bluuebunny/crossref_metadata_2025 huggingface dataset:
CHARACTERIZATION OF MUTANT V2 RECEPTORS ASSOCIATED WITH PARTIAL CONGENITAL NEPHROGENIC DIABETES INSIPIDUS
Authors: Faerch Mia , Christensen Jane Hvarregaard , Corydon Thomas Juhl , Gregersen Niels , Johansson Jan‐Ove , de Zegher Francis , Rittig Søren
Published: June 2021

X‐linked recessive congenital nephrogenic diabetes insipidus (CNDI) is a disease causing a defective renal response to the effects of the antidiuretic hormone arginine vasopressin (AVP). Two mutations (Arg104Cys and Ser329Arg) associated with partial CNDI were characterized by expression in human embryotic kidney cells. The Arg104Cys mutation in the first extracellular loop and the Ser329Arg mutation in the intracellular C‐terminal demonstrate that despite differences in localization, they have the same effect on receptor processing. Western blotting showed equal amount of receptor protein in WT and mutants. Confocal laser scanning microscopy established that WT receptors were localized on the cellular surface whereas mutant receptors accumulate in a diffuse pattern in the cells. Ligand binding assay demonstrated that the Bmax for the Arg104Cys and the Ser329Arg mutant receptors were 10‐fold and 2‐fold lower than the WT. There was no difference in AVP affinity (1/Kd) between WT and Ser329Arg mutant receptors, while affinity for Arg104Cys mutant receptors was improved. cAMP assay revealed that the Arg104Cys mutation located in the first extracellular loop produced a lower amount of cAMP compared to WT, but still 4‐fold higher than the Ser329Arg mutation. The mutation Arg104Cys and Ser329Arg of the AVPR2 gene seems to have completely different effects on receptor functionality despite similar phenotypes.
YC‐1 stimulates heme oxygenase‐1 gene expression in vascular smooth muscle cells
Authors: Liu Xiao‐ming , Peyton Kelly J. , Tulis David A. , Durante William
Published: June 2021

The benzyl‐indazole derivative YC‐1 enhances the biological actions of heme oxygenase (HO) in vascular smooth muscle cells (SMC) by sensitizing soluble guanylate cyclase (sGC) to the stimulatory effect of HO‐derived carbon monoxide (CO). However, the regulatory impact of YC‐1 on HO and/or CO is not known. In this study, the effect on YC‐1 on HO‐1 expression was investigated in rat cultured aortic SMC. Treatment of SMC with YC‐1 (5‐100μM) stimulated HO‐1 protein expression in a time and concentration‐dependent manner. A significant rise in HO‐1 protein was first detected after 4 hours and HO‐1 levels progressively increased over 24 hours of YC‐1 exposure. Increases in HO‐1 protein were preceded by marked elevations in HO‐1 mRNA, and transient transfection experiments showed that YC‐1 stimulated HO‐1 promoter activity approximately 2‐fold. Interestingly, the induction of HO‐1 by YC‐1 was unchanged by the sGC inhibitor ODQ or by the protein kinase G inhibitor KT5823. Moreover, YC‐1‐mediated HO‐1 induction was not duplicated by the NO‐independent sGC stimulator, BAY 41‐2272. In conclusion, this study demonstrates that YC‐1 stimulates the transcriptional activation of the HO‐1 gene in a cGMP‐independent fashion. The distinct ability of YC‐1 to amplify the beneficial vascular effects of CO at both the level of CO synthesis and cellular response underscores the therapeutic potential of this agent. Supported by NIH.
Isoflurane Preconditioning Delays Opening of Mitochondrial Permeability Transition Pore via Protein Kinase C Signaling Pathway
Authors: Pravdic Danijel , Sedlic Filip , Mueller Samantha J , Bosnjak Zeljko J
Published: June 2021

Isoflurane protects the myocardium from ischemia and reperfusion (I/R) injury via protein kinase C (PKC) signaling pathway. It has been suggested that the mitochondrial permeability transition pore (mPTP) is the end effector of I/R injury, whereas PKC has a protective effect by blocking mPTP opening. Here we investigated whether anesthetic preconditioning (APC) with isoflurane delays mPTP opening and whether the PKC plays a role in isoflurane‐induced delay in mPTP opening. Isoflurane preconditioned rat cardiomyocytes were loaded with fluorescent probe tetramethylrhodamine ethyl ester (TMRE) and subjected to a simulated I/R injury by laser‐induced ROS production. Using the laser‐scanning confocal microscopy, we measured the time to 50% decrease in TMRE fluorescence, which coincides with depolarization of the mitochondria and represents an opening of mPTP. To confirm the role of mPTP, we used cyclosporine A (CsA). The role of PKC was tested using the non‐specific PKC inhibitor chelerythrine (CHEL). APC with isoflurane prolonged the mean depolarization time of the control fluorescence of 216±8 s, to 261±16 s. A similar time delay of 279±22 s was found in cells pretreated with CsA. Isoflurane‐induced delay in an mPTP opening was abrogated to 219±7 s in cells treated with CHEL. In conclusion, our results indicate that APC with isoflurane delays the opening of mPTP in a PKC‐dependent manner.

