homologous protein (CHOP) and glucose response protein 78 (GRP78) and phosphorylations of MAP kinases in TG or BFA‐treated HT22 cells. We also showed that TG or BFA increased intracellular ROS accumulation and apigenin blocked ROS accumulation induced by TG or BFA. Moreover, we found that TG or BFA decreased the mitochondrial membrane potential (MMP) and apigenin inhibited this decrease in MMP by TG or BFA. These results suggest that apigenin inhibits ER stress‐induced apoptosis by reducing ROS accumulation, mitochondrial dysfunction, and CHOP induction in HT22 hippocampal neuronal cells.This work was supported by grants from KOSEF of Korean Government.
Cerebrospinal fluid proteomics in tuberculous meningitis patients
Authors: KATARIA JITENDER , Rukmangadachar Lokesh Ankenahally , Hariprasad Gururao , Kaur Punit , Tripathi Manjari , Singh Tej P , Srinivasan Alagairi 
Published: June 2021

BackgroundTuberculous meningitis is a grave extra‐pulmonary complication of tuberculosis caused by Mycobacterium tuberculosis group of bacteria. Tuberculous meningitis comprise of ~5% of all cases of tuberculosis worldwide. The diagnosis is conventionally made by Acid fast bacilli (AFB) smear, culture and polymerase chain reaction (PCR) of cerebrospinal fluid for tb bacilli gene sequence. We used proteomic technologies to discover protein biomarkers for the disease.MethodsWe compared the cerebrospinal fluid of patients of tuberculous meningitis with normal healthy controls using proteomic technologies. All the patients were HIV negative and culture positive, while controls had no neurological deficits and no active tuberculosis. Cerebrospinal fluid samples were separated using 2D DIGE. Biological variations of the protein expression levels were analysed with DeCyder 6.5 software and evaluated for reproducibility and statistical significance. Differentially expressed proteins were identified by tandem mass spectroscopy.ResultsThe profile of differentially expressed protein revealed that between patients and controls serum albumin and its precursors, arachidonate 5‐lipoxygenase and immunoglobulin kappa chains are up regulated and apolipoprotein E and its precursor are down regulated in disease state.ConclusionsIdentification of differentially expressed proteins gives us the opportunity to understand the pathogenesis of tuberculous meningitis and these proteins can be potential biomarkers for non invasive diagnosis and prognosis of disease under treatment.The work has been supported from grant from Department of Biotechnology, Government of India to AS.
Characterization of genotype ‐ phenotype correlations in Adenylosuccinate Lyase (ADSL) Deficiency
Authors: Zikanova Marie , Skopova Vaclava , Hulkova Helena , Kmoch Stanislav 
Published: June 2021

ADSL deficiency is inherited disorder of purine metabolism affecting mainly central nervous system. Three forms differing in severity of symptoms and correlating negatively with ratios of accumulating SAdo/SAICAr in body fluids were established. The mechanisms leading to selective neuronopathy and underlying the clinical and biochemical heterogeneity are unknown.We analysed 22 patients, identified 16 ADSL mutations, expressed and characterized catalytic properties of recombinant wild type and mutant proteins, investigated expression patterns of ADSL in available tissues from 3 patients and correlated these data with patients' clinical and biochemical presentation.We found that phenotype severity corresponds with residual enzyme activity (calculated as a mean of homoallelic activities), but the mutant enzymes displayed proportional decrease in activity to both of its substrates, so no ground for the varied SAdo/SAICAr ratios was found.Western blot and immunohistochemistry analyses showed in patients generally reduced amounts of ADSL in investigated tissues.Our results suggest that selective affection of central nervous system is caused by toxic effects of accumulating SAdo‐SAICAr. Phenotype's heterogeneity may be explained by differential activity of metabolite transport across blood‐brain barrier in individual cases.This work was supported by grants GACR 301070600 and MSM CR 20620806.
How Neurons Internalize Alzheimer's Disease Amyloid β Protein
Authors: Worthy Patrice N , Olenych Scott G , Fulzele Smita , Davidson Michael W , Poduslo Joseph F , Kandimalla Karunya K 
Published: June 2021

