bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of extracts rich in phytosterols, flavonoids and saponins from black bean (Phaseolus vulgaris L.) seed coats on gene expression involved in β‐oxidation and lipogenesis
Authors: Chavez‐Santoscoy Rocio Alejandra , Tovar Armando , Granados Omar , Ortiz‐Ortega Victor Manuel , Torres Nimbe , Serna‐Saldivar Sergio , Gutiérrez‐Uribe Janet Alejandra
Published: June 2021

Liver X receptors (LXR) α and β are transcription factors that regulate the expression of key genes involved in hepatic fatty acid synthesis (FAS, SREBP‐1c) and reverse‐transport cholesterol(ABCG5/G8) that are activated by their natural ligands the oxysterols. Thus, the aim of the present work was to evaluate the effects of two extracts, one rich in flavonoids and saponins (FSE) and other rich in phytosterols (PHE) associated to black bean seed coats, on the expression of LXR‐regulated hepatic genes (ABCG5, SREBP‐1c and FAS) as well as CPT1, the rate‐limiting enzyme of the β‐oxidation of long‐chain fatty acids in primary rat hepatocytes. The results showed that FSE decreased the LXR‐regulated hepatic gene expression in a dose‐dependent manner. Interestingly, CPT1 was overexpressed when PHE was tested. Both extracts were able to suppress the effects of T090131, a synthetic LXR ligand that increased the expression of genes involved in hepatic lipogenesis. Our data suggest that addition of bioactive compounds from black bean has the capacity to decrease the activity of LXR, particularly the alpha isoform, and increased the expression of genes involved in β‐oxidation. The research support was provided by the Nutrigenomics chair at Tecnológico de Monterrey.
Fish consumption and cognitive function in older adults
Authors: Yang Yoon Jung , Yu Areum , Kim Jihye , Choi Bo Youl , Nam Jung Hyun , Kim Mi Kyung , Oh Dong Hoon
Published: June 2021

Fish consumption has been reported to lower the risk of cognitive impairment, but results are inconsistent. This study was conducted to investigate the association between fish consumption and cognitive function in Korean older adults. The subjects were 808 older adults aged 60 years and older participating in the Yangpyeong Cohort Study between July 2009 and August 2010. Dietary intake including fish consumption was estimated by using a food frequency questionnaire. Cognitive function was assessed by Korean version Mini‐Mental State Examination (MMSE‐KC). Multiple logistic regression analysis was used to determine the association between fish consumption and the risk of cognitive impairment. The mean of fish consumption was 0.57serving per day. The high fish intake group showed higher cognitive function scores than the low fish intake group. Fish consumption was inversely related to the risk of cognitive impairment after adjusting for exercise, supplement, intakes of folate, vitamin B12, fruits and vegetables (5th v. 1st quintile, OR 0.44, 95% CI 0.24–0.78, P for trend=0.002). Fish consumption may help in preventing or postponing cognitive impairment in older adults. Further studies are needed to confirm this association in different research settings. This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2012R1A1A1041792).
Longitudinal determination of total body creatine pool size and skeletal muscle mass in rats by D3‐creatine dilution
Authors: Stimpson Stephen A. , Leonard Michael S. , Clifton Lisa G. , Poole James C. , Turner Scott M. , Shearer Todd W. , Remlinger Katja S. , Clark Richard V. , Hellerstein Marc K. , Evans William J.
Published: June 2021

We recently described a new method to determine total body creatine pool size and skeletal muscle mass based on dilution of an oral dose of D3‐creatine in urinary creatinine. Enrichment of D3‐ creatinine was measured by isotope ratio mass spectrometry. Here we adapt the method to LC‐MS/MS, and investigate the potential for repeated application over time. Rats were given an oral tracer dose of D3‐creatine (1.0 mg/kg body weight) at 10 weeks of age. Urine D3‐creatinine enrichment was determined 72 hr after tracer administration and creatine pool size was calculated. Lean body mass (LBM) measured by quantitative magnetic resonance correlated with creatine pool size (r = 0.92, P = 0.0002). The rate constant for decline in urinary D3‐creatinine enrichment was slow and linear (2.73 ± 0.06 %/day). Subtracting background urinary D3‐creatinine enrichment from the enrichment following a second dose of D3‐creatine at 17 weeks permitted repeat calculations of creatine pool size. As at 10 weeks, 17 week LBM correlated with creatine pool size (r = 0.98, P < 0.0001). In addition, the change in creatine pool size was correlated with the change in LBM during the 7 weeks of somatic growth between measurements (r = 0.96, P < 0.0001). Thus, the LC‐MS/MS‐based D3‐creatine dilution method can be applied repeatedly to measure skeletal muscle mass change in longitudinal studies. These studies were funded by GlaxoSmithKline.
Nitric Oxide Inhibits the Production of a Soluble Form of Endothelin Converting Enzyme‐1.
Authors: Kuruppu Sanjaya , Rajapakse Niwanthi W , Smith Ian
Published: June 2021

We have recently identified a soluble form of Endothelin Converting Enzyme‐1 (ECE‐1) in endothelial cells, which has the potential to enhance the systemic production of endothelin‐1 in health and disease. In the present study, we determined the effect of nitric oxide (NO) on the production of a soluble form of ECE‐1. Endothelial cells were incubated with the NO donor sodium nitroprusside (0.1 – 0.5μM), Nitric oxide synthase (NOS) substrate L‐arginine (100 – 1000μM) alone or in the presence of NOS inhibitors L‐glutamine or L‐Lysine (100μM). ECE‐1 activity of the media (concentrated 5‐fold), was measured using a bradykinin based quenched fluorescent substrate assay. Production of soluble ECE‐1 was inhibited in the presence of 0.5μM sodium nitroprusside (reduction of 37.69 % compared to control, P<0.001) and 1000 μM L‐arginine (reduction of 45.88 % compared to control, P<0.001). L‐arginine mediated reduction in soluble ECE‐1 activity was prevented by L‐Lysine and L‐Glutamine (P≤0.01). Sodium nitroprusside (0.5 μM) alone had no effect on the activity of recombinant human ECE‐1.

