bluuebunny/crossref_metadata_2025 huggingface dataset:
Conjugated linoleic acid (CLA) prevents age associated skeletal muscle loss in mice by maintaining redox balance during aging
Authors: Fernandes Gabriel , Rahman Md Mizanur , Halade Ganesh , Williams Paul John
Published: June 2021

Age related skeletal muscle sarcopenia is a major health problem. Currently, there is no safe and effective therapies that selectively increase skeletal muscle mass. Previously, we have shown that CLA maintains higher skeletal muscle mass in mice. This study was designed to elucidate the molecular mechanisms of muscle loss prevention by CLA using 12 mo old C57BL/6 mice fed 10% corn oil (CO) or a diet supplemented with 0.5% c9t11 CLA (CLA 1), t10c12 CLA (CLA 2) or c9t11 CLA+t10c12 CLA (CLA mix) for 6 months. Dual energy X‐ray absorptiometry (DXA) scan was performed at baseline, and end of dietary intervention. CLA 2 and CLA mix groups showed significantly higher muscle mass as compared to CO and CLA 1 groups by DXA and muscle wet weight. Production of muscle mitochondrial ATP, and H2O2 as reactive oxygen species and muscle antioxidant enzymes (catalase and glutathione peroxidase) has been analyzed. Enhanced production of ATP, and antioxidant enzymes accompanied by slight increase in H2O2 level was noted in CLA 2 and CLA mix groups as compared to that of CO and CLA 1 groups. Finally, we have analyzed serum malondialdehyde (MDA) level. The lower level of serum MDA in CLA 2 and CLA mix groups indicate less oxidative stress in these groups. These data suggest that CLA 2 and CLA mix groups may preserve muscle mass by enhancing muscle mitochondrial energy production as well as by maintaining the redox balance during aging. Research support: NIH
Apolipoprotein A5 polymorphisms interact with dietary fat intake in association with markers of metabolic syndrome in the Boston Puerto Rican Health Study
Authors: Mattei Josiemer , Demissie Serkalem , Tucker Katherine L , Ordovas Jose M
Published: June 2021

The apolipoprotein A5 (APOA5) ‐1131T>C and S19W single nucleotide polymorphisms (SNPs) have been associated with plasma lipids concentration and metabolic syndrome (MetS), independently and in modulation by dietary factors. Puerto Ricans have high frequency of the two APOA5 variants, suggesting a role of the SNPs on such outcomes in this population. We aimed to determine the association of APOA5 ‐1131T>C and S19W with plasma lipids and markers of MetS, alone and in interaction with total fat intake, in Puerto Ricans living in the Boston, MA area (n=802, 45‐75 yrs). After adjusting for potential confounders, the APOA5 S19W variant was associated with HDL‐C (P=0.044); minor allele carriers had lower HDL‐C than those with the common variant (mean ± SE = 43.3 ± 1.0 versus 45.5 ± 0.5), even after adjustment for plasma triglycerides (TG), (P=0.012). Neither polymorphism was associated with other plasma lipids. Interaction of the ‐1131T>C SNP with total fat intake was observed for plasma TG and total cholesterol (P=0.032 and P=0.034, respectively). APOA5 S19W showed an interaction with total fat intake in association with systolic and diastolic blood pressure (P=0.002 and P=0.007, respectively). Neither SNP was associated with MetS in overall analysis or after stratifying by total fat intake. In conclusion, dietary fat intake seems to play a role in the mechanism of APOA5 SNPs on lipid and metabolic markers in Puerto Ricans.
Oxidative stress and hypoxia‐related stem cell death
Authors: Peterson Karen M , Abdelrhaman Aly , Rodriguez‐Porcel Martin
Published: June 2021

Background

Stem cells undergo significant death after transplantation, but the mechanisms underlying this deleterious response remain unclear. Increased oxidative stress has been shown to be present in states of hypoxia or cellular stress. Thus, we hypothesize that oxidative stress blockade would ameliorate the increased cell death when exposed to hypoxia.

Methods

Cardiac stem cells were exposed to hypoxic conditions in cell culture (1% O2 for 24 hours). Oxidative status (by measuring reactive oxygen species‐ROS‐) and cell survival (by the MTT assay) were measured. In a separate group of cells, oxidative stress was blocked using a superoxide dismutase mimetic (Tempol, 1‐10µm/L).

Results

Hypoxia resulted in increased expression of the oxidative stress enzyme NAD(P)H oxidase (subfraction 47phox) and in the amount of ROS (increased in the conversion of dihydroethidium ‐DHE‐ and dichlorofluorescein ‐DCF‐) what led to a decrease in stem cell viability. Pre‐conditioning of stem cells with antioxidants (Tempol) decreased the amount of cellular ROS and improved stem cell survival. The increased expression of NAD(P)H p47phox seen in hypoxia was unaltered by oxidative stress blockade.

Conclusion

Increased oxidative stress plays a role in the early cell death after hypoxia. Understanding of the mechanisms underlying the fate of stem cells will be critical for the advancement of the field of stem cell therapy.
Unmasking a role for nitric oxide in acetylcholine‐induced vasodilation in diseased human coronary arterioles.
Authors: Wittenburg April Lee , Mendoza Suelhem A , Zhang David X , Gutterman David D
Published: June 2021

Background

Acetylcholine (ACH) dose‐dependently constricts human coronary atrial arterioles (HCA) but dilates ventricular arterioles in patients with coronary disease. Whether NO mechanisms participate in Ach‐induced dilation in atrial arterioles remains unclear. Since CAD and its risk factors generate ROS that can quench NO, we hypothesized that acetylcholine‐induced dilation can be restored in HCA by restoring redox balance and supporting exogenous NO synthesis.

Methods

Arterioles (~150 μm) from patients with coronary artery disease were prepared for videomicroscopy. HCA's were pre‐incubated with indomethacin (10‐5), sepiapterin (10‐4), L‐Arginine (10‐3), and Peg‐SOD (300units/ml) for 30 minutes. After preconstruction with endothelin‐1, vasomotor responses to exogenous ACH (10‐9 to 10‐4 M) were evaluated in control or in the presence and absence of N‐nitro‐L‐arginine methyl ester (L‐name; 10‐4)).

