bluuebunny/crossref_metadata_2025 huggingface dataset:
Central angiotensin‐converting enzyme 2 overexpression decreases blood pressure and enhances baroreflex function in mice with chronic heart failure
Authors: Xiao Liang , Gao Lie , Lazartigues Eric , Zucker Irving H
Published: June 2021

Central angiotensin‐converting enzyme 2 (ACE2) contributes to the regulation of cardiovascular function through modulating other components of the renin‐angiotensin system (RAS) in the brain. Since chronic heart failure (HF) is associated with sympathoexcitation, impaired baroreflex function and activation of the central RAS, we hypothesized that overexpression of ACE2 decreases blood pressure (BP) and improves baroreflex sensitivity in HF. Transgenic mice overexpressing human ACE2 selectively in the brain (Syn‐hACE2) and their wild type littermates (WT) were used, both of which developed HF (ejection fraction <45%, echocardiography) four weeks after coronary artery ligation. 24‐hour BP was monitored by radiotelemetry. Compared with WT HF group, Syn‐hACE2 HF mice exhibited lower mean BP, and ameliorated the spontaneous baroreflex sensitivity (SBRS, sequence method). These data indicate a beneficial role of central ACE2 overexpression during HF, and we speculate that ACE2 overexpression attenuates the augmented sympathetic outflow through decreasing angiotensin (Ang) II and/or increasing Ang 1–7 in the brain.

WT Sham (n=5)
Syn‐ACE2 Sham (n=5)
WT HF (n=4)
Syn‐ACE2 HF (n=4)

Mean BP (mmHg)
113±3.2
113.7±5.0
109.2±4.7
88.9±4.7#

Heart Rate (bpm)
599.5±18.9
578.1.5±21.2
647.2±20.8
644.8±10.8

SBRS (ms/mmHg)
2.21±0.24
2.91±0.51
0.82±0.15*
1.80±0.19#

p<0.05 vs. WT sham;

p<0.05 vs. WT HF.
Mutation of Tyr985 of the leptin receptor differentially alters regional sympathetic activation to leptin
Authors: Harlan Shannon Marie , Morgan Donald , Dellsperger David , Myers Martin , Mark Allyn , Rahmouni Kamal
Published: June 2021

Leptin is an adipocyte derived hormone which acts in the brain to regulate food intake and energy expenditure. Leptin action in the brain also increases sympathetic nerve activity (SNA) to several beds including brown adipose tissue (BAT) and kidney. There is mounting evidence that the distinct signaling pathways downstream of the leptin receptor (LRb) may mediate selective leptin actions. We assessed the regional SNA response to leptin in mice (l/l) that carry a point mutation in LRb at Tyr985, which is involved in ERK activation by leptin. Multifiber recording of SNA was performed under α‐chloralose anesthesia in mice equipped with intracerebralventricular (ICV) cannula. As expected in wild type mice, ICV injection of leptin (2 μg) significantly (P<0.05 vs. vehicle) increased SNA subserving BAT and kidney. In contrast, l/l mice had a significantly (P=0.005) attenuated BAT SNA response to leptin (100±38%) as compared to wild type mice (456±130%). Renal SNA responses to leptin did not differ in l/l mice (154±80%) as compared to wild type controls (143±39%; P=NS). These data demonstrate the pivotal role of leptin receptor signaling emanating from the Tyr985 in the control of thermogenic sympathetic outflow. Our data also suggest that signaling from Tyr985 of the leptin receptor is not involved in leptin‐induced renal sympathetic activation. Together, these data demonstrate selectivity in leptin action.

This work was funded by a Cardiovascular Interdisciplinary Research Fellowship, HL007121 to Shannon Harlan.
Superoxide formation induced by angiotensin II in the rostral ventrolateral medulla is enhanced by high salt diet in rats
Authors: Braga Valdir A
Published: June 2021

Here, we investigated the association of dietary salt and angiotensin II (Ang‐II) infusion on hypertension and superoxide formation in the RVLM. Male Wistar rats were subcutaneously implanted with 14‐day osmotic minipumps containing either Ang‐II (150ng/Kg/minute) or saline (0.9% NaCl). In addition, starting at 7 days before drug infusion and continuing throughout the infusion period, rats had free access to drinking water containing 0.4% or 2.0% NaCl. For ROS accumulation measurements in the RVLM, brains were harvested at 15 days of infusion and were submitted to the dihydroethidium technique. Rats that received Ang‐II + water with 0.4% NaCl (G1) became hypertensive after 14 days of Ang‐II infusion (118±5 mmHg vs 98±4 mmHg, n=9, P<0.05). Rats that received Ang‐II + water with 2% NaCl (G2) had a significantly greater hypertension compared to G1 (165±7 mmHg vs 118±5 mmHg, n=10, P<0.05). Rats treated with saline infusion + 2% NaCl‐containing water (G3) or saline infusion + water with 0.4% NaCl (G4) did not become hypertensive (99±3 vs 98±4 mmHg, n=8 and 96±5 mmHg vs 98±4 mmHg, n=7, respectively). Furthermore, administration of hexamethonium (30 mg/Kg i.v.) reduced hypertension in G1 and G2 groups. The magnitude of superoxide formation in the RVLM was G2>G1>G3=G4 (G2= 123±10 Δ %; G1= 67±9 Δ%, P<0.05; and G3=5±3 Δ%, P>0.05), normalized by the G4 group (control). We suggest that dietary salt potentiates Ang‐II‐derived superoxide formation in the RVLM, due to an increase in the inputs within the forebrain‐PVN‐RVLM axis, leading to further increases in sympathetic nerve activity, resulting in a more severe hypertension.