These data provide evidence that increasing NO can inhibit the production of soluble ECE‐1 in endothelial cells. This likely represents an in direct mechanism by which NO regulates the production of the potent vasoconstrictor Endothelin‐1.
Activation of α1‐adrenergic receptors causes thromboxane‐prostanoid receptor desensitization in the aorta of the spontaneously hypertensive rat
Authors: Zhao Yingzi , Leung Susan WS , Vanhoutte Paul M
Published: June 2021

In the aorta of male spontaneously hypertensive (SHR), but not in that of normotensive Wistar‐Kyoto (WKY) rats, previous exposure to phenylephrine (PE) inhibits subsequent contractions to prostaglandin E2 (PGE2). The present study examined the mechanism underlying this inhibition. Aortic rings of adult male SHR and WKY were suspended in organ chambers for the measurement of isometric tension. In the SHR aorta, previous exposure to PE inhibited subsequent contractions to PGE2, but not those to PE or U46619. When the SHR aorta was incubated with S18886, at a concentration that only partially blocked the thromboxane‐prostanoid (TP) receptors, pre‐exposure to PE caused a significant depression of the subsequent contractions to U46619. In the WKY aorta, there were no significant differences in contraction to PGE2, PE or U46619 after previous exposure to PE. Previous exposure to PDBu also inhibited subsequent contractions to PGE2 in the SHR aorta. Incubation with calphostin C before pre‐exposure to PE abolished its effect. Thus, in the aorta of SHR but not that of the WKY, activation of α1‐adrenergic receptors causes TP receptor desensitization through phosphorylation of TP receptors by protein kinase C (PKC). Differences in expression of PKC isoforms in SHR and WKY may explain the TP receptor desensitization caused by PE in the aorta of the former (supported by the Hong Kong Research Grant Council).
S100A1 modulates inflammation and PI3/Akt signaling
Authors: Zimmer Danna B , Keeling David , Campbell Kerry , Campbell Katie , Afanador Lauriaselle
Published: June 2021

The Ca2+sensor protein S100A1 regulates numerous processes involved in Alzheimer's disease (AD) that include Ca2+ homeostasis, APP expression, tau phosphorylation, Aβ toxicity, synaptogenesis, neuronal survival and cognitive function. Nonetheless, S100A1's contribution to AD pathobiology has not been elucidated. This study uses genetic ablation in an AD mouse model (PSAPP) and neuronal cell line (PC12) to ascertain the effects of S100A1 inhibition on AD pathobiology. In PC12 cells, S100A1 ablation resulted in a 2‐fold increase in phospho‐Akt and phsopho‐GSK3β levels. Since Akt has an S100 binding motif, these results are most likely attributable to a direct interaction between S100A1 and Akt. In the PSAPP mouse model, S100A1 ablation also increased phospho‐Akt levels but decreased plaque‐associated Akt staining. These changes were accompanied by beneficial reductions in microgliosis (Iba1 burden), astrocytosis (GFAP burden) and plaque load/burden. This is the first report that S100A1 regulates PI3/Akt signaling in neuronal cells and is pro‐inflammatory in AD. Studies are underway to identify other downstream events regulated by S100A1‐Akt‐GSK3β signaling in neuronal cells and AD. Collectively, these data suggest that S100A1 inhibition may be a novel strategy for AD drug development.

Supported by the Center for Biomolecular Therapeutics.
A Physiologically Based Pharmacokinetic Model Incorporating Extrahepatic Metabolism Explains Voriconazole Pediatric Bioavailability Differences
Authors: Zane Nicole Rachele , Thakker Dhiren R.
Published: June 2021

Voriconazole, a potent antifungal, is cleared predominantly via oxidative metabolism. Clearance is 3‐fold higher and bioavailability is nearly one‐half in children compared to adults. To explain these differences, a physiologically based pharmacokinetic model was developed employing Simcyp. Physiochemical and hepatic in vitro data, derived from adult and pediatric tissues, were utilized in the model. Simulated populations contained 100 patients (10 trials with 10 patients each). Adult and pediatric clearance values were within 20 and 25% of observed values, respectively. Adult PK parameters were within reasonable estimates of published values and the bioavailability 95% confidence interval was 69–93%, well within the clinically observed range. Pediatric IV parameters were well predicted, demonstrating that the model could predict hepatic clearance. However, the oral model over predicted PK values and the bioavailability 95% confidence interval was nearly 2‐fold higher than observed. Incorporating intestinal metabolism improved PK predictions and the 95% confidence for bioavailability was 27–76%, better encompassing the clinically observed range of 44–66%. Overall, pediatric bioavailability improved when intestinal metabolism was included in the model providing an explanation for PK differences between adults and children which, until now, has not been reported.
Redox properties of mitochondria from C57BL/6J mice that lack NADP+‐transhydrogenase activity due to spontaneous NNT mutation
Authors: Figueira Tiago Rezende , Ronchi Juliana Aparecida , Ravagnani Felipe Gustavo , Oliveira Helena Coutinho , Vercesi Anibal Eugenio , Castilho Roger Frigerio
Published: June 2021

NADPH homeostasis is critical for mitochondrial H2O2 detoxification. Nicotinamide nucleotide transhydrogenase (NNT) is located in the inner mitochondrial membrane and contributes to an elevated NADPH/NADP+ ratio. This enzyme catalyzes the reduction of NADP+ at the expense of NADH and H+ reentry to the matrix. A spontaneous NNT mutation in C57BL/6J mice arose nearly 3 decades ago but was only discovered in 2005. We aimed to study redox functions of mitochondria from C57BL/6J mice. Liver mitochondria were isolated from both an NNT wild‐type C57BL/6 substrain (C57BL/6/JUnib) and from C57BL/6J mice. Refined NAD(P)H autofluorescence evaluations in respiring mitochondria revealed abnormalities in C57BL/6J mice, including an absence of transhydrogenation between NAD and NADP, the spontaneous oxidation of NADPH, and the very poor ability to metabolize organic peroxide (t‐BOOH). In addition, the mitochondria of C57BL/6J mice displayed a higher ratio of oxidized:reduced glutathione as compared to C57BL/6/JUnib wild‐type mice. The activity of mitochondrial IDH2 (a coexisting source of NADPH) was similar between both groups. Overall, our findings indicate that NNT loss due to mutation greatly affects mitochondrial NAD(P) redox homeostasis, especially upon peroxide challenge. Given these alterations, the potential scientific drawbacks of using C57BL/6J mice in biomedical research should not be overlooked.
Induction of the rat hepatic aryl hydrocarbon receptor nuclear translocator by glucocorticoids
Authors: Hunter Sarah R , Riddick David S
Published: June 2021

