bluuebunny/crossref_metadata_2025_split huggingface dataset:
Alpha‐2 Adrenergic Receptor Homology Models using Rhodopsin or Beta‐2 Adrenergic Receptor Templates
Authors: Montgomery Megan D , Bylund David B 
Published: June 2021

Alpha‐2 adrenergic receptor agonists are used as adjuncts to general anesthetics and in the pharmacological treatment of many disorders, including hypertension and attention‐deficit hyperactivity disorder. Clinically available alpha‐2 agonists are not subtype‐selective and their use is limited by adverse effects resulting from actions at non‐targeted subtypes. To develop subtype‐selective ligands for alpha‐2A, ‐2B, or ‐2C receptors, more binding site structural information is needed; however, the only high‐resolution crystal structures of GPCRs are of bovine rhodopsin and modified human beta‐2 and turkey beta‐1 adrenergic receptors. Our goal was to develop homology models of the alpha‐2 receptor subtypes, using either the rhodopsin or beta‐2 receptor crystal structure as a template, that are capable of identifying characteristics important in ligand binding. Alpha‐2A and ‐2C amino acid sequences were each aligned with each template sequence and homology models were created using Sybyl7.1. Fifteen alpha‐2 agonists, including catecholamines and imidazoline derivatives, were docked into the models using FlexiDock. A comparison of calculated binding energies with experimental binding affinities resulted in poor correlations in models of alpha‐2A (r2 = 0.396 and 0.606 for rhodopsin‐ and beta‐2‐based, respectively) and alpha‐2C (r2 = 0.349 and 0.338).Supported by a gift from Procter and Gamble.
Unusual reactions to 5‐HT3 receptor antagonists in a child with rhabdomyosarcoma
Authors: Lee Candace Y.W. , Ratnapalan Savithiri , Thompson Margaret , Nathan Paul , Closs Jordan , French Amy , Punnett Angela , Garcia‐Bournissen Facundo , Ito Shinya , Koren Gideon 
Published: June 2021

Ondansetron and granisetron are 5‐HT3 receptor antagonists commonly used for prevention of chemotherapy‐induced nausea and vomiting. Rare acute dystonic reactions to ondansetron have been reported previously. We report a case of abnormal facial movements associated with granisetron.A boy with prostatic rhabdomyosarcoma and lung and bone metastases developed abnormal facial movements while receiving ondansetron 4 mg po q8h (5 mg/m2/dose). These movements consisted of involuntary blinking of the eyes, pursing of the lips, forward thrusting of the jaw and protrusion of the tongue. After excluding other potential causes, the diagnosis of ondansetron‐induced movement disorder was made. These movements decreased when the dose of ondansetron was reduced and resolved with its discontinuation. On admission for a subsequent cycle of chemotherapy, the patient was treated with granisetron 480 µg iv q12h (20 µg/kg/dose). On day 3, the patient developed repeated forward thrusting of the jaw and simultaneous blinking of the eyes, which were self‐limiting and less severe than those associated with ondansetron use. As vomiting was effectively prevented, granisetron was continued until completion of therapy. Health professionals need to be aware of these possible, albeit rare, reactions to ondansetron and granisetron in paediatric oncology patients.Supported by Hospital for Sick Children Research Institute (CL, GK)
Platelets enhance neutrophil transendothelial migration
Authors: Lam Fong , Burns Alan , Smith C. Wayne , Rumbaut Rolando 
Published: June 2021

