bluuebunny/crossref_metadata_2025 huggingface dataset:
Angiopoietin‐2 inhibition impairs blood flow recovery during hindlimb ischemia
Authors: Tressel Sarah L , Jo Hanjoong
Published: June 2021

Controlling the growth of blood vessels could be a useful therapy for ischemic diseases or atherosclerosis. Previously, we found that angiogenesis was mediated by shear stress through Angiopoietin‐2 (Ang2); however the pathophysiological significance in vivo is not known. Therefore, we used a mouse model of hindlimb ischemia to investigate the role of Ang2 in blood flow recovery. We hypothesized that inhibiting Ang2 would impair blood flow recovery after femoral artery ligation by inhibiting angiogenesis and arteriogenesis. Mice underwent femoral artery ligation and were injected with an Ang2 inhibitor for 10 days. Laser Doppler perfusion imaging (LDPI) was performed to assess blood flow recovery. The adductor muscle was whole mount immunostained for endothelial cells, smooth muscle cells, or macrophages. Ang2 inhibition significantly inhibited blood flow recovery by 5 days. Ang2 inhibition decreased smooth muscle cell coverage of vessels but had no effect on capillary formation, suggesting Ang2 plays an important role in arteriogenesis but not angiogenesis. Ang2 inhibition also decreased macrophages suggesting another mechanism by which Ang2 inhibition decreases blood vessel formation. Our data suggests that Ang2 plays an important role in collateral formation and blood flow recovery after an ischemic event and could be an important therapy for diseases involving impaired blood vessel formation.
Alpha methyl tyrosine inhibits rotenone‐ and staurosporine‐induced apoptosis of adrenal chromaffin cells
Authors: Woodman Ryan , Lockette Warren
Published: June 2021

The increased synthesis of catecholamines following traumatic brain injury (TBI) is accompanied by the generation of reactive oxygen species (ROS) that damage neuronal tissues. We hypothesized that inhibition of catecholamine synthesis and ROS formation in catecholamine‐containing neurons with alpha‐methyl tyrosine (AMT) could improve outcome in TBI. Rotenone and staurosporine induce ROS‐dependent apoptosis in adrenal chromaffin cells that synthesize catecholamines. We determined whether or not this inhibitor of catecholamine synthesis could block rotenone‐ and staurosporine‐induced apoptosis of cultured PC12 cells. Cells were incubated for 24 h with either rotenone (10 uM) or staurosporine (0.1 uM), with or without AMT (10 uM); apoptosis was quantified using a luminescent caspase activity assay, and cell viability was determined with cell counts (n = 56 for each intervention, * p < 0.01).

AMT significantly reduced rotenone‐ and staurosporine‐induced apoptosis in cultured chromaffin cells and may ameliorate ROS‐induced damage in catecholaminergic neurons. (These studies were supported by Naval Medical Center, San Diego.)
Identification of proteins interacting with GTP cyclohydrolase I
Authors: Du Jianhai , Xu Hao , Wei Na , Wakim Basam , Pritchard Kirkwood A , Shi Yang
Published: June 2021

GTP cyclohydrolase I (GCH‐1) is the rate limiting enzyme for biosynthesis of tetrahydrobiopterin (BH4) that is an essential cofactor for Nitric Oxide Synthase and aromatic amino acid hydroxylase. To explore the cell biology of GCH‐1 and determine the proteins interacting with GCH‐1, we cloned rat GCH‐1 linked with FLAG and established a stable HEK293 cell line expressing GCH‐1. We confirmed GCH‐1 expression by western blot. FLAG‐GCH‐1 associated proteins were immunoprecipitated and analyzed by ESI‐nano‐LC/MS/MS. 38 proteins were found to interact with GCH‐1. Analysis revealed that many proteins associated with GCH‐1 were involved in cellular signaling, organization, protein expression/trafficking, metabolism and apoptosis. 21 of 38 Proteins were related to cellular signaling pathways including receptors (olfactory receptors), protein kinases (MRCKƒ8 4, LATS1) and transcription factors (SNF2L1, ATF‐6ƒ8 4). GCH‐1 was found to associate with dihydropyrimidine dehydrogenase, directly related to its function. Surprisingly p47‐phox was also observed to associate with GCH‐1. Although GCH‐1 is considered cytosolic, the proteins associated with GCH‐1 were also found in the membrane, nucleus and other subcellular compartments (mitochondria and Golgi). This study is the first to provide a comprehensive analysis of GCH‐1 interactome and offer new insight into potential mechanisms of diseases related to GCH‐1.
Cardioprotective role of sodium thiosulfate on chronic heart failure by modulating endogenous H2S generation
Authors: Sen Utpal , Vacek Thomas , Kumar Munish , Moshal Karni S , Tyagi Neetu , Metreveli Naira , Hayden Melvin R , Tyagi Suresh C
Published: June 2021

Sodium thiosulfate (STS) has been shown to be an antioxidant and calcium solubilizer, but the possible cardioprotective role of STS remains unclear. We created heart failure (Arterio‐venous fistula; AVF) in mice and supplemented those animals with STS in drinking water. Groups were: Sham, AVF, Sham + STS and AVF + STS. M‐mode echocardiograms showed ventricular contractile dysfunction with reduced aortic blood flow in AVF mice, whereas STS treatment prevented the decline in cardiac function. Ventricular collagen, MMP‐2, ‐9 and TIMP‐1 were found to increase with a decreasing trend in ACVI expression; however, STS supplement reversed these effects in AVF mice. Among two endogenous H2S producing enzymes, the CSE expression attenuated in AVF mice with no changes in CBS expression. Analyses of ventricular H2S production revealed that AVF mice had less H2S production compared to control; however, the H2S production was normalized with STS supplement. Moreover, cardiac tissues were more responsive to H2S when AVF mice were supplemented with STS compared to AVF alone. All these results suggest that volume overload‐induced cardiac dysfunction and extracellular matrix degradation can be modulated by STS supplement. This possibly rescues failing heart, in part, by increasing ventricular H2S generation.
2‐aminoethoxydiphenyl borate (2‐APB) and gentamicin inhibit thromboxane A2 induced calcium responses in cardiac myocytes
Authors: Kosloski Lisa M. , Gilbert William J. R. , Orr James A. , Moore David S. , Wacker Michael J.
Published: June 2021

