bluuebunny/crossref_metadata_2025 huggingface dataset:
Insertions in xanthene dehydrogenase genes suppress cell death due to stabilization of topoisomerase IA covalent complex
Authors: Annamalai Thirunavukkarasu , Tse‐Dinh Yuk‐Ching
Published: June 2021

Bacterial type IA or IIA topoisomerases modify DNA topology by transiently cleaving and rejoining either one or two strands of DNA respectively. When topoisomerases cleave the DNA, a covalent complex between the cleaved DNA and the enzyme is formed. Stabilization of the type IIA topoisomerase complex by antibacterial drugs such as fluoroquinolones leads to decrease in DNA rejoining, accumulation of DNA strand breaks which in turn leads to cell death. There is an urgent need for new antibiotics due to the emerging drug resistance and topoisomerase IA is a promising target. It has been shown that a recombinant type IA Yersinia pestis topoisomerase with a conserved glycine residue in the TOPRIM motif mutated to serine accumulates the covalent complex and causes rapid bacterial cell killing. The main goal of this work is to study the mechanism of cell killing resistance for the stabilized type IA topoisomerase covalent complex. Insertional mutagenesis was used to identify and characterize the genes in Escherichia coli that influence the cell killing by the mutant Y. pestis topoisomerase I. We observed that insertions in the xanthene dehydrogenase operon (xdhABC) and homolog xdhD resulted in resistance to topoisomerase IA covalent complex mediated cell killing by ~4 log10 fold. Currently, studies are being conducted to elucidate the effect of these insertions on SOS response and other DNA damaging agents.
CD40 disruption of autoaggressive (CD4+CD40+) to regulatory (CD4+CD25+FoxP3+) T cell homeostasis promotes diabetes
Authors: Wagner David H , Vaitaitis Gisela M , Waid Dan M , Pennock Nathan D
Published: June 2021

While regulatory T cells (Treg) are well described, identifying autoaggressive effector T cells has proven more difficult. However, we identified CD4loCD40+ (Th40) cells as directly diabetogenic in NOD mice. NOR mice have an identical T cell developmental background, yet are diabetes‐resistant. The BDC2.5.TCR transgenic mouse is on the NOD background; possesses the highly diabetogenic TCR, BDC25; but also is diabetes resistant. The seminal issue is how these mice remain tolerant to self‐antigens. Autoaggressive T cells develop in NOR mice and are confined to the Th40 subset. Interestingly, when Th40 cells and Tregs are depleted, the remaining cells are not diabetogenic, demonstrating that Th40 cells are required for diabetes transfer. We suggest that the determinant factor for autoimmunity is the Th40 to Treg ratio that is significantly disrupted in NOD mice. When compared to BALB/c mice the actual number of Tregs from NOD mice is statistically identical while Treg numbers in NOR or BDC2.5.TCR.Tg mice are slightly higher. The ratio of Th40 cells to Tregs from NOD but not NOR, BALB/c or BDC2.5.TCR.Tg mice is substantially increased. Mechanistically, NOD Th40 cells have low susceptibility to induced cell death and unlike cells from NOR and BALB/c mice, have low Fas expression. CD40 engagement of Th40 cells induces Fas expression but further confers resistance to Fas – mediated cell death in NOD mice. Only at very high Fas concentrations are Th40 cells susceptible to induced cell death. In the BDC2.5.TCR.Tg system we surprisingly found that Th40 cells are abundant but are completely anergic, expressing very high levels of FoxP3. In this instance, these T cells are not typical Tregs, but rather FoxP3 is suppressing the ability of these T cells to be pathogenic.
Vaccination to treat persistent viral infection
Authors: Brooks David , McGavern Dorian , Oldstone Michael
Published: June 2021

Therapeutic vaccination is a potentially powerful strategy to establish immune control and eradicate persistent viral infections. Yet, despite numerous different approaches and for reasons that are unclear, vaccines administered during an established persistent viral infection fail to substantially elicit immunity or to control viral replication. Our hypothesis to explain this failure is that although vaccines are designed to stimulate T cells, they do not alleviate the immunosuppressive environment. As a result, vaccine‐activated T cells rapidly succumb to the same constraints that previously limited T cell responses during persistent infection. Using the lymphocytic choriomeningitis virus (LCMV) model of infection we found that the immunosuppressive environment (and not the vaccinating agent, level of virus replication or an inherent inability of exhausted T cells to respond to stimulation) is indeed responsible for the failure of therapeutic vaccines. Antibody blockade of the key suppressive factor interleukin‐10 rendered otherwise ineffective therapeutic vaccines highly efficient at restoring T cell immunity leading to immune‐mediated control of an established persistent viral infection. Thus, our findings help solve one of the great enigmas in infectious disease and vaccine design by demonstrating why therapeutic administration of a vaccine agent is unsuccessful whereas prophylactic administration of the same vaccine to pathogen naïve individuals (in which no immunosuppressive environment exists) is highly effective.
Ready‐to‐eat cereal consumption and cognitive function in late life: Cache County Study on Memory, Health, and Aging
Authors: Wengreen Heidi , Nelson Chailyn , Corcoran Chris , Munger Ron
Published: June 2021

Ready‐to‐eat breakfast cereal (RTEC) is a major source of many nutrients with potential benefits for cognitive health, but little is known about the association between RTEC consumption and cognitive function of aging men and women. We prospectively examined associations between frequency of RTEC consumption and cognitive function and decline among elderly men and women of the Cache County Study on Memory and Aging in Utah. In 1995, 3831 resident men and women 65 years of age or older completed a baseline survey that included a food frequency questionnaire. Cognition was assessed using an adapted version of the Modified Mini‐Mental State examination (3MS) at baseline and three subsequent interviews spanning approximately 7 years. Multivariable‐mixed models were used to estimate the effect of RTEC consumption on average 3MS score over time. Eating RTEC 1–6 times per week was associated with higher baseline 3MS scores than those consuming RTEC more or less frequently (≤ 3 times per month, ≥ 1 time per day; a difference of 0.36 and 0.64 3MS points, respectively; p‐value = 0.0078). This effect was maintained over 7 years of follow‐up (p‐value = 0.028). RTEC consumption of ≥ 1 time per day may be associated with less healthy dietary and lifestyle behaviors. RTEC is a nutrient dense food and when included as part of a varied diet may help to attenuate age‐related cognitive decline.
Isolation of cucurbitacin E from pumpkin seed and analysis of its anti‐cancer and anti‐inflammatory activities
Authors: Jang Byeong‐Churl , Sim Hu‐Sung , Jeong Byeong‐Yong , Park Hye‐Min , Oh Man‐Jin
Published: June 2021

