bluuebunny/crossref_metadata_2025_split huggingface dataset:
Functional domains of the mouse β3‐adrenoceptor associated with differential G‐protein coupling
Authors: Sato Masaaki , Hutchinson Dana S , Evans Bronwyn A , Summers Roger J 
Published: June 2021

Alternative splicing of the mouse β3‐adrenoceptor (AR) produces two isoforms of the receptor: the β3a‐AR couples to Gs and the β3b‐AR couples to both Gs and Gi. A truncated receptor that lacks the C‐terminus of the β3a‐ or the β3b‐AR showed pertussis toxin (PTX) sensitivity in a cyclic AMP (cAMP) accumulation assay, suggesting that the β3a‐AR is restrained from coupling to Gi by residues in the C‐terminus. This view was supported by studies using a cell‐penetrating peptide transportan‐10 (Tp10) coupled to the C‐terminal tail of the β3a‐ARτhat caused the β3a‐AR to become PTX sensitive.Site‐directed mutagenesis was used to identify residues in the C‐terminus of the β3a‐AR responsible for inhibition of coupling to Gi. Mutation of a putative caveolin binding site caused β3a‐AR mediated cAMP accumulation to become PTX sensitive (control, 1.00±0.16; PTX, 1.75±0.14, n=4, P<0.0001). Furthermore, treatment with filipin III, an agent that disrupts lipid rafts, also caused the wild type β3a‐AR to become PTX sensitive (control, 1.00±0.19; PTX, 1.56±0.04, n=4, P<0.0001), but in addition concentration‐response curves to CL316243 were markedly shifted to the right (pEC50 control: 9.76±0.03, +filipin III: 8.73±0.21). These observations suggest that β3a‐ARs are restricted to caveolae and that localization of the receptor may play a specific role in G‐protein mediated signalling.This research was supported by National Health and Medical Research Council Project Grant 236884.
Use of 4‐[(2‐bromoethyl)methyl‐amino]‐2‐butynyl N‐(3‐chlorophenyl)carbamate (BR 384), to study allosteric interactions at M2 muscarinic receptor
Authors: Suga Hinako , Ehlert Frederick James 
Published: June 2021

We explored the interaction of a nitrogen mustard derivative of McN‐A‐343 (BR 384) with the human M2 muscarinic receptor expressed in CHO cells using the muscarinic radioligand, [3H]N‐methylscopolamine ([3H]NMS). BR 384 caused a concentration‐dependent, first order loss of [3H]NMS binding at 37°C, with the half maximal rate constant occurring at 8.6 μM BR 384. We examined the effects of various ligands on the rate of alkylation of M2 receptors by BR 384. NMS competitively slowed the rate of alkylation, whereas the inhibition by gallamine and McN‐A‐343 reached a plateau at high concentrations, indicating an allosteric inhibition. In contrast, WIN51708 had no effect. We also measured the inhibition of [3H]NMS binding by BR 384 at 0°C, conditions under which covalent binding is reduced. In these experiments, the dissociation constants of NMS, gallamine, McN‐A‐343 and the aziridinium ion of BR 384 for their binding sites were 0.0001, 0.64, 18 and 3.7 μM, respectively. In contrast, the parent mustard and alcoholic hydrolysis product of BR 384 were without effect. Our results show that the aziridinium ion of BR 384 binds covalently to the orthosteric site of the M2 receptor, whereas McN‐A‐343 binds to an allosteric site. Measurement of the effects of ligands on the rate of inactivation of muscarinic receptors by a covalent orthosteric ligand is a powerful method for detecting allosterism.Supported by a NIH Grant GM 69829.
Inhibition of cyclic AMP production is not reduced in HEK293 cells expressing the alpha‐2C adrenergic receptor Del322–325 polymorphism
Authors: Montgomery Megan D. , Bylund David B. 
Published: June 2021

