bluuebunny/crossref_metadata_2025 huggingface dataset:
Oxidative stress and aldehyde generation by monoamine oxidase cause mitochondrial dysfunction in cardiac myocytes
Authors: Kaludercic Nina , De Mario Agnese , Paolocci Nazareno , Di Lisa Fabio
Published: June 2021

Monoamine oxidases (MAO) have been shown to play a major role in cardiac injury following ischemia/reperfusion or pressure overload. Mitochondrial dysfunction occurs in both conditions, yet there is no definitive proof that MAO activity directly jeopardizes mitochondrial function. We stimulated MAO activity with dopamine (DA) in neonatal rat cardiomyocytes, monitoring reactive oxygen species (ROS) production by Mitotracker Red and mitochondrial function with TMRM. DA administration induced a significant increase in mitochondrial ROS production (+50%, p<0.05) and MAO inhibitor pargyline completely prevented it (p<0.05). Mitochondrial membrane potential was unchanged in the same experimental conditions (100±0 in ctrl vs 106±4.5 % in DA‐treated cells). However, when cells were co‐incubated with DA and ATP synthase inhibitor oligomycin, membrane potential dropped by 50%. Pargyline offset this change suggesting that MAO activation induces mitochondrial dysfunction causing mitochondria to hydrolyze ATP. Furthermore, inhibition of aldehyde dehydrogenase (a DA catabolic enzyme downstream of MAO) in the presence of DA, also determined a 50% loss in membrane potential (p<0.05) and pargyline completely prevented this (p<0.005). Here we show that ROS and aldehyde intermediates deriving from MAO activity can directly target mitochondria leading to organelle dysfunction and eventually cell death.
Study for cancer suppression activity of Artemisia lactiflora
Authors: Cheng Jing‐Jy , Chen Chien‐Chih
Published: June 2021

Components from Artemisia lactiflora showed cancer cell cytotoxicity and also synergistically enhanced the effect of doxorubicin. Angiogenesis inhibition was also observed. During cancer cell progression, NotchIC (a cleavage form of Notch intracellular domain) activation and £]¡Vcatenin accumulation were observed. £]‐catenin regulates cell proliferation including cyclin D1 activation. More, Notch inhibition results in Bcl‐2 suppression and induces apoptosis. Activation of Notch also implicates in cancer angiogenesis by translocation of NotchIC to bind with CBF‐1 and regulates Angiopoietin‐1 and Tie2 expression in endothelial cells. Our results in H460 cells showed significant inhibition of £]‐catenin followed by a suppression of cyclin D1 expression, but increased E‐cadherin protein expression. Dose‐dependently inhibition of NotchIC and Bcl‐2 expressions were observed. AL‐EA treatment inhibited NotchIC, Ang‐1, and Tie2 expression in ECs which were related to angiogenesis progress. Pure compounds with the same skeleton structure showed differential inhibition on NotchIC expression. These results indicated AL might participate in attenuation of cancer progress and angiogenesis process.
A convenient, fast and improved immunoprecipitation solution –Magnetic Bead Antibody Conjugates
Authors: Liu Haizhen , Hatler Michele , Huryn Karyn , Liu Selena , Butler Ann Marie , Armstrong Luke
Published: June 2021

Traditional immunoprecipitation using protein A or protein G agarose beads has long been a popular research method. Recently, scientists have replaced the agarose beads with magnetic beads for easier retrieval of the immunoprecipitated sample. Millipore's new PureProteome magnetic beads offer easy sample retrieval and the added benefit of faster immunoprecipitation. Side‐by‐side comparisons of agarose and magnetic beads in an immunoprecipitation assay demonstrated that the use of PureProteome beads yielded equivalent results while reducing clean up time from minutes to seconds. The immunoprecipitation can be done with the following advantages comparing to the traditional Protein A agarose bead antibody conjugate. 1). High capacity, 2). Fast processing time and able to handle multiple samples (mid‐throughput), 3). Complete recovery, 4) No sample loss, 5). Consistent results, 6). Minimize the IgG light chain contamination in the subsequent immunodetection (western blot).
MODIFYING CELLULAR PROPERTIES USING ARTIFICIAL APTAMER‐LIPID RECEPTORS
Authors: O'Donoghue Meghan B , Xiong Xiangling , Tan Weihong
Published: June 2021

Proteins found on a cell's surface are crucial for cell recognition and signaling. These events are often precipitated by capture of proteins onto the cell surface by membrane receptors. Here we present a quick, one‐step method to add artificial receptors onto cell membranes. Aptamer‐lipid conjugates acting as artificial receptors quickly integrate into the cell membrane, allowing the cell to capture the aptamer's target protein on the cell membrane. Using streptavidin‐aptamer artificial receptors, fluorescently labelled streptavidin was reversibly captured on cell membranes. In addition thrombin modified cells, capable of clotting a buffer containing the thrombin substrate fibrinogen were made by functionalizing the live cells with thrombin‐aptamer artificial receptors. This technology can be translated to modifying a cell with any protein that has an aptamer.
Association of p53 and mdm‐2 Polymorphisms and Expression pattern with Breast Cancer in Saudi Arabia
Authors: Alshatwi Ali A , Hasan Tarique N , Shafi Gowhar , Al‐Hazzani Amal A , Alsaif Mohammed A , Lei Kai Y
Published: June 2021

