bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of pannexin isoforms in the murine arterial network
Authors: Lohman Alexander W , Billaud Marie , Straub Adam C , Best Angela K , Lee Monica , Barr Kevin , Penuela Silvia , Laird Dale , Isakson Brant E
Published: June 2021

Pannexins form ATP‐release channels that have both physiological and pathological roles in cells such as regulation of vascular tone and apoptosis, respectively. However, distribution of pannexins across the vasculature is poorly documented. Therefore, we aimed to detail their distribution across different vascular beds. We first tested antibodies against pannexin 1 (Panx1), Panx2 and Panx3 on HEK cells (which do not endogenously express pannexins) transfected with plasmids for each pannexin isoform. Each of the pannexin antibodies was found to be specific. By comparing expression in C57Bl/6 and Panx1−/− mice, we demonstrate that in the systemic arterial network, Panx1 is the only isoform detected. In the large arteries and coronary circulation, Panx1 was primarily found in the endothelial cells, whereas in small arteries and arteioles Panx1 localizes to the smooth muscle cells. In the kidney, pulmonary and hepatic circulations, there was more variability in the pannexin isoforms detected. Pannexins were not detected at myoendothelial junctions across multiple vascular beds. We conclude that the specific localized expression of pannexin channels throughout the vasculature under physiological conditions points towards an important role for these channels in the regulation of ATP release throughout the arterial network.
Mast cells in proximity of adult and aged mesenteric lymphatic vessels
Authors: Chatterjee Victor , Gashev Anatoliy A
Published: June 2021

Aging impairs mesenteric lymph flow, which is crucial for fluid and macromolecule homeostasis, fat absorption, and immunity. Differences in contractile behavior of aged isolated mesenteric lymphatic vessels (MLV) and those observed in situ suggest an important regulatory involvement of some cellular elements in the immediate surrounding environment of aged MLV. We used whole mount immunohistochemistry and vital staining to identify and characterize mast cells (MCs) surrounding MLV in adult and aged Fischer‐344 rats. Additionally, we investigated the aging‐associated differences in MCs activation in the mesenteric bed in control and treated conditions. We used mesenteric tissue segments for acute treatments with 48/80, and biological activators, and used Ruthenium Red staining to identify activated MCs. Degree of activation was assessed by the image analysis of activated cells in control and treated conditions. We found a greater degree of pre‐activation of MCs in control conditions in aged rats compared to adult animals. Such pre‐activated status of aged MCs reflects lesser ability of aged MCs and adjacent MLV to adequately react to acute stimulation in elderly. These findings serve as first step in investigations of the aging‐associated mechanisms that potentially link MCs function, inflammation, and functional contractile disturbances in aged MLV. NIH R01 AG030578, HL094269.
Mitochondrial handling of excess Ca2+ is substrate‐dependent with implications on ROS generation
Authors: Aldakkak Mohammed , Stowe David F , Dash Ranjan K , Camara Amadou KS
Published: June 2021

Mitochondrial complex III is a major source of ROS during ischemia and reperfusion (I/R). Several mechanisms modulate ROS production such as mitochondrial Ca2+ uptake. Here we sought out the mechanisms of ROS generation from complex III due to excess extra matrix Ca2+. Guinea pig heart mitochondria were suspended in respiration media containing different concentrations of Ca2+, and either amplex red/horseradish peroxidase, rhodamine 123, or calcium green to measure H2O2 generation, membrane potential (ΔΨ), or [Ca2+], respectively. Either pyruvate or succinate (10 mM) was added next, followed by antimycin A (AA; 5 μM) to inhibit complex III. Mitochondria established a fully polarized ΔΨ under excess extra matrix Ca2+ with pyruvate, whereas they remained partially depolarized with succinate as the substrate. AA caused Δ Ψ depolarization for both substrates, and matrix Ca2+ extrusion which was more with succinate than pyruvate and coincided with a larger H2O2 generation that was significantly decreased with cyclosporine A, an inhibitor of permeability transition pore (PTP) opening. Succinate accumulates during I/R and becomes the major substrate utilized by mitochondria. Mitochondrial inability to maintain a polarized Δ Ψ under excess extra matrix Ca2+ when succinate, but not pyruvate, is the substrate may lead to PTP opening and enhancing ROS formation from complex III during I/R.
Mucosal repair after tissue injury requires innate immune mediated neutrophil and macrophage recruitment via IL‐6 and MIP‐2 induction by symbiotic commensal bacteria
Authors: Meng Di , Zhu Weishu , Nanthakumar Nanda
Published: June 2021

Intestinal injury leads to loss of epithelial barrier integrity and sepsis caused by microbial infiltration. The mucosal repair process includes accelerated epithelial restitution with concurrent induction of specific immune responses. To determine which arm of the immune system is responsible for tissue restoration, we used the mouse models of DSS, TNBS or OAA‐induced injury. The mouse raised in conventional facility was able to recover form injury, but the recovery from mucosal injury was compromised in bacteria‐depleted (BD) mice treated with DSS but not by TNBS or OAA. Also the ability to recruit neutrophils and macrophages after injury was significantly compromised in DSS‐treated BD mice. Gram (−) bacteria (B. fragilis and E. coli) or lipoplysaccharide (LPS) was sufficient to rescue the morbidity and mortality (40%) observed in DSS‐treated BD mice. But, Gram (+) bacteria were unable to rescue these mice. In addition, mucosal induction of MIP2 and IL6 were significantly compromised in DSS‐treated BD mice. Exogenous MIP2 was sufficient to rescue BD mice from DSS‐induced injury and to restored neutrophil and macrophage recruitment. However the ability to repair the mucosa after DSS‐induced injury was lost in IL‐6−/− mice. This suggests a previously unknown role for IL6 and MIP2 in restoring mucosal integrity by regenerating the epithelial barrier and by recruiting specific innate immune cells.