The human alpha‐2C adrenergic receptor inhibits norepinephrine release in the sympathetic nervous system. A four residue deletion in the third intracellular loop of this receptor, Del322–325, has been reported to exhibit reduced signaling in vitro (Small, et al., 2000) and be a potential risk factor for heart failure, presumably due to excess norepinephrine release and action. We attempted to replicate the reduced signaling, with the hypothesis that decreased signaling is due to decreased coupling of Del322–325 to Gi proteins. Our objective was to compare cyclic AMP inhibition of alpha‐2C wild type and Del322–325 receptors stably expressed in HEK293 cells. Cyclic AMP production was stimulated by 30 μM forskolin, and alpha‐2 agonists were used to inhibit this production in clones with high or low receptor expression levels. WT and Del322–325 clones with high expression levels (Bmax = 1500 and 2300 fmol/mg) did not differ in their percent inhibition (94 and 98%) or in their potencies (0.5 and 0.4 nM) for UK‐14,304. Clonidine also showed no difference between WT and Del322–325 clones with high Bmax (93 and 97%, 2.2 and 1.7 nM EC50s). Similar results were obtained in clones with low Bmax (20 and 20 fmol/mg). Therefore, in HEK293 cells, Del322–325 does not have a reduced ability to inhibit cyclic AMP production in comparison to WT.Supported by a donation from Procter and Gamble.
Tempol, a SOD‐mimetic, improves rat pharyngeal dilator muscle performance during hyperoxia and hypoxia
Authors: Skelly James Richard , Mitchell Gordon S , Bradford Aidan , O'Halloran Ken D. 
Published: June 2021

Agents that improve respiratory muscle performance may be useful as an adjunct treatment for obstructive sleep apnoea. The aim of this study was to examine the effects of antioxidants on rat pharyngeal dilator muscle contractile and endurance properties.Isometric contractile and endurance properties of isolated strips of sternohyoid muscle were examined in tissue baths under hyperoxic (95%O2/5%CO2) or hypoxic (95%N2/5%CO2) conditions in the absence (control) or presence of the antioxidants: N‐acetyl cysteine (NAC, 10mM), Tiron (10mM) or Tempol (10mM). Specific force (Sf) was measured at stimulus frequencies from 10–100Hz and throughout a standard fatigue trial (40Hz for 300msec every 2 seconds for 2 minutes).Under hyperoxic conditions, Tempol had an inotropic effect over initial 90s of fatigue (Sf at 90s was 3.7 ± 0.5 vs. 7.7 ± 0.4 N/cm2, control (n=9) vs. Tempol‐incubated (n=9) muscles, P<0.05 ANOVA). In hypoxia, Tempol significantly increased force over the initial 50s of fatigue (Sf at 50s was 1.3 ± 0.2 vs. 2.9 ± 0.5 N/cm2, control (n=9) vs. Tempol‐incubated (n=9) muscles, P<0.05 ANOVA). NAC had no significant effect and Tiron had weak inotropic effects on sternohyoid muscle function.This study illustrates that the SOD mimetic, Tempol, has inotropic effects on sternohyoid muscle during fatiguing and non‐fatiguing contractions. We conclude that antioxidants may be beneficial in the treatment of OSA.
Nitric oxide modulates rat pharyngeal dilator muscle force and endurance during hypoxia
Authors: McMorrow Clodagh , Mitchell Gordon S , Bradford Aidan , O'Halloran Ken D 
Published: June 2021