Supported by NIH (HL 034708 and GM 066730)
Post‐translational modification of matrix metalloproteinase‐2 by peroxynitrite
Authors: Nicolescu Adrian C , Viappiani Serena , Holt Andrew , Schulz Richard
Published: June 2021

Matrix metalloproteinases (MMPs) are key enzymes in the development and remodeling of tissues. Disruption of the interaction between a conserved Cys residue in the propeptide domain and Zn2+ in the catalytic site activates MMPs. Enhanced biosynthesis of peroxynitrite (ONOO‾) is implicated in heart failure and myocardial ischemia/reperfusion (I/R) injury, where activated MMP‐2 was shown to degrade important intracellular targets, e.g. troponin I (TnI), myosin light chain‐1 and α‐actinin. We investigated the mechanisms by which ONOO‾ may activate full length 72 kDa MMP‐2 in vitro. Challenging MMP‐2 with ONOO‾ resulted in a biphasic change in MMP‐2 activity, with low μM ONOO‾ increasing and high μM reducing its activity. Changes in the Vmax of the enzyme and no significant effect on the KM were observed using a synthetic fluorogenic substrate. Addition of glutathione (GSH) did not potentiate the effect of ONOO‾, but prevented the loss of MMP‐2 activity induced by higher concentrations of ONOO‾. Mass spectrometry analysis of the reaction of ONOO‾ with either MMP‐2 or synthetic peptide KPRCGNPDVANYNFFPR identified several structural modifications, including sulfhydryl oxidation and, in the presence of GSH, reversible glutathiolation of the critical Cys. These data suggest that ONOO‾ can modify the critical Cys of the propeptide domain in MMP‐2 and that may be responsible for its activation in the heart.
AMPK regulation of cardiac growth‐related signaling in ob/ob mice following 2 weeks of AICAR treatment
Authors: Williamson David L , Alway Stephen E
Published: June 2021

The aim of this study was to determine how AMP‐activated protein kinase (AMPK) regulates cardiac growth‐related pathways in lean and obese mice. 12 wk old male ob/ob (body weight (BW)=57.9g) and lean wild type (BW=30.3g) mice received and injection of 0.5 mg/g BW AICAR (AIC) in saline or a vehicle control (CON) for 2 wks. All mice were sacrificed 24 hrs after the last injection, including a 12 hr fast. Heart weights did not change for any of the groups. Western analysis of heart lysate from fasted ob/ob CON mice displayed reduced AMPK T172 and ACC S79 phosphorylation compared with lean CON, which increased in both groups after the 2 wk AIC treatment. Immunoprecipitations (IP) of tuberin displayed a reduced association with hamartin in ob/ob CON versus the lean CON, which relatively increased in both groups following AIC treatment. IPs for mTOR displayed comparable levels of raptor association in the CON groups, and increased similarly following AIC treatment. Fasted ob/ob CON mice displayed higher S6K1 T389, ribosomal protein S6 S240/244, and 4E‐BP1 T37/46 phosphorylation than lean CON, which was attenuated by AIC treatment. These data suggest that obese mice display elevated cardiac growth‐related signaling that precedes cardiac hypertrophy, and acute treatment with an AMPK activator attenuates growth‐signaling and the progression toward cardiac hypertrophy. Support: WVU Research Development Grant NT10023W (to DLW)
Strain‐Dependence in Susceptibility to Heart Failure: Role of Mitochondria
Authors: Ascah Alexis , Marcil Mariannick , Matas Jimmy , Oliver‐Dussault Christelle , Deschepper Christian , Burelle Yan
Published: June 2021

The purpose of this study was to compare two genetically different strains of rats submitted to overload‐induced ventricular remodelling to examine factors that predispose to the progression toward heart failure. Volume overload was induced by an aorto‐caval fistula (ACF) in SD and WKHA male rats, and hearts were studied at 4, 8 and 12 weeks after surgery. Compared to WKHA, ACF‐induced LV hypertrophy and dysfunction progressed more rapidly in SD rats, and functional recovery of SD hearts to I‐R was poorer vs WKHA during the compensated phase of disease. At later times (up to 40 weeks after ACF), SD rats (but not WKHA rats) died progressively of acute cardiac decompensation. Both strains developped a vulnerability to opening of the PTP, as measured in situ (in ischemic‐reperfused hearts using the mitochondrial [3H]DOG entrapment method) or in isolated mitochondria submitted to in vitro stress. Increased vulnerability to PTP opening was observable beginning at 4 weeks but no differences in severity was apparent between strains. The % of collagen‐cross linking increased progressively after ACF in both strains, but to a lower extent in SD vs. WKHA hearts. These results suggest that, while mitochondrial dysfunction may participate to the negative consequences of ACF‐induced LVH in both strains, it is not responsible for the more the rapid progression to decompensation observed in SD rats. Further experiments are underway to test the differences in cardiac outcome may be due to differences in the levels of collagen‐cross linking.

Funded by: CIHR, FRSQ
Bigger Muscles and Smaller Tendons ‐ Tendons of Myostatin Deficient Mice are Small, Brittle, and Hypocellular
Authors: Mendias Christopher L , Bakhurin Konstantin I , Faulkner John A
Published: June 2021

Tendons play a significant role in the modulation of forces transmitted between bones and skeletal muscles and consequently protect muscle fibers from contraction‐induced, or high strain, injuries. Myostatin (MSTN) is a negative regulator of muscle mass. Inhibition of myostatin not only increases the mass and maximum isometric force of muscles, but also increases the susceptibility of muscle fibers to contraction‐induced injury. Due to the protective role of tendons in preventing contraction‐induced injury, and the greater susceptibility of muscles from MSTN−/− mice to contraction‐induced injury, we hypothesized that myostatin would regulate the morphology and mechanical properties of tendons. The expression of myostatin and the myostatin receptors, ACVR2B and ACVRB, were detectable in tendons. Surprisingly, compared with MSTN+/+ mice, the tendons of myostatin‐deficient mice MSTN−/− were smaller, had a decrease in fibroblast density and a decrease in the expression of type I collagen. Tendons of MSTN−/− mice also had a decrease in the expression of two genes that promote tendon fibroblast proliferation, scleraxis and tenomodulin. Treatment of tendon fibroblasts with myostatin activated the p38 MAPK and Smad2/3 signaling cascades, increased cell proliferation and increased the expression of type I collagen, scleraxis and tenomodulin. Compared with the tendons of MSTN+/+ mice, the mechanical properties of tibialis anterior tendons from MSTN−/− mice had a greater peak stress, lower peak strain and a increased stiffness. We conclude that in addition to the regulation of muscle mass and force, myostatin regulates the structure and function of tendon tissues.
The effects of resistance exercise on fiber‐type specific GLUT4 and insulin‐like growth factor‐I receptor expression in human skeletal muscle
Authors: Gallagher Philip , Tehel Michelle , McCabe Everlee , Gandy Nicole , Wacker Michael
Published: June 2021

