bluuebunny/crossref_metadata_2025 huggingface dataset:
Ovariectomy decreases expression of transcription factors for mitochondrial biogenesis in mouse cerebrovascular endothelial cells
Authors: Kemper Martin F , Duckles Sue , Krause Diana
Published: June 2021

Estrogen has protective effects on the cerebrovasculature in physiological as well as pathological states such as stroke. However teasing out effects on individual cell types remains elusive. Thus we adapted Dynabeads (Invitrogen) technology using PECAM1 antibodies to isolate brain vascular endothelial cells. We used appropriate cellular markers to demonstrate enrichment of brain endothelial cells as well as decreased presence of other cell types. We developed a method to correct for variations in endothelial enrichment among different preparations. We found that the level of eNOS mRNA is two‐fold higher in endothelial cells from intact females compared to ovariectomized mice. This agrees with our previous observation of higher eNOS in brain vessels from estrogen‐exposed mice. We also evaluated levels of mitochondrial transcription factors in brain endothelial cells. We observed 61% and 38% higher mRNA levels of NRF1 and TFAM, respectively, in endothelial cells from intact females relative to ovariectomized mice. These two factors are essential in maintenance of respiration as well as mitochondrial biogenesis. The findings extend our previous observations that estrogen impacts cerebrovascular mitochondria. This is the first time that female reproductive competency has been shown to influence endothelial cell master regulators of mitochondrial function.
Monoamine oxidase activity promotes oxidative stress and cell death in cardiomyocytes exposed to high glucose
Authors: Kaludercic Nina , De Mario Agnese , Paolocci Nazareno , Di Lisa Fabio
Published: June 2021

Mitochondrial respiratory chain and NADPH oxidase are described as major sources of reactive oxygen species (ROS) in diabetic cardiomyopathy. Oddly, the role of monoamine oxidase (MAO) has been overlooked so far. These mitochondrial flavoenzymes are responsible for oxidative stress and cell death in failing and ischemic/reperfused hearts. Here we assessed whether MAO contribute to high glucose (HG) induced cell damage. Neonatal rat cardiomyocytes were cultured in high glucose (HG, 30 mM) for 24 or 48 hours in the absence or presence of MAO inhibitor pargyline and compared to those cultured in normal (NG, 5 mM) or high mannitol (HM, 30mM) containing media. Mitochondrial ROS production, detected by Mitotracker Red, was significantly increased in HG myocytes (+40%, p<0.001) as compared to other cells. Pargyline completely prevented this increase. Apoptosis and necrosis, assessed by bisbenzimide and propidium iodide staining respectively, were 1.8 and 2‐fold higher in HG (p<0.05). MAO inhibition significantly reduced cell death both after 24 and 48 hours of culture in HG. In conclusion, here we demonstrate that MAO play a major role in the oxidative stress and cell death in cardiomyocytes exposed to HG. MAO may amplify the effects of existing ROS triggers or HG may directly enhance MAO activity. Both phenomena are likely to be relevant for the onset and progression of diabetic cardiomyopathy.
Apocynin derivatives as potential inhibitors of NADPH oxidase
Authors: Macias Martha Edith , Martinez Federico , Padilla Itzia Irene , Correa Jose , Rosales Martha Cecilia
Published: June 2021

NADPH oxidase (nicotinamide adenine dinucleotide phosphate oxidase), is the major source of superoxide anion (O2.‐), wich is a free radical and reacts with Nitric Oxide (NO) decreasing its bioavailability in endothelial cells producing severe cardiovascular diseases such as hypertension. It had been reported that Apocynin inhibit NADPH oxidase, however, has not been used as a therapeutic treatment, due to Apocynin needs to be converts to its dimeric form by Mieloperoxidasa (MPO) and this enzyme has low affinity for Apocynin. Therefore, in this work were synthesized a set of Apocynin ethers with the main to compounds as obtain better NADPH oxidase inhibitor than Apocynin and avoiding the MPO dimerization. The inhibitory activity of the compounds was evaluated by Electron Paramagnetic Resonance (EPR) employed homogenized of aortic tissue contained NADPH oxidase of spontaneously hypertensive (SHR/NHsd) and normotensive (WKY/NHsd) rats.
CYP Pathway Modulators Alter Development and Angiogenesis in Zebrafish Embryos
Authors: Pace Jonathan Robert , Leigh Noah , Ramchandran Ramani , Imig John
Published: June 2021

Epoxyeicosatrienoic acids (EETs) are CYP metabolites of arachidonic acid that have vascular actions including vasodilation. Arachidonic acid is also metabolized by CYP hydroxylase enzymes to 20‐hydroxyeicosatetraenoic acid (20‐HETE) that causes vasoconstriction and increases vascular reactivity. These vascular properties make CYP metabolites attractive targets for therapeutics. Thus, we determined the actions of chemical compounds that modulate EETs or 20‐HETE on cardiovascular development. A zebrafish embryo model system was utilized to compare heart and caudal vascular plexus development. Tg(kdr:EGFP) zebrafish embryos were exposed to compounds at 10 hours post fertilization (hpf), and incubated until 48hpf when images were taken, quantified, and analyzed using a 2‐tailed t‐test to determine significance. Heart development was affected significantly with a 20‐HETE antagonist (n=19), eliciting a heart width of 176.7 ± 8.2 μm compared with 114.5 ± 17.1 μm in the control fish (n=4). (p<0.05). A soluble epoxide hydrolase inhibitor (sEHI), EET analogs, and combined EET analog/sEHI all caused a narrowing of the caudal vascular plexus. All compounds affecting the caudal vascular plexus caused a 13–21% narrowing compared with control fish (p<0.05). These data demonstrate that chemical compounds that modulate EETs and 20‐HETE have distinct actions on heart and vascular plexus development in the zebrafish embryos. Therefore, we conclude that 20‐HETE antagonism alters heart and vascular development whereas sEHI and EET analogs significantly alter vascular development in zebrafish.
Changes in in vivo renal artery hemodynamics after exercise training
Authors: Gurovich Alvaro N , Moningka Natasha , Baylis Chris , Delp Judy M.
Published: June 2021