In order to improve the use of pumpkin seed, the present study was performed to isolate compositions of the bitter substances which were not seen in pumpkin seed itself but biosynthesized during germination of the seed. The compositions isolated were then further purified by silica gel, TLC, and HPLC in which a fraction with Rf 0.73 and RT 10.30 was obtained. Cucurbitacin E with molecular weight of 557 was identified by subsequent structural analysis of the fraction by LC‐MS/MS. The production of cucurbitacin E peaked with 224.7 mg/Kg at 7 days of germination at 20–23C with the water supply at intervals of 48 hr in the darkness. While that of cucurbitacin E reached 146.7 mg/Kg in the brightness. In vitro‐cell based assays demonstrated that cucurbitacin E isolated and purified had not only an apoptotic effect on prostate and lung cancer cells but also an anti‐inflammatory activity by inhibiting expression of cyclooxygenase‐2, an inflammatory protein, suggesting that cucurbitacin E may be useful as a potential anti‐cancerous and anti‐inflammatory agent.
A novel mechanism of translational control by RGS2
Authors: Nguyen Chau Huynh , Ming Hong , Hugendubler Lynne , Kimball Scot , Chidiac Peter
Published: June 2021

The Regulator of G protein Signaling (RGS) proteins serve as GTPase accelerating proteins. We have identified a novel role for RGS2 in the control of protein synthesis that is independent of its established RGS domain function. The objective of this research was to characterize further this new aspect of RGS2 signaling, with the promise of identifying a novel therapeutic target for treating diseases characterized by impaired protein synthesis. RGS2 was found to bind to the eukaryotic initiation factor 2 ε‐subunit (eIF2Bε) in a yeast two‐hybrid screen, and this interaction was confirmed at the protein level by co‐immunoprecipitation. RGS2 was able to inhibit de novo protein synthesis in an in vitro translation assay, whereas RGS1 and RGS4 did not interact with eIF2Bε or have an effect on protein synthesis. The ability of RGS2 to interact with eIF2Bε and inhibit protein synthesis was dependent on a subdomain within its RGS domain, spanning residues 79–116. An RGS2 peptide based on the eIF2Bε‐interacting region was capable of inhibiting protein synthesis in the translation assay. This work was supported by the Canadian Institutes of Health Research and the Heart and Stroke Foundation of Ontario.
Interaction of M3 receptors and calcium channels in feline bladder smooth muscle.
Authors: Dalisky Ryan , Theobald Robert
Published: June 2021

It is known that muscarinic receptors and calcium channels play roles in smooth muscle contraction, but it is unclear which receptor and which calcium channel types are most important. Recent studies have shown that M3 receptors may have a direct interaction with L‐type calcium channels. It is hypothesized that if stimulus evoked activation of muscarinic M3 receptors causes an influx of extracellular calcium by opening of L‐type calcium channels, then administration of L‐type calcium channel inhibitors will attenuate evoked contractions of bladder smooth muscle. This hypothesis was tested using an M3 specific antagonist, DAU 5884 (DAU), and an L‐type calcium channel inhibitor, verapamil (Ver). Using an anesthetized cat model, bladder contractions, evoked by pelvic nerve stimulation (PNS) and exogenous administration of acetylcholine, ATP or beta, gamma methylene ATP (APPCP), before and after administration of DAU, or Ver, were measured using DATAQ data acquisition hardware and software. The results show that DAU decreased cholinergic, but not purinergic contractions. Ver after DAU had no effect on any evoked contractions. Ver alone decreased cholinergic contractions, but not purinergic contractions. DAU after Ver decreased cholinergic, but not purinergic contractions. This data suggests that there is an interaction between M3 receptors and calcium channels, as indicated by the effect of these agents on cholinergic evoked bladder contractions.
Estrogen receptor‐mediated regulation of UDP‐glucuronosyltransferase (UGT) 1A4 by estrogen
Authors: Jeong Hyunyoung , Chen Huiqing , Choi Suyoung , Fischer James
Published: June 2021

The objective of this study is to characterize estrogen‐mediated regulatory mechanisms of UGT1A4, the major enzyme responsible for elimination of lamotrigine, in view of increased oral clearance of lamotrigine in pregnancy and oral contraceptive users. To this end, luciferase promoter reporter assays, semi‐quantitative real‐time (RT)‐PCR, and deletion assays were conducted in HepG2 and MCF7 cells. Treatment of HepG2 cells with estradiol exhibited a concentration‐dependent increase in the activity of UGT1A4 promoter when ERα was co‐transfected. In MCF‐7 cells, pretreatment of the cells with ICI 182,780 (antiestrogen leading to degradation of ERα that are constitutively expressed in MCF‐7 cells) abolished estrogen‐mediated induction of UGT1A4 promoter activity. Endogenous UGT1A4 expression, determined by RT‐PCR, was also induced by 2.3‐fold by estrogen treatment in HepG2 when ERα was co‐transfected. Results from deletion assays in HepG2 cells further indicated that ‐1667 to ‐863 bp region of UGT1A4 promoter is responsible for the ERα‐mediated regulation. In conclusion, the data demonstrate that estrogen positively regulates UGT1A4 expression at the transcriptional level by a mechanism involving ERα. This regulatory mechanism may be responsible for the increased clearance of lamotrigine in pregnancy. The study was supported by University of Illinois at Chicago.
Absorption, Disposition and Elimination of 3 Ionic Liquids: Bmim‐Cl, NBuPy‐Cl, and BmPy‐Cl in male F‐344 rats
Authors: Knudsen Gabriel Arther , Kuester Robert K , Cheng Yao‐feng , Sipes I Glenn
Published: June 2021