There has been little research on the effects of resistance exercise on fiber‐type specific gene expression of glucose transporter 4 (GLUT4) and insulin‐like growth factor‐I receptor (IGFIR) expression. Thus, the purpose of this investigation was to measure the effects of an acute bout of resistance exercise (RE) on GLUT4 and IGFIR expression in human skeletal muscle. Six college‐aged males performed a bout of knee‐extensor RE. Individual fibers were isolated from muscle biopsy samples obtained from the vastus lateralis muscle prior to and 4‐hours following RE. Myosin heavy chain analysis was performed on 1/3 of each fiber and 2/3 of the fiber was directly placed in individual tubes containing buffer for the reverse transcription (RT) step of a two‐step RT‐PCR kit (Invitrogen). After RT, the cDNA of all slow‐ (MHC I, N=20 per subject) and fast‐ (MHC IIa and IIa/x, N=30 per subject) twitch fibers were combined for each subject. PCR reactions for GLUT4, IGFIR, and GAPDH (housekeeping gene) were then performed on the samples. ΔΔCT analysis revealed that there were no differences in GLUT4 (slow‐twitch = 0.81, fast‐twitch = 0.72) or IGFIR (slow‐twitch = 0.49, fast‐twitch = −0.11) expression between the pre‐RE and 4‐hour post RE time‐points. These data show that GLUT4 and IGFIR gene expression are not elevated four hours after resistance exercise; however, these genes may be elevated at other times post‐exercise.
Activation of ionotropic excitatory amino acid (EAA) receptors modulates both eupnea and gasping in the arterially‐perfused adult rat preparation
Authors: Warren Kelly A. , Solomon Irene C.
Published: June 2021

Previous work from our laboratory in an arterially‐perfused adult rat preparation has shown that blockade of ionotropic EAA receptors reduces or abolishes basal phrenic nerve discharge and markedly enhances inspiratory neural network complexity. Further, we have demonstrated that during EAA receptor blockade, gasping can still be elicited. The effect of activation of ionotropic glutamate receptors on phrenic motor discharge requires clarification. We examined the effects of perfusion with the glutamate analog DLH on phrenic burst timing and inspiratory neural network complexity in the arterially‐perfused adult rat preparation. DLH (500 μM, n=8) transiently increased phrenic burst frequency by 41% (P=0.005) mediated by decreasing the duration between bursts by 55% (p<0.001), followed by a 36% increased stabilization of burst frequency (P=0.074). Post‐inspiratory activity was also increased. DLH decreased the duration of the peripheral chemoreflex from 23.8±1.7 to 13.2±1.4 s (P<0.001), the duration of ischemia‐induced gasping from 58.6±5.3 to 33.4±2.3 s (P<0.001), and the onset latency to gasping by 79% (P<0.001). These findings support a role for ionotropic EAA neurotransmission in establishing the temporal dynamics of basal phrenic nerve discharge and suggest that it also has a role in determining the onset and duration of the peripheral chemoreflex and gasping. Supported by NS045321 and NS049310
Analysis of cardiopulmonary dynamics using wavelet analysis methods: removing the influence of respiration and prediction of emphysema
Authors: Bartels Matthew N , Petrock Anne Marie , Reisman Stanley S , Rockland Ronald H , Rebmann Rebecca A , De Meersman Ronald E
Published: June 2021

Respiratory effects are significant in standard frequency analysis of heart rate variability (HRV). This limits evaluation of sympathovagal balance in exercise and other dynamic states. We developed a method using wavelet statistical analyses combined with cluster analysis to reduce this. Methods: Ten emphysema subjects (ES) and 5 matched controls (CS) were evaluated using wavelet entropy (WE) of heart rate data during rest and exercise. The model included wavelet source separation via a discrete wavelet decomposition of heart rate and respiration signals. Data were analyzed blinded, and prediction for group separation (ES versus CS) was performed using cluster analysis. Results: Using WE, the source separation model was able to predict actual classification of individuals with 93% accuracy at rest (4/5 CS, 10/10 ES) and 100% accuracy at exercise. The most significant factors for principal component analysis were identified for resting and exercise states. Conclusion: Novel utilization of wavelet analysis in combination with principal component analysis and wavelet analysis methods can remove the influences of respiration on the time frequency analysis of HRV. Support: VIDDA Foundation
In vitro and in silico experiments to identify the influence of temperature on skeletal muscle calcium and force dynamics
Authors: Groenendaal Willemijn , Jeneson Jeroen , van Riel Natal , Eikelder Huub ten , Nicolay Klaas , Hilbers Peter
Published: June 2021

Temperature has been shown to influence skeletal muscle performance. To be able to understand and quantify the influence of temperature on local calcium and force dynamics, in vitro and in silico experiments were performed in mouse EDL.

The spatiotemporal model of Baylor and Hollingworth (J Gen Physiol. 1998 112:297–316) was adapted in such a way that it was able to describe local calcium dynamics in EDL at different temperatures. Most processes in the sarcomere were temperature dependent and were scaled with Q10 values based on literature. Model simulations were performed for a stimulation frequency of 1–10–30–60–90–120 Hz and muscle temperatures of 15–20–25–30–35C. In parallel, the mechanics of isometric contractions of isolated mouse EDL were measured for the same conditions as the simulations (ter Veld et al. 2006 Pflugers Archiv 452: 432–8).