The aryl hydrocarbon receptor nuclear translocator (ARNT) is a shared partner between the hypoxia signaling pathway and the dioxin‐responsive aryl hydrocarbon receptor signaling pathway. Dexamethasone (DEX), a synthetic glucocorticoid known to activate both the glucocorticoid receptor (GR) and the pregnane X receptor (PXR), was shown previously to increase rat hepatic ARNT mRNA levels. To examine the roles of GR and PXR in this response, we treated male rats with the GR‐selective agonist triamcinolone acetonide (TA), the PXR‐selective agonist pregnenolone‐16α‐carbonitrile (PCN), and several doses of DEX. At 6 h following TA treatment, there was a 7.5‐fold induction of ARNT mRNA and a 4.5‐fold increase in levels of an unidentified lower molecular weight protein reacting with ARNT antibody. There was a trend for increased ARNT protein levels at 24 h after TA exposure. PCN treatment had no effect on these ARNT parameters. A low dose of DEX (1 mg/kg), shown to activate GR but not PXR, caused a 10‐fold induction of ARNT mRNA at 6 h along with a 6.5‐fold increase in levels of the unidentified ARNT antibody‐reactive protein. A trend for increased ARNT protein levels at 24 h was observed, but only at higher doses of DEX (10 to 50 mg/kg), shown to activate both GR and PXR. These findings suggest that the GR is a key mediator of the induction of rat hepatic ARNT expression by glucocorticoids. [Support: CIHR]
Adhesive Interaction of Functionalized Particles and Endothelium in Idealized Microvascular Networks
Authors: Lamberti Giuseppina , Tang Yuan , Prabhakarpandian B. , Wang Yi , Pant Kapil , Kiani Mohammad F. , Wang Bin
Published: June 2021

Objective

Leukocytes response to tissue injury/infection is mediated by a series of interactions between receptors and ligands on the endothelium and the leukocytes. Here, we characterize the adhesion profile of functionalized particles under physiological flow conditions in an idealized synthetic microvascular network (SMN) characterized by a bifurcation.

Methods

Functionalized particles were prepared by coating their surfaces with different ratios of antibodies against ICAM‐1 and E‐selectin (aICAM‐1:aE‐selectin = 100:0, 70:30, 50:50, 30:70, 0:100). The adhesion of functionalized particles to 4hr TNF‐α activated HUVECs under shear flow (0.5, 2, and 4 dyn/cm2) in bifurcating SMNs was then quantified.

Results

The level of adhesion of 50:50 aICAM‐1:aE‐selectin particles was significantly higher compared to other particles in the bifurcating SMNs (~1.5–4 folds higher). Furthermore, the adhesion of particles in junction regions was about 3–18 folds higher than that in straight sections of the SMNs. Particle adhesion did not change significantly with decreasing shear at the junction regions of SMNs for all functionalized particles.

Conclusion

The differential adhesion patterns of particles between straight sections and junction regions of bifurcating SMNs, indicates that adhesion profile of particles is highly dependent on the vascular geometry of the system used.
A comparison of Doxorubicin and Doxorubicinol concentrations in rat heart and liver tissue following anthracycline administration
Authors: Chenard Pamela , Fabris Sergio , Anderson Lindsay , MacLean David
Published: June 2021

Doxorubicin (DOX) is an anti‐cancer chemotherapeutic and although it is widely used, its accumulation over time in different tissues has not been thoroughly investigated. The purpose of this study was to examine the concentration of both DOX and its metabolite (Doxorubicinol, DOXol) in the heart and liver of male Sprague‐Dawley rats. Two doses (1.5 mg/kg and 4.5 mg/kg) were injected intraperitoneally and tissues were collected at 24, 48, 72 and 96 hrs post‐injection. In the heart, at all time points, DOX concentrations were higher (p<0.05) following the 4.5 vs 1.5 mg/kg injection (0.78±0.07 vs 1.81±0.26 nmol/g at 24 hrs, 0.37±0.06 nmol/g vs 0.96±0.06 nmol/g at 96 hrs). It was observed that DOX heart concentrations steadily decreased (p<0.05) over time for the 4.5 mg/kg dose, whereas for the 1.5 mg/kg dose, concentrations remained constant for the first 72 hrs and then decreased (p<0.05) at 96 hrs. In the liver, at all time points and for both doses, DOX concentrations were higher (p<0.05) as compared to the heart (3.85±1.1 and 5.29±0.87 nmol/g in the liver vs 0.78±0.07 and 1.81±0.26 nmol/g in the heart, at 24 hrs). While DOXol was detected in small concentrations in both heart and liver tissue, no accumulation was observed over the 96 hrs. These data suggest that in both heart and liver that DOX accumulates in a dose dependent fashion. In contrast, DOXol does not accumulate to any degree in either tissue, suggesting that it is rapidly degraded or eliminated from the tissues.

