bluuebunny/crossref_metadata_2025_split huggingface dataset:
Ubiquitination of glycogen phosphorylase by the malin‐laforin complex regulates glycogen catabolism (663.17)
Authors: Dukhande Vikas , Paasch Bradley , Shaikh Nazeer , Gentry Matthew 
Published: June 2021

Lafora disease (LD) is fatal progressive myoclonic epilepsy characterized by pathological aberrant glycogen accumulations in brain and various other organs. Autosomal recessive mutations in genes encoding malin or laforin cause LD. Our group identified laforin as a novel glycogen phosphatase essential for maintaining glycogen in a soluble form. Malin, a RING‐type E3 ubiquitin ligase, is known to ubiquitinate proteins involved in glycogen metabolism such as laforin, glycogen synthase, protein targeting to glycogen and glycogen debranching enzyme. However, malin’s role in LD pathogenesis is not clear. In this study, we aimed to discover substrate/s of malin and its function in LD pathogenesis. We used malin‐ and laforin knockout mice, affinity pull‐down and mass spectrometry, immunoprecipitation, western analysis, fluorescence microscopy, glucan‐binding assay, and enzyme activity measurements. We discovered glycogen phosphorylase (GP) as a novel substrate of malin. GP is vital in catabolism of cellular glycogen. The expression and phosphorylation of GP was decreased in tissues from malin knockout mice. Similarly, overexpression of LD mutations of malin in HEK‐293 cells decreased GP expression. Overexpression of malin in HEK‐293 cells increased the endogenous expression of GP. Malin interacted with and ubiquitinated GP, and presence of malin increased nuclear localization of GP. Malin also altered glucan‐binding of GP. Thus, we have identified a novel control mechanism of glycogen catabolism. Further exploration into the mechanisms of this regulation will unravel insights in glycogen metabolism, physiological functions of the malin‐laforin complex and will aid in betterment of LD pharmacotherapy.Grant Funding Source: Supported by NIH grants R00NS061803, R01NS070899 and University of Kentucky College of Medicine star
Fullerene C60 chemically linked for biocompatible polymers for in vivo radical trapping (663.2)
Authors: Tegegn Cheregn , Megos Welay , Simpson Duane , Volkis Victoria 
Published: June 2021

Free radicals can be formed in living tissues under UV irradiation and/or when oxygen interacts with certain bio‐molecules. Once formed these highly reactive particles are capable to start number of chain reactions damaging living tissues and are believed to be a reason for some kinds of cancer. It is thus important to capture and study free radicals in live biological tissues and samples, which is complicated due to the short‐lived life span of free radicals. Mostly indirect methods focus on studying tissues affected by free radicals instead of direct observation and trapping of the latter. Here we present a method for the synthesis of biocompatible polymers based on polycyclic esters, in which fullerene C60, is chemically linked to the polymeric matrix. Fullerene is biocompatible and also known as a good radical scavenger capable to trap multiple small radicals from solutions. In such a way, radical trapping from living tissues and blood can happen on the surface of a biocompatible polymer which is in contact with physiological sample (for example as a filter built‐in into infusion device) allowing in‐vivo studying of free radical effects. In this project we first use the Diels‐Alder reaction to connect C60 to amino‐anthracene. Next we use the Schiff‐base or reductive amination reactions of carbonyl groups of poly‐cyclic esters with primary amines in order to link the fullerene radical trap to the polymeric matrix. Like pure fullerene C60 this biocompatible polymeric hook for free radicals may be used in tissues and physiological liquids in in‐vivo experiments. The synthesis, product isolation and characterization and preliminary trapping experiments will be presented.Grant Funding Source: Supported by Grant T34GM008411, from the National Institute of General Medical Sciences
Nrf2 mutation eliminates vasoprotective effects of low dose angiotensin II infusion in Sprague‐Dawley rats fed high salt diet (664.12)
Authors: Priestley Jessica , Weinberg Brian , Geurts Aron , Lombard Julian 
Published: June 2021