For increasing muscle temperatures, shorter rise and decay times of the Ca2+ pulse were predicted by the model. Similar trends were obtained for the rise and decay time of the measured force. For increasing stimulation frequency, the model predicted an increase in baseline and maximal Ca2+ concentration, while the measurements showed similar trends for the produced force. On top, the experiments showed that stimulation frequency influenced the decay rate at 35C.

Furthermore, the experiments will be used to validate Q10 values in the model as well as the total amount of Ca2+.
A multilevel open source integrative model of human physiology
Authors: Hester Robert L. , Coleman Thomas , Summers Richard
Published: June 2021

We have developed Quantitative Human Physiology (QHP), an open source Windows‐based mathematical model of integrative human physiology containing over 3000 variables. QHP describes human physiology using whole‐body properties, organs, and multi‐organ systems. Users can view time‐dependent solutions and interactively alter parameters to investigate new hypotheses. All aspects of the model, including the mathematical equations describing the physiological processes have an XML open source, text‐readable description of the physiology. Model structure is based upon documented physiological responses within the peer‐reviewed literature. The model can be used to understand proposed physiological mechanisms and physiological interactions that may not be otherwise intuitively evident. Uses of this model include the analyses of renal control of blood pressure, the central role of the liver in creating and maintaining insulin resistance, and the mechanisms causing orthostatic hypotension in astronauts. Additionally the open source aspect of the modeling environment allows any investigator to add detailed descriptions of human physiology to test new concepts. QHP provides scientists a modeling environment to understand the complex interactions of integrative physiology. The model is freely available at http://physiology.umc.edu/themodelingworkshop. Supported by HL 51971 and NSF EPSCOR
Regulation of Dimethylarginine Dimethylaminohydrolase (DDAH) in Rat Preglomerular Vascular Smooth Muscle Cells (PGVSMCs) by Reactive Oxygen Species (ROS)
Authors: Luo Zaiming , Gill Primohinder S. , Welch William , Jose Pedro A. , Wilcox Christopher S
Published: June 2021

DDAH degrades asymmetric dimethylarginine (ADMA) which is an endogenous NOS inhibitor and risk factor for cardiovascular disorders. We tested the hypothesis that ROS regulate the transcription, translation or activity of DDAH. We created a stable rat PGVSMC line overexprissing p22phox (S‐p22phox) using a p22phox‐pcDNA4/HisMax construct (a generous gift from Kathy Griendling). Quantitative real time PCR and Western blotting confirmed a marked increase in p22phox mRNA and protein of 4.7±0.6 and 2.3±0.5 fold, (p<0.01), compared to wild type cells (WT). S‐p22phox cells had elevated superoxide and hydrogen peroxid generation (2.3±0.09 fold and 1.8±0.1 fold p<0.01). The mRNAs for DDAH ‐1 and ‐2 were increased by 2.8 ± 0.2 and 1.8 ± 0.1 fold in S‐p22phox cells p< 0.01). DDAH‐2 promoter activity, determined by transient transfection and luciferase assay, was 4.1 fold increased in S‐p22phox cells (p < 0.01). Interestingly, both DDAH‐1 and ‐2 proteins were significantly lower in S‐p22phox cells (0.9±0.2 and 0.9±0.1 fold; p<0.01), yet β‐actin was not different. DDAH activity measured by a colorimetric assay (Kidney Int. 72:886–9, 2007) was reduced by 62±2% in S‐p22phox(p<0.01). The results reveal for the first time an important effect but complex of p22phox – dependent ROS generation on DDAH. Thus, ROS upregulate the expression of DDAH‐1 and ‐2 genes via activating a promoter, at least for DDAH‐2, yet downregulate both DDAH‐1 and ‐2 proteins and their enzyme activity. This is a specific effect since ROS do not change the expression of cell structure proteins. These findings in PGVSMCs provide a novel regulatory pathway whereby ROS may modulate renal hemodynamics, and thereby BP and salt homeostasis.
Evidence that NBCe1‐A Monomers are Functional
Authors: Azimov Rustam , Abuladze Natalia , Kao Liyo , Pushkin Alexander , Newman Debra , Liu Weixin , Kurtz Ira
Published: June 2021

Previous studies from our laboratory have shown that the oligomeric structure of NBCe1‐A is predominantly a dimer. We also demonstrated that oligomerization of NBCe1‐A is absolutely dependent on S‐S bond formation. Whether NBCe1‐A monomers are functional is currently unknown. To address this question, we took advantage of our recent finding that the NBCe1‐AT442C mutant functions normally but can be completely inhibited by the MTS reagents MTSEA, MTSES, and MTSET. In contrast these agents have no effect of wt‐NBCe1‐A. The following concatameric constructs of wt‐NBCe1‐A and NBCe1‐AT442C were studied: wt‐NBCe1‐A‐linker‐wt‐NBCe1‐A; wt‐NBCe1‐A‐linker‐NBCe1‐AT442C; NBCe1‐AT442C‐linker‐wt‐NBCe1‐A; and NBCe1‐AT442C‐linker‐NBCe1‐AT442C. The length of the linker varied inversely with the baseline concatamer function, and concatamers with the shortest linker (Thr‐Gly) functioning optimally. Flux through the four concatamers was measured in HEK‐293T cells using BCECF and quantitating the cell buffer capacity. Non‐denaturing gel electrophoresis demonstrated that each of the four concatameric constructs is monomeric. The function of the four concatamers was similar to wt‐NBCe1‐A and NBCe1‐AT442C. In the presence of the MTS reagents MTSES and MTSET, the function of both mixed‐concatamers was decreased to 40–53% of normal. The function of the wt‐NBCe1‐A homo‐concatamer was unaffected, whereas both reagents blocked the function of the mutant NBCe1‐AT442C homo‐concatamer. Our results demonstrate that the NBCe1‐A monomer is functional.