Studies were conducted to characterize the effects of dose and route of administration on the absorption and dispositional fate of three ionic liquids (organic quaternary amines) in male Fischer‐344 rats. The compounds tested were 1‐butyl‐3‐methylimidazolium‐Chloride (Bmim‐Cl), N‐butylpyridinium‐Chloride (NBuPy‐Cl), and 1‐butyl‐3‐methylpyrrolidinium‐Chloride (BmPy‐Cl). Following IV administration (5 mg/kg), radioactivity in blood decreased rapidly in a biphasic manner for all compounds (elimination was >85% of dose in urine). Following oral administration (50 mg/kg), urinary elimination accounted for 78±6% (Bmim‐Cl), 63±11% (NBuPy‐Cl), or 37±10% (BmPy‐Cl) of dose, suggesting differences in the oral absorption among the three compounds. Comparison of blood AUCoral/AUCIV for parent compound revealed systemic oral bioavailabilities of 62.1% (Bmim‐Cl), 67.7% (NBuPy‐Cl), and 43.4% (BmPy‐Cl) of dose. Oral doses of 5 or 0.5 mg/kg resulted in similar elimination patterns. Serial daily oral administration (50 mg/kg, 5 days) of each compound did not alter the absorption or elimination of that compound. Co‐ or pre‐administration of BmPy‐Cl with [14C]‐Bmim‐Cl did not notably alter the rate or route of elimination of [14C]‐Bmim‐Cl. Pharmacokinetic analyses following IV administration for each compound indicated that clearance from blood exceeded the glomerular filtration rate which suggests a role for renal organic cation transporters. It was concluded that while the structures varied, that which was absorbed is extracted by the kidney and eliminated in the urine as parent compound, independent of dose, number of doses, or route of administration. This research was supported by the NIEHS NTP Grant No. N01‐ES‐45529 and NIEHS‐sponsored Southwest Environmental Health Science Center Grant Number P30‐ES‐06694.
Purinergic control of apical plasma membrane PI(4,5)P2 levels sets ENaC activity in cultured and native principal cells
Authors: Pochynyuk Oleh , Bugaj Vladislav , Stockand James David
Published: June 2021

Activity of the epithelial sodium channel (ENaC) is limiting for Na+ reabsorption at the distal nephron. Phosphoinositides, such as phosphatidylinositol 4,5‐biphosphate (PI(4,5)P2) modulate the activity of this channel. Activation of purinergic receptors triggers multiple events including activation of protein kinase C (PKC) and phospholipase C (PLC) with the latter depleting plasma membrane PI(4,5)P2. Here, we investigate regulation of ENaC in renal cultured and native principal cells by purinergic receptors via PLC and PI(4,5)P2. Purinergic signaling rapidly decreases ENaC open probability and apical membrane PI(4,5)P2 levels with similar time courses. Moreover, inhibiting purinergic signaling with suramin rescues ENaC activity. The PLC inhibitor, U73122, but not its inactive analogue, U73343, recapitulates the action of suramin. In contrast, modulating PKC signaling failed to affect purinergic regulation of ENaC. Unexpectedly, inhibiting purinergic receptors and PLC in resting cells dramatically increased ENaC activity above basal levels indicating tonic activation of purinergic signaling in this cultured and native renal epithelia. Increases in ENaC activity were associated with an increase in apical membrane PI(4,5)P2 level. Subsequent treatment with ATP in the presence of inhibited purinergic signaling failed to decrease ENaC activity and apical membrane PI(4,5)P2 levels. Dwell time analysis reveals that depletion of PI(4,5)P2 forces ENaC toward a closed state. In contrast, increasing PI(4,5)P2 levels above basal values locks the channel in an open state interrupted by brief closings. Thus, our results suggest that purinergic control of apical membrane PI(4,5)P2 levels is a major regulator of ENaC activity in cultured and native renal epithelial cells.
Histone deacetylase 2 plays an important role in the development and progression of diabetic renal injury
Authors: Oh Eun Young , Noh Hyunjin , Seo Ji Yeon , Yu Mi Ra , Lee Hi Bahl , Ha Hunjoo
Published: June 2021

Diabetic nephropathy is the leading cause of end‐stage renal disease and characterized by excessive accumulation of extracellular matrix (ECM) in the kidney. The role of epithelial‐mesenchymal transition (EMT) in the development of renal fibrosis is well known. Histone deacetylase (HDAC) inhibitors have been shown to exert antifibrotic effect but the role of HDAC in diabetic renal injury remains incompletely understood. Here we demonstrate that trichostatin A (TSA), a nonselective HDAC inhibitor, prevents albuminuria, EMT, and enhanced ECM expression in streptozotocin‐induced diabetic rat kidney without significant effect on blood glucose. Hyperacetylation of histone (H3 and H4) by TSA is associated with downregulation of ECM mRNAs and reversal of EMT in TGF‐β1‐stimulated normal rat kidney tubular epithelial (NRK52E) cells. TGF‐β1 significantly increases activity of HDAC 2, a class I HDAC, and HDAC 2 silencing using small interference RNA represses TGF‐β1‐induced ECM upregulation and EMT in NRK‐52E cells. These results support an important role for HDAC 2 in the development and progression of renal fibrosis and suggest HDAC 2 as a novel therapeutic target for the attenuation of diabetic renal injury. (Supported by R01‐2006‐000‐10829‐0s and R15‐2006‐020 from KOSEF and BK 21)
Cortisol and Catecholamine stress hormone decrease is associated with the behavior of perceptual anticipation of mirthful laughter
Authors: Berk Lee S , Tan Stanley A , Berk Dottie
Published: June 2021