Results

ACH dilated pretreated compared to control intact arterioles [%max dilation (MD): 80±8% vs. ‐36.4±7%]. Treatment with L‐name abolished dilation (MD:‐0.5±9%).

Conclusions

Reducing endogenous oxidative stress and providing NOS substrate converts an , ACH‐induced constriction to an NO ‐dependent vasodilation in HCA. There may be a greater sensitivity to ROS in endothelium of atrial vs. ventricular arterioles.
Metabolic and cardiovascular responses to walking in fructose‐fed male rats
Authors: Machi Jacqueline Freire , Bernardes Nathalia , Irigoyen Maria Claudia , de Angelis Kátia , Wichi Rogério Brandão
Published: June 2021

The aim of this study was to investigate the metabolic and cardiovascular responses to walking in fructose‐fed male rats. Male Wistar rats were divided into 3 groups (n=6): control (C), sedentary fructose (SF) and walking fructose (100g/L in drinking water at 18 wk) (WF). The walking was performed on a treadmill (1 h/day; 5 days/wk for 8 wk). The arterial pressure (AP) signals were directly recorded (CODAS, 2Khz). Baroreflex sensitivity was evaluated by the tachycardic (TR) and bradycardic (BR) responses induced to AP changes. The tryglicerides concentrations and the insulin tolerance test were performed to evaluate the metabolic profile. The results showed that walking decreased the adipose tissue (SF: 6.5±0.4; WF: 2.8±0.1; C: 3±0.3 g), blood triglycerides level (SF: 291±6.5; WF: 150±8.1; C: 103±4.5 mg/dl) which were observed after fructose overload. The WF group showed increased insulin sensitivity (3.3±0.32 mg/dl/min) compared to SF group (2.5±0.2 mg/dl/min). BR was significantly reduced in WF group (1.95±0.33 vs. 1.40±0.14 bpm/mmhg in SF). However, the walking not changed the increase in AP (SF: 137±2; WF: 129±1; C: 115±6 mmhg). These findings suggest that walking is especially effective for treating the metabolic disturbances. The exercise should be prescribed as appropriate and individualized to be as effective in the management of cardiovascular risk in metabolic syndrome.
Intravascular Pressure Initiates Cerebral Arterial Constriction by Inducing Voltage‐Independent Calcium Waves
Authors: Karen Bradley , Brett Suzanne , Tran Cam Ha , Hunt Donald , Jones Peter , Chen Wayne , Welsh Donald
Published: June 2021

The study examined whether elevated intravascular pressure induces asynchronous Ca(2+) waves in cerebral arterial smooth muscle cells and if their generation contributes to myogenic constiction. Rat cerebral arteries mounted in a customized arteriograph were pressurized (20‐100 mmHg) and examined under a variety of experimental conditions. Diameter and membrane potential (Vm) were assessed using conventional techniques whereas Ca(2+)waves were monitored by measuring Fluo‐4 fluorescence with a spinning disk confocal microscope. Elevated pressure increased the proportion of cells firing Ca(2+) waves (30 to 97%) and their frequency (3 to 10 waves/cell). Ryanodine, an agent that depletes the sarcoplasmic reticulum (SR) of Ca(2+) blocked Ca(2+) wave generation. Interestingly, Ca(2+)waves were voltage‐independent as Diltiazem (L‐type Ca(2+) channel blocker) along with changes in Vm had little effect on these pressure‐induced events. Functional experiments also revealed that Ca(2+) waves contribute to myogenic tone development through a mechanism independent of arterial Vm. In summary, this study revealed that like agonists, mechanical stimuli elicit Ca(2+) waves in arterial smooth muscle and that such events facilitate tone development through myosin light chain kinase activation.
Interleukin‐6 and Formation of the Coronary Vasculature
Authors: Bowers Stephanie Kidder , Banerjee Indroneal , Fuseler John , Souders Colby A , Baudino Troy A
Published: June 2021

Defects in the vasculature of the heart accounts for a large portion of cardiac‐related illness. Alterations in cell signaling factors, which can occur via changes in cytokine expression or biomechanical load, can affect vessel patterning in the heart and, therefore, the pressure‐flow network and availability of oxygen and critical growth factors to cardiac cells. Interleukin‐6 (IL‐6) is a pleiotropic cytokine involved in the regulation of several key biological processes, including wound repair, inflammation and vasculogenesis; circulating levels of IL‐6 can serve as clinical indicators for several cardiac pathologies. We observe an increase in IL‐6 secretion in fibroblast‐myocyte co‐cultures in vitro, as well as altered ratios of cardiac cell populations in IL‐6 knockout hearts. Using immunolohistological and cardioangiographic analyses of hearts from wild type (WT) and IL‐6−/− mice, we have found that IL‐6 deficiency alters vessel density and vascular distribution. Using non‐Euclidean geometrical fractal analyses, we observe a marked increase in the fractal dimension of IL‐6−/− mice, indicating that IL‐6 loss is closely associated with vascular patterning. Characterization of pathways involved in IL‐6‐dependent vasculogenesis and vascular remodeling may lend insight into factors critical to cardiac function.
The Effect of Hairtail Hydrolysate on Hydrogen Peroxide‐induced Apoptosis in Human Vascular Endothelial Cells
Authors: Kim Jung‐Lye , Choi Jung‐Suk , Park Sin‐Hye , Bae Ji‐Young , Lee Eun‐Sook , Han Seon‐Young , Kang Young‐Hee
Published: June 2021

