bluuebunny/crossref_metadata_2025_split huggingface dataset:
Measuring and modeling dynamics of regulators of G protein signaling to facilitate discovery of allosteric inhibitors of protein‐protein interactions (843.13)
Authors: Higgins Colin , Fuentes Ernesto , Roman David 
Published: June 2021

Ischemic stroke morbidity and disability stem from neuronal death largely driven by glutamatergic excitotoxicity following hypoxia in brain tissue deprived of blood flow. Pharmacological inhibition of RGS4 with chemical probes significantly potentiates endogenous neuroprotection against excitotoxicity in vivo. The only known RGS4 inhibitors are unsuitable as clinical leads because they are relatively nonspecific covalent modifiers that inhibit through an uncharacterized, though putatively allosteric, mechanism. We seek to determine the structural mechanism by which current small‐molecule chemical probes allosterically inhibit RGS4. We hypothesize that inhibitors, binding to Cys148, trap a stochastically minor open conformation of RGS4 and disrupt a structurally key helical bundle to both prevent a unique conformational shift in RGS4 required to bind G‐alpha and to shift the population dynamics toward increasingly extended and unstable conformations. Structural changes in RGS proteins upon binding to inhibitory compounds measured with structural biological techniques including nuclear magnetic resonance, circular dichroism, light scattering, small‐angle X‐ray scattering, and atomistic molecular dynamics simulations support our hypothesis. Our investigations further show that RGS8, a homolog of RGS4, differs significantly in biophysical properties and we conclude that that these differences underly specificity of inhibitors for RGS4.Grant Funding Source: Supported by 5T32GM067795‐08 and pre‐doctoral fellowship from AFPE.
GPCR‐Gs‐mediated cyclic AMP signaling for neuritogenesis, growth arrest, and cell survival is parcellated among the cAMP sensors NCS/Rapgef2, Epac, and PKA (843.7)
Authors: Emery Andrew , Eiden Maribeth , Mustafa Tomris , Eiden Lee 
Published: June 2021

Ligands that elevate cAMP through Gs‐coupled GPCR activation have profound effects on the differentiation of neuroendocrine cells. Using the PC12 cell subclone NS‐1, we found that cAMP‐induced neurite extension, cAMP‐induced growth arrest, and cAMP‐dependent cell survival are all dissociable at the level of the cAMP sensor through which signaling occurs. Previously, we showed that the protein product of the Rapgef2 gene (NCS/Rapgef2) is activated by cAMP and is coupled to the activation of the Rap1‐BRAF‐MEK‐ERK signaling cascade necessary for cAMP‐dependent neuritogenesis (Emery and Eiden, FASEB J. 26, 3199, 2012; Emery et al., Sci. Signal. 6, ra51, 2013). Cyclic AMP elevation also causes Epac (Rapgef3/4) activation, which we now find leads to activation of both Rap1 and the MAP kinase p38. Surprisingly, these two downstream effectors of Epac appear to be arranged in separate signaling pathways, since p38‐dependent growth arrest initiated by Epac was not affected by inhibition of Epac‐dependent Rap1 activation. Thus, in NS‐1 cells, cAMP‐dependent growth arrest is wholly dependent on Epac‐p38 signaling and does not require NCS/Rapgef2‐ERK or PKA‐CREB activity. Finally, we found that cAMP protects NS‐1 cells from serum withdrawal‐induced cell death, also by a wholly separable signaling mechanism, which is PKA‐dependent and does not require the activation of either NCS/Rapgef2 or Epac.Grant Funding Source: Supported by NIMH‐IRP project ZO1‐MH002386
A “heavy metal sponge” for selective removal and recovery of toxic or precious metals from water (844.8)
Authors: Nguyen Phuc , Madera Paul , Rendon Adrian , Ong Aaron , Mao Herbert , Trujillo Nancy , Acey Roger 
Published: June 2021