Supported by the NIH
EGF Stimulates Murine Duodenal Mucosal Bicarbonate Secretion via CFTR Channels
Authors: Ko Kwang Hyun , Lee Jonathan , Dong Hui
Published: June 2021

Epithelial growth factor (EGF) is a 53 amino acid residue peptide that is made in the salivary glands and Brunner's glands of the duodenum. EGF receptors are extensively expressed in the gastrointestinal tract. It has been reported that EGF can inhibit cysteamine‐induced duodenal ulcers in rats; however, the underlying mechanisms are poorly understood. Since duodenal mucosal bicarbonate secretion (DMBS) protects the duodenum against acid‐induced injury, our aims were to test whether EGF affects duodenal ion transport in general and DMBS in particular. Duodenal mucosae from C57BL mice were stripped of seromuscular layers and mounted in Ussing chambers. Both short‐circuit current (Isc) and HCO3− secretion were measured in these tissues. DMBS was also measured in vivo. EGF (100 ng/ml) did not alter basal duodenal Isc, but inhibited CCh‐induced Isc (n=8–10, p<0.05). EGF at the same concentration stimulated DMBS, but did not affect CCh‐induced DMBS (n=8–11, p<0.05). In the whole animal study, EGF (200 ng/ml) stimulated DMBS in CFTR wild type mice, but failed to stimulate DMBS in CFTR knock out mice (net peak: 4.26 ± 0.84 vs. 0.41 ± 0.17 μmol/cm.h, n=4–5, p<0.01). Taken together, these data suggest that EGF stimulates DMBS via duodenal epithelial CFTR channels, which may contribute to its action against duodenal ulcer (Supported by Cystic Fibrosis Foundation).
Inhibition of alveolar liquid secretion by lipopolysaccharide
Authors: Lindert Jens , Islam Mohammad N. , Bhattacharya Jahar
Published: June 2021

In the adult lung, the surfactant subphase formed by the alveolar wall liquid (AWL) determines alveolar patency. We reported that the AWL is established by alveolar liquid secretion (Am J Resp Cell Mol Biol. 2007. 36:688–96). To determine whether lung inflammation affects AWL secretion, we intra‐tracheally instilled saline or lipopolysaccharide (LPS) (1 mg/kg) in lungs of anesthetized rats. After 24h we isolated the lungs and perfused (14 ml/min) them at constant pressures of 10, 3 and 5 cmH2O held at respectively, the pulmonary artery, the left atrium and the airway. Then, we viewed the lungs by two‐photon microscopy. To determine AWL secretion rate (JAWL), we micropunctured single alveoli and microinfused FITC‐dextran (20 kD) to fluorescently label the AWL. We quantified FITC fluorescence in the AWL in 0.6 μm optical sections. In saline‐treated lungs AWL fluorescence intensity decreased exponentially to 61±3 % of initial in 20 min (mean±SE, n=3, P<.05), reflecting progressive fluorophore dilution by alveolar secretion. From the exponential regression of the fluorescence decay, we determined JAWL as 4.8±0.7 x10−8 ml/(cm2.sec). However, in LPS‐instilled lungs JAWL was zero (n=3, P<.05), indicating absence of liquid secretion. We suggest that in septic lungs, impaired AWL secretion might underlie surfactant dysfunction, thereby impairing lung function. (HL78645, HL64896).
Chromium silences nickel‐induced vascular endothelial growth factor (VEGF) expression in human airway epithelial cells
Authors: Nemec Antonia A , Barchowsky Aaron
Published: June 2021

Co‐exposure to chromium(VI) (Cr(VI)) and nickel (Ni) is a widely recognized pulmonary health hazard. Chronic inhalation of either metal promotes lung disease and co‐exposures to metal mixtures may pose even greater risks. VEGF is essential in lung injury and repair since loss of VEGF expression causes apoptosis and emphysema. The current study investigates the hypothesis that Cr(VI) may contribute to airway injury by silencing Ni‐induced VEGF expression in human bronchial airway (BEAS‐2B) epithelial cells. VEGF mRNA levels were measured in cells left untreated or exposed to a non‐cytotoxic concentration of Cr(VI) (5μM) prior to the addition of 200μM Ni. Exposure to Cr(VI) alone had no effect on basal VEGF expression, but inhibited Ni‐induced VEGF mRNA transcripts. Ni induction of VEGF requires HIF‐1α transactivation. Pretreatment with Cr(VI) blocked Ni stabilization of HIF‐1□ protein levels and stimulation of a hypoxia response element (HRE)‐driven luciferase reporter. These data suggest that Cr(VI) inhibits Ni‐induced VEGF mRNA expression by disrupting signaling events mediating Ni stimulation of the HIF‐1α pathway. This study provides mechanistic insight into potential epigenetic interactions of metal mixtures and support future investigation of the role of these interactions in the severity of pulmonary injury and the etiology of lung diseases. Supported by ES10638.
Peer‐interactive instructional video in Team‐Based Learning (TBL): Assessment of gender on self‐reported attitudes and behaviors
Authors: Vasan Nagaswami , DeFouw David , Compton Scott
Published: June 2021

We assessed the role of gender on first year medical students’ self‐reported attitudes and behaviors regarding team‐based learning (TBL) following a TBL peer‐interaction instructional video and educational module addressing acceptable and unacceptable team behaviors. Attitudes and behaviors were described and compared between male (n=92) and female (n=84) ratings using independent samples t‐tests, and chi‐square and Fisher's exact tests, as appropriate. The overall percentages of students are presented that agreed the video encouraged them to: understand responsibilities to learning (99.4%); feel comfortable teaching peers (95.5%); feel comfortable learning from peers (98.3%); come prepared for the TBL (98.3%); come to TBL on time and stay until the end (97.7%); evaluate own behavior during TBL (97.2%); respect and value other members’ views (99.4%); share resources (97.7%); ask team members for help (96.6%); provide constructive criticism (93.8%); and receive constructive criticism (95.5%). However, 14 (8.0%) rated that the video had at least some negative impact. There were no significant differences between males and females on any item. We conclude that the peer‐interaction instructional video educational module was well received by both male and female students, and resulted in highly positive self‐reported attitudes and behaviors regarding TBL.
Using an online text messaging communication tool as an instructional supplement in an undergraduate anatomy course
Authors: Brueckner Jennifer , Challman Sandra , MacPherson Brian
Published: June 2021

