bluuebunny/crossref_metadata_2025 huggingface dataset:
A far upstream and genetically polymorphic alternative promoter drives expression of the human microsomal epoxide hydrolase (EPHX1) gene
Authors: Yang Xi , Su Shengzhong , Omiecinski Curtis
Published: June 2021

Microsomal epoxide hydrolase (EPHX1) plays an important role in the bioactivation of polycyclic aromatic hydrocarbons (PAH). The genetics and regulation of EPHX1 in humans is likely an important determinant of interindividual differences in toxicity outcomes related to chemical exposure. Previously, we identified two EPHX1 genetic polymorphisms that alter the amino acid structure of the enzyme. Subsequently, a number of epidemiology investigations have associated EPHX1 coding region polymorphisms as a risk factor for lung cancer. The initially defined EPHX1 exon 1, E1, is derived from the use of a promoter directly proximal to exon 2 of the EPHX1 coding region. Remarkably, the E1 promoter directs expression only in the liver. We have now identified an alternative EPHX1 promoter, E1‐b, localized ~18.5 kb upstream of exon 2. Recently, real‐time RT‐ PCR assays were specifically designed for E1 and E1‐b, and the expression of each transcript was quantified relative to a standard curve. The results demonstrate that the E1‐b promoter is the primary driver of EPHX1 gene expression in liver as well as extrahepatic tissues, with the expression of E1‐b in lung comparable to levels in liver. Additionally, E1‐b promoter region polymorphisms at −85 and −137, as well as the genetically variable presence of Alu repetitive elements appear to function as determinants of interindividual differences in EPHX1 transcript expression.
Down‐regulation of CNS cyclooxygenase‐2 message by inhibitors of soluble epoxide hydrolase lead to anti‐hyperalgesia in inflammatory pain
Authors: Inceoglu Bora , Ulu Arzu , Hegedus Christine , Georgi Katrin , Jinks Steve L. , Hammock Bruce D.
Published: June 2021

Epoxyeicosatrienoic acids (EETs), metabolites of arachidonic acid produced by cytochrome P450s, have recently been shown to possess anti‐inflammatory and anti‐hyperalgesic activities. Through inhibition of soluble epoxide hydrolase (sEH), the major enzyme that inactivates these metabolites, we have shown that plasma concentrations and residence times of EETs can be increased dramatically. Interestingly, in models of inflammation, inhibition of sEH leads to decreases in circulating pro‐inflammatory lipid mediator concentrations, most prominently prostaglandin E2. We previously showed that sEH inhibitors transcriptionally down‐regulate cyclooxygenase (COX2) message in mouse liver. Indeed co‐administration of sEH inhibitors and selective COX‐2 inhibitors lead to a synergistic reduction in circulating prostaglandin concentrations. Here we show that the analgesic potency of inhibitors of sEH are comparable to that of morphine and that sEH inhibitors penetrate the blood brain barrier and down‐regulate the spinal induction of COX2 mRNA in response to peripheral local inflammation.
Pharmacokinetic studies of four novel soluble epoxide hydrolase (sEH) inhibitors and anti‐inflammatory efficacy of the most promising one t‐AUCB
Authors: Liu Junyan , Tsai Hsing‐Ju , Hwang Sung Hee , Jones Paul D. , Morisseau Christophe , Hammock Bruce D.
Published: June 2021

Earlier sEH inhibitors such as AUDA are effective anti‐hypertensive and anti‐inflammatory agents in multiple animal models. However, their poor metabolic stability and water solubility make them difficult to use pharmacologically. For obtaining a compound easy to use in vivo, we present the PK profiles of four conformation‐restricted sEH inhibitors and anti‐inflammatory efficacy of the most promising one.

Better PK parameters (higher Cmax, longer t1/2, and greater AUC) were obtained from the four tested inhibitors than AUDA with t‐AUCB showing the best result. Oral bioavailability of t‐AUCB (0.1 mg kg−1) was 75±12% (n=4). Furthermore, when mice were administrated t‐AUCB in drinking water at 4 mg L−1, a fairly stable blood concentration of ~70 nM t‐AUCB was maintained since one day administration, and was totally cleared from blood after 3 days of drug withdrawal. Finally, oral gavage of 0.5 and 1.0 mg kg−1 t‐AUCB were effectively anti‐inflammatory evidenced by reversing LPS‐induced hypotension in a dose‐dependent manner, and significantly shifting the ratio of lipid epoxides to corresponding diols in plasma. t‐AUCB appears around 10 times more orally active than AUDA in this model. Thus t‐AUCB is promising for further in vivo studies related to the biology of sEH and its potential therapeutic applications.
Expansion of the Eicosanoid Profiling Methodology by the Addition of w‐3 Fatty Acid Metabolites
Authors: Georgi Katrin , Hwang Sung‐Hee , Nording Malin , Yang Jun , Zivkovic Angela , Pedersen Theresa , Newman John W , Hammock Bruce D
Published: June 2021

Eicosanoids, a large group of arachidonic acid (AA) metabolites, play an important role in several physiological processes such as inflammation or hypertension. They are produced via three different pathways by cytochrome P450, lipoxygenase and cyclooxygenase enzymes. It has been established that these enzymes metabolize not only AA but also other poly unsaturated fatty acids (PUFAs), i.e. eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA).