p53 and its regulator mdm‐2 play a crucial role in maintaining genomic stability. Despite the importance of p53 in breast cancer development and progression, little is known about the association of functional SNPs in the p53 and mdm‐2 genes and breast cancer aggressiveness in Saudi population. The present study has assessed p53 and mdm2 transcripts based on genotype pattern in breast cancer patients to understand their relative expression profile. To investigate if p53 and mdm‐2 SNPs regulate their expression, we analyzed association between each SNP and their expression in breast cancer. We found association between SNP status and tumor p53 overexpression. 24 cases of the 50 cases for p53 SNP as having the CC, 15 were CT and 9 had TT genotype. Genotypes were correlated with p53 and mdm‐2 transcript levels to check their independent association. Genotype versus expression analysis has shown that p53 level was 9.65 fold, significantly over expressed in cases with CC genotype (13.81) as compared to CT and TT genotypes, which is 5.58 (p<0.03). Interestingly, p53 SNP is associated with mdm‐2 levels, with 7.68 fold higher expression of mdm2 levels with CC genotype when compared to TT and CT genotypes (3.47). Our results suggest that elevated levels of mdm2 contributes advantage to mdm2 mediated signal transduction events resulting in over expression of genes such as p53 in breast cancer of Saudi population.
DepoFoam® Bupivacaine (EXPARELTM) is Compatible Following Lidocaine: Pharmacokinetic Study in Mini‐pigs
Authors: Richard Brigitte M , Rickert Doug E , Doolittle Danette , Mize Amy , Liu Jason , Lawson Charles F
Published: June 2021

DepoFoam bupivacaine (DB; bupivacaine extended‐release liposome injection) is in development for prolonged postsurgical analgesia via wound infiltration (WI). Lidocaine with epinephrine (lido/epi) is commonly used for WI prior to surgery. DepoFoam product, DepoDur® can exchange its drug load with lido (ref. DepoDur® USPI) and we explored this potential with DB. Sixty (60) male Yucatan Swine were randomized into 20 groups (N=3/group). DB (2 or 4 mg/kg) and/or lidocaine HCl solution 1% or 2% (with epinephrine 1:200,000) were injected subcutaneously along a 5‐cm virtual incision line. Time intervals (5, 10, 20 or 40 minutes) between administration of lido/epi and DB were tested. Plasma concentrations of bupivacaine (bup) and lido were measured simultaneously by a LC‐MS/MS assay. Systemic exposure to lido was increased (AUC0–24hr by 48%; Cmax by 1,640%) when lido (4 mg/kg) was followed 5 minutes later by DB (4 mg/kg) compared to lido/epi alone. The systemic exposure to bup was increased (AUC0–24hr by 50 – 95%; Cmax by 67 – 1000%) when lido (4 mg/kg) was followed 5 or 10 minutes later by DB (4 mg/kg) compared to DB alone. While DB should not be admixed with lidocaine, this study shows that DB may be locally administered after at least 20 minutes following local administration of lido with minimal potential for a clinically relevant interaction. Source of Research

Support: Pacira Pharmaceuticals, Inc.
A STUDY ON CLINICAL PROFILE OF THE PATIENT PRESENTED WITH CEREBRAL VENOUS THROMBOSIS
Authors: nagarajan elanagan
Published: June 2021

Introduction

Purpose of this study was to evaluate the clinical profile of the patients with cerebral venous thrombosis and to assess the risk factors involved.

Methods

This study was a retrospective study performed at Sri Ramachandra Medical Centre, Porur, Chennai and a total of 25 patients were reviewed. Clinical features, mode of onset, predisposing factors and CT/MRI findings were noted. All patients who were diagnosed to be having cerebral venous thrombosis were included in this study.

Results

Mean age was 35.7 years (range, 15 to 70 years) with a male to female ratio of 13:12. Headache (84%) and convulsions (47%) were the most common clinical features. 44% of the patients had altered sensorium and 9.1% were comatose at the time of presentation. Focal neurological deficits were observed in 60% of the patients and hemiparesis was the common deficit, seen in 28% of the cases. Subacute presentation (52%) was the most common mode of onset followed by acute presentation (40%). Predisposing factors were identified in 72% of patients. Infection was identified as a risk factor in 28% cases. CT scans showed hemorrhagic infarcts in 32% of the patients. Superior sagittal sinus was the most frequent sinus involved, 68% had partial or complete occlusion of the superior sagittal sinus. There was no mortality in the present series.

Conclusion

This study reports that cerebral venous thrombosis in developing countries particularly in rural area are no different from developed countries and needs to follow similar prevention and management.
Ovarian Cycle‐Related Neurosteroid Regulation of GABA‐A Receptor Subunit Plasticity in the Hippocampus Subfields
Authors: Gangisetty Omkaram , Reddy Doodipala Samba
Published: June 2021

Progesterone (P) plays a key role in epilepsy via modulation of GABA‐A receptors in the brain. Seizures decrease in the mid‐luteal phase (high P) and increase during the perimenstrual period (low P) in women with epilepsy. However, the mechanisms underlying the menstrual cycle‐differences in seizure susceptibility are not clear. Here we tested the hypothesis that ovarian alterations in GABA‐A receptor plasticity contributes to seizure susceptibility. The TaqMan assay was used for quantification of GABA‐A (α1‐4, β2, γ2,δ) subunit mRNAs in the CA1, CA3 and dentate gyrus (DG) at different estrous cycle stages. The δ‐subunit was strikingly (4‐folds) increased during the diestrous stage than the estrous stage in the DG region. No such change was noted in the CA1 or CA3 regions. There was a marked increase in α1 and α2 subunits in the DG during diestrous stage. In contrast, no changes in levels of γ2 subunit expression were observed during the diestrous stage. The level of δ‐subunit was not affected by ovariectomy suggesting the role of ovarian cycle. The diestrous upregulation of δ‐subunit was undiminished in PR knockout mice, but it was significantly reduced in mice treated with finasteride, a neurosteroid synthesis inhibitor. These results suggest that GABA‐A δ subunit expression is regulated by ovarian P‐derived neurosteroids that control tonic inhibition and catamenial epilepsy. ** Supported by NIH grant NS051398 **
Nerve Growth Factor (NGF) and Its Receptors in Skeletal Muscle Signaling
Authors: Ettinger Keren , Mizrachi Shlomit , Marcinkiewicz Cezary , Nevo Yoram , Lazarovici Philip
Published: June 2021

The role of nerve growth factor (NGF) and its receptors in physiology of skeletal muscles has not been extensively studied. However, anti‐NGF transgenic mice expressed skeletal muscle damage resembling myopathy, implying that NGF plays an important role in muscle physiology. Our aim is to evaluate the role of NGF and its receptors in muscle, using C2C12 skeletal muscle cell line and wild type (WT) and homozygote dystrophic dy2J mice muscles.