Recent studies on the effects of exercise on the kidney have identified strain‐specific responses of shear dependent endothelial enzymes, endothelial nitric oxide synthase (eNOS) and extracellular superoxide dismutase (ECSOD). Here we investigated the effect of 12 weeks voluntary wheel running (VWR) (~3 km/day @wk 12) on in vivo renal artery hemodynamics in male Fisher 344 rat compared to sedentary controls (both n=6). We measured peak and mean flow velocity (PFV and MBV, respectively) using a 10 MHz Doppler probe and workstation 24h after withdrawal from VWR, under isoflurane anesthesia. After Doppler acquisition, the right renal artery diameter (RAD) was measured ex vivo to calculate peak and mean blood flow (PBF and MBF, respectively) and peak and mean shear rate. RAD, PBF, and MBF significantly decreased after ET (0.43±0.08 vs. 0.35±0.05 mm, 4.43±1.47 vs. 2.08±0.87 ml/min, 1.66±0.56 vs. 0.77±0.47 ml/min, respectively, p<0.05). However, despite the fall in PBF/MBF there were no changes observed in PFV, MBV, or shear rate. Thus, 12w of mild exercise (VWR) causes persistent reduction in renal blood flow and vascular remodeling in the renal artery. There was no fall in shear rate entering the kidney, however, which may contribute to the preservation/enhancement of renal cortical eNOS and ECSOD in the F344, which contrasts with the Sprague Dawley, where VWR reduces eNOS/ECSOD.

Funded by NIH R01 HL077224 and DK56843
The Effects of Aerobic Exercise Training on Arterial‐Ventricular Coupling in Patients with Metabolic Syndrome: A Pilot Study
Authors: Reger Brian , Donley David , Bonner Daniel , Failinger Conard , Olfert Mark , Chantler Paul
Published: June 2021

Arterial‐ventricular coupling (EA/Ees) is an important determinant of cardiovascular (CV) function, and can be indexed by the ratio of arterial elastance (EA; net arterial load), to left ventricular (LV) end‐systolic elastance (Ees; LV performance). Metabolic syndrome (MetS) patients have an abnormal CV function, which may be due to a suboptimal EA/Ees.

PURPOSE

To examine EA/Ees, at rest and peak exercise, in MetS patients before and after 8 weeks of exercise training.

METHODS

MetS patients (n=13, 46±8) were defined according to ATPIII. Using echocardiography we noninvasively characterized Ees (single beat elastance), and EA (end‐systolic pressure/stroke volume) from which EA/Ees was calculated, and compared to healthy controls (n=4, 48±7). MetS patients were separated into exercise or non‐exercising groups for 8 weeks.

RESULTS

At rest, EA/Ees was 21% lower in MetS vs. controls due to a 24% higher Ees in MetS vs. controls. The reduction in EA/Ees during exercise was blunted in MetS (31% vs. 52%, p<.01) because of a blunted increase in Ees in MetS vs. controls (102% vs. 234%, p<.05). 8 weeks of exercise training in MetS increased EA/Ees at rest, and enhanced the reduction in EA/Ees (25%, p=.05) during exercise due to a 61% (p<.05) greater increase in Ees.

CONCLUSION

The abnormal EA/Ees at rest and peak exercise in MetS patients was restored to similar levels noted in healthy controls after 8 weeks of aerobic training
Role of actin cytoskeleton and microtubules in passive diameter and viscoelastic charectistics of rat cremaster arterioles
Authors: Beig Mirza Irfan , Dolan Allison , Zhao Guiling , Hoover Erin , Martinez‐Lemus Luis A
Published: June 2021

In resistance arteries the role of actin fibers and microtubules on their passive viscoelastic charectistics are not clearly understood. Arterioles were isolated from rat cremaster muscles, and pressurized to 60mmHg and incubated at 34.5 °C. The arterioles were observed under a microscope where vessel diameter was recorded. After vessels developed tone they were washed and incubated in solution containing EGTA but not Ca++ for 30 minutes. After obtaining maximum passive diameter arterioles were subjected to different pressures before and after a 30‐minute incubation with Cytochalasin D, Latrunculin, or Demecolcine alone or in combination.

Results

Pressure diameter curves were obtained before and after actin cytoskeleton and microtubule disruption. There was no significant difference in the pressure diameter curve before and after administration of DMSO, Cytochalasin D, Latrunculin or Demecolcine alone or in combinations (p>0.05). We have previously shown that Demecholcine disrupts the microtubular network. Confocal images further indicate that treatment with Cytochalasin D and or Latrunculin successfully disrupts the actin cytoskeleton.

Discussion

Based on the above results, it can be considered that the actin fibers and microtubules do not play a major role in the passive viscoelastic characteristics of cremaster arterioles.
Structural basis for recognition of centromere histone variant CenH3 by the chaperone Scm3
Authors: Zhou Zheng , Feng Hanqiao , Zhou Bingrui , Ghirlando Rodolfo , Hu Kaifeng , Jenkins Lisa , Xiao Hua , Tjandra Nico , Wu Carl , Bai Yawen
Published: June 2021