Recent studies indicate that w‐3 fatty acids and their metabolites play an important protective role in multiple inflammatory conditions including IgAN. We are expanding our analytical method by adding separate platforms for lipid amides and leukotrienes but also by adding more targets to the oxylipin method. For example we incorporated metabolites of the three most important w‐3 fatty acids (EPA, DHA and a‐linolenic acid) in our existing eicosanoid profiling methodology. Thus, we developed a precise and sensitive method using LC‐MS/MS to identify and quantitate 88 oxylipin metabolites in plasma samples. This work was supported by NIEHS SBRP Grant P42 ES04699.
Attenuation of Cisplatin Nephrotoxicity by the Soluble Epoxide Hydrolase Inhibitor nbAUDA
Authors: Akintola Adebayo D , Parrish Alan , Burghardt Robert C , Watanabe Takaho , Morisseau Christophe , Hammock Bruce D
Published: June 2021

Cisplatin is a highly effective chemotherapeutic agent against many tumors, but it also a potent nephrotoxicant. Given that there have been no significant advances in our ability to clinically manage acute renal failure since the advent of dialysis, the development of novel strategies to ablate nephrotoxicity would represent a significant development. In this study, we investigated the ability of an inhibitor of soluble epoxide hydrolase (sEH), n‐butyl ester of 12‐(3‐adamantan‐1‐yl‐ureiido)‐dodecanoic acid (nbAUD), to attenuate cisplatin‐induced nephrotoxicity. nbAUDA is quickly converted to AUDA and produces higher blood levels of AUDA than administration of AUDA itself. Administration of 40 mg/kg of nbAUDA to C3H mice every 24 hr resulted in elevated levels of AUDA; this protocol was also associated with attenuation of cisplatin‐induced nephrotoxicity as assessed by BUN levels and histological evaluation of kidneys. This is the first report of the use of sEH inhibitors to protect against acute nephrotoxicity and suggest a therapeutic potential of these compounds.
Soluble epoxide hydrolase inhibition attenuates aortic plaque formation in a murine model of accelerated atherogenesis
Authors: Ulu Arzu , Davis Benjamin B. , Tsai Hsing‐Ju , Kim In‐Hae , Hammock Bruce D. , Weiss Robert H.
Published: June 2021

Epoxyeicosatrienoic acids (EETs) have hemodynamic and anti‐inflammatory effects. They can be stabilized in vivo by inhibiting the soluble epoxide hydrolase (sEH). Epoxide hydration appears to be the major route of metabolism. For this reason, sEH inhibitors (sEHIs) are being developed as potential pharmaceuticals for the treatment of cardiovascular and inflammatory diseases. To determine the effect of sEHIs on atherosclerotic lesion formation, we utilized an established mouse model of accelerated atherogenesis: 5 months old male Apo E (−/−) mice. ApoE(−/−) mice were treated with sEHI 1‐adamantyl‐3‐(5‐(2‐(2‐ethoxyethoxy) ethoxy) pentyl urea (AEPU) in drinking water (90μg/ml) for two months. During the last month of the study, all animals were fed an atherogenic diet and were simultaneously infused with angiotensin‐2 by osmotic minipumps to induce atherosclerosis. The descending aortas were examined for areas of plaque within their lumens. In animals that received the sEH inhibitor we observed a 53% reduction in atherosclerotic lesions as compared to control animals. A highly significant increase in plasma arachidonic and linoleic acid epoxides (EETs and EpOMEs) was observed in the AEPU treated group. Our data suggest that orally‐available sEHIs may be useful in the treatment of atherosclerosis by directly attenuating plaque formation.
A novel mechanism of Angiotensin II‐induced cardiac hypertrophy – The role of soluble epoxide hydrolase
Authors: Ai Ding , Pang Wei , Xie Xuefen , Xu Ming , Jones Paul , Zhang Youyi , Chiamvimonvat Nipavan , Shyy John Y‐J , Hammock Bruce D. , Zhu Yi
Published: June 2021

Pathophysiological cardiac hypertrophy is one of the most common causes leading to heart failure. Epoxyeicosatrienoic acids can induce dilation of coronary arteries and cardio‐protection, but they are hydrolysed by soluble epoxide hydrolase (sEH). In this study, we found that the protein level of sEH expression was elevated in the heart of both spontaneously hypertensive rats and Ang II‐infused rats and losartan could abolish this effect. This upregulation of sEH is Ang II specific, since it was not observed in swimming exercise‐induced physiological hypertrophy or norepinephrine‐induced hypertrophy. Moreover, the administration of a potent sEH inhibitor TUPS could prevent Ang II‐induced hypertrophy, demonstrated by a decrease of the left ventricular hypertrophy, reduction of the size of cardiomyocytes and expression of markers of hypertrophy. An in vitro study revealed that Ang II stimulation resulted in the upregulation of sEH and the markers of hypertrophy in neonatal rat myocytes. Those markers were elevated by adenovirus overexpressed sEH, but blocked by the treatment with TUPS. Thus, sEH is specifically upregulated by Ang II, which directly mediated Ang II‐induced hypertrophy. Pharmacological inhibition of sEH is a potential approach to protect Ang II‐induced cardiac hypertrophy.
Dietary soluble epoxide hydrolase inhibitors effect on hepatic gene expression of lipid metabolism in hamsters
Authors: Bartley Glenn , Tsai Hsing‐Ju , Hong Yun‐Jeong , Hammock Bruce , Yokoyama Wallace
Published: June 2021