Contact information: Dr. Nanthakumar; e‐mail: nanthaku@helix.mgh.harvard.edu

Supported by NIH grant RO1HD059126
Estrogen antagonizes LPS‐induced monocyte adhesion to endothelial cells
Authors: Holm Anders , Nilsson Bengt-Olof
Published: June 2021

The biological actions of estrogens are supposed to be mediated by the two classical estrogen receptors ERα and ERβ, and one putative G‐protein coupled receptor, GPER1. Estrogen exerts both pro‐inflammatory and anti‐inflammatory effects but the mechanisms are not identified. Here, we investigate if estrogen affects recruitment and adhesion of monocytes to endothelial cells (EC) using a co‐culture system. The microvascular endothelial cell line bEnd.3 was incubated for 6–24 hours with lipopolysaccharide (LPS) from E. coli. Some wells were preincubated with 17β‐estradiol (E2), the ERα‐specifc agonist DPN or the ERβ‐specific agonist PPT. Adhesion of [3H]‐thymidine labeled human THP‐1 monocytes to bEnd.3 was determined by measuring radioactivity of DNA from co‐culture homogenates. Preincubation with E2 reduced monocyte adhesion by about 25%, whereas preliminary results show that neither DPN nor PPT had any effect. Expression of inflammation‐associated genes, including MCP‐1, VCAM‐1 and IL‐6 was increased by LPS, but not significantly decreased by E2, DPN or PPT, suggesting that estrogen reduces adhesion to EC via another mechanism. Taken together, these data suggest that estrogen‐induced attenuation of monocyte adhesion to EC involves other molecules than MCP‐1, VCAM‐1 and IL‐6 and is mediated through another mechanism than via ERα and ERβ. Supported by the Swedish research council.
Platelet Derived TLR4 Enhances Microvascular Thrombosis Independent of a Systemic Inflammatory Response
Authors: Stark Ryan , Aghakasiri Niloufar , Rumbaut Rolando
Published: June 2021

Lipopolysacchride (LPS) initiates systemic inflammatory responses and enhances microvascular thrombosis through Toll‐like receptor 4 (TLR‐4). Since platelets express TLR‐4, we examined whether platelet‐derived TLR‐4 was sufficient to mediate LPS‐enhanced thrombosis. We transfused platelets from wild‐type (WT) mice into TLR‐4‐deficient mice and exposed them to either LPS or saline. We then assessed the kinetics of photochemical injury‐induced thrombosis in mouse cremaster venules. LPS enhanced the rates of microvascular thrombosis (times to thrombotic occlusion were reduced by 40–50%) in two strains of TLR‐4‐deficient mice transfused with WT platelets (n=12 per group, p<0.05), but not in control mice transfused with platelets from TLR‐4‐deficient mice (n=12 per group, N.S.). We found no difference in platelet aggregation between WT and TLR‐4‐deficient to account for the results. Further, while LPS increased pro‐inflammatory cytokines by approximately 2 orders of magnitude in wild‐type mice, the responses were absent in TLR4‐deficient mice, despite transfusion of WT platelets. These studies provide evidence that platelet derived TLR‐4 is sufficient to mediate LPS‐enhanced microvascular thrombosis independent of a systemic inflammatory response. Support: T32 HL007939‐07, HL‐079368, and a Merit Review Grant from The Department of Veterans Affairs.
Water drinking‐induced pressor response in sinoaortic denervated rats
Authors: Abe Chikara , Iwata Chihiro , Morita Hironobu
Published: June 2021

Arterial baroreflex is well known to have a significant role in arterial pressure (AP) regulation upon acute disturbance for AP. Previous study from our laboratory demonstrated that AP was well maintained during rear‐up, if the baroreflex operated, but AP decreased by 15 ± 1 mmHg in sinoaortic denervated (SAD) rats. However, the different AP response was observed during rear‐up behavior with water drinking: i.e., the AP increased by 60 ± 5 mmHg in SAD rats. In order to clarify this mechanism, we examined the both afferent and efferent pathways in this response with SAD rats. For the efferent pathway, the pressor response was completely abolished by prazosin administration. However, for the afferent pathway, the pressor response was not suppressed by denervation of oral cavity, pharynx, or esophagus. However, magnitude of pressor response depends on the water infusion velocity via the oral tube. Furthermore, powder food feeding‐induced pressor response was significantly larger than solid food feeding. Accordingly, it is possible that the motor behavior such as swallowing might participate in the pressor response.
Regional blood flow changes underlying the hypotensive action of 5‐HT:Studies using Doppler and Microsphere technologies
Authors: Seitz Bridget Mahon , Dorrance Anne , Fink Gregory D , Watts Stephanie
Published: June 2021

Serotonin (5‐hydroxytryptamine, 5‐HT) causes a sustained decrease in mean arterial pressure (MAP) and elicits a fall in total peripheral resistance (TPR) in normal conscious rats. We hypothesized that the decrease in TPR induced by 5‐HT is caused by relaxation (direct or indirect) of arteries serving one or more major regional vascular beds. Alzet pumps containing vehicle or 5‐HT (25 ug/kg/min) were implanted in male Sprague‐Dawley rats and 24 hours later the rats were anesthetized and underwent: 1) ear and tail blood flow measurements using Doppler to determine cutaneous vascular contribution; or 2) left ventricular injection of fluorescent microspheres (300,000 yellow green microspheres) to determine global organ blood flow. Infusion of 5‐HT increased ear (69.1±20.5% time‐controlled vehicle) and tail (23.5±2.5%) blood flow (Doppler) while elevating small intestinal (50% increase) flow (microsphere) over vehicle‐infused rats. In the microsphere experiments there was no alteration in blood flow by 5‐HT infusion (vs vehicle) in spleen, liver, lung, right or left kidney, leg muscle, abdominal skin, visceral fat or brain, and right and left kidney flows were statistically similar (R; 3.78±0.69 vs L: 4.00±0.74 ml/min/g tissue, p> 0.05). These findings define the cutaneous and splanchnic vasculature as regions of interest to study the mechanism of 5‐HT ability to lower MAP and TPR. NIH HL081115.
Exercise increases plasma AICAR (5‐amino‐4‐imidazolecarboxamide riboside) in the horse
Authors: Robinson Mary A , Liu Ying , Uboh Cornelius E , Soma Lawrence R
Published: June 2021

