bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of advanced glycation end products on dilation of rat skeletal muscle arterioles
Authors: Murphy Timothy Vincent , Jenkins Alicia J , Hill Michael A , Naser Nadim
Published: June 2021

Post translational protein modification, including the formation and accumulation of advanced glycation endproducts (AGE), may contribute to vascular dysfunction in diabetes. This study investigated the effect of AGE on dilation of arterioles possessing myogenic tone. AGE were manufactured by incubating 250 mM glucose‐6‐phosphate with 10 mg/ml BSA in PBS for 60 days. The effects of the modified protein were studied on isolated, pressurized (70 mmHg) first‐order arterioles from rat cremaster muscle. The AGE preparation inhibited dilation of the arterioles caused by both acetylcholine (ACh; endothelium‐dependent) and sodium nitroprusside (SNP), but had no effect on adenosine‐induced vasodilation. Iberiotoxin (0.1 μM), an antagonist of large‐conductance, Ca2+‐activated K+ channels (BKCa), inhibited ACh‐induced dilation. The combination of iberiotoxin and AGE did not cause a greater inhibition of ACh‐induced vasodilation than the application of iberiotoxin or AGE alone. The inhibitory effect of AGE on ACh‐ and SNP‐induced vasodilation was abolished by pre‐incubation of the arterioles individually with the NOS inhibitor L‐NAME (100 μM), the anti‐oxidant lipoic acid (2 μM) or the NADPH oxidase inhibitor apocynin (500 μM). Taken together, these observations suggest that AGE inhibit ACh‐ and SNP‐induced dilation of the arterioles by inhibiting BKCa, through generation of reactive oxygen species.
Morphological changes in the ductus arteriosus in response to development in hypoxia and hyperoxia
Authors: Dzialowski Edward M , Copeland Jennifer
Published: June 2021

The ductus arteriosus (DA) is an oxygen sensitive fetal blood vessel that allows for a right‐to‐left shunt of right ventricular outflow away from the lungs. Previously we have shown that the maturation of the oxygen induced contraction of the DA can be altered by incubation under varying O2 levels. The objective of this study was to characterize the influence of development in hypoxia or hyperoxia on the morphological changes occurring in the chicken DA during hatching. White leghorn chicken eggs were incubated in normoxia (21% O2), hypoxia (15% O2), and hyperoxia (30% O2) for the entire incubation period. The DA was removed from day 18, internally pipped, and externally pipped embryos and hatchlings for histological examination. The major finding was a significant effect of incubation oxygen levels on the DA lumen diameter during hatching. During hatching, the DA lumen diameter from hyperoxic incubated embryos was significantly smaller than that from control embryos. Incubation in hypoxia resulted in a decrease in the closure of the DA in comparison to the control embryos. A similar pattern was observed in the overall length of the right and left DA. The timing of morphological closure of the O2‐sensitive DA is accelerated by in hyperoxia and delayed in hypoxia. This work was funded by the American Heart Association South Central Affiliate and the National Science Foundation.
IL‐10 produced by B1 cells modulates inflammatory response to LPS
Authors: Theobaldo Mariana Cardillo , Barbeiro Denise Frediani , Barbeiro Hermes Vieira , Ariga Suely Kubo , Soriano Francisco Garcia
Published: June 2021

B1 cells (B1) migrate from the peritoneal cavity of mice and home to a distant site of inflammation to become macrophage‐like cells. We studied the role of B1 on inflammatory response to lipopolyssaccharide (LPS). TNFα, IL6 and IL10 (pg/ml) were measure in serum (S), lung (L) and intestine (I) of Xid mouse (a strain deprived of B1) and Balb/c in 1,5; 4, 6h after LPS injection. There was higher survival in Balb/c (100%) in relation to Xid (60%) after 16h of LPS injection. TNFα was increased in Xid compared to Balb/C in 1,5h (I 2045±356; 877±65) and 4h (S 6284±1469; 366±90, L 1135±270; 247±27). Higher IL6 levels in 1,5h (S 11422±545; 2494±144) and 4h (S 10038±38; 1947±384, L 12086±86; 6647±950; I 5638±1456; 745±260) and 6h (S 10365±365; 2469±115, L 13979±919; 8445±662; I 8508±1329; 864±350). Xid presented lower IL10 concentrations in 1,5h (S 102±13; 749±83, L 205±15; 331±29; I 184±11; 316±18), 4h (L 210±7; 297±12) and 6h (I 175±24; 334±37). For studies in vitro we used B1 from IL10 knockout mice (KO) in coculture with RAW 264.7 and the supernatants were collected 24 h after LPS challenge. We found increase of TNFα (7571±717; 2368±231) and IL6 (8477±274; 1473±58) production when B1 (KO) were cocultivated with macrophages compared to WildType. Our data shown that B1 absence increased TNFα, IL6 and reduced IL10 production, suggesting that B1 regulates inflammatory response through IL10. FAPESP
Requirement for labile Zn2+ in phagocytosis by murine macrophages
Authors: Williams Miguel , Kohler Jonathan E , Blass Amy L , Lederer James A , Kelly Edward , Soybel David I
Published: June 2021

Introduction

Systemic Zn2+ deficiency impairs the ability to fight infection. We hypothesized that one mechanism of immune dysfunction in the setting of Zn2+ deficiency is impaired phagocytosis by macrophages.