Chronic stress is correlated with increases in stress hormones cortisol and catecholamines. We have reported that a mirthful laughter humor experience (a eustress positive phenomenon that ameliorates biological effects of distress): 1) increases CD4 cells; 2) increases NKCA; 3) increases IFN‐γ and, in perceptual anticipation of a laughter eustress event increases positive psychological mood states along with β‐endorphin and HGH. Postulating that a decrease in neuroendocrine stress hormones may parallel these anticipatory changes, we studied 16 healthy fasting male subjects for cortisol and catecholamine plasma levels in anticipation of a eustress humor/laughter experience. Three days prior to the intervention subjects were notified of random assignment (experimental or control). Blood was drawn before the intervention, four times during and three times post. One sided independent t‐tests showed anticipatory baseline decreases for cortisol, epinephrine and dopac (dopamine catabolite) in the experimental laughter group (p<0.01). Trend analysis showed progressive patterns of decrease (p<0.01). We suggest the perception of anticipating a positive eustress event decreases stress hormones cortisol and catecholamines, concomitant with positive mood state changes. These neuroendocrine findings have immune modulation implications beneficial in stress reduction for wellness & prevention.
Angiotensin‐(1‐7) ICV Chronic Infusion Attenuates DOCA‐Salt Hypertension
Authors: Campagnole‐Santos Maria J , Guimarães Priscila
Published: June 2021

Ang‐(1‐7)‐Mas receptor axis is an important counter‐regulator of Ang II‐AT1 on cardiovascular actions. This study evaluated ICV chronic infusion of Ang‐(1‐7) on the progression of DOCA‐Salt hypertension. Sprague‐Dawley rats (3–4 months of age, 300–450g) were submitted to right cerebral ventricle cannulation (ICV), 7 days after deoxycorticosterone‐acetate (DOCA) implantation (200 mg/Kg, sc). ICV infusion was performed with ALZET osmotic pumps filled with Ang‐(1‐7) (200 ng/0.5 μl/h) or saline (0.5 μl/h; CTL) for 14 days. On the 14th day of infusion, in conscious rats, baseline arterial pressure (AP), the sensitivity of the baroreflex bradycardia (BRS) and the cardiac autonomic tonus were evaluated. Ang‐(1‐7) ICV infusion reduced baseline mean AP (135 ± 8 mmHg vs 163 ± 3 mmHg, CTL), reduced baseline heart rate (325 ± 15 beats/min vs 383 ± 17 beats/min, CTL), increased the cardiac parasympathetic tonus (74 ± 4 beats/min vs 51 ± 8 beats/min, CTL), decreased the cardiac sympathetic tonus (57 ± 15 beats/min vs 101 ± 6 beats/min, CTL) and increased the BRS (1,49 ± 0,25 ms/mmHg vs 0,56 ± 0,10 ms/mmHg, CTL). These results suggest that increasing central Ang‐(1‐7) levels may contribute to the attenuation of arterial hypertension, at least in part, by altering the central integration of autonomic control of HR. Support Fapemig, CNPq, CAPES.
Contribution of the Carotid Body (CB) to Respiratory Stability During Sleep in Developing Inbred Mice
Authors: Balbir Alexander , Martone P. , Lande B. , O’Donnell C. P. , Shirahata Machiko
Published: June 2021

We previously reported the ventilatory responses to hyperoxia and hypoxia during sleep develops at 1‐week (1W) of age and matured differently at 4‐weeks (4W) of age in the DBA/2J and A/J strains of mice. Because the CB develops differently in these strains, we speculated that the CB differentially contributed to the control of respiration. The purpose of the current study is to determine the contribution of the CB to respiratory stability during sleep. Whole‐body flow‐plethysmography was used to assess breath‐to‐breath variability [coefficient of variation (CV) of Ttot] and behavioral indices were employed for sleep‐wake detection. Measurements were made at 30s intervals of hyperoxia (100% O2) and hypoxia (15% O2). At 1W, the CV during hyperoxia, relative to baseline, was significantly greater in the DBA/2J (average±SE: 23.3±2.53% vs. 42.4±6.36%). Further, the interquartile range (IQR) of Ttot during hyperoxia is significantly greater relative to baseline. In the 1W A/J, CV or IQR during hyperoxia did not change significantly. Hypoxia did not elicit changes in CV in either strain. However the distribution of CV values for the A/J was more variable. At 4W neither strain demonstrated significant changes in CV in response to either gas. However, IQR during hyperoxia (0.09±0.014) was significantly increased relative to baseline (0.04±0.006) in the DBA/2J. This study suggests that resting input from the CB contributes significantly to respiratory stability in the 1W DBA/2J and to a lesser extent in the 4W DBA/2J. In the A/J, resting input from the CB has insignificant effects. Supported by HL72293 and HL081345.
Differential effect of aerobic exercise training on cardiovagal baroreflex and carotid arterial compliance
Authors: Komine Hidehiko , Sugawara Jun , Hayashi Koichiro , Yoshizawa Mutsuko , Yokoi Takashi
Published: June 2021

The purpose of this study was to examine the hypothesis that short term aerobic exercise training increase cardiovagal baroreflex sensitivity without altering compliance of the barosensitive carotid artery. To examine this hypothesis, we estimated cardiovagal baroreflex sensitivity and carotid arterial compliance in elderly subjects before and after 2 weeks of exercise training. The training was walk/jog for a period of 30–45 min/day (3–5 days/week) at an intensity of 65–75% of their individual maximum heart rate. Cardiovagal baroreflex sensitivity was assessed by beat‐to‐beat analysis of arterial blood pressure and heart rate during phase ? of Valsalva maneuver. Carotid arterial compliance was determined from systolic and diastolic carotid arterial diameter measured by ultrasound imaging and determined from carotid arterial blood pressure obtained using applanation tonometory. After 2 weeks of exercise training, arterial baroreflex sensitivity increased but arterial compliance did not change. These results suggest that the change in cardiovagal sensitivity caused by short‐term aerobic exercise training does not always depend on the alteration of compliance of the barosensitive carotid artery.
TRPV4 channel mediates flow‐induced dilation in mouse small mesenteric arteries
Authors: zhang david , bubolz aaron , mendoza suelhem , gutterman david
Published: June 2021