Supported by CNPq.
Central hypertonic NaCl increases cytokine expression in the hypothalamic paraventricular nucleus
Authors: Shi Peng , Dong Ying , Shan Zhiying , Lin Fan , Raizada Mohan K , Sumners Colin
Published: June 2021

Recent studies indicate that brain‐derived cytokines are involved in sympathetic activation and hypertension. Our objective was to determine the relationship between hyperosmotic stimulus (HS), which enhances sympathetic outflow, and cytokine expression in the CNS. SD rats received an intracerebroventricular (ICV) injection of 2M NaCl (HS) or 0.9% NaCl, and 3 hours later PVN tissues were dissected for RT‐PCR analyses. PVN levels of interleukin (IL) 1β, IL6, IL10 and tumor necrosis factor α mRNAs were significantly increased in response to 2 M NaCl compared with the 0.9% NaCl treatment. Levels of CD11b mRNA, a marker of microglial activation, were also increased in the PVN of 2M NaCl injected rats. Pretreatment of rats with minocycline, an inhibitor of microglial activation, via s.c. infusion for 7 days, abolished the HS stimulated increase in IL1β, IL6 and CD11b expression. Angiotensin II type 1 (AT1) receptor mRNA levels were significantly increased by HS, and this response was abolished by minocycline. In summary, our data show that central administration of hypertonic NaCl increases expression of cytokines and AT1 receptors in the PVN, and these responses were completely inhibited by minocycline. This suggests that cytokines in the PVN are involved in the regulation of AT1 expression in response to hypertonic NaCl challenge. (NIH: HL33610 & HL76321 [M.K.R.], HL 076803 [C.S.]; AHA: 09POST2230164 [P.S.])
A long‐term high salt intake involves reduced small conductance Ca2+‐activated K+ (SK) current and increased excitability of RVLM projecting PVN (PVN‐RVLM) neurons
Authors: Chen Qing‐Hui , LaGrange Lila P , Andrade Mary A , Calderon Alfredo S , Toney Glenn M
Published: June 2021

Accumulating evidence indicates that long‐term high salt (HS) intake is involved in the activation of sympathetic nerve system. The neural mechanism responsible for the sympathetic activation in rat on a HS diet was investigated. Recently, we reported that SK channels regulate membrane properties of PVN‐RVLM neurons and limit in vitro excitability. Here, we determined whether 5 weeks of HS diet (2% NaCl) alters the SK current among PVN‐RVLM neurons and whether this change affects their intrinsic excitability. In voltage‐clamp recordings, the amplitude of SK current in neurons from HS treated rats (13±5 pA, n=6) was significantly (p<0.05) less than that from normal salt (NS, 0.4% NaCl) treated animals (63±11 pA, n=6). In current‐clamp recordings, graded current injections evoked graded increases in discharge frequency among neurons of both groups. Maximum discharge evoked by +200 pA current injection in HS group was 43±4 Hz (n=8). This response was significantly greater (P<0.01) than that of NS controls (22±1 Hz, n=9). The SK channel blocker apamin (100 nM) significantly increased (p<0.05) discharge in NS controls (47±4 Hz, n=6), but had no effect in HS group (45±2 Hz, n=5). Our data indicate that reduced SK current in PVN‐RVLM neurons contributes to the increased excitability in neurons from rats on a HS diet, which may be a contributing factor in the sympathetic activation that appears to accompany elevated salt intake. Support: AHA0865107F (CQH) & HL076312, HL088052 (GMT).
Coupled Activity‐dependent Trafficking of SK2 Channels and Glutamate Receptors
Authors: Lin Mike T , Lujan Rafael , Watanabe Masahiko , Frerking Matthew E , Adelman John P , Maylie James
Published: June 2021

Small conductance Ca2+ activated K+ type 2 (SK2) channels are expressed in the postsynaptic density (PSD) of CA1 neurons where they are activated by synaptically evoked Ca2+ influx, providing a local inhibitory current. Upon the induction of long‐term potentiation (LTP), synaptic SK2 channels undergo protein kinase A (PKA)‐dependent endocytosis, thus contributing to the increased excitatory postsynaptic potential (EPSP). Under basal conditions, SK2 channels and α‐amino‐3‐hydroxyl‐5‐methyl‐4‐isoxazole‐propionate receptors (AMPAr) populate distinct sets of endosomes. Surprisingly, inhibiting LTP‐dependent AMPAr exocytosis also blocks SK2 channel endocytosis. Following LTP and internalization, SK2 channels repopulate the PSD within ~2 hours. This time course is similar to the accumulation of N‐methyl‐D‐aspartate receptors (NMDAr) that rebalances the AMPA‐to‐NMDA ratio, and during this time SK2 channels and NMDAr co‐localize to the same vesicles. Thus, the trafficking of spine SK2 channels and the two subtypes of glutamate receptors are tightly and differentially coupled in response to LTP.
Adolescent stress alters synaptic plasticity: Is PKMzeta the mediator?
Authors: Serrano Peter A , Hernandez Alejandro Ivan , Schrott Lisa M
Published: June 2021