Nuclear factor erythroid‐derived 2‐like 2 (Nrf2) is a master transcriptional regulator of antioxidant and cytoprotective genes. In the Sprague‐Dawley (S‐D) rat, suppression of angiotensin II (ANG II) by high salt (4.0% NaCl) diet leads to endothelial dysfunction and oxidant stress, which can be prevented by low‐dose ANG II infusion to restore normal plasma ANG II levels. To understand the role of Nrf2 in maintaining normal vascular function, the MCW genomics group developed a colony of S‐D Nrf2 mutant rats using transcriptional activator‐like effector nuclease (TALEN) technology targeting the first exon of the Nrf2 gene. This resulted in a frame shift mutation of 43 base pairs, removing the initiation codon from the resultant mRNA. We hypothesized that this mutation would prevent the restoration of vasodilation phenotypes in salt‐fed rats by ANG II infusion. In isolated, cannulated middle cerebral arteries from salt‐fed rats, diameter change (ΔD) in response to 10 µM acetylcholine was restored in WT animals receiving ANG II (ΔD=9.67 ± 0.33 µm), but not in WT rats receiving saline infusion, or mutant animals receiving either ANG II or saline (ΔD=0.33 ± 0.67 µm, ΔD=2.00 ± 2.47 µm, or ΔD=1.75 ± 3.09 µm, respectively; p < 0.001 for each comparison). These data support the hypothesis that physiological levels of ANG II help preserve vascular function by preserving redox balance in an Nrf2‐dependent manner.Grant Funding Source: NIH HL065289
Orally‐delivered, tetrahydrobiopterin‐containing nanoparticles targeting gastrointestinal lymphatics modulate nitric oxide synthesis by aortic endothelial cells (666.4)
Authors: Meininger Cynthia , Stees Melinda , Kelly Katherine , Heaps Cristine , Wu Guoyao , Labhasetwar Vinod 
Published: June 2021

A deficiency of tetrahydrobiopterin (BH4), a critical cofactor for endothelial cell (EC) nitric oxide (NO) synthase, may be a common basis for vascular dysfunction in many diseases. Our goal was to modulate BH4 levels in ECs to increase NO bioavailability and improve vascular function in diabetes. We previously showed that BH4 encapsulated in poly(D,L‐lactide‐co‐glycolide) nanoparticles (NPs) and injected i.v. significantly improved vascular function in diabetic rats. In the current study, we synthesized solid lipid NPs (SLNPs) containing BH4. Our objective was to deliver BH4 to the systemic circulation via uptake and transit of SLNPs by the gastrointestinal lymphatics. SLNPs were administered via oral gavage once per day for 2 days (SLNPs) and endothelium‐dependent vessel relaxation assessed 24 hours later by measuring the response to increasing concentrations of acetylcholine. BH4 levels were determined using a reversed phase high‐performance liquid chromatography method. BH4‐loaded SLNPs taken up by cultured ECs increased cellular BH4 levels significantly, compared to vehicle/control NPs. Importantly, BH4‐loaded SLNPs increased vasodilatory responses of aortic rings taken from gavaged diabetic rats (maximal relaxation 75% vs. 55% in rats receiving control NPs). These data support the feasibility of oral NPs to deliver therapeutic agents to ECs in the general circulation via the lymphatic system.Grant Funding Source: Supported by AHA 11GRNT7930004 and NIH HL093689
Pannexin 1‐dependent ATP release from venous endothelium promotes acute vascular inflammation (669.8)
Authors: Lohman Alexander , Isakson Brant 
Published: June 2021