Hydrogen peroxide (H2O2) which is one of the reactive oxygen species has been seen to cause various disease, various cellular disinfections, gene transformation and cell death. Much same unsaturated fatty acid as linoleic acid, taurine is included in a hairtail, and effect on adult disease prevention. The present study elucidated apoptosis pathways, through which hairtail hydrolysate exerted anti‐apoptosis in endothelial cells exposed to H2O2. Hairtail hydrolysate substantially diminished the H2O2‐induced cytotoxicity and Hoechast 33258 staining suggesting the inhibition of accumulation of H2O2‐triggered ROS and consequent apoptosis. The western blot data identified that hairtail hydrolysate at 200 μg/ml promptly attenuated phosphorylation of JNK and p38 MAPK by H2O2. Furthermore, this study also attempted to explore caspase activation in H2O2‐induced endothelial apoptosis. It was found that 200 μg/ml hairtail hydrolysate rapidly attenuated the cleaved of caspase‐3, ‐6, ‐7 within 18 h. Thus, hairtail hydrolysate may acts as an anti‐apoptotic agent blocking H2O2‐induced endothelial apoptosis. This study was supported by grants of KRF F00072 and Brain Korea 21 from Korea Research Foundation and from Korea Science and Engineering through the Silver Biotechnology Research Center at Hallym University.
IL‐10 suppresses pro‐fibrotic and matrix remodeling genes in normal and myxomatous mitral valves
Authors: Hagler Michael A , Roos Carolyn M , Hadley Thomas A , Schaff Hartzell V , Suri Rakesh M , Miller Jordan D
Published: June 2021

Myxomatous mitral valve disease (MMVD) is associated with increases in valvular fibrosis, matrix remodeling, and transforming growth factor beta (TGF β) signaling. While interleukin 10 (IL‐10) can suppress tissue fibrosis and matrix metalloproteinase (MMP) expression, its role in MMVD is unknown. We tested the hypotheses that reduced IL‐10 expression contributes to increased tissue fibrosis in MMVD, and that IL‐10 tonically suppresses TGF‐β signaling in normal mitral valves. We used qRT‐PCR to measure expression of IL10, TGF‐β1, MMP 2 and 9, and connective tissue growth factor (CTGF) in normal and myxomatous human mitral valves and in mitral and tricuspid valves from wild‐type and IL‐10 knockout mice. IL‐10 expression was significantly reduced in human myxomatous valves compared to normal valves (53± 12%), and was associated with increases in TGFβ, CTGF, and MMP2. Mitral valve gene expression patterns in IL‐10−/− mice favored fibrosis and matrix remodeling compared to wild‐type littermates, with increases in TGF β (132±7%), CTGF (154±3%), and MMP9 (259±7%) (p< 0.05 for all), but not MMP2. Interestingly, expression of these genes was not altered in tricuspid valves from IL‐10−/− mice. Collectively, these data are the first to show that IL‐10 tonically suppresses TGF β signaling in normal mitral valves and that deficiency of IL‐10 is likely to contribute to fibrosis and matrix remodeling in MMVD.

NHLBI
Ozone Exposure Induces Beta‐Adrenergic Insensitivity
Authors: Abston Eric , Fairweather DeLisa
Published: June 2021

The association between ambient ozone (O3) and adverse cardiopulmonary outcomes has long been recognized. Acute O3 classically induces bradycardia, hypotension, and increases heart rate variability – all of which are affected by the heart's response to sympathetic output. To test whether O3 exposure changes the sensitivity of the heart to beta adrenergic stimulus, C57BL/6 mice (n=10) were exposed to either Room Air (RA) 24 hours of 0.3 ppm O3 and then underwent in‐vivo hemodynamic assessment and isoproterenol (Iso) challenge involving I.V. bolus doses at half log intervals. Mice exposed to O3 displayed decreased left ventricular systolic function as characterized by decreased heart rate 565 bpm vs. 595 bmp in RA; systolic pressure decreased from 106 mmHg in RA mice to 87 mmHg in O3 exposed mice; dP/dT max decreased 10% with O3 exposure. Diastolic function was also depressed by O3 exposure: dP/dT min increased by 25% and Tau (Log) increased by 30% with O3 exposure. O3 exposure resulted in a decreased response to Iso as indicated by a leftward shift in the dose‐response curve. At the maximal dose (6 ng Iso in 10 μL PBS) heart rate increased by 60 bpm in RA controls and 35 bpm in O3 treated mice; dP/dT max increased by 6000 mmHg/s in control and only increased by 3000 mmHg/s in O3 exposed mice. In addition to confirming the systolic and diastolic dysfunction typically associated with O3, this study demonstrates that acute O3 exposure induces beta adrenergic insensitivity – a condition thought to contribute to heart failure in patients with heart disease that may be important in understanding the deleterious cardiopulmonary effects of inhaled O3.

NIH R01 HL087033
Synaptic pathology of outer retina in Ccl2/Cx3cr1 deficient mice
Authors: Zhang Jun , Tuo Jingsheng , Cao Xiaoguang , Shen Defen , Li Wei , Chan Chi‐Chao
Published: June 2021

Age‐related macular degeneration (AMD) is the leading cause of blindness among the elderly. Although various AMD mice models exhibit retinal pathology, there are limited reports about synaptic morphology in these models. We investigate the synaptic pathology of outer retina in C57BL/6 (wild type) and Ccl2−/−/Cx3cr1−/− (DKO) mice, a model with AMD‐like retinal lesions, at various ages by light and electron microscopy (EM). The labeling of CtBP2, a presynaptic ribbon protein, was discontinuous and interrupted by areas with few punctas. Some CtBP2 punctas were visible in the outer nuclear layer (ONL) at one month old. Consistently, a few calbindin‐labeled horizontal processes and occasionally PKC‐labeled rod bipolar dendrites extended aberrantly into the ONL. These aberrant ribbons and processes increased in numbers with ages. Double labeling studies showed close associations of synaptic ribbons with either horizontal or rod bipolar processes. EM showed a significant decrease of cone synapses in DKO mice compared to the WT mice. Furthermore, it confirmed that aberrant horizontal and bipolar processes made synaptic contacts with photoreceptor terminals. We conclude that, in addition to presynaptic photoreceptor terminals degeneration, horizontal and bipolar processes sprout to form ectopic synapses in the ONL of Ccl2−/−/Cx3cr1−/− mice, suggesting a neuronal remodeling in the retinal lesions.
Irradiation induces vessel rarefaction by differential regulation of Ang‐1, Tie‐2, Ang‐2, and VEGF in rat brain
Authors: Lee Won Hee , Cho Hyung Joon , Sonntag William E. , Lee Yong Woo
Published: June 2021