PKMζ has a fundamental role in the retention of long‐term memories and synaptic plasticity. We examined the behavioral consequences of stress and the concomitant changes in PKMζ protein expression. Our results show that adolescent (38 day‐old) male rats given an acute stress significantly elevated corticosterone in blood sera, increased PKMζ protein expression within the hippocampus, and produced a translocation of the GluR2 subunit to the synaptic membrane. Immediately following an acute stress condition (45 min of elevated platform stress) PKMζ protein increased within the synaptic and cytosolic fractions. The GluR2 receptor subunit increased significantly in the synaptic fraction (p>.05). In contrast, seven days following acute stress PKMζ levels continued to be significantly elevated in the cytosolic fraction but not in the synaptic fraction compared to age matched, unstressed controls. Hippocampal slices made from rats immediately after acute stress show depressed levels of synaptic potentiation (long‐term potentiation; LTP) in area CA1 of the hippocampus, but increased levels of depression (long‐term depression; LTD) as compared to unstressed controls. These results showing an increase in PKMζ and the GluR2 subunit in the synaptic region are consistent with PKMζ induced trafficking of this receptor during synaptic plasticity and LTP.
The Effects of Chronic Social Defeat Stress on Mouse Grooming Behavior Patterning
Authors: Tien David , Duncan Ashley , Chung Amanda , Wong Keith , Tan Julia , Cachat Jonathan , Goodspeed Jason , Grimes Chelsea , Elegante Marco , Suciu Christopher , Elkhayat Salem , Jackson Andrew , Rosenberg Michael , Chung Kyung Min , Badani Hussain , Kadri Ferdous , Roy Sudipta , Gaikwad Siddharth , Gilder Thomas , Zapolsky Ivan , Bartels Brett , Kalueff Allan V.
Published: June 2021

Animal social defeat stress is a strong modulator of anxiety and depression‐like states. Although grooming is a frequent behavioral marker of stress, little is known about the effects of social stress on grooming. This study explores the effects of chronic social defeat stress on mouse self‐grooming behavior. After exposing mice to 13–15 days of social defeat, grooming behavior was measured in both winner and loser mice in the novelty test. The occurrence of transitions between paw, head, body, tail and genital regions was recorded. In loser mice, chronic social defeat increased latency to grooming, and produced fewer correct transitions, grooming bouts, and vertical rears. No differences were found between winners and losers for serum corticosterone levels, duration of grooming, distance travelled, or velocity. Overall, this study describes how the stress induced by social defeat leads to an increase in anxiety‐like disruption of grooming sequencing, thus illustrating its importance as a model for high‐throughput behavioral studies.
Reversible inactivation of the medial prefrontal cortex causes antidepressant‐like effects of in rats
Authors: Scopinho América Augusto , Scopinho Michele , de Aguiar Corrêa Fernando Morgan , Guimarães Francisco Silveira , Joca Sâmia Regiane Lourenço
Published: June 2021

Reversible inactivation of the prelimbic (PL) and infralimbic (IL) parts of the mPFC with cobalt (CoCl2, a synaptic blocker) produced anxiolytic‐like effects. The aim of the present study was to verify if the same treatment would also induce antidepressant‐like effects in the forced swimming test (FST). Male Wistar rats were stereotaxically implanted cannulae in the PL or IL were submitted to the FST consisting of an initial 15 min swimming session (pre‐test) followed, 24 h later, by the 5 min test session. The animals (n=5–7) received bilateral microinjections of vehicle (200 nL) or CoCl2 (1 mM) into the PL or IL at different periods of the experimental procedure. In the PL CoCl2 induced a decrease in IT that was independent of the moment of the injection (before pre‐test, V: 187.3±25.8 s, CoCl2:157.7±16.2 s, after pre‐test, V: 207.1±6.3 s, CoCl2: 129.6±19.2 s, before test, V: 188.4±20.4 s, CoCl2: 88.1±29.7 s). In the IL, the treatment was effective when administered immediately before pre‐test or before test (before pre‐test, V: 186±21.1 s, CoCl2: 123±4.7 s; before test, V: 186.8±7.8 s, CoCl2: 8.5±2.6 s) but not after the pre‐test (V:161±9.6 s, CoCl2: 178.8±27.1 s). These results suggest that mPFC synaptic activity produces antidepressant‐like effects in this test. Also indicate that, although both the PL and IL cortices seem to be involved in these effects, they may play different roles. Financial support: CAPES, CNPq.
Direct Inhibition with Aliskiren Normalizes Blood Pressure in Cyp1a1‐Ren2 Transgenic Rats with Inducible ANG II‐Dependent Malignant Hypertension
Authors: Howard Catherine G , Mitchell Kenneth D
Published: June 2021

The present study was performed to determine the effects of acute direct renin inhibition with aliskiren on blood pressure and renal hemodynamics in Cyp1a1‐Ren2 transgenic rats with ANG II‐dependent malignant hypertension. Male Cyp1a1‐Ren2 rats (n=6) were fed a normal diet containing 0.3% I3C for 9–11 days to induce malignant hypertension. Mean arterial pressure (MAP) and renal hemodynamics were measured in pentobarbital‐anesthetized male Cyp1a1‐Ren2 rats during control conditions and following administration of the renin inhibitor, aliskiren (10 mg/kg, iv). Rats induced with I3C had higher MAP (196±4 vs. 142±3 mmHg, P<0.01) and lower renal plasma flow (RPF; 1.98±0.32 vs. 3.86±0.26 ml/min.g, P<0.01) than non‐induced rats (n=6). There were no differences in glomerular filtration rate (GFR) between the two groups (1.22±0.13 vs. 0.85±0.15 ml/min.g). Aliskiren administration decreased MAP (196±4 to 126±6 mmHg, P<0.001) and increased RPF (1.98±0.32 to 2.67±0.46 ml/min.g, P<0.05) in the hypertensive rats. GFR remained unaltered following renin inhibition in the hypertensive rats. Aliskiren administration did not alter MAP or GFR, but increased RPF (3.86±0.26 to 4.53±0.33 ml/min.g, P<0.05) in the normotensive rats. The present data demonstrate that acute renin inhibition with aliskiren normalizes MAP and improves RPF in Cyp1a1‐Ren2 transgenic rats with malignant hypertension. The normalization of MAP and the increased RPF following acute renin inhibition with aliskiren indicate that renin generated as a consequence of expression of the Ren2 gene is responsible for the development of malignant hypertension and the associated reduction of renal hemodynamic function in Cyp1a1‐Ren2 transgenic rats.
Dietary sodium and the renal responses to Angiotensin1–7 (ANG1–7)
Authors: Johns Edward J , Corbett Alan , Albulushi Arif , O'Neill Julie
Published: June 2021