Soluble epoxide hydrolase inhibitors (sEHI) block the conversion of the vasodilatory epoxyeicosatrienoic acids to their dihydroxy derivatives and consequently reduce blood pressure. In this study we investigated the effects of three sEHI on lipid metabolism in fat fed male Syrian hamsters. Hamsters were fed a diet containing 20% fat and 40 to 200 mg/Kg diet of the test sEHI compounds. Plasma and liver lipids, body weight, and organ weights were not significantly different between sEHI fed animals and controls. However, relative expression of SCD1 (stearoylCoA desaturase) and PPARalpha in the liver were reduced by sEHI feeding. PPARgamma and HL were not different from controls. Fibrates were also fed in reference diets. The results show that in addition to inhibition of endothelial epoxide hydrolases, sEHI may target PPARalpha and affect fatty acid biosynthesis in liver.
Polymorphisms in the human soluble epoxide hydrolase gene EPHX2 are linked to cardiomyocyte survival following oxygen and substrate deprivation
Authors: Merkel Matthias Johannes , Liu Lijuan , Cao Zhiping , Packwood William , Koerner Ines P. , Alkayed Nabil J. , Van Winkle Donna M.
Published: June 2021

Epoxyeicosatrienoic acids (EETs) protect against myocardial ischemic injury in vivo. EPHX2 encodes soluble epoxide hydrolase (sEH), the enzyme that breaks down EETs. We tested the hypothesis that EPHX2 polymorphisms alter cardiomyocyte survival after simulated ischemia. Cultured neonatal murine cardiomyocytes were transduced with five human EPHX2 variants linked to protein transduction domain TAT. Cultures were subjected to 1.5h of oxygen & glucose deprivation followed by 3h re‐oxygenation, with or without sEH substrate 14, 15‐EET. Cell death was assessed by propidium iodide (% of all cells, mean±SEM, n=5–9 replicates). Successful transduction was confirmed by immunoblot. All mutants reduced cell death compared to the human wild‐type EPHX2 (rh‐sEH WT, 63±1%), but only the Lys55Arg and Arg287Gln mutations were different from untransduced controls (54±1% & 45±1% vs. 58±1%, p<0.05 & p<0.001). The Arg287Gln mutation was previously shown to exhibit reduced hydrolase activity. Excess 14, 15‐EET improved cell survival in all polymorphisms tested except for Arg287Gln (45±1% vs. 45±1%). These data may explain genetic variability in sensitivity to ischemic injury. Support: VA Merit Review 317 (DMVW) and RO1 NS44313 (NJA).
Delayed addition of dexamethasone suppresses chemokine mRNA during ongoing inflammation
Authors: Horton Devin , Remick Daniel G.
Published: June 2021

This study aimed to examine the effects of delayed therapy on the inflammatory response. Heparinized whole blood was collected, stimulated with 50ng/ml LPS, incubated at 37°C with 5% CO2 and continuous rotation. Results showed peak protein levels of the pro‐inflammatory cytokines TNF (1.8 ± 0.38 (3h)), IL‐6 (62.6 ± 8.9), and IL‐1β 5.3 ± 1.7) by 3–6 hours post‐stimulation, while chemokine (CXCL1 from 1.8 ± 0.72 to 10.9 ± 4.6; CXCL8 from 4.9 ± 1.4 to 18.0±4.6*) levels rose over 24 hours. Published data suggest that cytokines signal through a common pathway via NF‐κB transcription factors, however the present data suggest possible differential regulation. To investigate this possibility, dexamethasone (dex; 10−6M) was added to the system 6 hours post‐LPS, and its effect on protein kinetics assayed. Delayed dex had little effect on cytokines, but significantly suppressed chemokine protein levels (CXCL1 6.3 ± 1.5; CXCL8 4.3 ± 1.8*). To determine the level of regulation, the effects of delayed dex on mRNA kinetics was assessed. Delayed dex noticeably, but insignificantly, suppressed chemokine mRNA (CXCL1 from 2.8 ± 1.1 to 0.80 ± 0.59 and CXCL8 from 90.7 ± 55.3 to 47.8 ± 20.6), further implying a difference in regulation of cytokine and chemokine expression during ongoing inflammation. While additional experimentation is necessary, these preliminary data are key to understanding the regulating ongoing inflammation.
HMGB1 mediated LPS‐induced myocardial dysfunction in mice
Authors: Wu Jun , Yang Min , Martin Claudio , Rui Tao
Published: June 2021

The mechanism involved in the endotoxin‐induced myocardial dysfunction is not fully understood. The purpose of the present study was to demonstrate that myocardial dysfunction in endotoxinemia is mediated via an increase in myocyte production of high mobility group box 1 protein (HMGB1). In vivo, mouse model of endotoxinemia in mice was induced by i.p. injection of LPS (10 mg/kg) and myocardial function was evaluated 24 hrs later. LPS induced a decrease in myocardial contractility (end‐systolic and end‐diastolic volume relation). The decrease in myocardial contractility was diminished when the mice were received either a HMGB1 antagonist (A‐box) or an inhibitor of HMGB1 (glycyrrhizic acid). Expression of myocardial HMGB1 was increased in the heart of mice treated with LPS (immunoflorescence staining). Further, recombinant HMGB1 (10–20 μg/mouse, i.p.) resulted in a dose dependent decrease in myocardial contractility. To confirm that the source of HMGB1 was the cardiac myocytes in the hearts, isolated cardiac myocytes were exposed to LPS (10 μg/ml). Treatment of cardiac myocytes with LPS increased in intracellular levels of HMGB1 and release HMGB1 by myocytes to the supernatant (Western). Taken together, our study suggests LPS‐induced myocardial dysfunction is a result of increase in myocyte HMGB1. (CIHR MOP 81303).
The Mechanistic Basis for the Disparate Effects of Ang II on Coronary Collateral Growth
Authors: Rocic Petra
Published: June 2021