It has been proposed that radiation‐induced brain injury is mediated by cerebrovascular rarefaction. In the present study, we investigated the molecular mechanisms of radiation‐induced vessel rarefaction in the brain. Four month old F344×BN rats received either whole brain irradiation with a single dose of 10Gy γ‐rays or sham‐irradiation, and were maintained for 4, 8, and 24 h following irradiation. A series of immunofluorescence staining revealed that whole brain irradiation significantly reduced levels of CD31‐immunoreactive cells in rat brains compared with sham‐irradiated controls. Significantly suppressed endothelial cell (EC) proliferation and increased EC apoptosis were also detected in irradiated rat brain. In addition, a significant decrease in mRNA and protein expression of angiopoietin‐1 (Ang‐1), endothelial receptor tyrosine kinase (Tie‐2), and vascular endothelial growth factor (VEGF) was observed in irradiated rat brains by real‐time RT‐PCR, ELISA, and immunofluorescence staining. In contrast, whole brain irradiation significantly up‐regulated angiopoietin‐2 (Ang‐2) expression in rat brain. These data provide the novel evidence that whole brain irradiation differentially regulates expression of Ang‐1, Tie‐2, Ang‐2 and VEGF, which may affect vessel rarefaction by decreasing EC proliferation and increasing EC apoptosis in brain. (This work was supported by NINDS R01NS056218)
An Unusual Presentation of Ischemic Stroke as Brain Mass lesion
Authors: Dabi Alok , Kurukumbi Mohankumar , Adams Alice , Jayam‐Trouth Annapurni
Published: June 2021

Background

Ischemic stroke is one of the common neurological disorders; however, its presentation as a brain mass lesion is rare.

Case Presentation

45 year gentleman of Afro‐American origin presented with complaints of right sided weakness, progressing over at least 3 weeks prior to admission. He was found to have no risk factors for stroke except history of substance abuse. He had right sided weakness involving face, and upper limbs more than lower limbs on exam. MRI with gadolinium of the brain revealed a moderately large left fronto‐parietal lesion that showed cortical enhancement. The primary differential was a neoplastic lesion. The patient's clinical condition was progressively worsening, and a brain biopsy was performed. He was simultaneously investigated for primary malignancy elsewhere in the body but had a negative workup. Brain biopsy was reported as ischemic tissue necrosis with no evidence of neoplasm/vasculitis. His course was complicated by development of subdural hematoma after initiation of oral anti‐coagulation for his ischemic stroke. He responded well to surgical evacuation of the hematoma.

Conclusion

This rare presentation of a stroke as brain mass lesion signifies the importance of being vigilant about such a possibility. This case is reported for the rarity of its presentation and its impact on the diagnostic and therapeutic management strategies.
The Accuracy of Intraoperative Frozen‐Section Diagnosis in Clinical Staging of Endometrial Carcinomas According to the AJCC 7th Edition Guidelines
Authors: ZULFIQAR MUHAMMAD IMRAN , AHMED YASIN , SHAHAB ASIF , TASHJIAN RANDY , MAZZARA PAUL
Published: June 2021

Introduction

Endometrial carcinoma is the most common invasive cancer of the female genital tract accounting for 6% of all invasive cancers in women. The prognosis and surgical staging depends on the histologic grade, depth of myometrial invasion and cervical involvement. Surgical staging consists of total abdominal hysterectomy, bilateral salpingo‐oophorectomy, peritoneal cytology, and pelvic lymphadenectomy. Intraoperative frozen section analysis followed by formalin fixed permanent section analysis is used for surgical staging. We compared the accuracy of frozen section staging with the accuracy of permanent section staging.

Methods

A retrospective analysis was performed on 46 consecutive patients with endometrial carcinoma between February 2005 and June 2010 at St. John Hospital and Medical Center in Detroit, Michigan. Frozen section staging was performed on all the cases followed by permanent section staging using the AJCC 7th edition staging manual.

Results

Comparison between frozen section and permanent section examination did not show a significant difference in the depth of myometrial invasion (p = 0.7). A greater difference was observed with respect to cervical involvement, but this finding was not statistically significant (p = 0.2).

Conclusion

Although sampling of the specimen at frozen section has its limitations, the stage rendered through intraoperative examination generally correlates well with the pathologic stage reported upon permanent section examination.
Parameters indicative of reversible proximal tubule injury in rats following administration of cisplatin
Authors: McDuffie James Eric , Sablad Marciano , Sonee Manisha , Ma Jing , Varacolli Lynn , Louden Calvert , Guy Antonio , Cuellar Jennifer
Published: June 2021

Qualification of selected renal biomarkers indicative of the progression of acute kidney injury (AKI) in rats has been reported, however, little is known about the reversibility of this condition. In a 28‐day reversibility study, rats (n=4/group) received either a single dose of cisplatin (1 mg/kg, i.p.) or vehicle. Sample collections and necropsy time points were 6‐ and 72‐hours, 1‐week, 1.5‐, 2‐, 3‐, and 4‐weeks post dosing. The severity of cisplatin‐related histopathologic damage (proximal tubule regeneration, degeneration, dilatation, vacuolization and/or interstitial inflammation) increased significantly at 72‐hours post dosing and decreased thereafter. IHC labeling showed increased renal kidney injury molecule‐1 (KIM‐1) expression at 72‐hours, lower expression at 1‐week post dosing, and decreased to insignificant levels of expression thereafter. Concentrations of urinary albumin, lipocalin‐2, osteopontin, KIM‐1, glutathione S‐transferases (αGST and GSTYb1), renal papillary antigen‐1 (RPA‐1), clusterin, β2‐microglobulin, cystatin‐C, gamma glutamyl transferase (GGT), glucose (GLU), N‐acetyl‐beta‐D‐glucosaminidase (NAG), and total protein (TP) were measured and normalized to the concentration of urinary creatinine. Both urinary KIM‐1 and αGST were significantly elevated at 72‐hours post dosing and decreased at subsequent times. This work demonstrated various parameters that may be used to positively identify reversible cisplatin‐induced AKI in rats.
Evidence of impaired neuronal plasticity in response to inactivation of the small GTPase Rac1
Authors: Bongmba Odelia Yuh Ngala , Martinez Luis Alberto , Butler Karlis , Elhardt Mary , Tejada‐Simon Maria Victoria
Published: June 2021

