bluuebunny/crossref_metadata_2025 huggingface dataset:
TRPA1 is part of muscle reflex
Authors: Koba Satoshi , Hayes Shawn G. , Sinoway Lawrence I.
Published: June 2021

This study was undertaken to elucidate the role played by transient receptor potential A1 channels (TRPA1) in evoking the muscle reflex, a sympathoexcitatory drive originating from contracting muscle. In the first set of experiments, we tested if stimulation of TRPA1 on muscle afferents reflexly increases sympathetic nerve activity (SNA). In 13 decerebrate rats, allyl isothiocyanat (AITC, 50 µg·kg−1), a TRPA1 agonist, was injected into the hindlimb muscle circulation, and led to a 46% increase in renal SNA (RSNA). The response was prevented by intra‐arterial injection of HC‐030031 (3 mg), a TRPA1 selective blocker (n=8). The effect of AITC was a reflex because the response was prevented by section of the sciatic nerve (n=5). In the second set of experiments, we tested if blockade of TRPA1 reduces RSNA response to contraction. In 8 decerebrate rats, 30 s static contraction of hindlimb muscle, induced by electrical stimulation of the cut ends of L4 and L5 ventral roots, increased RSNA and blood pressure. The increase in RSNA seen during the late period of contraction (10‐30 s after onset of contraction) was reduced by HC‐030031 injected intra‐arterially (32±5 vs. 14±3 arbitrary units; before vs. after HC‐030031; P<0.05). Taken together, we suggest that TRPA1 located on muscle afferents is part of the muscle reflex. Physiological stimulants/mediators of TRPA1 need to be explored. Supported by AHA BGIA 0865416D (SK).
Non ‐ adrenergic receptor mediated tonic vasoconstriction in skeletal muscle does not change with age
Authors: DeLorey Darren S , Buckwalter John B , Mittelstadt Scott W , Kluess Heidi A , Clifford Philip S
Published: June 2021

Sympathetic co‐transmitter release may increase with aging. Thus, non‐adrenergic receptor mediated vasoconstriction may assume a greater role in the control of skeletal muscle vascular tone with aging. The purpose of this study was to investigate the effect of age on neuropeptide Y ‐ Y1 (NPY) and purinergic (P2X) receptor mediated restraint of skeletal muscle blood flow. Young (n = 6; 22 ± 3 months) and old (n = 7; 119 ± 22 months) beagles were studied. Selective NPY (BIBP‐3226; 2.5 mg bolus) and P2X receptor (PPADS; 40 mg bolus) antagonists were infused at rest, and during treadmill running at 2.5 miles/h and 4 miles/h with 2.5% grade. Hindlimb blood flow (HBF) and mean arterial pressure (MAP) were monitored continuously and vascular conductance (VC) was calculated. Infusion of BIBP‐3226 resulted in similar increases in VC in young and old beagles at rest (Young 137 ± 64 % ; Old:, 96 ± 54 % ) and at 2.5 miles/h (Young 56 ± 39 % ; Old:, 52 ± 24 %) and 4 miles/h with 2.5% grade (Young: 45 ± 24 %; Old: 39 ± 35 %). PPADS infusion also produced similar increases in VC in young and old beagles at rest (Young: 88 ± 35 %; Old: 164 ± 143 %) and at 2.5 miles/h (Young: 47 ± 43 %; Old: 31 ± 27 %) and 4 miles/h with 2.5% grade (Young: 26 ± 33 %; Old: ‐13 ± 21 %). These results demonstrate that NPY and P2X receptor mediated restraint of skeletal muscle blood flow does not increase in older compared to young beagles at rest or during dynamic exercise.
CLIC5 is required for appropriate thermogenesis during fasting
Authors: Bradford Emily , Shull Gary
Published: June 2021

CLIC5 (Chloride Intracellular Channel 5) is an intracellular protein that has been shown to associate with the actin cytoskeleton of placental microvilli and plays a role in the maintenance of hair cell stereocilia in the inner ear. Mice that are homozygous for the mutant allele of Clic5, designated jbg, exhibit hyperactivity and circling behavior. In these studies we show that the jbg/jbg mice have an impaired ability to regulate appropriate thermogenesis during fasting. Although their basal core body temperatures and surface temperatures are not different from wild type mice, during prolonged food restriction the mutant mice become severely hypothermic. Furthermore, the mutant mice lose a greater percentage of their body weight, and tend to exhibit vasodilation and piloerection during fasting. Although their stomach morphology and gastric acid secretion are normal under basal conditions, the jbg/jbg mice also experience gastric hemorrhaging, the rate and severity of which increase with the duration of fasting. These hemorrhages are completely reversible when food is replaced. These data suggest a role for CLIC5 in the maintenance of body temperature during fasting, either by directly influencing non‐shivering thermogenesis or by affecting energy metabolism.
Effect of whole‐body heat stress on peripheral vasoconstriction during engagement of the venoarteriolar response
Authors: DEL COSO JUAN , Brothers R. Matthew , Wingo Jonathan E. , Crandall Craig G.
Published: June 2021

The aim of this study was to test the hypothesis that whole‐body heat stress attenuates venoarteriolar response (VAR) mediated vasoconstriction of the cutaneous and whole‐leg circulations. During normothermia (NT), cutaneous blood flow (CBF) and femoral artery blood flow (FBF) were obtained during supine and leg dependent conditions. These measurements were repeated following whole‐body passive heat stress (WBH) that increased internal temperature by 1.4±0.2 °C. Cutaneous (CVC) and femoral vascular conductances (FVC) were calculated by dividing CBF and FBF by mean arterial pressure. During leg dependency, the absolute decrease in CVC (NT: 0.22 ± 0.1; WBH: 0.19 ± 0.3 arbitrary units·mmHg−1; P=0.97) and FVC (NT: 1.90 ± 1.7; WBH: 0.70 ± 2.1 ml·min−1·mmHg−1; P=0.71) were similar between thermal conditions. However, since WBH substantially increased CVC and FVC (P<0.001 for both variables), the percent reduction in CVC (NT: 58 ± 9%; WBH: 9.4 ± 9.1%, P<0.001) and FVC (NT: 33 ± 21%; WBH: 6.0 ± 17%; P=0.02) during leg dependency were attenuated during WBH relative to NT. These data suggest that heat stress reduces the vasoconstrictive response in the cutaneous and whole leg circulations during engagement of the VAR via leg dependency. These attenuated responses may contribute to reduced blood pressure control in hyperthermic subjects leading to impaired orthostatic tolerance.
The role of kisspeptin and GPR54 in the hippocampus
Authors: Arai Amy C. , Orwig Nathane , Xia Yan‐Fang
Published: June 2021

