bluuebunny/crossref_metadata_2025 huggingface dataset:
The hydrogen sulfide donor IK‐1001 improves the outcome of acute respiratory distress syndrome in murine and ovine models
Authors: Esechie Aimalohi , Enkhbaatar Perenlei , Djukom Clarisse , Atsumori Hamahata , Bolanowski David , Kiss Levente , Horvath Eszter , Traber Lilian , Traber Daniel , Szabo Csaba
Published: June 2021

The novel biological mediator hydrogen sulfide (H2S) has beneficial effects as a vasodilator, a cytoprotectant and as an anti‐inflammatory agent. Using IK‐1001, an aqueous formulation of H2S, here we tested the effects of hydrogen sulfide in models of acute respiratory distress syndrome (ARDS) in mice and sheep. In the mouse model of 40% third degree burn and smoke inhalation injury, median survival was 22 hours after injury in the vehicle group. At 24h, 33.3% of the control animals were alive. There was a significant increase in median survival in the mice that received hydrogen sulfide treatment after injury (p<0.02; 75 hours). In the same group, 8 out of 12 of the animals were alive during the first 24h. IK‐1001 significantly reduced the level of the pro‐inflammatory cytokine (pg/mg ± SE) in the lung by 50% when compared with the control animals, as measured at 12h in lung homogenates. In the presence of acute lung injury and saline treatment, IL‐10 concentration was unaffected compared with the sham (without burn and smoke) group. However, there was a 2‐fold increase in IL‐10 concentration in the lung after injury and treatment with IK‐1001 (p < 0.05). IK‐1001 was next tested in a clinically relevant LD100 sheep model of ARDS. IK‐1001 significantly prolonged survival and improved PaO2/FiO2. Our results demonstrate the protective effects of hydrogen sulfide in two different experimental models of ARDS.
Cardioprotection of glutathione S‐transferase P against ischemia‐reperfusion injury
Authors: Conklin Daniel J. , Hill Bradford G. , Guo Yiru , Prough Russell A. , Prabhu Sumanth D. , Bolli Roberto , Bhatnagar Aruni
Published: June 2021

Oxidative stress is a key determinant of cardiac injury associated with acute ischemia and reperfusion (I/R). Yet, the mechanisms that regulate the outcome of redox changes in I/R‐heart remain unclear. Because glutathione‐linked metabolism is a major pathway for the detoxification of electrophiles generated during I/R, we examined the role of glutathione S‐transferase P (GSTP) – the major extrahepatic GST. To test the hypothesis that GSTP is cardioprotective, GSTP1/P2 wild‐type (WT) and null mice were subjected to left coronary artery ligation (30 min) and 4 h reperfusion in situ. Infract size due to I/R was significantly increased to 73.1 ± 3.7% of risk region (n = 8) in null mice vs WT mice (55.6 ± 3.9%; n = 8; P < 0.01). Similarly, increased levels of oxidative stress markers were observed in isolated I/R‐hearts of GSTP‐null vs WT mice. Moreover, isolated cardiomyocytes of GSTP‐null mice were more sensitive than those of WT mice to hydrogen peroxide‐ and acrolein‐induced hypercontracture. In contrast, no differences in basal levels of cardiac oxidative stress markers (e.g., GSH level), expression of the GSTs A and M, and cardiac phenotype were observed between WT and null mice, although GST and peroxidase activities were significantly decreased in null vs WT hearts. These findings indicate GSTP is a novel cardioprotective gene against I/R. This work funded by NIEHS and NIH grants ES11860 (AB) and HL78825 (RB).
Acute Estrogen Receptor‐α Activation Improves Ischemic Tolerance in the Aged Female Rat Heart Through a PKCε‐Dependent Mechanism
Authors: Korzick Donna H , Novotny Jennifer L , Lancaster Timothy S , Tomicek Nanette J
Published: June 2021

Introduction: The contribution of non‐genomic estrogen receptor (ER) signaling to ischemia and reperfusion (I/R) injury is poorly understood and has not been directly examined with aging. Hypothesis: Acute ERα activation would restore membrane ER targeting and reduce infarct size with aging. Methods: Adult (4 mo) and aged (23 mo) female F344 rats with ovaries intact (n=16) or removed (n=16; OVX) were subjected to I/R (47 min global I), and s.c. injection of Propyl pyrazole triol (PPT) or vehicle 45 min prior to euthanasia (5 μg/kg). Infarct size was assessed with TTC staining, ERα, Akt, and PKCε levels with western blotting, and ERα‐mediated gene expression through real time PCR. Results: Infarct size was greatest in aged OVX > aged > adult OVX > adult, suggesting a greater effect of E2 deficiency with age. PPT reduced infarct size in aged and aged OVX (p<0.05), but had no effect in intact adults. With PPT, particulate ERα increased, with reversal of age‐related reductions in nuclear and mitochondrial PKCε and pAkt (p<0.05). PPT also increased mRNA levels for RACK2 (p<0.05). Conclusions: Selective ERα activation reduces I/R injury in the aged, E2‐deficient heart through a mechanism involving non‐genomic redistribution of ERα and PKCε translocation. Our data further suggest, for the first time, a feed‐forward transcriptional mechanism for ERα‐mediated delayed cardioprotection to enhance PKCε‐RACK2 interactions.
Exogenous ghrelin suppresses cardiac sympathetic tone following acute myocardial infarct in rats
Authors: Schwenke Daryl Owen , Tokudome Takeshi , Kishimoto Ichiro , Cragg Patricia , Kangawa Kenji
Published: June 2021