The formation and maintenance of dendrites and dendritic spines are very critical for synaptic development and plasticity, and require a very dynamic actin cytoskeleton. Rac1, a Rho GTPase largely known for its involvement in the actin cytoskeleton remodeling, is also associated with neuronal development. Our laboratory and others have recently revealed that Rac1 might have a role in plasticity, bringing us to hypothesize that Rac1 is involved in the morphological changes observed at neuronal synapses during hippocampal learning and memory as well as long‐term plasticity. To investigate our hypothesis, we used the Cre/Lox‐P system to develop a tissue‐specific mutant mouse in which Rac1 was ablated from the hippocampal area CA1. We then demonstrated that Rac1 is necessary for normal dendritic morphology and long‐term synaptic plasticity. We further showed that Rac1 is also critical for hippocampus‐dependent learning and memory tasks. Thus, regulation of Rac1 may provide a functional link between deficient neuronal morphology, aberrant synaptic plasticity and cognition impairment, phenotypes often associated with certain autistic disorders.
Neuroprotective effects of various flavonoids alone and in combination on lipopolysaccharide‐induced neurotoxicity in PC‐12 cells
Authors: Jobaliya Chintan Deepakkumar , Benzeroual Kenza E.
Published: June 2021

Pro‐inflammatory cytokines have been detected in Alzheimer's disease (AD) brain suggesting that neuroinflammation also plays a role in the pathogenesis of AD. PC‐12 cells in culture respond to LPS stimuli by upregulating the levels of cytokines TNF‐alpha, IL‐1beta, nitric oxide (NO), and caspase activation thereby causing neuronal cell death. The present study investigated the neuroprotective effects of flavonoids such as fisetin, diosmetin and robinetin when administered alone and in combination, on the proinflammatory responses and apoptosis induced in LPS‐stimulated PC‐12 cells. Our studies showed that all the flavonoids alone and in combination markedly suppressed the production of TNF‐alpha, interleukin (IL)‐1beta, and nitric oxide (NO) in a dose –dependent manner, respectively. In parallel, cell apoptosis triggered by LPS and characterized with caspase‐3 activity, pro‐apoptotic gene Bad and anti‐apoptotic gene Bcl‐2 expressions were also significantly inhibited by these flavonoids. The results indicate that the inhibitory activity of flavonoids was greater when taken in combinations compared to those when taken alone. The present study suggests that flavonoids alone and in combination, might offer therapeutic opportunities to delay the progression of neuroinflammatory diseases such as AD.
NAD+ Induces Autophagy of Neuro2a Cells by Increasing Oxidative Stress
Authors: Han Jin , Shi Shengtao , Shao Jiaxiang , Xia Weiliang , Ying Weihai
Published: June 2021

NAD+ plays important roles in various biological processes. Previous studies have shown that NAD+ treatment can significantly decrease genotoxic agent‐induced death of primary neurons and astrocytes, and NAD+ administration can markedly reduce ischemic brain damage in rodents. However, whether NAD+ treatment affects cell autophagy remains unknown. In this study we have found that treatment of NAD+ at concentrations from 100 μM to 10 mM can induce autophagy of Neuro2a cells, as assessed by the immunoblotting of LC 3 I/II conversion,, real‐time qPCR quantification of beclin‐1 expression, and monodansylcadaverine (MDC) staining. Our study has also indicated that oxidative stress mediates the effects of NAD+ on the autophagy of the cells: NAD+ induced increases in the superoxide levels in Neuro2a cells, and treatment of the cells with antioxidants attenuated the effects of NAD+ on the autophagy. We have further indicated the NAD+‐induced autophagy contributes to the NAD+‐induced decrease in the survival of the cells. Our experimental results have also argued against the possibility that NAD+ affects the autophagy through its degradation product ‐‐‐ nicotinamide. In summary, our study has provided the first evidence that NAD+ treatment can induce autophagy of such tumor cells as Neuro2a by generating oxidative stress, which contributes to the NAD+‐induced decrease in cell survival (Supported by a Key Research Grant of Shanghai Municipal Scientific Committee #08JC1415400, a National Key Basic Research ‘973 Program Grant’ #2010CB834306, a Shanghai Engineering Center Grant of Equipment and Technology of Physical Therapy for Major Diseases #08DZ2211200).
Methamphetamine exposure affects α‐synuclein expression in the cortex
Authors: Gamble‐George Joyonna Carrie , Khoshbouei Habibeh
Published: June 2021

Methamphetamine (METH) is a highly addictive psychostimulant that is becoming a global public health problem. Previous studies have suggested that METH exposure is neurotoxic that may induce long‐lasting cognitive and motor impairments after cessation of drug use. α‐synuclein is implicated in neurodegenerative diseases. In this study, we propose that METH exposure affects the levels of α‐synuclein in the brain. Few studies have investigated the molecular mechanism of METH modulation of α‐synuclein in the brain. The present study sought to examine the effect of METH exposure via intraperitoneal injection (24 mg/kg/day) and an osmotic pump (1 mg/kg/hr) on α‐synuclein levels in the brains of juvenile C57BL/6J mice. We found that juvenile mice treated with METH for two weeks via osmotic pump showed an increase in α‐synuclein levels compared to saline‐treated control mice. This increase in α‐synuclein levels may be accountable for METH‐induced neurotoxicity. Since overexpression of α‐synuclein has been implicated in the dysregulation of DA homeostasis, we propose that METH‐induced α‐synuclein changes are dependent upon the pattern of METH administration. Currently, we are examining whether a correlation exists between alterations in α‐synuclein levels and additional molecular targets involved in DAergic homeostasis in the brain that are induced by METH exposure.