The granule cells of the dentate gyrus form the input stage of the hippocampal trisynaptic circuit and their function is strongly influenced by peptidergic systems. GPR54 is highly and discretely expressed in these cells. We have found that activation of GPR54 with kisspeptin‐10 causes a rapid increase in the amplitude of excitatory synaptic responses in granule cells, without changing membrane properties. The effect was suppressed by a G‐protein inhibitor and a calcium chelator, and analysis of miniature EPSCs revealed an increase in mean amplitude but not event frequency, indicating that GPR54 and the mechanisms for enhancing EPSCs are postsynaptic, possibly involving changes in AMPA receptor number or conductance. The synaptic potentiation was abolished by inhibitors of ERK1/2, tyrosine kinase, and CaMKII. RT‐PCR experiments showed that KiSS‐1 is expressed in the dentate gyrus. KiSS‐1 mRNA was increased by seizure activity in rats and when neuronal activity in organotypic hippocampal slice cultures was enhanced by kainate or picrotoxin, while mRNA for GPR54 remained essentially unchanged. These results suggest that kisspeptin may be locally synthesized and act as an autocrine. Hippocampal KiSS‐1 mRNA in male rats was increased after gonadectomy. In summary, kisspeptin is a novel endogenous factor which is dynamically regulated by neuronal activity and which, in marked distinction from other neuropeptides, increases synaptic transmission in dentate granule cells through signaling cascades possibly linked to the MAP kinase system. This novel peptide system may play a role in cognition and in the pathogenesis of epilepsy. Supported by Whitehall Foundation.
Neural cells and acetylcholine inhibit secretion of glial cell line‐derived neurotrophic factor by skeletal muscle cells in culture
Authors: Vianney John‐Mary , Spitsbergen John M.
Published: June 2021

The long term aim of this study is to understand the regulation of glial cell line‐derived neurotrophic factor (GDNF) at the neuromuscular junction (NMJ). GDNF has been identified as a potent survival factor for both central and peripheral neurons. Although the effects of exogenous administration of GDNF at the NMJ have been extensively studied, little is known about factors that regulate intracellular GDNF expression and secretion. In this study a coculture system of the C2C12 skeletal muscle cell line and the NG108‐15 neural cell line was used. GDNF protein within cells and in conditioned cell culture medium was analyzed by Enzyme‐Linked Immunosorbant assay. GDNF localization was examined by immonohistochemical analysis. The results showed that addition of acetylcholine (ACh) to cultured skeletal muscle cells inhibited GDNF secretion by skeletal muscle. The nerve‐muscle coculture study indicated that the addition of motoneurons to skeletal muscle cells also reduced the secretion of GDNF by skeletal muscle. The results suggest that motoneurons may be regulating their own supply of GDNF being produced by skeletal muscle, possibly via ACh release. This work was supported by NIH grant 1 R15 AG022908‐01A2, Western Michigan University, and MSU‐KCMS.
Repetitive acute intermittent hypoxia increases neurotrophic and growth factor expression in non‐respiratory motor neurons
Authors: Satriotomo Irawan , Dahlberg Jenny M , Dale‐Nagle Erica A , Mitchell Gordon S
Published: June 2021

Repetitive acute intermittent hypoxia (AIH) elicits respiratory motor plasticity and increases growth/trophic factor expression in respiratory motor neurons (Satriotomo et al., 2007; Dale et al., 2007). Here, we tested the hypothesis that repetitive AIH similarly affects neurons involved in non‐respiratory motor behaviors. Growth/trophic factor protein levels were examined via immunofluorescence in sections from perfused rats exposed to thrice weekly AIH (3xwAIH; 10 episodes,10.5% O2, 5 min duration, 5 min interval, 3x per week, 10 weeks). Upper motor‐neurons of primary and secondary motor cortex (M1‐M2) and alpha motor neurons in the cervical (C7) and lumbar (L3‐4) ventral horns were investigated. Compared to sham rats, neurons in all three areas exhibited increased expression of: 1) brain derived neurotrophic factor (BDNF) and its high affinity receptor (TrkB); 2) vascular endothelial growth factor (VEGF) and VEGF receptor‐2; and 3) an important VEGF gene regulator, hypoxia‐inducible factor‐1α. Thus, 3xwAIH regulates endogenous levels of important growth/trophic factors critical for motor neuron survival and plasticity, even in regions of the CNS associated with somatic (versus respiratory) motor control. 3xwAIH may be a useful tool in the treatment of disorders leading to partial paralysis, including spinal injury and motor neuron disease.

