bluuebunny/crossref_metadata_2025 huggingface dataset:
Hsp27 regulates p53 transcriptional activity in ultraviolet C‐induced DNA damage of MCF‐7 cells
Authors: Kanagasabai Raghu , Krishnamurthy Karthikeyan , Wani Altaf , Zhu Qianzheng , Ilangovan Govindasamy
Published: June 2021

Transcriptional activation of the p53 target genes plays a critical role in the cellular response to DNA damage by ultraviolet C (UV‐C), which causes either cell cycle arrest or apoptosis. However, what determines the choice between cell cycle arrest or apoptosis is not clear. In this report we show that Hsp27 level increases after UV‐C irradiation which leads to the protection of human carcinoma cells (MCF‐7) from UVC‐induced DNA damage by increasing the accumulation of p21, MAPKAP‐2, p38 MAPK and Akt. The results also show that the increase in Hsp27 enables cells to proliferate 48 hr after DNA damage, which prevents cell senescence. Hsp27 silencing by siRNA causes more apoptosis and enhances cell death, induced by UV‐C irradiation. We conclude that the p53 and Hsp27 interaction plays an important role in determination of UV‐C induced apoptosis and / or cell growth.
Androgens induce mitochondrial dysfunction and apoptosis in dopamine neurons
Authors: Cunningham Rebecca Lynn , Centonze Victoria E. , Lechleiter James , Giuffrida Andrea , Roberts James L.
Published: June 2021

Parkinson's disease (PD) is a neurodegenerative condition associated with oxidative stress and dopamine neuronal loss in the nigrostriatal pathway. Aged men have a greater incidence of PD than women. While estrogens have been shown to play a neuroprotective role in PD, there is little information on androgens' actions on dopamine neurons. In this study, we examined the effects of androgens on mitochondria function and its downstream signaling pathways in a dopaminergic cell line (N27 cells). Our results show that physiological levels of testosterone (100 nM) caused mitochondrial dysfunction and apoptosis, as evidenced by increased free radical formation, decreased mitochondria membrane potential, and decreased levels of reduced thiols ‐ an indicator of reactive oxygenated species (ROS) generation. We also found that androgens activate the apoptotic PKCδ pathway via a caspase‐3 dependent mechanism. To determine the specificity of androgen‐induced apoptosis, we used the same experimental protocol on the non‐dopaminergic GnRH cell line. Androgens did not induce apoptosis in these neurons, suggesting that androgen‐induced apoptosis is not a generalized phenomenon and that it may be specific to dopamine neurons. These results show that androgenic influences may underlie the gender bias in Parkinson's disease. NIH grants F32NS061417‐01 to RLC.
Prolonged Activation of P2X7R Leads to Alveolar Epithelial Type I Cell Death by Suppressing Wnt/beta‐catenin Pathway
Authors: GUO YUJIE , MISHRA AMARJIT , WENG TINGTING , WANG YANG , GOU DEMING , LIU LIN
Published: June 2021

Hyperoxic acute lung injury (ALI) is characterized by pulmonary endothelial and epithelial injury. This work is aimed to understand the mechanisms of cell death of alveolar epithelial type I cells (AEC I) during ALI. We have previously shown that purinergic P2X7 receptor (P2X7R) is specifically expressed in AEC I. We hypothesized that the activation of P2X7R causes AEC I cell death through the suppression of the Wnt/beta‐catenin pathway and thus contribute to the development of ALI. To test this hypothesis, an immortal AECI cell line E10 and a primary alveolar epithelial type I‐like cell were used as models. The prolonged activation of P2X7R by 2′‐3′‐O‐(4‐benzoylbenzoyl)‐ATP (BzATP) caused cell death in both types of cells. Beta‐catenin was released from the plasma membrane and degraded by the increased activity of glycogen synthase kinase‐3beta in the BzATP‐treated cells. The downstream genes of Wnt/beta‐catenin pathway were also significantly suppressed. The activation of Wnt/beta‐catenin pathway by the inhibition of glycogen synthase kinase‐3beta or the addition of Wnt3a completely blocked the P2X7R‐medaited cell death. These data support an important role of P2X7R and Wnt/beta‐catenin pathway in the pathogenesis of ALI. Strategies to interfere with the activities of P2X7R or Wnt/beta‐catenin signaling may therefore represent potential therapeutic method to limit the damage of ALI.
Caspase‐8 activation requires functional mitochondria in camptothecin‐induced apoptosis in intestinal epithelial cells
Authors: Jin Shi , Ray Ramesh M. , Johnson Leonard R.
Published: June 2021

Both TNF‐alpha and camptothecin (CPT) activate caspase‐8 in IEC‐6 cells, but the degree of activation is lower in response to CPT compared to TNF‐alpha. The mechanism by which TNF‐alpha induces receptor‐mediated caspase‐8 activation is well established, but that of CPT which damages DNA is unknown. The present study explored the mechanisms by which caspase‐8 is activated in CPT‐induced apoptosis. IETD‐FMK, an inhibitor of caspase‐8, attenuated activation of caspase‐9 and effector caspases, blocked poly (ADP‐ribose) polymerase (PARP) cleavage, and inhibited apoptosis. Mitochondrial electron transport chain inhibitors (antimycin A, rotenone), and an inhibitor of protein synthesis (cycloheximide) prevented CPT‐induced activation of caspase‐8, ‐9 and ‐3. DEVD‐FMK, an inhibitor of caspase‐3, significantly inhibited CPT‐induced activation of caspase‐9, but had no effect on activation of caspase‐8. Inhibition of caspase‐9 by LEHD‐FMK, an inhibitor of caspase‐9, decreased caspase‐8 activation. Together, these data show that mitochondrial function is essential to the activation of caspase‐8 in the absence of receptor‐mediated signaling in CPT‐induced apoptosis. (Supported by NIDDK grant DK‐16505)
Chronic hypoxia decreases arterial compliance in rat lungs
Authors: Jin Yi , Calvert Thomas J , Chicoine Louis G , Nelin Leif D
Published: June 2021