This study investigated how the renal excretory actions of ANG1–7 were altered when the renin‐angiotensin system (RAS) was activated or suppressed following a period of low or high dietary sodium intake. Male Wistar rats were fed a low (0.03%), normal (0.3%) or a high (3.0%) sodium diet for 2 weeks. The animals were anaesthetised with chloralose/urethane and prepared for the measurement of blood pressure (BP), iv infusion of saline (0.9%NaCl, 3.0 ml/h). Cannulae were placed in the left ureter for urine collection and at the cortico‐medullary border for saline or ANG1–7 infusion at 1.0ml/h. Four 20 min clearances were taken, two before and two 45min after either saline or ANG1–7 (9×10−7M) infusion. Data, means ±SEM were analysed using Student's t test. Infusion of saline had no effect on BP or any renal functional parameter. ANG1–7 infusion in rats on the normal diet did not change BP, at 99±4mmHg, but urine flow, at 18.1±1.3μl/min/kg, and fractional sodium excretion (FENa), at 0.6±0.1, were increased by 24 and 25% (both P<0.05). In the low salt rats, ANG1–7 caused a greater response in FENa, of some 2–3 fold (P<0.05) whereas in the high salt rats, it was blunted, being only 60% of the response in normal rats. These findings demonstrate a dependency of the excretory responses to ANG1–7 on the endogenous levels of RAS.
Cadmium (Cd)‐induced E‐cadherin (E‐cad)/β‐catenin (β‐cat) disruption triggers Wnt signaling and β‐catenin/TCF4‐dependent upregulation of proliferation and survival genes c‐Myc, cyclin‐D1 and ABCB1 in renal proximal tubule cells (PTC)
Authors: Chakraborty Prabir Kumar , Lee Wing‐Kee , Molitor Malte , Wolff Natascha A , Thévenod Frank
Published: June 2021

The class 1 carcinogen Cd disrupts the E‐cad/β‐cat complex of epithelial adherence junctions (AJs). Deregulation of E‐cad adhesion and changes of Wnt/β‐cat signaling are known to contribute to carcinogenesis. We investigated Wnt signaling after Cd‐induced E‐cad disruption in cultured PTC. Cd (25μM, 3–9 h) caused nuclear translocation of β‐cat and triggered a Wnt response by TOPFLASH reporter assays. Cd reduced the interaction of β‐cat with AJ components and increased binding to the transcription factor TCF4 of the Wnt pathway, which was upregulated and translocated to the nucleus. While Wnt target genes (c‐Myc, cyclin‐D1 and ABCB1) were upregulated by Cd, electromobility shift assays showed increased TCF4 binding to cyclin‐D1 and ABCB1 promoter sequences. Overexpression of wild‐type and mutant TCF4 confirmed Cd‐induced Wnt signaling. Wnt signaling triggered by Cd was more prominent in proliferating subconfluent than confluent cells, which showed increased E‐cad expression. Overexpression of E‐cad reduced Wnt signaling, PTC proliferation and Cd toxicity. Cd also induced ROS dependent expression of the pro‐apoptotic ER stress marker and Wnt suppressor CHOP which, however, did not abolish Wnt response and cell viability. Hence Cd may facilitate carcinogenesis of PTC by inducing the Wnt pathway to promote proliferation and survival of pre‐neoplastic cells. Funded by DFG TH 345/10‐1
Microvascular rarefaction precedes the decline in renal function in the streptozotocin (STZ)‐induced diabetic rat
Authors: Maric Christine , Flynn Elizabeth , Sawyer R Taylor , Marbury David C , Chade Alejandro R
Published: June 2021

Diabetic nephropathy is a generalized vasculopathic condition associated with abnormal angiogenesis. The aim of the study was to correlate changes in the 3‐dimensional renal microvasculature density (MV) with changes in renal function and vascular endothelial growth factor (VEGF).

The study was performed in male non‐diabetic (ND) or STZ‐induced diabetic (D) Sprague‐Dawley rats followed for 4 (4w) or 12 (12w) weeks. Renal MV density was quantified ex vivo using a 3D micro‐CT reconstruction of the kidney. Urine albumin excretion (UAE) and renal VEGF levels were measured by ELISA and glomerular filtration rate (GFR) by 125I‐[iothalamate] clearance. 4w of diabetes was associated with a 34% reduction in MV density, no changes in UAE and augmented renal VEGF levels. At 12w, the reduction in MV density was even more pronounced (75%) and correlated with increased UAE; however, renal VEGF remained unchanged.

ND
D
P value

4w
12w
4w
12w

MV density/cm2
69.3±8.8
210±40.2*
45.5±12.3
52.7±15.6#
*0.05 vs ND at 4w #0.05 vs ND at 12w $0.05 vs D at 4w

UAE (mg/day)
1.2±0.2
2.3±1.3
2.9±0.3*
6.0±1.0#,$

GFR (ml/min/kidney)
1.4±0.2
1.3±0.2
2.1±0.3*
2.2±0.4#

VEGF (ml/min/kidney)
1.5±0.07
1.7±0.2
1.9±0.2*
1.7±0.2

The study suggests a novel concept of MV rarefaction preceding the decline in renal function in STZ‐induced diabetic rats. This effect may be related to changes in renal VEGF bioavailability with progression of diabetic renal injury.
Not towing the allometric line: morphology and physiology of the respiratory system during postnatal development in marsupials
Authors: Simpson Shannon J , Frappell Peter B
Published: June 2021