Tumor necrosis factor α (TNFα) activates endothelial cells (EC) promoting EC‐leukocyte interactions in the venous circulation which is mediated, in part, by purinergic signaling. However, it is currently not known if ECs release ATP to TNFα. Employing in vitro ATP release assays, we report that TNFα significantly increased ATP release from venous ECs (Human Umbilical and Saphenous) which was not observed in arterial ECs (Human Coronary and Aortic). ATP release was inhibited by WP9QY, a peptide antagonist of the TNF receptor 1 (TNFR1), indicating receptor activation in the initiation of this event. Pharmacological inhibition of Pannexin 1 (Panx1) channels in venous ECs with CBX or 10panx1 and siRNA knockdown of Panx1 significantly inhibited ATP release, whereas inhibition of connexin hemichannels, CALHM1 channels and exocytosis had no effect. This indicates that Panx1 channels are the conduit for ATP liberation. Additionally, we show that Src family kinases mediate Panx1 channel activation, as the Src inhibitor PP2 significantly reduced TNFα‐induced ATP release. Functionally, both Panx1 inhibition and degradation of extracellular ATP attenuated leukocyte‐venous EC interactions to TNFα stimulation. Utilizing ex vivo cannulated mesenteric venules, luminal perfusion of TNFα significantly increased ATP release into the perfusate, supporting a role for TNFα‐mediated ATP release in intact venules. Finally, we developed an EC‐specific, inducible Panx1 knockout mouse (VEcadCreERT2+/Panx1fl/fl) to determine the role of Panx1 in vascular inflammation in vivo. In conclusion, TNFα activates Panx1 channels in venous ECs inducing ATP release to promote leukocyte‐EC interactions.Grant Funding Source: Supported by NIH, AHA
Vasodilators of active hyperaemia effect one another’s action: potassium attenuates the vasodilation produced by nitric oxide and adenosine (677.15)
Authors: Lamb Iain , Murrant Coral 
Published: June 2021

Potassium (K+), nitric oxide (NO) and adenosine (ADO) are present contemporaneously during active hyperaemia, thus, we sought to test if interactions occur between these vasodilators. Using intravital microscopy of the hamster cremaster in situ, we assessed arteriolar diameter response to the presence of dilators added in succession or simultaneously. In succession experiments a dose response (10‐8–10‐4M) of NO donor, S‐nitroso‐N‐acetyl penicillamine (SNAP) was preformed and repeated after 5‐minute pre‐exposure and continuous presence of 10mM KCl. Separately, a dose response of KCl (10 and 20mM) was preformed and repeated after 5‐minute pre‐exposure and continuous presence of 10‐7M SNAP. A similar protocol was used using the remaining combinations of KCl, ADO and SNAP. In simultaneous experiments 10‐7M SNAP and ADO were applied independently, then each were simultaneously applied with 10mM KCl. In succession experiments KCl attenuated the dilatory response of both ADO and SNAP at all concentrations (average decreases of 43.2±9.7% and 39.8±11.7%, respectively). In simultaneous experiments KCl reduced the dilatory response of SNAP and ADO (by 69.1±3.7% and 75.1±7.1%, respectively), KCl‐induced attenuation occurred after 5 and 7 minutes for SNAP and ADO, respectively. Therefore, K+ reduces dilatory action of ADO and NO implying dilators released during active hyperaemia interact.Grant Funding Source: Supported by NSERC Canada
Sex differences in the fetal programming of hypertension: role of adrenal phenylethanolamine N‐methyltransferase gene expression (680.21)
Authors: Khurana Sandhya , Grandbois Julie , Nguyen Phong , Peltsch Heather , Lo Suet , Venkataraman Krishnan , Tai T.C. 
Published: June 2021