Methods

RAW 264.7 murine macrophages were incubated for three hours with E. coli cell walls labeled with Vybrant and pHrodo fluorescent reporters. Co‐incubation occurred in the presence or absence of two intracellular Zn2+ chelators (high affinity TPEN 10μM, low affinity BAPTA 10μM) and one extracellular zinc chelator (DTPA 10 μM). Confirmatory experiments were performed with mouse peritoneal macrophages.

Results

Intracellular zinc chelators caused reduced phagocytosis vs control (TPEN: 28% ± 8; BAPTA 54% ± 14%, p < 0.02, N = 5). In contrast, chelation of extracellular zinc with DTPA had no effect.

Conclusion

Free cytoplasmic Zn2+ is required for effective internalization of extracellular bacterial particles by both cultured and primary murine macrophages. Under acute conditions, intracellular stores are sufficient to optimize function.
Characterization of regression of exercise‐induced cardiac hypertrophy
Authors: Stricker Janelle C , Nichols Cody E , Katz Andrew , Fancher Ibra , Cuppett Tiffany C , Miletti Kady , Vasisko Corey , Dabkowski Erinne R , Baseler Walter A , Hollander John M , Morissette Michael R
Published: June 2021

The heart is capable of changing size in response to changes in chronic stress or demand. Exercise and hypertension are classic examples of stimuli that promote physiologic and pathologic hypertrophy respectively. Although initially beneficial for maintaining function with increased stress, pathologic hypertrophy can progress to heart failure if the inducing stress is not mitigated. Additionally, in contrast to physiologic hypertrophy, reverse remodeling of pathologic hypertrophy is often incomplete in respect to returning to normal size and function. In order to understand the molecular mechanisms involved in the reversal of hypertrophy we subjected mice to a three‐week swim protocol followed by 0, 3, and 6 days of rest. Swim‐training induced a 25.4±2.0% (n=19, p<0.05) increase in heart weight/body weight compared to sedentary controls (n=13). Hypertrophy regressed by 31.8% and 90.0% after 3 and 6 days of rest respectively. Interestingly, western blot analysis showed an increase in cardiac LC3B II in rested mice, suggesting the involvement of autophagy in reverse remodeling. Unexpectedly the phosphorylation of Akt (308, 473), GSK3β, and S6 were not increased in rested hearts, but actually decreased in hypertrophied hearts. These data suggest that the observed increase in LC3B II is not mediated by the Akt‐mTOR pathway. Support: NCRR 5P20RR016477 (WV INBRE), NIA K01AG026337 (MRM), AHA (MRM)
Characterization of the DNA binding of Ikaros protein using three dimensional modeling and rapid prototyping technology
Authors: Dovat Elanora , Toomey Dan , Dovat Sinisa
Published: June 2021

Human hematopoiesis is controlled by the coordinated activity of various genes. The Ikaros gene encodes a DNA‐binding protein that is essential for normal hematopoiesis and acts as a tumor suppressor. The absence of Ikaros leads to the severe impairment of lymphocyte development. Deletion of the Ikaros gene, or the loss of Ikaros activity, has been associated with human B and T cell acute lymphoblastic leukemia (ALL). Ikaros binds DNA in a sequence‐specific manner with four C2H2 zinc finger motifs that are located at the N‐terminal end of the protein. DNA binding is an essential part of Ikaros’ function – to regulate expression of the genes that are involved in cellular differentiation and proliferation. Ikaros binds to the upstream regulatory elements of its target genes and regulates their transcription via chromatin remodeling. The Edgewood Campus School SMART (Students Modeling a Research) Team has built an accurate three‐dimensional model of the zinc finger motifs of the Ikaros protein binding to DNA using 3D printing technology. By modeling the Ikaros‐DNA interaction, we hope to provide new insights into the mechanism of Ikaros’ function, as well as to the specificity of its protein‐DNA recognition. This information will help in understanding the role of Ikaros in normal hematopoiesis and in preventing the development of leukemia. Supported by grants from HHMI Pre‐College Program and NIH‐NCRR‐SEPA.
Beneficial effect of melatonin on improvements of neurological function in focal cerebral ischemia
Authors: Lee Seunghoon , Lee Sang‐kil , Chang Kyu‐tae , Hong Yonggeun
Published: June 2021