Acute myocardial infarction (MI) initiates an increase in cardiac sympathetic nerve activity (SNA), which exacerbates ventricular remodelling and cardiac dysfunction. Ghrelin, a growth hormone‐releasing peptide, is an effective treatment for improving cardiac function in chronic heart failure. Ghrelin may also have important therapeutic benefits during the early stages of MI, since studies have shown that ghrelin reduces sympathetic tone. In this study we hypothesised that ghrelin would prevent the increase in cardiac SNA that normally occurs following MI. Cardiac SNA was continuously recorded in urethane‐anaesthetized rats for five hours following ligation of the left anterior descending coronary artery. Rats received either saline or ghrelin (150 μg/kg, s.c.) one minute, or two hours, after infarction. SNA remained stable in sham rats. Myocardial infarction induced a maximal 110% increase in SNA, which was completely prevented in those rats that also immediately received an injection of ghrelin. When ghrelin was injected two hours after the MI (when SNA had increased by ∼85%), SNA significantly fell to pre‐MI activity. These results show that ghrelin acutely prevents the increase in cardiac SNA following MI. Further studies are now essential to determine whether this prophylactic benefit of ghrelin will aid in attenuating subsequent cardiac dysfunction. A Departmental Grant supported this research.
Influence of aging on the interaction of the PI3K survival pathway and the p53‐mediated apoptosis pathway in a rat model of myocardial ischemia‐reperfusion
Authors: Liu Peitan , Xu Baohuan , Cavalieri Thomas A. , Hock Carl E.
Published: June 2021

Cardiomyocyte death induced by myocardial ischemia‐reperfusion (MI/R) can occur via apoptosis and/or necrosis, depending on the ischemic intensity and available survival signals. Aging may alter the balance between survival and apoptotic signals, thereby influencing cell fate. A rat model of MI/R has been used to investigate the cross‐talk between the PI3K survival pathway and the p53‐dependent apoptosis pathway. Our results indicate that inhibition of p53 transcriptional function by pifithrin‐α improved cardiac index (79±11 vs 38±9 ml/min/100g BW, p<0.05) and reduced myocyte apoptosis (96±11 vs 158±17 cells/20 HPF, p<0.05). Moreover, inhibition of PI3K by LY294002 worsened cardiac index (46.3±7 vs 68.3±9 ml/min/100g BW, p<0.05) and increased DNA fragmentation (344±57 vs 118±21 Vmax %, p<0.05), when compared to rats treated with vehicle. Interestingly, treatment of aged MI/R rats with LY increased the expression of p53, compared to young MI/R rats given LY. These results suggest that an interaction exists between the PI3K survival pathway and the p53‐mediated apoptosis pathway during MI/R, and that this interaction may be influenced by aging.
Cardioprotective effect of bicyclol against ischemia‐reperfusion induced injury in anesthetized rats
Authors: Cui Jie , Qian Ling‐bo , Wang Jue , Gao Qin , Xia Qiang
Published: June 2021

The aim of the present study was to investigate the protective effect of bicyclol against myocardial injury induced by ischemia‐reperfusion (IR) and its underlying mechanism. Bicyclol was orally administrated to rats at the does of 50, 100 or 200 mg/kg/d for 3 days. Six hours after the last administration, myocardial injury was induced in anesthetized Sprague‐Dawley rats by coronary artery ligation for 1 h and then reperfusion for 3 h. The myocardial infarct size was measured by 2, 3, 5‐triphenyltetrazolium chloride staining. Homodynamic parametres, the levels of serum lactate dehydrogenase (LDH) and myocardial lipid peroxidation were also determined. Myocardial mitochondria membrane fluidity was measured by diphenylhexatriene. We found that pretreatment with bicyclol significantly reduced infarct size and LDH release induced by IR, and also improved left ventricular developed pressure compared with IR group. Futhermore, pretreatment with bicyclol markedly attenuated the decreases of myocardial mitochondria membrane fluidity and lipid peroxidation caused by IR. These results indicate that bicyclol has significant cardioprotective effect against ischemia and reperfusion.
Hyperglycemia induced oxidative stress is attenuated in cardiac tissue by 670nm photobiomodulation in an experimental model of diabetes
Authors: Gopalakrishnan Sandeep , Lark Daniel S , Sandoval Chris L , Snyder Ann C , Eells Janis T
Published: June 2021

Oxidative stress contributes to the pathogenesis of diabetic cardiomyopathy. Photobiomodulation by light in the red to near‐infrared (NIR) range (630–1000 nm) using low energy lasers or light‐emitting diode (LED) arrays accelerates wound healing, improves recovery from ischemic injury and attenuates degeneration in the injured optic nerve.

Purpose

To investigate the efficacy of 670 nm NIR‐LED treatment in attenuating diabetes‐induced oxidative stress in cardiac tissue.

Methods

Adult Wistar rats were made diabetic by streptozotocin treatment (STZ, 50 mg/kg, ip). One group of animals (NIR‐STZ) was treated daily with 670 nm LED irradiation. (5 min/day, 6 J/cm2) 5 days/week for 14 weeks. Other treatment groups were untreated control (CON) and STZ‐treated (STZ). At week 14, animals were euthanized, perfused with cold PBS and the heart was rapidly harvested and flash frozen. Glutathione concentrations were determined by an enzymatic recycling method.

Results

The GSSG/GSH ratio in heart tissue was increased nearly four fold in STZ‐treated rats relative to control animals (CON = 0.08 ±0.01, vs. STZ=0.31± 0.08). 670 nm LED treatment significantly attenuated GSH oxidation (NIR‐STZ = 0.13± 0.03).

Conclusion

NIR photobiomodulation reduces intracellular oxidative stress and may attenuate cardiomyopathy in diabetes.