AICAR (5‐amino‐4‐imidazolecarboxamide riboside) is endogenously produced from an intermediate in the purine biosynthetic pathway. In human athletes, urine [AICAR] correlates with gender (males > females), athletic season (in competition > out of competition), and sport (weight lifting > soccer). Plasma AICAR was hypothesized to increase with exercise. An LC‐MS‐MS method was developed to quantify AICAR in plasma collected from race horses before and immediately after competition. Plasma AICAR was increased in the majority of horses after racing (n = 52, p < 0.0001). [AICAR] did not correlate with age, gender, or performance in this population. When administered intravenously, AICAR (1.0 mg/kg/min; 10 min) acutely (minutes) decreased mean arterial blood pressure (132 to 103 mmHg; n = 1) despite a compensatory increase in cardiac output (36 to 85 L/min; n = 1) and heart rate (41±4 to 72±14 bpm; n = 4). Exercise‐induced increases in AICAR may facilitate vasodilation. This work was supported by the Racing Medication and Testing Consortium, the PA Racing Commissions, the Pennsylvania Harness Horsemen Association, the Meadows Standardbred Owners Association, and the Pennsylvania Horsemen Benevolent and Protective Association.
Acute antioxidant therapy restores baroreflex sensitivity in spontaneously hypertensive rats
Authors: Guimarães Drielle Dantas , Oliveira-Monteiro Nayanny Morais , Braga Valdir Andrade
Published: June 2021

During hypertension there is impairment in baroreflex sensitivity (BRS). Here we tested the hypothesis that acute superoxide scavenging would restore the depressed BRS in spontaneously hypertensive rats (SHR). Serum samples from SHR (n=20) and WKY (n=20) were subjected to TBARS assay before and after the administration of tiron (30 mg/Kg, i.v.), a superoxide dismutase mimetic, or apocynin (30 mg/Kg), a NADPH oxidase inhibitor. Cardiovascular parameters were recorded in male 10‐week‐old SHR (n=14) and their controls (WKY, n=14). In addition, BRS was tested using phenylephrine (8 mg/Kg, i.v.) and sodium nitroprusside (25 μg/Kg, i.v.) before and after tiron or apocynin administration. The SHR group had considered oxidative stress compared to WKY (36.2±3.0 vs. 13.3±2.6 nmol/L), SHR + APO group showed a reduction in oxidative stress when compared to SHR (36.2±3.0 vs. 21.5±3.0 nmol/L) so was the SHR + Tiron (37.2±3.9 vs. 21.9±1.6 nmol/L). Regarding BRS, SHR presented diminished BRS when compared to WKY (−1.34±0.11 vs. −2.91±0.20 bpm.mmHg−1). Acute administration of tiron improved BRS in SHR (−2.26±0.21 vs. −1.34±0.11 bpm.mmHg−1). Furthermore, acute apocynin improved BRS in SHR (−2.14±0.23 vs. −1.34±0.11 bpm.mmHg−1). Both tiron and apocynin had no effect on BRS in WKY. These data suggest that acute scavenging of NADPH oxidase‐derived superoxide improves baroreflex sensitivity in SHR.
Smaller cerebrovascular arteries have a greater age‐related endothelial dysfunction and a blunted response to life‐long caloric restriction
Authors: Walker Ashley E , McCullagh Joesph P , Lesniewski Lisa A , Donato Anthony J
Published: June 2021

Life‐long caloric restriction (CR) protects against age‐associated diseases, but its effect on cerebrovascular arteries of varying size is unknown. We measured ex vivo endothelium‐dependent dilation (EDD, response to acetylcholine [ACh]) of larger (carotid) and smaller (middle cerebral artery [MCA]) arteries from young (Y‐AL, 6 mo, n=12) and old (O‐AL, 29–31 mo, n=12) ad libitum fed and old CR (O‐CR, 29–31 mo, n=19, 40%<ad libitum) B6D2F1 mice. Aging leads to an increased systolic and diastolic blood pressure that was prevented by CR. In the carotid artery, O‐AL had lower EDD compared with Y‐AL and O‐CR (72±6 vs 95±2 and 91±2%, P<0.05). The reduction in EDD with age was greater in the MCA (O‐AL: 30±6 vs Y‐AL: 71±5%, P<0.01) compared to the carotid, but the preservation of EDD with CR was less (50±8%, P<0.05). Inhibition of nitric oxide synthase (L‐NAME) abolished the differences between groups (P>0.05). TEMPOL (superoxide dismutase mimetic) restored carotid artery EDD in O‐AL (91±3%, P<0.05), but had no effect in Y‐AL and O‐CR (P>0.05). In the MCA, TEMPOL restored EDD partially in O‐AL (43±16%, P<0.05), but diminished ACh‐induced dilation in O‐CR and Y‐AL (P<0.05). Thus, smaller cerebrovascular arteries have a greater age‐related reduction in endothelial function. Life‐long CR preserves endothelial function with age in larger arteries, but this response is blunted in smaller cerebrovascular arteries.