Previous work in our laboratory has shown that accelerated idioventricular arrhythmias are induced by the administration of a thromboxane A2 (TxA2) mimetic, U46619 (U4), into the left atrium of the anesthetized rabbit. Preliminary experiments indicate that application of U4 to cultured myocytes increases intracellular calcium. Therefore, we hypothesize that TxA2 may produce arrhythmias by directly stimulating cardiac myocytes and altering intracellular calcium via activation of the IP3 pathway. Ventricular cardiac myocytes from the rabbit heart were isolated and treated with the fluorescent indicator fluo‐4 in 3 series of experiments to examine the effects of U4 and 2 inhibitors of IP3 mediated calcium release, 2‐APB and gentamicin. In series 1, 5 μM U4 was added to cells that had not been pretreated resulting in an average increase in calcium of 33.7 ± 65.7% (n = 24). In series 2, cells that were pretreated with 1.5 μM 2‐APB exhibited an average calcium increase of 8.1 ± 11.7% upon addition of U4 (p = 0.07; n = 15). In series 3, cells that were pretreated with 1.5 μM gentamicin exhibited an average calcium increase of 6.4 ± 8.5% after addition of U4 (p = 0.03; n = 21). In all 3 series, KCl (50 μM) was administered after U4 as a positive control; cells that did not respond to KCl were discarded from further analysis. These data support the hypothesis that TxA2 induces a calcium response in cardiac myocytes that involves the IP3 pathway.
MuRF1 Inhibits JNK Signaling in Cardiac Ischemia Reperfusion injury by Degrading Phosphorylated cJun
Authors: Willis Monte S , Li Hui‐Hua , Rodriguez Jessica E , Li Luge , Rojas Mauricio , Lockyer Pamela , Patterson Cam
Published: June 2021

We recently identified that increased expression of the cardiac Muscle Ring Finger‐1 (MuRF1) is cardio‐protective in ischemia reperfusion (I/R) injury in vivo. To begin to understand the underlying mechanism, we investigated how cardiac MuRF1 regulates JNK signaling, a key regulator of cell survival/death during I/R injury. Hearts from transgenic (Tg) mice over‐expressing cardiac MuRF1 underwent global I/R and were assayed for total and phosphorylated JNK and cJun. Compared to littermate wild type mice, MuRF1 Tg hearts had significant reductions in total and phospho‐cJun (but not JNK) after I/R injury. To determine how MuRF1 specifically interacts with phosphorylated cJun, H9C2 cardiomyocytes were transfected with MuRF1 and cJun expression plasmids and subjected to hypoxia/reoxygenation (H/R). MuRF1 immunoprecipitation pulled down phospho‐cJun specifically, and significant increases in cJun ubiquitination were identified. Phosphorylated cJun levels induced by H/R were blunted when MuRF1 was present, which was reversed by treatment with a proteasome inhibitor (MG132). These studies identify MuRF1 as the first E3 ligase that recognizes phosphorylated cJun, ubiquitinates it, and targets it for degradation by the proteasome. We hypothesize that cardiac MuRF1 over‐expression is cardio‐protective due in part to its ability to inhibit pro‐apoptotic signaling through the JNK pathway.
A Protective effect of Taikong Xieli decoction, a traditional Chinese medicine, on the damage of HPAA in the rat induced by weightlessness and irradiation
Authors: Ming Xie , Lin Zhang
Published: June 2021

The weightlessness and irradiation are the major factors that regulate physiological adaptive responses under the spaceflight environment. The aim of the study is to investigate the effect of Taikong Xieli decoction(TKXLD), a traditional Chinese medicine, on the alteration of HPAA in the rats induced by both weightlessness and irradiation. Wister rats were randomly divided into four groups, each with 12 rats control (C), tail suspended (S), tail suspended and irradiated (SAI) and TKXLD group. Simulated weightlessness and irradiation was induced by treatment with 4.5Gy of 60Co‐gamma rays on 8th day of tail suspended. The TKXLD group were orally administrated at a dose of 7g •kg‐1•d‐1. Rats were killed on the 21st day of tail suspended. The contents of CRH in hypothalamus, ACTH in pituitaria, and CRH, ACTH and CORT in blood were measured. The results showed that the contents of CRH in both hypothalamus and blood were markedly reduced (P<0.01), whereas ACTH in pituitaria ACTH (P<0.05) and COR (P<0.01) in blood significantly enhanced in S and SAI groups as compared with the C group. The concentration of CRH in blood was clearly attenuated (P<0.05), but ACTH in pituitaria had a tendency to increase and ACTH in blood markedly augmented (P<0.01) in SAI group as compared with the S group. The production of CRH in both hypothalamus (P<0.05) and blood (P<0.01) were significantly increased, and ACTH and CORT in blood attenuated (P<0.01) in the TKXLD group as compared with the SAI group. The result demonstrated that the combination of tail suspended and irradiation exaggerated the disorder of HPAA in the rat, mainly manifested by dyspituitarism. It suggests that TKXLD treatment could improve the deleterious effect on the function of HPAA of weightlessness and irradiation during spaceflight.
Feasibility of employing galvanic vestibular stimulation for examining vestibulo‐cardiovascular reflex in human subjects
Authors: Morita Hironobu , Abe Chikara , Tanaka Kunihiko , Awazu Chihiro
Published: June 2021