The centromere is a unique chromosomal locus that ensures accurate segregation of chromosomes during cell division by directing the assembly of a multiprotein complex, the kinetochore. The centromere is marked by a conserved variant of conventional histone H3 termed CenH3 or CENP‐A. A conserved motif of CenH3 called CATD is necessary and sufficient for specifying centromere functions of CenH3. The structural basis of this specification is of outstanding interest. Yeast Scm3 and human HJURP are conserved nonhistone proteins that are required for the deposition of CenH3 at centromeres in vivo. Here we have elucidated the structural basis for recognition of budding yeast CenH3 (Cse4) by Scm3. We solved the structure of the Cse4‐binding domain (CBD) of Scm3 complexed with Cse4 and H4 in a single chain model. An α‐helix and an irregular loop at the conserved N‐terminus and a shorter α‐helix at the C‐terminus of Scm3‐CBD wraps around the Cse4‐H4 dimer. Four Cse4‐specific residues in the N‐terminal region of helix 2 are sufficient for specific recognition by conserved and functionally important residues in the N‐terminal helix of Scm3 through formation of a hydrophobic cluster. Scm3‐CBD induces major conformational changes and sterically occludes DNA binding sites in the structure of Cse4 and H4. These findings have implications for the assembly and architecture of the centromeric nucleosome.
Correlates of cardiovascular Risk Factors with Blood Pressure in College Adolescents in Texas
Authors: Agu Ngozi , Oloyo Ahmed Kolade , Oyekan Adebayo
Published: June 2021

The incidence of cardiovascular diseases (CVDs) is beginning to appear in children. Identifying CV risk factors at an early stage of life can provide direction for managing CVDs. This study tested the hypothesis that an association exists between CV risk factors and blood pressure (BP) in young adults. 91 young (18 – 36 years) adults (36 males, 55 females) from ethnically diverse backgrounds at Texas Southern University were evaluated for CV risk factors: race, gender, family history, residence (urban/rural), BMI, waist circumference (WC) and correlated with BP (sitting and standing) and pulse rate (PR). Results indicate that BMI correlated positively with WC and BP across all ethnic groups. The correlation was greater in males (p<0.05) and in Asians compared with other races (p<0.05). BP was higher (p<0.05) in Blacks than Asians and Whites but not different between Blacks and Hispanics. Urban dwellers have higher (p<0.05) BP compared with suburban and/or rural settlers. Family history of hypertension revealed no significant effect on BP and PR across all the races, gender, and age groups. Similarly, there was no significant difference in CV parameters across the different age groups in this study. We conclude from these data that BMI and WC are the most predictive CV parameters for BP in young adults and this is gender‐dependent. Contrary to popular belief, these CV risk factors are most predictive in Asians not Blacks.
Examination of microRNA (miRNA) dysregulation in the type 1 diabetic heart and its functional implications
Authors: Thapa Dharendra , Baseler Walter A , Croston Tara L , Lewis Sara E , Hollander John M
Published: June 2021

Dysfunctional mitochondria are central in the pathogenesis of type 1 diabetes mellitus. Previous data suggests mitochondrial proteomes are altered during diabetic insult, with impact primarily on nuclear‐encoded proteins. However, the mechanisms accounting for the dysregulation are unclear. The goal of this study was to determine whether changes in cardiac mitochondrial proteomes resulting from diabetic insult, occur through microRNA (miRNA) dysregulation. Five weeks following streptozotocin injection, 376 of the most prevalent mouse miRNAs were screened. Ten of the indentified miRNAs were statistically increased in diabetic heart, including miR‐141 (p<0.05). Using Target Scan, miR‐141 was identified as a potential regulator of the inner membrane phosphate transporter, solute carrier family 25 member 3 (slc25a3), which is essential for ATP production. Western blot analyses for slc25a3 indicated a decrease in diabetic cardiac interfibrillar mitochondria (IFM), compared to control (p<0.05). Overexpression of miR‐141 in HEK 293 cells revealed a decrease in slc25a3 protein content as compared to control, which was similar to the diabetic pathology (p<0.05). Together, these results indicate that diabetes‐induced dysregulation of miRNA may influence mitochondrial proteomes. Further, miRNA‐141 may be specifically involved via its effect on the regulation of slc25a3. (Support: NIH DP2 DK083095)
Vasomotor regulation of the human coronary circulation by hydroxyepoxyeicosatrienoic and trihydroxyeicosatrienoic acids
Authors: Larsen Brandon T , Mendoza Suelhem A , Campbell William B , Gutterman David D
Published: June 2021

12‐ and 15‐lipoxygenases (LOs) metabolize arachidonic acid (AA) to vasoactive products including hydroxyeicosatetraenoic (HETE), hydroxyepoxy‐ (HEETA), and trihydroxy‐eicosatrienoic (THETA) acids. In human coronary arterioles (HCAs), we previously observed dilation to HETEs, indicating a role for LOs in vasomotor regulation, but the most plentiful lipids observed by HPLC analysis were in a pattern suggestive of HEETAs and THETAs. We hypothesized that HEETAs/THETAs also contribute to LO‐dependent vasodilation in the human heart. AA metabolism was assayed by HPLC, 12/15‐LO expression by western blot, and HCA dilation by videomicroscopy. Human coronary artery endothelial cells (HCAECs) metabolized AA to products that coeluted with HEETA/THETA standards, and synthesis was enhanced by the endothelium‐dependent agonist ADP and the pro‐inflammatory cytokine TNFα. ADP induced vasodilation (47.5±7.1% at 10 nM, n=4) that was attenuated by the LO inhibitor CDC (11.8±6.7%). Expression of 12‐ and 15‐LO in HCAECs was increased by TNFα (4.02‐ and 1.51‐fold, respectively, at 30 ng/ml, n=2), suggesting that LO products may be particularly important in vascular inflammatory states. We conclude that HEETAs and THETAs are produced by HCAECs and play a role in endothelium‐dependent dilation. These findings indirectly support a novel role for these lipids as modulators of vasomotor tone in the diseased human heart.
Nitric oxide prevents cardiovascular collapse during shock
Authors: Cabrales Pedro
Published: June 2021