Metallothionein (MT) is a low molecular weight metal binding protein. It has the unique ability to selective bind trace amounts of toxic heavy metals such as lead and cadmium. It does not bind essential metals such as sodium or calcium. We are developing a novel patented “heavy metal sponge” technology for the removal and recovery of toxic, e.g., lead and mercury, and precious metal, e.g., gold and platinum, from water. MT also has the ability to bind the lanthanides lanthanum and europium. Recombinant MT was cloned into pET vectors with and without a Pel secretory sequence, pPMpel, and pCODAmt, respectively. The protein is expressed in bacteria and purified to near homogeneity from cell lysates by a simple and cost effective method using FPLC anion exchange chromatography. The purity of the preparation was confirmed by SDS‐PAGE. Expression of MT and secretion of the protein using the pPMpel construct is pH dependent. The level of expression from both constructs was the same for IPTG ranging from 0.5 to 2.0 mM. The purified MT was used to determine the potential for removing trace amounts of heavy metal from seawater. The protein was mixed with seawater containing 100 ppt (trillion) of 109Cd. The protein/metal complex was then collected using a membrane filter centrifugal device and the flow through assayed for radioactivity. The efficiency of metal removal was determined from the radioactivity in the flow through. Under these conditions, 88% of the metal was removed from the seawater after a single centrifugation.Grant Funding Source: Funded by a grant from CSUPERB
Aldehyde dehydrogenase 2 null mice as an oxidative stress‐based model of age‐related cognitive impairment with Alzheimer’s disease‐like pathologies (845.3)
Authors: D'Souza Yohan , Elharram Ahmed , Frith Margaret , Andrew David , Bennett Brian 
Published: June 2021

Most current mouse models Alzheimer’s disease (AD) exhibit pathological changes dependent on overexpression of mutant human genes linked to familial AD, and animal models that mirror late‐onset/age‐related AD (95% of AD cases) are lacking. Oxidative stress is considered a causative factor in age‐related AD, and Aldehyde dehydrogenase 2 (ALDH2) is important for the detoxification of endogenous aldehydes, such as 4‐hydroxynonenal (HNE), a lipid peroxidation product formed during periods of oxidative stress that can form protein adducts, altering cell function. HNE adducts accumulate in the brains of AD patients and are associated with AD pathology. Given this linkage, manipulations that increase HNE levels might be expected to result in biochemical and cognitive changes that mirror those found in AD. Accordingly, we followed the age‐related changes of a number of relevant AD markers over a 12 month period in hippocampal homogenates from Aldh2 null mice. Marked increases in HNE adduct formation occurred as early as 3 months, and there were age‐related increases in monomeric amyloid β (Aβ), phospho tau protein, and activated caspases 3 and 6. Also observed were age‐related decreases in PSD95 and synaptophysin (indicating synaptic loss) and of total and phosphorylated cAMP‐response element binding protein (CREB). Aldh2 null mice exhibit a graded, age‐related decrease in performance in recognition and spatial memory tasks beginning at 3 months of age and maximal at 6 months. These mice also exhibit dystrophic neurites and loss of dendritic spines, defects in cholinergic agonist‐induced CREB phosphorylation, endothelial dysfunction, and monomeric and oligomeric Aβ deposition in cerebral microvessels. Aldh2 null mice exhibit a wide spectrum of AD‐like pathological changes that are rarely observed together in current AD animal models, and may prove useful for assessing the efficacy of therapeutic agents for improving memory and for slowing, preventing, or reversing disease progression.Grant Funding Source: Botterell Foundation
SCM‐198 protects neurons from microglia‐induced neurotoxicity and improves cognitive performances in Alzheimer’s disease models (846.3)
Authors: Hong Zhen , Shin Ru , Zhu Kai , Wu Ting , Zhu Zhun 
Published: June 2021

Background: Chronic inflammation in central nervous system is a conspicuous hallmark in Alzheimer’s disease (AD) and microglial activation is now accepted as a key factor in neuroinflammation. SCM‐198 is an alkaloid extracted from Herba leonuri. In the present study, we investigated its direct and indirect neuroprotective effects in neurons and in animal AD models.Methods and Results: (1) Pretreatment of microglia with SCM‐198 could reduce nitric oxide elevation and inhibit iNOS, TNF‐α, IL‐1β mRNA and protein expressions induced by LPS or Aβ1‐40. NF‐κB translocation induced by LPS was effectively mitigated by SCM‐198 in BV‐2 cells. Western blot results showed that IκBα degradation, NF‐κB p65 activation, phosphorylations of JNK1/2 and tau protein were also inhibited in BV‐2 cells. (2) Microglia/neuron co‐culture assay showed that pretreatment of activated microglia with SCM‐198 could significantly increase neuronal survival and alleviate excessive tau phosphorylation and synaptophysin loss in neurons. (3) Pretreatment of primary neurons with SCM‐198 before Aβ1‐40 challenge could reduce lactase dehydrogenase release, inhibit Bax protein elevation, prevent caspase‐3 and ‐9 activations, increase Bcl‐2 protein expressions in neurons. Besides, neuritic morphology was largely preserved under the intervention of SCM‐198. (4) Animal studies demonstrated that SCM‐198 could ameliorate cognitive deficits of both SD rats injected with Aβ1‐40 and APP/PS1 mice in Morris water maze and novel object recognition tests. Suppression of microglial activation could be observed in brain slices of SD rats and less neuronal loss in hippocampus and cortex were found in APP/PS1 mice.Conclusions: Our findings are the first to report that SCM‐198 could directly protect neurons from apoptosis and indirectly protect neurons by inhibiting microglial overactivation through JNK and NF‐κB pathways. Therefore, SCM‐198 might be a new potential drug candidate for AD therapy in the future.Grant Funding Source: This study was supported by the National Science fund for Distinguished Young Scholars of China
Cognitive performance and homecage activity in female cynomolgus monkeys: relationship to social rank and cocaine reinforcement (848.1)
Authors: Kromrey Sarah , Czoty Paul , Rowe Michael , Nader Michael 
Published: June 2021