We hypothesized that reported positive or negative effects of AT1 receptor (AT1R) blockers on coronary collateral growth (CCG) are due to altered activation of AT1R‐dependent signaling in normal (WKY) vs. metabolic syndrome (JCR) rats. We stimulated CCG by repetitive ischemia (RI), and measured blood flow and kinase activation in normal (NZ) and collateral‐dependent (CZ) zones. RI induced CCG in WKY rats (CZ flow 83±5% vs. 18±6% of NZ), associated with redox‐sensitive p38, Akt and Src and redox‐insensitive ERK1/2 activation. p38, ERK1/2, Akt and Src blockade abrogated CCG in WKY (CZ flow 42±7%, 52±5%, 48±13% and 13±2% of NZ, respectively), indicating their requirement in RI‐induced CCG. CCG in WKY was associated with sustained Akt and transient p38 activation. RI‐induced CCG was compromised in JCR (CZ flow 12±3% of NZ), associated with lack of p38, ERK1/2, Akt and Src activation. AT1R blocker, candesartan, partially restored CCG in JCR (CZ flow 45±4% of NZ) and restored p38, ERK1/2, Akt and Src activation; however, while able to elicit transient p38 activation, candesartan failed to induce sustained Akt activation. Candesartan abrogated CCG in WKY (CZ flow 62±5% of NZ) and decreased p38, ERK1/2, Akt and Src activation. We conclude that RI‐induced CCG critically depends on extent and duration of redox‐sensitive and ‐insensitive signaling activation, which is altered in the metabolic syndrome. Support: AHA 0630285N.
Physical, Cellular & Molecular Mechanisms of Collateral Artery Growth
Authors: Heil Matthias
Published: June 2021

The compensatory growth of blood vessels after major arterial occlusions has been termed arteriogenesis. Arteriogenesis describes the remodelling of small interconnecting arterial anastomoses with almost no net blood flow to large functional arteries. It has been shown that growth of these collateral arteries is triggered by physical forces, but does not require hypoxia as a stimulus. Using a hind limb ischemia model in combination with an arterio‐venous shunt the role of chronically increased fluid shear stress (FSS) was investigated. FSS initiates the activation of endothelial cells and modulates processes, which control attraction of circulating cells to the collateral wall. On the molecular level, a number of genes was found to be induced by FSS. Moreover, monocytes were shown to have a pivotal role for arteriogenesis. After entering the vascular wall they function as micro‐bioreactors producing cytokines, thereby controlling cell proliferation and remodelling. Cell proliferation coincides with the transient dismantling of extracellular structures by proteases. After the re‐arrangement of wall structures collaterals with large calibres represent functional arteries
Vascular niches in fetal kidneys
Authors: Hyink Deborah
Published: June 2021

Vascularization is essential to form a functional kidney. In addition to providing nutrients and oxygen, vascular cells can influence growth and development of surrounding cells by production of growth factors and extracellular matrix components. Thus, the vasculature is capable of generating microenvironments, also called vascular niches, which support survival, growth and differentiation of stem and progenitor cells. In this talk, we will examine the evidence that vascular niches exist in the kidney. To detect the location of these niches, we used three‐dimensional imaging of fetal and adult kidneys to show spatial relationships between the blood vessels, extracellular matrix components, and hematopoitic cells. Understanding how these niches are regulated could provide insights into how the kidney responds to injury.
Effect of immobilized glycosaminoglycans on megakaryocyte expansion, apoptosis and platelet release
Authors: Kishore Vipuil , Eliason James F. , Matthew Howard W.T.
Published: June 2021

Umbilical cord blood transplantation in adults is limited due to delayed platelet recovery. Transfusion of expanded megakaryocytes (MKs) derived from hematopoietic stem cells (HSCs) has been suggested as a potential solution. Glycosaminoglycans (GAGs) have been shown to influence the proliferation and differentiation of HSCs. Our objective in this study was to examine the effects of various immobilized GAGs on the expansion, apoptosis, ploidy, and platelet release of CD41+ MK progenitors in vitro. Cultures were conducted in serum free media supplemented with TPO. Freshly isolated HSC were seeded onto 24 well plates coated with GAG‐derivatized chitosan membranes. Controls consisted of plastic and un‐derivatized chitosan membranes. Wells were demidepopulated at intervals and analyzed by flow cytometry. At day 11, GAG surfaces like heparin, dermatan sulfate, hyaluronan and heparan sulfate showed significantly lower (p < 0.05) MK apoptotic fractions compared to plastic. The apoptotic MK fraction on GAG surfaces increased with time and was comparable to plastic by day 17. However, the reduced early apoptosis resulted in overall higher MK proliferation on GAGs. MKs on all surfaces had a ploidy level below 8N. On all surfaces, platelet numbers correlated closely with the number of apoptotic MKs at all time points. Results show that GAG surfaces may be useful tools to enhance in vitro production of MKs. NIH Supported.
Deletion of smooth muscle alpha‐actin in mice alters neovascularization response to oxygen‐induced retinopathy
Authors: Tomasek James J , Haaksma Carol J , Hu Yang , Ma Jian‐xing
Published: June 2021

Retinal neovascularization, growth of new abnormal blood vessels, is part of proliferative diabetic retinopathy resulting in blindness. Neovascularization can be mimicked using the oxygen‐induced retinopathy (OIR) model; neonatal mice are placed into hyperoxia (P7‐12) resulting in avascularization of the central retina with rapid and abnormal neovascularization upon return to normoxia. Smooth muscle α‐actin (SMAA) null mice were used to determine effects reduction in contractile function of pericytes and smooth muscle cells (SMC) have upon neovascularization. Lack of SMAA significantly enhances increased retinal vascular permeability observed in response to OIR. WT mice exposed to OIR have significantly greater pre‐retinal tufts, a measure of neovascularization, compared to SMAA null mice. Similarly, SMAA null mice have reduced vessels in the central avascular zone after return to normoxia compared with WT mice. These results suggest that reduced contractility in pericytes and SMCs may reduce OIR‐induced neovascularization due to a reduced response to or reduced levels of angiogenic factors which are critical in diabetic retinopathy. Supported by the Juvenile Diabetes Research Foundation
Regulation of Degradation of Histone mRNA
Authors: Marzluff William F. , Mullen Thomas E. , Kaygun Handan
Published: June 2021