Nitric Oxide (NO), a highly reactive second messenger, plays an important role in skeletal muscle physiology. The aim of this study was to investigate the effects of the NO synthase inhibitor, N‐nitro‐L‐arginine (L‐NNA) on the contractile and endurance properties of the sternohyoid (upper airway dilator) muscle under hyperoxic and hypoxic conditions in vitro.Adult male Wistar rats were killed humanely and sternohyoid muscle strips were mounted isometrically in an organ bath containing aerated physiological salt solution maintained at 30degC. Four groups were assessed: control muscle strips exposed to hyperoxic (95%O2, 5% CO2; n=9) or hypoxic (95%N2, 5%CO2; n=8) conditions and L‐NNA‐treated strips (1mM) studied under hyperoxic (n=9) or hypoxic conditions (n=8). Force‐frequency relationship was assessed.L‐NNA had no effect on sub‐maximal forces but significantly decreased peak tetanic force at 100Hz under both hyperoxic (17+/−1.5 vs. 13.8+/−1.2 N/cm2; mean+/−SEM, control vs. L‐NNA, P<0.05 ANOVA) and hypoxic conditions (9.3+/−0.9 vs. 6.6+/−0.7, control vs. L‐NNA, P<0.05).We conclude that NO facilitates rat sternohyoid muscle contractile function especially under hypoxic conditions. Although NO is reported to inhibit diaphragm contractile function, a positive inotropic effect of NO donors has been observed in limb muscle.
3‐Iodothyronamine is a novel suppressor of fetal sheep cardiomyocyte proliferation in vitro
Authors: Chattergoon Natasha N , Louey Samantha , Grandy David K , Scanlan Thomas S , Hohimer Roger , Giraud George D , Thornburg Kent L 
Published: June 2021

OBJECTIVES:Fetal cardiomyocyte (CMC) maturation results in cells losing ability to divide. In late‐gestation sheep CMCs thyroid hormone (T3) decreased proliferation and changed signaling protein levels in favor of cell cycle arrest (Chattergoon et al, 2007). 3‐Iodothyronamine (T1AM) is a naturally occurring relative of T3 that does not bind classical thyroid receptors (TRs). We hypothesized that T1AM may depress CMC proliferation similar to T3.METHODS:Isolated right and left ventricular fetal sheep CMCs were cultured with T1AM (1nM, 10nM) and Bromodeoxyuridine (BrdU; 10μM) for 48 hours. Two ages were studied—100 days gestational age (dGA) and 135dGA—to determine developmental variations. BrdU uptake (index of proliferation) in fixed cells was analyzed.RESULTS:Basal BrdU uptake in serum free medium (SFM) was 10–12% in 100dGA CMCs versus 1–2% in 135dGA CMCs (p<0.01). BrdU uptake in response to 10% serum medium (SM) increased to 20% in 100dGA cells versus 8–10% in 135dGA CMCs (p<0.01). Similar to T3, T1AM does not affect cells exposed to SFM but decreases BrdU uptake by CMCs exposed to SM by 50% (p<0.05).CONCLUSIONS:These data suggest T1AM is as potent an inhibitor of fetal CMC proliferation as T3. Because T1AM does not activate TRs, non‐classical signaling of T1AM and T3 may be important in CMC growth and maturation.Grant support: NICHD (P01HD34430) and Minority Supplement (3PO1 HD034430‐09S1).
Induction of NOS and PDE5 isozymes in resistance arteries under chronic high and low flow
Authors: Lee Hyon Jae , Zhang Haiying , Fisher Steven 
Published: June 2021

Myosin phosphatase (MP), the primary effector of smooth muscle relaxation, is a key target of signaling pathways that regulate vascular tone. Using the mesenteric artery ligation high flow/low flow model (HF/LF), we previously showed changes in MP isoform expression associated with increased sensitivity of VSM to NO/cGMP signaling. We hypothesized that altered expression of genes proximal in this pathway also influence NO/cGMP signaling under sustained HF/LF.Methods: Every other second order mesenteric artery (MA2) was ligated to induce LF in upstream MA1s and HF in adjacent MA1s. Rat MA1s were harvested 1‐28 days after ligation and assayed for gene expression by real time PCR.Results: Fold change vs. Control, Day4Changes in NOS and PDE5 expression were confirmed at the protein level by Western blotting.Conclusion: This and the functional studies presented in the accompanying abstract suggest that induction of PDE5 de‐sensitizes vascular smooth muscle to NO signaling under sustained HF/LF conditions. These results provide a novel mechanism for the dynamic changes in NO signaling in HF and LF disease states.Supported by NIH R01 HL‐66171 and Case School of Medicine Crile Fellowship.
Hyperglycemia induced oxidative stress is attenuated in cardiac tissue by 670nm photobiomodulation in an experimental model of diabetes
Authors: Gopalakrishnan Sandeep , Lark Daniel S , Sandoval Chris L , Snyder Ann C , Eells Janis T 
Published: June 2021