Platelets are increasingly recognized as important mediators of inflammation in addition to thrombosis. While platelets have been shown to promote neutrophil (PMN) adhesion to endothelium in various inflammatory models, it is unclear whether platelets enhance neutrophil transmigration across inflamed endothelium. To test this hypothesis, we studied the effects of platelets on PMN transendothelial migration both in vitro and in vivo, using interleukin‐1 β‐stimulated human umbilical vein endothelial cells (HUVEC), and a well‐characterized corneal injury model in C57BL/6 mice. In the HUVEC model, PMN in the presence of platelets demonstrated enhanced transendothelial migration under stationary conditions (% transmigration at 500 sec was 57±6% vs. 33±5%; p<0.01). Further, in a parallel‐plate chamber under physiological flow conditions, platelets increased the speed of PMN transmigration following firm adhesion (duration of transmigration was 34.4±2.7 vs. 51.4±2.9 sec; p<0.01). In vivo, platelet depletion reduced neutrophil emigration from limbal vessels 60 minutes after injury (280±12 vs. 513±44 PMN; p<0.05). These data support the notion that platelets enhance neutrophil migration across inflamed endothelium both in vitro and in vivo. The mechanisms involved remain to be elucidated.Support: T32 HL07747‐15; RO1 EY018239
A Murine Model of Acute Hindlimb Ischemia/Reperfusion Injury
Authors: TU HUIYIN , LI YULONG , TRAN PAUL 
Published: June 2021

Introduction15% of preventable combat fatalities are due to hemorrhage from extremity wounds, making emergency tourniquet one of the first‐line treatments. While lifesaving, tourniquets can cause serious ischemia‐reperfusion (IR) injury. Fundamental to IR injury is mitochondrial production of superoxide. We refined a murine model of acute hind limb IR to simulate the clinical use of tourniquet.MethodsGroups of C57/BL6 mice underwent an IR protocol of three hours of ischemia and four hours of reperfusion. IR was achieved by placement and release of a rubber tourniquet around the hind limb, at the greater trochanter level. Muscle blood flow (MBF) was measured using a Doppler probe. Muscle necrosis was measured using TTC stain. Superoxide production was measured using chemiluminescence and DHE staining. MnSOD protein expression and activity were measured using Western blot and a modified nitrite method. Mitochondrial respiratory chain function was measured via spectrophotometry.ResultsSignificant ischemia and microcirculatory dysfunction were achieved: MBF during IR was < 10% and < 41% of baseline, respectively (n=8), accompanied by >40% necrosis (n=9). Superoxide production was increased (0.05 vs 0.13, p<.05 sham vs IR)accompanied by concomitant increases in MnSOD activity and protein expression (1.51 vs 2.26 , p<.05,sham vs IR, n=8). Mitochondrial complexes I‐IV showed signs of dysfunction (Complex I‐ IV: 0.13, 0.31, 1.37, 2.76 sham vs 0.08, 0.21, 0.7, 1.97 IR, p<.05 ).ConclusionsWe have demonstrated an animal model of clinical tourniquet use. IR produces significant mitochondriopathy which is associated with elevated superoxide production and antioxidant activity. The model can be used to mechanistically evaluate antiischemic molecules in efforts to minimize the morbidity and mortality of battlefield injury.
Characterization of VIP‐induced contractile inhibition in lymphatic vessels
Authors: von der Weid Pierre‐Yves , Rehal Sonia , Dyrda Peter 
Published: June 2021

Lymphatic vessels actively remove interstitial fluid through rhythmical, phasic contractions of the muscle layer in the vessel wall. This activity is critical during inflammation to prevent excessive edema. Importantly, lymphatic pumping is modulated by inflammatory mediators. In particular, it is inhibited by vasoactive intestinal peptide (VIP), a neuro‐ and immuno‐modulator with anti‐inflammatory properties, detected in lymphatic innervation and in immune cells patrolling the lymph. Using electrophysiology, pharmacology and molecular biology techniques we aimed at characterizing the mechanisms involved in the inhibitory effect of VIP in lymphatic vessels of the guinea pig mesentery. RT‐PCR analysis revealed expression of VPAC1, VPAC2 and PAC1 receptor mRNAs in lymphatics. VIP‐induced pumping inhibition was shown to be concentration‐dependent (EC50 15 nM) and accompanied by a hyperpolarization (range 4‐15 mV, n=4‐22). The PAC1 antagonist PACAP(6‐38) most effectively blocked VIP‐induced responses (n=4). VIP‐induced responses were not affected by nitric oxide synthase and cyclooxygenase inhibitions (n=4), but depend on protein kinase A and KATP channel activation (n=4‐9). This inhibitory action of VIP needs to be considered during inflammation since high VIP concentrations in the vicinity of lymphatics may compromise lymph drainageSupported by the Crohn's and Colitis Foundation of Canada
Temperature controlled small animal platform for in vivo microcirculation studies
Authors: Scheider Linda , Berger Brian , Torres Filho Ivo 
Published: June 2021