(NIH HL080209 and NS057778).
Inhibition of O‐GlcNAcase in perfused rat hearts by NAG‐thiazolines at the time of reperfusion is cardioprotective in an O‐GlcNAc dependent manner
Authors: Laczy Boglarka , Marsh Sue A , Brocks Charlye A , Marchase Richard B , Chatham John C
Published: June 2021

Increased O‐linked‐N‐acetylglucosamine (O‐GlcNAc) levels of cardiac proteins afford protection against ischemia‐reperfusion (I/R) injury. One strategy to increase cellular O‐GlcNAc levels is inhibition of O‐GlcNAcase, which catalyzes O‐GlcNAc removal. Here we tested the cardioprotective efficacy of two novel and highly selective O‐GlcNAcase inhibitors, NAG‐Bt (Bt) and NAG‐Ae (Ae). Isolated perfused rat hearts were subjected to 20 min no‐flow ischemia followed by 60 min reperfusion. At the time of reperfusion hearts were assigned to 4 groups 1) untreated control (CON); 2) 50µM Bt; 3) 100µM Bt or 50µM Ae. All treatment groups significantly increased O‐GlcNAc levels and improved functional recovery compared to CON group. O‐GlcNAc increased in a dose dependent manner with Bt and was highest in Ae group. There was a strong correlation between O‐GlcNAc, improved functional recovery and reduced troponin I release. Immunohistochemistry of non‐ischemic hearts showed higher O‐GlcNAc intensity at the Z‐lines and intense nuclear staining. There was a marked loss of nuclear O‐GlcNAc and disruption of the striated Z‐line structures following I/R. Bt treated hearts exhibited less structural disruption and higher overall O‐GlcNAc. These data further support the notion that increasing cardiac O‐GlcNAc by inhibition of O‐GlcNAcase may be a clinically relevant approach for ischemic cardioprotection. (NIH HL079364)
Ranolazine, a late sodium current inhibitor, reduces ischemia‐induced superoxide emission and improves functional recovery in guinea pig isolated hearts
Authors: Aldakkak Mohammed , Heisner James S , Camara Amadou KS , Haumann Johan , Stowe David F
Published: June 2021

Ranolazine (RAN) is believed to block a late Na+ (NaL) current that may be activated by reactive oxygen species (ROS) via peroxidation in cardiac cells during ischemia. Na+ loading contributes to cell Ca2+ loading via Na+/Ca2+ exchange (NCE) and cell damage on reperfusion. We tested if RAN treatment during ischemia improves cardiac function on reperfusion and reduces superoxide (O2 ?−) emission. Left ventricular pressure (LVP) and coronary flow (CF) were measured in guinea pig isolated hearts perfused with Krebs‐Ringer's solution; O2 ?− emission was assessed in the LV wall, on‐line, by fluorescence spectrophotometry using DHE. RAN (5 µM), infused for 1 min before 30 min global ischemia, itself decreased heart rate by 20% and LVP by 30% and increased CF by 20%. During late ischemia O2▸− emission increased less with RAN (20% vs. 40% control). At 15 min reperfusion O2▸− emission was reduced 20% by RAN vs. control. At 120 min reperfusion the RAN group (vs. control) exhibited higher LVP (68% vs. 55%) and CF (72% vs. 58%), and reduced incidence of V fibrillation (16% vs. 100%). Ischemia and consequent ROS release are believed to induce a persistent NaL current so blocking this current may reduce Ca2+ loading via NCE. These studies suggest a link between ROS‐induced Na+ loading via NaL and LV function during reperfusion. The decrease in O2▸− emission with RAN during ischemia is likely secondary to improved mitochondrial bioenergetics.
Which preconditioning‐associated G protein‐coupled receptors are expressed on the sarcolemma?
Authors: Xin Wenkuan , Cohen Michael V. , Rich Thomas C. , Downey James M.
Published: June 2021

It has been assumed that G protein‐coupled receptors associated with preconditioning reside on the sarcolemma. We tested that assumption by infecting rabbit ventricular myocytes with adenovirus encoding cyclic nucleotide‐gated channels. Increasing cAMP opens the channel and increases ionic currents measured using whole cell patch clamp. Peak current was determined by exposure of cells to a phosphodiesterase inhibitor cocktail (PDEI) plus 50nM isoproterenol (ISO) at end of experiments. Current after 50nM ISO binding to Gs‐coupled β‐adrenergic receptors was 56±9% of peak. 500nM Bradykinin or 20nM δ‐opioid agonist DADLE reduced ISO current to 19±5 and 11±3% of peak, respectively, indicating activation of these Gi‐coupled receptors. 200nM CCPA and 200nM IB‐MECA, adenosine A1 and A3 agonists, respectively, also attenuated responses to ISO with maximal currents of 25±11% and 24±5% of peak. A2 subtypes are normally Gs‐coupled. Neither 1μM BAY60‐6583, A2b agonist, nor 300nM CGS21680, A2a agonist, increased current themselves, but CGS21680 could after PDEI indicating a low level of A2a. Neither attenuated ISO's current. We conclude that all four Gi‐coupled receptors and the Gs‐coupled A2a are present but not A2b. If there really is a role for the A2b in preconditioning's protection, then the signaling must involve a different cell type in the heart such as vascular smooth muscle. HL‐20648, HL‐74278
Comparison of a rat and rabbit hemorrhagic shock model in response to administration of supplemental oxygen following hemorrhage
Authors: Gunther Robert A. , Talken Linda , Davis Jessica
Published: June 2021

Previous studies have shown that giving oxygen (O2) post hemorrhage results in an increased mean arterial pressure (MAP) in the anesthetized rat. This study was done to determine if in the larger anesthetized rabbit model the same effect would be observed with supplemental oxygen. Changes in MAP, cardiac output (CO), and heart rate (HR) were observed.

Methods

Six rats and five rabbits were studied. Rats (443±10 gm) were anesthetized with 1.5 to 2.0% isoflurane and allowed to spontaneously breath. Initial the inspired O2 content was the same for each group, air at 21% O2. Rabbits (3.8 ± .4 kg) were similarly anesthetized and intubated. Post hemorrhage (PH), both groups were switched to100% oxygen.