Funding Source

Biomedical Technology Alliance Award and UWM Research Growth Initiative
Hypergravity exercise training on a human‐powered centrifuge
Authors: Blank Jason Michael , Yang Yifan , Hargens Alan R. , Macias Brandon R. , Caiozzo Vincent J. , Hicks James W.
Published: June 2021

Exposure to microgravity during spaceflight results in changes in cardiovascular function that impair normal function on return to a 1G environment. The Space Cycle is a short‐arm human‐powered centrifuge that couples exercise with hypergravity as a potential countermeasure for loss of exercise capacity and orthostatic tolerance in microgravity. Healthy, sedentary men (n = 5) trained for 4 weeks by pedaling the Space Cycle 4 days/week for 45 minutes/session. Target training intensity was set at 80% max heart rate and 2.5 +Gz measured at the subject's feet. Training intensity during the workout was monitored by the subject using a commercial heart rate monitor. VO2max was measured before and after the training program using an incremental bicycle ergometer test to volitional fatigue. Orthostatic tolerance was tested using simultaneous 60 degree head‐up tilt and graded levels of lower‐body negative pressure to presyncope. Space Cycle training increased VO2max from 38 ± 7 to 43 ± 6 ml/kg/min (p<0.01). Four of five subjects improved orthostatic tolerance time following training. All subjects completed the training program and noted increasing comfort with the rotational motion of the Space Cycle as the training program progressed. Funded by NSBRI MA00403 (VJC) and MA00801 (JWH).
Responses of cleaved caspase‐8 and −12 apoptotic pathways to 12 week treadmill exercise in aging rat skeletal muscle
Authors: Kwak Hyo‐Bum , Kim Jong‐Hee , Lawler John M
Published: June 2021

Previously our laboratory demonstrated that exercise training (ET) attenuated age‐induced muscle fiber atrophy, caspase‐3 cleavage, and pro‐apoptotic Bcl‐2 signaling in the rat white gastrocnemius. However, cleavage of caspase‐3 and apoptosis may also be stimulated by inflammatory cytokines (e.g. TNF‐α), the fas/cytokine pathway, and ER/Ca2+ stress pathway via caspase‐8 and caspase‐12 activation, respectively. Therefore, the purpose of this study was to test the hypothesis that 12 weeks of exercise training would protect against caspase‐8 cleavage, upstream fas/cytokine signaling, and caspase‐12 cleavage in the rat white gastrocnemius. Three and 31 mo. old Fischer 344 x Brown Norway rats were assigned to young sedentary (YS), young exercise (YE), old sedentary (OS), and old exercise (OE) groups. ET groups ran on a treadmill for 45 min/day, 5 d/wk for 12 wks. TNF‐α, TNFR1, cleaved caspase‐8, cleaved caspase‐12, hydroperoxides, and Hsp70 levels were assesssed. Cleaved caspase‐8 levels were elevated (+91.7%) in the OS group, but not in the OE group. Upstream TNFR1 was lower in ET young (−62%) and old (−25%) rats, without a reduction in TNF‐α. In contrast, cleaved caspase‐12 was not affected by aging or ET. ET decreased hydroperoxide levels (−19.8%) and increased Hsp70 (+46.7%) in the OE group vs. OS. These data indicate that protective effects of ET against age‐induced apoptosis in white muscle were related to upstream modulation of oxidative stress, Hsp70, and possibly the fas/cytokine pathway, but not the ER/Ca2+ stress pathway.
Exercise Training Upregulates Mitochondrial Survival Proteins BAG‐4 and Thioredoxin‐2 in the Aging Rat Heart
Authors: Lawler John M , Kwak Hyo‐Bum , Kim Jong‐Hee
Published: June 2021

Recently, we showed that in the rat heart exercise training (ET) reduces age‐induced oxidative stress, apoptosis, caspase‐3 cleavage, and upstream modulation of mitochondrial Bcl‐2 signaling by increasing anti‐apoptotic Bcl‐2 and decreasing pro‐apoptotic Bax. BAG‐4 and thioredoxin‐2 (Trx2) are mitochondrial survival proteins that promote protection against apoptosis by binding or stimulating Bcl‐2 and Bcl‐XL. The purpose of this study was to test the hypothesis that 12 weeks of endurance exercise training would upregulate levels of survival proteins BAG‐4 and Trx2 in the aging rat heart. Three and 31 mo. old Fischer 344 x Brown Norway F1 rats were assigned to young sedentary (YS), young exercise (YE), old sedentary (OS), or old exercise (OE) groups. ET groups ran on a treadmill at 20 m/min (young) or 10 m/min (old), 18% grade, 45 min/day, 5 day/wk for 12 wks, and left ventricle (LV) samples were extracted from all groups after ET. Active BAG‐4 levels decreased by 66% with aging in the LV, but were increased (+121%) by exercise in the OE group. ET‐induced upregulation of active BAG‐4 in OE rats was directly related a 139% increase in the pro‐form of BAG‐4. Trx2 protein levels were 75% and 78% higher in LV from the YE and OE groups, compared to the YS and OS groups. Our data indicate the mitochondrial survival proteins BAG‐4 and Trx2 are inducible with endurance training in the old rat heart, and offer putative therapeutic targets.
Respiratory‐related pacemaker neurons in isolated turtle brainstems in vitro
Authors: Chapman Mark A. , Alexander Michael J. , Johnson Stephen M.
Published: June 2021

For adult vertebrates, it is not known whether respiratory‐related pacemaker neurons exist and play a critical role in rhythm generation. To address this question, brainstems from adult turtles were isolated in vitro and respiratory motor bursts were recorded on hypoglossal (XII) nerve roots. Extracellular recordings of respiratory‐related neurons were acquired using multichannel silicon electrodes. While bathed in artificial cerebrospinal fluid, neurons (n=87) exhibited phasic behavior in relation to the XII motor bursts. Synaptic transmission was blocked with antagonists for GABAA (bicuculline), glycine (strychnine), AMPA (CNQX), and NMDA (APV) receptors. During synaptic transmission blockade, 14/87 neurons continued to fire with rhythmic bursts of action potentials and were considered putative pacemaker neurons. When [K+] was increased to 6.0–9.0 mM during ongoing synaptic blockade, 9/10 pacemaker neurons increased burst frequency and action potential firing rates within the bursts. Application of a mu‐opioid agonist (DAMGO) decreased burst frequency in 3/4 pacemaker neurons. These results suggest that respiratory pacemaker neurons, which respond to increased [K+] and mu‐opioid receptor activation, exist in adult turtles. We hypothesize that these pacemaker neurons may be critical for rhythm generation.
Influence of extracellular [K+] on inspiratory‐phase synchronization in arterially‐perfused adult rat
Authors: Shen Tabitha , Perkins Matthew H. , Warren Kelly A. , Solomon Irene C.
Published: June 2021