Supported by AG029337, AG040297, AG033196
Profound decrease in myogenic tone of parenchymal arterioles in a genetic model of cerebral ischemic small vessel disease
Authors: Dabertrand Fabrice , Bonev Adrian D , Ingalls Jill E , Brayden Joseph E , Joutel Anne , Nelson Mark T
Published: June 2021

Dominant mutations in NOTCH3 gene induce the most common heritable cause of stroke and vascular dementia, named Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL). Recently, a mouse genetic model for CADASIL was developed, exhibiting the main features of the disease. Importantly this model established that cerebrovascular dysfunction is an early event that precedes white matter lesions (Joutel et al., JCI 2010). Here we report the effects of CADASIL on arterioles within the brain. Elevation of intravascular pressure to 20 mm Hg constricted isolated parenchymal arterioles (PAs) from WT and CADASIL brains to the same extent (34%). However, above 30 mm Hg, CADASIL PAs lost myogenic tone. At 40 mm Hg, PAs from WT and CADASIL constricted 39% and 25%, respectively, and the membrane potential of the CADASIL arterioles was 10 mV more hyperpolarized. Consistent with these finding, the current density of voltage‐dependent K+ channel currents was 30% greater in myocytes from CADASIL vs WT. These results indicate that the CADASIL mutation has a profound effect on PAs tone and point to a critical role for Kv channels. The present work provides new insights about the earliest events that initiate brain lesions in this model of cerebral ischemic small vessel disease. Supported by AHA 09POST2290090 (FD), the NIH HL095488, HL44455, HL58231 and the Totman Trust for Medical Research.
Pancreatic triglyceride levels: implications for type 2 diabetes development in ethnic minorities
Authors: Szczepaniak Lidia S. , Mathur Ruchi , Szczepaniak Edward , Nelson Michael D. , Chen Ida , Victor Ronald G. , Lingvay Ildiko
Published: June 2021

The exact role of pancreatic fat in the development of human impaired glucose tolerance remains unclear. Basic research using rodent models of type 2 diabetes has identified pancreatic steatosis and lipotoxicity as a leading cause of beta cell dysfunction. We sought to translate these mechanistic studies into the clinical population. The purpose of this investigation was to determine differences in pancreatic fat content by localized 1H MR Spectroscopy across three major ethnic groups: African American (AA), Hispanic (HA), and White (WA); and to explore the association between pancreatic fat content and insulin secretion. We found differences in pancreatic fat content between AA, HA and WA. Pancreatic TG levels were higher inWA and HA, compared to AA (P < 0.05). Importantly, the level of pancreatic TG content was associated with insulin secretion. Our data suggest that pancreatic steatosis may identify a subset of asymptomatic individuals who are at high risk for development of type 2 diabetes. 1H‐MRS and measurement of pancreatic TG content may constitute a new therapeutic target. Our data also highlight a potential need for ethnically appropriate preclinical biomarkers.
Renal vascular relaxation response to glucagon‐like peptide‐1 (GLP‐1) is impaired in spontaneously hypertensive rats (SHR)
Authors: Crajoinas Renato Oliveira , Savignano Fernanda Alcantara , Nakamuta Juliana Sanajotti , Girardi Adriana C
Published: June 2021

Research in our laboratory and others have established that GLP‐1 causes diuresis and natriuresis in normotensive animals by means of tubular and hemodynamic mechanisms. Interestingly, we have recently observed that GLP‐1 does not affect renal hemodynamics in SHR. The aim of the present study was to evaluate the renal vascular effects of GLP‐1 on normotensive and hypertensive rats. By means of immunofluorescence, we verified that the GLP‐1 receptor (GLP‐1R) colocalized with endothelial and smooth muscle cell markers in renal arteries from both rat strains. However, it was noted that 100 nM GLP‐1 induced vasodilation in isolated renal arterial rings through an endothelial‐independent mechanism only in Wistar rats, and did not alter vascular reactivity in renal arteries from SHR. The lack of a vasodilator effect of GLP‐1 was not due to decreased expression of GLP‐1R in renal arteries from SHR. Besides, we observed that the activity and expression of the enzyme dipeptidyl peptidase IV (DPPIV), responsible for GLP‐1 degradation, was significantly increased in the renal artery of SHR vs. Wistar. Furthermore, although there was no difference in basal concentrations of cAMP between renal arteries of Wistar and SHR, incubation of these vessels with GLP‐1 significantly raised cAMP concentration only in renal arterial rings from Wistar, while in the SHR cAMP concentration remained similar to baseline. These data suggest that GLP‐1 has a vasodilator effect in the Wistar renal artery through binding to its receptor and elevation of vascular smooth muscle cAMP. In addition, renal arteries from SHR do not respond to GLP‐1‐mediated vasorelaxation, at least in part due to higher levels of DPPIV activity and expression, and/or by an alteration in the signaling processes that link GLP‐1R activation with the stimulation of adenylyl cyclase.

Support: FAPESP
Role of Glyoxalase System on Methylglyoxal Induced Peritoneal Thickeness in Glyoxalase Transgenic Rats
Authors: Ooba Ikuko , Mori Takefumi , Sato Emiko , Yoneki Yoshimi , Akao Kento , Ishikawa Mayuko , Kisu Kiyomi , Ito Hiroko , Saito Mai , Nakamichi Takashi , Kiyomoto Hideyasu , Miyazaki Mariko , Miyata Toshio , Sato Hiroshi , Ito Sadayoshi
Published: June 2021

Methylglyoxal (MG) has been shown to involve in the peritoneal fibrosis in peritoneal dialysis, which is metabolized by the glyoxalase system. The present study was designed to determine the role of glyoxalase system on peritoneal thickness induced by MG.