Results

Data are mean ± SD. In the rat baseline MAP was 76.6 ± 5.2 mmHg, HR 335 ± 30 bpm, CO 154 ± 22 ml/min and in rabbit MAP was 74.8 ± 17, HR 257 ± 42 bpm, CO 841 ± 239 ml/min. At PH in rat MAP was 54.5 ± 9 mmHg, HR was 302 ± 24 bpm, CO 114 ± 29 ml/min, and in rabbit MAP was 32 ± 5 mmHg, HR 277 ± 39 bpm, CO 361 ± 281 ml/min. With oxygen in rat MAP was 85.7 ± 10 mmHg, HR 308 ± 23, CO 110 ± 23 ml/min, and in rabbit MAP was 29 ± 7 mmHg, HR 290 ± 32, CO 303 ± 112 ml/min.

Conclusion

Administering O2 following hemorrhage significantly (P = 0.002) increased MAP in the rat hemorrhage model but did not increase MAP in the rabbit model. This data suggest that oxygen may have a unique role in regulating arterial pressure in the hypovolemic rat compared to the rabbit.
Microarray Analysis of Embryonic Kidneys Following Inactivation of GATA3
Authors: Reese James , Mauch Teri Jo , Paul William , Nelson Raoul , Miller R Lance
Published: June 2021

Mutations of GATA‐3 lead to Hypoparathyroidism, Sensioneural Deafness, and Renal Hypoplasia Syndrome (HDR). The mechanism by which GATA‐3 causes developmental abnormalities remains poorly understood because murine knockouts are embryonic lethal. To overcome this limitation we inactivated GATA‐3 using the floxed exon‐4 GATA‐3 mouse and the temporally regulated Cre‐recombinase CreERT2 transgenic mouse. GATA‐3 was inactivated in embryos by administering a single dose of tamoxifen to the pregnant dams at E11.5 and removed at E14.5. Inactivation of GATA‐3 resulted in smaller kidneys with a 35% reduction in terminal ureteric bud tips; cell proliferation and apoptosis were not significantly different. To better understand the role of GATA3 in renal development cDNA microarray analysis was performed; results show that expression of 191 genes was altered by 2‐fold or more in embryonic kidneys expressing the mutant GATA3 compared to control, with an average fold change of 2.24 ± 0.34. Additional analysis shows that the majority of altered genes in the ureteric bud tip (UB) were down regulated (2.7:1), while the majority of genes in the UB trunk and metanephric mesenchyme were up regulated (18:1 and 2.6:1, respectively). This project discusses the altered gene expression pattern and the novel signaling pathways.
Multiple Blood Pressure loci on rat Chromosome 18 attenuate the development of hypertension in the Dahl S Hypertensive rat
Authors: Moreno Carol , Rogge Roberta , Tschannen Michael , Barreto Nadia , lazar Jozef , Jacob Howard J.
Published: June 2021

The aim of the present study was to identify the region within Chr 18 involved in the development of salt‐induced hypertension in the Dahl salt‐sensitive (SS) rat. Previous linkage and chromosomal substitution studies suggest the presence of a locus for salt‐sensitive hypertension in chr 18. Consomic SS‐18BN rats were backcrossed to the SS rat, and the offspring intercrossed to generate eight overlapping congenic strains covering the entire length of chr 18. Blood pressure and protein excretion were measured by telemetry in male rats during low salt and three weeks of high salt diet (8% NaCl) in parental (SS and SS‐18BN) and congenic strains. Mean arterial pressure (MAP) did not differ significantly between any of the strains studied during low salt diet. After three weeks of high salt, SS rats developed hypertension (MAP 1734 mmHg), while in the SS‐18BN consomics the development of hypertension was significantly attenuated (MAP 1485.5 mmHg). Characterization of the overlapping congenic strains suggests that there are at least five regions in chr 18 that are involved in the response of blodd pressure to a high salt diet in the SS rats.
Differential effect of two haplotypes of the human angiotensinogen gene on blood pressure in transgenic mice
Authors: kumar ashok ‐
Published: June 2021

Angiotensinogen (AGT) gene locus is associated with human essential hypertension and over‐expression of the AGT gene increases blood pressure in transgenic mice. Human AGT gene has A/G polymorphic sites at ‐6 and ‐217 positions in its promoter. The frequency of AA haplotype of the human AGT gene containing ‐6A:‐217A is significantly increased in African‐American hypertensive patients as compared to the AG haplotype‐6A:‐217G. Reporter constructs containing AA haplotype of the AGT gene promoter have increased promoter activity on transient transfection in human liver cells as compared to the AG haplotype. In order to understand the role of AA and AG haplotypes on hAGT expression in an in vivo situation, we have generated double transgenic mice containing either haplotype AA or AG of the hAGT gene (using knock‐in strategy at the HPRT locus) and human renin gene. Our studies show that transgenic mice containing AA haplotype have 1.6 fold increase in the AGT mRNA level in the liver as compared to the transgenic mice containing AG haplotype. In addition, Western blot and immunohistochemistry shows that liver and kidney of transgenic mice containing AA haplotype have increased hAGT protein level as compared to AG haplotype. In addition, blood pressure of three months old male transgenic mice containing AA haplotype is increased by about 8‐10 mmHg as compared to the transgenic mice containing AG haplotype. Supported by NHLBI.
Kidney Specific Regulation of Blood Pressure through Nedd4‐2
Authors: Yang Baoli , Shi Peter P , Cao Renee X , Sweezer Eileen M , Kinney Thomas S , Williams Nathan R , Husted Russell F , Stokes John B
Published: June 2021