Ischemic stroke involves oxidative damage caused by the cellular accumulation of calcium; thus, in the present study, we investigated the effect of neurological dysfunction on various markers related to anti‐oxidant and anti‐inflammatory processes in brain tissue obtained from focal cerebral ischemic rats, and we assessed the influence of long‐term melatonin treatment. Male Sprague‐Dawley rats (n=25, eight‐week‐old) were divided into five experimental groups: group‐A (untreated control), group‐B (vehicle, treated with 0.9% saline for eight weeks), and groups‐C through ‐E (MCAo‐induced focal cerebral ischemic rats treated subcutaneously with melatonin at 10 mg/kg/day at 07:00, 19:00, and 07:00 plus 19:00, respectively). After eight weeks of treatment, the rats were sacrificed by decapitation, and their brains collected for RNA isolation. The differential expression of such inflammation‐ and neurotrophic‐related genes as iNOS, nNOS, and BDNF was then examined. The expression of iNOS and nNOS was decreased in groups‐C and ‐E, as compared to that in the vehicle group, while expression of the neurotrophic factor BDNF was significantly increased in the melatonin‐treated groups. Our results indicate that melatonin decreases the expression of anti‐inflammatory and anti‐oxidant‐related genes.

Funding: BioGreen21 Program (Code No. 20070401‐034‐006‐009‐02‐00), Rural Development Administration, Korea
Elucidating The Cellular Markers In The Extended Amygdala Associated With Acute Morphine‐Induced Withdrawal
Authors: Brown Michael Kameron , Radke Anna K , Meisel Robert L. , Gewirtz Jonathan C.
Published: June 2021

Anxiety is a classic symptom of withdrawal that often occurs in humans and animals. Preliminary studies show that anxiety in animals occurs four hours after acute morphine exposure. However, the question of the neurological structures associated with this phenomenon remains unanswered. Structures such as the central nucleus of the amygdala (CeA) and the bed nucleus of the stria terminalis (BNST) have been shown to have crucial overlapping roles in mediating anxiety. Therefore through the use of the immediate early genes (IEGs) c‐Fos and p‐CREB, which serve as molecular markers, we investigated whether structures in the extended amygdala are involved in acute morphine‐induced anxiety in spontaneous and precipitated withdrawal. Cellular activation was observed through c‐Fos staining of the CeA, two hours after withdrawal from morphine. Definitive conclusions could not be drawn from the c‐Fos staining in the BNST. The p‐CREB staining in the CeA suggests that this region was activated by spontaneous withdrawal from morphine. The p‐CREB staining in the BNST suggests that this region of the brain may be activated by both precipitated and spontaneous withdrawal. Together the data from this experiment supports the hypothesis that extended amygdala structures are involved in acute‐morphine induced withdrawal. Supported by NIH T34GM008411
Postprandial changes in GI‐tract peptide hormones in the teleost Atlantic salmon
Authors: Ronnestad Ivar , Valen Ragnhild , Murashita Koji , Jordal Ann‐Elise Olderbakk
Published: June 2021

The spatial distribution of two isoforms of Ghrelin (Ghrelin‐1 and ‐2), two isoforms of Cholecystokinin (CCK‐L and ‐N) and peptide YY (PYY) was assessed by Q‐PCR in four GI‐tract segments covering stomach, pyloric caeca, midgut and hindgut after feeding a single meal to Atlantic salmon. Both CCK‐L and CCK‐N levels increased within 1.5 hours past feeding (hpf). At this time the stomach was still ca 80% full (the gastric evacuation half‐time was 4.5 hrs), the gallbladder was almost completely empty and chyme had started to be transferred into the midgut. During the 24 hpf there were both temporal and spatial differences between the expression profile of the CCK isoforms. PYY expression increased at 1.5 hpf, and then decreased after 3 hpf. Feeding had only minor effects on the Ghrelin isoforms expression. Taken together this suggests that CCK and PYY have in part similar roles in regulating digestive processes to that observed in higher vertebrates. The two isoforms of CCK appear to have different functions. The data did not identify a role for Ghrelin related to digestion or appetite comparable to the orexigenic role described in humans.
Mice Deficient in Sympathetic Activation of Brown Fat Gain Excessive Weight when Housed at Thermoneutrality
Authors: McClure Elizabeth Ann , Swoap Steven John
Published: June 2021