This study investigated the systemic and microvascular hemodynamic changes after severe hemorrhage. Nitric oxide (NO) availability was increased by administration of NO releasing nanoparticles (NO‐nps). Hemodynamic responses to hemorrhagic shock were studied in the hamster window chamber. Acute hemorrhage was induced by arterial controlled bleeding of 50% of blood volume, and the resulting hemodynamic parameters were followed over 90 min. Exogenous NO was administered in the form of NO‐nps (5 mg/kg suspended in 50 μl saline) 10 min following induced hemorrhage. Control groups received equal dose of NO free nanoparticles (Control‐nps) and Vehicle solution (50 μl saline). Animals treated with NO‐nps partially maintained systemic and microvascular function during hypovolemic shock compared to animals treated with Control‐nps or the Vehicle. The continuous NO released by the NO‐nps reverted arteriolar vasoconstriction, partially recovered both functional capillary density and microvascular blood flows. Additionally, NO supplementation prevented cardiac decompensation. Paradoxically, peripheral vasodilation induced by the NO‐nps did not decrease blood pressure or vascular resistance. NO‐nps promoted intravascular pressure redistribution, increased blood flow and prevented ischemia. Therefore, NO released from NO‐nps can ultimately increase survivability and reduce shock sequelae.
Characterizing the effect of long‐range basepairing interactions on translational control of the unfolded protein response transcription factor Hac1
Authors: Bolinger Cheryl G , Dever Thomas E.
Published: June 2021

Under conditions of ER stress, the unfolded protein response (UPR) induces expression of the transcription factor Hac1. In contrast, under non‐stress conditions the HAC1 mRNA is constitutively expressed, but the protein is not detected. It has been proposed that HAC1 expression is regulated by a basepairing interaction between nucleotides in the mRNA 5′ UTR and intron. The implicit mechanism is that secondary structure created by the interaction blocks scanning ribosomes and that this block is alleviated by removal of the HAC1 intron through a non‐conventional splicing reaction. The goal of the current study is to define the mechanism of HAC1 translational control.

A series of HAC1 expression plasmids was created with mutations that disrupt 5′ UTR/intron basepairing. We have found that disruption of a single basepair within the 21 basepairs reported to block HAC1 mRNA translation is sufficient to allow Hac1 synthesis. Whereas the location and identity of the mutation within the 5′ UTR element resulted in varying levels of Hac1 expression, any mutation within the basepairing element in the intron derepressed Hac1 production. Additional studies are examining the association of HAC1 mRNA with ribosomes. The results indicate that Hac1 expression may be regulated through multiple mechanisms in addition to long‐range basepairing interactions.
Sensory and sympathetic nerve contributions to the cutaneous vasodilator responses to a noxious heat stimulus
Authors: Carter Stephen J , Hodges Gary J
Published: June 2021

We investigated the roles of sensory and sympathetic nerves on the cutaneous vasodilator response to a localized noxious heating stimulus. In a two‐part study, forearm sites were instrumented with microdialysis fibers, local heaters and laser‐Doppler probes. Skin sites were heated from 33°C to 42°C or rapidly to 44°C (noxious). In Part 1, we tested sensory nerve involvement using EMLA cream. Treatments were as follows: control 42°C, EMLA 42°C, control 44°C, and EMLA 44°C. At EMLA treated sites, the axon reflex was reduced compared to the control sites during heating to 42°C (p<0.05). There were no differences during the plateau phase (p>0.05). At both the sites heated to 44°C, the axon reflex and nadir became indistinct. The EMLA treated sites were lower compared to the control sites during the plateau phase (p<0.05). In Part 2, we tested the role of sympathetic nerves in response to the noxious heating using bretylium tosylate (BT). Treatments were as follows: control 42°C, BT 42°C, control 44°C, and BT 44°C. Treatment with BT at the 42°C sites resulted in a marked reduction in both the axon reflex and secondary plateau (p<0.05). At the 44°C sites the axon reflex and nadir became indistinct, and the plateau phase was reduced at the BT treated sites (p<0.05). These data suggest that both sensory and sympathetic nerves are involved during the vasodilator response to a noxious heat stimulus. Supported by University of Alabama.
Protease‐activated receptor ‐3 signals independently to phosphorylate endothelial nitric oxide synthase in human endothelial cells
Authors: Tillery Lakeisha C. , Epperson Tenille , Austin Anita , Motley Evangeline
Published: June 2021

Protease‐activated receptor‐3 (PAR‐3) is widely recognized as a non‐signaling elusive member of a G protein‐coupled receptor family known to regulate the endothelial nitric oxide synthase (eNOS)/nitric oxide (NO) signaling pathway via phosphorylation of its regulatory sites. Failure of the eNOS/NO pathway leads to endothelial dysfunction, a precursor to cellular induced hypertension and other cardiovascular diseases. Using human umbilical vein endothelial cells (HUVECs) as a model for human endothelium, we demonstrated that PAR‐3 is highly co‐expressed with PAR‐1 and ‐2 and can be activated using a synthetic peptide, SFNGGP‐NH2. Therefore, we hypothesize that PAR‐3 can signal independently of PAR‐1 and ‐2 to regulate eNOS. Immunoblot analysis using eNOS specific antibodies showed that 25uM of SFNGGP lead to the phosphorylation of the positive site of eNOS at ‐Ser1177 and the negative site ‐Thr495 in a time‐dependent manner similar to that observed with thrombin. After HUVECs were pretreated with 10uM of SCH79797, a selective PAR‐1 inhibitor, then stimulated with SFNGGP for 5 minutes, we observed an increase in phosphorylation at both eNOS regulatory sites. Thrombin‐induced phosphorylation of these sites was inhibited in the presence of SCH79797. Our data suggests that PAR‐3 is able to regulate eNOS/NO signaling via site specific phosphorylation that is independent of the activation of PAR‐1. Cardiovascular disease is the leading cause of death among Americans, where hypertension can be a silent aggressor. Studying the signaling pathways responsible for eNOS‐induced NO production will lead to a better understanding of endothelial dysfunction in cardiovascular diseases such as hypertension.