Studies examining central respiratory control using reduced preparations commonly increase the K+ concentration ([K+]o) in the artificial cerebral spinal fluid (aCSF) supplied to the preparation in order to increase excitability and maintain stable respiratory output. Although studies have evaluated the influence of varying the [K+]o on temporal and spectral characteristics of inspiratory motor discharge in the in vitro transverse slice preparation, much less is know about the influence of [K+]o on these parameters in the arterially‐perfused adult rat preparation. Therefore, using this preparation, we examined the effects of perfusion with aCSF containing either 3 mM (n=9; low) or the typically used 6.25 mM (n=8; control) [K+]o on phrenic nerve discharge. During perfusion with low [K+]o, phrenic burst frequency was 16.6± 1.4 bpm, TI was 904±145 ms, and TE was 2.9±0.2 s; during perfusion with control [K+]o, phrenic burst frequency was 17.7± 1.7 bpm, TI was 741±47 ms, and TE was 2.9±0.3 s. Perfusion with low [K+]o also resulted in a broadening of the high frequency oscillation (HFO) spectral peak and HFO activity was dispersed across much of the inspiratory burst, with a shift in the onset of spectral activity to an earlier point in inspiration. These findings suggest that low [K+]o reduces inspiratory‐phase synchronization. Supported by NS045321 and NS049310
Nonlinear analysis of breathing patterns in development of acute and chronic lung injury
Authors: Palacios Mariana Yvonne , Jacono Frank J. , Ceco Ermelinda , Fishman Mikkel , Loparo Kenneth A , Dick Thomas E
Published: June 2021

Objective: Determine whether nonlinear analysis can identify breathing pattern trends that are indicative of the progression and outcome of acute lung injury (ALI). Methods: Male Sprague‐Dawley rats (n=10) received intratracheal instillation of either bleomycin (3–4 units/120–160μL PBS) or PBS (160μL). Baseline breathing was recorded in unanesthetized animals using whole‐body plethysmography every other day after surgery. Multiple epochs (600‐s) were selected and breath‐to‐breath variability of expiration (Te) was assessed systematically by linear (mean, coefficient of variation) and nonlinear (mutual information, sample entropy of intervals and successive differences) tools. At two weeks, lung collagen content was measured. Results: Linear analysis did not discriminate the severity or outcome of injury. In contrast, nonlinear methods showed a trend between decreased average entropy of successive differences of Te across time lags and acute injury. The increase in entropy of breath‐to‐breath magnitude changes of Te across time lags was significantly greater for the animals with fulminant injury compared to resolving injury. Lung collagen content was not different between groups. Conclusion: Nonlinear analysis identified subtle changes in breathing variability, indistinguishable by linear analysis, that correlate to injury acuity prior to development of fibrosis (HL080138, VA Research Service & T35 NHLBI Summer Research Program).
Bone strength is maintained after 8 months of inactivity in hibernating ground squirrels, Spermophilus lateralis
Authors: Utz Jenifer C. , Nelson Stacy , O'toole Brendan J. , van Breukelen Frank
Published: June 2021

Prolonged inactivity leads to disuse atrophy, a loss of muscle and bone mass. Hibernating mammals are inactive for 6–9 months a year but must return to full activity immediately after completing hibernation. This necessity for immediate recovery presents an intriguing conundrum as most mammals require 2–3 times the period of inactivity to recover full strength. To address this problem, we dissected femur and tibia bones from squirrels in various stages of the annual hibernation cycle and measured the amount of force required to fracture these bones. Three groups were investigated; summer active animals were captured during the summer and immediately sacrificed, animals in the 1‐month detraining group were captured in the summer and sacrificed following a 1‐month period of restricted mobility, hibernating animals were sacrificed following 8 months of inactivity. A 3‐point bend test was employed to measure the force required to break the bones. Additionally, flexural strength and flexural modulus (material stiffness) were calculated for femurs. There were no significant differences between groups for femur break force, femur flexural strength, or tibia break force. Femur flexural modulus for the 1‐month detraining group was significantly different from both the summer active and hibernating groups (ANOVA, p < 0.05). Thus, hibernators seem resistant to the deleterious effects of prolonged inactivity.
Studies of Psychrophilic Methionine Sulfoxide Reductases from Colwellia psychrerythraea
Authors: Feagans Mika , Cottrell Christopher A , Domanski Tammy L , Smith Virginia F , Schlessman Jamie L
Published: June 2021

Methionine sulfoxide reductases (Msr) function as anti‐aging proteins by providing organisms with a mechanism to defend against the damaging effects of oxidation. These ubiquitous enzymes restore activity to oxidized methionine side chains in proteins. Methionine sulfoxide S and R epimers are repaired stereospecifically by MsrA and MsrB, respectively. Genomic analysis of the psychrophilic bacterium Colwellia psychrerythraea revealed five distinct msr genes, two msrAs, two msrBs and an msrA‐msrB fusion. Putative residues involved in substrate binding and reduction were identified by amino acid alignment of Cp MsrB proteins to mesophilic and hyperthermophilic homologs. In an effort to understand the significance of Msr function in this psychrophile, msrB1 was amplified from the Cp genome using PCR, ligated into a vector and transformed into competent E. coli host cells. Following confirmation of the gene sequence, the plasmid was transformed into expression host cells. Soluble recombinant Cp MsrB1 was produced as a histidine fusion and purified by affinity chromatography. Msr function was observed upon incubation with the peptide substrate H‐Lys‐Lys‐[Met(O)]‐Val‐Glu‐Asn‐Lys‐Lys‐OH at 4°C and monitored using MALDI‐TOF mass spectrometry. Temperature limitations of MsrB1 activity and conformational stability were assessed using MALDI‐TOF MS and CD and fluorescence spectroscopies.
The olfactory isoform of adenylyl cyclase (AC3) in the renal macula densa serves as a key regulator of glomerular filtration rate
Authors: Pluznick J L , Zou D J , Zhang X , Yan Q , Rodriguez‐Gil D , Eisner C , Wells E K , Greer C , Schnermann J , Wang T , Firestein S , Caplan M J
Published: June 2021