Supported by NSERC.
An in vitro model of coronary artery disease and the changes in intracellular calcium regulation during its progression.
Authors: Dineen Stacey L , McKenney Mikaela L , Sturek Michael
Published: June 2021

Ca2+ is a ubiquitous second messenger and is tightly regulated in coronary smooth muscle (CSM) cells. In coronary artery disease (CAD) Ca2+ regulation will change as well as the CSM phenotype. CSM will dedifferentiate from a contractile to a synthetic/proliferative phenotype, which will lead to neointimal thickening and CAD progression. We studied these changes by inducing CAD progression in vitro via an organ culture model. Healthy coronary rings were harvested from lean Ossabaw miniature swine, isolated under sterile conditions, and stored at 37°C in low glucose Dulbecco's Modified Eagle's Medium (DMEM) + 1% penicillin/streptomycin for up to 11 days. On the day of collection (day 0), tissue was used for histology and fura‐2 fluorescence Ca2+imaging experiments. The same was performed on days 4, 7 and 11 to assess any changes over this time period. Histology supported our hypothesis of CSM dedifferentiation by demonstrating neointimal thickening, increased collagen deposition, and loss of structural organization in the tunica media. During fura‐2 experiments, cells were exposed to 80 mM K+ to assess voltage‐gated Ca2+ channel (VGCC) activity and 5 mM caffeine to assess ryanodine receptor (RyR)‐sensitive sarcoplasmic reticulum (SR) Ca2+ store release. A significant decrease in VGCC activity was consistent with CAD progression and CSM dedifferentiation (p<0.05). There was a trend toward increased SR Ca2+ release in cells from days 4 and 7 followed by a significant decrease by day 11 (p<0.05). This in vitro model of CAD progression mimics the same trends we observed from our in vivo models of CAD.
Characterization of LPS‐Induced Inflammation in Mice Expressing Human Cholestyl Ester Transfer Protein
Authors: Murphy Beth Ann , Metzger Daniel , Gollapudi Anantha , Cumiskey Anne Marie , Johns Douglas G
Published: June 2021

Cholesteryl ester transfer protein (CETP) inhibitors increase plasma HDL and reduce LDL cholesterol in humans. In animal models, exogenous HDL ameliorates lipopolysaccharide (LPS) associated toxicities. In contrast, the ILLUMINATE trial showed that pharmacologically increasing HDL with the CETP inhibitor, torceptrapib, tended to increase infection in patients. Thus, the effect that elevating HDL has on the response to infection is unclear. To probe how HDL levels influence the toxic and inflammatory response to LPS, we injected human CETP transgenic mice and wild‐type littermates with LPS (5, 25 mg/kg). 100% of mice of both phenotypes succumbed to LPS‐induced toxicity at both doses. Despite this similarity, ex vivo LPS‐invoked TNFα release is significantly greater (p=0.005) in CETP‐tg mouse blood vs. WT controls. In addition, we showed in vivo that both circulating TNFα and endotoxin levels of CETP‐tg mice were significantly greater compared to that of WT mice injected with the same amount of LPS (5 mg/kg). Taken together these data suggest that less efficient endotoxin clearance could account for the enhanced cytokine response of CETP‐tg mice. Therefore, we hypothesize that increasing HDL via pharmacological CETP inhibition should not result in an increased incidence of infection. However, the effect of CETP inhibitors on human morbidity and mortality/outcomes, is being tested in phase III clinical trials.
Delayed Sevoflurane Preconditioning Enhances Endothelial Progenitor Migration in the Ischemia/Reperfusion (I/R) Injured Myocardium
Authors: Munteanu Adelina Vlad , Popescu Mihaela , Isvoranu Gheorghita , Ceafalan Laura , Pavel Bogdan , Marinescu Bogdan , Zagrean Leon
Published: June 2021

We aim at proving in a rat model of anesthetic preconditioning (APC) and heart I/R injury the involvement of endothelial progenitor cells (EPCs) in the protection conferred by sevoflurane. Wistar rats were treated with 1 MAC of sevoflurane; the I/R lesion was performed immediately afterwards (early preconditioned group, EAPC) or after 24 h post exposure (late preconditioned group, LAPC). Control group was ventilated with room air. Significantly raised levels of VEGF and G‐CSF evaluated with Luminex were recorded at 3 respectively 6 h post exposure. An important increase of CD34+ and CD34+/Flk1+ cells in the peripheral blood, assessed by flow cytometry, was observed at 24 h after sevoflurane administration. The number of c kit+ cells, measured by immunofluorescence one day after the I/R lesion increased considerably (3 ± 0.5) in the LAPC group compared to the control group (0.8± 0.1, p< 0.0001), but not in the EAPC group. Also, the levels of IL‐1alpha and IL‐6, two cytokines involved in resistance to ischemia, were significantly increased at 6 and 24 h post exposure. Cardioprotection conferred by APC was proved by decreased apoptosis and area of fibrosis in rats exposed to sevoflurane versus control. Thus, exposure to sevoflurane increases the levels of plasma cytokines in the early hours, and enhances mobilization and recruitment of EPCs in the damaged area during the late phase. Financed with private founds.
Endothelin‐1 Expression In Vascular Adventitia
Authors: Di Wang Hui , Rätsep Matthew , Nguyen Huy , Adams Michael
Published: June 2021

Background and hypothesis

Emerging evidence indicates that the vascular adventitia is a dynamic tissue and may play a critical role in the development and progression of vascular disorders. Endothelin‐1 (ET‐1) is a potent vasoactive peptide, mediating both vascular contractile and growth responses. Endothelial cells are a major source of ET‐1, but the possibility that vascular adventitial fibroblasts generate ET‐1 has not been explored. We hypothesized that aortic adventitial fibroblasts have the ability to produce ET‐1.

Methods

For studies in cultured cells, vascular adventitial fibroblasts were isolated from the mouse (15 weeks of age) thoracic aorta and incubated with various concentrations of angiotensin II (Ang II). mRNA levels of preproET‐1 were detected by RT‐PCR. ET‐1 levels in the culture medium were measured by ELISA. In another set of study, after being anesthetized and euthanized by exsanguinations, the aortas were isolated, formalin fixed, embedded in paraffin, and then serially sectioned. The expressions of ET‐1 were determined by immunofluorescence staining.