Nedd4‐2 has been proposed to play a critical role in regulating epithelial Na+ channel (ENaC) activity through ubiquitination mediated degradation, and blood pressure (BP) regulation. To further understand the in vivo role of Nedd4‐2, we generated mice with global and kidney collecting duct (CD)‐specific inactivation of Nedd4‐2. The global knockout mice had higher BP on a normal diet, and a further increase in BP when on a high‐salt (HS) diet. The hypertension was mediated through ENaC, as both expression level and activity were increased in Nedd4‐2 null mice, and hypertension was substantially reduced in the presence of amiloride. However, unlike mice with global inactivation of Nedd4‐2, mice with CD‐specific inactivation did not have spontaneously elevated BP. When the mice were placed on HS diet, mice with CD‐specific inactivation of Nedd4‐2 had increased BP, although it was significantly lower than that of mice with global inactivation of Nedd4‐2. Overall, our results demonstrate that in vivo Nedd4‐2 is critical regulator of ENaC activity and BP, and suggest that kidney (CD) expression of Nedd4‐2 is necessary but not sufficient for the general increase in BP. Supported by NIH DK52617 to BY and JBS.
Protein Kinase C and PI3 Kinase Mediate Ang II‐Dependent Generation of Reactive Oxygen Species in Renal Nuclei
Authors: Pendergrass Karl D , Gwathmey TanYa M , Grayson Jason M , Chappell Mark C
Published: June 2021

We previously reported that nuclei isolated from rat renal cortex contain a high density of AT1 receptors (AT1R) that are functionally linked to reactive oxygen species (ROS) generation. Moreover, the protein kinase C (PKC) mimetic, PMA, stimulated ROS to a similar extent as Ang II. Therefore, the present studies elucidated the signaling pathways responsible for Ang II‐AT1R dependent generation of ROS in renal nuclei. Ang II (1 nM, 5 min) increased fluorescence of the ROS indicator dichlorofluorescein (DCF) [39±6% above control, P≤0.05, N=8], which was abolished by the oxidase inhibitor DPI [‐5±5%, P≤0.01 vs. Ang II, N=4]. The selective PKC inhibitor GF109203X also attenuated Ang II‐dependent ROS generation [‐8±8%, P≤0.01 vs. Ang II, N=3]. Immunoblot screening of five PKC isoforms revealed a predominant band for PKC‐δ (80 kDa) in renal nuclei consistent with the inhibitory spectrum of the PKC agent. As activation of PKC is typically downstream from PI3‐kinase (PI3K), addition of the highly selective PI3K inhibitor LY294002 abolished Ang II‐stimulated ROS [2±2%, P≤0.01 vs. Ang II, N=3] that was consistent with protein expression of the PI3K subunits p85 and p110 in isolated nuclei. Finally, immunoblots revealed NOX4 (65 kDa) in isolated nuclei and flow analysis localized NOX4 to 65±4% of nuclei [N=4]. We conclude that the nuclear Ang II‐AT1R‐ROS axis may involve NOX4, as well as PKC‐ and PI3K‐dependent pathways.
The effect sex chromosomal complement (SCC) on the expression of sodium transporters is dependent on gonadal sex
Authors: Tiwari Swasti , Shi Min , Arnold Arthur , Sandberg Kathryn , Verbalis Joseph G. , Ecelbarger Carolyn M.
Published: June 2021

The sex chromosome complement (SCC) has been demonstrated to have sex‐steroid‐ independent effects on the male versus female phenotype. To examine the role of the SCC in blood pressure (BP), we created a mouse model by translocation of Sry gene, a testes‐determining gene, onto an autosome in both XX and XY mice. Recently, we showed that ovariectomized mice with the XX SCC had higher angiotensin II (Ang II)‐induced BP as compared to the XY SCC. This effect appeared dominant in mice that were gonadally female at birth. The expression of renal sodium transporters may affect BP. To avoid the activational effect of steroidal hormones, mice were either ovariectomized (OVX) or castrated (CAS) depending upon their gondal sex at birth. All mice were infused with Ang II (A, 800 ng/kg•bw/min, n=5/group) for 7 days. Western blotting revealed that OVX‐XY mice had significantly reduced protein abundance for NaKATPase α‐1subunit and γENaC relative to OVX‐XX mice (band density % XX; for NaKATPase were 63 ± 0.01 vs. 100 ± 0.09 %; for γENaC 72.6 ± 0.03 vs. 100 ± 0.09% ). However, no significant difference due to SCC in these protein levels was observed in the CAS XXSry and XY− Sry male (at birth) mice. Moreover, AQP‐3 was significantly reduced in XY− Sry mice relative to XXSry (62.6 ± 0.12 vs. 100 ± 0.07 %). These data demonstrate that the SCC may influence expression of renal sodium and water transporters independently of activational effects of the gonadal steroids. Increased expression of Na‐K‐ATPase or γENaC could play a role in the increased BP observed in the XX SCC mice. The fact that the XX SCC appeared to increase BP suggests that without the overlay of sex steroids, female mice may be have a greater susceptibility to high BP. This may have particular relevance in menopause. NIH
cGMP/PKG‐mediated regulation of lymphatic contractility
Authors: Gasheva Olga Yu. , Gashev Anatoliy A. , Zawieja David C.
Published: June 2021

Recently we demonstrated a principal role of nitric oxide (NO) in the endothelium/shear‐dependent regulation of contractility in thoracic duct (TD). We tested the hypothesis that cGMP/PKG is involved in the intrinsic & extrinsic flow‐dependent modulation of lymphatic contractility. Diameters & indices of lymphatic pumping of isolated, cannulated & pressurized segments of rat TD were measured. The influences of incrementally increased transmural pressure (1 to 5 cm H2O) and imposed flow (1 to 5 cm H2O transaxial pressure gradient) were examined before & after treatment with cGMP analog and antagonist. We found that the cGMP analog ‐ 8pCPTcGMP (1‐100 µM) mimics the extrinsic flow‐induced relaxation in a dose‐dependent manner. Treatment of TD by the cGMP/PKG inhibitor ‐ Rp‐8‐Br‐PET‐cGMPS (10‐50 µM) eliminated the NO‐dependent intrinsic flow‐dependent relaxation, and largely prevented the extrinsic flow‐dependent relaxation. Western blotting demonstrated the presence of both PKGα and β isoforms in TD, with ~10‐times greater expression of the PKGα protein in TD compared with aorta and vena cava. The PKGβ isoform expressed equally in TD and vena cava, both being ~2 times higher than that in the aorta. These findings demonstrate that cGMP is critical to the flow‐dependent regulation of TD contractility; and also suggests important involvement of PKG in these processes. NIH HL70308
Selective targeting of cAMP signaling components and adhesion molecules to caveolin‐enriched fractions of pulmonary microvascular endothelial cells (PMVECs)
Authors: Sayner Sarah L. , Stevens Troy , Cooper Dermot MF
Published: June 2021