We previously found that TRPV4 channel is an important mediator of flow‐induced, endothelium‐derived hyperpolarizing factor (EDHF)‐mediated dilation in human coronary arterioles. Here we further investigated the role of the mechanosensitive TRPV4 channel in flow‐induced dilation using the TRPV4 knockout (TRPV4−/−) mouse model. Using RT‐PCR and Western blots, TRPV4 mRNA and protein were detected in mesenteric arteries. Small mesenteric arteries from TRPV4−/− mice and their wild‐type (WT) control were constricted with U46619 and subjected to step‐wise increase in luminal flow. Relaxation response to flow was markedly reduced in TRPV4−/− vs. WT mice (maximal relaxations of 32±8% vs. 51±6% respectively; n=7, p<0.05). Ruthenium red, a TRPV4 blocker, inhibited flow‐induced dilation in mesenteric arteries from WT mice (maximal relaxation of 11±8%; n=5, P<0.05) but not in TRPV4−/− mice (maximal relaxation of 26±10%, n=3). Papaverine‐induced relaxations were similar between WT and TRPV4−/− mice. In WT mice, flow‐induced dilation was inhibited by the NO synthase inhibitor L‐NAME (maximal relaxation of 25±7%; n=7, P<0.05). We conclude that TRPV4 channel is involved in flow‐induced, endothelium‐dependent (possibly NO‐mediated) vasodilation of murine small mesenteric arteries. TRPV4 channel may represent a mechanical‐chemical signal transduction mechanism linking shear and vasomotor response.
TRPC6 and Orai1 expression increase with mitogen augmented store‐operated Ca2+ entry in rat aortic smooth muscle
Authors: Emter Craig A. , Shaw Rebecca L. , Bowles Douglas K.
Published: June 2021

Transient receptor potential canonical (TRPC), Orai1, and STIM1 proteins are proposed components of store‐operated Ca2+ (SOC) channels. Increases in SOC entry (SOCE) may play a role in phenotype modulation of smooth muscle cells via Ca2+‐dependent signaling mechanisms. The purpose of this study was to determine if mitogen‐induced phenotype modulation alters SOCE or expression of components comprising SOC channels. 24 hour treatment of cultured rat aortic smooth muscle cells (RASMC) with platelet‐derived growth factor‐BB (PDGF‐BB) increased intracellular Ca2+ levels and rate of SOCE (p<0.05) induced by 10 minute exposure to 10 μM cyclopiazonic acid (CPA). CPA exposure increased SOC currents (ISOC) in the PDGF‐BB treatment group and reversal potential shifted from 0 mV to approximately +10 mV (whole‐cell voltage clamp). TRPC6 and Orai1 protein and mRNA expression increased (p<0.05) and STIM1 decreased (p<0.05) in PDGF‐BB treated RASMC. Our results show treatment with PDGF‐BB increases SOCE and ISOC in cultured RASMC with concurrent increases in TRPC6 and Orai1 and decreased STIM1 expression. In conclusion, increased SOCE is characteristic of mitogen‐induced phenotype modulation in RASMC. We speculate that a TRPC6‐Orai1 complex may form the components of the SOC channel following PDGF‐BB treatment. Research support: NIH HL52490.
The Mechanism of Phenotypic Modulation of Vascular Smooth Muscle Cells: Role of extracellular matrix and PDGF‐BB/IL‐1b
Authors: Yeh Yi‐Ting , Chen Cheng‐Nan , Chien Shu , Chiu Jeng‐Jiann
Published: June 2021

The phenotype of vascular smooth muscle cells (SMCs) play important roles in atherogenesis. We studied roles of extracellular matrix (ECM) and platelet‐derived growth factor (PDGF)‐BB/interleukin (IL)‐1b in modulating SMC phenotype. Human aortic SMCs on monomeric collagen (MC) showed lower expression of contractile markers, with a higher percentage in the S‐phase, than the cells on polymerized collagen (PC). SMCs on PC had lower expressions of cyclin A, D1, and E, cyclin‐dependent protein kinase (Cdk) 2, 4, and 6, and higher expressions of p27 and p21, as compared with the cells on MC. PDGF‐BB/IL‐1b inhibited the contractile marker expression and induced cell cycle progression, with an increase in Cdk6 expression, in SMCs on PC. Transfection of SMCs with Cdk6‐siRNA inhibited the PDGF‐BB/IL‐1b‐induced cell cycle progression. Wortmannin and LY294002 inhibited the PDGF‐BB/IL‐1b‐induced Cdk6 expression in SMCs on PC. SMCs on MC showed higher levels of avb3 and lower levels of a2b1 integrins than the cells on PC. PDGF‐BB/IL‐1b inhibited the b1 and b3 expressions in SMCs on PC. Our findings provide insights into the molecular basis for the roles of ECM and PDGF‐BB/IL‐1b in modulating SMC phenotype.
Proteomic analysis of brainstem micro vessels in angiotensin II induced hypertension
Authors: Toward Marie Ann , Heesom Kate J. , Kasparov Sergey , Paton Julian F.R.
Published: June 2021

Previously, we have compared brainstem micro vessel tissue from spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto (WKY) rats using microarrays; the results suggested that the SHR brainstem may be hypoxic. With its known raised activity in hypertension, we infused pressor (800ng·kg−1·min−1) and non‐pressor (50ng·kg−1·min−1) doses of angiotensin II (ANGII) into WKY rats (male, 12–14 weeks) and ran 2D‐gels of the brainstem vessel fraction to identify protein changes between rats infused with peptide or vehicle via osmotic mini pump. After 10 days, a brainstem vascular fraction was isolated. The pressor dose increased mean arterial pressure from 91 ± 3 to 150 ± 3 mmHg by day 7 (mean ± S.E.M., p<0.05; n=12) with no change in the low dose and vehicle groups. The Ettan™ DIGE 2D‐gel system (GE Healthcare) with DeCyder™ software and mass spectrometry were employed to detect protein changes. ANGII altered proteins were identified as having roles in energy metabolism with many regulated by hypoxia. These data suggest that increased levels of ANGII may lead to hypoxia and a switch in oxidative metabolism in brainstem areas controlling blood pressure and that this may be a common mechanism underlying increased sympathetic activity in neurogenic hypertension. BBSRC, Pfizer and British Heart Foundation funded research.
Sustained hypertension‐induced dynamic transcriptional regulation in the nucleus tractus solitarius
Authors: Miller Gregory M , Hao Haiping , Vadigepalli Rajanikanth , Schwaber James S
Published: June 2021