We hypothesized that the arterial remodeling seen with chronic hypoxic exposure would result in lower arterial compliance. Sprague‐Dawley rats were exposed to either normoxia (N) or hypobaric hypoxia (H) for 2 weeks, and then the lungs were isolated and perfused with a Kreb's Ringer solution containing 4% albumin. Vascular occlusions were performed and modeled using a 3 compliance (C), 2 resistor (R) model, such that arterial (Ca), capillary (Cc) and venous (Cv) compliances, as well as arterial (Ra) and venous (Rv) resistances could be determined. In the N lungs, Ca was 0.034 ± 0.004 ml/torr while in the H lungs, Ca was 0.015 ± 0.005 ml/torr (p<0.05). The distribution of vascular C was different (p<0.05 for all 3 compliances) between N and H lungs, with the vast majority of the vascular compliance residing in Cc in the H lungs (Ca 26 ± 3%; Cc 46 ± 6%; Cv 28 ± 3% N versus Ca 11 ± 4%; Cc 76 ± 6%; Cv 13 ± 2% H). Vascular resistance was greater in the H lungs and this was due to greater Ra (Ra 19.6 ± 1.8 torr/ml/min N versus 36.8 ± 9.8 torr/ml/min H), with little difference in Rv between the groups (Rv 19.9 ± 2.8 torr/ml/min N versus 25.6 ± 3.8 torr/ml/min H). These results demonstrate that the vascular remodeling associated with chronic hypoxia results in lower arterial compliance. These results have implications for right heart function in pulmonary hypertension.
pH‐sensitive K+ current in glial cells of the retrotrapezoid nucleus (RTN)
Authors: Mulkey D K , Kreneisz O , Sun Y , Chen X , Nishiyama A
Published: June 2021

The RTN contains pH‐sensitive neurons that sense changes in arterial pH to dive breathing (i.e., chemoreceptor). RTN glial cells are also thought to contribute to chemoreception by an unknown mechanism. We hypothesize that select glial cells in the RTN are intrinsically pH sensitive. To test this we made whole cell voltage clamp recordings from glial cells in transverse brain slices (300 ìm) from rats (P7‐12). Glial cells were defined based on a membrane potential <‐75 mV and an inability to generate action potentials. We found a subset of RTN glial cells feature a pH‐sensitive current; alkalization increased outward current whereas acidification decreased outward current. The I‐V relationship of the pH‐sensitive current reveals a voltage‐independent current which reverses near EK (‐90 mV, N=14). Duel current clamp recordings from pH‐sensitive glial and RTN chemoreceptors identify an interaction between glia cells and neurons (cross correlation coefficient, 0.8); glial cell depolarization initiates neuronal depolarization with a delay of ~50 ms. These results indicate that i) RTN glial cells can respond to physiologically relevant changes in pH; ii) a voltage‐independent K+ channel likely confers pH‐sensitivity to RTN glia; iii) pH‐sensitive glial cells can interact with RTN chemoreceptors. These results identify a potential mechanism by which RTN glial cells can contribute to central chemoreception.
Ventilation of hypoxic gas results in changes in whole nerve vagal recordings in the newborn rat
Authors: Qaqish Robert T , Vincent Sandra , Fisher John T , Yeger Herman , Cutz Ernest
Published: June 2021

Pulmonary neuroepithelial bodies (NEBs) form clusters of specialized epithelial cells situated within the airway mucosa. They express a unique membrane delimited oxygen (O2) sensor (Nature. 1993; 365(6442):153‐5). NEBs are innervated by afferents within the vagus nerve and synthesize serotonin (5HT) that is released as a neurotransmitter of hypoxia stimulus. NEBs and their vagal afferents express the ionotropic 5HT3 receptor (5HT3‐R) involved in hypoxia chemotransduction. We hypothesized that hypoxia, via NEB activation, would increase vagal nerve activity. Newborn rat pups between days 1 to 10 were anesthetised, paralyzed (pancuronium 0.25 mg/kg) and mechanically ventilated. Muscarinic receptor blockade was achieved with atropine (0.25 mg/kg). The right cervical vagus was exposed and whole nerve activity was recorded using a platinum bipolar electrode. Ventilation with hypoxic gas (10% O2) caused an increase (P<0.001 ANOVA) in the integrated nerve activity (8.66 ± 4.96 %) compared to control (100% O2). Preliminary studies using the 5HT3‐R blocker (ICS 205‐930) resulted in a partial inhibition of the hypoxia‐induced increase in whole nerve vagal activity. We conclude that ventilation with hypoxic gas resulted in gross changes in integrated vagal nerve activity. We propose that these gross changes in global vagal activity as a result of ventilation with 10% O2 are the result of NEB stimulation via its membrane bound oxygen‐sensor. (Supported by CIHR Grants MOP‐12742 and MPG‐15270)
Thymic Stromal Lymphopoietin (TSLP) and Airway Smooth Muscle
Authors: Smelter Dan , Thompson Michael , Meuchel Lucas , Townsend Elizabeth , Ryu Alexander , Pabelick Christina , Vassallo Robert , Prakash Y S
Published: June 2021

TSLP is a newly‐identified cytokine thought to be involved in airway inflammation. TSLP levels have been shown to significantly increase in airway smooth muscle (ASM) with chronic obstructive pulmonary disease and other inflammatory diseases. This study evaluated the expression of TSLP in human ASM cells and tissues exposed to cigarette smoke extract (CSE) or to pro‐inflammatory cytokines TNF‐α and IL‐13. In parallel studies, TSLP expression was evaluated in house dust mite sensitized mice with or without cigarette smoke exposure. The presence of TSLP and TSLP‐receptors were verified using Western blot analysis. Exposure to TNF‐α and IL‐13 (50 ng/mL each) for 24 hours resulted in increased TSLP and TSLP‐R expression. Simultaneous exposure to functional TSLP antibody blunted cytokine induced TSLP‐R expression. Interestingly, acute CSE (30 min) down‐regulated TSLP expression, while chornic exposure increased expression. Similarly, [Ca2+]i responses of human ASM to histamine were blunted with chronic CSE exposure, effects reversed by TSLP and TSLP‐R antibodies. Sensitized mice demonstrated an increase in TSLP expression in whole lung samples. This effect was even further enhanced in dust mite sensitized mice exposed to chronic cigarette smoke. These results indicate a complex role for TSLP in ASM inflammation with or without cigarette smoke exposure.