Prenatal exposure to high levels of glucocorticoids (GCs) predisposes to hypertension in later life. GCs regulate catecholamine biosynthesis and are critical for blood pressure homeostasis. This study examined whether prenatal exposure to elevated GCs influences the development of adult hypertension via alterations in phenylethanolamine N‐methyl transferase (PNMT), a gene that is linked to hypertension. Pregnant Wistar‐Kyoto dams were injected with 100 μg/kg/day of the synthetic GC dexamethasone (DEX) in their third trimester. Blood pressure and weights of the offspring were measured from week 3‐18, at which point the animals were sacrificed. Total RNA and protein were extracted from the adrenals, and the expression of PNMT and its regulatory transcription factors analyzed by qPCR and Western blot. Results reveal that in utero exposure to DEX promotes the development of hypertension in the offspring. A more pronounced increase in blood pressure was seen in males than females. The mRNA transcripts for PNMT, and its transcriptional regulators Egr‐1, Sp1, GR and AP‐2, were elevated in the adrenals of these animals compared to saline controls, with differences in both sexes. Preliminary Western data also shows upregulation of PNMT and the transcription factor Egr‐1. These results suggest that prenatal GC exposure increases adrenal PNMT gene expression via altered transcriptional regulatory mechanisms.Grant Funding Source: Supported by CIHR
Dietary NaCl alters several sympathoexcitatory and sympathoinhibitory reflexes: dependence on the ventral lamina terminalis (683.4)
Authors: Simmonds Sarah , Stocker Sean 
Published: June 2021

Elevated dietary NaCl intake enhances both sympathoexcitatory and sympathoinhibitory responses evoked from the rostral ventrolateral medulla. These enhanced responses are prevented by prior lesion of the LT. The purpose of the present study was to test whether increased dietary NaCl intake enhanced a number of sympathetic reflexes dependent on neurotransmission in the rostral ventrolateral medulla and whether this was prevented by LT lesion. Male Sprague‐Dawley rats were fed 0.1% (low) or 4.0% (high) NaCl for 14‐18 days. Electrical stimulation of sciatic afferent nerves (1ms, 1‐20Hz, 500 uA) produced greater increases in renal SNA (5 Hz: 125±9% vs 187±23%, n=6/group) and mean ABP (5Hz: 14±2mmHg vs 28±2mmHg, n=6/group) of rats ingesting high versus low NaCl, respectively. LT lesion prevented the enhanced responses of renal SNA (low: 129±13% vs high: 144±13%, n=3/group) and mean ABP (low: 17±2mmHg vs high: 14±5mmHg, n=5/group). Stimulation of the aortic depressor nerve (1ms, 1‐20 Hz, 500 uA) produced greater decreases in renal SNA (low: ‐53±10% vs high: ‐74±7%, n=6/group) and mean ABP (low: ‐16±3mmHg vs high: ‐32±3mmHg, n=6/group) of rats fed low versus high NaCl diets. Again, LT lesion prevented the enhanced responses of renal SNA (low: ‐58±10% vs high: ‐59±6%, n=3/group) and mean ABP (low: ‐18±5mmHg vs high: ‐15±3mmHg, n=3/group). Similar findings were observed for additional reflexes including activation of vagal afferents and intracerebroventricular infusion of 1M NaCl. These findings suggest dietary salt activates LT neurons to alter the responsiveness of hindbrain neurons and enhance sympathetic reflexes.Grant Funding Source: Supported by NIH R01HL113270 and AHA Established Investigator Award
Chronic isometric strength training reduces anxiety‐like behavior in Wistar rats (684.15)
Authors: Conceicao Rodrigo , Laureano‐Melo Roberto , Almeida Claudio , Pinto Guilherme , Cortes Wellington 
Published: June 2021

The objective of this study was to assess the anxiety‐like behavior in rats subjected to isometric strength training. For this purpose, the animals were randomized into 2 groups (n=10): control group and trained, the trained rats undergone a 8 week of isometric strength training. At the end of the training period, the rats underwent a behavioral assessment in an open field test, elevated plus maze test and marble burying test. Then, the animals were euthanatized and blood was collected for determination of serum corticosterone by radioimmunoassay method. Statistical analysis was performed by t student test, considering the significant differences between means at p <0.05. According to the results, in the elevated plus maze, it was demonstrated that trained group had higher time spent in the open arms (302%, p = 0.007) and greater percentage of entries into the open arms (288%, p = 0.007). Moreover the serum levels of corticosterone were significantly lower in the trained group (41%, p = 0.005). Together, these results suggest that to isometric strength training improves anxiety regulation by engaging inhibitory mechanisms related to stress responses.Grant Funding Source: FAPERJ/CNPq
IL‐1β microinjections into the nucleus tractus solitarius in rodents alter cardiorespiratory patterning consistent with endotoxemia (686.19)
Authors: Hsieh Yee‐Hsee , Nethery David , Jacono Frank , Dick Thomas 
Published: June 2021