Strongly adherent PMVECs interact to form the pulmonary capillary barrier, necessary to maintain efficient gas exchange in the lung. Tight cell‐cell junctions between adjacent pulmonary capillary endothelial cells and cytoskeletal elements control barrier integrity and are dynamically regulated by cAMP. Within PMVECs, calcium sensitive adenylyl cyclase (AC) activity has been resolved in a phosphodiesterase 4 regulated membrane fraction. However, compartmentation of AC and other signaling molecules involved in barrier regulation within PMVEC membranes has not been described. Utilizing detergent free discontinuous sucrose density gradient centrifugation to separate caveolae/lipid raft fractions from the bulk plasma membrane we detect AC5/6 in the caveolin rich light density fractions. TRPC4 a putative molecular component of the store operated calcium entry channel also resides in caveolin fractions. In addition, filamin, an actin binding protein that when PKA phosphorylated stabilizes the actin cytoskeleton, also localizes to caveolae with cell adhesion molecules N‐ and E‐cadherin, and ALCAM. In contrast, VE‐cadherin is found outside of lipid rafts in the bulk plasma membrane compartment. Therefore, it appears that AC6 compartmentalizes to caveolae with other signaling components that dynamically regulate endothelial barrier. Supported by the Welcome Trust, HL66299 and F32HL091651
Mechanistic Insights into Membrane Remodeling Through BAR‐Domain Proteins
Authors: Frost Adam , Mim Carsten , Perera Rushika , Spasov Krasimir , Egelman Edward , De Camilli Pietro , Unger Vinzenz M
Published: June 2021

Critical to cellular physiology, cells constantly manipulate the shape of their constituent membranes through processes whose mechanisms are poorly understood. A major limitation in the study of membrane remodeling is the lack of structural information that visualizes membrane‐remodeling domains in the context of their bilayer substrates. Overcoming this bottleneck, we obtained structural models for two membrane‐bound states of F‐BAR domains by electron cryomicroscopy and image reconstruction. The models suggest that F‐BAR domains can engage bilayers through two very different molecular surfaces that are aligned with functional states prior to, and after induction of membrane curvature. Moreover, the reconstructions reveal how curvature is generated through the cooperative assembly of a highly organized protein scaffold, and how subtle changes in the orientation of dimeric F‐BAR modules facilitate the generation of structures with a range of different curvatures.

Supported by PHS grant DA024101
Comparative morphology of the optic nerve in nocturnal and diurnal birds
Authors: Grady Shauna Marie , Perkins Ashley R , Hall Margaret I
Published: June 2021

Nocturnal and diurnal birds exhibit differences in retinal wiring, specifically with regard to "retinal pooling," or the number of photoreceptor cells providing information to a single ganglion cell in the retina. Diurnal birds have low retinal pooling, which acts to increase acuity, since each photoreceptor cell is responsible for a proportional angle of visual space. Nocturnal birds have high retinal pooling; if multiple photoreceptors provide information to a single ganglion cell, light sensitivity is increased. The optic nerve (CN II) is comprised of the ganglion cell axons delivering visual information to the brain. This study tests the hypothesis that nocturnal birds consistently exhibit a smaller optic nerve and chiasm than diurnal birds, based on area measurements. Whole bird heads are preserved in 4% paraformaldehyde, and the optic nerve and chiasm are dissected and mounted in modeling clay and photographed. Areas are measured from the photographs in SigmaScan. ANOVA and regression analysis of the comparative size of the optic nerve supports our hypothesis that nocturnal birds have consistently smaller optic nerve and optic chiasm areas.

Grant Funding Source

AAA Student Travel Award
Fibroblast Differentiation in Dermal Equivalents Affected by Tension, Age in Culture, and TGF‐β
Authors: Palmer Tiffany Robin , Vaughan Melville Bailey
Published: June 2021

The objective of this study was to determine if tension and/or transforming growth factor beta (TGF‐β) induce stress on human fibroblasts causing the development of stress fibers and differentiation into proto‐myofibroblasts (a transitional state) and myofibroblasts and if its affect varies with cell age. The study used three different age groups of normal dermal fibroblasts including young, old, and immortal, each individually mixed with collagen in the presence or absence of a tension‐maintaining ring. Each well was given media and half of each group was given TGF‐β‐treated media. The plate was then incubated at 37°C with new media on the fifth day. After seven days the cells were fixed and one from each treatment group was selected to be stained and photographed using immunofluorescence technology. Preliminary results showed the old and young cells reacted as expected as the percentage of myofibroblasts increased with tension and TGF‐β. However, the immortal line showed little differentiation which could be attributed to their proliferation in the lattice. The development of an epidermal equivalent by adding keratinocytes will follow.