Nonhuman primate social groups, which represent a continuum of experiences from environmental enrichment in dominant monkeys to chronic social stress in subordinates, have been used to study susceptibility and resilience to human diseases. We have shown that social rank influences sensitivity to cocaine and other dopaminergic drugs. However, it is not clear what biological and environmental factors determine what rank an individual monkey will attain. In females, novel object reactivity and CSF concentrations of 5‐HIAA predicted social rank; these factors did not change after hierarchies stabilized. The present studies examined whether other variables, including cognitive function (using CANTAB touchscreen software) and activity (using Actical monitors), predict eventual social rank and change after social hierarchies stablized. Prior to social housing 4 monkeys per pen, performance on a cognitive task was not different in eventual dominant and subordinate monkeys. After stable hierarchies were formed, subordinate monkeys demonstrated cognitive impairments. Furthermore, future subordinates had higher activity during the day which remained higher after hierarchies stabilized, and preliminary results suggest rank differences in nighttime activity. Additionally, the relationship between these behaviors and cocaine reinforcement is being investigated. Understanding behavioral phenotypes and individual differences in response to environmental manipulations, such as attainment and occupation of social ranks, and the relationship to cocaine self‐administration will aid in the development of behavioral and pharmacological treatment strategies for drug addiction.Grant Funding Source: Supported by DA017763
LPS induced LOX‐1 expression via TLR4/MyD88/ROS activated p38MAPK/NF‐κB pathway (851.15)
Authors: Chen Xiuping , Zhao Wenwen , Ma Guixin 
Published: June 2021

Lectin‐like receptor for oxidized low density lipoprotein (LOX‐1) plays a key role in endothelial ox‐LDL endocytosis, endothelial dysfunction and atherogenesis. In the present study, the effect of lipopolysaccharide (LPS) on LOX‐1 expression and the underlying molecular pathways were investigated. Human umbilical vein endothelial cells (HUVECs) were treated with LPS and the protein expressions of LOX‐1,TLR4, TLR2, MyD88, Nox4, Nox2, PI3K, p38MAPK, JNK, ERK, Nrf1, Nrf2 and p65 were examined by Western blotting. The intracellular reactive oxygen species (ROS) production was examined by flow cytometry with fluorescence probe DCFH2‐DA. The role of TLR4, MyD88 and Nox4 were determined with specific siRNA. The endothelial ox‐LDL uptake and the endothelial‐monocyte adhesion were evaluated with DiI‐ox‐LDL and Hoechst 33342 respectively. The effect of LPS on LOX‐1 expression in aorta tissue was also studies with Male C57/BL6 mice by intraperitoneal injection of LPS. The results showed that LPS induced LOX‐1 protein expression in a time‐ and concentration‐dependent manner. The mRNA expression of LOX‐1 was also upregulated. The protein expression of LOX‐1 and phosphorylated p38MAPK, p65 was significantly enhanced by LPS both in vitro and in vivo. LPS induced LOX‐1 expression was blocked by siRNA for TLR4, MyD88, and Nox4 and inhibitors for p38MAPK, NF‐κB and NADPH oxidase. Both LPS induced ox‐LDL uptake and endothelial‐monocyte adhesion were significantly inhibited by anti‐LOX‐1 antibody. LPS dramatically induced LOX‐1 protein expression in aorta tissues. In conclusion, our data suggested that LPS induces LOX‐1 expression via TLR4/MyD88/ROS activated p38MAPK/NF‐κB pathway, which put new insight on the role of LPS in atherosclerosis.Grant Funding Source: Supported by the National Natural Science Foundation of China (No. 81160048)
Comparing the effects of atorvastatin and vitamin E on endothelial function of sildenafil non‐responders (851.8)
Authors: abd Elkawy Khaled , El‐Sisi Alaa , Hegazy Ssahar , Salem Khaled 
Published: June 2021