When exposed to dietary excess, mice release norepinephrine (NE) onto brown fat, activating uncoupling protein‐1, and dissipating caloric excess as heat. Mice lacking dopamine beta‐hydroxylase(DBH) cannot produce NE, making them cold‐sensitive and unable to activate a sympathetic response to thermal stress or dietary excess. However, DBH−/− mice fed a high fat diet (HFD) and housed at 22°C gain weight normally due to compensatory thermogenic mechanisms which raise metabolic rate. The hypothesis that NE is essential for diet‐induced thermogenesis was tested by housing DBH−/− mice at thermoneutrality on a HFD or low fat diet (LFD) for 8 weeks. The thermoneutral zone and basal metabolic rate (BMR) of female DBH−/− mice and littermate controls was calculated. There was no difference in the mass specific BMR between DBH−/− mice and control mice (24.3 ± 0.6 vs. 24.6 ± 0.9 ml O2/min/g). A trend of a reduced lower critical temperature was observed in the DBH−/− mice in comparison to controls (28.4 ± 0.1 vs. 28.6 ± 0.1 °C). DBH−/− mice gained weight on either a HFD or a LFD (23.5 ± 7.0% and 19.2 ± 3.2% of body weight, respectively.) Control mice on either a HFD or LFD at thermoneutrality gained significantly less weight than the DBH−/− mice (11.5 ± 3.3% and 16.3 ± 2.6% of body weight, respectively). These results demonstrate that while DBH−/− mice have a normal BMR, they are more susceptible to weight gain at thermoneutrality than control mice.
Upregulation of adipogenesis induced by exogenous melatonin in vitro and in vivo
Authors: Kim Sunmi , Hong Yunkyung , Li Zichao , Hong Jeong‐wha , Hong Yonggeun
Published: June 2021

Melatonin, a major product of the pineal gland, controls many biological systems on a roughly 24‐h cycle. The regulation of energy homeostasis, including adipogenesis, is one example of a circadian clock‐controlled pattern. Here, we investigated the putative role of melatonin in lipogenesis in vitro and vivo. Undifferentiated 3T3‐L1 preadipocytes were cultured and treated with melatonin. On day 1 or 60, exogenous melatonin was injected subcutaneously into Sprague‐Dawley rats at 07:00 and 19:00. The expression of several markers of lipid metabolism, including PPARγ, perilipin, and Hmgcr, was analyzed by RT‐PCR. Oil red O staining was used to detect lipid droplets in vitro. Morphological changes were observed in 3T3‐L1 cells exposed to exogenous melatonin in vitro. A significant increase in adipogenesis marker expression was detected over time (p<.05), and oil red O staining was observed in cells treated with 1 mM or 10 nM melatonin. Exogenous melatonin treatment for one day significantly decreased cardiac Hmgcr mRNA expression, whereas treatment for 60 days resulted in a definite reduction in hepatic expression (p<.05). Significant changes in Hmgcr, PPARγ, and perilipin mRNA expression were detected in response to melatonin; thus, melatonin regulates lipid metabolism through transcriptional regulation.

Funding: BioGreen21 Program (Code No. 20070401‐034‐006‐009‐02‐00), Rural Development Administration, Korea
IMPROVED CARNITINE STATUS IN THE TRAINED RAT IS ASSOCIATED WITH INCREASED mRNA EXPRESSION OF LIVER γ‐BBH AND KIDNEY OCTN2
Authors: Tedeschi Jamie , Luna Branden , Broderick Thomas
Published: June 2021

Endurance training alters the oxidative capacity of tissues to favor the oxidation of fatty acids. In response to training, carnitine concentrations in plasma are increased suggesting that either carnitine biosynthesis is stimulated or renal uptake of carnitine from the urine is enhanced, or both. Carnitine is essential in the oxidation of fatty acids by permitting their transport into the mitochondria for oxidation. Carnitine is released into the circulation following its hydroxylation by γ‐butyrobetaine hydroxylase (BBH), the final enzyme in the biosynthetic pathway in liver. The organic cation transporter (OCTN2) is also important in the distribution of carnitine by facilitating renal reabsorption of carnitine from the urine. To investigate whether these proteins are implicated in carnitine homeostasis in response to exercise, BBH and OCTN2 expression were determined following 10 weeks of treadmill exercise training in male Wistar rats. Exercise training significantly increased the level of total carnitine in plasma, whereas in liver the level of total carnitine (sum of free, short‐chain acylcarnitine and long‐chain acylcarnitine) was not modified by exercise. In exercise‐trained rats, BBH expression in liver was significantly increased compared to sedentary rats, whereas in kidney a significant decrease was observed. OCNT2 expression in both liver and kidney were significant increased in exercise‐trained rats. Our results indicate that the improved plasma carnitine status in the trained rat can be explained, in part, by increases in both carnitine biosynthesis and renal reabsorption of carnitine.
A New Respirometry Technique for Room Calorimetry and Other Long‐Duration Recordings
Authors: Lighton John RB , Melanson Edward L
Published: June 2021