Oxidative stress contributes to the pathogenesis of diabetic cardiomyopathy. Photobiomodulation by light in the red to near‐infrared (NIR) range (630–1000 nm) using low energy lasers or light‐emitting diode (LED) arrays accelerates wound healing, improves recovery from ischemic injury and attenuates degeneration in the injured optic nerve.PurposeTo investigate the efficacy of 670 nm NIR‐LED treatment in attenuating diabetes‐induced oxidative stress in cardiac tissue.MethodsAdult Wistar rats were made diabetic by streptozotocin treatment (STZ, 50 mg/kg, ip). One group of animals (NIR‐STZ) was treated daily with 670 nm LED irradiation. (5 min/day, 6 J/cm2) 5 days/week for 14 weeks. Other treatment groups were untreated control (CON) and STZ‐treated (STZ). At week 14, animals were euthanized, perfused with cold PBS and the heart was rapidly harvested and flash frozen. Glutathione concentrations were determined by an enzymatic recycling method.ResultsThe GSSG/GSH ratio in heart tissue was increased nearly four fold in STZ‐treated rats relative to control animals (CON = 0.08 ±0.01, vs. STZ=0.31± 0.08). 670 nm LED treatment significantly attenuated GSH oxidation (NIR‐STZ = 0.13± 0.03).ConclusionNIR photobiomodulation reduces intracellular oxidative stress and may attenuate cardiomyopathy in diabetes.Funding SourceBiomedical Technology Alliance Award and UWM Research Growth Initiative
Enhancing Science Education at a Liberal Arts College ‐ A Model Approach by EPA and Livingstone College
Authors: Mack Cina M , Kodavanti Prasada R , Smith Edward G 
Published: June 2021

Current data indicate the United States will experience a critical short fall of skilled professionals in science and technology (STEM). This problem has multiple dimensions, which include improving the quality of science education at liberal arts institutions. In an effort to address this dilemma, a pilot project between the Neurotoxicology Division/US EPA and Livingstone College (LC), a small, historically black college, was initiated. Faculty from the biology, chemistry and psychology departments of LC participated in a 10 week fellowship at the National Health and Environmental Effects Research Laboratory (NHEERL) of US EPA. Faculty were involved in research projects with EPA investigators, and subsequently co‐authored abstracts for presentation at scientific conferences. Modules of biobehavioral research and environmental science were incorporated into the curricula at LC, and a physiology research seminar was added. Lastly, an NIH EARDA proposal, authored by an LC fellow, was submitted to fund an Office of Biomedical and Behavioral Research at LC. This pilot program demonstrates a cost‐effective mechanism ($40,000 NHEERL training grant) for direct improvement of science education at a small, liberal arts college serving a student population that is historically under‐represented in STEM majors.(This abstract does not necessarily reflect US EPA policy).
Defects in ATP Dependent Canalicular Transporter ABCC2. Mutation Study of ABCC2 Gene in a Large Family with Dubin‐Johnson syndrome.
Authors: Barnincova Lenka , Behunova Jana , Martasek Pavel 
Published: June 2021