Supported by NIH grants HL088029, HL090595, RR024150, and the Flight Attendants Medical Research Institute (FAMRI). Additional funding from the Department of Anesthesiology, Mayo Clinic.
Up‐regulation of the Ca2+ permeable TRPM7 channel in Atrial Fibrillation patients
Authors: Du Jianyang , Xie Jia , Silverman David , Liang Bruce , Yue Lixia
Published: June 2021

Cardiac fibroblasts play an important role in cardiac fibrogenesis which associated with a variety of heart diseases including atrial fibrillation (AF). However, the Ca2+ permeable channels which are responsible for cellular functions of fibroblasts remain unknown. Here, we investigated the potential role of TRPM7‐mediated Ca2+ entry is involved in fibroblast differentiation. With consent, fibroblasts were obtained from patients undergoing cardiac surgery. The isolated fibroblast cells were used for patch‐clamp, Ca2+ image and immunostaining. TRPM7 siRNA was used to knock‐down TRPM7 expression when it was necessary. A typical TRPM7‐like current obtained from isolated fibroblasts by patch‐clamping. TRPM7 siRNA decreased the current density by 76%, confirming it was TRPM7. Interestingly, the current in AF patients were 3 folds bigger than those in control patients suggest that TRPM7 play a role in AF. Immunostaining results showed basal level of differentiation was much higher in cells from AF patients than those from control patients. TRPM7 siRNA significantly decreased TGF‐β1 induced fibroblast differentiation in both control and AF patients. To conclude, our study provided a novel molecule (TRPM7) as well as a novel mechanism for cardiac fibrogenesis: TRPM7‐mediated Ca2+ signal is important in cardiac fibroblast differentiation, which largely contributes to cardiac fibrogenesis cascade and related heart diseases. Project supported by NIH # HL 078960
Imatinib mesylate presents a cytostatic effect on B16 cells in vitro and an apoptotic outcome in melanoma in vivo.
Authors: Pirraco Ana , Coelho Pedro , Rocha Ana , Costa Raquel , Soares Raquel
Published: June 2021

Melanoma is characterised by a general expression of platelet‐derived growth factor (PDGF) and its cognate receptor and by the down‐regulation of c‐Kit as the malignant potential increases. PDGF is also a powerful growth factor with autocrine and paracrine effects within melanoma. Both receptors are protein tyrosine kinases shown to be inhibited by imatinib mesylate.

The aim is to investigate the effect of imatinib in melanoma cells in vitro and in vivo.

Culture of B16 cells were incubated with imatinib either in the presence or absence of recombinant PDGF‐AA and assayed for proliferation (BrdU incorporation assay), apoptosis (TUNEL assay) and invasion (Double‐Chamber assay). C57BL/6 mice injected subcutaneously with B16 cells were treated daily with 100mg/Kg imatinib mesylate (n=10) or saline solution (n=6) for 2wk. Paraffin‐embedded tumour tissues were analysed for proliferation and apoptosis.

Results suggest that imatinib has an inhibitory effect on the proliferation and migration of melanoma cells with no significant increase in apoptosis. However, besides preventing proliferation, imatinib strongly induces apoptosis within the tumor.

In conclusion, these distinct in vitro and in vivo findings suggest that tumor cell behaviour is influenced by the action of imatinib in the tumor microenvironment. (supported by Novartis Farma, Portugal)
Modulation effect of catechin in angiogenesis and inflammation
Authors: Negrão Rita , Moura Liane , Duarte Delfim , Silva Rafael , Soares Raquel
Published: June 2021

Inflammatory process is closely related to angiogenesis. Diets rich in plant‐derived polyphenols seem to protect from pathologies which present angiogenesis and inflammation in common. Catechin (Cat) is an abundant polyphenol. The aim of this study was to investigate the effect of Cat in angiogenesis and inflammation. Human umbilical vein endothelial cell (HUVEC) and human aortic smooth muscle cell (HASMC) were treated with 0.01‐100 µM Cat for 24 h. Cat increased viability (MTT) and decreased apoptosis (TUNEL) and proliferation (BrdU assay) in both cell lines. It decreased invasion capacity (double‐chamber assay) of HASMC while increasing it in HUVEC. Cat treatment led to a slight reduction in HASMC cell migration (injury assay) and increased HUVEC migration in the same conditions. Incubation of HUVEC on Matrigel‐coated plates with 10 µM Cat led to the formation of ramified cord‐like structures while 100 µM did not change the number of these structures. 100 µM Cat did not change the vessel sprouting capacity (aortic ring assay). Cat treatments diminished NO released to the extracellular medium but did not change the activity of NFkB. Cat interferes with some steps of angiogenic and inflammatory process but in vivo experiments are needed in order to confirm these properties.