Because traditional respirometry measures metabolic rates as a function of the difference between incurrent and excurrent gas concentrations (usually air entering and leaving a sealed chamber such as a room calorimeter), the accuracy with which small changes in gas concentrations are measured can limit the overall accuracy of the technique. Repeated measurement of incurrent gas concentrations is necessary, but disrupts the continuity of metabolic recordings, resulting in a tradeoff between temporal continuity and accuracy. Differential gas analyzers, although an improvement on single‐channel analyzers, do not eliminate the baselining requirement. JL has developed a patent‐pending technique that allows baselining to occur in the background, and eliminates the requirement for water vapor scrubbers of any kind. Signal processing of the resultant raw data reassembles a complete, unbroken metabolic recording. We present details of the technique, and data from three case studies, including a 24‐hour response‐corrected room calorimeter record including episodes of activity and sleep, and demonstrating excellent temporal resolution.
Oxidative Gene Expression in Single Muscle Fibres after Continuous and Intermittent Exercise
Authors: WANG LI , Sahlin Kent
Published: June 2021

Fibre‐type heterogeneity is a potential problem in the interpretation of gene expression in mixed muscle. The current study aimed to investigate the fibre‐type variation of master genes involved in oxidative metabolism and signaling pathway as well as the influence of exercise. Untrained subjects (n = 8) cycled for 90 min either at a constant 60 % of VO2max (CE) or at an intensity alternating between 120 % (12 s) and 20 % of VO2max (18 s) (IE). Muscle biopsies were obtained before (Pre) and 3 h after (Post) exercise. Peroxisome proliferator‐activated receptor‐γ coactivator‐1α (PGC‐1α) and pyruvate dehydrogenase kinase 4 (PDK4) genes were measured with quantitative real‐time PCR in freeze‐dried muscle single type I or type II fibres. PGC‐1α mRNA showed large variation within the same fibre type (> 3‐fold). PGC‐1α and PDK4 mRNA was 2–3‐fold higher in type I fibres (P < 0.05 vs. type II) at Pre, but at Post the difference was reduced (IE) or abolished (CE). Acute cycling increased PGC‐1α and PDK4 mRNA in both fibre types. Exercise‐induced increases in mRNA of PGC‐1α and PDK4 were negatively correlated with the basal level. We conclude that human skeletal muscle has higher baseline mRNA level of oxidative genes in type I than in type II fibres, but this difference is reduced after exercise. This may in part be explained by the negative correlation between the basal mRNA level and the exercise‐induced increase in mRNA. This work was supported by grants from the Swedish Research Council (23998‐50466), GIH and the National Centre for Research in Sports (CIF).
Increased contractile acitivity induces autophagy in skeletal muscle
Authors: Lira Vitor A. , Okutsu Mitsuharu , Zhang Mei , Yan Zhen
Published: June 2021

Contractile activity is essential in maintaining and promoting the oxidative phenotype of fatigue resistance, metabolic flexibility, insulin sensitivity and resistance to oxidative stress in skeletal muscle. This requires constant remodeling of structural proteins and organelles (e.g. mitochondria). We therefore hypothesized that autophagy, a catabolic process involved in the degradation of long‐lived proteins and organelles, is enhanced by increased contractile activity (i.e. endurance exercise). We submitted adult mice (12‐wk old, C57BL6) to 28 days of voluntary wheel running and used sedentary mice as controls (n=10/group). The expression of proteins associated with autophagosome formation (Beclin 1, LC3‐I+LC3‐II), mitochondrial autophagy (Bnip3), and aggregate protein degradation (p62/SQSTM1) were measured in the exercise‐recruited plantaris muscles. Consistent with our hypothesis, exercise training resulted in significant induction of Beclin1 (33%), Bnip3 (41.5%), LC3‐I+LC3‐II (43%) and LC3‐II‐to‐LC3‐I ratio (210%), and reduction of p62 (24%). The same directional changes were observed when tonic, oxidative soleus muscles were compared with phasic, glycolytic white vastus muscles in sedentary mice. Our findings suggest a role for autophagy in contractile activity‐mediated maintenance and promotion of oxidative phenotype in skeletal muscle.