NIH‐NIGMS‐SCORE and NIH‐NHLBI‐T32
Polarized but Differential Localization and Recruitment of STIM1, Orai1 and TRPC Channels in Secretory Cells
Authors: Hong Jeong Hee , Muallem Shmuel
Published: June 2021

In secretory cells, polarized Ca2+ signals are determined by compartmentalized localization of Ca2+ signaling proteins at the apical pole. STIM1 and the Orai channels are critical components in store‐operated Ca2+ influx. STIM1 also gates the TRPC channels. We asked how cell stimulation affects the localization, recruitment, and function of these proteins in polarized secretory cells. Inhibition of Orai1, STIM1, or deletion of TRPC1 reduces Ca2+ influx and frequency of Ca2+ oscillations. Orai1 localization is restricted to the apical side. Cell stimulation causes polarized recruitment of STIM1 to the apical and lateral regions, thus to regions with and without Orai1. STIM1 shows higher co‐localization with E‐cadherin than does Orai1, while Orai1 showed higher co‐localization with ZO1. TRPC1 is expressed in apical and basolateral regions of the plasma membrane. Co‐IP of STIM1/Orai1/IP3 receptors/TRPCs is enhanced by cell stimulation and disrupted by 2‐APB. The polarized recruitment of these proteins results in preferred Ca2+ entry that is initiated at the apical pole. These results suggest that in addition to Orai1, STIM1 likely regulates other Ca2+ permeable channels, such as the TRPCs.
Microtubule‐associated protein EB3 regulates calcium signaling and facilitates increase in endothelial permeability
Authors: Komarova Yulia A , Vogel Stephen M. , Garcia Alexander N. , Gu Lianzhi , Malik Asrar B.
Published: June 2021

End binding proteins (EBs) are highly conserved microtubule plus‐end tracking accessory factors that bind to growing microtubule ends and suppress microtubule catastrophe events. We investigated the roles of EB1 and EB3 in regulating endothelial cytoskeletal dynamics and cell shape change, the primary determinants of the permeability of endothelial barrier. Protein depletion experiments showed that EB3, but not EB1, facilitates thrombin‐induced release of calcium from endoplasmic reticulum (ER), and thereby activates calcium entry from extracellular stores. We observed that EB3 interacts with inositol 1,4,5‐thriphosphate receptor type 3 (IP3R3) in lung microvasculature. Cell permeant variant of IP3R3 derivative peptide containing EB3 consensus motif reduced the interaction between EB3 and IP3R3 and mitigated increased endothelial permeability response associated with the cell shape changes. Furthermore, infusion of IP3R3 derivative peptide in ex vivo murine lung preparation attenuates the increase in lung vascular liquid permeability (Kf,c) in response to activation of the Protease‐Activated Receptor ‐1. Intravenous injection of this peptide prevents lung injury and reduces the lethality of polymicrobial sepsis in mice. These data indicate that EB3 regulates calcium signaling in endothelium and facilitates pulmonary vascular leak in disease states.
Regulation of heteromeric Kir4.1‐Kir5.1 channel but not the homomeric Kir4.1 channel by S‐glutathionylation
Authors: Jin Xin , Yu Lei , Zhang Shuang , Zhang Xiaoli , Jiang Chun
Published: June 2021

The Kir4.1 channel is expressed in the brainstem, retina and kidney. Its heteromerization with Kir5.1 leads to K+ currents with distinct properties such as single‐channel conductance, rectification, pH sensitivity and phosphorylation modulations. Here we show that Kir5.1 also enables S‐glutathionylation to the heteromeric channel. The studies were performed in Kir4.1 and Kir4.1‐Kir5.1 channels expressed in HEK‐293 cells. Exposure to the oxidant H2O2 or diamide (DIA) produced concentration‐dependent inhibitions of the whole‐cell Kir4.1‐Kir5.1 currents. In the inside‐out patches, the currents were inhibited by a combination of 100μM DIA and 100μM GSH (76.5±14.7%, n=4) or 5mM GSSG (57.5±6.6%, n=4). The Kir4.1‐Kir5.1 currents were also strongly inhibited by several 2‐pyridinedisulfides (2‐PDS), thiol modification reagents. In contrast, none of the DIA/GSH, GSSG and 2‐PDS exposures had effects on the homomeric Kir4.1 channel. The finding that the heteromeric Kir4.1‐Kir5.1 but not homomeric Kir4.1 channel is subject to the S‐glutathionylation modulation suggests a novel regulation mechanism that may be involved in the dysfunction of the heteromeric Kir4.1‐Kir5.1 channel in oxidative stress.
Characterization of Ca2+ oscillations in pulmonary artery smooth muscle cells from patients with idiopathic pulmonary arterial hypertension
Authors: Yamamura Hisao , Yamamura Aya , Yuan Jason X.‐J.
Published: June 2021

A rise in cytosolic Ca2+ ([Ca2+]cyt) in pulmonary artery smooth muscle cells (PASMC) is a major trigger for pulmonary vasoconstriction and an important stimulus for PASMC proliferation, two major causes for elevated pulmonary vascular resistance in patients with idiopathic pulmonary arterial hypertension (IPAH). The rise in [Ca2+]cyt is mainly induced by Ca2+ release and Ca2+ influx. Under the resting conditions, we observed spontaneous and periodic increases in [Ca2+]cyt, or Ca2+ oscillations, in some (<5%) normal PASMC. Interestingly, 50% of IPAH‐PASMC showed spontaneous Ca2+ oscillations. The amplitude of spontaneous Ca2+ oscillations in IPAH‐PASMC was 5‐fold larger than in normal PASMC. Removal of extracellular Ca2+ abolished the Ca2+ oscillations in IPAH‐PASMC, while 10 μM La3+ significantly decreased the amplitude and frequency of the Ca2+ oscillations. Furthermore, application of OAG (100 μM), a membrane‐permeable diacylglycerol analogue, slightly increased [Ca2+]cyt in 30% of normal PASMC, but elicited a large increase in [Ca2+]cyt followed, sometimes, by Ca2+ oscillations in most IPAH‐PASMC. In summary, human PASMC possess spontaneous and OAG‐induced Ca2+ oscillations. The amplitude and frequency of the Ca2+ oscillations are enhanced in IPAH‐PASMC, which may contribute to the sustained vasoconstriction and vascular remodeling in patients with IPAH. NIH grants: HL 066012 and HL 098053.
Association of M3 muscarinic receptors and Kir6.1 with human detrusor caveolae
Authors: Rippe Catarina , Ekman Mari , Dabestani Saeed , Mörgelin Matthias , Karbalaei Mardjaneh , Uvelius Bengt , Swärd Karl
Published: June 2021