The replication‐dependent histone mRNAs are the only eukaryotic mRNAs that are not polyadenylated, ending instead in a conserved stem‐loop. Histone mRNAs are present in high levels only in S‐phase, and they are regulated coordinately with DNA replication. A major regulatory step is the rapid degradation of histone mRNA when DNA replication is inhibited or at the end of S‐phase. We have elucidated the pathway of histone mRNA degradation. ATR, a kinase activated when DNA synthesis is inhibited, is required for histone mRNA degradation, although the intermediate steps in signaling that ultimately initiate histone mRNA degradation are not known. The stemloop must be close (25–75 nts) to the termination codon for efficient regulation of translation and the mRNA must be actively translated. The protein that binds the stem‐loop, SLBP, is involved in translation and is likely a target of the signaling pathway that activates histone mRNA degradation, as a result of inefficient translation termination. The inefficient termination leads to the recruitment of Upf1, the critical factor also required for nonsense mediated decay. A terminal uridyl transferase (TUTase) is then recruited and 8–12 U's added to the end of the histone mRNA. This provides a platform for binding Lsm1‐7, which in turn recruit the decapping enzymes, as well as the exosome to degrade individual histone molecules simultaneously from both ends.
A step or two towards solution of the RNA folding problem
Authors: Brenowitz Michael D , Laederach Alain , Mitra Somdeb , Schlatterer Joerg , Shcherbakova Inna
Published: June 2021

The RNA folding problem addresses the question of predicting the three‐dimensional structure of RNA molecules from their nucleotide sequences. It is well known that large RNA molecules may follow multiple pathways with significantly populated intermediate species during folding. Many RNA folding reactions are kinetically controlled. We collect ensembles of time – dependent local changes in solvent accessibility to construct ‘structural – kinetic’ models describing the folding of group I ribozymes. These models define the pathways that dominate folding, the rate constants that govern interconversion of the species present in solution and the structures of the reaction intermediates. The collection of local measures is acquired using ‘Fast Fenton’ footprinting, a laboratory based method for time‐resolved hydroxyl radical footprinting that follows folding with millisecond time and single nucleotide spatial resolution. Clustering of the 25 – 30 individual time – progress curves typically acquired in a Fast Fenton folding experiment and kinetic modeling is carried out using the KinFold software. Folding studies will be presented that utilize solution conditions, perturbation of tertiary contacts and phylogenetic relationships to illuminate the physical character of the individual steps of the group I ribozyme folding reactions. The work to be described in this presentation is supported by grant PO1–GM066275 from the National Institutes of Health.
Exploitation of cytoplasmic and nuclear actin by baculoviruses
Authors: Welch Matthew D , Ohkawa Taro , Goley Erin D , Volkman Loy E
Published: June 2021

Diverse bacterial and viral pathogens hijack the actin cytoskeleton of host cells to facilitate processes such as attachment, internalization, or spread. Among known pathogens, baculoviruses are unique in their ability to manipulate actin both in the cell cytoplasm and nucleus. We seek to determine how baculoviruses exploit actin to reveal new roles for actin in pathogenesis, and to illuminate the poorly understood function and regulation of nuclear actin. Using time‐lapse microscopy, we find that Autographa californica Multiple Nucleopolyhedrovirus (AcMNPV) undergoes actin‐based motility within the cell cytoplasm. Motility requires p78/83, a viral capsid protein that mimics host WASP proteins and activates actin assembly with the host Arp2/3 complex. As with other pathogens, moving AcMNPV are propelled into surface projections that may function in cell‐to‐cell spread. However, unlike other pathogens, motility also enhances viral collision with the nucleus, enabling translocation into the nucleus, where replication takes place. Once in the nucleus, AcMNPV induces nuclear actin accumulation, and then assembles nuclear actin using p78/83 to activate the host Arp2/3 complex. Nuclear actin assembly by p78/83 and Arp2/3 complex is essential for progeny production. Further study of this novel mode of pathogenesis will likely reveal elusive details about the regulation and function of nuclear actin.
Nuclear receptors in lipid metabolism and immunity
Authors: Tontonoz Peter
Published: June 2021

PPARγ is the master regulator of adipogenesis and the molecular target of the thiazolidinedione anti‐diabetic drugs. By screening for compounds that promote adipogenesis, we identified a small molecule that targets the PPARγ pathway by a distinct mechanism. This molecule, harmine, is not a ligand for the receptor; rather, it acts as a cell type‐selective regulator of PPARγ expression. Administration of harmine to diabetic mice mimics the effects of PPARγ ligands on adipocyte gene expression and insulin sensitivity. Unlike thiazolidinediones, however, harmine does not cause significant weight gain or hepatic lipid accumulation. Molecular studies indicate that harmine controls PPARγ expression through inhibition of the Wnt signaling pathway. This work validates phenotypic screening of adipocytes as promising strategy for the identification of bioactive small molecules and suggests that regulators of PPARγ expression may represent a complementary approach to PPARγ ligands in the treatment of insulin resistance.
Structure and Dynamics of the Human beta 2 Adrenoceptor
Authors: Kobilka Brian K
Published: June 2021