Supported by ERAB (EA0641), FCT (SFRH/BD/41888/2007) and iBeSa (BLSAD‐01/08, BLSAD‐2‐JUL/08)
Mineralocorticoid Receptor (MR) Inhibition Attenuates High Salt‐Aldosterone Induced Increases in Vascular Renin‐Angiotensin‐Aldoesterone System (RAAS) and Oxidative Stress
Authors: Habibi Javad , DeMarco Vincent G , Wei Yongzhong , Whaley‐Connell Adam , Schneider Rebecca I , Tilmon Roger , Shin Ji Young , Sowers James R
Published: June 2021

Previous studies indicate that high salt and high circulating aldosterone activates tissue RAAS and increases oxidative stress. To determine if MR blockade reverses salt/aldosterone increases in tissue RAAS and associated oxidative stress in the aorta of Sprague Dawley (SD) rats, we placed rats on a high salt diet and/or infused with aldosterone for 3 weeks. Aorta was harvested from 9 week old male SD rats following 21 day 4% high salt and/or aldosterone infusion with/without spironolactone treatment (5mg time released pellet for 21days). Paraffin sections were incubated with various primary antibodies to evaluate oxidative stress and remodeling. Images were obtained by confocal microscopy and gray scale intensities were quantified. Signal intensities of Nox2, p47phox, Rac1, MR, renin, angiotensinogen, and 3‐nitrotyrosine were elevated in the animals receiving high salt diet and aldosterone infusion even more with combination. Treatment with a low dose of spironolactone significantly diminished the vessel immunostaining for all components of RAAS as well as nitotyrosine. High‐salt fed and aldosterone treatments have additive effects on vascular RAAS and oxidative stress which are reversed by treatment with a MR blocker in doses not affecting blood pressure. Supported by NIH R01‐HL‐63904 and VA Merit (JRS).
Relative lack of β1‐subunit‐mediated regulation of BKCa in cremaster arteriolar smooth muscle
Authors: Yang Yan , Ella Srikanth R , Murphy Timothy V , Grayson T Hilton , Hwang Yun T , Haddock Rebecca , Peichun Gui , Andrew Braun P , Korthuis Ronald J , Davis Michael J , Hill Michael A
Published: June 2021

The relationships between membrane potential (Em) and myogenic tone in skeletal muscle and cerebral arteries suggest differences in the role of BKCa. To test this idea, whole cell K+ currents were measured by patch clamp. At 5 μM Ca2+, cremaster smooth muscle cells (SMC) showed lower current density compared to cerebral SMC (34.5±1.9 vs 45.5±1.7 pA pF‐1 at +70mV; p < 0.05). The selective inhibitor, iberiotoxin (0.1 μM), revealed the differences in K+ current to be due to BKCa. 17β‐Estradiol, reported to act via the BKCa β‐subunit, caused greater enhancement of K+ current in cerebral SMC compared to those of cremaster. The α subunit‐selective BKCa opener, NS1619, exerted similar effects in both types of SMC. Spontaneously transient outward currents (STOCs) were more evident and occurred at more negative Em in cerebral SMC. Also consistent with decreased STOC activity in cremaster SMC was an absence of Ca2+ sparks (0/76 cells) relative to cerebral SMC (76/105 cells). Measurements of mRNA and protein indicated a higher ratio of BKCa β:α‐subunit expression in cerebral SMC. The data suggest that decreased activity of BKCa in cremaster SMC is due in part to a relative lack β‐subunit regulation.
A multiscale computational model of microcirculatory vasoreactivity: Linking subcellular events to macroscale responses in health and disease
Authors: Kapela Adam , Gadkari Tushar , Tsoukias Nikolaos M.
Published: June 2021

Microcirculatory tone is regulated by an elaborate system of intracellular and intercellular signaling pathways. We developed an integrated mathematical model to elucidate how this regulation emerges from the nonlinear interaction of individual cellular components and mechanisms. Detailed descriptions of calcium dynamics and membrane electrophysiology in isolated endothelial and smooth muscle cells are validated against subcellular‐ and cellular‐level data. A multicellular vessel segment is formulated by integrating individual cells and accounting for intercellular communication. The passive and active biomechanical properties of the vessel are accounted in simulating diameter changes. The model can simulate pressure‐induced diameter changes and myogenic tone, agonist‐induced vessel constrictions and relaxations, and conducted vasoreactivity. The model also predicts altered vascular reactivity from cellular changes reported in pathological conditions. Thus, the model was successful in integrating cellular mechanisms to describe vasoreactivity and is useful for relating reported changes in subcellular components to physiological responses in health and disease.

[Supported by AHA grant NSDG043506N and NIH grant HL095101]
Protein Kinase G attenuates myofilament Ca++ sensitivity in ovine adult but not fetal cerebral arteries via mechanisms ablated by chronic hypoxia
Authors: Williams James Michael , Pearce William Julian
Published: June 2021

Chronic hypoxia modulates contractility and Protein Kinase G (PKG) activity differentially in fetal and adult ovine cerebral arteries. Because PKG can modulate contractility via inhibition of myofilament Ca++ sensitivity, the present study explores the hypothesis that chronic hypoxia induces changes in PKG activity that yield age‐dependent changes in myofilament Ca++ sensitivity. Middle cerebral arteries from non‐pregnant adults and term fetal sheep were prepared for measurements of contractile force and cytosolic Ca++ (Fura‐2) during responses to graded concentrations of 5HT in the presence and absence of 10 µM 8‐pCPT‐cGMP (a PKG activator). In normoxic adult arteries: 1) 8‐pCPT‐cGMP depressed 5HT‐induced increases in both [Ca++] and Ca++ sensitivity; 2) chronic hypoxia depressed Ca++ sensitivity but increased 5HT‐induced increases in [Ca++]; and 3) hypoxia ablated inhibition of Ca++ sensitivity by 8‐pCPT‐cGMP. In normoxic fetal arteries: 1) 8‐pCPT‐cGMP depressed 5HT‐induced increases in [Ca++] without effect on Ca++ sensitivity; 2) chronic hypoxia depressed Ca++ sensitivity but increased 5HT‐induced increases in [Ca++]; and 3) 8‐pCPT‐cGMP was without effect in hypoxic arteries. These results suggest that chronic hypoxia and PKG attenuate myofilament Ca++ sensitivity via a common mechanism that is absent in fetal cerebral arteries. Supported by USPHS Grants HL54120, HD31266, & HL64867.
Regulation of endothelial NO by DDAH1 and DDAH2 isoforms
Authors: Pope Arthur , Karuppiah Kanchana , Cardounel Arturo J
Published: June 2021