Endotoxemia increases heart (HR) and respiratory (fR) rates and disrupts cardio‐respiratory coupling (CRC). We previously identified expression of proinflammatory cytokines in the nTS during endotoxemia which was associated with increases in HR and fR, as well as uncoupling of cardio‐respiratory patterns. We hypothesized that microinjection of the proinflammatory cytokine IL‐1β into the nTS would increase HR, fR, and alter cardio‐ventilatory coupling (CVC), defined as the significant tendency for a heartbeat to occur at a preferred latency prior to the next breath. To test this hypothesis, we injected IL‐1β (0.5 ng, n=8) bilaterally into the nTS of anesthetized (isoflurane) spontaneously breathing adult male Harlan Sprague‐Dawley rats (n=12) and recorded diaphragmatic electromyogram (EMG) and electrocardiogram (ECG). 2h after focal injections of IL‐1β, fR increased 32% (from 42.9 ± 2.2 to 57.0±15 brths/min; p<0.05). CVC was analyzed with custom software and χ2 from multi‐ordered histograms was calculated. 2h post IL1‐β injection, the average χ2 value reduced from 40.76 ± 5.9 to 15.91 ± 3.6 (p<0.001) indicating loss of CVC. We conclude that exogenous IL‐1β injections into the nTS (reduced model of brainstem inflammation) reproduces some of the cardiorespiratory changes observed in experimental endotoxemia, and may contribute to altered cardio‐respiratory coupling.Grant Funding Source: T32 HL007913, NS069220, VA I01BX000873
Impaired PVN neuronal activity in response to acute sodium challenge drives persistent elevations in MAP in conscious rats lacking CNS Gαi2 proteins (686.6)
Authors: Carmichael Casey , Wainford Richard 
Published: June 2021

Aim: We have reported CNS Gαi2 proteins mediate the sympathoinhibitory responses to chronic high salt‐intake. These studies assessed the role of CNS Gαi2 proteins in the activity of PVN neurons in response to acute sodium challenge.Methods: 24‐h ICV Gαi2 or SCR oligodeoxynucleotide (ODN; 25μg/5μl)‐pretreated conscious Sprague‐Dawley rats were monitored for changes in MAP in response to IV bolus NaCl (3M; 0.14 ml/100g). Separate groups of rats were sacrificed at time of the peak MAP change (40 min post‐IV NaCl) and Fos IHC was performed (N=6/gp).Results: No difference was observed in sodium‐evoked peak change in MAP between treatment groups (IV 3M NaCl ΔMAP [mmHg] SCR: 13±4 vs. Gαi2: 10±3), however MAP returned to control levels by 100 min in SCR but not Gαi2 treated rats (MAP 100min post‐IV NaCl [mmHg] SCR: 134±2 vs. Gαi2: 146±3, P<0.05). In SCR, but not Gαi2, treated rats, an IV NaCl bolus produced significant reduction in Fos immunoreactivity ([PVN Fos positive cells] SCR control: 95±9 vs. post NaCl: 5±1, p<0.05, Gαi2 control 90±13: vs. post NaCl: 66±9).Conclusion: These data reveal a novel role of CNS Gαi2 proteins in the regulation of the neuronal response within the hypothalamic PVN to an acute sodium challenge that significantly elevates MAP. The sodium‐induced decrease in Fos‐positive PVN cells in SCR ODN rats likely reflects inhibition of neuronal activity facilitating the return of MAP to basal levels. In Gαi2 pretreated rats, failure to inhibit PVN neuronal activity represents a neural mechanism by which impairment of Gαi2 signal‐transduction pathways may contribute to the pathophysiology of salt‐sensitive hypertension.Grant Funding Source: Supported by RO1 HL107330, K02 HL112718, & T32 GM008541
Coenzyme Q supplementation or over‐expression of the yeast Coq8 putative kinase stabilizes multi‐subunit Coq polypeptide complexes in yeast coq null mutants (693.17)
Authors: He Cuiwen , Xie Letian , Allan Christopher , Clarke Catherine 
Published: June 2021