Results and Conclusion

Ang II induced an increase in preproET‐1 mRNA levels and the immunoreactive peptide ET‐1 levels. The increases in preproET‐1 mRNA expression and ET‐1 release were blocked by Losartan, an AT1‐receptor antagonist but not PD123319, an AT2‐receptor antagonist. In the aorta sections, although staining of ET‐1 was present throughout the vascular wall, the majority of its expression was observed in both the adventitial and endothelial layers. In conclusion, these findings demonstrate that the adventitia contributes to ET‐1 production which suggests the possibility for the adventitia to contribute to regulate vascular functions.

We would like to acknowledge our granting agency the Heart and Stroke Foundation of Canada.
Variations of the circumflex fibular artery
Authors: Canby Craig
Published: June 2021

The circumflex fibular artery is described in anatomy textbooks as frequently branching from the posterior tibial artery, or alternatively, from anterior tibial or, less frequently, the popliteal artery. The purpose of this study was to examine the variation incidence of the branching pattern of the circumflex fibular artery due to a dearth of studies in this area. Our study examined 82 lower extremities used over six years in our lower limb anatomy course. The anterior tibial artery was the most frequent source of the circumflex fibular artery (n=33; 40%) followed by the posterior tibial artery (n=26; 32%) and popliteal artery (n=19; 23%). Infrequently, it was observed stemming from the fibular artery (n=3; 4%). In one instance (1%), the circumflex fibular artery issued from the lateral inferior genicular artery. In summary, the study confirmed the three most frequent sources of the circumflex fibular artery (i.e., anterior and posterior tibial and popliteal arteries) though the incidence was greatest in the anterior tibial artery. Moreover, the fibular and lateral inferior genicular arteries may infrequently issue the circumflex fibular artery. Supported by the Anatomy Department, Des Moines University
Aminolevulinic Acid Attenuates Increases in Pulmonary Arterial Superoxide Elicited By Organoid Culture with Endothelin‐1 and Exposure of Mice to Chronic Hypoxia Through Mechanisms Potentially Involving Protoporphyrin IX‐Elicited Stimulation of Soluble Guanylate Cyclase and cGMP Protein Kinase Signaling
Authors: Alhawaj Raed O , Wolin Michael S.
Published: June 2021

Our studies have detected that treatment of mice with the heme biosynthesis precursor δ‐aminolevulinic acid (ALA, 50mg/kg/day) prevents the development of pulmonary hypertension (PH) in mice exposed to 21 days of hypoxia (10% O2). Since ALA may promote the accumulation of protoporphyrin IX, a direct activator of soluble guanylate cyclase, we investigated if the known beneficial effects of cGMP or inhibiting the actions of endothelin‐1 (ET) and Nox2 in treating PH could originate from an attenuation of ET‐elicited pulmonary arterial superoxide generation by ALA & cGMP. Organoid culture of bovine pulmonary arteries (PA) with ET (10 μM, 24hr) increased the detection of superoxide by 5uM lucigenin. This was attenuated by stimulation of protein kinase G (PKG) activation in the presence of 0.1mM ALA, 0.1mM 8‐bromo‐cGMP or 0.1mM dihydroxyepiandrosterone, or by Nox inhibition (0.1mM apocynin), respectively. ALA also activated PKG and lowered PA superoxide in mice exposed to 21D hypoxia.
Modulation of Purinoceptor mediated Ca2+ signals by β‐adrenergic receptor activation in Salivary Glands
Authors: Bhattacharya Sumit , Ampem Prince , Dartt Darlene , Giovannucci David
Published: June 2021

Crosstalk between Ca2+ signaling pathways has been demonstrated to facilitate secretory activity. However it is not known whether such a crosstalk exists between adrenergic receptor signaling pathway and a non‐cholinergic non‐adrenergic (NANC) pathway (purinergic pathway). In this study we use enzymatically isolated mouse parotid acini along with live cell Ca2+ imaging and pharmacological manipulations to assess crosstalk between these two pathways.

We showed that [Ca2+]i rises induced by selective activation of P2X4R and P2X7R were potentiated three to eightfold following treatment with the cAMP elevating compounds forskolin, 8‐cpt‐cAMP or 3‐isobutyl‐1‐methylxanthine. Importantly, rises in cAMP levels evoked by β‐adrenergic receptor activation using isoprenalin also resulted in a marked enhancement of P2X‐mediated [Ca2+]i signals.

Potentiation of P2X4R‐mediated [Ca2+]i signals following β‐adrenergic receptor activation was likely mediated by PKA because protein kinase inhibitor application in the presence of forskolin or isoprenalin abolished the enhancement of evoked [Ca2+]i signals. Interestingly, application of 2‐Aminoethoxydiphenyl borate during P2X7R stimulation also abolished the enhancement of [Ca2+]i rises following isoprenalin treatment.

The results from this study reveal a crosstalk between purinergic and β‐adrenergic signaling pathways and may provide novel targets for construction of therapies for salivary hypofunction patients.
Characterization of protein glycosylation in skeletal muscle of Type 2 diabetics after aerobic exercise training
Authors: Chen Dapeng , Wang Yan , Prior Steve J. , Ryan Alice S. , Ortmeyer Heidi K. , Blumenthal Jacob , Beans Jeffrey , Chin Eva R.
Published: June 2021

Post‐translational modification of proteins play a key role in signal transduction. We hypothesized that skeletal muscle protein glycosylation is a mechanism for explaining impaired glucose metabolism in Type 2 Diabetes Mellitus (T2DM). The aim of this study was: i) to develop a method for large‐scale analysis of N‐linked protein glycosylation in skeletal muscle; and ii) to determine whether aerobic exercise training (AEX) that improved glycemic control in subjects with T2DM would alter muscle protein glycosylation patterns. Older, sedentary T2DM subjects (75±7 yrs; HbA1c 7.1±0.5%) completed 12 mos. of AEX and skeletal muscle biopsy samples were obtained at baseline and 12 mos. training. Muscle biopsy samples were enriched for glycoproteins using lectin affinity columns and then analyzed by tandem mass spectrometry. 87 N‐linked glycoproteins were identified in skeletal muscle. By comparing spectral counts at baseline and after AEX, a decrease in number of glycosylation sites or reduced glycoprotein levels were observed. Five proteins had reduced glycan count, including isoform H17 of myeloperoxidase which was down‐regulated after AEX. This was associated with significant reduction in HbA1c of 0.8±0.1%. Our data support the notion that improved glycemic control is associated with alterations of skeletal muscle protein glycosylation patterns.
Single Muscle Fiber Power Generation with Non‐Weightbearing Conditions: Does Muscle of Origin Play a Role?
Authors: Kim Jong‐Hee , Torgerud Windy , Graber Ted , Liu Haiming , Thompson LaDora
Published: June 2021