Mouse models are becoming more representative of human studies with the complete mapping of the mouse genome. Anesthetized mice may easily become hypothermic and exposed tissues are highly affected by environmental temperature changes. In certain applications, superfusion is not necessary: a dry, compact, inexpensive, temperature controlled platform with a clear viewing field is needed for these studies. We describe two designs for animal platforms with these characteristics. The platforms include a body heater and heated pedestal for tissue observation under an upright microscope. In the fixation area for the exposed tissue, a metal cylinder heats an isolated water column. The platforms use feedback sensors and controllers to maintain a constant body temperature. One platform is very compact and is about 13 mm in height for optimal distance between the substage condenser and the microcirculation. The second is slightly taller but uses all readily available inexpensive parts for easy assembly. These platforms are ideal for studies with transillumination of small animal microcirculation. Testing of the platforms showed constant temperatures were held with time and the components are durable and reliable.SupportAD Williams Trust Funds, VCU.
Resonance Raman spectroscopy of human sickle cell hemoglobin from transgenic mice
Authors: Torres Filho Ivo P , Terner James , Scheider Linda 
Published: June 2021

We have previously developed a methodology to measure hemoglobin (Hb) oxygen saturation using Raman spectroscopy in vivo (AJP 289:H488, 2005; JAP 104:1809, 2008). However, the technique has not yet been used in sickle cell disease (SCD). We have established a colony of transgenic (Tg) SCD mice. This strain no longer expresses mouse Hba and Hbb, but express human sickle hemoglobin (HbS). It mimics the genetic, hematologic and histopathologic features found in humans with SCD. Whole blood samples were obtained from anesthetized Tg mice and normal control mice. Raman spectra from these samples were obtained in vitro using the exciting wavelengths in the range of 405 ‐ 532 nm emitted by a tunable laser. The scattered light was sampled at 90 degrees using a 0.5 m spectrometer and a nitrogen cooled CCD detector. In order to induce HbS polymerization, Raman spectra were collected from blood samples exposed to high and low oxygen environments. The findings suggest that strong Raman signals can be obtained from normal and SCD blood, opening the possibility for in vivo noninvasive measurements of hemoglobin Hb and HbS saturation in the microcirculation. The resonance Raman technique should provide additional worthwhile microscopic information in the study of the abnormal hemoglobins in vivo.SupportVCU.
Superoxide – NO interaction plays an important role in inhibition of hypoxic pulmonary hypertension by hypercapnia
Authors: Chovanec Milan , Hnilickova Olga , Smolkova Pavlina , Herget Jan 
Published: June 2021

Superoxide, nitric oxide (NO) and peroxynitrite play a pathogenetic role in the development of hypoxic pulmonary hypertension (HPH).Rats exposed to normobaric hypoxia with hypercapnia (10% O2+4.5% CO2, n=7) for 3 weeks had significantly lower (22.9 vs. 30.0 mmHg, p<0.01) PAP and right ventricle weight (RV/LV+S ratio, 34.4 vs. 54.0, p<0.0001) compared to rats exposed to hypoxia with no hypercapnia (10% O2, n=9).We tested the hypothesis that superoxide ‐ NO interaction plays role in inhibition of HPH by hypercapnia. We measured the expired amount of NO of living rats and the level of NOx in plasma by the chemiluminescent method and the plasma concentration of nitrotyrosine (a marker of peroxynitrite) by ELISA on male adult Wistar rats exposed to normobaric hypoxia ‐ 10% O2 (group H, n=6) or normobaric hypoxia ‐ 10% O2 with hypercapnia ‐ 4.5% CO2 (group H+CO2, n=6) for 4 days. Group N (n=6) were normoxic controls. The amount of expired NO (689.3 vs. 889.2 pg.min−1, p<0.05), the level of NOx in plasma (57.4 vs. 97.2 µM, p<0.01) and the plasma concentration of nitrotyrosine (7.9 vs. 9.9 µM, p<0.05) were significantly decreased in group H+CO2 compared to group H.Superoxide ‐ NO interaction plays an important role in inhibition of HPH by hypercapnia.Supported by Cardiovascular Research Centre MSMT 1M 0510 and Grant: GAUK 7780/2007
NATURALLY‐OCCURRING MISSENSE MUTATIONS IN THE CANINE CFTR GENE
Authors: Spadafora Domenico , Hawkins Eleanor C , Ballard Stephen T 
Published: June 2021