This study was funded by NIH Grant AR050429 (Zhen Yan).
Reinventing the Wheel: Voluntary Running Promotes Mitochondrial Adaptations in mtDNA Mutator Mouse Model of Aging
Authors: Stokl Andrew James , Safdar Adeel , Hettinga Bart P. , Ogborn Daniel I. , Kujoth Gregory C. , Prolla Tomas A. , Tarnopolsky Mark A.
Published: June 2021

The role of mitochondrial abnormalities and mitochondrial DNA (mtDNA) mutagenesis has been extensively characterized in aging. A causal role for mtDNA mutagenesis in aging is supported by recent studies demonstrating that the polymerase gamma (PolG) mutator mouse, harbouring a proofreading‐deficient copy of PolG, exhibits an accelerated aging phenotype, systemic mitochondrial dysfunction, multisystem failure, and reduced lifespan. Longitudinal studies in humans demonstrate that a physically active lifestyle reduces the risk of chronic diseases and increases life expectancy. We aimed to delineate if voluntary wheel running (VOL) can prevent mitochondrial respiratory chain dysfunction and attenuate mitochondrial ultrastructural abnormalities in skeletal muscle of PolG mice. At 3 months of age, 24 PolG mice (♀ = ♂) were randomly assigned to a sedentary (SED) or VOL group. VOL training attenuated the loss of muscle mass (19%) and prevented cardiac hypertrophy (20%) in PolG‐VOL vs. PolG‐SED (P<0.05). VOL training dramatically increased skeletal muscle mtDNA copy number (1.5‐fold), COX activity (75%), and complex‐IV subunit I (2‐fold) and subunit‐IV (1.7‐fold) protein content vs. PolG‐SED (P<0.05). Electron microscopic analysis revealed an atypical accumulation of swollen pleomorphic mitochondria with fragmented cristae and disrupted membranes in the skeletal muscle of PolG‐SED. VOL training reduced these mitochondrial morphological abnormalities in PolG mice. We propose that VOL training promotes mitochondrial capacity and offers a valuable therapeutic intervention for attenuating aging and related morbidity and mortality. (Funded by CIHR)
Mechanisms of Histone Chaperones and Chromatin Remodelers in Transcription Elongation
Authors: Kuryan Benjamin George , Carey Michael
Published: June 2021

It is established that nucleosomes pose a barrier to elongating RNA Polymerase II (Pol II), but the mechanisms by which this is overcome are still largely unknown. In vivo, the nucleosomal barrier is likely abrogated through the coordinated activities of various enzymes including ATP‐dependent chromatin remodelers (ATPases) and histone chaperones. We have developed a biochemical system whereby transcription through a nucleosome is dependent on both the activity of the ATPase complex “Remodels Structure of Chromatin” (RSC) and the histone chaperone “Nucleosome Assembly Protein 1” (Nap1). RSC alone stimulates elongation through the nucleosome, but depends on an acceptor DNA molecule to receive the octamer evicted from the transcribed template. We find that Nap1 stimulates RSC‐dependent transcription in the absence of an acceptor DNA molecule. However, Nap1 inhibits RSC‐mediated octamer transfer, but not remodeling. In reactions with RSC and Nap1, an H2A‐H2B dimer is lost. These data support a model where RSC evicts the octamer from the template to facilitate Pol II passage and Nap1 reassembles a hexasome in cis. Formation of this hexasome is consistent with previous studies of transcription through nucleosomes where transcription was dependent on high salt conditions to loosen the histone‐DNA contacts. We have now shown this occurs enzymatically through Nap1‐mediated RSC remodeling.
Endothelin‐converting enzyme‐1 (ECE‐1)‐dependent trafficking of corticotropin‐releasing factor receptor 1 (CRF‐R1).
Authors: Bhargava Aditi , Bunnett Nigel W , Hasdemir Burcu
Published: June 2021

CRF and Urocortin1 (Ucn1) regulate endocrine responses to stress via activated CRF‐R1. Whether CRF or Ucn1 differentially affects trafficking or signaling of CRF‐R1 is unknown. The metalloendopeptidase ECE‐1 degrades internalized neuropeptides in endosomes to promote recycling and resensitization. Trafficking was examined by labeling cell‐surface receptors on HEK‐CRF‐R1 cells and stimulating cells with CRF or Ucn1. Within 0.5 h, CRF‐R1 trafficked from the plasma membrane to the endosomes, where it co‐localized with ECE‐1. Ucn1 served as substrate for ECE‐1 at extracellular pH 7.4, but CRF was degraded by ECE‐1 at endosomal pH 5.5. Inhibition of ECE‐1 activity did not affect CRF or Ucn1‐mediated receptor endocytosis, but prevented Ucn1‐ but not CRF‐induced CRF‐R1 recycling. Resensitization of CRF‐R1 signaling was examined by measuring [Ca2+]i responses to repeated challenge with CRF or Ucn1. Each caused a similar increase in [Ca2+]i. Another challenge after 0 h of recovery yielded a minimal [Ca2+]i response, indicating desensitization, whereas, after 4 h, responses were mostly recovered, indicating resensitization. ECE‐1 inhibition strongly inhibited resensitization to Ucn1 (27±9% of control), but CRF‐mediated resensitization was unaffected (82±8% of control). Thus, ECE‐1 may regulate differential trafficking and signaling of CRF‐R1 upon binding different ligands.
Disrupted in schizophrenia 1 (DISC1) regulates ATM‐mediated NFκB activation during hypoxia and reoxygenation (H/R)
Authors: Liu Jia‐Ren , Ibla Juan Carlos , Liu Qian , Li Jing , Han Xiaohui , Khoury Joseph
Published: June 2021