ObjectiveThe objective is to investigate cellular mechanisms mediating the internalization of beta‐amyloid 40 (Aβ40) in part of the neurovascular unit, neurons, and blood‐brain barrier (BBB) endothelial cells.MethodsLocalization of fluorescein labeled Aβ40 (F‐Aβ40) and other endocytotic markers in wild type (WT) mouse brain slices, PC12 cells, rat primary hippocampal (RPH) neurons, and bovine brain microvascular endothelial (BBME) cells was investigated using laser confocal or fluorescence microscopy. The uptake of fluorophores in these cells was quantified by flow cytometry.ResultsLaser confocal micrographs of WT mouse brain slices treated with F‐Aβ40 demonstrated selective accumulation of the protein in cortical and hippocampal neurons via non‐saturable, energy independent, and non‐endocytotic pathways. A significant portion of internalized Aβ40 is located outside of the endosomal or lysosomal compartments, which may accumulate without degradation. In contrast, BBME cells exhibit energy dependent uptake of Aβ40, and accumulate the protein in acidic cell organelles, indicative of endocytotic uptake.ConclusionThe difference in internalization of Aβ40 between neurons and BBB endothelial cells may provide clues to understanding how various cells can regulate Alzheimer's disease (AD) proteins and help explain the vulnerability of cortical and hippocampal neurons to AD toxicity.
Insulin‐mediated redox regulation of cyclic AMP‐dependent protein kinase (PKA) in rat adipocytes
Authors: Riveros‐Rosas Héctor , Zentella de Piña Martha , Vázquez‐Meza Héctor , Pardo Juan Pablo , Rendón Juan Luis , Villalobos‐Molina Rafael , Piña Enrique 
Published: June 2021

Lipolysis and lipogenesis are two highly regulated pathways. Catecholamines promote lipolysis via cAMP, whereas insulin stimulates lipogenesis. H2O2 mimics insulin action inhibiting lipolysis, and insulin stimulates NADPH oxidase in adipocytes and transiently generates H2O2. Therefore, the possibility that H2O2 might regulate cAMP‐dependent protein kinase (PKA) was explored.Results show that H2O2 generated by insulin in rat adipocytes impaired cAMP‐mediated amplification cascade of lipolysis. Micromolar concentrations of H2O2 added before cAMP suppressed cAMP activation of both type IIß or type IIα cyclic AMP‐dependent protein kinase (PKA) holoenzyme. However, H2O2 was ineffective on reconstituted PKA holoenzyme preincubated with cAMP, or if added to the catalytic αsubunit alone. The same results were obtained if the catalytic αsubunit was substituted by its C199A mutant. H2O2 inhibition of PKA activation remained after H2O2 elimination but was reverted with thioredoxin. Electrophoresis of holoenzyme in SDS gels showed separation of catalytic and regulatory subunits after cAMP incubation but a single band after H2O2 incubation. These data strongly suggest that H2O2 promotes the formation of an intersubunit disulfide bond, impairing PKA activation. Phylogenetic analysis showed that Cys‐97 is conserved only in type II (but not type I) regulatory subunits. Supported by DGAPA‐UNAM grant IN208308
Searching for binding partners for the phosphorylase kinase variant PhK γ181
Authors: Polireddy Kishore , Rice Nancy A. 
Published: June 2021

Phosphorylase kinase (Phk), a key regulatory enzyme in glycogenolysis, is a hexadecameric protein that contains 4 different subunits with a stoichiometry (αβλδ)4. Alpha and βare regulatory subunits, γis catalytic and d is an intrinsic molecule of calmodulin. The gene which encodes for the γ subunit, PHKG1, undergoes alternative processing to produce a truncated form of γcontaining 181 residues (γ181) instead of the normal 387 residues. Although much of the kinase helical domain is absent, the truncated form is still catalytic with its activity being influenced by PKC, and its mRNA is expressed in high levels in brain. A natural substrate for γ181 has not yet been identified. In the present study, we are using the LexA based yeast two‐hybrid system to identify binding partners for γ181. Gamma181 was cloned into the pLexA vector and used as bait to screen a human fetal brain cDNA library. Interactions between γ181 and cDNA library proteins were monitored by reporter genes. Characterization of the bait, LexA‐γ181, shows that it is efficiently localized to the nucleus and does not activate the reporter genes. The cDNA library was amplified, titered, and then transformed into EGY48 yeast cells for screening. Following screening for leucine prototrophy and β‐galactosidase activity, we obtained 36 colonies that show positive interactions for leucine prototrophy although only 11 of these colonies are β‐galactosidase positive. Positive clones are currently being sequenced for identification. The discovery of novel binding partners for γ181 is critical in determining the physiological role of this unique kinase in brain.The work was supported in part by NIH grant P20RR16481.
Chitosan nanoparticles for siRNA‐mediated gene knockdown to promote neuronal regeneration
Authors: Mittnacht Ursula , Hartmann Hanna , Andersen Morten , Hein San , Howard Ken , Kjems Jorgen , Schlosshauer Burkhard 
Published: June 2021