To examine a role of vestibular system in controlling arterial pressure (AP) in animal studies, AP response to an adequate vestibular input has been compared between intact and vestibular lesioned (VL) animals. However, such invasive method as VL can not be used in human studies. Thus, alternative method for interrupting vestibular system has been required. Galvanic vestibular stimulation (GVS) is known to create an imbalance in vestibular inputs, thus it is possible that continuously applied GVS masks adequate inputs to the vestibular organs and impairs the vestibular‐mediated AP response. In the present study, feasibility of GVS for interrupting vestibular‐mediated AP response was examined in animal studies, and then GVS was applied for human subjects. In conscious rats, the effect of GVS on gravitational change‐induced AP response was qualitatively and quantitatively similar to that caused by VL. However, GVS had no effect on air jet‐induced AP response, suggesting that GVS eliminates vestibular‐mediated AP response to gravitational change like as VL. In human subjects, AP was well maintained upon head up tilt (HUT), however the AP with GVS rapidly decreased at the onset of HUT by 21 ± 3 mmHg. These results indicate that the vestibular system has a significant role in controlling AP upon posture change in humans, and GVS can be applied to human studies for acute interruption of the vestibular‐mediated AP response.
Expression of anabolic factors and extra‐cellular matrix related factors in rat tendon and skeletal muscle in response to different types of muscle contractions
Authors: Heinemeier Katja Maria , Olesen Jens Lykkegaard , Schjerling Peter , Langberg Henning , Kjaer Michael
Published: June 2021

It is not known 1) how loading‐induced collagen I synthesis in tendon and skeletal muscle is mediated and 2) whether the expression of anabolic and matrix related factors in tendon and muscle is associated with contraction type. Rats were subjected to 4 days of unilateral training of either shortening, lengthening or static gastrochnemius muscle contractions. In Achilles tendon tissue, expression of collagen I, transforming growth factor‐β‐1 (TGF‐β‐1), insulin like growth factor‐IE‐a and ‐b (IGF‐IEa and IGF‐IEb) increased substantially, and the response was equal with all types of training. In muscle, similar changes were seen, but the effect of lengthening contractions was significantly greater than that of shortening contractions. Thus, short‐term training induces collagen expression in both tendon and muscle, and TGF‐β‐1, and possibly IGF‐I isoforms, are potential mediators of this response. The superior effect of lengthening contractions in relation to the muscle response may be connected to a greater force production during lengthening contractions, or perhaps to a greater local shear stress in the muscle tissue. The changes seen in tendon were not dependent on training type, which indicates that this tissue is less sensitive than skeletal muscle to differences in force production and/or contraction type.
The influence of aging on the in vivo properties of human patellar tendon
Authors: Dickinson Jared M , Carroll Chad C , Haus Jacob M , Lee Gary A , Hollon Christopher J , Aagaard Per , Magnusson S Peter , Trappe Todd A
Published: June 2021

We investigated the in vivo mechanical properties of the patellar tendon in 37 men and women (11 Young, 27±1 y; 26 Old, 65±1 y). Patella and tibial displacement were monitored with ultrasonography during ramped isometric contractions of the knee extensors and magnetic resonance imaging was used to determine tendon cross‐sectional area (CSA), length, and signal intensity (SI). At maximal levels of force, deformation (15%, p=0.05), stress (23%), and strain (13%) were higher in Young compared to Old (p<0.05). However, aging did not alter the force‐deformation or stress‐strain characteristics of the patellar tendon. Tendon CSA (12%) and SI (24%) decreased with aging (p<0.05) but these differences were isolated to the mid‐region of the tendon. Independent of age, deformation (18%), stiffness (35%), stress (41%), CSA (28%), and length (11%) were greater (p<0.05) in men compared to women. After force normalization, stress (26%) and strain (22%) were higher in women compared to men (p<0.05). Declines in quadriceps muscle function with aging are not a result of changes in patellar tendon mechanical properties. The age‐associated decrease in SI indirectly suggests that the internal milieu of the tendon is altered with aging.

NIH R01 AG020532 (TT), APS Postdoctoral Initiative Award (CC)
Changes in EPC levels and gene expression of homing factors in exercising human skeletal muscle
Authors: Strömberg Anna , Rullman Eric , Jansson Eva , Gustafsson Thomas
Published: June 2021

In the present study the aim was to investigate the circulating level of EPCs defined by expression of VEGFR2 following an acute bout of exercise, as well as plasma levels and skeletal muscle gene expression of factors hypothesized to be involved in homing of EPCs.

10 healthy, moderately active males performed 1 h of cycle exercise. Muscle biopsies from the m. vastus lateralis of both legs were obtained before, directly after and 2 h after the exercise bout. Blood samples for flow cytometric measure of EPC numbers and ELISA measurements of plasma protein levels were retrieved before, directly after, 30 minutes after and 2 h after the exercise bout. The circulating level of EPCs increased robustly after the exercise bout. The plasma level of G‐CSF was significantly increased directly after the bout when adjusted for plasma albumin concentration. ICAM‐1 and VEGFR2 mRNA levels were increased 2 h post exercise. The present results indicate that exercise induces recruitment of EPCs to the circulation and prepares the tissue for homing of these cells.
Peak power is elevated in slow and fast‐twitch muscle fibers from competitive distance runners
Authors: Harber Matthew , Trappe Scott
Published: June 2021