H/R has been described to mediate cellular mechanisms involved in anti‐inflammatory responses. In cultured epithelia and lung tissue we describe a mechanism by which H/R results in the transcriptional induction of DISC1 protein which is involved in controlling diverse cell signaling mechanisms and potentially involved in regulating inflammatory responses. Using cell models of H/R and DISC1 exogenous overexpression we show a significant inhibitory effect on the ATM‐mediated NFκB activation cascade. We demonstrate that DISC1 overexpression both significantly attenuates hypoxia‐mediated ATM phosphorylation and prevents its cytoplasmic translocation where it functions to activate the Iκκ complex. DISC1‐inactivated ATM is then unable to phosphorylate Serine‐85 residue of the NFκB essential modulator (NEMO), Iκκ’s principal regulatory molecule. Wild type and cells overexpressing DISC1 demonstrate differential patterns of IκBα phosphorylation and degradation in the presence of hypoxia. DISC1 protein levels significantly modulate NFκB activation profiles and pro‐inflammatory gene expression (IL‐6). We propose a mechanistic model by which H/R regulates the expression of DISC1, modulating ATM activation and further controlling NFκB‐regulated pathways. Together these results implicate DISC1 in NFκB signaling and provide novel basis for understanding innate anti‐inflammatory responses.
Down‐regulation of autophagy induced by caveolin‐3 and myosin heavy chain in response to forced exercise in rats
Authors: Park Sookyoung , Hong Yunkyung , Lee Youngjeon , Hong Yonggeun
Published: June 2021

Although suppressing myostatin‐mediated signaling by caveolin‐3 (cav‐3) may results in reduction of muscular atrophy, the skeletal muscle phenotypic response following forced exercise is not fully defined. Therefore, we examined whether the changes of caveolins and myosin heavy chain (MHC) gene expression have relevance to autophagic signaling in forced exercised muscle. Male (n=16) and female (n=16) Wistar rats (10–12 weeks age, 280–300 g each) were divided into two groups: control (Con) and exercise (Ex). The rats in the Ex group were subjected to treadmill exercise at 25 m/min for 30 min, three times a day (07:00, 15:00, and 23:00). Stable expression of cav‐3 was detected in all tissues taken from the Ex group. In the gastrocnemius (GCM) muscle of the female rats, the expression of cav‐3 and MHC2a was significantly increased in the Ex group, as compared with the Con group (p<.05). Similarly, significantly greater MHC2x gene expression was detected in the GCM muscle of the male rats in the Ex group, as compared to the Con group (p<.05). Finally, increased cav‐3 gene expression and slightly decreased TGF‐β1 gene expression were noted in the muscles of the rats, suggesting that cav‐3 influences the expression of MHC isoforms and TGF‐β1. Furthermore, the conversion of LC3I to LC3II was significantly decreased in the GCM muscle of the male rats, indicating that protein degradation was reduced by forced exercise.

Funding: BioGreen21 Program (Code No. 20070401‐034‐006‐009‐02‐00), Rural Development Administration, Korea
LiCl causes an acute decrease in ataxia telangiectasia mutated (ATM) protein levels in L6 myotubes
Authors: Robinson‐Hill Rona , Ching James Kain , Fisher Jonathan S.
Published: June 2021