A similar mRNA expression for NGF and it receptors p75NTR and α9β1 integrin was found in C2C12 myoblasts and myotubes, while mRNA for trkA receptor was absent. In addition, C2C12 myoblasts secreted NGF to the media in an amount of 60pg/ml/24h, while NGF was not secreted by C2C12 myotubes. NGF treatment induced ERK1/2 stimulation in C2C12, an effect blocked by VLO5 (a disintegrin with relative selectivity towards α9 integrin). We also characterized mRNA expression of NGF, p75NTR, trkA and α9β1 integrin in WT and dy2J mice. We propose that NGF induced signaling in muscle cells is mediated by coupling of p75NTR and α9β1 integrin, as previously reported for glioma cells. Clarification of the role of NGF in muscle pathophysiology may provide novel understanding on the interaction between neurotrophins receptors and integrins towards possible development of novel drugs for muscle diseases.
HYPOXIA/REOXYGENATION DIFFERENTIALLY MODULATES ASTROGLIAL‐SOLUBLE EPOXIDE HYDROLASE EXPRESSION
Authors: Shivachar Amruthesh
Published: June 2021

Stroke is the third leading cause of death in the United States. Although severe ischemic stroke kills neurons in the penumbra region, astrocytes appear to adapt to ischemic stroke by expressing hypoxia response elements, mainly hypoxia‐induced factor‐1a, vascular endothelial growth factor, and cytochrome P450 ‐mediated vasoactive epoxides. Our previous studies demonstrate that astrocytes metabolize epoxides by the soluble epoxide hydrolase (sEH). Here we investigated the mechanisms regulating sEH expression in rat and human astrocytes under hypoxia by measuring cell viability and expression of sEH. Hypoxia was induced by oxygen glucose deprivation (OGD) for 20h followed by reoxygenation (3, 6 and 24h). OGD‐induced cell death 3‐fold higher in human astrocytes as assessed by double staining of cells with fluorescent dyes Hoechst/Propidium iodide. Quantitative western blot analyses revealed that OGD down‐regulated sEH expression completely in rat astrocytes and only partially in human astrocytes. Reoxygenation reversed the expression of sEH to their normoxic controls in 24hr suggesting a role for sEH against OGD‐induced astrocyte death and survival. These results suggest that down‐regulation of sEH may be linked to astrocytes ability to adapt to ischemia and stroke and protect neighboring neurons (support: Institutional seed grant).
Characterization of D3 dopamine receptor signaling in a PD mouse model
Authors: Cote Samantha Rae , Kuzhikandathil Eldo V
Published: June 2021

The dopamine D3 receptor is known to couple to many signal transduction pathways and has been implicated in Parkinson's disease (PD). The D3 receptor exhibits a tolerance property wherein repeated stimulation elicits a progressively decreasing response. In vitro studies have shown that among others, the tolerance is observed in the D3 receptor‐Mitogen Activated Protein Kinase (MAPK) signal transduction pathway. PD patients on chronic levodopa treatment develop Levodopa Induced Dyskinesia (LID). In this study our objective was to test whether the D3 tolerance property exists in the 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) mouse model of PD. Drd3‐EGFP mice that report the expression of endogenous D3 receptor were used in this study. D3 receptor induced activation of the MAPK kinase pathway was determined in the striatum of control mice or mice treated chronically with levodopa, MPTP or MPTP + levodopa. MPTP treated mice exhibited increased MAPK phosphorylation when challenged with a D3 selective dose of PD128907. However, the MPTP group chronically treated with L‐dopa showed a decrease in phospho‐MAPK after administration of PD128907. These results suggest that in the mouse PD model chronically treated with levodopa, the tolerance property of D3 receptor might be involved.

Supported by: Parkinson's Disease Foundation, NIH (MH82376) and the FM Kirby foundation grants
Effect of maternal immune activation on progeny behavior in the Drd3‐EGFP mouse model
Authors: Li Ying , Kuzhikandathil Eldo
Published: June 2021

Offspring of women who develop infections while pregnant are at a higher risk of developing mental disorders. Several rodent studies have shown that maternal immune activation (MIA) results in altered behavior in the progeny. The progeny also exhibit dopaminergic dysfunction which includes changes in D1 and D2 dopamine receptor expression. Among D2‐like receptors, the D3 dopamine receptor (D3R) has the highest affinity for dopamine and many commonly used antipsychotics. Our long‐term objective is to use the Drd3‐EGFP mouse model (which reports the endogenous expression of D3R) to determine the effect of MIA on D3R expression and function in the progeny. To this end, in this study, our objective was to use the Drd3‐EGFP mice strain to determine the effect of MIA on the behavior of the progeny. The viral mimic, Polyriboinosinic‐polyribocytidilic acid (PolyIC), was administered to pregnant Drd3‐EGFP mice to induce immune activation. The behavior of the resultant offspring was assessed in several tests. We found that 35‐ and 70‐day old offspring of maternally infected mothers showed increased anxiety in the Open Field test, reduced working memory in the Novel Object Recognition test, and exhibited prepulse inhibition deficits. These results validate the MIA experimental model in the Drd3‐EGFP mice and will facilitate studies that will examine changes in D3R expression and function. This research is supported by grants from NIH (MH82376) and the FM Kirby foundation.
Mice Expressing Cocaine‐Insensitive Norepinephrine Transporters Display Supersensitive Cocaine‐Induced Locomotor Activity
Authors: Martin Bradley Joseph , Wei Hua , Thirtamara‐Rajamani Keerthi , Menon Aravind , Hadjiconstantinou Maria , Gu Howard H
Published: June 2021