This research was supported by NIH Grants U54NS041071, U54RR026140, DA026947, and DA021471.
Effect of social isolation on the expression of GluR1 and GluR2 in rat hippocampus
Authors: Pereira Marina Teixeira Ramalho , Limonte Fábio Henrique , Iyomasa Melina , Oliveira Fabíola , Rosa Maria Luíza Nunes Mamede
Published: June 2021

This study aimed at evaluating the changes induced by social isolation (SI) on the expression of GluR1 and 2, isoforms flip and flop, in hippocampus. Two groups of rats (140g) were grouped (housed 4/cage and handled 3 times/week) orisolated (housed individually and handled once/week) for ten weeks. 40‐ƒÝm brain sections were used for immunohistochemistry. The immunopositive cells (IC) were quantified in hippocampus and data compared by t‐test (p<0.05). Gene expression of flip and flop isoforms of GluR1 and 2 were evaluated by qPCR and data compared by Mann‐Whitney test (p<0.05). SI induced an increase in GluR1‐IC in CA3 (90%, p=0.004) and CA1 (270%, p=0.001) while no change was found in the hilus. In contrast, a decrease was found in GluR2‐IC in CA3 (30%, p=0.01) but no changes were seen in the hilus or CA1. qPCR showed that SI resulted in upregulation of the genes encoding both isoforms of GluR1, flip (250%, p=0.03) and flop (280%, p=0.03) and only GluR2 flop (60%, p=0.03) in the whole hippocampus. The increased expression of GluR1, as revealed by immunohistochemistry and qPCR, suggest that flip and flop isoforms may underlie the hippocampal glutamatergic mechanism involved on the brain alterations induced by SI. The decreased expression of GluR2 may contribute to this mechanism due to the opposite kinetic properties of this subunit related to Ca permeability.
Interaction of Angio‐associated Migratory Cell Protein with the TPα and TPβ isoforms of the human Thromboxane A2 receptor
Authors: Mulvaney Eamon P , Reid Helen M , Kinsella B Therese
Published: June 2021

In humans, thromboxane (TX) A2 signals through the TPα and TPβ isoforms of its G‐protein coupled TXA2 receptor (TP) to mediate a host of (patho)physiologic responses. Herein, angio‐associated migratory cell protein (AAMP) was identified as a novel binding partner of both TPα and TPβ through an interaction dependent on common (residues 312–328) and unique (residues 366–392 of TPβ) sequences within their carboxyl‐terminal (C)‐tail domains. While the interaction was constitutive in mammalian cells, agonist‐stimulation of TPα/TPβ led to a transient dissociation of AAMP from immune complexes which coincided with a transient redistribution of AAMP from its localization in an intracellular fibrous network. Although the GTPase RhoA is a downstream effector of both AAMP and the TPs, AAMP did not influence TP‐mediated RhoA or vice versa. Small interfering RNA (siRNA)‐mediated disruption of AAMP expression decreased migration of primary human coronary artery smooth muscle cells (1° hCoASMCs). Moreover, siRNA‐disruption of AAMP significantly impaired 1° hCoASMC migration in the presence of the TXA2 mimetic U46619 but did not affect VEGF‐mediated cell migration. Given their roles within the vasculature, the identification of a specific interaction between TPα/TPβ and AAMP is likely to have substantial functional implications for vascular pathologies in which they are both implicated. Funded by SFI and HRB.
Increased Phosphodiesterase 4B (PDE4B) and decreased cellular cAMP regulate LPS‐inducible TNF‐α in glucose‐primed monocytes
Authors: Chambers Elaina , Gobejishvili Leila , Joshi‐Barve Swati , McClain Craig , Barve Shirish
Published: June 2021

Diabetes mellitus (DM) is a chronic disease with no cure. Diabetics experience increased gut permeability; therefore, lipopolysaccharide (LPS)‐inducible cytokines are highly relevant in this disease. Elevated levels of pro‐inflammatory cytokines, specifically Tumor Necrosis Factor‐α(TNF‐α), play a major role in the evolution of multi‐organ dysfunction. Our earlier work demonstrated that cAMP and monocyte/macrophage‐specific PDE4B play a key role in LPS‐inducible TNF‐α expression. The current data demonstrate that human monocytes exposed to high glucose (HG:15–25 mM) exhibit a “primed phenotype”, wherein they express elevated levels of TNF‐α following LPS stimulation. The data obtained show for the first time that HG exposure up‐regulates PDE4B expression leading to a decrease in cellular cAMP levels and a corresponding increase in TNF‐α in response to LPS stimulation. These data strongly imply that PDE4B plays a significant mechanistic role in the development of inflammation occurring in response to HG. Moreover, these data also suggest that PDE4B could serve as a therapeutic target in the treatment of Diabetes.
Functional selectivity of brain Gα‐subunit proteins in the cardiovascular and renal excretory responses evoked by central α2‐adrenoceptor activation in vivo
Authors: Wainford Richard David , Kapusta Daniel R
Published: June 2021

Hypothesis

Brain Gα‐subunit proteins signal in a functionally selective manner to mediate the integrated cardiovascular depressor and renal excretory responses evoked by pharmacological activation of central α2‐adrenoceptors in vivo.

Methods

The α2‐agonist guanabenz (50 μg) was administered by intracerebroventricular (i.c.v.) injection to conscious instrumented Sprague‐Dawley rats pre‐treated (24‐h) i.c.v. with saline or oligodeoxynucleotides (ODN) targeted to Gαi1,Gi2, Gi3, Gαo, Gαq, Gαz or a scrambled (S) sequence (25 μg, 24‐h, N=5/group). Mean arterial pressure (MAP) and heart rate (HR) were recorded and was urine collected for 150‐min (10‐min periods).