The purpose of this study was to examine how non‐weightbearing (NWB) alters power generation of myosin heavy chain (MHC) IIB, IIXB, and IIX fibers from the plantaris (PL), tibialis anterior (TA), extensor digitorum longus (EDL) muscles and MHCI fibers from the soleus (SOL) muscle. NWB was achieved in rats by the hindlimb suspension technique (1 to 3 weeks). Single fiber power, force, and velocity were determined from force‐velocity curves and MHC isoforms were identified by SDS‐PAGE (total 387 fibers). Peak power (PP), peak force (Po) and velocity (Vmax) of MHCIIB and IIXB fibers from the TA and EDL did not change with NWB. In contrast, PP and Po of MHCII fibers from the PL were significantly reduced by 25–60% with NWB. Because NWB‐induced atrophy (3–23%) was present in the MHCII fibers from the PL, normalized power (NP) and specific force (SF) did not change with NWB. The contractile parameters (PP, NP, Po, SF) of MHCI fibers from the SOL were significantly reduced with NWB by 30–69%. The results indicate that the contractile properties of MHCII fibers from the TA and EDL, both ankle dorsiflexor muscles, are maintained during NWB. However, the contractile properties of MHCII fibers from the PL, an ankle plantarflexor, show NWB‐induced reductions. Collectively, MHC type II fibers adapt to NWB; however, the extent of adaptation is muscle‐specific. These findings are important when designing therapeutic exercise interventions.
Cytoskeletal regulation dominates proteomic changes associated with hibernation in 13‐lined ground squirrels
Authors: Hindle Allyson , Epperson L Elaine , Martin Sandra L
Published: June 2021

13‐lined ground squirrels are obligate hibernators that transition from summer homeothermy to winter heterothermy – wherein they exploit episodic torpor bouts. To explore the elements of winter neuroprotection, we applied 2D gel electrophoresis coupled with LC‐MS/MS to brain extracts of squirrels in two summer, four winter and fall transition states. Only 88 protein spots differed significantly among groups. The abundance pattern of the brain proteome was reciprocal between cold‐torpid and euthermic animals. The brain proteome of fall warm‐, but not cold‐torpid squirrels strongly resembled the homeotherms, indicating that the changes observed in torpid hibernators are defined by body temperature, not torpor per se. Metabolic enzymes were largely unchanged despite varied metabolic activity across annual and torpor‐arousal cycles. Proteins elevated in summer and aroused winter squirrels represented pathway enrichments in neural growth/differentiation and synaptic transmission. Differential cytoskeletal regulation across torpor‐arousal cycles is revealed by changes in microtubule dis/assembly regulating protein abundance (STMN1, DPYSL2), suggesting a mechanism for cytoskeletal stabilization during torpor and rapid reorganization on return to euthermy. Funded by NIH R01HL089049.
The Effect of TYPE 1 DIABETES ON REPRODUCTIVE HORMONES (FOLLICLE STIMULATING, LUTEINIZING AND TESTOSTERONE HORMONES) IN HUMAN MALE SUBJECTS
Authors: Adegbesan Bukunola Oluyemisi , Ogunlabi Olugbenga O
Published: June 2021

Diabetes mellitus is a syndrome characterized by chronic hyperglycemia (high blood sugar) either because the body does not produce enough insulin (Type 1 or Insulin Dependent Diabetes Mellitus‐IDDM), or because body cells do not properly respond to insulin that is produced (Type 2 or Non Insulin Dependent Diabetes Mellitus‐NIDDM).Diabetes Mellitus has been recognized as a clinical syndrome since ancient times and remain a crippling global health problem till today (Wild et al.,2004).

This study was designed to determine the effect of Type 1 diabetes on reproductive hormones (Luteinizing hormone (LH), Follicle Stimulating Hormone (FSH) and Testosterone Hormone (TH) in human male subjects. Forty (40) human male subjects were used for the purpose of this experiment and were grouped into two ;Control group consisting of twenty (20) healthy subjects (confirmed non‐diabetic with fasting blood glucose level of < 110mg\dl) and the Experimental group consisting of 20 Type 1 diabetic patients. Blood samples were collected and immunoassay analyses were carried out to determine the levels of plasma FSH, LH and TH.

The results from this study showed significant reductions ( p<0.05) in the levels of FSH, LH and TH in the diabetic group when compared with the control or non‐diabetic group confirming that diabetic condition affects reproductive activities negatively.
The direct effect of incretin hormones on glucose metabolism
Authors: Karstoft Kristian , Mortensen Stefan Peter , Knudsen Sine Haugaard , Solomon Thomas Phillip James
Published: June 2021

Objective

To assess if glucagon‐like peptide (GLP‐1) and glucose‐dependent insulinotropic polypeptide (GIP) have a direct effect on glucose metabolism independent of their effect on the endocrine pancreas.

Methods

Five healthy males (22+/−1 years, BMI 21+/−1) with normal glucose metabolism (fasting glucose = 5.0+/−0.1 mM, HbA1c = 5.2+/−0.1%) were studied using the pancreatic clamp technique (100 ng/kg/min somatostatin infusion with basal replacement of insulin, glucagon and growth hormone) in 3 different trials (GLP‐1 [0.5 pmol/kg/min], GIP [1.5 pmol/kg/min] or saline infusion). A 2‐step clamp was performed at euglycemic (90 min) and hyperglycemic (90 min; euglycemic + 5.4 mM) levels. Peripheral blood flow and vascular function (forearm flow‐mediated dilation; FMD) were determined by ultrasound Doppler.