On the whole, marsupials are born small; the fat‐tailed dunnart produces one of the smallest and most developmentally immature newborn mammals, weighing ~13 mg. Newborn marsupials, to varying extent, rely on cutaneous gas exchange and in the extreme, e.g. the dunnart, typically do not breathe until 3–4 days postpartum. Surface area and a functional lung volume in neonatal fat‐tailed dunnarts and tammar wallabies were determined with phase‐contrast imaging using a synchrotron radiation source. Values for surface area and lung volume were both less than that expected from allometry, and remained low until the animals reached a body mass of approximately 1 gram. Interestingly, this is also the age/size where we first observe the presence of secondary septa in the lung and cutaneous gas exchange ceases. The short gestation, low birth weight and less than allometrically predicted values for lung surface area and volume (and associated implications for pulmonary diffusing capacity) and higher than predicted respiratory system resistance may all play a part in reducing ventilation and hence placing a reliance of the newborn marsupial on cutaneous respiration in the first few days of life.
Copper transport in gill cells of Ucides cordatus, a mangrove crab
Authors: GRANADO SÁ MARINA , ORTEGA PRISCILA , ZANOTTO FLAVIA PINHEIRO
Published: June 2021

For most aquatic animals, the gills are important site for respiration, excretion, acid‐base balance and ion regulation. The branchial epithelium is exposed to the environment, which is frequently polluted. The aim of this work was characterize copper (Cu) transport using a fluorescent dye, Phen Green, in gill cells of a mangrove crab, found in polluted regions of Brazil. The results showed that CuCl2 added in crescent concentrations (0,0.025, 0.15, 0.275, 0.55 and 1.11μM) showed the same kinetic uptake in gill cells and a strong regulation in levels of intracellular Cu, independent of Cu concentration. Verapamil and nifedipine, known to inhibit calcium channels in apical membrane, also inhibited Cu uptake. Amiloride caused a greater efflux of copper from intra to extracellular medium, and the same results were found using calcium ionophore, which creates artificial calcium channels in plasma membrane. Besides that, the gill cells were incubated in ATP, and there were no changes in copper uptake. Caffeine, on the other hand, inhibited Cu uptake. The results showed that copper uptake is dependent of calcium uptake in gill cells, regulated by Ca channels and by Na/Ca exchangers.

Supported by a CNPq fellowship to MGS, Fundo Mackenzie de Pesquisa (MACKPESQUISA) and a FAPESP Grant (06/52498‐9).
Identification of 4 charged amino acids essential for Slc4a10‐derived protein function
Authors: Damkier Helle Hasager , Aalkjaer Christian , Praetorius Jeppe
Published: June 2021

The slc4a10 gene product imports Na+ and HCO3−. The aim of this study was to investigate the functional importance of charged amino acids in mouse slc4a10 protein. Negatively charged amino acids, possibly involved in Na+ translocation, should be conserved among Na+‐dependent Slc4a‐derived proteins. Alanine mutations were introduced at two candidate amino acids in a proposed hinge‐like loop between TM9–10 of (E890A and E892A). E584A in the end of TM3 served as negative control. Positively charged amino acids are presumed important for HCO3− translocation. We mutated two amino acids corresponding to a proposed hinge‐like loop between TM11–12(H976L and H980G). NIH‐3T3 cells were transfected with the 5 single‐mutated slc4a10 forms, double E890A/E892A and H976L/H980 mutations as well as quadruple mutations. The cells were compared functionally to wild type slc4a10 cells. The net Na+‐dependent acid flux after acidification, in E890A and E892A mutants, was reduced to 36 and 31% of wild type, respectively. The Na+ dependent acid flux was reduced in H976L and H980G (42 and 30% of wild type). In both double and the quadruple mutants the net acid flux was undetectable. The Na+‐dependent net acid flux in E584A mutant cells was not changed (96% of wild type). In conclusion, the four amino acids are essential for normal slc4a10 protein activity. We speculate that these sites are involved in attracting or translocating the ions.
Dimerization of the plasma membrane Na+/H+ exchanger type 3 (NHE3)
Authors: Babich Victor , Bobulescu I. Alexandru , Fuster Daniel G. , Gisler Serge , Bonny Olivier , Hilgemann Donald , Stagljar Igor , Moe Orson W.
Published: June 2021

Many transmembrane transport proteins are known or inferred to function as homo or hetero oligomers. NHE3 has been reported to associate in oligomers. However, the direct NHE3‐NHE3 interaction, the oligomer configuration and the significance of oligomerization for NHE3 function and regulation are unknown. Using split‐ubiquitin yeast two‐hybrid system for membrane protein‐protein interactions and co‐immunoprecipitation in mammalian cells, we demonstrated that full‐length NHE3, its C‐terminal truncations, but not full‐length NHE1, interact with full‐length NHE3 prey and NHE3 exists as an oligomer in mammalian cells. We confirmed that NHE3 is a dimer on the plasma membrane by counting photobleaching steps in total internal reflection fluorescence images of NHE3‐mGFP expressed in NHE‐null cells. In its native state in opossum kidney cells, NHE3 exhibits highly cooperative Na+ kinetics compatible with two substrate binding sites. To show functional cross‐talk between two NHE3 molecules in a dimer we used two NHE3 mutants: S552A/S605G (abolished regulation by protein kinase A, PKA) and E213I (transport defective). The transport activity of NHE‐null cells expressing S552A/S605G NHE3 mutant was comparable to wild‐type NHE3, but it was not affected by PKA activation. Co‐expression of both S552A/S605G and E213I NHE3 mutants restored the exchanger activity along with the PKA regulation. These findings suggest that NHE3 is present at the plasma membrane as homo dimers and NHE3 dimers may constitute the functional units of the transport and affect its regulation.
Vascular endothelial growth factor antagonism inhibits breast cancer proliferation via Akt and Erk signaling
Authors: Nelson Adam Michael , Gingery Anne , Soldner Emma L B , Gilbert Jeffrey S
Published: June 2021