Results

i.c.v. ODN pre‐ treatment (24h; 5 μg)
Peak Δ HR (bpm)
Peak Δ MAP (mmHg)
Peak Δ urine output (μl/min)
Peak Δ UNaV (μeq/min)

Saline Vehicle
−92 ± 9
−19 ± 2
+136 ± 9
+6.2 ± 0.8

S
−94 ± 12
−18 ± 1.2
+132 ± 11
+5.9 ± 0.6

Gαi1
−88 ± 11
−19 ± 2
+153 ± 16
+6.1 ± 0.8

Gαi2
−90 ± 13
−1 ± 1**
+125 ± 11
+0.6 ± 0.3**

Gαi3
−66 ± 9
−17 ± 1.3
+143 ± 16
+6.2 ±0.9

Gαo
−47 ± 5*
−16 ± 1.6
+127 ± 10
+6.2 ± 0.5

Gαq
−106 ± 7
−15 ± 2
+214 ± 7**
+7.2 ± 1.2

Gαz
−92 ± 8
−16 ± 2
+110 ± 6*
+5.8 ± 1.2

p<0.05,

p<0.01 vs. S ODN group

Brain Gαi2 subunit protein‐gated signal‐transduction pathways selectively mediate the hypotensive and natriuretic, but not bradycardic or diuretic, responses evoked by central α2‐adrenoceptor activation in vivo. In contrast, the profound α2‐agoinst‐stimulated diuretic response is mediated, in part, via opposing Gαz and Gαq subunit protein gated pathways (stimulatory versus inhibitory, respectively).

Conclusions

These studies demonstrate that in vivo, brain GPCR Gα‐subunit protein pathways play an essential and physiologically important gating role in mediating functionally selective changes in systemic cardiovascular function and the renal excretion of water and sodium post‐ligand binding at central α2‐adrenoceptors. RDW‐AHA 2250585; DRK‐DK43337, HL071212, AHA 0855293E, and RDW/DRK‐ P20 RR018766
Differential signaling of PAR1 and PAR4 through 12‐hLO
Authors: Yeung Jennifer , Apopa Patrick , Vesci Joanne , Holinstat Michael
Published: June 2021

Platelet activation plays a central role in the formation of a thrombus and uncontrolled platelet activation often leads to vessel occlusion and stroke. Thrombin mediates its action through 2 receptors, PAR1 and PAR4. Our lab has previously shown that PAR1 and PAR4 have unique signaling cascades while maintaining common biochemical endpoints. The relative importance of positive reinforcement on PAR‐mediated activation is unclear. We hypothesized that 12‐lipoxygenase‐mediated eicosanoid formation may differentially regulate the extent and stability of PAR‐induced platelet activation. To address this question, biochemical and physiological endpoints including platelet aggregation, granule secretion (dense and alpha), and integrin activation (αIIbβ3) were measured in the presence of selective 12‐hLO inhibitors. Inhibition of 12‐hLO resulted in a significant shift in platelet aggregation αIIbβ3 activation and a complete inhibition of dense granule secretion with a more significant effect being observed following PAR4 activation. Additionally, PAR4‐mediated α‐granule secretion was more sensitive to inhibition of 12‐hLO relative to PAR1. These observations support a central role for 12‐hLO and its metabolites in PAR‐mediated platelet activation with PAR4 being highly sensitive to perturbations in 12‐hLO activity. This work was supported by NIH grant HL089457 (to MH).
Thrombin stimulated glioblastoma cell proliferation is mediated by RhoA and phospholipase D dependent Rap1 activation
Authors: Sayyah Jacqueline , Nogal Nekeisha , Dusaban Stephanie
Published: June 2021

We previously reported that stimulation of PAR1 receptors with thrombin activates RhoA and induces mitogenesis in 1321N1 glioblastoma cells. Signaling through RhoA is known to be required for cell transformation by Ras. Another Ras family GTPase, Rap1, plays an important role in cancer and constitutive activation of Rap‐GEFs are associated with aberrant cell growth. Accordingly we asked whether stimulation of PAR1 receptors with thrombin elicits Rap1 activation in 1321N1 cells. We found that thrombin induces a robust and sustained (up to 6 hours) increase in Rap1 activation that is dependent on RhoA signaling. Since RhoA is known to activate phospholipase D (PLD) and diacyclglycerol generated via PLD metabolism can activate GEFs that regulate Rap1 activity, we hypothesized that PLD is a mediator of thrombin‐induced Rap1 activation. Inhibition of the PLD pathway by 1‐butanol and FIPI attenuated thrombin‐induced Rap1 activation and downstream ERK1/2 signaling in 1321N1 cells, indicating that PLD is required for thrombin stimulated Rap1 and ERK1/2 activation. Moreover, blockade of Rap1 activity in these cells significantly suppressed thrombin induced ERK1/2 phosphorylation and cell proliferation. Collectively, our results suggest that PLD is an upstream mediator of thrombin elicited Rap1 activation and that Rap1 signaling is required for mitogenesis in glioblastoma cells.

(NIH T32 AG000216‐16 grant)
Differential effects of Go and Gi2 on seizure threshold
Authors: Kehrl Jason , Stern Matt , Charbeneau Raelene , Neubig Richard
Published: June 2021

The inhibitory G protein family, Gi/o, play important roles in neuronal excitability and epileptic pathways. The Gi/o family may protect from seizures by inhibiting neurotransmitter release via inhibition of N‐type calcium channels and activation ofhyperpolerizing potassium channels. However, increased activation of the Gi/o‐coupled GABAB receptor produces absence seizures. The Gi/o family member responsible for each of these physiological effects has not been established. We aim to understand the role of Go and Gi2, two Gi/o family members, in altering seizure thresholds. We utilize mouse models with enhanced signaling through Gi2 and Go due to a genomic knock‐in (G184S) within the α subunit that prevents negative regulation by the Regulators of G protein Signaling. Mice with enhanced Gi2 signaling, but not Go, show reduced seizure severity and death after administration of the competitive GABAA antagonist bicuculline. In a kindling model with repeated injections of subthreshold doses of pentylenetetrazol (35mg/kg) Go G184S mice, but not Gi2 G184S mice, show a more rapid onset of tonic‐clonic seizures and/or death. Together these data indicate opposing roles for these inhibitory G proteins in setting the murine seizure threshold. Supported by RO1‐GM39561 (RRN) and 5T32GM008322 (JMK)
Gβγ‐activated phosphoinositide 3‐kinase γ regulates airway smooth muscle contraction by modulating calcium oscillations
Authors: Jiang Haihong , Abel Peter W , Toews Myron L , Deng Caishu , Casale Thomas B , Xie Yan , Tu Yaping
Published: June 2021