ABSTRACT Endothelial dysfunction causes the failure of sildenafil response due to reduced nitric oxide (NO) . This study aimed to compare the biochemical and clinical effects of atorvastatin and vitamin E on endothelial function of sildenafil non‐responders with investigation to the underlying mechanisms.Methods: Sixty participants were randomly divided into three groups receiving atorvastatin 80 mg daily, or vitamin E 400 IU daily, or placebo capsules. Participants were examined both before and after six weeks for these biochemical tests; NO, glutathione peroxidase (GPO), interlukin 6 (IL‐6), testosterone, and endothelial nitric oxide synthase (eNOS) and for erectile function tests; international index of erectile function (IIEF ‐5) scores, rigiscan, and duplex l Results: in comparison to control group, the treated groups showed a significant increase in GPO, and a significant decrease in IL‐6. Atorvastatin only showed a significant increase in NO, eNOS, IIEF‐5 scores and rigiscan rigidity parameters. Other rigiscan parameters, testosterone and duplex ultrasound showed no difference in the three groups
Conclusions: Atorvastatin improves endothelial function of sildenafil non‐responders by increasing NO level attributed to enhancing eNOS activity (main mechanism) added to antioxidant and anti‐inflammatory activities. Atorvastatin is a real promising drug for sildenafil non‐responders.
Dynamic regulation of β1 subunits controls vascular contractility (853.7)
Authors: Leo M. , Bannister John , Narayanan Damodaran , Nair Anitha , Jaggar Jonathan 
Published: June 2021

Ion channels composed of pore‐forming and auxiliary subunits control physiological functions in virtually all cell types. Whether the multi‐subunit composition of surface channels is fixed following protein synthesis or flexible and open to acute and rapid modulation to control physiological cellular functions is unclear. Arterial myocytes express large‐conductance Ca2+‐activated potassium (BK) channel α and auxiliary β1 subunits that are functionally significant modulators of arterial contractility. Here, we show that native BK α subunits are primarily (~95%) plasma membrane‐localized, whereas only a small fraction (~10%) of total β1 subunits are located at the cell surface in human and rat arterial myocytes. Immuno‐FRET microscopy demonstrated that intracellular β1 subunits are stored within Rab11A‐postive recycling endosomes. Nitric oxide (NO), acting via protein kinase G, stimulated rapid (1 min) anterograde trafficking of β1 subunit‐containing endosomes. These β1 subunits associated with surface‐resident BKα proteins, elevating Ca2+‐sensitivity and inducing activation. Data also show that rapid β1 subunit anterograde trafficking is the primary mechanism by which NO activates myocyte BK channels and induces vasodilation. In summary, we show that rapid surface trafficking of β1 subunits controls functional BK channel activity in arterial myocytes and vascular contractility.Grant Funding Source: Supported by NIH‐HL67061
Effects of calf venous capacitance initiation rate on tolerance to lower body negative pressure in women with and without vasovagal syncope (858.5)
Authors: Skoog Johan , Lindenberger Marcus , Zachrisson Helene , Länne Toste 
Published: June 2021

Objective: Lower limb venous blood pooling has been considered an important factor in the pathophysiology of vasovagal syncope (VVS). However, not only the pooled volume, but also the rate by which the hypovolemic stimulus is instituted may be of importance. The aim of this study was to determine the influence of calf venous capacitance (Vcap initiation rate on tolerance to lower body negative pressure (LBNP) in women with and without VVS. Design and Methods: Graded LBNP to presyncope was used to determine LBNP tolerance index (LTI) in 13 young women with VVS and 15 matched controls. During LBNP, Vcap and the initiation rate of Vcap was evaluated with volumetric technique and overall cardiovascular responses were measured. Results: No differences in Vcap were found. In controls, the initiation rate of Vcap was negatively related to LTI (r = ‐0.54, P < 0.05), and a rapid initiation of Vcap was related to a lesser decline in stroke volume (r = ‐0.68, P < 0.05) and pulse pressure (r = ‐0.52, P < 0.05) during LBNP. In VVS, the initiation rate of Vcap was positively related to LTI (r = 0.82, P < 0.01), and a slower initiation was related to lesser decline in stroke volume (r = 0.61, P < 0.05). Conclusions: In healthy women, a rapid institution of Vcap was related to a more effective cardiovascular regulation and a higher LTI, possibly explained by greater baroreceptor deactivation due to a more rapid onset of the hypovolemic stimulus. However, the opposite was presented in VVS women, highlighting fundamental differences in cardiovascular regulation.Grant Funding Source: Supported by the Swedish Heart and Lung Foundation
Do kappa opioid receptors regulate cardiovascular responses to severe haemorrhage in conscious newborn lambs? (859.3)
Authors: Smith Francine , Samhan Mohamed , Qi Wei 
Published: June 2021