G protein coupled receptors (GPCRs) are remarkably versatile signaling molecules. The beta 2 adrenoceptor (beta2AR) is a prototypical Family A GPCR that mediates physiologic responses to adrenaline and noradrenaline. The function of the beta2AR can be modulated by a spectrum of synthetic ligands ranging from full agonists to inverse agonists. We have used crystallography to determine the three‐dimensional structure of the beta2AR, and fluorescence spectroscopy to map ligand‐induced conformational changes and characterize the structure of beta2AR dimers. I will discuss what we these studies have taught us about the structural basis of beta2ARs function.
Using Low‐Carbohydrate Diet to Teach Metabolic Pathways to Undergraduates and Medical Students
Authors: Pogozelski Wendy K. , Makowske Mary , Feinman Richard D.
Published: June 2021

The need to understand metabolic pathways has never been greater, given recent increases in obesity‐related illnesses. We previously described how discussion of low‐carbohydrate (low‐CHO) diets can assist undergraduates1 and medical students2,3in learning metabolism. Here we describe further benefits of this approach.

The medical establishment largely holds to low‐fat diets as a treatment for obesity, diabetes and metabolic syndrome but the method is under attack by a minority documenting benefit of CHO restriction as a preferred approach. We use this controversy to engage student interest and to provide context.

The underlying principle of low‐CHO diets lies in control of insulin and relief of insulin resistance. The role of insulin ties together many disparate pathways. Discussion of metabolic syndrome and its dependence on insulin response reinforces the approach. The method has been a highly effective tool for maintaining student interest in metabolism.
Teaching the Poiseuille's Law to Brazilian physiology students with challenging exercises
Authors: Primola‐Gomes Thales Nicolau , Roman‐Campos Danilo , Duarte Hugo Leonardo Lucio , Coimbra Candido Celso , Cruz Jader Santos
Published: June 2021

The students used two different tubes to measure the flow of water and oil through a system connected to a reservoir (600 ml): A (2mm diameter, 110cm length) and B (4mm diameter, 110cm length). Exercise tasks: 1) Measured the water flow we found in A = 90 ml/min and B = 125 ml/min. Discuss vessel diameter and blood flow. 2) Measured the water flow through the two tubes we found A + B = 180 ml/min. Discuss series and parallel resistances. 3) We encouraged the students to change the height of water column in B (75 to 90 cm) and find the pressure gradient, ΔP= Pend − Pinitial= pgh, where p is the density of water (998 kg/m3), g is the gravitational acceleration constant (9.8 m/s2) and h is height of the fluid column (cm). Result: ΔP75 = 55 mmHg and ΔP90 = 66 mmHg. Flow in B with ΔP90:150 ml/min. Discuss the intravascular pressure gradient. 4) Changed the length of tube A (110 to 187 cm) we found a flow of 35 ml/min. Discuss vessel length and blood flow. 5) Changed water by oil in A the flow decrease to 3 ml/min. Attention for liquids viscosities (1 and 35 x10‐6 m2/s, respectively). Discuss the influence of blood viscosity in vessels flow. Finally, we introduced the Poiseuille Equation [Q= (Nπr4/8nl)x ΔP] making analogies with the exercise values. This approach has been very attractive and challenging to Brazilian physiology students. Supported by CNPq.
Design, implementation and assessment of basic laboratory skills in an introductory molecular cell biology course
Authors: Daggett Melissa A. F.
Published: June 2021

Over the past three years changes in the design and implementation of laboratory exercises in the lower level molecular cell biology course at MWSU have been made with the aim of increasing the proficiency of basic laboratory skills acquired by our biology and biochemistry majors. Simultaneously, improvements in the methods for how to assess student performance have been designed and implemented. Previous results have shown that our students do not significantly increase specific laboratory skill proficiency as they progress from the lower level to upper level biology and chemistry courses. This suggests that skills introduced early, with appropriately administered rigorous assessment early in the curriculum, will have the best opportunity for impacting student performance in subsequent courses. Results from student surveys administered during the spring 2007 semester show a significant increase in what students think they learned in the revised laboratories during the course of the semester. These results however, have not yet been carefully correlated with an assessment of how these students actually performed in the laboratory. Work is currently underway to improve the correlation of how students evaluate their performance in the laboratory with their instructor‐assessed laboratory skill proficiency.
Teaching respiratory physiology to health science students using digital data acquisition systems at a community college
Authors: Fronhofer Van , Schaefer Peter A.
Published: June 2021

The teaching of complex physiological processes to health science students in a community college laboratory can be challenging because of limited time and resources. Hence, the development of meaningful experiments that effectively demonstrate physiological principles is essential. To enhance the understanding of concepts like airway resistance and lung compliance and how they contribute to respiratory disease, we implemented the use of iWorx digital data acquisition systems linked to laboratory laptop computers. Students attend traditional lectures on respiratory anatomy and physiology prior to the laboratory exercise. In the lab, the students perform pulmonary function tests using an intuitive software interface, followed by derivation of their individual static lung capacities and assessment of their dynamic respiratory performance. Based on student satisfaction surveys and scores on lab quizzes, comprehension and retention of respiratory physiology concepts increased by 86% as compared to previous methods with an average score increase of 15.2 points. These results support the concept that hands‐on laboratory‐based pedagogy improves comprehension of physiological principles.
Ubiquitin and TDP‐43 immunohistochemistry: how many types of frontotemporal lobar degeneration?
Authors: Spina Salvatore , Murrell Jill R , Ghetti Bernardino
Published: June 2021