MG was intraperitoneally administered daily to either rats overexpress human glyoxalase 1 (GLO1‐Tg rats) or control rats for two‐weeks. Saline was administered as sham group. Peritoneal equilibration test was performed at the end of the study, and thickness of peritoneum was determined.

Administration of MG did not change systolic blood pressure measured by tail‐cuff method in either GLO1‐Tg rats or control rats. Significant reduction in peritoneal glucose transport was observed in MG treated control rats but was not recovered in GLO1‐Tg rats. D4/D0 glucose was reduced by MG treatment but was not altered in GLO1‐Tg rats. D/P urea did not change by MG treatment. MG increased peritoneal thickness in control rats which was attenuated in those of GLO1‐Tg rats.

We conclude that MG induces peritoneal dysfunction associated with peritoneal thickness. Although enhanced glyoxalase system did not recover peritoneal dysfunction, these results indicate that glyoxalase is responsible for peritoneal thickness, which could be a target mechanism for peritoneal fibrosis in peritoneal dialysis. (Support: KAKENHI23659438)
Differential regulation of CLC anion channel by SPAK kinase ortholog mediated multisite phosphorylation
Authors: Miyazaki Hiroaki , Strange Kevin
Published: June 2021

The CLC channel CLH‐3b is activated and inhibited by cell swelling and shrinkage, respectively, and by changes in meiotic cell cycle progression. Channel inhibition requires concomitant phosphorylation of S742 and S747 mediated by the SPAK ortholog GCK‐3. Cell swelling‐induced dephosphorylation of either S742 or S747 alone by PP1 activates CLH‐3b. Glutamate substitution fully mimics phosphorylation. However, the S747E mutant activates and inactivates in response to cell volume changes much more slowly than either wild type or S742E channels. The slower activation/inactivation of the S747E mutant is not due to altered rates of PP1 mediated dephosphorylation or rates of dephosphorylation‐induced conformational changes. GCK‐3 binds to a 4 amino acid motif located on the channel C‐terminus between the CBS1 and CBS2 domains. The 176 amino inter‐CBS “linker” also contains S742 and S747. Co‐expression of wild type CLH‐3b and GCK‐3 with wild type, S742E or S747E linkers titrates GCK‐3 away from full length channels. However, channels co‐expressed with the S747E linker exhibit altered regulation compared to channels expressed with wild type or S742E linkers. Our data suggest that the phosphorylation state of S742 and S747 may modulate GCK‐3/channel interactions. Such modulation may allow distinct modes of channel regulation under different physiological conditions and may also play a role in GCK‐3 regulation.
Calcium‐activated potassium channels expressed in retinal pigment epithelial cell
Authors: Kim Na-Hyun , Chang Jae-Seung , Cha Seung-Kuy , Kong In Deok
Published: June 2021

Retinal pigment epithelium (RPE) cell serves very important functions to support vision. The aim of this study was to identify which the expression of KCa channels and ATP‐induced Ca2+ transient lead to the activation of SK channels as well as those of BK channels in the ARPE‐19 cells. We used reverse transcription polymerase chain reaction and electrophysiological patch clamp recordings to investigate the expression and characteristics of 3 types of the Kca channel subunits, BK, IK and SK.

The presence of Ca2+ activated K+ channels were identified by expression profile of transcripts encoding BK and SK channel subunits (i.e., BK α1, BK β3, BK β4, SK1, SK3 and SK4/IK). 1‐ethyl‐2‐benzimidazolinone (EBIO), a specific SK channel activator, increased the whole‐cell current amplitude to 440.9% of control (at 50 mV, n = 8, p < 0.01), whereas apamin, a specific SK channel blockers, attenuated the outward currents by to 72.5% of control when BK and IK channels were blocked with iberiotoxin (IBTX) and clotrimazole (CLT) (at 50 mV, n = 6, p < 0.05). The single channel conductance of SK channel was 17.5 ± 0.8 pS in the symmetrical high K+ solutions (145/145 mM, n = 6). ATP (100μM) increased the NPo of the channels in cell‐attached patch mode (from 0.11 ± 0.26 to 0.40 ± 0.10 at 0 mV, n = 6, p < 0.05).

We suppose that SK channels related closely to these purinergic signals may play a role of important functions in the physiology of RPE cells.
Mechanical stress activates ATP‐sensitive K+ channels in native pulmonary epithelium (Xenopus laevis)
Authors: Richter Katrin , Clauss Wolfgang , Fronius Martin
Published: June 2021

Objective

The pulmonary epithelium is permanently exposed to mechanical forces (e.g. shear force, strain), but knowledge about their impact on pulmonary epithelial ion transport is scarce. Initial studies identified the activation of K+ channels in response to the application of hydrostatic pressure, although the identity of these channels is unknown. This study focuses on the potential contribution of ATP‐sensitive K+ channels (KATP) as part of the mechanosensitive complex in the lung epithelium.

Methods

Dissected lungs of Xenopus laevis were used for transepithelial Ussing‐chamber measurements. The Ussing‐chamber enabled an increase of the water column by 5 cm (hydrostatic pressure, HP) on the apical side that was used to mechanically stimulate the tissues while the short‐circuit current (ISC) was recorded. RT‐PCR experiments were performed to verify the expression of KATP channel subunit transcripts.

Results

Under control conditions increased HP led to a decrease of the ISC (~20%, P<0.001). This effect was repeatable over a longer time scale of 30 min and sensitive to known KATP inhibitors glibenclamide and HMR1098. RT‐PCR experiments confirmed the expression of Kir6.1 and SUR1.