Previously, we showed that activation of kappa opioid receptors (KORs) influences systemic haemodynamics and the baroreflex control of heart rate (HR) in conscious lambs. The endogenous ligand for KORs, Dynorphin, is increased in response to severe blood loss however, any role for KORs in the haemodynamic responses to haemorrhage in the newborn is not known. In the present study, mean, systolic and diastolic arterial pressures (MAP, SAP, DAP) as well as HR were measured for 30 min before (Control) and 60 min after 30% haemorrhage in conscious lambs (aged ~one week, N=7) after pretreatment with vehicle (experiment one) or the selective KOR antagonist, GNTI (experiment two). After vehicle, MAP fell from 75±5 (Control) to 48±9 mmHg by 10 min after haemorrhage and remained decreased. This response resulted from a decrease in both SAP and DAP and was not altered by pretreatment with GNTI. HR decreased from 189±29 (Control) to 139±30 beats/min 10 min after haemorrhage; this response was attenuated by GNTI. Therefore, KORs do not appear to influence the blood pressure response to severe blood loss early in life but do abrogate the HR response to haemorrhage.Grant Funding Source: Supported by the CIHR.
Transient receptor potential vanilloid 2 is a mediator of left ventricular hypertrophy (862.5)
Authors: Rubinstein Jack , Naticchioni Mindi , Gao Xu , Koch Sheryl , Lasko Valerie , Lorenz John 
Published: June 2021

Background: Impaired calcium handling has been implicated in the development of left ventricular hypertrophy (LVH) and subsequent development of heart failure (HF). We have recently shown that the calcium channel Transient Receptor Potential Vanilloid 2 (TRPV2) is present in cardiomyocytes and regulates contractility on a beat to beat basis. This study was performed to elucidate if TRPV2 also modulates the development of LVH in a pressure overload mouse model via transverse aortic constriction (TAC).Methods: WT and TRPV2‐/‐ mice were subjected to pressure overload via TAC and followed echocardiographically for 8 weeks. A subgroup of WT mice were treated with a nonspecific TRPV2 antagonist (tranilast) for the same amount of time. At the conclusion, the hearts were collected for molecular and histological analysis of hypertrophy.Results: There was significantly less hypertrophy in TRPV2‐/‐ and in the WT mice treated with tranilast (n=5 and 2) in comparison to untreated WT mice (n=7) as noted via heart weight: body weight ratio and LV mass via echocardiography (P<0.05; TRPV2 vs. untreated WT). Furthermore, there was a trend towards improved contractility and less LV cavity dilation in the TRPV2‐/‐ and the WT treated mice in comparison to untreated mice.Conclusions: We have shown that TRPV2 modulates the development of LVH and is a potential target for the prevention and treatment of LVH and development of HF.
Changes in mTOR/4‐EBP1 pathway induced by a novel mutation in PRKAG2 gene (864.7)
Authors: Xu Yanchun , Tseng Yi‐Tang , Padbury James , Kobashigawa Laura C. , Phornphutkul Chanika , Hardie D. Grahame 
Published: June 2021

Objective: The genetics of hypertrophic cardiomyopathy (HCM) is heterogeneous. Mutations in PRKAG2 have been implicated in some previous cases of HCM. PRKAG2 encodes the γ2 isoform of AMPK, a heterotrimeric enzyme with major roles in regulation of energy metabolism in response to cellular stress. We have identified a K475E mutation which results in a neonatal HCM phenotype. We have demonstrated that overexpressing the K475E mutant in HEK‐293 cells leads to 1) a markedly increase in the basal phosphorylation of T172 and associated kinase activity; 2) a reduction in the sensitivity to AMP in allosteric activation. The aim of this study was to investigate changes in downstream signaling pathways induced by the mutation.Methods: Primary dermal fibroblast cultures of control and a patient with the K475E mutation were maintained under standard cell culture condition. Cell lysates were subject to Western blotting for downstream factors, including Akt, p70S6K, and 4E‐BP1.Results: K475E mutation results in an increase in the activation of p‐P70S6K as evidenced by increased phosphorylation at both the critical phosphorylation sites (T389 and T421/S424). Fibroblasts from the K475E mutation also show increases in phosphorylation of 4E‐BP1 (S65 and T70).Conclusions: Our study shows K475E mutation leads to activation of a signaling pathway that favors protein synthesis/growth, providing a logical mechanism for the HCM phenotype observed.Grant Funding Source: NIH