Increased levels of the endogenous eNOS inhibitor, ADMA, has been implicated in the endothelial dysfunction associated with cardiovascular disease. Endothelial ADMA and L‐NMMA are metabolized by the enzyme Dimethylarginine Dimethylamino hydrolase (DDAH). DDAH has two isoforms, both of which are expressed in the endothelium. However, the role of these isoforms in regulating vascular NO production is unclear. Therefore, studies were performed to study the effects of DDAH gene silencing and over‐expression on eNOS derived NO production using BAECs. DDAH‐1 over‐expression increased NO production by 21%, while DDAH‐2 over‐expression had no effect. DDAH‐1 gene silencing resulted in a marginal decrease in NO production while DDAH‐2 gene silencing resulted in a 58% decrease in NO production. The DDAH‐2 gene silencing effect on NO was partially restored with L‐arginine supplementation to 65% of the control. HPLC measurements of intracellular ADMA revealed no significant increase in ADMA levels following DDAH gene silencing. However, western blot analysis revealed a 40% reduction in eNOS expression following DDAH‐2 gene silencing. These results demonstrate that DDAH‐2 can modulate endothelial NO bioavailability through ADMA independent mechanisms involving decreased eNOS expression. These finding demonstrate a novel mechanism through which DDAH regulates endothelial NO generation.
Transcriptional analysis of skeletal muscle adaptations to marine life in king penguin juveniles (Aptenodytes patagonicus)
Authors: Dégletagne Cyril , Rey Benjamin , Keime Céline , de Dinechin Marc , Rouanet Jean Louis , Roussel Damien , Duchamp Claude
Published: June 2021

King penguin chicks, that are strictly terrestrial during first year after hatching, must depart to sea to reach nutritional emancipation. Juvenile survival therefore depends on their adaptation to the energetic challenges of marine life: 1) harsh thermal environment, 2) long distant food resources, 3) repeated, deep and prolonged nutritional divings.

To elucidate these adaptations, the transcriptome of pectoralis muscle, the main thermogenic and locomotor tissue, was analysed in juveniles that had never been to sea (n=4) and in naturally adapted juveniles (n=3) by using chicken Affymetrix DNA chips (Profilexpert, Lyon).

Results showed the differential expression of nearly 3600 genes after marine life. Genes encoding enzymes of carbohydrate metabolism were down regulated while those encoding lipid transport and catabolism were up regulated suggesting an increased use of lipids as energy substrates. Further, transcripts encoding enzymes involved in cellular defense against reactive oxygen species and the associated injuries were also up regulated, suggesting an adaptive response against oxidative stress.

This study opens large perspectives to elucidate how king penguins can survive in an energetically unfavorable and pro‐oxidative environment and what contributes to their remarkable longevity.

This study received the financial support of the French Polar Institut (IPEV).
Chromatin remodelling linked with heat acclimation memory suggests epigenetic programming
Authors: Tetievsky Anna , Horowitz Michal
Published: June 2021

Re‐induction of heat acclimation (AC) after its loss occurs markedly faster than in the initial AC session. We demonstrated that the underlying mechanisms involve continuous upregulation of cytoprotective and chromatin remodeler gene transcripts during deacclimation despite a return of the physiological AC phenotype to pre‐AC. Here we tested the hypothesis that faster re‐AC implicates 'molecular memory' linked to epigenetic modifications. Using our AC(30d)‐DeAC(30d)‐ReAC(2d) R.norvegicus model, histones acetylation (H4, H3) and phosphorylation (H3‐Ser10) at the regions of the heat shock response element (HSE) in HSP70 and HSP90 promoters were measured using Chromatin immunoprecipitation (ChIP). Non‐AC and AC for 2d served as controls. To assess accessibility to the HSE, HSF1‐ChIP at the region of the HSP70 promoter and hsp70 mRNA kinetics following heat stress (HS) were conducted. ChIP analyses provided substantial evidence of chromatin remodelling via histone H4 but not H3 acetylation during AC, DeAC and ReAC. The accessibility of the HSE to HSF1 was validated by its elevated binding in the HSP70 promoter region and by the demonstration of acclimatory mRNA kinetics posts HS in the AC, DeAC and ReAC groups. In conclusion, we provide evidence that long term acclimation induces long standing chromatin remodelling of H4, and so, activates epigenetic mechanisms.
Effects of exercise on miRNA expression levels in human neutrophils
Authors: Radom‐Aizik Shlomit , Zaldivar Frank , Oliver Stacy , Galassetti Pietro , Cooper Dan M
Published: June 2021

microRNAs (miRNA) are short non‐coding RNAs that play a role in post‐transcriptional regulation of mRNA and have recently been implicated in development of immune cells. Since we recently showed that brief bouts of exercise alter immune related gene expression in human neutrophils, we hypothesized that exercise would influence neutrophil miRNA expression as well. Eleven healthy men (19‐30 yr old), performed 10 consecutive 2‐min bouts of cycle ergometry (~80% peak VO2) interspersed with 1‐min rest intervals. Neutrophils were isolated before and after the exercise. RNA was hybridized to Agilent Human miRNA Microarray v2.0. Significant changes in expression levels were observed in 14 of 799 miRNA (p<0.01, FDR=0.1). 9 had lower expression level after exercise (e.g., miR‐20a, miR‐20b, miR‐22, miR‐17, miR‐106a, miR‐18a, miR‐18b, miR‐185, and miR‐130b) whereas, 5 had higher expression (e.g., miR‐505, miR‐1225‐5p, miR‐145, miR‐125a‐5p and miR‐940). Several of these miRNAs are implicated in leukocyte differentiation and maturation. This is the first study to demonstrate in vivo, and only 30 min after stimulation, an effect of exercise on neutrophil miRNA, and may prove useful in understanding the role of neutrophils in pathological conditions like exercise induced asthma.
Fish smell: an exercise in finding, analyzing, and interpreting physiological data
Authors: Anderson Margaret
Published: June 2021