Cocaine exerts its psychostimulant effects by inhibiting the function of the membrane transporters for dopamine, serotonin, and norepinephrine. Although the role of the dopamine transporter in cocaine addiction is well‐established, the role of the norepinephrine transporter (NET) is unclear. Our laboratory has shown that mutating amino acid residues F101C‐A105G‐N153T within transmembrane 2 of NET results in a functional NET that has a reduction in cocaine sensitivity (i.e. cocaine IC50 reduced by 37 fold). To explore the role of NET in cocaine addiction, we produced knockin mice expressing this mutant cocaine‐insensitive NET (NET‐CI mice). In this preliminary study, we used these animals to investigate cocaine‐induced locomotor stimulation and reward. NET‐CI mice had reduced locomotor activity in general as estimated in an open filed assay, but they were 50% more responsive to the locomotor stimulating effects of cocaine (10 and 20 mg/kg) than control mice. NET‐CI mice did not appear to show significant differences in cocaine reward‐related behaviors as measured by conditioned place preference. These data show that without NET inhibition cocaine's stimulating effects are enhanced while its rewarding effects appear to remain unaltered. The NET‐CI mice may offer an improved tool for studying the role of NET in cocaine addiction.
A Novel Peripheral CB1 Antagonist and Its Effect on Metabolic Parameters
Authors: Hung Ming‐Shiu , Vijayakumar Ramasamy S. , Lin Yinchiu , Chang Chun‐Ping , Yeh Yen‐Nan , Hsiao Wen‐chi , Chao Yu‐Sheng , Shia Kak‐Shan
Published: June 2021

Activation of cannabinoid receptor 1 (CB1), a G protein‐coupled receptor, mediates a positive energy balance by decreasing the energy expenditure, meanwhile enhances appetite and anabolic pathways via both central and peripheral actions. As the function of CB1 receptor favors the energy influx and antagonization of its physiological effect becomes a potential therapeutic target for treating obesity and metabolic diseases. However, prior studies have indicated the psychological side effects resulted from the central action of CB1 receptors, which limits the development of centrally acting CB1 antagonists. Thereof, identification of peripheral CB1 compounds is actively pursued. Compound 1 (IC50 = 25.8 nM, EC50 = 89.8 nM) is an orally active aryl alkynylthiophene analog. While administered at 20 mg/kg, it exhibited a unique profile with significant weight loss but failed to suppress food intake in rats. Very low brain/plasma (0.04) ratio of this compound denoted that it is less permeable to brain tissue. In addition, compound 1 at a dose as high as 50 mg/kg did not reverse CB1 agonist‐induced hypothermia and analgesia in mice, thus discriminated the central CB1‐dependent effects. Furthermore, compound 1 treatment to postprandial rats increased the level of plasma free fatty acids and ketone bodies which accompanied elevated rate of lipolysis and lipid oxidation. These results suggest that compound 1 is a peripheral acting CB1 antagonist and can be served as a promising lead compound for later drug development.
Venoarteriolar reflex and heart rate variability
Authors: Kurtev Angel
Published: June 2021

The venoarteriolar reflex is a local vasomotor response to a gravitational stimulus depending on both myogenic and neural mechanisms. The neural component is considered to be mediated by an axon reflex and as such should have little central effects. The afferent sensory input however may still reach systemic vasomotor regulatory centers and eventually result in a more generalized response. To evaluate such a possibility we studied heart rate variability while inducing a venoarteriolar reflex in the right arm. The reflex was elicited by lowering the arm 40 cm below heart level while the subject was in supine position after initial rest. To document the reflex a photoplethysmography was continuously recorded from the index finger of the same arm. Precordial single channel ECG was also recorded. Both recordings were obtained by using MP100 system, PPG100C transducer, and TSD200 and ECG100C amplifiers (Biopac Systems Inc). To obtain time‐frequency analysis of the heart rate variability we used AcqKnowledge 4.0 software and Matlab based software (HRVAS). All our subjects were young healthy adults. The results showed changes in the heart rate variability similar to those obtained by standard orthostatic tests, although not as pronounced and more variable. We conclude that even though the venoarteriolar reflex is a local phenomenon it is integrated within the overall cardiovascular regulation.
Modulation of N‐type calcium currents by presynaptic imidazoline receptor activation in rat superior cervical ganglion neurons
Authors: Chung Seungsoo , Ahn dukc‐sun , Kim Young Hwan
Published: June 2021

Presynaptic imidazoline receptors (Ri‐pre) are found in the sympathetic axon terminals of animal and human cardiovascular systems, and they regulate blood pressure by modulating the release of peripheral noradrenaline (NA). The cellular mechanism of Ri‐pre‐induced inhibition of NA release is unknown. We, therefore, investigated the effect of Ri‐pre activation on voltage‐dependent Ca2+ channels in rat superior cervical ganglion (SCG) neurons. Cirazoline (30 μm), an Ri‐pre agonist as well as an α‐adrenoceptor (Rα) agonist, decreased Ca2+ currents (I Ca) by about 50% in a voltage‐dependent manner with prepulse facilitation. In the presence of low‐dose rauwolscine, which blocks the α2‐adrenoceptor (Rα2), cirazoline still inhibited I Ca by about 30%, but prepulse facilitationwas significantly attenuated. This inhibitory action of cirazoline was almost completely prevented by high‐dose rauwolscine, which blocks Ri‐pre as well as Rα2. In addition, pretreatment with LY320135, another Ri‐pre antagonist, in combination with low‐dose rauwolscine, also blocked the Rα2‐resistant effect of cirazoline. Addition of guanosine‐5_‐O‐(2‐thiodiphosphate) to the internal solutions significantly attenuated the action of cirazoline. However, pertussis toxin did not significantly influence the inhibitory effect of cirazoline. Moreover, cirazoline suppressed Mcurrent in SCG neurons cultured overnight. Finally,ω‐conotoxin (ω‐CgTx) GVIA obstructed cirazolineinduced current inhibition, and cirazoline significantly decreased the frequency of action potential firing in a partly reversiblemanner.
Regulation of Arsenic Triglutathione [As(GS) 3] Transport by the Human Multidrug Resistance Protein 1 (MRP1/ABCC1) Through Post‐translational Modification
Authors: Shukalek Caley , Conseil Gwenaelle , Cole Susan PC , Leslie Elaine M
Published: June 2021