While recent epidemiological studies report preeclampsia has long‐term protective effects against breast cancer, the mechanisms and the factors responsible for these observations remain unknown. We have previously shown that proliferation of breast cancer cells in vitro is decreased when treated with serum from rats with placenta ischemia‐induced model of preeclampsia. We hypothesize that anti‐angiogenic factors elaborated by the ischemic placenta such as soluble fms‐like tyrosine kinase‐1 (sFlt‐1), a vascular endothelial growth factor antagonist, inhibits proliferation of breast cancer cells by reducing Erk 1/2 and Akt signaling. To this end, we treated MCF‐7 and LCC9 breast cancer cells with 15 ng/mL sFlt1 for up to 24 hrs and measured proliferation and Akt and Erk signaling. sFlt‐1 reduced MCF‐7 (66.8% ‐ 24h) and LCC9 (98.8% ‐ 24h) proliferation, and decreased Akt activation 44.9% within 5 minutes of treatment and 73.0% 24 hours post‐treatment. Erk1/2 phosphorylation was reduced by 92.6% after 30 minutes of treatment and by 24 hours Erk1/2 activation was minimal in both the control and treated cells. Taken together sFlt‐1 may act on key regulators of cell survival and proliferation at early time points to regulate breast cancer cell proliferation and survival. Understanding the mechanisms responsible for these observations may reveal novel therapeutic approaches to cancer prevention and treatment.
A high fat diet induces glucose intolerance and decreases GLUT4 and estrogen receptor alpha in adipose tissue of female rats
Authors: Gorres Brittany K , Bomhoff Gregory L , Gupte Anisha A , Geiger Paige C
Published: June 2021

Clinical studies suggest that postmenopausal women are at an increased risk for type 2 diabetes (T2D). While estrogen replacement therapy (ERT) can ameliorate this risk, the molecular mechanisms of estrogen's beneficial effects remain unknown. We hypothesize that a HF diet decreases ERα and GLUT4 in adipose tissue, leading to a decrease in whole body glucose tolerance. Five month old rats were fed a chow (10% fat) or HF (60% fat) diet for 6 weeks, and a subset of animals from each diet group was ovariectomized (OVX) or sham OVX (intact ovaries). Our results show that a HF diet increases abdominal fat, decreases GLUT4 protein in adipose tissue, and decreases whole body glucose tolerance. In addition, a HF diet and OVX alone and in combination decreased ERα protein levels in adipose tissue, and there was a trend towards decreased GLUT4 protein in adipose tissue with OVX, suggesting that a decrease in ERα either by a HF diet and/or OVX may decrease GLUT4. A HF diet and OVX alone and in combination decreased ERβ in the soleus muscle, suggesting that ERβ may play a role in skeletal muscle glucose regulation. Our study shows that the decrease in whole body glucose tolerance due to a short‐term HF diet results from decreased GLUT4 protein in adipose tissue. Furthermore, this decrease in GLUT4 may be modulated by a decrease in ERα. ERT may benefit postmenopausal women by increasing GLUT4 protein through ERα.
Estrogen is a modulator of Tumor Necrosis Factor responses in the human vascular endothelium
Authors: Chakrabarti Subhadeep , Davidge Sandra T
Published: June 2021

Premenopausal women are protected against cardiovascular diseases compared to age‐matched men. However, these women are also at an increased risk of inflammatory and autoimmune diseases. Higher circulating levels of estrogen may exert an immunomodulatory effect. Given this background, we hypothesized that chronic estrogen exposure alters the endothelial response to inflammatory stimulation. We treated confluent monolayers of second passage human umbilical vein endothelial cells (HUVECs) for 24 hours with physiologically relevant doses of 17‐beta estradiol (E2) prior to stimulation with the pro‐inflammatory cytokine tumor necrosis factor (TNF). We found that E2 treatment increased TNFR2 levels without affecting TNFR1. There was a trend towards aggravation of TNF induced upregulation of leukocyte adhesion molecules such as intercellular adhesion molecule‐1 (ICAM‐1) and vascular cell adhesion molecule‐1 (VCAM‐1) on E2 pre‐treatment. There was also a change in the activation profile of nuclear factor kappa B on TNF stimulation between E2 treated and control cells. In summary, chronic E2 treatment altered the endothelial responses to TNF stimulation, involving modulation at the levels of receptors, transcriptional pathways and adhesion molecule expression. This work provides a better understanding of the interplay between estrogen and the endothelial inflammatory response in health and disease.
Functional estrogen receptors in the nucleus and plasma membrane of TM3 Leydig cell line
Authors: McGuinness Michael P , Shaw Richard A
Published: June 2021

Estrogen receptor (ER) expression and function were examined using the TM3 Leydig cell line. Both ERα and ERβ were detected using Western blot analysis and immunocytochemistry. Fluorescence was detected in both the nucleus and non‐nuclear regions. To determine if the nuclear receptors were functional, cells were transfected with a reporter gene containing an estrogen response element. There was a significant decrease in luciferase activity in cells treated with estradiol (10, 100, and 1000 nM) compared to controls. To determine it the non‐nuclear fluorescence seen by immunocytochemistry was on the plasma membrane, cells were incubated with fluorescent labeled estradiol or non‐labeled estradiol at 4°C, rinsed with buffer, and levels of fluorescence measured by fluorometer. Significant levels of fluorescence were detected on the surface of these cells. To determine the function of these receptors, TM3 cells were treated with 17β‐estradiol for 5 minutes and protein kinase C activity measured using PepTag assay kit (Promega, Madison, WI). PKC activity was significantly decreased at 100 nM and 200 nM 17β‐estradiol compared to control. In summary, the TM3 Leydig cell line expresses both ERα and ERβ. These receptors may function as nuclear receptors that can down regulate transcription as well as putative plasma membrane receptors capable of decreasing protein kinase C activity.
A phenotypic study of intraflagellar transport and FLA10 in the lf4 mutant of Chlamydomonas reinhardtii
Authors: Meek Bill , Wilson Nedra , Pargeter Kevin
Published: June 2021