The purpose of this study was to characterize the influence of a long‐term (8‐yr) history of high‐intensity endurance training on the contractile properties of slow‐ and fast‐twitch myofibers. Muscle biopsies were obtained from 8 competitive distance runners (RUN) and 8 recreationally active individuals (REC). Slow‐twitch (MHC I) and fast‐twitch (MHC IIa) myofibers were analyzed for diameter, peak force (Po), contractile velocity (Vo), and power. MHC I and MHC IIa fibers from RUN subjects were ~20% larger (P<0.05) than REC. Peak force of the MHC IIa fibers was 31% higher (P<0.05) in RUN while Po of MHC I fibers was not different between groups. No differences for specific tension (Po/CSA) were present between groups for either fiber type. Vo was higher (P<0.05) in MHC I (+70%) and MHC IIa (+18%) fibers from RUN subjects. Peak absolute power of both fiber types was greater (P<0.05) in RUN. Normalized power of the MHC I fibers was 64% higher in RUN while no differences were noted for MHC IIa fibers. These data suggest that performing long‐duration endurance training modifies myofiber contractile function, resulting in higher peak power production of slow‐ and fast‐twitch fibers. Furthermore, these data suggest that long‐term training may result in adaptations in contractile physiology that are more pronounced than alterations previously observed in short‐term exercise interventions.
Evidence suggesting three respiratory oscillators in frogs (Rana catasbeiana): implications for the Oscillator Homology Hypothesis and the evolution of breathing.
Authors: Duchcherer Maryana , Wilson Richard J. A.
Published: June 2021

Recent data in mammals and frogs suggest at least two separate respiratory oscillators, one who's activity coincides with the powerstroke of lung inflation (the preBötzinger Complex and the lung oscillator, respectively) and a second priming oscillator, who's activity appears to precede the powerstroke (the PreI and buccal oscillators, respectively). According to the Oscillator Homology Hypothesis, the oscillators in mammals evolved from the same ancestors as those in lower vertebrates. However, the priming oscillator in mammals and frogs maybe located in different rhombomeric segments and their rostral‐caudal location relative to the powerstroke oscillator may be reversed. Using the “sheep‐dip” technique, a new method for systematically surveying respiratory rhythm promoting sites in lower vertebrates, we provide evidence suggesting that the lung oscillator in late‐stage metamorphic frogs can, itself, be subdivided into a powerstroke and a novel priming component. As in mammals, the novel lung‐priming component in frogs appears to extend more rostrally than the powerstroke component. These data suggest the Oscillator Homology Hypothesis has to be revised: the mammalian PreI and PreBötzinger Complex oscillators may have evolved from the same two components that gave rise to the frog lung oscillator and not from the buccal oscillator as previously thought. Work funded by NSERC.
Localization of multiple neurotransmitters and neuromodulators in fish gills
Authors: Ciuahndu Cosima S. , Coolidge Emily H. , Milsom William K.
Published: June 2021

Carotid body chromafin cells in mammals contain a plethora of different neurochemicals and several hypotheses exist to explain their roles in O2‐chemosensing. In the present study we assessed the distribution of several of these neurochemicals in the gills of trout and goldfish using immunohistochemistry. In both species, catecholamines and acetylcholine were co‐localized to cells that were distinct from those expressing serotonin. Although, some the catecholamine/acetylcholine cells contain receptors for serotonin. Neuroepithelial cells containing serotonin have been postulated to be the putative O2 sensing cells, but our results, combined with the known effects of acetylcholine and serotonin on gill afferent discharge, lead us to suggest that serotonin participates in O2 chemoreception by modulating the release of acetylcholine as the primary neurotransmitter. (Funded by NSERC of Canada)
Effects of oviposition on jump performance in the American locust (Schistocerca americana)
Authors: Arntzen Kaitlin , Kirkton. Scott D.
Published: June 2021

Life history changes, such as ontogeny or gravidity, can alter energetic demands associated with animal locomotion. By studying how locomotory performance in the American locust (Schistocerca americana) varies with development, we are investigating how physiological systems cope with these fluctuating conditions. In particular, we examined how the jumping muscle can accommodate dramatic body mass changes. During four weeks of adult development, the body mass of older female grasshoppers increased by 95% but the femur mass only increased by 44%. When induced to jump repeatedly, older adult females had reduced endurance but increased muscle‐specific power outputs. However, these data do not control for reproductive state. We found that body mass in reproductive female grasshoppers fluctuated by approximately 20% due to gravidity and oviposition. By comparing repeated jumping performance, we show that age‐matched, unmated females had a greater jump endurance than gravid animals. Current studies are using high‐speed video analysis to compare the maximal jump performance of females before and after oviposition. Our initial results suggest that life history changes, such as gravidity, can significantly reduce locomotory performance. This work was supported by the Union College IEF fund.
In Vivo Neuronal Injury in Painted Turtles Forcibly Submerged at 20°C
Authors: Warren Daniel E. , Robertson Treena , Quindor Rhea , Bickler Philip
Published: June 2021

Painted turtles, Chrysemys picta, are regarded as the most anoxia‐tolerant air‐breathing vertebrates. However, if and when neuronal injury occurs during forced submergence is not known. In the present study, painted turtles were forcibly submerged for 36, 48, or 60 hours at 20°C and, after transcardial paraformaldehyde fixation, the extent of neuronal injury was assessed in 15 μm cerebrocortical sections using the following two histological methods: 1) staining with Fluoro‐Jade C, a marker that specifically labels degenerating neurons and 2) co‐immunolabeling of activated caspase‐3, a protein found in apoptotic cells, with NeuN, a nuclear protein expressed only in adult neurons. After 60 h of submergence at 20°C, plasma lactate was increased to 100 mmol l−1. Fluoro‐Jade C and activated caspase‐3 staining were present in neurons throughout the medial and dorsal cortices, indicating apoptosis‐mediated neuronal injury resulting from long‐term anoxia. Despite this, several animals showed visible signs of life and required anesthesia prior to fixation. All of the animals’ hearts were visibly contracting at the time of sampling. Future studies should assess the extent of neuronal injury during anoxia in the cold, a more ecologically relevant condition, and the possibility that injured regions might undergo repair.
Cerium oxide nanoparticles decrease inflammation in J774 cells
Authors: Hirst Suzanne Marie , Peairs Abby D , Gogal Robert , Seal Sudipta , Reilly Christopher M
Published: June 2021