Conclusion

KATP channel subunits are expressed in Xenopus lung and activation of KATP by HP indicates that they are part of a mechanosensory complex in pulmonary epithelial cells.
Cardiovascular deconditioning augments baseline breathing as well as peripheral and central chemoreflex responses
Authors: Zhou Zijin , King T Luise , Kline David D , Heesch Cheryl M , Hasser Eileen M
Published: June 2021

Cardiovascular deconditioning (CVD) results in altered central autonomic regulation including baroreflex dysfunction. However, little is known regarding the effects of CVD on chemoreflex function. We hypothesized that CVD increases baseline breathing and peripheral (hypoxic ventilatory response, HVR) and central chemoreflex responses (hypercapnic ventilatory response, HCVR). Hindlimb unloaded (HU; 2 wks) rats were used to simulate CVD. Breathing and O2 saturation were evaluated (5 min) during normoxia, increasing severity of hypoxia (12%O2, 10%O2, 8%O2), and hypercapnia (5%CO2) to determine HVR and HCVR. Under normoxic conditions, O2 saturation and breathing rate were similar while tidal volume index (VT) was increased in HU vs. control rats. Increasing the intensity of hypoxia produced a progressive decrease in O2 saturation that was greater in HU vs. control rats. Hypoxia produced an intensity dependent augmentation of breathing rate, VT and minute ventilation (VE) in both groups. Increases in breathing rate during hypoxia were similar between groups. However, the VT response was augmented in HU rats. Hypercapnia also increased VE in both groups and VE was greater in HU vs. control rats due to increased VT. Preliminary data suggest that CVD due to HU increases VT under basal conditions. Furthermore, HU enhances the peripheral chemoreflex HVR and central chemoreflex HCVR due to increased VT. HL55306
Adeno‐Associated Virus Labeling of Catecholaminergic neurons in Nucleus of Solitary Tract (NTS) of Rats
Authors: Bathina Chandra , Mifflin Steve
Published: June 2021

A2 catecholaminergic neurons located within caudal and sub‐postremal NTS play a critical role in modulating responses to physiological stress and project to multiple brain stem and forebrain hypothalamic regions. A2 neurons by definition express tyrosine hydroxylase, the rate‐limiting enzyme in catecholamine synthesis. In vitro analyses of A2 neurons in the rat have been limited as identification of A2 neurons requires labeling of neuron and post‐mortem immunohistochemistry. As a result, one does not know the phenotype of the neuron at the time of study. To overcome this challenge we have used commercially available adeno ‐ associated virus (AAV) vector mediated delivery of green fluorescent protein (GFP) labeled TH promoter (AAV‐GFP‐TH), which will incorporate into the TH genome and express GFP with the TH expression. To verify that GFP expression was restricted to catecholaminergic neurons, conventional immunohistochemistry for TH was performed on sections from AAV‐GFP‐TH injected animals. We found that, 78.4 ± 3.07% TH immunoreactive neurons to be GFP labeled and 95 ± 2.40% (n=3 sections) of the GFP expressing neurons of caudal NTS to be TH‐immunoreactive, making our technique a reliable and accurate way to label A2 neurons prior to in vitro study. Brain slices from these rats can be used for direct visualization of TH‐immunoreactive neurons without any need for further processing.
Novel effects of folinic acid supplementation in embryonic zebrafish, danio rerio
Authors: Slavnic Bojan , Miller Stefanie , Sacks Jonathan , Kosmin Sarah , Puryear T.K. , Saszik Shannon
Published: June 2021

Although periconceptional folate supplementation has lead to a decrease in the incidence of neural tube defects (Rosano et al., 1999), studies suggest that there is also a relationship with the increased incidence of autism spectrum disorders (Beard et al., 2011). The purpose of the current study was to examine the effect of folinic acid (FA) on embryonic development. Adult zebrafish, Danio rerio, were maintained using standard procedures and breed in house. After light onset, embryos were sorted into either FA (10 mM for 24 hrs) or Control. Assessments (time to hatch, gross morphology, startle response (SR), and development of swim behavior (ST)) were made at 2, 3, and 5 DPF. FA was found to alter the timing of development, with an increase in the percentage hatched prematurely to 2 DPF. Embryos were also found to be smaller at 3 DPF (M = 3.22, SD = 0.26) than the control (M = 3.37, SD = 0.19). However, by 5 DPF the FA group had larger overall body size (FA: M = 3.67, SD = 0.17 Control: M = 3.46, SD = 0.20). While both SR and ST behaviors were similar at 3 DPF, at 5 DPF the FA had lower swim time (higher anxiety). The obtained results indicate that FA exposure had an effect on both anatomical developmental and locomotor behavior. Embryos exposed to FA hatched approximately 24 hours before the control group and showed higher level of anxiety in both the startle response and swim behavior.
Cell Transplantation & Biotherapeutics‐ a course linking bio‐therapies with survival surgery & experimental assessment
Authors: Cardinal Trevor
Published: June 2021

Laboratory mice are the most commonly used model in biomedical research and often the first in vivo preclinical model used in the medical device, pharma, and biotech industries. Despite the wide‐spread use of mice in basic investigation and preclinical product evaluation, few universities offer formalized courses on experimental and surgical approaches in rodents. To address this unmet need, I developed a new course entitled Cell Transplantation & Biotherapeutics. The primary goals of the laboratory‐intensive course are to train students in: animal handling and anesthesia, aseptic surgery, and experimentation. To accomplish these goals, 24 students perform 48 femoral artery ligation surgeries to produce a model of chronic peripheral ischemia. At the time of surgery, collagen gels and collagen gels containing fibroblasts are implanted deep to the gracilis muscles. One to two weeks after surgery and cell transplantation, hindpaw pulse oximetery, gastrocnemius weight, and gastrocnemius force production is measured to determine the effectiveness of the cell transplantation therapy. The students use their results for preliminary data in a mock grant application, which they present during a mock study section. This course is well‐received by the students and provides them with an opportunity to gain skills of great value to both basic research and bio‐medical industry.
SEGMENTATION OF RENAL PARENCHYMA IN THE CASE OF PRESENCE OF ADDITIONAL RENAL ARTERIES
Authors: MATUSZ Petru , BORDEI Petru , SAPTE Elena , ILIESCU Dan , BULBUC Ionut
Published: June 2021