Recent work shows that olfactory receptors (ORs) are functionally expressed in non‐olfactory tissues. We find that key components of the OR signaling pathway, olfactory G protein (Golf) and the olfactory adenylyl cyclase (AC3), are detectable in the kidney by RT‐PCR and western blot. AC3 and Golf colocalize with one another and with markers of the distal convoluted tubule. Most notably, AC3 is expressed in the macula densa (MD). The MD regulates glomerular filtration rate (GFR) through both the tubuloglomerular feedback (TGF) and renin secretion pathways. Renal function studies using AC3−/− mice and wild‐type littermates demonstrated that GFR in AC3−/− mice is significantly decreased (AC3−/−:0.42 ± 0.08 ml/min/100g BW, n=8; AC3+/+: 0.87 ± 0.06, n=6, p<0.01). This is not due to alterations in TGF, as TGF assayed by micropuncture is normal in AC3−/−; however, AC3−/− have a ~50% reduction in plasma renin concentration (p<0.001). By RT‐PCR, we have identified six ORs in mouse kidney, suggesting that the chemosensors that participate in this pathway are also present in renal epithelia. These studies indicate that the olfaction machinery present in the kidney is necessary for proper control of GFR, presumably through modulation of renin secretion to regulate efferent arteriolar diameter. This OR machinery is found in the MD, an ideal localization for ORs to sense the chemical composition of the forming urine.
Effect of iron oxide perfusion on renal micro vessel morphology, and dilatory function and endothelial mRNA expression of mouse aorta
Authors: Rettig Juliane , Schmidt Sebastian , Schildroth Janice , Paliege Alexander , Martinka Peter , Sendeski Mauricio , Bachmann Sebastian , Persson Pontus B. , Patzak Andreas
Published: June 2021

Renal microvessels can be selectively isolated using a combined iron oxide‐sieve technique. Here we investigate a potential damaging effect of the iron oxide perfusion on microvessel morphology and endothelial cells. C57Bl6 mice were perfused with PBS (1% albumin) or PBS (1% albumin) with 1% iron oxide. Histology of the kidney was investigated using thin and hemi‐thin sections. Endothelium mediated vasodilatation was tested in rings of abdominal aorta under isometric conditions. Relative mRNA content of endothelial nitric oxide synthase (eNOS), endothelin 1 (ET‐1), and the platelet endothelial cell adhesion molecule‐1 (PECAM‐1) was determined using quantitative PCR analysis in aorta. The histological investigation did not show damage of microvessels in iron oxide perfused kidneys. There was no difference in the dilatation to acetylcholine in aortas of both groups. The quantitative PCR revealed no differences in the expression of eNOS and ET‐1 comparing iron oxide perfused and PBS perfused vessels. PECAM‐1 expression was enhanced in iron oxide perfused vessels. The study shows that iron oxide perfusion may have a slight effect on the analysis of endothelial mRNA expression as indicated by significant changes in PECAM‐1 expression. Similar dilatory function of aortic rings and similar expression of eNOS and ET‐1 mRNA suggest integrity of endothelial cell layer after iron oxide perfusion in aorta.
(Pro)renin Receptor Activation Causes Acute Production of Macula Densa Prostaglandins
Authors: Meer Elliott , Kang Jung Julie , Vargas Sarah Laurin , Toma Ildiko , Peti‐Peterdi Janos
Published: June 2021

Prostaglandins (PG) are known to be mediators of juxtaglomerular (JG) renin expression and release. The macula densa (MD) is a critical source of these local JG PGs, and MD PG production is known to vary in pathological conditions. In MD cells, the (pro)renin receptor has recently been shown to activate COX‐2, a key enzyme in PG synthesis. The (pro)renin receptor has been localized to the basolateral membrane of MD cells, with direct access to the renin granular cells and interstitium of the JG region. Therefore, we hypothesized that activation of the (pro)renin receptor in MD cells may play a role in MD control of JG renin synthesis and release through intracellular signaling promoting PG synthesis. We utilized cultured mouse MD‐derived cells (MMDD1) and found that the addition of 10 nM renin activated the MAP kinase ERK1/2 within 15 minutes (n=3), assessed by Western blot. Since MAPK activation is known to activate COX‐2, a critical enzyme implicated in PG synthesis, we investigated acute rises in MD PG production in response to renin. Utilizing fura 2‐loaded biosensor HEK cells expressing the PGE2 EP1 receptor, cuvette‐based spectrofluorometry detected significant increases in PG release from MMDD1 cells in response to 10 nM renin (n=4). In summary, activation of the (pro)renin receptor in MD cells rapidly activates MAPK signaling and causes PGE2 release, which may be an important feedback mechanism for JG (pro)renin synthesis.
Annotated virtual microscope laboratory units for teaching general and oral histology to dental students
Authors: Heidger Paul M. , Finkelstein Michael , Leaven Timothy , Dee Fred
Published: June 2021