Grant Funding Source

NIH Idea Network of Biomedical Research Excellence P20 RR016478‐06
Effects of ubiquitylation on the temporal progression of Nucleotide Excision Repair
Authors: Lalimar Danielle , Fischhaber Paula
Published: June 2021

This study is examining whether ubiquitylation of Rad4 influences the temporal progression of Nucleotide Excision Repair (NER). To investigate this, a Rad7SOCS mutant has been constructed in which amino acids L168 and C172 have been mutated to alanines thus rendering Rad7 deficient in its ubiquitylation capability. In this ubiquitylation deficient background it will be possible to determine if there is a change in the timed recruitment of NER and therefore if ubiquitylation regulates this timing. The Rad7SOCS mutant was prepared by site‐directed mutagenesis. The Rad7SOCS plasmid has been transformed into yeast and screening and sequencing efforts are currently underway to select for the Rad7SOCS yeast strain. After transforming into yeast, the Rad7SOCS strain will be combined genetically with strains containing the fluorescently labeled Rad10‐YFP and Rad14‐CFP genes. These strains, as well as the isogenic Rad7 wild type strain, will be UV‐irradiated and temporal changes in the appearance or disappearance of Rad10 and Rad14 will be observed.
SINGLE‐PASS CLASSIFICATION OF ALL NON‐CODING SEQUENCES IN A BACTERIAL GENOME USING PHYLOGENETIC PROFILES
Authors: Marchais Antonin , Naville Magali , Bohn Chantal , Bouloc Philippe , Gautheret Daniel
Published: June 2021

Identification and characterization of functional elements in the non‐coding regions of genomes is an elusive and time consuming activity whose output does not keep up with the pace of genome sequencing. Hundreds of bacterial genomes lay unexploited in terms of non‐coding sequence analysis although they may conceal a wide diversity of novel RNA genes, riboswitches or other regulatory elements. We describe a strategy that exploits the entirety of available bacterial genomes to classify all non‐coding elements in a reference species in a single pass. This method clusters non‐coding elements based on their profile of presence among species. Most non‐coding RNAs display specific signatures that enable their grouping in distinct clusters, away from sequence conservation noise and other elements such as promoters. We submitted 24 ncRNA candidates from Staphilococcus aureus to experimental validation and confirmed the presence of 7 novel small RNAs or riboswitches. Besides offering a powerful method for de novo ncRNA identification, the analysis of phylogenetic profiles opens a new path towards the identification of functional relationships between co‐evolving coding and non‐coding elements.
Accurate SHAPE‐directed RNA structure prediction
Authors: Deigan Katherine E , Li Tian W , Mathews David H , Weeks Kevin M
Published: June 2021

Almost all RNAs can fold to form extensive secondary structures. Many of these structures then modulate numerous elements of gene expression. Deducing these structure‐function relationships requires that it be possible to predict RNA secondary structure accurately. However, RNA secondary structure prediction for large RNAs, such that a single predicted structure reliably represents the correct structure, has remained an unsolved problem. Here we demonstrate that quantitative, nucleotide‐resolution information from a SHAPE experiment can be interpreted as a pseudo‐free energy change term and used to determine RNA secondary structure with high accuracy. We use three metrics to evaluate the prediction accuracy for E. coli 16S rRNA (1542 nts). Taking the structure determined by comparative sequence analysis as the standard, we correctly predict 90% of all phylogenetically supported base pairs. Allowing for experimentally supported local refolding relative to the phylogenetic structure, the prediction accuracy is 95%. As judged by the ability to identify helices of 3 base pairs or greater, and thus the overall topology of the RNA, the prediction accuracy is again 95%. This work demonstrates that, given sufficient quantitative in‐solution information, it is possible to predict the structure of an important subset of RNAs with accuracies comparable to those achievable by comparative sequence analysis.
Specific interaction between Lin28 and pri‐let‐7g
Authors: Huang Ying , Ding Lei , Ma Jin‐Biao
Published: June 2021

MicroRNAs (miRNAs) are a highly conserved class of small (18‐24 nucleotides) non‐coding RNAs that regulate a broad spectrum of biological processes. Aberrations or corruptions of miRNA functions may lead to mis‐regulated cell proliferation, tumorgenesis, and ultimately, cancer. Recent evidences indicate that certain miRNAs are specifically regulated at post‐transcriptional level by RNA‐binding proteins, such as Lin28. The interactions of Lin28 or its retroviral‐type CCHC zinc finger motif (ZFM) with RNA fragments derived from pri‐let‐7g have been investigated by electrophoretic mobility shift assay (EMSA) and NMR spectroscopy. We have measured the binding affinities of Lin28 or ZFM domain and pri‐let‐7g or its fragments. The specific RNA‐binding sites of ZFM were investigated by EMSA and NMR spectroscopy. The molecular mechanism and substrate selectivity for blocking the pri‐miRNA processing by Lin28 are further studied by mutagenesis and biochemical assays.
Identification of MicroRNA Targeting MYC
Authors: Takwi Apana Agha Louis , Lu Zhongxin , Li Yong
Published: June 2021

c‐Myc is a transcription factor that regulates numerous processes including cell cycle, apoptosis, cellular differentiation, cellular metabolism and genomic instability. In normal cells, expression of c‐Myc is tightly regulated by external signals such as growth factors and extracellular matrix. However in stressed and cancerous cells, c‐Myc is generally over‐expressed by chromosomal translocation, gene amplification, or stabilization of its mRNA. We hypothesize that MYC is a target of microRNA (miRNA). miRNAs are 20‐22bp short RNAs that regulate gene expression by binding target mRNA 3′‐UTR, leading to mRNA degradation or translational repression. We used a reporter construct carrying c‐Myc binding sites to screen miRNAs targeting MYC. The results demonstrated that miR‐212 (30%), miR‐203 (40%), miR‐33b (50%) and miR‐33a (50%) down‐regulated reporter activity. Next, we cloned the 3′‐UTR of MYC into a reporter vector and performed assays in cell transfected with miRNAs. We found that miR‐33a (60%), miR‐33b (50%), miR‐203 (40%) down‐regulated the expression of the reporter gene located upstream of the MYC 3′‐UTR. Finally, we performed colony formation assay with rat RK3E‐cmyc cells, which constitutively express c‐Myc. Our results showed that miR‐212 (85%), miR‐33a (50%), miR‐33b (60%) and miR‐203 (17%) reduced colony formation. We will continue to investigate whether MYC is an authentic target of these miRNAs.
A Bacterial Regulatory Small RNA Perturbing LPS Modification
Authors: Moon Kyung , Gottesman Susan
Published: June 2021