Results

Insulin was maintained at basal levels during euglycemia in all 3 trials, with equal decreases in C‐peptide levels. During hyperglycemia, insulin and C‐peptide levels increased in all trials (P<0.05). Glucose infusion rates were higher during euglycemia in the GLP‐1 trial as compared to the saline trial (P<0.05), also when adjusted for insulin values (P=0.10). Peripheral blood flow and vascular function did not differ between trials.

Conclusion

These data suggest a direct insulin/glucagon‐independent effect of GLP‐1 on glucose metabolism during euglycemia.
Chenodeoxycholate (CDCA) activates Cl− transport via CF transmembrane conductance regulator in T84 and HEK cells
Authors: Domingue Jada , Ao M , Sarathy J , Shanker S , Nelson D , Alrefai W , Rao M
Published: June 2021

The mechanism by which high levels of colonic luminal bile acids (BAs), such as CDCA, cause secretory diarrhea in pathologies like inflammatory bowel disease, is not fully elucidated. We have previously shown that BA act rapidly (in mins) to increase cAMP and activate CFTR in human colonic T84 cells. We hypothesize that BAs act via membrane receptors to stimulate Cl− secretion. T84 cells express protein and transcript for the BA G‐protein coupled receptor, TGR5. The multidrug resistant protein (MRP) inhibitor MK571 (20μM) enhances the CDCA response in T84 cells 2.6 fold, suggesting the involvement of the cAMP transporter, MRP4. T84 cells express MRP4 transcript. While BA stimulation of colonic proliferation involves M3 muscarinic and epidermal growth factor (EGF) receptors (Biochem Pharm 70:1035,’05), we report that CDCA action in T84 cells is attenuated by the EGFR inhibitor AG1478 (CDCA: ΔIsc 16±5; +AG1478 (1μM): 5.2±1.2, n>;3), but not by atropine (M3 antagonist). CDCA (dose‐dependent) or forskolin (FSK) increase Cl− transport [measured by I‐ efflux] in HEK cells stably transfected with CFTR (HEK‐CFTR), but not in untransfected cells. MK571 potentiated this FSK response. HEK‐CFTR express MRP4 transcript and possess TGR5 transcript and protein, making them a useful model. These data indicate that membrane proteins, such as EGFR, MRP4 and perhaps TGR5 are involved in CDCA's action in stimulating CFTR. Supp. by NIH.
Conditional deletion of biliverdin reductase in macrophages determines the sensitivity to acetaminophen injury.
Authors: Wegiel Barbara , Gallo David , Csizmadia Eva , Nemeth Zsuzsanna , Kelly Vincent , Mantle Tim , Alam Jawed
Published: June 2021

Biliverdin reductase (BVR) is a multifunctional enzyme, kinase, receptor as well as exhibiting transcription factor activity. We have recently elucidated mechanisms by which BV and BVR regulate the innate inflammatory response in vitro and in vivo. In this study we aimed to elucidate the role of BVR in vivo in response acetaminophen‐mediated liver injury using newly generated BVR conditional knockout mice.

We confirmed lack of BVR in the Cre‐Lyz‐BVR‐fl/fl macrophages in vivo as well as in bone‐marrow derived macrophgaes. We validated decreased bilirubin levels in BVR deficient mice. Importantly after challenge with LPS+D‐Gal Cre‐Lyz‐BVR‐fl/fl mice show greater injury as compare to BVR‐fl/fl control mice. However, Cre‐Lyz‐BVR‐fl/fl mice were resistant to acetaminophen induced liver injury. Lack of BVR in macrophages led to low inflammatory responses to TLR9 ligands (CpG rich region) in vitro and in vivo. We showed that BVR binds specific DNA sequences in the membrane and may be a novel mediator of TLR9 signaling.

In summary, BVR in macrophages is critical for acetaminophen mediated liver injury due to its interaction with DNA released from necrotic cells and amplification of inflammation.
Down‐regulation of TLR‐4 in Hypoxic Myoblast Cells
Authors: Thompson Jennifer Anne , Webb R. Clinton
Published: June 2021

Intrauterine hypoxia is a common complication of human pregnancy. Intima hyperplasia and increased expression of inflammatory markers in aortae of chronically hypoxic fetuses were demonstrated previously.

Objective

To determine the effect of hypoxia on inflammatory markers and Toll‐like receptor (TLR‐4) expression in myoblast cells.

Methods

Myoblast cells derived from embryo rat aorta (A10, ATCC) were placed in an anaerobic incubator set to provide hypoxic atmospheres of 1% O2 (5% CO2, balance N2) for 2 days. Control cells remained in 21% O2. Additional cells were treated with lipopolysaccharide (LPS, 100 ng/ml) or vehicle control. mRNA levels were measured using Real‐time PCR and expressed relative to control (S15 and ubiquitin C). Protein expression was measured by Western blot and expressed relative to β‐actin.

Results

TGFβ1 mRNA levels were increased 5.2 fold and 1.9 fold on day 1 and 2 of hypoxia, respectively. There were no changes in interleukin‐6 (IL‐6) mRNA levels in response to hypoxia. mRNA levels of IL‐6 were increased 2.4 fold on day 1 and 15.3 fold on day 2 of LPS treatment. TLR‐4 expression was reduced by 33%, MyD88, a downstream adaptor protein of TLR‐4, was decreased by 27% and P‐ERK was decreased by 23%, on day 2 of hypoxia. Stimulation of TLR‐4 by LPS also decreased expression of TLR‐4 and MyD88 by 18% and 31%, respectively.