The 190‐kDa ATP‐binding cassette transporter, MRP1, is responsible for exporting a diverse array of substrates from the cell including glutathione conjugates. Arsenic, a high priority environmental carcinogen and clinical anti‐cancer agent, is a substrate of MRP1 in its trigluathione form [As(GS) 3]. We initially characterized As(GS) 3 transport by MRP1 using membrane vesicles isolated from a doxorubicin‐selected small cell lung cancer cell line (H69AR) and found it to be a high apparent affinity substrate (Km 0.3 μM). In the current study, we show that the Km of MRP1 for As(GS) 3 depends upon the cell line it is expressed in. Thus, wild‐type MRP1‐enriched membrane vesicles exhibit a >10‐fold difference in Km for As(GS) 3 (0.3 μM vs 4.4 μM) when prepared from transfected HeLa vs HEK293 cells, respectively. On the other hand, mutant MRP1 lacking all N‐linked glycosylation (sugar‐free) expressed in either cell line exhibits a low Km (0.3 – 0.6 μM). Furthermore, when prepared in the presence of phosphatase inhibitors, both wild‐type and sugar‐free MRP1‐enriched membrane vesicles from either HeLa or HEK293 cells exhibit a high Km for As(GS) 3 (3 – 6 μM). Together, these results suggest that the affinity of MRP1 for As(GS) 3 is influenced by cell type, phosphorylation, and glycosylation and elucidate potential implications for the relative detoxification of arsenic by different cell types and tissues.
Cav1.2 Ca2+ channel knock‐down alters colonic smooth muscle function
Authors: Duffey Michael E , Gugino Sylvia F , Russell James A , Kinne Laura N , Bett Glenna CL , Rasmusson Randall L
Published: June 2021

Timothy Syndrome (TS) arises from a point mutation in the voltage‐gated Cav1.2 L‐type Ca2+ channel in exon 8 (TS1) or exon 8a (TS2). We used a point mutation knock‐in technique to create a TS2 mouse (TS2 NEO) which contains the TS2 G406R mutation in exon 8a, and a NEO cassette‐induced Cav1.2 knock‐down. In vitro Topo cloning determined 17% of colonies expressed exon 8a (0% mutant) in distal colon of TS negative mice, and 30% of colonies (35% mutant) in TS2 NEO mice. To determine the effects of the mutation on function, we measured isometric force generation in cross sectional rings (circular muscle) or longitudinal strips (longitudinal muscle) of proximal colon using force‐displacement transducers. In circular muscle in Ca2+‐free, high‐K+, bath less tension was generated during a Ca2+ dose‐response in TS2 NEO mice compared to negative littermates. Electric field stimulation (EFS) of longitudinal muscle from TS negative mice produced frequency‐dependent relaxation followed by rebound contraction (EFS off), consistent with release of multiple neurotransmitters; muscle from TS2 NEO littermates produced a strong EFS off contraction, but only a weak relaxation response. TS2 NEO is a Cav1.2 knock‐down mouse, consistent with reduced Ca2+ entry into colonic circular smooth muscle and reduced enteric nerve‐stimulated relaxation of longitudinal smooth muscle. (DOD‐USAMRMC support)
Mitochondrial respiratory capacity is decreased in muscle fibers from tumor‐bearing mice
Authors: Gilliam Laura A. A. , Cathey Brook , Lin Chien‐Te , Pittman H. Keith , Verbanac Kathryn M. , Neufer P. Darrell
Published: June 2021

Debilitating muscle weakness and fatigue are common symptoms in cancer patients, the underlying cause of which is unknown. Compromised mitochondrial function can lead to persistent weakness in skeletal muscle. To determine if cancer alters skeletal muscle mitochondrial respiratory control/capacity and/or reactive oxygen species (ROS) production, C57BL6 male mice were injected with syngeneic Lewis lung carcinoma cells subcutaneously. Permeabilized fibers were prepared from the red gastrocnemius muscle 30 days post‐injection. Compared to controls, TB mice showed a significant (p<0.05) reduction in both complex I‐linked (mM: 5 glutamate/2 malate) basal (state 4) respiration (−54 ± 8 % change, mean ± SE) and maximal ADP‐stimulated (State 3) respiration (−51 ± 6 %, n=5). To examine the kinetics of complex I mediated respiration, we titrated in glutamate under maximal state 3 conditions. Surprisingly, the Km for glutamate was slightly decreased in TB mice (TB: 2 ± 9, CTRL: 4 ± 1, p<0.05), indicating improved sensitivity. In response to succinate titration during state 4 conditions supported by glutamate, preliminary data suggests mitochondrial H2O2 emitting potential is elevated in skeletal muscle from TB mice (+58 ± 23 %, n=4). Our findings suggest cancer compromises metabolic function in skeletal muscle, leading to a mitochondrial environment that increases the potential for electron leak and ROS generation.