Naturally‐occurring, cystic fibrosis‐causing mutations in the CFTR have not been identified in any non‐human animal species. Since domestic dogs are known to develop medical conditions associated with atypical CF in humans, (e.g. idiopathic bronchiectasis, pancreatitis, and aplasia of the vas deferens), we hypothesized that some dogs also carry CFTR mutations. Temporal temperature‐gradient gel electrophoresis (TTGE) was used to screen dogs for CFTR mutations. Canine whole blood was obtained from veterinary clinics and diagnostic laboratories. We screened DNA from 290 dogs that fell into one of three categories: dogs diagnosed with pancreatitis, dogs diagnosed with bronchiectasis, and dogs admitted to clinics for any illness (at‐large dogs). Thus far, 24 dogs have been identified with one of four missense mutations: R812W, P1281T, R1456W and P1464H. P1281T and P1464H mutations occur in relatively unconserved residues. R1456W is analogous to the human R1453W mutation, which has about 20% of normal CFTR function and is associated with pancreatitis and panbronchiolitis. R812W is in a highly conserved region of the regulatory domain. Mutation frequencies in pancreatitis and bronchiectasis animals were found at about twice the frequency of at‐large dogs. We conclude that naturally occurring CFTR mutations are relatively common in domestic dogs and can be detected with TTGE.Supported by NIH HL633002 and Cystic Fibrosis Foundation grant BALLAR07G0
Mechanotransduction and signaling in wounded airway epithelial cells
Authors: Waters Christopher M. , Desai Leena P. , White Steven R. 
Published: June 2021

Epithelial repair following injury to the airways occurs upon a substrate undergoing cyclic mechanical distention. The purpose of this study was to examine how mechanical stretch alters signaling pathways involved in wound healing of airway epithelial cells. We used a scratch‐wound model of human bronchial epithelial cells (16HBE) subjected to cyclic stretch (20% strain, 30 cycles/min) using the Flexercell device. As demonstrated previously, stretch caused inhibition of wound closure over 9 hr. We also found decreased PI3‐kinase activity, decreased membrane‐associated Tiam‐1, and decreased Rac1 activity in stretched cells. Adenoviral expression of a constitutively active form of PI3‐kinase stimulated wound closure of cyclically stretched cells, but not to the level of unstretched cells. Since stretch also decreased focal adhesion kinase (FAK) phosphorylation, we co‐expressed FAK, constitutively active PI3‐kinase, and JIP3 in stretched cells, and restored wound closure to the level of unstretched cells. Mechanical stretch alters multiple pathways that are important in restoration of epithelial integrity.Supported by NIH grants HL064981 (CMW) and HL080417 (SRW).
Voluntary aerobic exercise abolishes age‐associated arterial stiffening in mice: relation to collagen subtype expression in the medial and adventitial layers
Authors: Fleenor Bradley S , Lesniewski Lisa A , Durrant Jessica R , Connell Melanie L , Donato Anthony J , Seals Douglas R 
Published: June 2021