Non‐coding small RNAs play a major role in post‐transcriptional regulation of gene expression. Thus far, about 100 small RNAs have been identified by genome‐wide searches in E. coli; more than 30 depend on the RNA chaperone Hfq. Hfq immunoprecipitation studies in both E. coli and Salmonella identified a novel Hfq‐dependent RNA, called here Spk. Spk is located in an intergenic region between ydeH and ydeE. Expression of Spk is dependent upon the PhoQ/PhoP two component system; the PhoP regulator is active under low Mg++ concentrations and is an important virulence regulator in Salmonella. Negatively regulated targets of Spk, identified using microarrays,included eptB, involved in LPS modification, and ygdQ encoding a hypothetical protein. Cell sensitivity to the antimicrobial Polymyxin B is affected by LPS modifications. Cells carrying an spk deletion were approximately 10 times more resistant to Polymxyin than wild type. Thus, in E. coli, changes in Mg concentrations, sensed by PhoQ/PhoP, lead to expression of Spk, changing LPS; this is likely to play a significant role for bacterial survival in some environments. Other physiological roles of Spk are being explored.
Determining the function of nuclear Bmp4
Authors: Loos Trina J , Bridgewater Laura C
Published: June 2021

Bone morphogenetic protein 4 (Bmp4) is a well known growth factor that regulates gene expression through the SMAD signaling pathway. Bmp4 is involved in many developmental processes and has been identified as an important factor in several cancers, including melanoma, ovarian cancer, and colon cancer. Surprisingly, Madoz‐Gurpide et al. (2006) observed Bmp4 in the nuclei of a minor percentage of cells in colon cancer tissues. In addition, our lab has recently discovered a nuclear variant of Bmp2 (nBmp2), the TGF‐β family member most closely related to Bmp4. These observations led us to hypothesize that a nuclear variant of Bmp4 (nBmp4) also exists. Preliminary analyses have been performed that verified nuclear localization of Bmp4 in several cancer cell lines. Subsequent analyses suggest that nBmp4 is translated from an alternative start codon downstream of the signal peptide sequence, which allows a bipartite nuclear localization signal to direct translocation of nBmp4 to the nucleus. In an effort to determine the function of nBmp4, a yeast two‐hybrid screen was performed using a cDNA library from MCF‐7 breast cancer cells. Several proteins were identified as potential interacting partners. These interacting proteins suggest a role for nBmp4 in protein degradation, reactive oxygen species modulation, and apoptosis. Research supported by NIH grant AR48839 and the Brigham Young University Cancer Research Center.
Deletion of PERK in Liver Impairs the Hepatic ER Stress Response to Tunicamycin.
Authors: Bunpo Piyawan , Brueck Danielle , Cavener Douglas R , Wek Ronald C , Anthony Tracy G
Published: June 2021

Endoplasmic reticulum (ER) stress activates PKR‐like ER kinase (PERK), increasing phosphorylation of eukaryotic translation initiation factor 2 (p‐eIF2). PERK is one arm of the tripartite Unfolded Protein Response (UPR), which functions to alleviate protein misfolding in the ER. In this study, mice homozygous for the LoxP allele of PERK and expressing albumin‐driven Cre recombinase (liver‐specific PERK null; AlbCre+) and Cre‐negative controls (AlbCre−) were treated with tunicamycin (Tun; 1mg/kg body weight i.p.), an inhibitor of protein glycosylation, or excipient. Livers were harvested 6h after injection. AlbCre− mice treated with Tun demonstrated increased p‐eIF2 in the liver. In contrast, AlbCre+ mice showed no increase in p‐eIF2. Induction of the UPR was monitored using RT‐PCR to evaluate mRNA splicing of the transcription factor, X‐box binding protein‐1 (XBP‐1), and quantitative Real‐Time RT‐PCR to evaluate mRNA expression of C/EBP homologous protein (CHOP) and the molecular chaperone, BiP/grp78. In AlbCre− livers, Tun increased XBP‐1 mRNA splicing 5‐fold and hepatic expression of BiP and CHOP 35‐fold and 350‐fold, respectively. On the other hand, activation of the UPR in the livers of AlbCre+ mice treated with Tun was significantly dampened. Specifically, Tun induced XBP‐1 mRNA splicing 3‐fold whereas BiP and CHOP mRNA expression increased only 7‐fold and 5‐fold, respectively. In summary, liver‐specific PERK null mice demonstrate impaired or delayed UPR activation following Tun injection. These data suggest that PERK is solely responsible for Tun‐mediated p‐eIF2 and serves an important role in early activation of the UPR to ER stress in liver.
Purification and characterization of mutant exon 1 huntingtin polyglutamine expressed in a mammalian system
Authors: Feng Xia , Porat Yair , Ross Christopher A. , Poirier Michelle A.
Published: June 2021

Huntington's disease is a neurodegenerative disorder characterized by neuronal degeneration and inclusions containing N‐terminal fragments of mutant huntingtin (htt). Polyglutamine aggregation is a complex process, involving the assembly of multiple intermediates prior to the formation of inclusions. Recent studies have suggested that soluble htt oligomeric or protofibrillar forms may be more toxic than late‐stage fibrils. Previously, we expressed recombinant mutant htt exon‐1 fragment in bacteria. We monitored aggregation over time and showed that globular and protofibrillar species comprised of β‐structure participate in the genesis of mature fibrils. In the present study, we have established a mammalian system for expression of GST‐tagged htt exon‐1 in HEK293 cells. Our preliminary aggregation data indicate that recombinant mutant htt exon‐1 produced in a mammalian system aggregates more slowly than the protein generated in bacteria, possibly resulting from post‐translational modifications. Future experiments will be aimed at isolating mammalian htt exon‐1 monomer and aggregation species and further investigating morphological and structural features of each form.

Grant Support: NIH/NINDS