Objective: Frontotemporal lobar degeneration with ubiquitin (Ub)‐immunoreactive (ir) inclusions (FTLD‐U) has been classified into four different pathological subtypes. Aim of this study is to assess the applicability of this classification to our series of FTLD‐U cases. Methods: Ub and TDP‐43‐immunohistochemistry (IHC) were carried out on tissue from 53 cases with pathological diagnosis of FTLD‐U, ALS or dementia lacking distinctive histopathology (DLDH). Results: 25 cases were classified according to the proposed criteria (type I: 5; Type II: 7; type III: 9; type IV: 4). Among the other 28 cases, we identified 6 distinct groups: a) ALS with Ub and TDP‐43 pathology confined to the motor neurons only (5); b) ALS with Ub‐ir/TDP‐43‐negative inclusions (4); c) FTD with Ub‐ir and TDP‐43‐ir inclusions limited to the dentate gyrus (DG) (4); d) FTD with Ub‐ir and TDP‐43‐negative inclusions limited to the DG (4); e) FTD with FTLD‐U type II‐like pathology with Ub‐ir/TDP‐43‐negative inclusions (2); f) DLDH (9). Conclusions: The use of Ub and TDP‐43 IHC allowed the classification of 47.1% of cases from our series, according to the criteria. 17% had a pathological diagnosis of DLDH. Among those cases not meeting criteria for inclusion in one of the proposed subtypes, new possible subtypes, potentially associated with novel biological mechanisms of neurodegeneration were observed. Supported by AG10133
High‐throughput assays for gingival fibroblast in vitro wounds treated with nicotine, and/or TGFβ1
Authors: Svoboda Kathy K H , Cline Rob , Cowan Cameron
Published: June 2021

Nicotine treated gingival fibroblast cells have inhibited wound and TGFβ1 responses leading to altered phosphorylation of signaling proteins. Over 600 signaling proteins were screened for changes in phosphorylation when treated with nicotine and/or TGFβ1 and wounded in vitro and a high‐throughput cell culture assay was developed to screen effects on wound closure. Primary human fibroblasts were grown to confluence in 30mm dishes and 96 well plates, starved for 24 hours and treated with nicotine or TGFβ1 2 hours before wounding. Four treatment groups: controls (C), nicotine (N), TGFβ1 (TGF), and N+TGF were divided into wounded and unwounded groups. Cells were lysed in ice cold buffer with phosphatase inhibitors, frozen and transported to Kinexus for phosphorylated protein screening. A 96 well plate pin array was used to uniformly wound each well. Plates were fixed and stained at 0, 4, 7, 14, and 24 hours. Plates were imaged with a light scanner at 1200 dpi. Wound areas were measured with Metamorph. Proteins involved in cell migration including p38 MAP kinase, MEK1, and PCTK1 were increased in C and TGF, but decreased in N and TGF treated groups. N treatment increased phosphorylation of CDK1/2 and PKCd. The pin array wounded the cells in a uniform and predictable manner. Wounds had closed 50–56% by 4hr. Nicotine treated wounds were larger at 7 and 14 hr and closed at 24 hr.
Neuropathological phenotype of a new type of lissencephaly associated with mutations of TUBA1A
Authors: Fallet‐Bianco Catherine , Loeuillet Laurence , Loget Philippe , Chapon Françoise , Poirier Karine , Chelly Jamel , Francis Fiona
Published: June 2021

In 2007, Keays et al identified in an ENU‐induced mutant mouse, a mutation in Tuba1a, a gene coding for alpha 1 tubulin protein, associated with behavioral disorders and a disturbance of the cortical cytoarchitectony. Subsequently, mutations of TUBA1A were found in children with mental retardation and lissencephaly with abnormalities of the corpus callosum and cerebellum, on MRI. Recently, mutations of TUBA1A were found in 4 fetuses, with a prenatal diagnosis of cerebral dysgenesis leading to a medical termination of pregnancy according to French laws.

These fetuses, not mutated for LIS1, DCX and ARX, were studied at 23, 25, 26 and 35 gestational weeks respectively. The neuropathological study demonstrated a phenotype including abnormalities affecting constantly the neocortex, hippocampus, corpus callosum, cerebellum and brainstem, and inconstantly, basal ganglia, olfactory bulbs and subventricular germinal zones. This phenotype was different from that of LIS1, DCX, ARX lissencephalies, and in agreement with the pattern of expression of TubA1A studied in the mouse embryo. Compared with children mutated for TUBA1A, fetal cases are located at the most severe end of the spectrum, suggesting that prenatally diagnosed cases are probably the most severe forms of TUBA1A lissencephaly.
ATP induces proliferation and neuroprotection in Swiss Webster mouse olfactory epithelium
Authors: Hegg Colleen , Doherty James , Crudgington Sean , Jones Eric
Published: June 2021

The olfactory epithelium (OE) is a good model to study neuroregeneration. Throughout life, progenitor cells proliferate, differentiate into neurons, and undergo apoptosis. In the CNS, ATP activation of purinergic receptors (P2R) induces proliferation. We tested the hypothesis that ATP can initiate proliferation in the OE. We measured BrdU incorporation in neonatal OE slices and adult mice 48 hours following treatment with ATP or ATPγS and/or P2R antagonists PPADS + suramin. Mice treated intranasally with ATPγS had significantly more BrdU‐immunoreactive cells compared to control. Pre‐treatment with P2R antagonists reduced the ATP‐induced BrdU incorporation to control levels. Similar results were obtained with OE slices. These data suggest that P2R activation induces proliferation. To determine if the proliferation is related to cell survival, OE slices were treated with ATP or suramin and exposed to CaspACE FITC‐VAD‐FMK, a marker for activated caspase‐3. 18 hr exposure to ATP decreased apoptosis, and suramin increased apoptosis, reversing the protective effect of ATP. These data suggest that ATP and P2R activation have a proliferative and a protective effect in the OE. Supported by NIDCD DC006897.
Association of the causative malignant hyperthermia RyR1‐Thr2206Met‐mutation with discrete clinical and muscular biopsy changes
Authors: Schober Ralf , Ogunlade Vera , Wehner Markus , Rüffert Henrik
Published: June 2021