Hepatobiliary excretion of conjugated bilirubin is mediated by an ATP dependent canalicular transporter, the multidrug resistence associated protein 2 (MRP2/ABCC2). MRP2 is responsible for biliary excrection of glucuronide and glutathione conjugates of endogenous and exogenous compounds. Before this excretion, bilirubin is conjugated by uridine‐diphosphoglucuronosyl transferase 1A1 (UGT1A1) with glucuronic acid.Dubin‐Johnson syndrome (DJS) is an inherited autosomal recessive disorder characterised by the absence of functional protein MRP2 at the canalicular membrane of hepatocytes. Known mutations in this gene cause impaired maturation and trafficking of the mutated protein from the endoplasmic reticulum to the Golgi complex. As a result, conjugated hyperbilirubinemia is found. Defects of UGT1A1 gene cause deficient glucuronidation manifested in unconjugated hyperbilirubinemia – Gilbert syndrome.Here we present a study of a large gypsy family with mixed congenital hyperbilirubinemia Dubin‐Johnson syndrome and Gilbert syndrome. The novel mutation 1012delGT in MRP2 gene, which causes a frameshift was found. Twenty nine subjects were investigated, two of them were found to be homozygous for both named hyperbilirubinemia. This is the first large molecular study of mixed hyperbilirubinemia with a description of both homozygous forms of mutations in UGT1A1 and MRP2 genes.
Overexpression of YTCa, a novel gene can promote growth of NIH3T3 cells
Authors: Hwa KuoYuan , Tseng Chia‐Jen , Cheng Wei , Chang Chun‐Chi , Lee Yu‐may 
Published: June 2021

YTCa, a novel human gene was first discovered in our studies for Dopa‐responsive dystonia by differential display. A unique cell model was established with the characteristic of the dominant‐negative effect of the GCH G201E mutation. By PCR‐based differential display, YTCa and other proteins were found to be involved in regulating cellular mRNA and protein half‐life. From bioinformatics analysis, we found that YTCa is located nearby chromosome 20q13. Although tissue distributions of YTCa mRNA were found in normal pancreas, muscle and heart, the protein can only be detected in brain by Western analysis. Moreover, normal tissue did not have detectable amounts of YTCa protein. In order to understand the functional role of YTCa, we have performed many experiments. First, overexpression of YTCa full‐length cDNA on NIH3T3 cell allowed cells to grow faster in tissue cultures under a low serum condition. Furthermore, nude mice injected with genetically altered NIH3T3 cells, stably transfected with YTCa formed tumors in 4 weeks.Acknowledgement: This work is supported by the grant from Academia Sincia, Taiwan, ROC (No: AS‐94‐TP‐B17).
The Role of Fc receptor‐like 2 in B cell Signaling
Authors: Jackson Tanisha Anne , Haga Christopher L. , Ehrhardt Goetz R. A. , Davis Randall S. , Cooper Max D. 
Published: June 2021

The Fc receptor‐like protein 2 (FCRL2) is a member of a family of receptors identified on the basis of sequence similarity with previously identified Fc receptors. The human FCRL2 transmembrane protein has 4 Ig‐like extracellular domains and a cytoplasmic tail containing one potential ITAM (immunoreceptor tyrosine‐based activation motif) and two ITIMs (immunoreceptor tyrosine‐based inhibitory motifs). To define its signaling potential in B cells, we constructed a chimeric receptor containing the extracellular and transmembrane domain of FcγRIIb fused to the intracellular domain of FCRL2 and expressed it in an Fc receptor deficient B cell line. Our functional analysis shows that, when coligated with the B cell receptor (BCR) by intact antibody, the FcγRIIb‐FCRL2 chimeric receptor is tyrosine phosphorylated, recruits the SHP‐1 phosphatase, and inhibits whole cell tyrosine phosphorylation. Preliminary data suggest that ERK activation and calcium mobilization are also inhibited upon crosslinking of FCRL2 with the BCR. These results indicate that FCRL2 may serve an inhibitory function in B cells.NIH/NIAID 5 T32 AI07051; 5 T32 GM08111‐20
Natural glucocorticoids induce survival and proliferation of murine bone marrow granulocytes without altering function
Authors: Trottier Mark Daniel , Newsted Matthew M. , Williams Olatundun , King Louis E. , Fraker Pamela J. 
Published: June 2021