Flagellar length is a controlled mechanism in the biflagellate alga Chlamydomonas reinhardtii. Several long flagellar mutants have been isolated and used to demonstrate that this is a dynamic process. We characterized the morphology of the lf4 mutant using immunofluorescence (IMF) and scanning and transmission electron microscopy (TEM). Using IMF, the FLA10p and LF4p localized to flagella in a punctate pattern, likely indicating intraflagellar transport (IFT) particles. No bulbous tips were observed in lf4, unlike the lf2 and lf3 mutants as reported by Tam and coworkers (2003, 2007). Multiple IFT particles were observed in cross‐sectional and longitudinal flagellar ultrastructure of lf4 using TEM. Plus, an as yet unidentified electron‐dense structure in an lf4 cross‐section was observed to connect the outer doublet microtubules to the cell membrane. Quantification of IFT particles per length, area and volume of flagella revealed that there were twice as many IFT particles in cross sections of lf4 vs. wild‐type cells. The regulation of flagellar length appears to involve not only regulation of flagellar assembly and disassembly by IFTs but also other signal pathways operating in flagellar assembly and disassembly.
Notch and transforming growth factor‐beta signaling pathways cooperatively regulate vascular smooth muscle cell differentiation
Authors: Tang Yuefeng , Liaw Lucy
Published: June 2021

Notch and transforming growth factor‐beta (TGFβ) play pivotal roles during vascular development and the pathogenesis of vascular disease. The interaction of these two pathways is not fully understood. The present study utilized primary human smooth muscle cells (SMC) to examine molecular cross‐talk between TGFβ1 and Notch signaling on contractile gene expression. Activation of Notch signaling induced smooth muscle alpha‐actin (SM actin), smooth muscle myosin heavy chain (SM‐MHC), and calponin1, and the expression of Notch downstream effectors hairy‐related transcription factors (HRT). Similarly, TGFβ1 treatment of HASMC induced SM actin, calponin1, and smooth muscle protein 22‐α (SM22α) in a dose and time‐dependent manner. HRT proteins, which antagonize Notch activity, also suppressed the TGFβ1‐induced increase in SMC markers, suggesting a general mechanism of inhibition. We found that Notch and TGFβ1 cooperatively activate SMC marker transcripts and protein through parallel signaling axes. Moreover, the combination of Notch activation and TGFβ1 treatment led to synergistic activation of a TGFβ1 responsive SM specific gene promoter by the interaction of CBF1 and pSmad2/3. Thus, Notch and TGFβ1 coordinately induce a molecular and functional contractile phenotype by co‐regulation of Smad activity at SMC promoters.

Grant Funding Source: NIH NCI
Myocardin‐related transcription factor‐A (MRTF‐A) is a key regulator of myofibroblast formation and function
Authors: Crider Beverly , Risinger George , Howard Eric , Haaksma Carol , Tomasek James
Published: June 2021

Myofibroblasts (MFs) are specialized contractile cells that arise from fibroblasts and facilitate wound closure and can promote pathological contractures. MFs have “super‐mature” focal adhesions and express α‐smooth muscle actin (ASMA) and other smooth muscle specific cytoskeletal proteins (SMCPs). Myocardin‐related transcription factor A (MRTF‐A/MAL/MKL‐1) is a putative mechanical stress‐induced co‐activator of serum response factor that activates expression of contractile proteins. We hypothesize that MRTF‐A is a key regulator of these contractile genes which allows for the formation and function of MFs. Rat embryonic fibroblasts (REF‐52) transfected with a constitutively active MRTF‐A acquired the MF phenotype as determined by increased expression of ASMA, SM22α, and smooth muscle γ‐actin (SMGA), as well as, an increased ability to contract a deformable silicone substrate. REF‐52 cells were treated with transforming growth factor‐β1 to promote MF formation. Small interfering RNA specific to MRTF‐A was used to knockdown expression of MRTF‐A in MFs, and resulted in decreased expression of ASMA, SM22α, and SMGA and decreased ability of MFs to contract a wrinkling substrate. These results suggest MRTF‐A functions as a regulator of MF formation by playing a critical role in regulating the expression of SMCPs and the resultant contractile activity. Funded by NIH grant R01 GM60651.
Functional heterogeneity of muscle satellite cells in both somite‐derived and branchiomeric muscles
Authors: Ono Yusuke , Boldrin Luisa , Knopp Paul , Morgan Jennifer E. , Zammit Peter S.
Published: June 2021

Muscle satellite cells are responsible for postnatal muscle growth and regeneration. Most skeletal muscles of the trunk and limb are derived from somites, but head muscles originate from cranial mesoderm. Here we first investigated the range of functional properties of satellite cells and then determined whether they were different in cells with separate embryonic origins, before examining how satellite cells respond to aging. Cultured satellite cells from head muscles proliferated more and differentiated later than those from limb muscles. Although some gene expression profiles in head‐derived satellite cells were different from limbs, head cells were able to regenerate limb muscles when transplanted. Surprisingly, clonal analysis showed that functional properties differed markedly between satellite cells: ranging from cells that proliferated extensively and gave rise to both differentiated and self‐renewed progeny, to other clones that divided only a few times before then differentiating completely. This heterogeneous distribution in satellite cell properties was preserved in both limb and head muscles from old mice. In conclusion, satellite cells exist as a functionally heterogeneous population, with many occupants of the satellite cell niche exhibiting stem cell characteristics in both somite‐derived and branchiomeric muscles. This study is supported by The Muscular Dystrophy Campaign.