An integrated approach to learning laboratory concepts and skills in general medical histology has been reported previously (Heidger, et al, 2002; Harris, et al, 2001) which employs both traditional and virtual imaging of microscope slides. This methodology has been extended recently to teaching both general and oral histology to dental students. The annotated images comprising the University of Iowa's Virtual Slidebox of Histology, together with newly prepared virtual slides from our oral histology slide collection, constitute the core of required material for individual virtual laboratory study. Elective opportunities for small‐group interaction with faculty over traditional glass slides are scheduled, as well. In contrast to previous years of laboratory study that relied heavily upon labeled 35 mm slides, the expanded Virtual Slidebox (see www.path.uiowa.edu/virtualslidebox) now offers our dental students a means for individual, comprehensive laboratory study of both general and oral histology preparations. Student reception of this option has been overwhelmingly positive, scores in practical exams have risen, and the quality of the learning experience in dental histology enhanced in the view of faculty and student evaluators, alike. Our histology offerings for dental and medical students at Iowa are now closely linked with common lectures, but with distinct laboratory experiences. This has facilitated desirable cross‐collegiate interactions among students, and better appreciation for the goals and emphases of the respective professional school curricula. Supported by a grant from the National Library of Medicine to FD, and Dental and Medical Educational Development funds to MF and PH.
Enhancing Science Education at a Liberal Arts College ‐ A Model Approach by EPA and Livingstone College
Authors: Mack Cina M , Kodavanti Prasada R , Smith Edward G
Published: June 2021

Current data indicate the United States will experience a critical short fall of skilled professionals in science and technology (STEM). This problem has multiple dimensions, which include improving the quality of science education at liberal arts institutions. In an effort to address this dilemma, a pilot project between the Neurotoxicology Division/US EPA and Livingstone College (LC), a small, historically black college, was initiated. Faculty from the biology, chemistry and psychology departments of LC participated in a 10 week fellowship at the National Health and Environmental Effects Research Laboratory (NHEERL) of US EPA. Faculty were involved in research projects with EPA investigators, and subsequently co‐authored abstracts for presentation at scientific conferences. Modules of biobehavioral research and environmental science were incorporated into the curricula at LC, and a physiology research seminar was added. Lastly, an NIH EARDA proposal, authored by an LC fellow, was submitted to fund an Office of Biomedical and Behavioral Research at LC. This pilot program demonstrates a cost‐effective mechanism ($40,000 NHEERL training grant) for direct improvement of science education at a small, liberal arts college serving a student population that is historically under‐represented in STEM majors.

(This abstract does not necessarily reflect US EPA policy).
Thiol‐Reactive Inhibitors of Cdc48/p97 AAA ATPase as Potential Anti‐Cancer Therapeutics
Authors: Chou Tsui‐Fen , Jones Amanda C , Stoltz Brian M. , Deshaies Raymond J.
Published: June 2021

Cdc48/p97 is an important AAA ATPase not only due to its diverse cellular functions but also because it has been implicated in mediating turnover of proteins involved in tumorgenesis, such as cyclin E and IkBa, the inhibitor of nuclear factor‐kB. We have set out to develop small‐molecule inhibitors of Cdc48/p97 to use as tools to study Cdc48/p97 function and to evaluate whether it is a viable therapeutic target. Using p97 crystal structures as a guide, we first derivatizated a PP1 like compound to incorporate electrophiles that are likely to be targeted by an active‐site cysteine of wild type p97. One of the inhibitors (ACJI‐99C) identified from our initial efforts exhibited an IC50 ~0.6 μM for wild type murine p97 and ~300 μM for wild type yeast Cdc48 and ~100 μM for hamster NSF in an in vitro ATPase activity assay. ACJI‐99C‐induced inactivation of p97 was protected by cysteine and DTT. Cys522 of p97 was shown to form a covalent bond with ACJI‐99C by tandem mass spectroscopy. Furthermore, ACJI‐99C exhibited anti‐proliferation activity toward cancer cell lines with sub‐micromolar GI50. After 1 hr incubation with ACJI‐99C, accumulation of poly‐ubiquitinated proteins was observed in HeLa and RPMI‐8226 cancer cells, which recapitulated the effect of p97 depletion by siRNA‐mediated knockdown. In conclusion, we have identified a cell‐permeable covalent inhibitor of p97 and demonstrated its potential to be an anti‐cancer agent.
Paradoxical effect of the long chain omega‐3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), on nuclear transcription in vitro
Authors: Yu Fu‐Li , Bender Wanda , Berbeka Katarzyna
Published: June 2021

Long chain unsaturated fatty acids are intimately involved in both the cause and prevention of various human diseases, e.g. obesity and diabetes, cardiovascular diseases, inflamation and allergy, and cancers including breast cancer. However, the long chain omega‐3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are believed to have protective effects against the above listed diseases. The mechanism(s) is not clear. But it is reasonable to believe that these beneficial effects are most likely through a common pathway deeply rooted at the gene level. This is to report the effect of EPA and DHA on nuclear transcription in vitro. We found that when EPA or DHA was added to the isolated rat liver nuclei, the nuclear RNA synthesis was stimulated over 2‐fold. This effect was determined to be a result of an increased rate of elongation of the nuclear RNA synthesis, and the rate at the initiation was not affected. Surprisingly, we found also that both EPA and DHA were able to strongly inhibit the enzyme activity of the isolated “free” nuclear RNA polymerase per se. We believe this is the first report on the paradoxical effect of EPA and DHA on the stimulation of the “engaged” and inhibition of the “free” nuclear RNA polymerase activity [Yu, FL. (1974) Two functional states of the RNA polymerases in the rat hepatic nuclear and nucleolar fractions. Nature 251, 344–346]. We hope that this finding will inspire further investigations into the mechanism of this paradoxical effect.
Comparative studies of human fatty acid transport proteins 1, 4 and 6 in the vectorial acylation of exogenous long chain fatty acids
Authors: Arias‐Barrau Elsa , Melton Elaina M. , Singer Diane , DiRusso Concetta C. , Black Paul N.
Published: June 2021