Natural glucocorticoids (Gc) produced during stress have profound effects on cells of the immune system. It is known that Gc induce apoptosis in precursor T and B cells markedly altering lymphopoiesis; however, it has been noted that bone marrow myeloid cells expanded both in proportion and absolute numbers in the mouse marrow after Gc treatment. In this study, increased serum Gc and atrophied thymus were observed in mice implanted with corticosterone tablets compared to sham operated mice. Additionally, blood neutrophil counts were elevated dramatically, while lymphocyte counts sharply declined. In the bone marrow, nucleated cell number was unchanged, however the distribution of cell type was dramatically shifted. As expected, marrow lymphocytes declined more than 40% after 3 days, while monocytes and granulocytes increased in number by at least 40%. Further, all granulocyte developmental stages showed dramatic increases in both total number and percent of nucleated marrow. Gc‐exposure had little to no effect on the ability of granulocytes to produce superoxide, undergo chemotaxis or phagocytose bacteria. These results indicated that Gc treatment drastically shifted bone marrow composition and provided evidence that granulocytes and their progenitors were selectively preserved under stressful conditions without losing function.This research was supported by NIH grant DK 52289‐29.
The recurring AGCT motif in S region DNA specifically recruits 14‐3‐3 adaptor proteins that are critical for the unfolding of CSR
Authors: Xu Zhenming , Fulop Zsolt , Zhang Jinsong , Al‐Qahtani Ahmed , Pone Egest , Pal Zsuzsanna , Park Seok‐Rae , Zan Hong , Fu Haian , Yates John , Casali Paolo 
Published: June 2021

Somatic hypermutation (SHM) and class switch DNA recombination (CSR) are central to the maturation of the antibody response and occur mainly in germinal centers (GCs). To identify critical switch (S) region cis‐elements, we analyzed S region DNA sequences and identified AGCT motifs occurring at a high frequency in Sμ and all other downstream S regions in human, mouse and Xenopus laevis. 14‐3‐3 adaptor proteins were the major AGCT‐binding proteins and preferentially localized to S regions in switching B cells. Expression of 14‐3‐3 was induced in GC B cells in vivo and B cells stimulated to undergo CSR in vitro. The differential induction of the seven 14‐3‐3 isoforms in these B cells suggests an isoform‐specific role of 14‐3‐3 in CSR. This was confirmed by different degrees of CSR inhibition in B cells that were null in 14‐3‐3γ or expressed a semi‐dominant 14‐3‐3σ mutant or reduced levels of 14‐3‐3τ or 14‐3‐3ε. Further, 14‐3‐3 interacted with critical CSR factors, such as AID and Ku80/Ku86. Finally, difopein, a pan‐14‐3‐3 isoform peptide inhibitor that disrupts interaction between 14‐3‐3 and its protein ligands, reduced the level of resected double strand DNA breaks (DSBs), the obligatory intermediates in CSR. Thus, our findings demonstrate that the recurring AGCT motif in S regions specifically recruit 14‐3‐3 adaptor proteins that are critical for the unfolding of CSR.Supported by NIH grants AI 45011, AI 60573 and AR 40908.
BG1, a candidate gene affecting the occurrence of herpesvirus–associated avian lymphomas
Authors: Wang Yujun , Goto Ronald M. , Miller Marcia M. 
Published: June 2021

Oncogenic herpesvirus infections contribute to the occurrence of lymphomas in the human population. Genetic makeup may have a role in susceptibility. Through genetic mapping with recombinant haplotypes we have identified BG1, a member of the extended butyrophilin gene family, as a gene having a significant role in MHC‐associated resistance to the occurrence of T‐cell lymphomas that often follow infection with gallid herpesvirus 2 in the chicken animal model. Significant differences in tumor incidence occur among congenic birds differing only at the BG1 locus. Two alleles, BG1*R2 and BG1*R4, are respectively associated with tumor resistance and susceptibility. The alleles differ only in the 3′‐untranslated region (3′‐UTR) with the longer 3′‐UTR of the BG1*R4 allele containing unique sequence. In dual Firefly/Renilla luciferase reporter assays we found that the 3′‐UTR of the BG1*R4 suppresses PMA‐stimulated translation of the firefly reporter gene, while the 3′‐UTR of BG1*R2 has a far less inhibitory influence. With further constructs we have narrowed the effective region of the BG1*R4 3′‐UTR to a region relatively rich in CG dinucleotides.Supported in part by NCI R21 CA105426.