In this study, we focus on the transcriptional regulation mediated by the factors AP‐1 and CREB in the adaptive response of the nucleus tractus solitarius (NTS) to sustained hypertension. Analysis of a gene expression time series data revealed extensive differential regulation in NTS in response to elevation of systemic blood pressure. We have pursued a bioinformatics approach to analyze the upstream promoter sequences of these differentially regulated genes for the presence of AP‐1 and CREB binding sites. Our analysis resulted in a total of 500 differentially regulated genes. We have experimentally validated a focused subset of neuronal function relevant genes (e.g., ion channels and signaling kinases) through a novel Fast Carrier Chromatin Immunoprecipitation approach. Our results reveal dynamic changes in binding of AP‐1 and CREB at these promoters, specifically phospho‐CREB binding at the c‐fos and Tyrosine Hydroxylase (TH), and AP‐1 binding at the TH and Angiotensin II receptor, type 1 gene promoters. These results indicate the combinatorial regulation by AP‐1 and CREB in shaping the neural adaptation in NTS in response to elevated blood pressure. Research Support: NIH/HLB R33 HL087361 to JSS.
Hyperhomocysteinemia causes cardiac rhythm disturbances due to a shift in atrial and ventricular gap junction protein distribution
Authors: Rosenberger Dorothea Susanne , Rodriguez‐Alvarez Walter E , Tyagi Neetu , Steed Mesia B , Moshal Karni , Sen Utpal , Metreveli Naira , Tyagi Suresh C
Published: June 2021

Homocysteine (Hcy) is an amino acid byproduct in the metabolism of methionine. Hyperhomocyseinemia (HHcy) is associated with thromboembolic events. Hcy has been identified to correlate with atrial fibrillation and stroke in humans. The role of Hcy and its influence on atrial and ventricular gap junction proteins has been investigated. We hypothesize that changes in the Connexin (Cx) distribution and localization are responsible for cardiac arrhythmia. ECG was monitored in freely moving mice (Hcy‐diet for 12 weeks) with a subcutaneously implanted telemetric ECG probe. 2D transthoracic echocardiography was performed to assess regional wall motion abnormalities and ventricular diameter changes in M‐Mode. Immunoblotting was used for quantification, immunohistochemistry was used to visualize the localization of the gap junction proteins Cx 40, 43 and 45. A shift of Cx 43 to Cx 45 was seen in the ventricular myocardium, a loss of Cx 40 was detected in the atrial myocardium in HHcy animals. Arrhythmia, sudden cardiac death and left ventricular dysfunction along with right ventricular diameter changes were detected in the HHcy animal group.
Mechanisms underlying Endothelin‐1 effects on ischemic ventricular tachycardia in canine myocardium
Authors: Gopinathannair Rakesh , Xing Dezhi , Chaudhary Ashok , Martins James B
Published: June 2021

Background: Endothelin‐1(ET‐1) induces ventricular tachycardia (VT), possibly by promoting triggered activity (TA). We studied ET‐1 and the ET‐A receptor blocker, BQ123, in a model of focal or reentrant VT, to test the hypothesis that focal VT and TA were selectively affected. Methods: Anesthetized dogs (n=38) with 1‐3 hours of coronary artery occlusion underwent 3‐D activation mapping to identify mechanisms of VT. If VT was not inducible, ET‐1 (0.15–0.45 mcg/kg IV) was infused. If VT was reproducibly induced at baseline, BQ123 (2.5 mcg/kg IV) was given. Effects on action potential (AP), delayed and early afterdepolarizations (DADs and EADs), and TA of ischemic endocardium were studied in vitro with microelectrodes. Results: Of 15 dogs without VT, ET‐1 promoted induction of VT of mixed reentrant and focal origin in 5 (p<0.05 (*) vs. saline alone). In 11 dogs, BQ123 prevented reentrant VT in 4 of 6* vs. 0/5 with focal VT. ET‐1 and BQ123 infusion did not change effective refractory period, pacing threshold, mean arterial pressure, or infarct size. In vitro, ET‐1 infusion did not induce and BQ123 did not prevent EADs, DADs, or TA but ET‐1 superfusion with rapid pacing produced AP alternans in 8 of 15 tissues. Conclusions: ET‐1 promotes focal and reentrant VT but ischemic sites in vitro did not show TA. BQ123 preferentially blocked reentrant VT. The effect of ET‐1 on ischemic reentrant VT may be mediated by triggering AP alternans.
Crystal structure of human thymine DNA glycosylase bound to DNA elucidates sequence‐specific mismatch recognition
Authors: Maiti Atanu , Pozharskiy Edwin , Drohat Alexander C
Published: June 2021

Cytosine methylation at CpG dinucleotides is central to transcriptional regulation and genome stability and is implicated in genetic disease, cancer, and ageing, a prominent mechanism involving 5‐methylcytosine deamination to give mutagenic G•T mispairs. Human thymine DNA glycosylase (hTDG) excises T from G•T mismatches, and removes other damaged bases, and has specificity for lesions paired with guanine and located at CpG sites. The molecular basis of these important catalytic properties has remained unknown. Here, we report a crystal structure of hTDG (catalytic domain) in complex with abasic DNA reveals that it binds as a protein dimer, one subunit at the abasic site and the other at an undamaged (nonspecific) site. The structure also reveals interactions that provide selectivity for guanine versus adenine as the pairing partner of the target base, and interactions that confer sequence specificity, a highly unusual capability for a DNA glycosylase. We find striking and unexpected differences between hTDG and its prokaryotic ortholog, mismatch‐specific uracil DNA glycosylase (MUG), despite high (32%) sequence identity. This work was supported by a grant from the National Institutes of Health and University of Maryland Greenebaum Cancer Center.
Epithelial Sodium Channel and Acid Sensing Ion Channel Subunits Interact With Each Other To Play A Role In Glioblastoma Whole Cell Current
Authors: Kapoor Niren , Bartoszewski Rafal , Fuller Catherine M , Palmer Cheryl A , Benos Dale J
Published: June 2021