Malignant hyperthermia is a potentially fatal neuromuscular disease with hypersensitivity against anaesthetic agents, in the majority of cases based on mutations of the ryanodin receptor 1 gene (RyR1) in various loci. We have examined 17 out of 62 members of a family over 3 generations that had some accidents of narcosis and/or mild muscular symptoms in their anamnesis. In 8 of them there was a positive in‐vitro‐contracture‐test, and direct sequencing of the RyR1‐gene revealed the c6617C>T (Thr2206Met) mutation exclusively in these patients. Histological, histochemical and immunohistological examination of the muscle biopsies revealed mild architectural changes and changes in the oxidative intermyofibrillar network of type 1 muscle fibers, being exclusively present in the malignant hyperthermia patients and most pronounced in the maternal grandfather. The changes were best visualized in NADH‐stains and did not correspond to classical central cores or multi‐minicores. Since a variety of genetic and epigenetic changes with clinical presentations different from the ones found by us have been described in these conditions, the report represents a novel aspect in the spectrum of malignant hyperthermia and will be helpful for the classification of RyR1 mutations and for genotype‐phenotype correlations.
Structural studies of RecF
Authors: Korolev Sergey , Makharashvili Nodar , Koroleva Olga
Published: June 2021

RecF, together with RecO and RecR, belong to a ubiquitous group of recombination mediators (RMs) that include eukaryotic proteins such as Rad52 and BRCA2. RMs help maintain genome stability in the presence of DNA damage by loading RecA‐like recombinases and displacing single‐stranded DNA binding proteins. Here we present the crystal structure of RecF from Deinococcus radiodurans. RecF exhibits a high degree of structural similarity with the head domain of Rad50, but lacks its long coiled‐coil region. The structural homology between RecF and Rad50 is extensive, encompassing the ATPase subdomain and the so‐called “Lobe II” subdomain of Rad50. The pronounced structural conservation between bacterial RecF and evolutionarily diverged eukaryotic Rad50 implies a conserved mechanism of DNA binding and recognition of the boundaries of double‐stranded DNA regions. The RecF structure, mutagenesis of conserved motifs and ATP‐dependent dimerization of RecF are discussed with respect to its role in promoting presynaptic complex formation at DNA damage sites.
Structural basis for base discrimination by RB69 DNA polymerase
Authors: Wang Mina , Klimenko Dmitry , Steitz Thomas A , Wang Jimin
Published: June 2021

Replicative DNA polymerases (pol) copy genomes efficiently with high base selectivity. Kinetic studies have shown that in the RB69 pol triple mutant, L561A/S565G/Y567A, in which residues in the nucleotide binding pocket that contact the nascent base pairs, were reduced in size, base selectivity drops dramatically but the kinetic parameters for incorporation of correct bases was unaltered relative to the wild type enzyme. To provide the structural basis for this kinetic discrimination, we solved crystal structures of the RB69 pol triple mutant in the presence and absence of dA‐dTTP and in complex with mismatched base pairs. The apo form of the mutant RB69 pol was captured in a unique, half‐closed state. Helix P in the apo form of the pol mutant was straight, eliminating the necessity of the kinked‐to‐straight conversion that occurs in wild type pol when it forms the ternary complex. Also, the phenyl ring of Y567, at the kink of helix P, which contacts the templating base, rotates 25° during conversion to the ternary complex. The extent of this rotation is limited by its interaction with Y416, Y391 and I586. This constraint is eliminated in the triple mutant and, as a consequence, base selectivity is reduced. L561 and S565 also contact the templating base, restricting its flexibility. When these two residues are replaced in the triple mutant, base discrimination is further reduced.
Stimulation of the human mitochondrial replicase by deletion mutants of mitochondrial single‐stranded DNA‐binding protein
Authors: Oliveira Marcos Tulio , Kaguni Laurie S
Published: June 2021

Biochemical studies of the human mitochondrial replisome demonstrate that the mitochondrial replicase (pol γ) and the mtDNA helicase Twinkle are stimulated by the mitochondrial single‐stranded DNA‐binding protein (mtSSB). mtSSBs share similar physical and biochemical properties with Escherichia coli SSB, and a high degree of conservation in the central region of their amino acid sequences; however, the roles of their variable N‐ and C‐termini are not understood. The aim of this work is to evaluate the stimulation of the human mitochondrial replicase by its cognate mtSSB (HsmtSSBwt) and mutants lacking the N (HsmtSSBΔN), the C (HsmtSSBΔC), and both termini (HsmtSSBΔNΔC). In a reaction that mimics mitochondrial lagging DNA strand synthesis, we show that an N‐terminally His‐tagged version of HsmtSSBwt and the untagged HsmtSSBΔN stimulate pol γ ∼ 10 fold, in a manner similar to Drosophila pol γ stimulation by its cognate mtSSB. Interestingly, both human proteins show stimulatory effects that are ∼ 2 fold higher than that of the untagged HsmtSSBwt. Although differing physical interactions may account for these observations, we are examining these and various biochemical properties of the mutants to understand the mechanism by which mtSSB stimulates DNA synthesis by pol γ. This work is supported by grant GM45295 from the National Institutes of Health.