Voluntary aerobic exercise reduces large elastic artery stiffness with aging in humans, but it is unknown if reductions in collagen subtypes in the medial and/or adventitial layers of the arterial wall could be involved. Young (Y, 7 mo) and old (O, 31 mo) adult B6D2F1 mice underwent cage‐control (C) or voluntary wheel running (VR) for 12 weeks with carotid artery in vitro stiffness measures (n=9‐10/group) and immunohistochemical assessment of collagen (n=4‐8/group). VR (OVR: 3.7 ± 1.1 AU) abolished the age‐associated increase in passive stiffness in C animals (YC: 5.3 ± 0.8 vs. OC: 8.2 ± 1.0, P < 0.05). Collagen type I and III densities were 40‐55% greater in adventitia of OC vs. YC (P < 0.05), whereas OVR mice had medial and adventitial levels < OC (P < 0.05) and < YC. Collagen type IV was 30‐70% greater in media and adventitia of OC vs. YC (P < 0.05), whereas OVR had medial and adventitial levels < OC (P < 0.05) and < YC. Relative stiffness was not different in YC vs. YVR, and collagen expression was similar or lower in YVR. Short‐term voluntary aerobic exercise started late in life abolished the increase in carotid artery stiffness observed with sedentary aging in mice. Our data are the first to suggest a role for adventitial collagen deposition in age‐associated arterial stiffening, and that regular exercise may reduce stiffness in old mice in part by reducing collagen I, III and IV in the medial and adventitial layers of the arterial wall.NIH AG013038, AG006537, AG015897, AG029337, AG000279
Chronic aerobic exercise opposes age‐ and high fat diet‐associated vascular endothelial dysfunction: relation to IKKβ and AMPK
Authors: Durrant Jessica R , Folian Brian J , Connell Melanie L , Russell Molly J , Seals Douglas R , Lesniewski Lisa A 
Published: June 2021

Age and high fat, ie. Western, diet (WD) are associated with inflammation and impaired endothelium‐dependent dilation (EDD), whereas aerobic exercise has opposite effects. Activation of the pro‐inflammatory signaling molecule inhibitor of kappa B kinase beta (IKKβ), as modulated by adenosine monophosphate activated protein kinase (AMPK), may contribute to these differential effects. Old (O: 29‐31 mo) B6D2F1 mice fed either normal chow (NC) or WD (40% fat, 8 weeks) either were sedentary (SED) or performed voluntary wheel running (VR). In SED, carotid artery EDD to acetylcholine was reduced after WD (max dilation: 62 ± 8 vs. NC: 76 ± 2%, P < 0.05) and this was associated with increased aortic IKKβ activation (phosphorylated (pSer177/181):total IKKβ: 2.1 ± 0.8 vs. 0.5 ± 0.1 AUs, P = 0.01) and reduced AMPK activation (pThr172:total AMPKα: 0.7 ± 0.1 vs. 1.0 ± 0.1 AUs, P = 0.05). VR improved EDD in both NC (96 ± 2%, P < 0.01) and WD (91 ± 7%, P < 0.05) fed mice, while reducing IKKβ activation by ~60% (P < 0.01 in NC, P = 0.1 in WD) and increasing AMPK activity by 30‐40% (P = 0.1 in NC, P < 0.05 in WD). Here we demonstrate that habitual voluntary aerobic exercise may reverse age‐ and WD‐associated vascular endothelial dysfunction by increasing AMPK activity and inhibiting IKKβ‐linked pro‐inflammatory signaling.NIH AG013038, AG006537, AG015897, AG022241, AG000279
CDK‐dependent assembly of replication proteins at the initiation step of chromosomal DNA replication
Authors: Araki Hiroyuki , Hirai Kazuyuki , Li Yan , Tanaka Tamon , Muramatsu Sachiko , Tanaka Seiji 
Published: June 2021