An important goal of my intermediate‐level Animal Physiology class is to help students develop skills in using primary literature in conjunction with learning physiological principles. Using publications on olfaction in the Crucian carp, I developed a written exercise that required the students to find and download two articles (Hamdani and Doving, 2007 and Hamdani et al., 2008), answer guided questions about the studies, and relate what they learned about chemosensory physiology from these papers to information provided in other sources. Some questions tested the students' ability to follow the narrative of the papers and others the ability to interpret data and consider experimental design. A review of the students' response sheets showed that 34 of 56 students made errors on one or more selected factual questions; 16 showed weakness in interpreting a bar graph; and 34 did not understand how to interpret a set of micrographs. Ten students had difficulty with both the graph and micrographs. Correcting the exercise gave me insights regarding which skills and knowledge to emphasize in future assignments, and my feedback to the students alerted them to the areas on which they need to work.
Teaching research skills to undergraduates: Designing and implementing original research projects in a senior capstone course in animal physiology.
Authors: Hancock Thomas Vincent , Ruppenthal Brittany , Gregory Brandee , Morrow Krista , Ewing Alicia
Published: June 2021

Engaging students in research during their undergraduate experience is critical to encourage and train students to enter graduate programs in the sciences. In our capstone course in animal physiology, students design and implement original research projects, employ novel techniques, utilize appropriate statistical analyses, and present the project and results. Projects are reviewed by our animal care committee and students are challenged in their methodology. Our ten‐week quarter system requires that students rapidly initiate necessary methodology and design projects which can be implemented and analyzed successfully in a short amount of time. While the short duration is challenging from both a student and instructor perspective, results worthy of further investigation are often discovered and an example of a successful project measuring the effect of dietary capsaicin and exercise on body fat accumulation in young Sprague‐Dawley rats will be presented. Analysis using two‐way ANOVA revealed a significant decrease in fat accumulation with the addition of capsaicin over a five week period. Moderate swimming exercise reduced fat accumulation, though not statistically (P=0.051). While high levels of faculty and staff support must be employed to implement this course design, immersive experiences such as this provide the student with a real world perspective of the difficulties and rewards of research.
FXR Promotes Endothelial Cell Motility through Reciprocal Regulation of FAK and MMP‐9
Authors: Das Amitava , Yaqoob Usman , Mehta Dolly , Shah Vijay H
Published: June 2021

Farnesoid X Receptor (FXR) mediates important signaling functions of bile acids in diverse cell types including those residing in the vascular wall. Indeed, recent work has identified FXR as a potential regulator of vascular structure and function in part through transcriptional activation of MMP‐9. However, the signal transduction pathways linking bile acids to changes in actin cytoskeleton that are responsible for bile acid induced vascular cell migration remain unexplored. The FXR agonist and prototypical bile acid, chenodeoxycholic acid (CDCA), significantly increased endothelial cell (EC) motility, as analyzed by time lapse video microscopy, and tube formation, an in vitro correlate for angiogenesis. Increased cell motility was associated with prominent increases in focal adhesion (FA) plaques and was inhibited by FXR or MMP‐9 siRNA, indicating a FXR‐MMP‐9‐dependency of this signaling pathway. Mechanistically, incubation of cells with CDCA was associated with phosphorylation of a key FA protein, Focal Adhesion Kinase (FAK) at Y397 but not at Y576/577, or Y925. Studies using a site‐specific phosphodefective FAK mutant revealed that FAK phosphorylation at tyrosine residue ‐397 was required for CDCA induced activation of the downstream FA assembly protein, paxillin. Lastly, siRNA based silencing of FAK as well as phosphodefective FAK mutant both inhibited CDCA induced upregulation of MMP‐9, cell motility and tube formation. Thus, this study demonstrates a pivotal role for FAK in the process of FXR‐induced and MMP‐9 ‐dependent EC motility and vascular tube formation. Funded by NIH Grant # DK059615
Differential Gene and Protein Expression is Associated with Hypoxia in the Human Abdominal Aortic Aneurysm (AAA) Wall
Authors: Vorp David Alan , Xavier Anton E. , Chew Douglas W. , Cleary Deborah A. , Morgan Melissa A. , Shah Samarth R. , Rhee Robert Y. , Marone Luke K. , Dillavou Ellen D. , Abu‐Hamad Ghassan S. , Makaroun Michel S.
Published: June 2021

The goal of this work was to investigate the hypothesis that AAA wall exposed to hypoxia as a result of being adjacent to a thick intralumenal thrombus (ILT) exhibits impaired remodeling, leading to further degeneration. Pairs of AAA wall, selected based on disparate thicknesses of adjacent ILT, were snap frozen. QPCR was used to compare the gene expression. Protein levels were measured with Western blotting. MMP‐2 and ‐9 levels were measured with gelatin zymography. In separate paired samples, total wall thickness was measured on H&E‐stained sections. We found the activity, expression, and amounts of some genes and proteins to vary with ILT thickness within a given AAA, while others were at least spatially variable at the current sample number. For example, iNOS gene expression was up‐regulated in regions of AAA wall adjacent to thick ILT. Some protein levels were correlated to fold changes in ILT thickness. For example, calponin protein levels are negatively correlated to the fold increase in ILT thickness. AAA wall thickness was positively correlated to ILT thickness. The results support our hypothesis, suggesting that compared to AAA wall near thin ILT, the AAA wall adjacent to thick ILT has variable protein expression and impaired remodeling mechanisms. This work was supported by NIH R01‐HL79313.
The characterization and distribution of microcirculation in the mouse spleen
Authors: Shimizu Kazuhiko , Ezaki Taichi
Published: June 2021