The presence of additional renal additional arteries represents the most often renal vascular variation. The analysis of the segmentation of the renal parenchyma renal in the case of the presence of additional renal arteries was performed on a number of 75 renal corrosion casts. The renal corrosion casts with the presence of additional renal arteries represented 20% from the whole studied trial (375 renal corrosion casts). The corrosion casts were realized by injecting the renal vasculo‐ductal systems with plastic mass, followed by the corrosion of the renal parenchyma with hydrochloric acid. There have been revealed 66 corrosion casts (88%) with 2 renal arteries and 9 casts with 3 renal arteries (12%). 68% of the corrosion casts presented 5 renal segments; 25.33% presented 6 renal segments; 4% of the casts presented 7 segments, 2.67% presented 8 renal segments. The main renal artery of the kidney is: the superior renal artery in 64% of the cases; the middle renal artery in 5.33% of the cases and the inferior renal artery in 30.67%. The knowledge of these morphological aspects is important for the urologic surgery of resection and transplantation (Supported by UEFISCDI 42097/2008).
The dirk‐tooth Smilodon and the cookie‐cutter Xenosmilus, both saber‐tooth ambush predators, show differences in killing bite strategy, forelimb musculoskeletal proportions and shoulder and elbow joint ranges of movement, demonstrating there is more than one suite of adaptations that allows for the killing of large prey
Authors: Naples Virginia L.
Published: June 2021

Dirk‐tooth Smilodon and scimitar‐tooth Xenosmilus sabercats were ambush predators. With short legs, plantigrade hind feet and heavy musculature both were adapted for capturing and killing large‐bodied prey. The Smilodon skeleton was described previously; that of Xenosmilus recently. The latter included a functional reconstruction with muscle maps, showing that muscle scars reveal more than one way to capture large prey. Each species emphasizes different musculoskeletal characters. Specifically, Smilodon's humeral head is ovate, that of Xenosmilus rounded, suggesting lateral glenohumeral movement restriction in Smilodon. In contrast, the humeroulnar articulation is less restricted, giving Smilodon a greater forearm range of motion, although distal limb movements are primarily anteroposterior. Limb muscle proportions in both also differ. Although each has an equally robust brachialis origin, it is more proximal in Smilodon, i. e., this cat emphasized humeroulnar joint flexion speed. Collectively, anatomic differences suggest that Smilodon had a more stereotyped prey killing method than Xenosmilus, a conclusion that agrees with requirements for extremely precise killing bites in the dirk‐tooth in comparison to the scimitar‐tooth. These results also demonstrate that prey‐capture demands outweigh phylogenetic history, resulting in generally similar ecomorphs that differ in anatomic detail.
Is the phrenic nerve myelinated fiber distribution affected in short term experimental diabetes?
Authors: Alcântara Adriana Cristina Licursi , Tanaka Natália Massumi , Fazan Valéria Paula Sassoli
Published: June 2021

Previous reports from our laboratory showed that chronic experimental diabetes affects the shape of myelinated fiber normal distribution histogram in the phrenic nerve [1]. We aimed to investigate if this distribution is also affected in the short term experimental diabetes. Male Wistar rats (N=6) were rendered diabetic for 15 days, by a single injection of streptozotocin. Control rats received vehicle. Under pentobarbital anesthesia, rats were killed and phrenic nerves were prepared for semi‐thin transverse sections investigated by light microscopy and morphometry. Our results show that control nerves myelinated fiber distributions are bimodal, with peaks at 2.0 and 5.0 μm. Diabetic nerve distributions were skewed to the right, with peaks at 2.5 and 5.5 μm, showing reduction of the frequency of the large fibers. Control nerves myelinated axon distributions are unimodal, with peak at 3.0 μm and diabetic nerve distributions were skewed to the right, with peak at 3.5. G‐ratio distributions were unimodal in both groups, being the diabetic nerves distribution skewed to the left, suggesting the presence of axonal atrophy. Our results confirm previous reports [1] of axonal neuropathy in phrenic nerves of diabetic rats, adding to the literature that this neuropathy has a very early onset.
Sampling method for morphometry of the myelinated fibers of sural nerves in adult rats
Authors: Silveira Fernando Braga Cassiano , Sanada Luciana Sayuri , Fazan Valéria Paula Sassoli
Published: June 2021

Morphometric analysis of peripheral nerves is time consuming, tedious, difficult to perform correctly and subject to many sources of errors. Researchers try to develop sampling methods to reduce the number of structures evaluated, particularly the myelinated fibers. Nevertheless, there is no consensus on which is the best sampling method or even if there is a method that reproduces with reliability the complete evaluation of the endoneural space of a determined nerve, investigated without any sampling method. We aimed to investigate a sampling method that is able to reproduce the data obtained by the integral morphometric analysis of the sural nerves in adult rats. Sural nerves were prepared for light microscopy and computer morphometry. Microscopic scanning of the endoneural space was completed by the automatically shifting of digitalized frames under the 100× lenses on the way that the sequenced digitalized frames, when put together, reproduce the complete image of the fascicle. Morphometric analysis was performed on 30%, 50% and 70%, and compared to 100% of the frames digitalized from the fascicle. The One Way ANOVA test was performed and no significant differences were found in any of the investigated parameters. The sampling methods were efficient in representing the evaluation of 100% of sural nerve fibers, being 30% enough on the morphometric evaluation of sural nerve in adult rats.