Caveolae are 50–100 nm large membrane invaginations that represent a plasmalemmal sub‐compartment of relevance for cellular signaling. We aimed to assess whether the muscarinic receptor M3 and the K‐ATP channel Kir6.1 are associated with human detrusor caveolae and to probe the functional relevance of this organization. Muscle strips were dissected from human detrusors and used in ultrastructural, biochemical and mechanical studies. Caveolae were ablated by cholesterol desorption with methyl‐β‐cyclodextrin. The subcellular distribution of M3 muscarinic receptors and Kir6.1 was examined using sucrose density fractionation and immunoelectron microscopy. Desorption of cholesterol right‐shifted the concentration‐response curve for the muscarinic receptor agonist carbachol. Using a K‐ATP channel inhibitor (glibenclamide) and activator (levcromakalim), we were able to inhibit and mimic this effect, respectively. Sucrose density fractionation revealed co‐fractionation of detrusor M3 receptors and Kir6.1, in partial overlap with caveolin‐1. Immunoelectron microscopy showed M3 in caveolae and in sub‐plasmalemmal caveolae‐like structures. Thus, M3 receptors are enriched with Kir6.1 in caveolae, and this organization underpins cholinergic detrusor activation in man.

Funded by Swedish Research Council (K2009‐65X‐4955‐01‐3), ALF, Crafoord Foundation.
Methylxanthine‐evoked seizure‐like perturbation of isolated newborn rat hippocampal and cortical networks
Authors: Kantor Chase M , Panaitescu Bogdan , Kuribayashi Junya , Ruangkittisakul Araya , Mildenberger Eva , Sharopov Salim , Luhmann Heiko J , Kilb Werner , Ballanyi Klaus
Published: June 2021

Treatment of (preterm) infants with caffeine and theophylline for stabilization of breathing can evoke seizures. Here, we studied in brain slices from 1–10 days‐old rats whether these methylxanthines affect early network oscillations (ENOs) in hippocampal and cortical areas that occur at rates of 15–45/min and 0.5–3/min, respectively, and presumably promote connectivity. Bath‐applied caffeine or theophylline did not substantially perturb either type of ENOs at <0.5 mM whereas 0.5–10 mM transformed ENOs into slow (0.5–5/min) large amplitude seizure‐like discharges resembling those upon GABAA receptor blockade using gabazine (1–5 μM). Both methylxanthines also provoked spontaneous and electrically‐evoked epileptiform activity in an in‐toto hippocampus preparation from 1–10 days old rats, with theophylline being more effective at low millimolar concentrations similar to its more potent action on ENOs. Findings suggest that >0.5 mM theophylline (and higher doses of caffeine) perturbs both spontaneous bursting and evoked synaptic processes in hippocampal and cortical neural networks. If such methylxanthine levels were reached in these brain structures during clinical administration, which can last for several weeks, development of connectivity may be hampered.
An anti‐bystin antibody labels hypertrophic astrocytes in zebrafish following optic nerve injury: possible marker for reactivity
Authors: Neve Luis Daniel , Savage Alissa A , Koke Joseph R , Garcia Dana M
Published: June 2021

Following nerve injury, teleost fish are capable of initiating robust signaling cascades leading to the repair of damaged tissue and a functional recovery of nerves: a phenomenon absent in amniotes. Microarray analysis of an injured zebrafish optic nerve revealed 120 genes significantly differentially expressed at one or more time points. Atf3, a member of the cAMP‐response element binding protein family, was among these genes and has been previously characterized to become upregulated in response to various stressors in nervous tissue. In the present study, we have been able to employ immunohistochemical techniques and eGFP reporter constructs to show ATF3 labeling in the bundled fiber layer and retinal ganglion cells; however, the lack of a marker for reactive astrocytes has made analyzing ATF3 expression in the glial cells of injured nerves difficult. Using antibodies against ATF3 and the protein bystin, an essential component of astrocyte development and a novel marker for the astrocyte transition from a quiescent to a reactive state in mammals, we show colocalization of bystin like protein and ATF3 in cells morphologically suggestive of hypertrophic astrocytes. These results suggest that ATF3 is expressed by reactive astrocytes in response to injury. Reactive astrocytes are the major cellular components of the glial scar in mammals, the formation of which prevents sprouting neurites from reaching their synaptic targets. The finding that the anti‐bystin antibody labels cells which morphologically appear to be reactive astroctyes supports using zebrafish as a model organism with relevance to human health.
Zinc (Zn) accumulation and metallothionein (MT‐1) expression in choroid plexus of spiny dogfish shark (S. acanthias)
Authors: Villalobos Alice R , Perry Deborah , Taylor Robert J , Young Robin K
Published: June 2021