Replication proteins assemble onto origins in two successive steps; formation of the pre‐RC in late M and G1 phases and assembly of other replication proteins including DNA polymerases to initiate DNA synthesis when CDK is activated from G1/S boundary. Sld2 and Sld3 are phosphorylated by CDK and bind to N‐terminal and C‐terminal tandem BRCT domains of Dpb11, respectively. Their interactions are essential and sufficient for the CDK‐dependent initiation of chromosomal DNA replication. However, how these interactions promote initiation of chromosomal DNA replication has not been elucidated.We have argued that the CDK‐dependent interaction between Sld2 and Dpb11 facilitates formation of the pre‐LC containing Dpb11, Sld2, Polε and GINS. The pre‐LC is a fragile complex that is detected only with the aid of a cross‐linking agent in vivo and is formed in a CDK‐dependent and pre‐RC‐independent manner. We can now reconstitute the pre‐LC from purified proteins. To recruit replication proteins, such as GINS and Polε, to origins, the pre‐LC may bind to phosphorylated Sld3, which associates with origins in a Cdc45‐dependent manner. Moreover, Sld3 seems to facilitate formation of an active replicative helicase, the CMG complex consisting of Cdc45, MCM and GINS. We, thus, propose that the interactions between Sld2, Sld3 and Dpb11 recruit replication proteins to origins and remodel them to form an active helicase at origins.
Inhibition of ataxia telangiectasia mutated (ATM) prevents the prolonged increase in phosphorylation of Akt substrate of 160 kDa (AS160) subsequent to activation of the AMP‐activated protein kinase (AMPK)
Authors: Lewis LaQuinta R. , Davis Jennifer , Akinola Ayotola , Fisher Jonathan S. 
Published: June 2021

We hypothesized that activation of AMPK would result in a lingering increase in AS160 phosphorylation (P‐AS) and that inhibition of ATM, a reported regulator of AMPK and Akt, would prevent this effect. L6 myotubes were incubated for 30 min with DMSO (vehicle) or the ATM inhibitor KU55933 (KU) at a concentration (1 μM) that would inhibit ATM but would be unlikely to interfere with the related kinases PI3K or mTOR. Then, myotubes were incubated 60 min ± 2 mM AICAR (an AMPK activator) and ± KU, after which cells were incubated 1 h ± KU, then 1 h without additives, and finally 20 min ± 100 nM insulin. There was a ~40% increase in AMPK phosphorylation (P‐AMPK) immediately after 1 h of AICAR treatment, but this had worn off by 140 min later. In myotubes not exposed to KU, P‐AS tended to be elevated by ~70% in myotubes previously exposed to AICAR. Insulin also tended to increase P‐AS by about 2‐fold, and effects of insulin and prior exposure to AICAR tended to be more than additive. KU caused an overall elevation in P‐AS, but prevented any further effects of insulin or prior exposure to AICAR on P‐AS. KU increased Akt phosphorylation (P‐Akt, S473 and T308) in the absence of insulin but did not interfere with insulin‐stimulated increases in P‐Akt. Previous exposure to KU depressed P‐AMPK by about 40%. It appears that exposure to an AMPK activator leads to long‐term elevation of P‐AS, and ATM may play some role in this effect.NIH DK08437 supported this work.
Ataxia telangiectasia mutated (ATM) is required in insulin‐like growth factor‐1 (IGF‐1) signaling through the PI3K/Akt pathway
Authors: Ching James Kain , Luebbert Stephen H. , Zhang Zhihong , Marupudi Nandhini , Banerjee Sankha , Hurd Robin , Collins Roy L. , Ralston Lyle , Fisher Jonathan S. 
Published: June 2021

Reports that ATM is required for full activation of Akt raise the hypothesis that ATM plays a role in IGF‐1 signaling through the PI3K/Akt pathway. Differentiated C2C12 cells harboring either ATM‐targeting shRNA or non‐targeting shRNA and myotubes from a C2C12 lineage previously exposed to empty vector lentivirus were incubated in the presence or absence of 10 nM IGF‐1. Western blot analysis showed that IGF‐1 stimulated phosphorylation of Akt S473, Akt T308, and p70 S6 kinase (S6K) in cells expressing non‐targeting shRNA and in empty vector controls, but the IGF‐1 effects were significantly reduced in cells with shRNA‐mediated ATM knockdown. Consistent with these findings, the ATM inhibitor KU55933 (10 μM) prevented stimulation of S6K phosphorylation in C2C12 myotubes exposed to IGF‐1. Furthermore, IGF‐1‐stimulated phosphorylation of Akt S473, Akt T308, and S6K was lower in isolated soleus muscles from mice expressing only one functional ATM allele (ATM +/‐) compared to muscles from wild‐type (ATM +/+) mice. Our data suggest that ATM is a modulator of IGF‐1 signaling through the Akt pathway.This work is supported by NICHD/NCMRR through a Pilot Project award from the National Skeletal Muscle Research Center.