Grant Funding Source : FAPESP, CNPq, CAPES and FAEPA
The Effects of Glucocorticoids and Voluntary Running on Spinal Curvature Deformation in Mdx Mice
Authors: Plochocki Jeffrey H , An Daniel , Simons Erin L.R. , Brereton Daniel S
Published: June 2021

In a previous experiment we found voluntary running exercise accelerated the progression of thoracolumbar kyphosis in mdx mice, the model for human Duchenne muscular dystrophy. In the present study, we test the hypothesis that glucocorticoid treatment concurrent with voluntary running exercise slows the progression of spinal deformation in mdx mice. Both glucocorticoid treatment and exercise have been shown to prolong muscle function in this animal model, although neither treatment has been thoroughly investigated in regard to spinal curvature deformation. In this experiment, mice were treated with twice weekly injections of the glucocorticoid methylprednisolone (MP, 5 mg/kg) or treated with voluntary exercise combined with MP for four weeks. We found no difference in kyphotic index between treatment groups or with controls from our previous study (P > 0.05). Histological morphometric analyses revealed no difference in paravertebral muscle fibrosis or vertebral bone area between groups (P > 0.05). Our findings suggest glucocorticoid treatment mitigates the acceleratory effects of voluntary running activity on pathological kyphosis we previously identified in dystrophic mice.
Footprinting analysis of the interactions between E. coli RNA polymerase and promoter DNA that produce very long abortive transcripts (VLATs)
Authors: Thandar Mya , Lee Ahri , Hsu Lilian M
Published: June 2021

T5 phage N25 is a prototypical strong Eσ70 promoter whose transcription initiation is rate‐limited at the promoter escape step, leading to extensive abortive initiation. The position of promoter escape depends not only on the promoter‐polymerase affinity but also on the initial transcribed sequence (ITS). Thus, N25 aborts to +11 and escapes at +12 whereas its variants with ITS changes— N25anti (+3 to +20) and DG203 (+3 to +10)—aborts to +15 and +19 and escapes at +16 and +20, respectively. Studies with GreB showed that while all abortive transcripts longer than 4 nt can be rescued (via cleavage‐reelongation)—suggesting they arose by RNAP backtracking, a fraction of the VLATs (16–19 nt) is resistant—indicating that they are formed by a mechanism different from RNAP backtracking (Chander et al. 2007 Biochemistry 46, 12687). We proposed that GreB‐resistant VLATs are the products of RNAP hyper‐forward translocation during escape. We tested this mechanism by placing EcoRI roadblock at various locations in DG203 downstream. The results indicate that GreB‐resistant VLATs are produced only when RNAP can forward translocate (via scrunching) to the 16th –19th positions and, during subsequent escape, hyper translocate when a spatial allowance of 2 bp (optimally 4–5 bp) downstream exists. Additional proofs are derived from KMnO4 and Exo III footprinting analyses. Supported by NSF‐RUI Grant (MCB‐ 0841452) to L. M. Hsu
A role for stem‐loop 4 of U1 snRNA in splice site pairing
Authors: Sharma Shalini , Wongpalee Somsakul , Black Douglas
Published: June 2021

Critical steps in the assembly of spliceosome are the association of the U1 snRNP with the 5′ splice site and of the U2 snRNP with the branch point/3′ splice site to form the prespliceosomal A complex. Interactions that pair the two splice sites within the prespliceosome are critical in committing the reaction to a particular splicing choice. These choices must occur with a high degree of specificity and fidelity for the appropriate expression of functional mRNAs. Candidate factors for mediating contact between the 5′ and 3′ splice site complexes include the U2 snRNP associated DEAD box protein Prp5 and SR related proteins. However, it is likely that additional interactions occur.

Using a U1 snRNP suppression/complementation assay, we show that the stem‐loop 4 of U1 snRNP (U1‐SL4) is essential for splicing. Results show that the G‐C rich region at the base of the stem is important. Using a combination of Stable Isotopic Labeling of Amino Acids in Culture (SILAC), biotin/Neutravidin affinity pull down, and mass spectrometry we find that U1‐SL4 interacts with proteins of the SF3a complex, which is a component of the U2 snRNP. Addition of free SL4 to the splicing reaction inhibits splicing and blocks complex assembly at the transition from E to A complex, indicating a role for this region of U1 snRNA in splice site pairing and commitment to splicing.
ANALYSIS OF DIFFERENTIALLY EXPRESSED PROTEINS IN ESCHERICHIA COLI WHEN EXPOSED TO L‐TELLUROMETHIONINE
Authors: Broderick Kathleen Marie , Boles Jeffrey O , Brinkley Sarah
Published: June 2021

Analyzing the three‐dimensional structure of a protein using x‐ray crystallography can be improved by incorporating unnatural amino acids into that protein. Most naturally occurring atoms do not have sufficient mass to diffract electrons. Using unnatural amino acids provides a solution to this problem. Bioincorporation of L‐Telluromethionine can provide residues with sufficient mass for electron scattering using a very simple procedure. However telluromethionine has cytotoxic effects which reduce cell growth and uptake. A proteomic analysis of telluromethionyl Escherichia coli was carried out using the methionine auxotroph E. coli DL41(DE3) (pCock) expression system. Both telluromethionyl and methionyl exposed cultures were analyzed with the objective of identifying differentially expressed proteins. Analysis was carried out using 2D gel electrophoresis followed by tryptic digestion, and peptide fragment analysis using nano‐ESI/qTOF/MS. Cell cultures harvested at the beginning of the stationary phase of growth are compared to cells harvested at the maximal expression (with and without exposure to TeMet). In addition, the proteome is also examined using ion exchange chromatography to fractionate the proteome prior to 2D gel analysis. The biological roles of differentially expressed proteins will be elucidated and applied to improving methods for the bioincorporation of telluromethionine.