The fatty acid transport protein (FATP) family consists of six members that are involved in the transport of exogenous long chain fatty acids (LCFAs) and the activation of very long chain fatty acids. Genetic and biochemical studies support the idea that several of the FATPs function in transport with a long chain acyl CoA synthetase by a process named as vectorial acylation. Two lines of experiments are being completed to define the role of selected FATP isoforms in vectorial acylation. In the first, we have expressed FATP1, FATP4 and FATP6 in a yeast strain devoid of both transport and activation activity. Our initial studies demonstrate FATP1 and 6 fail to function in fatty acid transport; FATP4 confers very low levels of it. Additional studies are evaluating complementation patterns relying on exogenous fatty acid transport and defining acyl CoA synthetase profiles using different fatty acid substrates. Second, we have cloned FATP1, 4 and 6 into pcDNA4/TO/myc‐His‐A and a tetracycline‐inducible promoter drives their regulated expression when transfected into T‐Rex 293 cells. We have generated stable lines expressing FATP1, 4 and 6 and the kinetic parameters of fatty acid transport are being determined using a live cell assay and the fluorescent fatty acid analog C1‐BODIPY‐C12. Additional studies are addressing localization of each of these proteins by immunofluorescence. Supported by a grant from the NIH (GM56850).
Stearoyl‐CoA desaturase‐1 and Hedgehog signaling in the small intestine
Authors: Worple Ericha Lee , Paton Chad , Ntambi James M
Published: June 2021

Stearoyl‐CoA desaturase‐1 knockout (SCD‐1−/–) mice are hyperphagic compared to WT and have elongated small intestines (SI). However, it is unknown whether the longer SI is causative or a result of hyperphagia. Therefore, we sought to determine the mechanism by which SI proliferation occurs. First, activation of the developmentally regulated Hedgehog pathway was assessed in the proximal SI from WT and SCD‐1−/– mice. The expression of mouse Sonic, Indian, and Desert Hedgehog (Shh, Ihh, Dhh), Patched‐1 and Smoothened, as well as Hh target transcription factors Gli‐1, ‐2, and ‐3 were assessed. We found that SCD‐1−/– mice display increased Hh signaling that is evidenced by increased expression of Patched‐1 and Gli‐1 in SI. Next, we attempted to determine the role of SCD‐1 in Hh signaling in vitro. SCD‐1 overexpressing HeLa cells were treated with either stearate (18:0) or oleate (18:1) for 12‐hours and Hh activation was assessed. We found that SCD‐1 overexpression increased Gli‐1 independent of treatment and Gli‐2 increased with 18:0 and 18:1. Control HeLa cells demonstrated a decrease in Gli‐2 and an increase in Gli‐1 expression. We can conclude that regulation of Hh signaling appears to depend on the presence of SCD‐1 expression. While it remains unknown how these two pathways display coordinated expression, there is evidence to suggest that dietary factors modify Hh signaling through SCD‐1. NIH RO1 DK62388 JM Ntambi (PI)
Defects in ATP Dependent Canalicular Transporter ABCC2. Mutation Study of ABCC2 Gene in a Large Family with Dubin‐Johnson syndrome.
Authors: Barnincova Lenka , Behunova Jana , Martasek Pavel
Published: June 2021

Hepatobiliary excretion of conjugated bilirubin is mediated by an ATP dependent canalicular transporter, the multidrug resistence associated protein 2 (MRP2/ABCC2). MRP2 is responsible for biliary excrection of glucuronide and glutathione conjugates of endogenous and exogenous compounds. Before this excretion, bilirubin is conjugated by uridine‐diphosphoglucuronosyl transferase 1A1 (UGT1A1) with glucuronic acid.

Dubin‐Johnson syndrome (DJS) is an inherited autosomal recessive disorder characterised by the absence of functional protein MRP2 at the canalicular membrane of hepatocytes. Known mutations in this gene cause impaired maturation and trafficking of the mutated protein from the endoplasmic reticulum to the Golgi complex. As a result, conjugated hyperbilirubinemia is found. Defects of UGT1A1 gene cause deficient glucuronidation manifested in unconjugated hyperbilirubinemia – Gilbert syndrome.

Here we present a study of a large gypsy family with mixed congenital hyperbilirubinemia Dubin‐Johnson syndrome and Gilbert syndrome. The novel mutation 1012delGT in MRP2 gene, which causes a frameshift was found. Twenty nine subjects were investigated, two of them were found to be homozygous for both named hyperbilirubinemia. This is the first large molecular study of mixed hyperbilirubinemia with a description of both homozygous forms of mutations in UGT1A1 and MRP2 genes.
N‐terminal but not C‐terminal sequence determines the polarized distribution of sodium dependent vitamin C transporters (SVCT) in MDCK cells
Authors: Varma Saaket , Kuo Shiu‐Ming
Published: June 2021

EGFP fused hSVCT1 and hSVCT2 localize differentially in the apical and the basolateral surface respectively, in MDCK cells. A 66% identity between hSVCT1 and hSVCT2 with most of the homology in the TM region led us to hypothesize that dissimilar N and C terminus cytoplasmic regions harbor trafficking motifs. Domain swapped hSVCT211‐EGFP, in which N‐terminus of hSVCT1 was replaced with hSVCT2, localized in the basolateral instead of the apical membrane and was functional. The truncated form of hSVCT211 (starting M13) without the N‐terminal first 12 amino acids showed normal basolateral sorting. However further truncation to M57 resulted in its accumulation throughout the cell suggesting the importance of the N‐terminal amino acids 13–56 for basolateral sorting. In contrast to hSVCT211, hSVCT112, in which C‐terminus of hSVCT1 was replaced with hSVCT2, remained as a functional protein in the apical surface. From these results we concluded that the N‐terminus but not the C‐terminus dictates differential sorting in hSVCTs. C‐terminal sequence, nevertheless, is likely essential for membrane localization. Deletion of hSVCT1 C‐terminal sequence was previously shown to lead to intracellular localization of protein. Deletion of the corresponding SVCT2 sequence from amino acids 617–634 of hSVCT2 also disrupted trafficking to the membrane. Supported by Mark Diamond Research Fund.
Overexpression of YTCa, a novel gene can promote growth of NIH3T3 cells
Authors: Hwa KuoYuan , Tseng Chia‐Jen , Cheng Wei , Chang Chun‐Chi , Lee Yu‐may
Published: June 2021