The use of nanomaterials in biological systems has gained attention due to the unique properties that these materials possess. Cerium oxide (CeO2) is a rare earth oxide that, on the nanomolar scale, has been reported to exhibit antioxidant properties. Cerium oxide acts as a regenerative free radical scavenger due to its valence and oxygen defects. In this study, we investigated the ability of CeO2 nanoparticles to reduce reactive oxygen species (ROS) and inflammatory mediator production in J774 murine macrophage cells in vitro. J774 cells were pretreated with various concentrations of CeO2 nanoparticles 24 hr prior to LPS/IFN‐γ stimulation. Our results showed that J774 cells internalized CeO2 particles as assessed by electron microscopy and atomic force microscopy. The cerium particles localized around the mitochondria, a major producer of ROS in the cell. Treatment with CeO2 at concentrations under 1 uM caused no toxicity. Cerium oxide pretreated cells, when stimulated with LPS/IFN‐γ, showed decreased oxidative stress. Furthermore, CeO2 pretreatment decreased inflammatory cytokines IL‐6 and TNF‐α in LPS/IFN‐γ stimulated cells. Taken together, these studies suggest that cerium oxide nanoparticles are effective in reducing ROS and inflammatory mediator production, and may constitute a novel therapeutic tool for the treatment of inflammation.
Xanthine Oxidase and NADPH Oxidase Induce Cardiovascular Endothelial Dysfunction During Hyperuricemia
Authors: Zhang Suhua , Huang Harer , Serpillon Sabrina , Wang Ziping , Yu Xiaohan , Hintze Thomas H.
Published: June 2021

Hyperuricemia(HU) induces cardiovascular diseases associated with O2− production through the activation of xanthine oxidase(XO). We hypothesized that HU can induce cardiovascular endothelial dysfunction and that both XO and NADPH oxidase are involved. Three groups of SD male rats, control(C), oxonic acid(OA‐ a uricase inhibitor) and OA + allopurinol(OAA) were studied for five weeks. In the OA group, serum uric acid (SUA, mg/dl) was significantly increased (2.5 ± 0.3 vs. 0.8 ± 0.1 in C, 1.9 ± 0.1 in OAA); Systolic blood pressure(SBP, mmHg) was elevated (170 ± 4.4 vs. 144 ± 1.1 in C, 157 ± 2.8 in OAA). There was a linear correlation between SUA and SBP (R2 = 0.82). The total peripheral resistance by echocardiography was increased (P<0.05). The ability of the NO agonists BK and Cch to reduce myocardial O2 consumption (MVO2) was impaired (to 15% for BK or 20% for Cch vs.25% in C) in vitro. However, this was reversed by allopurinol administered in vivo, and restored in the presence of allopurinol, apocynin or tempol in vitro. Western blots of left ventricle showed that the protein p47, nitrotyrosine and P‐p38/p38 ratio were upregulated (P < 0.05, respectively). HU enhanced bioactivity of XO and also NADPH oxidase, by a mechanism involving the activation of p47 protein and p38 kinase. NO is consumed via reaction with O2− and subsequently the ability of NO to control MVO2 and endothelium‐dependent vasorelaxation was attenuated.

Supported by HL‐PO1‐43023 grant.
Can renal blood flow response to a mechanically‐induced step change in blood pressure be derived using a numerical approach?
Authors: Siu Kin Lung , Chon Ki H.
Published: June 2021

Mechanically‐induced step changes in blood pressure (BP) are often used to fully elicit renal autoregulatory mechanisms as well as to characterize their steady‐state responses. However, induction of step changes in BP is not always feasible in experimental conditions. To determine if mathematically‐derived step response from spontaneous BP and renal blood flow (RBF) can provide a good alternative to explicit induction of step changes in BP, experiments were conducted on three isoflurane anesthesized Sprague‐Dawley rats (SDR). A series of spontaneous BP and RBF recordings were directly followed by step changes in BP (−20 mmHg) and the resultant RBF was measured in all rats. Numerical estimates of the step response were achieved by integrating the impulse response functions which were estimated via the Laguerre function expansion technique on the spontaneous recordings of BP and RBF. The preliminary results show that numerical estimates from the spontaneous recordings and the step‐induced BP and RBF results are comparable, with the duration of the myogenic response (MYO) to be 7.00±1.00 vs. 9.33±1.07, respectively, and tubuloglomerular feedback (TGF) response to be 32.67±2.14 vs. 38.33±2.34 seconds, respectively. While these values differ, as they are obtained with and without induction of step changes in BP, they are all within the prescribed durations of the MYO and TGF responses, suggesting that numerically derived step response is a good alternative.
Impaired autoregulation and TGF responses in NKCC1‐deficient mice
Authors: Oppermann Mona , Lorenz John , Wall Susan , Schnermann Jurgen
Published: June 2021

To explore the role of NKCC1 in the regulation of renal hemodynamics we determined tubuloglomerular feedback (TGF) and renal blood flow (RBF) autoregulation in wild type (WT) and NKCC1‐deficient mice.