The spleen has been regarded as a blood filtration organ and been studied in the aspects of immunology and hemodynamics. However, there were very few reports on lymphatic vessels (LVs) in the spleen. In this study, we used an immunohistochemical 3D‐imaging technique to characterize LVs in the mouse spleen and successfully demonstrated their spacial relationship to the blood vascular system for the first time by the use of various lymphatic markers, such as LYVE‐1 and podoplanin. The LYVE‐1(+) LVs ran reversely along the central arteries in the white pulp and trabecular arteries, and exited the spleen from the hilus. Furthermore, podoplanin was expressed not only LVs but also stromal cells in the white pulp. These podoplanin(+) cells formed mesh works surrounding the LVs and the central arteries. Intravenous transplantation of GFP(+) lymphocytes into normal recepient mice revealed that the donor cells appered in the mesh works as early as 1 hr and gathered in the splenic LVs at 6 hrs of post injection. These results suggest that the mesh works may act as an extravascular systems, and together with ordinary LVs play a primary role in the cell traffic as a special lymphatic route in the mouse spleen.
Selective blockade of the vasoactive intestinal peptide (VIP) receptors affect formation and development of coronary arteries in quail embryonic heart
Authors: Krieger Tatyana , Dedkov Eduard I
Published: June 2021

We have previously demonstrated that VIP receptors are morphogenic modulators of embryonic coronary vessel tube formation in vitro. This study tested the hypothesis that a selective blockade of VIP receptors will affect formation and development of the coronary vessels in vivo. At embryonic day (ED) 5, 6, 7, and 8, a selective VIP receptor antagonist, VIP (6‐28), was injected in ovo into the vitelline vein of the Japanese quail embryos at a final concentration of 10 µmol/L. Six embryos per each ED were sacrificed 4 days after the injection and the base of their hearts was fixed in 4% paraformaldehyde/PBS, and processed for frozen or paraffin‐embedded histological cross‐sections. The serial sections through the origin of the coronary vessels were studied by H&E staining or by immunostaining with the antibodies against α‐smooth muscle actin and quail endothelium (QH1). We found that the blockade of VIP receptors did not alter the formation of left and right coronary arteries in all experimental embryos. However, when the injection was done on ED8 (the time when multiple endothelial channels penetrate the aortic wall in all three aortic sinuses), an additional coronary artery originating from a non‐coronary sinus had been detected on ED12. Furthermore, the embryos from this experimental group showed a delay in the maturation of the tunica media of coronary arteries. Our findings demonstrate that VIP receptors are involved in morphogenic modulation of coronary artery development in the embryonic quail heart.

Grant Funding Source

Start‐up funding
Multicellular sprouting during vasculogenesis
Authors: Czirok Andras , Rupp Paul A , Lansford Rusty , Filla Michael , Szabo Andras
Published: June 2021

Vasculogenesis, the de novo assembly of vessels from endothelial precursors, is a fundamental process common to both embryonic development and certain pathophysiologies. We studied vasculogenic sprouting, the multicellular patterning mechanisms of vasculogenesis, with time‐lapse microscopy in avian embryos, and in cultures of endothelial cells invading 3D collagen I gels. In quail embryos cell‐autonomous endothelial cell motility and tissue movements were distinguished based on a new method utilizing digitally filtered displacements of immuno‐labeled extracellular matrix components fibrillin and fibronectin. Recently developed TIE1‐GFP transgenic quail lines enabled us to investigate the motility of endothelial cells inside the forming vessels. We correlate the degree of in situ cell‐autonomous motility with the level of TAL1 expression, the morphology of the surrounding vessel network and the structure of the extracellular matrix environment.
CT comparative study of the nasolacrimal angle as a surrogate measure of prognathism in primate and non‐primate species
Authors: Gregg Michael , Palma Douglas , Delman Bradley N. , Marquez Samuel
Published: June 2021

Previous studies suggested the path of the nasolacrimal duct (NLD) to be strongly influenced by the shape of the skull. While other studies have focused on intra‐species variation, this comparative study used a wide spectrum of mammalian species that include human, Old World monkeys, domesticated cow, pig and four dogs. Normal and CT with contrast studies were performed on the human and dog sample with patients presenting with normal and pathologic nasal conditions. The course of NLD was charted radiographically with gross dissections made upon the other cadaveric material. External NLD angle was determined by the intersection of a line that overlapped NLD path to the line represented by hard palate. Results showed non‐primate species (NPS) exhibited the largest positive NLD angles reaching values of positive 140o versus negative 37 o seen in humans. The highly prognathic NPS groups has a NLD that passes through the lacrimal bone ‐ maxilla‐ but can rostrally emerge from its bony canal deep to the nasal mucosa at various openings in the nasal region ‐ cow and pig opening occurs near mucocutaneous junction of nares; dog occurs near ventral nasal meatus. Primates presented with a NLD common drainage opening at the inferior meatus probably due to the relative prognathic reduction shared among this group. Results suggest that NLD angles may be used for determining the degree of prognathism exhibited in living and extinct primates.