Axons in the adult vertebrate central nervous system (CNS) exhibit almost no regeneration after injury, partly because of the presence of neurite outgrowth inhibitors in the CNS. The aim of this study is to enhance functional recovery and axon regrowth using siRNA. Thereby, we want to transiently inhibit the intracellular signalling pathway of central components into which various repulsive inputs converge. An effort concerning this objective is to find an appropriate carrier matrix for binding, stabilization, and promoting cellular uptake of siRNA.Chitosan is a biocompatible and biodegradable material which enables a strong electrostatic binding of oligonucleotides. In this study we present the effective binding of siRNA to chitosan under formation of nanoparticles. Analysis of different kinds of chitosan‐siRNA nanoparticles using gel‐retardation assays demonstrated high stability of those complexes abrogated only after addition of competing agents. Cell transfection experiments with fluorescently labelled siRNA showed efficient uptake of chitosan‐siRNA nanoparticles into different cells which play an important role in the nervous system.Our results therefore highlight the potential application of chitosan in siRNA‐mediated gene knockdown to promote neuronal regeneration after spinal cord injury.This project is supported by the Landesstiftung Baden‐Württemberg.
Inhibition of platelet aggregation by capsaicin is not mediated by the TRPV1 receptor
Authors: Nelson Richard A. , Daanen Jerome F. , King Andrew J. , Kort Michaele E. , Kym Philip R. , Lubbers Nathan L. , Lynch James J. , Cox Bryan F. , Mittelstadt Scott W. 
Published: June 2021

Capsaicin is an agonist of the TRPV1 receptor which appears to play a role in the integration of the nociceptive response to a variety of noxious stimuli. In addition to neuronal effects, capsaicin can affect non‐neuronal tissue. Previous in vitro studies have demonstrated that capsaicin can inhibit the platelet aggregation response induced by collagen. However, these studies have not investigated if this occurs through the TRPV1 receptor. The purpose of the present study was to investigate if capsaicin induced platelet aggregation could be inhibited with A‐993610, a selective TRPV1 antagonist.MethodsVenous blood obtained from dogs was incubated with vehicle, capsaicin, and/or A‐993610 for 10 minutes followed by the application of collagen (2 µg/ml). An aggregometer was used to quantify the platelet response.ResultsCapsaicin caused a significant inhibition of the collagen‐induced platelet aggregation at concentrations of 10 & 30 µg/ml. A‐993610 alone had no effect on platelet aggregation. In addition, A‐993610 did not have an effect on the inhibition of platelet aggregation by capsaicin.DiscussionThe current results agree with previous findings demonstrating that capsaicin inhibits platelet aggregation. However, the effects of capsaicin on platelet aggregation are not mediated via the TRPV1 receptor since the inhibition could not be blocked by a specific TRPV1 antagonist.
Anti‐anxiety effects of β3‐adrenoceptor agonists may be due to selective enhancement of lateral paracapsular GABAergic interneurons in the basolateral amygdala
Authors: Silberman Yuval , Ariwodola Olesegun J , Yorgason Jordan T , Chappell Ann M , Weiner Jeff L 
Published: June 2021

Extensive research suggests that stress‐related stimuli increase norepinephrine (NE) levels in brain regions that regulate anxiety, like the basolateral amygdala (BLA). NE, via α and β adrenoceptor (AR) activation, is thought to increase BLA excitability, thus contributing to increased anxiety. Surprisingly, recent studies have shown that systemic β3‐AR agonist exposure decreases anxiety measures, suggesting that β3‐ARs may actually inhibit BLA excitability. Therefore, we tested the hypothesis that β3‐AR agonist‐mediated anxiolysis may be due to increased BLA GABAergic inhibition. We first examined the effects of BRL37344 (BRL, selective β3‐AR agonist) on BLA GABAergic synapses arising from local feedback and lateral paracapsular (LPCS) (feed‐forward) interneurons in rat BLA slices using whole‐cell patch methods. BRL (0.5‐10μM) selectively increased LPCS‐evoked IPSCs (eIPSCs) with no effect on local eIPSCs. BRL potentiation of LPCS eIPSCs was blocked by SR59230A (β3‐AR antagonist) or by including Rp‐CAMPS in the patch pipette solution. BRL had no effect on spontaneous IPSCs or LPCS paired‐pulse ratios. Finally, bilateral intra‐BLA injection of BRL significantly reduced anxiety‐like behaviors on the elevated plus‐maze and open‐field assay. Collectively, these data suggest that β3‐AR activation selectively enhances LPCS‐mediated BLA inhibition and that increased LPCS inhibition is sufficient to decrease anxiety‐like behaviors.Funded by NIH grants AA17039 and AA17056