Supported by DK 073488
Neuromodulation of the intrinsic cardiac nervous system attenuates the formation of neurally‐induced atrial arrhythmias
Authors: Ardell Jeffrey L. , Gibbons David D. , Southerland E. Marie , Armour J. Andrew
Published: June 2021

The objective was to determine whether neurally‐induced atrial fibrillation (AF) differentially activates intrinsic cardiac neurons (ICN) and whether electrical or pharmacological modulation of ICN activity can reduce the arrhythmogenic potential. Trains of 5 electrical stimuli (1ms) were delivered during the atrial refractory period to mediastinal nerves (MSNS) on the right atria at the pericardial reflection to evoked AF. Recordings of neuronal activity were obtained from right atrial ganglionated plexus (RAGP) in response to MSNS prior to and following neuromodulation involving either pre‐emptive spinal cord stimulation (SCS) or ganglionic blockade (hexamethonium). MSNS evoked a ~4‐fold increase in RAGP neuronal activity, which SCS reduced by 43%. Hexamethonium blocked the MSNS evoked increase in RAGP neuronal activation. MSNS evoked atrial fibrillation/flutter in 78% of right‐sided nerve sites stimulated, which SCS reduced to 33% and hexamethonium reduced to 7%. MSNS induced bradycardia was maintained with SCS, but mitigated by hexamethonium. We conclude that MSNS activates subpopulations of intrinsic cardiac neurons resulting in the formation of atrial arrhythmias leading to atrial fibrillation. Stabilization of ICN local circuit neurons with SCS or local circuit and autonomic efferent projections with hexamethonium reduces the arrhythmogenic potential. (HL71830)
PLCε‐dependent activation of TRPC6 channels in kidney podocytes, murine embryonic fibroblasts (MEFs) and human embryonic kidney cells (HEK 293): A general mechanism?
Authors: Dietrich Alexander , Kalwa Hermann , Mayer Tim , Fahlbusch Meike , Storch Ursula , Offermanns Stefan , Smrcka Allan , Hildebrandt Friedhelm , Gudermann Thomas
Published: June 2021

Gain of function mutations of TRPC6 as well as loss of function mutations in PLC ε were identified in patients suffering from focal segmental glomerular sclerosis (FSGS), a disease displaying increasing proteinuria due to a defect in the glomerular filtration process of the kidney. Along these lines, we have previously shown that PLC ε physically interacts and activates TRPC6 via production of diacylglycerol (DAG) in kidney podocytes to increase the barrier function of the glomerular slit diaphragm. Moreover, down‐regulation of G‐protein α13‐subunits significantly reduced ATII‐mediated TRPC6 activation, whereas siRNAs directed against G protein αq‐subunits did not. By employing Gαq−/− murine embryonic fibroblasts, we were able to dissect Gα12/13‐PLC ε‐mediated from Gαq‐PLCβ‐induced TRPC6 activation using lysophosphatidic acid (LPA) receptor stimulation. Most interestingly, application of LPA resulted in a significantly larger increase in the intracellular Ca2+ concentration ([Ca2+]i) in Gαq−/− cells expressing TRPC6 in comparison to Gαq−/− control cells. Therefore, Gα12/13‐mediated activation of PLC ε via RhoA leading to TRPC6‐induced cation influx might be a general, but commonly‐ignored signal transduction pathway in many cell types.
Structural basis of HLA‐DO inhibition of HLA‐DM catalyzed peptide exchange on MHC class II
Authors: Guce Abigail I , Mortimer Sarah , Karlsson Lars , Mellins Elizabeth , Stern Lawrence J
Published: June 2021

HLA‐DO (DO) is a non‐polymorphic, non‐classical MHC class II that is known to inhibit MHC class II peptide loading mediated by DM. The mechanism of inhibition by DO as well as the interactions between DO and DM remain unknown. Here, we present the 3D structure of the DM and DO complex determined by x‐ray crystallography at 3.2 Å resolution which is also the first resolved structure of DO. DO exhibits close structural similarity to the MHC class II molecule. The crystal complex shows DO residues mainly at the N‐terminal side of the α1 peptide‐binding groove interact with DM residues located at a concave surface. Some interactions are also observed involving DO residues from the IgG‐like domain. DM concave surface includes an acidic patch where residues in and surrounding this acidic patch and residues in the IgG‐like domain interact with DO. Interestingly, the interaction surface on DM to DO is spatially close to DM's interaction surface for DR that was suggested by molecular modeling and mutagenesis studies. The novel structure of the DM/DO complex presented here provides an insight on the possible mechanism of inhibition of DO on DM as well as the mechanism of how DM catalyzes peptide loading to peptide free DR.
The Spliceosome is a Dynamic RNP Machine
Authors: Guthrie Christine
Published: June 2021

Our long‐term goal is to understand at a molecular level the mechanisms responsible for the fidelity and specificity of mRNA processing, with a particular focus on the ATP‐dependent steps. We previously established that the spliceosome is a highly dynamic RNA‐protein machine that relies on RNA‐dependent ATPases of the so‐called DEAD‐box family to promote multiple conformational rearrangements. Recent advances showing that mRNA processing in vivo is largely, if not exclusively co‐transcriptional, have now broadened our perspective. Evidence will be presented that implicates important new parameters from our studies in budding and fission yeast, including the structure and modification states of chromatin, as well as the elongation rate of RNA polymerase II.
High throughput identification of compounds targeting influenza RNA dependent RNA polymerase activity
Authors: Su Ching-Yao , Wong Chi-Huey
Published: June 2021

We developed an antiinfluenza screening strategy for a HTS campaign against a large compound library. The screening of a large library resulted in the identification of several classes of unique compounds that appear to inhibit influenza at different targets. Two of the identified antiinfluenza compounds inhibit influenza RdRP activities by targeting different subunit proteins. The compound 3061 (FA‐2) interacts with the NP, while the other antiinfluenza inhibitor, 367, prevents PB1 functions. It is conceivable that RdRP could be targeted at many different sites. Optimization of these compounds could result in the development of new antiinfluenza agents.