The Millennial generation college student relies on technology to stay socially and academically connected. To enhance interaction and communication with undergraduate students enrolled in our online anatomy course (ANA 209), the authors piloted an optional text messaging program during the Fall 2007 semester. Text messaging uses SMS, or Short Message Service, a feature enabled in most cell phones, allowing users to receive and send short written messages. To sent text messages to ANA 209 students, faculty logged into the University's Sharepoint™ system, which contains a mobility portal through which messages (email, SMS and/or voice) may be created and distributed. Messages were sent in order to notify students when a grade is posted, a new assignment made, or to remind students about an upcoming quiz. In addition, a pertinent “anatomy fact of the day” was text messaged to students as a reminder to review the assigned reading. Preliminary student feedback indicates that the text messaging is a useful way to enhance communication in this online anatomy class. This pilot program proved to have great potential for supporting a social context for online learners that fosters a greater sense of community than is typically reported in virtual classes.
Activation of Transforming Growth Factor β(TGFβ)by interaction of Matrix Metalloproteinase 13 (MMP‐13) with the large latent complex
Authors: Dragann Brendan N , Routson Stephanie M , Selim Abdulhafez , D’Angelo Marina
Published: June 2021

Dysregulated TGFβ activation has been indicated in the pathology of osteoarthritis. We have reported that hypertrophic chondrocytes produce large quantities of activated TGFβ and a large latent complex of TGFβ that contains MMP‐13 in non‐covalent association. Bioinformatics revealed that the hemopexin domains of MMP‐13 can non‐covalently interact with the calcium‐EGF‐like domains of Latent TGF Binding Protein (LTBP1), the extracellular matrix anchor protein for the TGFβ large latent complex. The catalytic domain of MMP‐13 is in alignment with the protease‐sensitive hinge region of LTBP1, the site of release of this molecule from the extracellular matrix, suggesting a novel mechanism of TGFβ large latent complex release. To test this proposed interaction, we designed peptides of the MMP‐13 hemopexin domain that are most likely to interact with LTBP1. Tissue extract of avian hypertrophic cartilage bound five times more MMP‐13 peptide than extracts from resting zone cartilage. In addition, rat tibial growth plate cartilage immunoprecipitated with antibody to LTBP1 demonstrated four times more binding affinity for MMP‐13 peptide than the scrambled peptide control. These data support the specific interaction between MMP‐13 and LTBP1 predicted in our bioinformatics model and our proposed model of the mechanism of release of the TGFβ large latent complex by MMP‐13. Sponsored in part by a grant from the CCDA.
A model of GAA•TTC repeat expansion in human cell lines
Authors: Ditch Scott , Banerjee Ayan , Sammarco Mimi C. , Grabczyk Ed
Published: June 2021

Friedreich ataxia (FRDA) is a neurodegenerative disorder caused by GAA•TTC repeat expansion in the first intron of the frataxin gene (FXN). Expanded GAA•TTC repeat tracts reduce FXN gene expression in FRDA patients. The mechanisms responsible for GAA•TTC repeat expansion remain unclear. The development of a human cellular model of GAA•TTC repeat instability will provide an experimental system in order to better understand the mechanisms leading to GAA•TTC repeat expansion in FRDA. Our system employs reporter constructs containing uninterrupted GAA•TTC repeat tracts that have been integrated into a unique location in the genome of human embryonic kidney (HEK) 293 cell lines. PCR and Southern blot analysis of the GAA•TTC repeat sequences within these cell lines has revealed substantial instability, with a bias towards repeat expansion over time. Continued GAA•TTC repeat expansion in FRDA may contribute to the unrelenting progression of this disorder. The expansion of GAA•TTC repeat sequences in our cell lines reflects the dynamics of GAA•TTC repeat instability observed in FRDA, and could therefore serve as an experimental model to dissect the mechanisms contributing to this instability. This work was supported by NIH grant number R01NS046567 and by a grant from the Friedreich's Ataxia Research Alliance (FARA).
A novel reporter system to investigate the molecular cause of Friedreich Ataxia
Authors: Banerjee Ayan , Ditch Scott , Sammarco Mimi C. , Grabczyk Ed
Published: June 2021

Friedreich ataxia (FRDA), is a neurodegenerative disease accompanied by hypertrophic cardiomyopathy. FRDA patients suffer an unrelenting progressive ataxia and most eventually succumb to cardiac failure. Most FRDA patients are homozygous for large GAA•TTC repeat expansions in the intron 1 of the FXN gene. Disease severity directly correlates to the size of the repeat expansion, which represses FXN gene expression by a mechanism that is still unclear. In order to investigate this inhibition by expanded GAA•TTC repeats, we designed a splicing FXN minigene construct carrying two reporters under the control of a bi‐directional tetracycline inducible promoter. We then used the Invitrogen Flp‐In system to integrate our constructs carrying GAA•TTC repeats of varying sizes into a unique chromosomal location in cell lines. Surprisingly, repeats as large as 352 do not cause decreased transcription in our system. However, repeat lengths between 700–1000 showed marked transcription inhibition, suggesting a larger disease threshold than presently believed. We are using this tool to better define the disease threshold and the possible role of co‐transcriptional events in transcription inhibition. These cell lines will also serve as a high throughput assay for testing therapeutic interventions. This work was supported by NIH grant number R01NS046567 and by a grant from the Friedreich's Ataxia Research Alliance (FARA).
TRICHOSTATIN A INDUCED DEREPRESSION OF THE HUMAN LUTEINIZING HORMONE RECEPTOR GENE BY CELL‐SPECIFIC PHOSPHATASE RELEASE FROM THE PROMOTER
Authors: Zhang Ying , Liao Mingjuan , Dufau Maria L.
Published: June 2021