Our objective is to determine whether Zn deficiency elicits changes in Zn accumulation and expression of the Zn‐binding protein, MT‐1 in isolated CP of spiny dogfish shark. Endogenous Zn in lateral CP, IVth CP, plasma and CSF from 5 sharks was analyzed by inductively coupled plasma mass spectrometry (ICP‐MS) and expressed in parts per million (ppm) of tissue wet weight (± 0.1 mg). Zn concentrations (mean ppm ± SE) were 0.22 ± 0.06 and 0.18 ± 0.02 in plasma and CSF but 14.16 ± 0.06 and 10.92 ± 1.78 in lateral CP and IVth CP. Zn accumulation and MT‐1 expression were examined in isolated lateral and IVth CP incubated with 0 or 10 μM ZnCl2 in elasmobranch L‐15 medium for 24 h. As per ICP‐MS, total Zn was 20 ppm in Zn‐treated CP and 9 ppm in control CP. As per real‐time RT‐PCR, MT‐1 gene expression in Zn‐treated CP was 425‐fold greater than that in control. Separate tissues were incubated with 10 μM Zn or a Zn chelator N,N,N′,N′,‐tetrakis‐2 pyridylmethyl‐ethylenediamine (TPEN, 2 μM) for 24 h. TPEN did not alter total Zn accumulation but suppressed MT‐1 gene expression by 80%. These data suggest that as extracellular Zn increases, CP accumulates additional Zn, but up‐regulation of MT‐1 may serve to bind Zn and regulate intracellular free concentrations. However, despite Zn depletion total intracellular Zn is not markedly decreased; however, decreased MT‐1 expression may in affect increase intracellular Zn availability.
Breathing pattern, pulmonary and intrabuccal gases of the South American lungfish Lepidosiren paradoxa
Authors: Minto Walter José , Giusti Humberto , da Silva Glauber , Glass Mogens L.
Published: June 2021

Genetic and physiological studies favor the lungfish as a probable sister group to the land vertebrates (Tetrapoda), and some fossil evidence is also available. As a physiological aspect, land vertebrates and lungfish possess both central and peripheral respiratory chemoreceptors. However, ventilatory pattern as well as buccal and pulmonary gases of the South American lungfish Lepidosiren paradoxa is not well studied. Therefore, our objective was to characterize respiratory pattern along with pulmonary and intrabuccal gases in the South American lungfish. Experiments were performed with five animals (n=5). Animals were submitted to surgical procedures to implant a cannula into pulmonary and buccal compartments for gases sampling (PO2 and PCO2). Pulmonary Ventilation (Ve) was measured using pneumotachograph method. The average Ve was 90.38 ± 24.59 ml ‐1 BTPS Kg h ‐1. The respiratory cycle of L. paradoxa begins with expiration followed by inspiration. The expiratory phase is marked by a series of small events. The PCO2 in the lung and buccal compartments had average values of 2.78 ± 0.3% and 3.14 ± 0.35% respectively. PO2 values were 13.78 ± 0.72% for pulmonary and 12.57 ± 1% for the buccal compartment. These results suggest a close similarity of breathing pattern between L. paradoxa and Amphibian group. Financial support: FAPESP and CNPq
Non‐radioactive RNA labeling and detection of RNA:protein interactions using a modified ribonucleotide
Authors: Opperman Kay , Etienne Chris , Schultz Jean‐Samuel , Meier Scott , Hommema Eric , Kaboord Barbara
Published: June 2021

The regulation of cellular processes is highly dependent on the interactions of RNA with proteins; however, these interactions are challenging to isolate, frequently use radioactivity for detection, and are highly dependent on the integrity of the RNA and the protein. We have developed non‐radioactive tools for end‐labeling RNA for in vitro detection of RNA:protein interactions. To achieve non‐radioactive RNA labeling, we modified cytidine to contain a 3′5′ bisphosphate for RNA ligation and a linker that can accommodate a variety of detectors. We used a biotin detector and achieved greater than 75% efficiency using either synthetic RNA or in vitro‐transcribed RNA. The biotin‐labeled RNA was able to enrich for associated proteins as assessed electrophoretic mobility shift assays (EMSA) and as bait for RNA‐binding proteins using an immobilized streptavidin sensor chip or streptavidin affinity resin. The flexibility and robustness of the labeling method, in combination with the adaptability of the chemical synthetic method, allow modification of additional nucleotides for downstream applications. These tools will enable RNA researchers to isolate RNA:protein complexes critical to cellular function, especially in the emerging field of miRNA.
Evidence Of An Inflammatory State In A Mouse Model Of Polycystic Ovarian Syndrome
Authors: Marino Joseph S , Dowling Abigail R , Qiu Xiaoliang , Nedorezov Laura , Mueller Laura. F.C , Hill Jennifer W
Published: June 2021

Pro‐opiomelanocortin (POMC) expressing neurons of the hypothalamus play an integral role in energy balance and glucose regulation. POMC neurons modulate metabolic function largely through central leptin and insulin receptor signaling. Mice lacking insulin and leptin receptor expression on POMC neurons develop obesity, insulin resistance, and infertility. The objective of this project was to test whether deletion of leptin and insulin receptors from POMC neurons leads to an altered lipid profile and a state of chronic inflammation. Serum was analyzed for triglycerides, total cholesterol, HDL, LDL/VLDL, C‐Reactive Protein (CRP), and cytokines. Adipose tissue was labeled for macrophage antigen F4/80 to investigate the accumulation of macrophages. Real time PCR was performed to quantify the expression of genes associated with an inflammatory response in adipose, liver, ovary and skeletal muscle. Serum lipids, free fatty acids, cytokines and CRP were unaltered in female mice lacking leptin and insulin receptors on POMC neurons (POMCcre+IRLepR) compared to controls (POMCcre‐IRLepR). Adipose, liver, and ovary showed evidence of macrophage accumulation and elevated mRNA of several inflammatory cytokines in POMCcre+IRLepR mice. Future studies will investigate the temporal relationship between the onset of low grade inflammation and the PCOS phenotype in POMCcre+IRLepR mice.
Role of Endogenous Angiotensin‐(1‐7) in the Regulation of Prostaglandin Synthetic Enzymes in the Uteroplacental Unit in Late Gestation
Authors: Bharadwaj Manish S , Brosnihan K. Bridget
Published: June 2021