Grant Funding Source: Academia Sinica
Characterization of E. coli mutants resistant to LpxC inhibitors
Authors: Zeng Daina , Zhou Pei , Raetz Christian R.H.
Published: June 2021

The emergence of multidrug resistant Gram‐negative bacteria requires the search for antibiotics with novel targets. One such target is lipid A, the anchor of lipopolysaccharide on the surface of Gram‐negative bacteria. Several potent and bactericidal inhibitors have been developed recently targeting LpxC, the deacetylase that catalyzes the second and committed step of lipid A biosynthesis. Here, we study mechanisms of resistance to these inhibitors by generating spontaneously resistant mutants that are ~200 fold resistant to all known LpxC inhibitors. These resistant mutants are isolated at a low rate and in two‐stages. All resistant mutants have two chromosomal point mutations that account for all resistance additively: one in thrS, the Thr‐tRNA ligase and the other in fabZ, a dehydrase in fatty acid biosynthesis that shares a common substrate with LpxA, the enzyme preceding LpxC in lipid A biosynthesis. The point mutations in thrS and fabZ decrease enzymatic activity. We speculate the mutation in thrS decreases protein synthesis and therefore slows cell growth, allowing the toleration of decreased lipid A biosynthesis, and the mutation in fabZ increases the amount of LpxC substrate that could compete with inhibitors. Understanding these resistance mechanisms has important implications in drug development and membrane homeostasis. This research was funded by N.I.H. grant GM51310 to C.R.H. Raetz.
Factors Associated with Low Water Intake among U.S. High School Students
Authors: Park Sohyun , Blanck Heidi M , Sherry Bettylou , Brener Nancy , O'Toole Terrence
Published: June 2021

Drinking water instead of sugar drinks is one approach for reducing energy intake. This cross‐sectional study examined associations of demographic characteristics, weight status, and behavioral factors with water intake among a nationally representative sample of high school students. The 2010 National Youth Physical Activity and Nutrition Study data for 11,049 students in grades 9–12 were used. The outcome measure was self‐reported water intake. Multivariable logistic regression was used to calculate adjusted odds ratio (OR) for variables associated with low water intake (<3 times/d). Nationwide, 54% of high school students reported drinking water <3 times/d. Variables significantly associated with a greater odds for low water intake were age ≤15 y (OR=1.1), consuming <2 glasses/d of milk (OR=1.5), regular soda ≥1 time/d (OR=1.6), other SSB ≥1 time/d (OR=1.4), fruits/100% fruit juice <2 times/d (OR=1.7), vegetables <3 times/d (OR=2.3), eating at fast‐food restaurants 1–2 d/wk and ≥3 d/wk (OR=1.3 and 1.4, respectively), and being physically active ≥60 min/d on <5 d/wk (OR=1.6). Being obese was significantly associated with a reduced odds for low water intake (OR=0.7). The findings of these associations of low water intake with poor diet quality, frequent fast‐food restaurant use, and physical inactivity may be used to tailor intervention efforts to increase water intake and to promote healthy lifestyles.
Spliceosome dynamics in the catalytic center
Authors: Tseng Chi-Kang , Liu Hsueh-Lien , Cheng Soo-Chen
Published: June 2021

Eight DExD/H‐box proteins are involved in the splicing pathway to mediate structural changes of the spliceosome. Among them, Prp2 and Prp16 are required for the first and second catalytic step, respectively. Recent studies have revealed the function of Prp2 and Prp16 to be associated with the displacement of SF3a/b and Yju2/Cwc25 from the spliceosome. SF3b and Cwc25 bind to the branch site at the pre‐catalytic and the first catalytic step during the splicing reaction. Their removal from the spliceosome may create an open space in the catalytic center, providing structural flexibility to allow repositioning of splice sites, which is facilitated by the binding of Yju2/Cwc25 and Slu7/Prp18/Prp22 for the first and second reaction, respectively. We have found that the affinity‐purified spliceosome arrested at specific stages is highly flexible in its structure and catalytic potentials. Not only both transesterification reactions are reversible, the spliceosome can also catalyze hydrolytic spliced‐exon reopening reaction (SER) and debranching reaction. The mode of the reaction catalyzed by the spliceosome is influenced by the pH, the presence of KCl, the concentration of divalent cation, and also by the structure of Cwc25 and Slu7 that binds to the spliceosome, suggesting that the spliceosome can be directed to catalyze versatile reactions by subtle changes in the conformation of the catalytic center.
Leucine and calcitriol modulation of human airway inflammation and hyper‐reactivity
Authors: Bruckbauer Antje , Biggerstaff John , Zemel Michael B
Published: June 2021

Obesity alters the balance of adipose tissue‐derived cytokines and increases airway inflammation and hyper‐reactivity. We previously found calcitriol and leucine to exert opposing effects on adipose tissue cytokine production, systemic inflammation, and monocyte adherence to vascular endothelial cells. We now propose that dietary calcium (via suppression of calcitriol) and leucine attenuate airway inflammation and reactivity by suppressing adipose tissue TNF‐α and enhancing adiponectin release. Human adipocytes were treated with calcitriol (10 nM) and leucine (0.5 mM) for 48 h and the conditioned medium (CM) were applied to human airway smooth muscle cells (HASMC). Calcitriol CM induced a 7‐fold increase in human leukocyte adhesion to HASMC (p<0.001) which was associated with increased TNF‐α receptor, VCAM and NFκB expression (~50%, p<0.02). In contrast, leucine CM decreased leukocyte adhesion (~50%, p<0.001) and NFκB (24%, p<0.02) and VCAM (21%, p<0.05) and increased adiponectin receptor 2 expression (29%, p<0.05). HASMC reactivity was measured as contractile responses to KCl and histamine under physiological flow and shear. Calcitriol CM augmented contractile responses to both agonists by 50 – 70% (p<0.001), while leucine inhibited the contractile response to KCl by 43% (p<0.001). These data suggest dietary calcium and leucine may suppress airway inflammation and reactivity.
Dose and time dependent effects of leucine or carbohydrate supplements to reduce AMPK and eEF2 phosphorylation and extend postprandial muscle protein synthesis in rats
Authors: Wilson Gabriel J , Layman Donald K , Moulton Christopher J , Garlick Peter J
Published: June 2021