The histone deacetylase inhibitor, Trichostatin A (TSA), induces de‐repression of the human Luteinizing Hormone receptor (hLHR) gene by de‐recruitment of the pRB homologue p107 repressor from the promoter in JAR and MCF‐7 cancer cells. TSA initiates a mechanism whereby the phosphatidylinositol 3‐kinase/PKCzeta cascade phosphorylates Sp1 at Ser641, which is essential for the release of the repression of LHR transcription. These studies have revealed a critical role of serine/threonine protein phosphatases PP2A and PP1 in TSA‐induced activation of LHR gene transcription in a cell type‐specific manner. Changes in chromatin structure induced by TSA cause the release of PP2A in JAR cells, or of PP1 in MCF‐7 cells, which is associated with Sp1 directly or through HDAC, respectively at the promoter. This favors the phophorylation of Sp1 mediated by the PI3K/PKCzeta pathway which in turn causes the release of the p107 inhibitor from SP1 and marked transcriptional activation of the LHR. These findings reveal the importance of phosphatases in the control of LHR transcription, where the balance between PI3K/PKCzeta and phosphatases could be critical for up‐ and down‐regulation of LHR gene expression in physiological and pathological settings.
Zinc deficiency response in Saccharomyces cerevisiae: Characterization of the Zap1‐responsive regulon
Authors: Wu Chang‐Yi , Bird Amanda , Winge Dennis , Eide David
Published: June 2021

The yeast Zap1 transcription factor is a central player in zinc homeostasis. To identify genes important for low zinc growth, DNA microarrays were used to identify genes directly regulated by Zap1. A previous study indicated that Zap1 activates expression of 46 genes under moderate zinc deficiency. Our new experiments identified Zap1 targets that respond only to severe deficiency. We also applied motif identification tools to find candidate Zap1 binding sites (ZREs) in the promoters of these genes. Using these methods, we found 31 new Zap1 target genes. We confirmed the expression levels of several by S1 nuclease protection assay and verified Zap1 binding to putative ZREs by EMSA. We also identified 36 genes that are repressed in a Zap1‐responsive manner. Among these genes, MET3/14/16 encode enzymes of sulfur assimilation. It was known that these genes are regulated by the Met4 transcriptional activator via ubiquitination by Met30, a component of SCF ubiquitin ligase. We found that MET30 is a Zap1 target up‐regulated under severe zinc deficiency. We also showed that less Met4 protein accumulates in zinc‐limited cells and in cells expressing a constitutive Zap1 allele. These results suggest that MET3/14/16 genes repression is due, at least in part, to Met4 down‐regulation following MET30 induction by Zap1. Hence, Zap1‐mediated repression may control sulfur assimilation during the zinc deficiency.
Mapping Post Translational Modifications of the Yeast Repressor Activator Protein, Rap1p
Authors: Tripathi Manish Kumar , Ham Amy Joan , Weil Peter Anthony
Published: June 2021

Repressor Activator Protein (Rap1p), DNA binding transcription factor, plays a critical role in yeast physiology by activating the expression of the most vigorously transcribed mRNA‐encoding genes, such as those encoding ribosomal proteins, translation factors, and glycolytic enzymes. Indeed, Rap1p drives ∼40% of all RNA Polymerase II‐mediated transcription initiation events. Rap1p appears to utilize different mechanisms of activation, through different regulatory domains, for each of these coordinated, complex regulatory events. Previously it has been shown that phosphorylation of Rap1p modulates its ability to bind enhancer DNA. However, the relevant Rap1p phosphorylation sites have never been mapped. In an effort to gain further insights into Rap1p function, we have used MS‐based approach to identify phosphorylation sites in Rap1p, and three potential phosphorylation sites, T:482; T:486 and S:616 were identified by our analyses. They reside within the DBD and C terminus (activation and silencing domain) of Rap1p. We are developing strains with all permutations of mutations at these phosphorylated residues in order to systematically test by biochemical and genetic analyses the contribution of phosphorylation to Rap1p function. These studies on Rap1p will give us critical information for understanding important aspects of the biology of this essential protein.
Repression of the Polycystic Kidney Disease‐1 (PKD1) Promoter by MEKK1 is mediated by p53
Authors: Islam M. Rafiqul , Rodova Marianna , Puri Sanjeev , Magenheimer Brenda , Maser Robin , Widmann Christian , Calvet Janes , Jimenez Tamara
Published: June 2021

We found that PMA and constitutively active MEKK1 (CAM) significantly reduced transcriptional activity (upto 90%) from the 200 bp proximal PKD1 (Polycystic Kidney Disease 1) promoter in HEK293T. Semi‐quantitative RT‐PCR showed decreased levels of endogenous PKD1 mRNA in PMA treated and CAM transfected HEK293T cells. ChIP‐PCR assay suggested MEKK1 interacts with the proximal promoter, however, the binding is indirect as purified CAM‐GST was unable to bind in EMSA assay. JNK inhibitors had no effect in the CAM‐mediated repression, rather surprisingly, a kinase‐inactive MEKK1 repressed the proximal promoter similar to CAM. The suppression of the transcriptional reduction by CAM was observed in HEK293T cells co‐transfected with p53siRNA or dominant negative p53. Furthermore, in HCT116 cells lacking p53, the 200 bp proximal promoter could not be repressed by either CAM. Co‐precipitation of p53 with CAM, or vice‐versa and their co‐nuclear localization indicated their interaction. Taken together, these data indicate that repression of the PKD1 promoter by CAM is mediated through p53, and suggest that PMA‐MEKK1 pathway may play a role in PKD1 regulation. The work is supported by NIH fund.
Dissecting the role of two activation domains in Zap1 in Saccharomyces cerevisiae
Authors: Frey Avery Glenn , Eide David
Published: June 2021