Cyclo‐oxygenase (COX) derived prostanoids (PG) regulate numerous maternal/fetal interactions during pregnancy and have been implicated in several pregnancy disorders. Previous studies from our lab have identified the presence of Ang‐(1‐7) and its processing enzyme, ACE2, in the fetal membrane (FM), uterus (U) and placenta (Pl) during late (L) gestation (G) in pregnant (Pr) rats. The objective of this study was to determine the contribution of endogenous Ang‐(1‐7) on the regulation of PG synthetic enzymes during LG. Pr SD rats at day 11 of G were implanted with a mini‐osmotic pump for the intravenous delivery of D‐(Ala)‐Angiotensin‐(1‐7) [D‐Ala], a specific receptor antagonist for Ang‐(1‐7), for 8 days. On day 19 of G, rats were sacrificed by decapitation and FM, Pl, and U tissues were quickly snap‐frozen on dry‐ice. COX‐1 and 2, PGI synthase (syn) [PGIS], PGE syn (PGES), and thromboxane syn (TXAS) were measured with Western blot hybridization. D‐Ala treatment reduced COX‐1, COX‐2, PGIS, PGES, and TXAS in FM; up‐regulated COX‐1 and down‐regulated TXAS in the PL; and up‐regulated COX‐1 and down‐regulated COX‐2 and PGES in the U. In conclusion, endogenous Ang‐(1‐7) exerts a positive regulation on COX‐2 in FM and U in association with a specific up‐regulation of PGES, most likely increasing the production of PGE2. In the Pl, on the other hand, Ang‐(1‐7) up‐regulates TXAS. This research was supported by NHLBI PO1‐HL051952.
Photoperiod‐dependent regulation of carboxypeptidase D and E effects the selective processing of neuropeptides in the seasonal Siberian hamster (Phodopus sungorus)
Authors: Helwig Michael , Herwig Annika , Heldmaier Gerhard , Mercer Julian G , Klingenspor Martin
Published: June 2021

Energy balance in seasonal animals such as the Siberian hamster is regulated by a complex hypothalamic neuropeptidergic network. Most of the neuropeptides within this network derive from biologically inactive peptide‐precursors and have to undergo extensive post‐translational processing including C‐terminal cleavage by carboxypeptidases D and E (CPD, CPE). We investigated the effect of photoperiod on the C‐terminal activation of energy balance‐related neuropeptides, alpha‐melanocyte stimulating hormone (α‐MSH) and β‐endorphin (β‐END), within the hypothalamus of short day (SD, 8h/16h light/dark) and long day (LD, 16h/8h light/dark) acclimated Siberian hamsters. We compared hypothalamic distribution of CPD, CPE, α‐MSH and β‐END using immunohistochemistry and measured enzyme activity of CPE and concentrations of its product, C‐terminally‐cleaved α‐MSH, by radioimmunoassay in SD and LD acclimated animals. In SD, immunoreactivity (‐ir) of CPD and CPE as well as CPE‐activity was up‐regulated along with a substantial increase in C‐terminally cleaved α‐MSH and β‐END‐ir. Our results suggest that in the Siberian hamster, CPE‐mediated cleavage of neuropeptides is tightly regulated by photoperiod and may therefore be a key event in the regulation of seasonal energy balance.
The effect of dietary fat on the expression of appetite‐regulating hormones in zebrafish, Danio rerio
Authors: Tryon Sarah E , Might Natalie E. , Baker Dianne M.
Published: June 2021

We hypothesized that dietary fat levels during juvenile development affect the central expression of the appetite‐stimulating neurohormone ghrelin. To test this hypothesis, zebrafish were fed high fat (HF) or low fat (LF) diets from 5 weeks to 12 weeks post‐fertilization. Total RNA was extracted from whole heads in fed and fasted conditions, and ghrelin mRNA levels were measured by quantitative PCR (qPCR) of synthesized cDNA, and normalized to GAPDH levels. We found that fish fed a LF diet had higher ghrelin mRNA levels than fish fed a HF diet, in both fed and fasted conditions. Ghrelin expression increased significantly (p<0.05) in LF diet fish after an overnight fast, but did not increase significantly in HF diet fish. To test whether fat levels during development have long‐lasting effects on ghrelin expression, fish reared on high and low fat diets over the same period were all fed a HF diet for an additional 7 weeks, and ghrelin mRNA levels were measured in fed fish. Ghrelin expression did not significantly differ between fish reared on LF and HF diets switched to HF diets as adults (p=0.06). We conclude that dietary fat levels affect central ghrelin expression, but these effects are not permanent. effects. This project was funded by a UMW Undergraduate Research Award to ST.
Experience with Sephadex G 50 in Crude Gastrin Purification
Authors: Babatunde Adelaiye Alexander
Published: June 2021

Animal Crude Gastrin has been a major source of gastrin for gastric acid stimulant in experimental studies. The objective of this study was to evolve a method of crude gastrin purification that could yield a better gastric acid output. The Gastrin was extracted by the standard method of Gregory and tracy(1964) nodified by Adelaiye and Amure(1990)

Extraction and purificatio of Crude Gastrin has been a physiological problem since Edkins first extracted Gastrin from the antral mucosa of animals (Edkins 1905) in 1954 Tauber and Medison described a new method for gastrin purification using the cell gel chromotography process. In this experiemnt crude gastrin extract was obtained from the antrum of the Camel, Sheep and Goat and purified using Sephadex G 50. The crdue extract solution was passed through Sephadex G50( Upsala Phamacia) column chromotography. The effluent was then adminsistered to albino rats in at O.5ml. The basal gastric acid output rose from 0.23 meq/lt to 2.32meq/l in 20minutes in sephadex purified crude gastrin as compared to 0.23 to 0.27 meq/lit for the same period. This was found to be statististically significant P<0.02.

Sephadex G50 column chromotography purifies crude gastrin extract and improves the gastric stimulant affect.