High grade gliomas like glioblastoma (GBM) characteristically display a basally active amiloride sensitive cation current not seen in normal human astrocytes. We hypothesize this current to be mediated by a hybrid channel composed of a mixture of Epithelial Sodium Channel (ENaC)/ Degenerins (Deg) subunits. Using semi‐quantitative RT‐PCR we have shown higher expression of ASIC1, αENaC, and γENaC in primary GBM cells compared to primary human astrocytes. We have also shown the expression of these subunits in GBM biopsies and on the plasma membrane of GBM cells using immunocytochemistry. To test our hypothesis we made dominant negative (DN) cDNAs for ASIC1, αENaC, γENaC, and δENaC. D54‐MG cells transfected with the DN constructs for ASIC1, αENaC or γENaC showed 50‐ 70% reduction in protein expression and significant inhibition of whole cell patch clamp current compared with untransfected D54‐MG. Knocking down δENaC in D54‐MG cells reduced δENaC protein expression by 50%, but had no effect on the whole cell patch clamp current. Using co‐immunoprecipitation we show interactions between ASIC1, αENaC, and γENaC, consistent with these subunits interacting with each other to form an ion channel in glioma cells. These data suggest that different ENaC/Deg subunits combine to form a hybrid channel that likely underlies the amiloride‐sensitive current in human gliomas. This study was supported by NIH Grant CA101952.
TRPM4 is a regulator of Ca2+ dependent cell functions
Authors: Vennekens Rudi , Shimizu Takahiro , Owsianik Grzegorz , Mathar Ilka , Freichel Marc , Flockerzi Veit , Nilius Bernd
Published: June 2021

Using a Trpm4 knockout mouse model, we have shown that TRPM4 proteins constitute Ca2+‐activated non‐selective cation channels in mast cells. TRPM4 critically determines the driving force for Ca2+ influx upon FcepsilonRI receptor stimulation, i.e. Trpm4‐/‐ mast cells display a significantly hyperpolarized membrane potential and increased Ca2+ entry after antigen‐mediated activation compared to WT mast cells. Consequently, degranulation and release of histamine from BMMC's is agente and Trpm4‐/‐ mice display a more severe IgE‐mediated acute passive cutaneous anaphylactic (PCA) response. These results established for the first time the physiological role of TRPM4 channels as critical regulators of Ca2+ entry in mast cells and for the development of acute hypersensitivity reactions in mice. Additional experiments have shown that selected insecticides that ameliorate allergic reactions interact with TRPM4. TRPM4 also critically determines migration of mast cells. In TRPM4‐/‐ mast cells, antigen induced migration and SCF induced chemotaxis are defective. Thus, TRPM4 is a critical regulator of Ca2+ dependent mast cell function. We will extend these results to other cell types expressing TRPM4.
Acetaldehyde and Hexanaldehyde in Cultured White Cells
Authors: Shin Hye‐Won , Umber Brandon J , Meinardi Simone , Leu Szu‐Yun , Zaldivar Frank , Blake Donald R , Cooper Dan M
Published: June 2021

We hypothesized that human white blood cells generate gas profiles, in particular, aldehydes, that are likely products of substrate degradation. To test this, we developed a bioreactor and analytical GC/MS system to accurately quantify trace gases emitted solely from cell/media culture. A human promyelocytic leukemia cell line, HL60, grown in serum‐free medium was used as a model system in this study. Non‐stimulated HL60 cells released acetaldehyde and hexanaldehyde to the gas phase in a time‐dependent manner. The mean (SD) of acetaldehyde and hexanaldehyde concentration following 4‐, 24‐ and 48‐hour incubation from non‐stimulated HL60 cells were 157 (13) ppbv, 490 (99) ppbv, 698 (87) ppbv, and 1 (0.3) ppbv, 8 (2) ppbv, and 11 (2) ppbv, respectively. Our experimental system permitted us to distinguish gases likely originating in the cell culture from contaminants such as styrene. This study demonstrates that non‐stimulated human immune cells produce volatile gases that can be measured in vitro in trace amounts.

(R01‐HL‐080947 and P01‐HD‐048721, D.M.C and Physical Sciences Dean's Innovation fund, D.R. B.).
Osteoprotegerin (OPG) and its ligand RANKL in vascular tissue
Authors: Olesen Malene , Skov Vibe , Ledet Thomas , Rasmussen Lars Melholt
Published: June 2021

Osteoprotegerin (OPG) is increased in arterial tissue from diabetic patients. OPG knock out mice develop vascular calcifications indicating that OPG is an inhibitor of the development of calcification. However, the observed calcifications may be secondary to osteoporosis in these animals. Furthermore, OPG neutralize the function of receptor activator of nuclear factor‐¿B ligand (RANKL).

The project aim is to investigate direct functions of OPG and RANKL in vascular cells, including whether OPG is an inhibitor of the development of vascular calcification in vitro, without the interference of bone metabolism.

OPG was knocked down in primary human vascular smooth muscle cells (HVSMCs) using siRNA. Microarray analysis was performed to investigate whether lack of OPG upregulates the expression of calcification‐associated genes. Pathway analysis (GenMAPP) showed no significant upregulation of the calcification‐associated gene group. Therefore, it was not possible to show that OPG is an inhibitor of calcification as suggested in animal studies.

HVSMCs treated with OPG siRNA following stimulation with RANKL resulted in downregulation of the insulin signaling pathway (GenMAPP). The effect of RANKL on insulin signaling is a novel observation, which may provide an interesting putative link between the OPG‐RANKL balance and insulin signaling in diabetic patient.