Airway smooth muscle (ASM) contractile hyper‐responsiveness plays a critical role in asthma. Muscarinic receptors in ASM convey bronchoconstrictor signals by dissociating G‐proteins into active Gα and Gβγ subunits. We found that expression of a Gβγ scavenger peptide suppressed acetylcholine (ACh)‐stimulated mouse ASM cell contraction. Gβγ plays prominent roles in signal transduction through its downstream effectors including PI3Kγ. Immunohistochemistry staining and Western blot indicated expression of PI3Kγ in ASM cells of mouse trachea, lung and cultured ASM cells. Inhibitors of PI3Kγ, but not PI3Kα, β, or δ, attenuated ACh‐stimulated airway contraction of mouse lung slices. It is noteworthy that airways in lung slices pretreated with PI3Kγ inhibitors still exhibited an ACh‐induced initial contraction, but the sustained contraction was significantly reduced. Furthermore, PI3Kγ inhibitors or silence of endogenous PI3Kγ by siRNA had a small inhibitory effect on the ACh‐stimulated initial Ca2+ transient in mouse ASM cells but significantly attenuated the sustained Ca2+ oscillations that are critical for sustained airway contraction. Thus, Gβγ/PI3Kγ signaling pathway controls contractility of airways through regulation of Ca2+ oscillations in ASM cells. Targeting this pathway may directly impact airway hyper‐responsiveness associated with asthma. Supported by Nebraska State LB595 and 692.
Spatiotemoporal regulation of Gβγ‐signaling and function by a novel Gβγ‐interacting protein, WDR26
Authors: Chen Songhai , Sun Zhizeng , Tang Xiaoyun
Published: June 2021

Heterotrimeric G protein βγ subunits play a critical role in many important cellular processes such as leukocyte migration. The essential role of Gβγ in directing leukocyte migration has been well established, but how it activates downstream effectors to generate a highly polarized intracellular signal that acts as an internal “compass” to drive directional cell migration remains unclear. Here, we characterized a novel interaction between Gβγ and WDR26, a WD40 protein that was identified from a yeast two‐hybrid screen and has unknown function. We showed that WDR26 associates with Gβγ following receptor activation, and is enriched at the leading edge of a polarized neutrophil. Inhibition of WDR26 expression by siRNAs abrogated directed leukocyte migration in vitro and T cell homing to lymphoid tissues in mice. Moreover, Gβγ‐mediated PI3K and PLCβ activation is alleviated by WDR26 suppression, suggesting that WDR26 is required for Gβγ signaling. Additional studies show that WDR26 binds and facilitates the activation of Gβγ effectors such as PLCβ2, probably by forming a large signaling complex containing both Gβγ and its effectors at the plasma membrane. Together, our data suggest that WDR26 is a novel interacting protein of Gβγ that may orchestrate spatiotemporal activation of downstream effectors of Gβγ required for precise control of directional cell migration.
β‐adrenergic receptors stimulate IL‐6 production in human bronchial smooth muscle cells: enhancement by non‐raft AC2 but not lipid raft AC6
Authors: Bogard Amy , Ostrom Rennolds
Published: June 2021

In human bronchial smooth muscle cells (hBSMC) various isoforms of adenylyl cyclase (AC) are localized to different compartments: AC6 in lipid rafts and caveolae, AC2 outside of rafts. This localization of AC isoforms associates them with different receptors, G proteins and downstream signaling proteins. We hypothesized that specific AC isoforms overexpressed in hBSMC might have differing effects on gene expression. We overexpressed AC2 and AC6 and measured a variety of genes whose expression is influenced by cAMP. Cells were treated with vehicle or the AC activator, forskolin (Fsk). Interleukin 6 (IL‐6) mRNA levels were enhanced by Fsk, particularly when AC2 was overexpressed. Fsk‐stimulated IL‐6 mRNA levels (qRT‐PCR) peaked in 1 hr while protein levels (ELISA) peaked at 24 hr. Agonists selective for Gs‐coupled GPCR were tested for their ability to stimulate IL‐6 in AC2 or AC6 overexpressing cells. Increases in cAMP and IL‐6 were observed in response to isoproterenol, PGE2, butaprost and beraprost. AC2, but not AC6, overexpression increased IL‐6 production by these agonists. Increasing cAMP levels via Gs/Fsk synergy led to no increase in IL‐6 as compared to agonists alone. These data imply that AC2 signaling can lead to IL‐6 production by hBSMC but that little correlation exists between cellular cAMP levels and the level of this pro‐inflammatory cytokine produced. Supported by NIH (HL079166).
Increased contractility in transgenic mice with overexpression of the targeting subunit B56a of PP2A
Authors: Kirchhefer Uwe , Boknik P. , Heinick A. , Kucerova D. , Neumann J. , Schmitz W. , Stümpel F. , Müller F. U.
Published: June 2021

Protein phosphatase (PP2A) is a heterotrimeric protein consisting of a catalytic (C), a scaffolding (A), and a regulatory (B) subunit. The B subunit anchors the C subunit to subcellular locations and therefore defines, at least in part, substrate specificity. To reveal new insights in the regulation of the main cardiac B subunit, we generated transgenic mice with heart‐directed overexpression of human B56α under the control of the α‐MHC promoter (TG). TG hearts exhibited a 2‐fold overexpression of B56α compared to wild‐type (WT) mice. This was associated with a 2‐fold higher protein expression of C (n=5, P<0.05). Consistently, total phosphorylase a‐PP activity was increased by 25% in TG (n=5, P<0.05). Overexpression of B56α reduced the efficacy of PP2A inhibition by okadaic acid (OA), a PP2A selective PP inhibitor, at low concentrations. The application of 0.3 nmol/L OA decreased total PP activity by 18% and 33% in TG and WT, respectively (n=5, P<0.05). The protein expression of Aα remained unchanged between TG and WT, as observed for PP1‐C. Force of contraction was increased after application of 10 μmol/L isoproterenol by 63% in TG compared to WT isolated left atria (n=6, P<0.05). We conclude that overexpression of B56α led to a higher contractility in TG after β‐adrenergic stimulation. Thus, our data suggest a critical role of PP2A targeting by B56α in the regulation of cardiac contractile function.

This study was supported by the DFG and the IMF Münster.