Grant Funding Source

Do not have funding.
Immunohistochemical and DNA microarray analysis of rat retinal Müller cells
Authors: Atsuzawa Kimie , Fukasawa Motoaki , Usuda Nobuteru
Published: June 2021

Gene expression and localization of nutrient‐metabolizing enzymes in retinal Müller cells were examined in order to clarify the meaning of fatty acid beta‐oxidation in the retina. Adult rat retinal tissues and isolated Müller cells from the retina were subjected to western blot analysis, immuno‐fluorescent microscopy and immuno‐electron microscopy for mitochondrial fatty acid beta‐oxidation enzymes. Gene expression of fatty acid beta‐oxidation enzymes and related nutrient‐metabolizing genes were analyzed by DNA microarray. Western blot analysis on adult retina and DNA microarray analysis showed that all the fatty acid beta‐oxidation enzyme examined and their gene expressions are present in the retina but small amount about 1/10 of liver tissue. Immuno‐histochemistry at light and electron microscopy showed all these are present only in Müller cells in the retina and isolated Müller cells, in contrast, they are not present in retinal neurons. Mitochondrial fatty acid beta‐oxidation system was shown to be present in retina, and the Müller cells were sole localization of mitochondrial fatty acid beta‐oxidation system in the retina.
Characterization of the nutrient‐metabolism in Sertoli cells by immunohistochemical and DNA microarray analyses
Authors: Fukasawa Motoaki , Atsuzawa Kimie , Matsuzawa Ayami , Usuda Nobuteru
Published: June 2021

Sertoli cells are sustentacular cells which support spermatogenesis for nutritional metabolism in seminiferous epithelium, however, entire picture has not been clarified yet. The localization and gene expression of enzymes for mitochondrial fatty acid beta‐oxidation and related metabolism in Sertoli cells were examined, in order to characterize the nutrient metabolism of seminiferous epithelium. Seminiferous tubules and Sertoli cells, isolated from adult rat testes, were subjected to light and electron microscopic immunohistochemistry and DNA microarray. Immunohistochemistry of the seminiferous epithelium and isolated Sertoli cells revealed the localization of fatty acid beta‐oxidation enzymes only in Sertoli cells but not in spermatogenic cells. DNA microarray analysis showed that gene expression of enzymes for fatty acid beta‐oxidation and ketone body metabolism were low in the seminiferous tubules but relatively high in Sertoli cells. These data suggested that Sertoli cells can do fatty acid beta‐oxidation and ketone body catabolism and anabolism. Sertoli cells were suggested to produce ketone bodies as metabolites of fatty acid beta‐oxidation and to give nutritional support by supplying ketone bodies as an energy source to spermatogenic cells, which may not be able to produce energy from fatty acid beta‐oxidation.
FoxO1negatively regulates sterol regulatory element binding protein‐1c (SREBP‐1c) gene
Authors: Deng Xiong , Howell George , Yellaturu Chandrahasa , Wilcox Henry , Park Edwards , Raghow Rajendra , Elam Marshall , Unterman Terry
Published: June 2021

The induction of genes involved in lipid biosynthesis by insulin is mediated in part by the sterol regulatory element‐binding protein‐1c (SREBP‐1c) and this effect is mediated in part through a mechanism that is dependent on the liver X receptor alpha (LXRα). FoxO1, a forkhead box class‐O transcription factor, is an important target of insulin action. We performed experiments to study the effects of FoxO1 on SREBP‐1c gene expression. Our studies show that over‐expression of constitutively active FoxO1 protein (CA‐FoxO1) inhibits, and small interference RNA against FoxO1 enhances both basal and insulin‐stimulated activity of the rat SREBP‐1c promoter. CA‐FoxO1 suppresses the activities of the SREBP‐1c promoter in a dose‐dependent fashion, and antagonizes the effect of insulin and TO901317 (a synthetic ligand of LXRα) on promoter activity. CA‐FoxO1 significantly reduces the effect of LXRα ?on basal and TO901317‐stimulated activity of a luciferase reporter construct driven by 3 LXR‐response elements, and decreases the transactivational ability of a Gal4‐LXRα fusion protein. These results indicate that FoxO1 plays an important role in regulating basal and insulin‐stimulated SREBP‐1c promoter activity and may exert this effect by antagonizing the function of LXRα.

This work was supported by a grant from the Office of Research and Development, Department of Veterans Affairs.
PROX1 regulation of endothelial cell cycle progression
Authors: Baxter Shannon A , Herbert Krista , Kim Hae‐Kwang , Cheung David Y.C. , Wigle Jeffrey T.
Published: June 2021

The homeobox transcription factor PROX1 is necessary for proper lymphatic vessel development. PROX1 upregulates the expression of CYCLIN E1 in both Drosophila melanogaster and mammalian cells. Expression of CYCLIN E1 is controlled by two E2F sites located on either side of the transcriptional start site. We have shown that PROX1 activates a 1 kb Cyclin E1 promoter via a DNA‐binding independent mechanism that requires both E2F sites.

We used reporter gene assays to assess PROX1 regulation of Cyclin E1 promoter versions in which the E2F sites were mutated. We infected Human Umbilical Vein Endothelial Cells (HUVEC) with adenovirus encoding PROX1, and used bromodeoxyuridine, proliferating cell nuclear antigen and phosphorylated histone H3 staining to quantify changes in cell cycle progression. We used western blotting to measure protein levels of CYCLIN E1.

We observed a decrease in the ability of PROX1 to activate a 206 bp Cyclin E1 promoter when either E2F site was mutated. We noted an increase of CYCLIN E1 in PROX1 infected HUVECs as well as an increased number of cells in S phase. In contrast, there was not a corresponding increase of cells in M phase.

We have established that both E2F sites are essential for PROX1 mediated activation of the Cyclin E1 promoter. Our studies have discovered a novel role for PROX1 as a transcriptional co‐activator.

The National Cancer Institute of Canada and the Manitoba Health Research Council support this project.