Conclusions

Hypoxia increases TGFβ1 mRNA expression but not IL‐6 and reduces expression of TLR‐4 and its signaling components.
ICAM‐1 expression by skeletal muscle cells augments stages of myogenesis
Authors: Goh Qingnian , Dearth Christopher L. , Pizza Francis X.
Published: June 2021

We previously reported that intercellular adhesion molecule‐1 (ICAM‐1), an important protein of the inflammatory response, was expressed by satellite cells and myofibers of hypertrophying muscles. As skeletal muscle cell expression of ICAM‐1 contributed to the formation of regenerating fibers, as well as hypertrophy to mechanical overload, we speculate that ICAM‐1 expression by skeletal muscle cells facilitates myogenesis. Therefore, the purpose of the current study was to establish phenotypic alterations associated with skeletal muscle cell expression of ICAM‐1. Through stable transfection of C2C12 myoblasts with an ICAM‐1 plasmid, we found ICAM‐1 expression augmented stages of myogenesis in which myotubes are forming, adding nuclei, aligning, synthesizing proteins, and hypertrophying. As the cytoplasmic domain of ICAM‐1 is capable of activating several intracellular signaling pathways, we selectively inhibited the function of the cytoplasmic domain with a cell permeable peptide to reveal underlying mechanisms involved in ICAM‐1 mediated myogenesis. Here, we showed that the ICAM‐1 cytoplasmic domain contributed to enhanced rates of protein synthesis via increased Akt/p70s6k signaling. Hence, our findings extend knowledge of the immunobiology of skeletal muscle cells by revealing a novel mechanism through which the inflammatory response facilitates myogenesis.
Azumolene administration during prolonged mechanical ventilation does not prevent mitochondrial reactive oxygen species emission or diaphragm atrophy
Authors: Talbert Erin E , Smuder Ashley J , Kwon Oh Sung , Sollanek Kurt J , Wiggs Michael P , Powers Scott K
Published: June 2021

Although mechanical ventilation (MV) is a life‐saving measure in patients suffering from respiratory failure, prolonged MV causes the rapid development of diaphragm atrophy. MV‐induced diaphragm atrophy is due, in part, to increased production of reactive oxygen species (ROS) in diaphragm mitochondria. Moreover, increased cytosolic calcium in diaphragm fibers, perhaps from leakage of the ryanodine receptor (RyR), may be responsible for this increase in mitochondrial ROS production during MV and the subsequent activation of calpain and caspase‐3. Therefore, we hypothesized that blocking the RyR in diaphragm fibers with azumolene (Az) would prevent MV‐induced increases in mitochondrial ROS production, protease activation, and diaphragm atrophy. However, our results reveal that Az treatment did not attenuate the MV‐induced increase in mitochondrial ROS emission or prevent the MV‐induced activation of calpain and caspase‐3 in the diaphragm. Further, Az treatment did not prevent MV‐induced diaphragmatic fiber atrophy. We conclude that inhibition of the RyR in diaphragm muscle fibers is not sufficient to prevent MV‐induced diaphragm atrophy.

Supported by NIH R01 HL087839 (SKP) and AHA 11PRE6520001 (EET).
Functional evidence for increased nitric oxide involvement in cardiovascular and autonomic modulation by the PVN in conscious HU rats
Authors: De Andrade Ozahyr , Friskey Sarah A , Martins‐Pinge Marli Cardoso , Hasser Eileen M
Published: June 2021

PVN is a pivotal hypothalamic integrative center for circulatory control and may play a role in autonomic regulation after cardiovascular deconditioning. Previous work demonstrates that neuronal nitric oxide (NO) synthase activity and protein expression is increased in the PVN of hindlimb unloaded (HU) rats, an animal model of cardiovascular deconditioning, compared to control. However, the functional role of increased nNOS is unknown. In this study we tested the hypothesis that tonic effects of NO in the PVN are augmented in HU rats. We studied the cardiovascular and autonomic responses to NOS inhibition in the PVN after return of HU rats to upright posture. Rats underwent 14 days HU or cage control (CC). Guide cannulas to the PVN were implanted on Day 9. Artery and vein catheters and renal nerve electrodes were implanted on Day 13 to record mean arterial pressure (MAP), heart rate (HR) and renal sympathetic nerve activity (RSNA). Baseline MAP was similar but resting HR was increased in conscious HU compared to control. Inhibition of NOS with L‐NMMA (2mM/100nL) into the PVN produced greater increases in MAP (CC: 13±1 vs. HU: 20±2 mmHg) and RSNA (CC: 155±34 vs. HU: 309±49% Baseline) in HU compared to control. Changes in HR to L‐NMMA were similar between groups. Data indicate that tonic NO function in the PVN is enhanced after HU in conscious rats and it may be part of the adjustments required for microgravity environment. HL55306
Thimet Oligopeptidase Forms Angiotensin‐(1–7) in the Nucleus of NRK‐52E Cells
Authors: Alzayadneh Ebaa M , Pirro Nancy T , Chappell Mark C
Published: June 2021

An intracellular renin‐angiotensin system (RAS) is evident in multiple tissues. Functional angiotensin (Ang) receptors are present intracellularly on kidney cortical nuclei; however, the intracellular pathways that contribute to Ang II or Ang‐(1–7) expression are not resolved. We sought to identify a proximal tubule epithelial cell model to elucidate the regulation of this intracellular system. Utilizing the rat‐derived NRK‐52E epithelial cell line, we demonstrate the nuclear expression of angiotensinogen (des‐Ang I Aogen), renin, AT7/Mas and prorenin receptors. Nuclear extracts of NRK cells expressed Ang‐(1–7) (51 ± 39 pg/mg protein; n=3) and immunostaining for Ang‐(1–7) was primarily confined to the nucleus. The downstream processing of 125I‐Ang I as substrate was examined in solubilized nuclear fractions by HPLC‐coupled γ‐detection. This analysis revealed the predominant conversion of Ang I to Ang‐(1–7) [41 ± 6 fmol/min/mg protein, n=4]. Both thimet oligopeptidase and thiol protease inhibitors essentially abolished Ang‐(1–7) formation [CPP: 83%; n=3 and PCMB: 85%; n=4; p<0.01 respectively]. In contrast, additional studies revealed minimal metabolism of 125I‐Ang II in the nuclear fraction [>;70 % intact at 2 hours]. Collectively, these data suggest that the localization of Aogen, renin and thimet oligopeptidase in the nucleus of NRK cells may constitute one source of intracellular Ang‐(1–7).