Tissue‐specific basal renal blood flow (RBF) was similar in NKCC1−/− and WT mice (5.82 ± 0.27 vs. 6.20 ± 0.42 ml/min/g; p=.43), but absolute RBF was lower in NKCC1−/− as a result of a reduced kidney weight (479 vs. 603 mg; p=.003). Maximum TGF responses of stop flow pressure were markedly lower in NKCC1−/ − than WT mice (2.9 ± 0.4 vs. 12.4 ± 0.8 mmHg, p<.001) while blood pressure under anesthesia was identical (90 mm Hg). In vivo perfused loops of Henle (6 nl/min) showed unaltered distal [Cl] and absorption rates in NKCC1−/ − and WT mice (54.4 vs. 56.3 mEq/l; 719 vs. 728 pEq/min). Renal vascular resistance (RVR) of WT responded to a step increase of renal arterial pressure with a fast component (onset at 1 s, duration 6 s) and a slow component (onset at 7 s, duration 10 s) establishing full autoregulation (restoration of 100% of initial RBF) after 17s. In contrast, RVR adjustments in NKCC1−/ − mice were slower in onset and longer in duration reaching completion after 58 s.

In summary, markedly reduced TGF and slower autoregulatory responses in NKCC1−/ − mice suggest that NKCC1‐mediated ion fluxes are important for maintaining the contractility of renal resistance vessels.
Thrombin induces calcium independent phospholipase A2 (iPLA2) activity and neutrophil adherence in human small airways epithelial cells
Authors: Rastogi Prerna , Young Dawn M , McHowat Jane
Published: June 2021

The presence of polymorphonuclear leukocytes (PMN) in the airways is a hallmark of many inflammatory conditions including ARDS, COPD and cystic fibrosis. In the event of a breach in the epithelial or endothelial cell barrier, PMN can migrate from the circulation to the airway lumen. This involves a series of steps including PMN adherence and transmigration through the vascular endothelium into the interstitial tissue, followed by a similar sequence of events used to cross the airway epithelium and enter the lumen. We have determined previously that stimulation of human small airway epithelial cells (HSAEC) with thrombin results in increased iPLA2 activity that is inhibited by the iPLA2 selective inhibitor bromoenol lactone (BEL). Further, activation of iPLA2 results in increased arachidonic acid and prostacyclin release. We also found that PMN adherence to thrombin‐stimulated HSAEC is increased 25 fold over adherence to unstimulated cells. Pretreatment with of HSAEC with BEL or PMN with CV3988, a PAF receptor antagonist, leads to inhibition of thrombin‐stimulated PMN adherence. This indicates that inhibition of iPLA2 or blocking the PAF receptor may be effective tools in limiting the cascade of neutrophil adherence, inflammatory mediator production and tissue destruction in cases of acute lung inflammation.
Impact of oxidative stress on epithelial repair and alveolar oedema, within in vitro model of lung injury
Authors: Chupin Cécile , Privé Anik , Brochiero Emmanuelle , Berthiaume Yves
Published: June 2021

Repair of lung injury is a complex process including epithelial growth, alveolar clearance and down‐regulation of inflammatory response. The impacts of oxidative stress in this process are still unknown. To study this question, we developed an in vitro model of lung injury to characterise the molecular and cellular mechanisms of distal lung epithelial repair. Alveolar epithelial cells in primary culture were submitted to oxidative stress with bleomycin (B : 12,5–150mU/ml) or DMNQ (D : 2,5–15μM). Bleomycin and DMNQ inhibit the epithelial repair (24–48h) following a mechanical injury in a time and concentration dependent manner (p<0,0001). These agents also decrease by 60% and 80% respectively the cell migration in Boyden's chamber (p<0,0001) and decrease cell proliferation measured as thymidine 3H incorporation by 95% and 70% (p<0,0001). These results show that oxidative stress has an impact on alveolar epithelial cell repair by inhibiting the cell migration and proliferation processes. The impact of bleomycin on the ionic transport was studied in Ussing chamber. The treatment decreases by 37% the total as well as amiloride‐sensitive transepithelial current (p<0,05). Basolateral permeabilisation in presence of a Na gradient shows that the epithelial Na channel (ENaC) is not involved in this decrease. The oxidative stress therefore in addition to inhibiting epithelial repair also decreases the Na transport involved in lung liquid clearance.

Supported by RSR‐FRSQ, CCFF, CIHR.
Unilateral doubling of the radial artery in the antebrachium observed in the cadaver
Authors: Norton Neil S. , Jergenson Margaret A. , Barritt Laura C.
Published: June 2021

The brachial artery branches into radial and ulnar arteries in the cubital fossa. Within the fossa, the radial artery descends distally towards the wrist. At the level of the wrist, the radial lies lateral to the tendon of the flexor carpi radialis and continues laterally to enter the anatomical snuff box prior to passing through the first dosal interosseous muscle. The path of the radial artery in the antebrachium is superficial except for the proximal portion, which is slightly overlapped by the brachioradialis. Proximal branching of the brachial artery into the radial and ulnar arteries at mid brachium are common anomalies. We report a unilateral variation in the radial vasculature observed in a female cadaver. A superficial radial artery branched from the brachial artery at mid brachium. Initially, this was thought to be a proximal bifurcation of the brachial into the radial and ulnar vessels. Further dissection revealed a normal bifurcation of the brachial into the radial and ulnar arteries occurred in the cubital fossa. The radial artery from the bifurcation was unusually small. The superficial radial paralleled the normal radial distally along the antebrachium. The normal radial artery was very small in the wrist and the superficial radial continued to enter the anatomical snuff box. There was no other variation associated with either radial artery beyond this point.
Crooked crania and lop‐sided larynges: New insights from CT imaging of the cranio‐cervical region in odontocetes (toothed whales, dolphins, porpoises)
Authors: Reidenberg Joy S. , Arruda Julie , Cramer Scott , Ketten Darlene R. , Laitman Jeffrey T.
Published: June 2021