Zinc homeostasis in Saccharomyces cerevisiae is mediated by the transcriptional activator Zap1. Under zinc‐limiting conditions, Zap1 activates expression of over 40 genes involved in zinc acquisition and utilization. When zinc is abundant, Zap1 function is inactivated. Zap1 has two activation domains, AD1 and AD2, that increase gene transcription. Intriguingly, both of these domains are independently regulated by zinc. Furthermore, the presence of two activation domains is a feature evolutionarily conserved among Zap1 orthologs in other species suggesting the importance of both domains to cell physiology. In this study, we are assessing the contribution of AD1 and AD2 to Zap1 target gene regulation and zinc homeostasis. Using a Zap1 allele deleted for AD1, we demonstrated that AD2 varied greatly in its ability to drive transcription of different Zap1 target genes under low zinc conditions. While some genes were still highly expressed in cells expressing Zap1 lacking AD1, other genes were expressed at only low levels. Moreover, cells bearing the AD1 deletion allele showed a severe growth under zinc limiting conditions. These results demonstrate that AD1 is critical for normal growth under zinc limiting conditions and for the expression of many Zap1 target promoters. The contributions of AD2 to zinc homeostasis are currently being assessed using similar approaches.
NMR structural characterization of the homodimerization domain of the translational repressor GLD‐1
Authors: Beuck Christine , Szymczyna Blair , Columbus Linda , Williamson James
Published: June 2021

GLD‐1 is a prototypical member of the STAR class of proteins, which are highly conserved among higher eukaryotes and seem to play a general role in posttranscriptional gene regulation. In the C. elegans hermaphrodite germ line, GLD‐1 is essential for inducing meiosis. Furthermore, it triggers the spermatogenesis‐to‐oogenesis switch by regulating the differentiation of germ line stem cells. GLD‐1 translationally represses the expression of proteins promoting oogenesis by binding to the 3′ untranslated region of their messenger RNAs (mRNAs). GLD‐1 homodimers recognize 28 nucleotide repeats within the mRNA. Each repeat can accommodate one GLD‐1 dimer.

Even though the structures of a few homologous STAR protein RNA binding subdomains have been determined, no structural information is available on the homodimerization subdomain. Our structural characterization of the GLD‐1 homodimerization domain by CD, NMR and EPR spectroscopy points towards a α‐helical, antiparallel coiled‐coil dimeric structure.
Triacsin C is a time dependent inhibitor of Long Chain Fatty Acyl CoA Synthetase (Acsl) in endothelial cells
Authors: Pillai Venkateswaran C , Chen Yi Qing , Crumley Jason , Bazinet Richard P , Rapoport Stanley I , Weis Margaret T
Published: June 2021

The literature indicates that triacsin C is a competitive inhibitor of Acsl. We found that, in partially purified rat brain endothelial Acsl, maximum inhibition by triacsin C was only 82 ± 2%. Solubilized human coronary endothelial cells (HCEC) treated with triacsin C for one minute lost about 86 ± 2.6% of their activity, without further decline as triacsin C exposure time increased. HCEC were treated with 5 μM triacsin C for up to 8 hours, then washed, solubilized and assayed for Acsl activity. Activity in treated cells declined with a half‐time of 19 ± 2.9 minutes. HCEC pre‐treated with triacsin C for 2 hours were washed, re‐fed, and allowed to recover. At 24 hours post‐treatment, activity was only about 39 ± 5% of that of untreated HCEC. After 2 hours triacsin C treatment and 2 hours recovery, HCEC were exposed to [14C]‐palmitic acid for 2 hours. Radioactivity was 15 ± 5.7% of control in the fatty acyl CoA fraction, and 39 ± 2.6% of control in total extractable lipids. The weight of total extractable lipids (μg/well) was 44 ± 2.3 for control and 43 ± 4.6 for treated HCEC (not significant). These data indicate that triacsin C is likely a partial competitive inhibitor of Acsl in vitro and may be an irreversible inhibitor in situ. Supported by AHA 06552254 and NIH R21AG028901.
Characterization of trinitrophenylated (TNP) adenosine nucleotide binding to bovine phenol sulfotransferase (bSULT1A1)
Authors: Macdonald Jane A. , Sawin Andrew , Lapham David , White Judy , Turk Jeffrey A. , Beckmann Joe D.
Published: June 2021

Sulfate transfer from 3′‐phosphoadenosine‐5′‐phosphosulfate (PAPS) to various acceptors is catalyzed by sulfotransferases (SULTs). SULTs also bind other nucleotides, although measuring these affinities can be difficult. We have previously observed binding of TNP‐ATP to purified recombinant bSULT1A1 by fluorescence, and we have now investigated this in greater detail. First, a molecular model using human SULT1A1 coordinates has identified a feasible docking site for a 3′‐TNP group. Second, ionic strength increased the Kd for TNP‐ATP from 1.1 nM at 16 mM I up to 185 nM at 516 mM I. This suggested an important role of charged phosphoryl groups in binding to the enzyme. Third, this role was confirmed by determination of Kd values for TNP‐ATP (1.1 nM), TNP‐ADP (9.3 nM), and TNP‐AMP (175 nM). Next, the effect of temperature on TNP‐ATP binding was determined from 5–45°C, and greatest affinity was observed at 35°C. The positive Arrhenius plot slope from 5–35°C revealed enthalpy‐driven binding (ΔH = ca. −25 kJ/mol), which is being tested by titration microcalorimetry. Finally, Kd values for biologically‐relevant nucleotide binding to bSULT1A1 are being determined by competitive titrations into SULT:TNP‐AD/TP complexes. These measurements will provide a meaningful view of SULT regulation and mechanism.