Grant Funding Source: Muscular Dystrophy Campaign. Medical Research Council. Wellcome Trust
Anatomical education: bringing a "dead" science to life through a hybrid online approach
Authors: Bee Mary Tracy , Grabowski Greg , Montante James
Published: June 2021

Educating students in the field of anatomy has been a challenge for decades. Bringing a “dead” science to “life” has been problematic for even the greatest of educators. We sought an approach to teaching anatomy that incorporated multiple teaching styles and formats to keep student learning engaging, stimulating, and self‐motivating. We achieved our goal by incorporating an online component for both the lecture and lab for our graduate and upper‐level students, including team‐based learning. Students attended weekly in‐class lectures and labs, which included team‐based learning. Each week they were involved in interactive on‐line lectures with voice and quizzes. To help students understand the nuances of human dissection, students conducted dissections through layers of the body via a virtual anatomy dissector, which prepared them well for their weekly cadaver dissection. Grades increased 12% and student satisfaction increased 64% with this format. The highest rated component was the on‐line lectures with voice, which we will demonstrate. We highly recommend this approach for successfully teaching anatomy.
Evaluation of computer visualizations developed for anatomy education
Authors: Nguyen Ngan , Nelson Andrew , Wilson Timothy D.
Published: June 2021

Introduction

The use of computer visualizations in anatomy courses is expanding, but there is little evidence available suggesting that these visuals enhance learning of spatial anatomical information.

Purpose

To evaluate computer visualizations of the superior mediastinum developed with different display modes and interactive control features.

Methods

Participants will be students from the University of Western Ontario. General information about the participants, including their gender, program of study, and past experiences with spatial tasks will be collected in a baseline questionnaire. A mental rotation task that has been standardized for university students will be used to determine participants' spatial ability. Next, participants will be randomly allocated to one of three visual groups: stereoscopic 3D, 3D, or 2D. Participants in each visual group will be subdivided into one of two interactive groups: active control or no control. Each participant will complete an identical standardized electronic learning module pertaining to anatomy of the superior mediastinum. The module will have clearly defined learning objectives and will differ only by the visualization and manipulation capabilities of the model. Learning will be assessed by a post‐knowledge test consisting of 50 multiple‐choice questions – half spatially related and half non‐spatially related. The spatial questions are assumed to require manipulation of mental representations, while the non‐spatial questions involve the recognition and identification of anatomical structures.

Significance

The results will help establish guiding principles that will facilitate the design and implementation of effective and efficient computer visualizations that can be adapted to the individual learner's level of spatial ability.

Grant Funding Source: n/a
Promoting Student Scholarship: A Web‐Based Peer‐Reviewed Educational Resource
Authors: Pearson William G. , Zumwalt Ann , Hoagland Todd
Published: June 2021

Background

Fourth year medical students enrolled in the Teaching in Anatomy elective serve as prosectors in the BUSM gross anatomy course. Their clinical experience combined with the opportunity to revisit anatomy creates fresh insight into the clinical relevance of anatomy. Writing a case study for educational purposes allows them to engage in the scholarly process, synthesize their clinical experience with anatomical knowledge, and share this understanding with first year medical students.

Methods

During the elective students developed a case study in an area of interest to be published as a web‐based clinical anatomy educational module. They presented and reviewed their cases in groups of four to six. Powerpoint™ files are converted into Quicktime™ and posted via Apple iWeb™ to anatomycases.com. First year students are encouraged to use the site for optional study material.

Results

In the past two years fourth year students have generated 34 case‐based teaching modules on various topics. In fall semester 2009, 47% of first year students accessed the website as an optional exercise. 73% of first year students accessed the website when promised a bonus question would be derived from this optional study material.

Discussion

Students are a remarkable resource for developing educational materials if anatomy educators are available to facilitate the scholarly process. Students in turn find that doing scholarly work is more enjoyable than initially perceived and have rated this as a useful exercise (mean=4.3 out of 5). Future plans include developing a global educational component to this project to elevate social consciousness about global health needs.

Grant Funding Source: none
The development of a web‐based module on Gestational Diabetes Mellitus for patients and healthcare professionals
Authors: Clancy Alicia , Johnson Marjorie , Watson Andrew , Paul Terri , Mottola Michelle
Published: June 2021

Gestational Diabetes Mellitus (GDM), the most common endocrine disorder during pregnancy, poses short‐term and long‐ term risks for mother and baby. However, intense management of GDM has been shown to reduce negative maternal and fetal outcomes. Such management requires that both healthcare providers and patients are capable of making informed decisions in the control of GDM. Reports from local diabetes clinics and medical professionals suggest healthcare providers’ knowledge of GDM is unsatisfactory. The objective of this project was to create an interactive web‐based module to educate medical students and to provide an effective resource for healthcare providers. The case‐based, self‐study tool was designed using Adobe Dreamweaver Creative Suite 3 and Articulate Studio 09. The modular content draws from the current Clinical Practice Guidelines established by the Canadian Diabetes Association, and will be available to medical students, practicing physicians, and women with GDM. Formative feedback from a questionnaire of the module is anticipated for the spring. We expect the GDM module will educate healthcare providers and patients on the prevalence and risk factors, as well as, the importance of early screening, treatment and prevention of GDM. This empowerment tool should encourage educated decision making, facilitate the management of GDM, and curtail known risks associated with poorly managed GDM.

Grant Funding Source: Internal