Cranio‐hyolaryngeal relationships are important in regulating respiration, vocalization, deglutition, and regurgitation/reflux. In marine mammals, this area has critical adaptations for these functions underwater. This study explores the in situ configuration of the cranio‐cervical region through CT imaging of 45 specimens from 9 genera of odontocetes (toothed whales, dolphins, porpoises). Analyses of shape of the skull and position of hyoid and larynx show extent of cranial and hyolaryngeal asymmetry is related. Asymmetry is predominately left directed (most pronounced in sperm and beaked whales) and marked by an enlarged left nasal aperture and tilted hyoid. The right side of the hyoid is depressed ventrally, resulting in a larger right piriform sinus. Deviations from typical terrestrial mammal symmetry may be linked to aquatic constraints. Pharyngeal asymmetry may facilitate swallowing larger prey. Lateral displacements of respiratory and digestive tracts assure their complete separation, thus protecting the respiratory tract from fluid entry during feeding. Asymmetry of larynx and pharyngeal spaces allows simultaneous airflow and swallowing, consistent with generating echolocation or communication sounds in tandem with open‐mouthed prey capture.
CD40 ligand reversal of dexamethasone‐induced osteoblast apoptosis as measured by a colorimetric caspase‐3 assay
Authors: Jones Daniel R. , Jones Amber L. , Bainbridge Carolyn R. , Hoffman Jolie A.
Published: June 2021

Long‐term glucocorticoid therapy is one of the leading causes of osteoporosis. Glucocorticoid‐induced osteoporosis (GIOP) is investigated by treating osteoblasts with dexamethasone, a synthetic glucocorticoid known to produce subsequent apoptosis in the MC3T3‐E1 bone cell model. It is also known that dexamethasone‐induced apoptosis in MC3T3‐E1 cells can be significantly reversed in the presence of CD40 ligand. The mechanisms of this reversal to apoptosis are presently unknown. In order to determine if activated caspase‐3, a key mediator of apoptosis, was affected by CD40 ligand, MC3T3‐E1 cells were assayed for caspase‐3 activity following treatment with either 10−6M dexamethasone or 10−6M dexamethasone subsequent to pre‐treatment with 1.25 micrograms/ml CD40 ligand. Caspase‐3 activity determinations in cell extracts were made by measurement of absorbance at 405 nm following cleavage of the colorimetric Ac‐DEVD‐pNA substrate. Results indicate a 2‐fold induction of caspase‐3 activity on dexamethasone treatment (p<0.05) with CD40 ligand reducing that activation by 50%. These findings validate our in vitro model for GIOP and allow us to further investigate the mechanism of CD40 ligand's protective role. This research was funded through an Indiana Academy of Science senior research grant and an Indiana Wesleyan University‐administered Lilly Faculty Scholarship Award.
The IRES‐Mediated Translation of Cytoplasmic Serine Hydroxymethyltransferase (cSHMT) is Stimulated by UV‐Induced DNA Damage
Authors: Fox Jennifer , Stover Patrick
Published: June 2021

Cytoplasmic serine hydroxymethyltransferase (cSHMT) is a key regulator of folate‐mediated one‐carbon metabolism. By preferentially directing one‐carbon units to thymidylate synthase (TS), cSHMT gives purine synthesis priority over methionine synthesis, resulting in elevated levels of thymidylate for DNA replication and repair. The 5′ untranslated region (UTR) of the cSHMT transcript contains an internal ribosome entry site (IRES) that regulates cSHMT expression through interactions with the IRES trans‐acting factors BrunoL2 and heavy chain ferritin (HCF). In this study, we investigate the physiological function of the cSHMT IRES. We show that cSHMT IRES activity is stimulated in response to a non‐lethal dose of UV irradiation, resulting in a four‐fold increase in cSHMT protein levels despite a global inhibition of cap‐dependent translation. Furthermore, we show that the mechanism of IRES stimulation involves the UV‐induced activation of HCF transcription and the relocalization of BrunoL2 to the cytoplasm. This response appears to be specific to DNA damage‐induced cellular stress, as nocodazole‐induced cell cycle arrest does not produce the same results. The UV‐induced increase in cSHMT expression is accompanied by an increase in the nuclear localization of cSHMT and TS, suggesting that cSHMT IRES activity functions to provide thymidylate for the repair of DNA lesions arising from UV irradiation.
Identification and Characterization of microRNAs modulated by Oxidative Stress
Authors: Wei Huei‐Tsun , Chen Hua‐Chien , Chen Shu‐Jen , Chiu Daniel Tsun‐Yee
Published: June 2021

Reactive oxygen species (ROS) play important roles in regulating cellular functions. Some growth factors, such as PDGF and EGF, have been discovered to signal through ROS and activate the MAP kinase cascades to regulate the expression of transcription factors. Several laboratories have found that ROS could directly regulate the binding of transcription factors, like AP‐1, NF‐κB, P21 and P53, to DNA and thus influence the downstream gene expression. MicroRNAs (miRNAs) are a class of regulatory RNAs of approximately 21∼25 nucleotides that posttranscriptionally regulate gene expression by directing mRNA cleavage or translational inhibition. The relationship between oxidative stress and miRNAs has not been clearly defined. Toward this end, we used colo205 as a cell model to explore the effect of oxidative stress on miRNA expression. Cells were exposed to 200 μM H2O2 for 1hr and the expression levels of 225 human miRNAs were determined by quantitative RT‐PCR. H2O2 treatment caused a 5‐fold increase in the level of has‐miR‐145. Similar increase was detected in the level of primary transcript of miR‐145 (pri‐miR‐145). These results indicate that the transcription level of miR‐145 is stimulated by ROS in Colo205. The biological significance of miR‐145 up‐regulation is currently under investigation.
Isolation and Characterization of a Novel Phospholipase A1 Secreted from Human Platelets
Authors: Bolen Alyssa Lynn , Niessen Sherry , Cravatt Benjamin , Beranova‐Giorgianni Sarka , Tigyi Gabor
Published: June 2021