Research support: Novo Nordisk
Effects of food form and resistance training on postprandial appetitive sensations and ghrelin, cholecystokinin, and glucagon‐like peptide‐1 in older adults
Authors: Apolzan John W , Leidy Heather J , Mattes Richard D , Campbell Wayne W
Published: June 2021

This study assessed the effects of food form (FF) and resistance training (RT) on appetitive sensations and postprandial (PP) plasma hormones in 16 RT and 18 sedentary (Sed) persons: age 62‐84 y; physical activity 425 ± 33 vs. 272 ± 28 kcal/d (*, p=0.05), mean ± SE; BMI 24.0 ± 0.6 vs. 25.7 ± 0.5* kg/m2; and whole body maximum strength 8.3 ± 0.3 vs. 6.7 ± 0.3* kg/kg fat free mass. On 2 randomized days, each subject consumed an isoenergetic macronutrient‐matched beverage (B) or solid (S) product. 4‐h PP measurements were assessed using area under the curve. Fullness was lower for B (218 ± 94) vs. S (475 ± 96* AU·240 min) and was lower in RT (230 ± 118) vs. Sed (450 ± 106*). Desire to eat was higher for B (‐65 ± 97) vs. S (‐331 ± 114* AU·240 min) but unaffected by RT. The hunger response tended to be higher for B vs. S (p=0.07). FF and RT did not affect ghrelin. CCK was lower in B (6.2 ± 8.9) vs. S (33.4 ± 8.9* ng/mL·240min) and higher with RT (29.0 ± 12.7) vs. Sed (12.2 ± 7.7*). GLP‐1 tended to be higher in B (37.1 ± 19.2) vs. S (0.9 ± 11.3 ng/mL·240min, p=0.06) and was lower in RT (3.9 ± 3.2) vs. Sed (31.6 ± 22.4*). GLP‐1 and ghrelin were negatively correlated with fullness (r=‐0.15*, r=‐0.18*). Ghrelin was positively correlated with hunger (r=0.16*) and desire to eat (r=0.15*). These results suggest that FF and RT influence appetitive sensations and hormonal responses. Older adults may benefit from incorporating RT and B meals to potentially reduce the anorexia of aging.

Grant Funding Source

NIH R01 AG021911, The Solae Company, Purdue Bilsland Fellowship
Genetic deficit of SK3 and IK1 channels abolishes EDHF‐type vasodilation and elevates blood pressure.
Authors: Koehler Ralf , Brähler Sebastian , Kaistha Anuradha , Woelfle Stephanie , Kaistha Brajesh P , de Wit Cor , Wulff Heike , Adelman John P. , Hoyer Joachim
Published: June 2021

In blood vessels, the Ca2+‐activated K+ channels SK3 (KCa2.3) and IK1 (KCa3.1) are expressed in the endothelium and their activation has been proposed to play a role in the arteriolar dilation attributed to the action of an "endothelium‐derived hyperpolarizing factor" (EDHF). However, the precise understanding of the roles of SK3 and IK1 channels in the EDHF‐dilator response and in blood pressure control remains incomplete. To clarify the roles of SK3 and IK1 channels in the EDHF‐dilator response, we generated mice deficient of both channels. Here we show that endothelial IK1/SK3 deficiency disrupted smooth muscle hyperpolarization and EDHF signalling in conduit arteries and in resistance arterioles in vivo. As a consequence, SK3/IK1‐deficient mice exhibit elevated arterial blood pressure which is most prominent during physical activity. Over‐expression of SK3 in IK1‐/‐/SK3T/T‐mice partially restores EDHF‐mediated vasodilation and lowers blood pressure. Our study demonstrates that endothelial SK3 and IK1 channels are the major constituents of the EDHF‐pathway in vivo and reveals a significant contribution of the EDHF‐dilator system to arterial blood pressure. Therefore, these endothelial K+‐channels may represent novel therapeutic targets for lowering arterial blood pressure in hypertension.
Mast Cells Regulation of Homeostatic Intestinal Barrier Function is dependent on the Human Chymase Homologue, mcpt4.
Authors: Hogan Simon Patrick , Ahrens Richard , Osterfeld Heather , Rothenberg Marc Elliot , Finkelman Fred , Abrink Magnus , Pejler Gunnar , Groschwitz Katherine
Published: June 2021

Disruption of the intestinal epithelial barrier has been proposed to be a central predisposing factor to intestinal diseases, however the molecular mechanisms involved in the maintenance of homeostatic intestinal epithelial barrier integrity has not been defined. We demonstrate a decrease in paracellular and transcellular intestinal permeability in mast cell (MC)‐deficient KitW‐sh/W‐sh mice as compared to C57Bl/6 WT mice (paracellular [Dextran‐FITC]: 172.3 ± 35.3 vs 378.1 ± 54.3 ng/ml; p < 0.005; transcellular [HRP]: 0.0079 ± 0.0036 vs 1.1129 ± 0.4798 ng/ml; p < 0.05). We confirm MC involvement in homeostatic intestinal barrier function as reconstitution of bone marrow derived mast cells (BMMC's) into KitW‐sh/W‐sh mice restored intestinal epithelial barrier integrity. Importantly, we identified that MC‐regulation of homeostatic intestinal barrier function was dependent on MC protease 4 (mcpt4). Moreover, mcpt‐4−/− mice intestinal epithelial barrier integrity was decreased as compared to WT mice ([Dextran‐FITC]: 115.3 ± 25.2 vs 378.1 ± 54.3 ng/ml; p < 0.005; [HRP]: 0.179 ± 0.1306 vs 1.1129 ± 0.4798 ng/ml; p < 0.05), to levels equivalent to that of KitW‐sh/W‐sh mice. Furthermore, reconstitution of mcpt4−/− BMMC's into KitW‐sh/W‐sh mice did not restore intestinal epithelial barrier integrity. Collectively, our data demonstrates a critical role for MC's in the regulation of homeostatic intestinal barrier function.