These experiments evaluated the potential for supplemental leucine (Leu) or carbohydrates (CHO) to prolong elevated muscle protein synthesis (MPS) after a meal. Male Sprague‐Dawley rats (~256 g) were food deprived for 12 h, and euthanized 0, 90, 155, 180, or 255 min after a standard 4 g test meal. At 135 min post‐meal, rats were gavaged with 270 mg Leu (Leu270), 80:40:40 mg Leu, isoleucine, and valine, respectively (Leu80), 2.63 g CHO (CHO2.6), 1 g CHO (CHO1.0), or water (Sham control). Following test meal consumption, MPS peaked at 90 min then declined at 180 min even though translation initiation signaling remained elevated. In contrast, rats administered Leu270 or CHO2.6 supplements at 135 min post‐meal maintained peak MPS through 180 min; while Leu80 and CHO1.0 supplements elicited intermediate responses. Leu and CHO treatments were able to raise MPS above sham values from 155–180 min post‐meal, while MPS for both treatments returned to sham values at 255 min post‐meal. Lastly, MPS was inversely associated with markers of cellular energy status and translation elongation. We conclude that the incongruity between MPS and translation initiation at 180 min reflects a block in translation elongation due to reduced cellular energy, and Leu or CHO supplements are able to enhance energy status and prolong the period of muscle anabolism.
Tryptophan requirement of young, growing sows during pregnancy
Authors: Moehn Soenke , Franco Danilo J , Josephson Jessica K , Pencharz Paul B , Ball Ron O
Published: June 2021

Accelerated conceptus growth in the last trimester may increase amino acid and energy requirements of pregnant sows. The objective of this study was to determine the Trp requirement in early (EG, d 35 to 53) and late (LG, d 92 to 111) gestation using the indicator amino acid oxidation (IAAO) method. Six 2nd parity sows received 6 diets each based on corn, corn starch and sugar in both EG and LG at a constant allowance of 2.4 kg/d. Diets in EG contained Trp at 20, 40, 60, 80, 100 and 120 % of the current Trp requirement (2.5 g/d) in EG and 60, 80, 100, 140, 160 and 180% in LG. Sows were fed 2 mg/(kg BW•h) of L[1‐13C]Phe over 4 h in 8 ½‐hourly meals. Requirements were determined as the breakpoint in IAAO using 2‐phase nonlinear models. Sows, 167.7 kg (SE 3.93) at breeding, gained 44.3 kg (SE 3.63) during pregnancy and had litters of 14.5 piglets (SE 0.43) weighing 19.0 kg (SE 1.41). The Trp requirement was greater (P = 0.002) by 52% in LG (2.6 g/d) compared to EG (1.7 g/d). The increase (P = 0.001) in Phe retention from EG (2.94 g/d) to LG (8.28 g/d) agreed with a gain of 1 g/d N per fetus in LG and indicated that maternal protein gain was similar in EG and LG. Heat production was greater (P = 0.008) by 3% in LG compared to EG. Lipid retention decreased (P < 0.01) from EG to below zero in LG. Young sows strive to maintain fetal and maternal protein growth even if lipid retention becomes negative. This shows the importance of meeting amino acid requirements in late pregnancy.
Generation of Branched chain α‐keto acid dehydrogenase enzyme complex E1α knockout mouse models
Authors: Taghavi Marzieh , Hutson Susan
Published: June 2021

The goal of this study was to generate murine models for Maple Syrup Urine Disease (MSUD), enabling elucidation of the biochemical basis for the pathology of this inborn error of branched chain amino acid (BCAA) metabolism. As expected, loss of the E1α subunit (E1α KO) of the E1 dehydrogenase enzyme resulted in a mouse that died 24hr after birth. KO pups showed elevated levels of BCAA and branched chain α‐keto acids (BCKA) in their plasma and body tissues. The heterozygote mice (Het) were viable with ~50% of E1 enzyme levels found in wild type (WT) mouse tissues. When fed a 60% protein diet (HPD), Het food intake was lower, whereas body weight, percent fat, and plasma BCAAs were significantly higher than WT mice. To determine if deletion of E1 in brain could reproduce the MSUD brain pathology, a neuronal E1α KO was generated using the nestin‐Cre promoter (Nes‐E1α KO). These mice did not exhibit an MSUD phenotype even though E1 enzyme protein was also lowered in tissues outside the brain. The Nes‐E1α KO mice were sensitive to a HPD exhibiting higher percent of body fat elevated plasma BCAA and BCKA and food aversion. The data suggest that BCAA and/or BCKA may play a role in food intake and fat metabolism. Elevated peripheral BCAA and BCKA are also required to induce the MSUD brain pathology. (Funding: Virginia Tech)
Effect of alkali extraction on the digestibility and bioavailability of rice protein
Authors: Kubota Masatoshi , Saito Yuhi , Masumura Takehiro , Kumagai Takehisa , Watanabe Reiko , Fujimura Shinobu , Kadowaki Motoni
Published: June 2021

Since a manufacturing method was developed for rice protein, a novel plant protein food material, its digestibility and bioavailability were investigated. The products from two different methods were compared: rice protein by alkali extraction (AE‐RP) and by starch degradation through heat‐stable alpha‐amylase (SD‐RP). To estimate their bioavailability, a growth study with male Wistar rats was performed for 4 weeks: AE‐RP was superior to SD‐RP and comparable to casein in PER. The digestibility of rice protein is not superb for prolamin, which is notoriously indigestible in rice, but AE‐RP was dramatically improved, compared to that of SD‐RP when analyzed by western blotting analysis of gastrointestinal contents and feces. Next, an in vitro digestion study with pepsin and pancreatin was conducted to clarify the mechanism improving prolamin digestibility in AE‐RP. It was clarified that particle structures of protein body‐I in which prolamin accumulates and the heat process affected prolamin digestibility. In conclusion, alkali extraction to prepare rice protein gives a higher digestibility and bioavailability, when compared to starch‐degradation by heat‐stable alpha‐amylase, which is typical for cooked rice. This study was supported by grants to MK from the Japan Science and Technology Agency Innovation Satellite Niigata, Nagaoka, Japan.