Lysophosphatidic acid (LPA) is a phospholipid mediator that plays a role in human diseases including thrombosis, arteriosclerosis, angiogenesis, cancer metastasis, and inflammation. LPA is present in blood at low nanomolar concentrations, whereas in serum it reaches several micromolar in concentration. The mechanisms responsible for LPA production during blood coagulation remain unknown. Because in mammalian phospholipids linoleoleic acid and arachidonic acid are esterified to the sn‐2 position of the glycerol backbone and 83% of LPA in serum carries these fatty acids, we hypothesized that a phospholipase A1 (PLA1) is involved in its production. We have purified a novel PLA1 from the microparticle‐free supernatant of thrombin‐activated human platelets that is distinct from known PLA1s and PLA2s. We have performed sequential chromatographic steps using Q‐Sepharose, Sephadex PD‐10, Butyl‐Sepharose, and MonoS columns and obtained a 150‐fold purified enzyme. Phospholipases are typically serine hydrolases and rhodamine‐ or biotin‐labeled flurorophosphonate probes abolished the PLA1 activity. These affinity labels will be used for mass spectrometric identification of the labeled proteins. Because PLA1 plays an important role in LPA production, further investigation of this enzyme may prove it to be an excellent therapeutic target for the inhibition of LPA production in disease.
Characterization of TrpE and PabB from Mycobacterium tuberculosis
Authors: Li Huei‐Jiun , Tonge Peter J.
Published: June 2021

While E. coli uses ubiquinone and menaquinone as electron shuttles in its aerobic and anaerobic respiration processes, respectively, Mycobacterium tuberculosis (Mtb) has menaquinone as its sole quinone. Since latent Mtb must respire to remain viable, inhibitors of menaquinone biosynthesis could potentially be active against both replicating and non‐replicating populations of bacteria. The initial step from chorismate to isochorismate in the pathway is carried out by the isochorismate synthase, MenF, in E. coli. However, there is no annotated menF gene in Mtb genome. We expressed Mtb MbtI, EntC, TrpE and PabB, the four enzymes with the highest similarity to E. coli MenF, in search of the isochorismate synthase that is utilized in Mtb for menaquinone biosynthesis. This report will focus on characterization of TrpE and PabB from Mtb. TrpE displays typical anthranilate synthase activities, including anthranilate production, Mg2+ and NH4+ dependence, and feedback inhibition by tryptophan. Unexpectedly, prephenate formation is also observed. PabB catalyzes a two‐step reaction which is consistent with an aminodeoxychorismate synthase activity. Thus the possibilities of TrpE or PabB being the E. coli MenF counterpart in Mtb can almost be ruled out. The study nevertheless adds new evidence to the catalytic specificity (or promiscuity) of these chorismate‐utilizing enzymes.
FRET Libraries for Protease Specificity Determination
Authors: Selby Thomas Lee
Published: June 2021

Specificity determination is an important step in characterizing novel proteases. Given the large number of proteases that are known to exist from genomic sequencing efforts, reasons that sensitive, reliable, and high‐throughput methods to determine protease specificity must be developed. This study describes the construction and initial characterization of a protein based FRET library to rapidly determine protease specificity using GFP and DsRed. A randomized DNA linker region was inserted between these donor‐acceptor FRET proteins to produce a library of protease substrates with varied amino acid sequences. Kinetic assays were then performed by monitoring the increase in GFP fluorescence as a function of time and substrate concentration to determine reaction velocities and specificity for a set of proteases. Our results demonstrate the ability of the proteases to discriminate between different linker regions as well as the resistance of GFP and DsRed to proteolysis. Screening methods utilizing color development of individual E. coli colonies and restriction enzyme digests, were used to eliminate those DNA samples in the library that contained stop (or rare) codons and/or deletions and a flow plan for efficient use of the library in high throughput and single molecule screening is also presented.
Aberrant signaling responses of cells lacking syndecan1 suggest differential metabolic regulation may underlie tumor resistance of syndecan1‐null mice
Authors: Wagner Caroline Ann , Alexander Caroline
Published: June 2021

Caloric restriction (CR) has been shown to inhibit tumorigenesis in a variety of animal models. CR suppresses the action of chemical carcinogens and reduces tumor formation in transgenic mouse models. Several mechanisms thought to underlie the effects of CR have been described. Levels of IGF‐1 are reduced in animals under caloric restriction, and animals show increased insulin sensitivity. CR reduces cell proliferation, enhances apoptosis, upregulates activation of AMPK (a low energy sensor) and downregulates mTOR signaling (a nutrient responsive pathway). Syndecan1 (Sdc1), a heparan sulfate proteoglycan, has been shown to be important for tumor formation. Mice lacking Sdc1 are protected from tumorigenesis in response to a chemical carcinogen and ectopic Wnt‐1 expression. To elucidate the mechanism of tumor resistance we study mammary epithelial cells (MECs). Sdc1‐null MECs in vitro show decreased population expansion in response to Wnt3A and insulin compared to wild‐type populations. Analysis of cell kinetics demonstrates that Sdc1‐null populations undergo reduced proliferation and increased apoptosis. Western blot analysis shows increased activation of AMPK in Sdc1‐null MECs. The characteristics we observe in Sdc1‐null mice and tissues suggest that metabolic alterations may be underlying the tumor resistance of Sdc1‐null mice. This research is supported by an RO1 grant and a DOD Era of Hope grant.