Coenzyme Q biosynthesis in yeast requires a multi‐subunit Coq polypeptide complex. Deletion of any one of the COQ genes leads to respiratory deficiency and decreased levels of the Coq4, Coq6, Coq7, and Coq9 polypeptides, suggesting that their association in a high molecular mass complex is required for stability. Over‐expression of the putative Coq8 kinase in certain coq null mutants restores steady‐state levels of the sensitive Coq polypeptides and promotes the synthesis of late‐stage Q‐intermediates. Here we show that over‐expression of Coq8 in yeast coq null mutants profoundly affects the association of several of the Coq polypeptides in high molecular mass complexes, as assayed by separation of digitonin extracts of mitochondria on two‐dimensional blue‐native gels. The Coq4 polypeptide persists at high molecular mass with over‐expression of Coq8 in coq3, coq5, coq6, coq7, coq9, and coq10 mutants, indicating that Coq4 is a central organizer of the Coq complex. Supplementation with exogenous Q6 increased the steady‐state levels of Coq4, Coq7, Coq9, and several other mitochondrial polypeptides in select coq null mutants, and also promoted the formation of late‐stage Q‐intermediates. Q supplementation may stabilize this complex by interacting with one or more of the Coq polypeptides. The stabilizing effects of exogenously added Q6 or over‐expression of Coq8 depend on Coq1 and Coq2 production of a polyisoprenyl intermediate. Based on the observed interdependence of the Coq polypeptides, the effect of exogenous Q6, and the requirement for an endogenously produced polyisoprenyl intermediate, we propose a new model for the Q‐biosynthetic complex, termed the CoQ‐synthome.Grant Funding Source: NIH GM45952, Ruth L. Kirschstein National Research Service Award GM007185, and USPHS National Resear
Short distance transportation stress in the adult domestic chicken as modulated by pretreatment with ascorbic acid and α‐tocopherol (693.18)
Authors: Azeez Odunayo , Oyagbemi Ademola , Oyewale Johson 
Published: June 2021

The effects of short distance transportation stress and pretreatment with non‐enzymic antioxidants ‐ ascorbic acid and α‐tocopherol on the haematology, erythrocyte osmotic fragility (EOF), Total plasma protein (TP), H2O2 generation, Malondialdehyde (MDA) and glutathione (GSH) levels were investigated using the domestic chicken.Thirty‐two, adult female domestic chickens of the Nera black strain were used. The birds were divided into four groups A‐D of 8 birds each. Groups A and B did not receive any medication. Group C received ascorbic acid (650mg/kg diet) while D, α‐tocopherol (270mg/kg diet) in feed for 2 weeks. Birds in groups B, C and D were transported through a distance of 200km. Blood samples were collected within 30 minutes after the journey for analysis. Haematocrit (PCV), red blood cell counts (RBC), haemoglobin, erythrocyte indices (MCV, MCH and MCHC) and EOF, TP, H2O2, LP and GSH levels were evaluated after transportation.Group B had lower EOF than the control, while those pretreated with Vitamins C and E were higher. Haemoglobin and MCHC values were higher in Group B while other haematological parameters were similar. There were no significant differences in the total protein, H2O2, MDA and GSH levels in Group B compared with the control. Group C however, had higher H2O2 and MDA levels but lower TP and GSH than the control. Whereas, Group D had higher H2O2 and GSH levels but lower protein than the control.This study demonstrated that nucleated erythrocytes of the domestic chicken respond to acute stress by increase in osmotic resistance and membrane stability in hypotonic solution, this was antagonized by pretreatment with Vitamins C and E. Furthermore, short distance transportation may not increase oxidative stress and lipid peroxidation in the chicken. Prolonged use of the antioxidants especially ascorbic acid may however lead to oxidative stress