YTCa, a novel human gene was first discovered in our studies for Dopa‐responsive dystonia by differential display. A unique cell model was established with the characteristic of the dominant‐negative effect of the GCH G201E mutation. By PCR‐based differential display, YTCa and other proteins were found to be involved in regulating cellular mRNA and protein half‐life. From bioinformatics analysis, we found that YTCa is located nearby chromosome 20q13. Although tissue distributions of YTCa mRNA were found in normal pancreas, muscle and heart, the protein can only be detected in brain by Western analysis. Moreover, normal tissue did not have detectable amounts of YTCa protein. In order to understand the functional role of YTCa, we have performed many experiments. First, overexpression of YTCa full‐length cDNA on NIH3T3 cell allowed cells to grow faster in tissue cultures under a low serum condition. Furthermore, nude mice injected with genetically altered NIH3T3 cells, stably transfected with YTCa formed tumors in 4 weeks.

Acknowledgement: This work is supported by the grant from Academia Sincia, Taiwan, ROC (No: AS‐94‐TP‐B17).
The Role of Fc receptor‐like 2 in B cell Signaling
Authors: Jackson Tanisha Anne , Haga Christopher L. , Ehrhardt Goetz R. A. , Davis Randall S. , Cooper Max D.
Published: June 2021

The Fc receptor‐like protein 2 (FCRL2) is a member of a family of receptors identified on the basis of sequence similarity with previously identified Fc receptors. The human FCRL2 transmembrane protein has 4 Ig‐like extracellular domains and a cytoplasmic tail containing one potential ITAM (immunoreceptor tyrosine‐based activation motif) and two ITIMs (immunoreceptor tyrosine‐based inhibitory motifs). To define its signaling potential in B cells, we constructed a chimeric receptor containing the extracellular and transmembrane domain of FcγRIIb fused to the intracellular domain of FCRL2 and expressed it in an Fc receptor deficient B cell line. Our functional analysis shows that, when coligated with the B cell receptor (BCR) by intact antibody, the FcγRIIb‐FCRL2 chimeric receptor is tyrosine phosphorylated, recruits the SHP‐1 phosphatase, and inhibits whole cell tyrosine phosphorylation. Preliminary data suggest that ERK activation and calcium mobilization are also inhibited upon crosslinking of FCRL2 with the BCR. These results indicate that FCRL2 may serve an inhibitory function in B cells.

NIH/NIAID 5 T32 AI07051; 5 T32 GM08111‐20
IL‐7 is required for B cell development from adult human bone marrow due to decreased B lymphoid generative capacity
Authors: Milford Terry‐Ann , Parrish Yasmin Khan , Baez Ineavely , Rogerio Jaqueline Willeman , Sahakian Eva , Kagoda Mercy , Benitez Abigail , Huang Grace , Payne Kimberly J.
Published: June 2021

Human B lymphopoiesis is thought to differ from that in mouse with respect to the requirement for IL‐7. Here we investigated the role of IL‐7 in human B cell development using an in vitro model based on co‐culturing human hematopoietic stem cells (HSCs) on primary stroma from adult human bone marrow (BM). Addition of IL‐7 to this co‐culture model increased B cell production by ∼40‐fold. IL‐7‐induced increases were dose‐dependent and specific to CD19+ cells. Flow cytometry analysis of STAT5 phosphorylation, IL‐7Rα, and the proliferation antigen, ki‐67, indicate that IL‐7 acts directly on B cell precursors where it increases proliferation, but not cell survival. The effects of IL‐7 were most profound in cultures with adult BM HSCs where few, if any, human B lineage cells are generated in the absence of IL‐7 activity. When co‐cultures initiated with cord blood (CB) and BM HSCs were compared, IL‐7‐induced increases were similar in magnitude, and B cell precursors responded similarly to IL‐7. However, a comparison of the generative capacity of CB and BM HSCs showed that the ability of BM HSCs to give rise to CD19+, but not CD19− cells, was 40 times less than that of CB HSCs. Our results provide evidence that IL‐7 is critical for B cell production from HSCs in adult BM due to their low B lymphoid generative capacity as compared to HSCs in CB. Supported by NIH K01 DK066163 and the Dept. of Pathology and Human Anatomy and the Center for Health Disparities and Molecular Medicine, Loma Linda University School of Medicine.
Natural glucocorticoids induce survival and proliferation of murine bone marrow granulocytes without altering function
Authors: Trottier Mark Daniel , Newsted Matthew M. , Williams Olatundun , King Louis E. , Fraker Pamela J.
Published: June 2021

Natural glucocorticoids (Gc) produced during stress have profound effects on cells of the immune system. It is known that Gc induce apoptosis in precursor T and B cells markedly altering lymphopoiesis; however, it has been noted that bone marrow myeloid cells expanded both in proportion and absolute numbers in the mouse marrow after Gc treatment. In this study, increased serum Gc and atrophied thymus were observed in mice implanted with corticosterone tablets compared to sham operated mice. Additionally, blood neutrophil counts were elevated dramatically, while lymphocyte counts sharply declined. In the bone marrow, nucleated cell number was unchanged, however the distribution of cell type was dramatically shifted. As expected, marrow lymphocytes declined more than 40% after 3 days, while monocytes and granulocytes increased in number by at least 40%. Further, all granulocyte developmental stages showed dramatic increases in both total number and percent of nucleated marrow. Gc‐exposure had little to no effect on the ability of granulocytes to produce superoxide, undergo chemotaxis or phagocytose bacteria. These results indicated that Gc treatment drastically shifted bone marrow composition and provided evidence that granulocytes and their progenitors were selectively preserved under stressful conditions without losing function.

This research was supported by NIH grant DK 52289‐29.