We have previously found that insulin‐like growth factor 1 (IGF‐1) causes an acute increase in levels of ATM in cultured myotubes and in mouse skeletal muscle and that serum starvation (SS) of myotubes causes concomitant increases in levels of the AMP‐activated protein kinase (AMPK) and ATM. Here, we show the validity of the SS effect on increase of ATM by running negative and positive controls for ATM and showing that ATM runs at the appropriate molecular weight (~350 kDa, between 460 kDa and 268 kDa markers). To determine the potential role of AMPK in the SS effect on ATM protein levels, we transduced L6 myotubes with either green fluorescent protein (GFP) or a dominant negative form of AMPK (AMPK‐DN). SS increased ATM protein levels by more than 2‐fold (P<0.05, n=6/group) in myotubes expressing GFP but not in myotubes expressing AMPK‐DN. Because IGF‐1 and AMPK both inhibit glycogen synthase kinase 3β (GSK3β), we hypothesized that the GSK3β inhibitor Li+ would cause an increase in ATM levels. Contrary to our hypothesis, one hour of incubation of L6 myotubes with 1 mM LiCl caused about a 50% decrease (P<0.05, n=12/group) in ATM protein levels compared to a 1 mM NaCl control. Thus, it appears that inhibition of GSK3β causes an acute decrease in ATM protein levels, and AMPK and IGF‐1 act through a GSK3β‐independent pathway to increase ATM protein levels.
Nuclear bone morphogenetic protein 2 mutant mice exhibit slowed relaxation and a shift in the force frequency relationship in skeletal muscle
Authors: Evanson Bradley G , Schmidt Alina D , Mayo Jaime L , Bridgewater Laura C , Hancock Chad R
Published: June 2021

Conventional bone morphogenetic protein 2 (Bmp2) is a secreted growth factor characterized by its ability to induce bone and cartilage formation, and by its role in developmental processes such as axis formation, cardiac development, and neuronal differentiation. Recently, however, we have identified a novel form of Bmp2 (nBmp2) that is translated from an alternative start codon and is localized to the nucleus rather than being secreted. To study the function of nBmp2 in the nucleus, we generated mice bearing an nBmp2 mutation that prevents nuclear localization. Here we evaluate muscle performance in these mutant mice using in situ stimulation of the gastrocnemius, plantaris, soleus (GPS) muscle complex via the sciatic nerve. Specific peak tetanic force of the GPS muscle group was slightly higher in nBmp2 deficient mice compared to control mice (3.3 ± 0.1 and 3.1 ± 0.1 gm tension/mg muscle in mutant vs. control muscles, respectively, p>0.05). No significant differences were observed in the specific peak twitch force between groups. Muscle fatigue was examined using 2Hz twitch contractions for 10 min. The fatigue profiles for the two groups were essentially identical, but half relaxation times were significantly prolonged by up to a 42% increase in mutant muscles compared to control muscles (p<0.05). Further, force frequency analysis showed significantly elevated force production in mutant muscles compared to controls from 10 to 80 Hz stimulation frequency (p<0.01). These results suggest that nBmp2 plays an important role in maintaining normal intracellular transport of calcium during skeletal muscle contraction and relaxation. Grant support: NIH AR48839
Development of siRNA strategy to knockdown the regulatory contractile proteins in lymphatic muscle
Authors: Chakraborty Sanjukta , Zawieja Scott , Davis Michael J , Gashev Anatoliy A , Zawieja David C , Muthuchamy Mariappan
Published: June 2021

The aim of this study was to develop a fast and efficient method to screen short interfering RNA (siRNA) sequences and incorporate them into recombinant adenoviruses to specifically knockdown regulatory myosin light chain 20, smooth muscle α‐tropomyosin (TM) and cardiac troponin C (TnC) in lymphatic muscle. siRNA sequences targeting each specific gene were either custom‐designed or purchased directly from Dharmacon. We used lymphatic muscle cells (LMCs) derived from rat mesenteric or thoracic duct lymphatics to test the efficiency of the siRNA sequences to knockdown a specific gene. After 48‐72 hrs, quantitative Western blot analyses were performed on the siRNA transfected or control LMCs to verify the percentage of knockdown of the targeted genes. Results demonstrated that myosin light chain kinase and troponin C were knocked down more than 90% in LMCs using the specific siRNAs. siRNA targeted to α‐TM SM isoform decreased the level of α‐TM SM up to 50–60% in the LMCs. We optimized the required siRNA concentrations and the transfection time for each specific gene. The steps required for design of a hairpin siRNA construct containing the target siRNA sequences and the generation of adenoviruses will be discussed. Our data demonstrate a strategy to screen siRNA sequences targeted to specific genes, which may play an important role in regulating lymphatic contractility. AHA 09POST2280005 SC; NIH RO1HL80526 MM.
Curcumin ((E,E)‐1,7‐bis(4‐Hydroxy‐3‐methoxyphenyl)‐1,6‐heptadiene‐3,5‐dione) activates and desensitizes the nociceptor ion channel TRPA1
Authors: Ghatta Srinivas , Leamy Andrew W , Mcalexander M Allen , Carr Michael J
Published: June 2021

Many TRPA1 ion channel activators are reactive electrophiles that form Michael adducts with cysteine and lysine residues in TRPA1’s intracellular N‐terminus. Curcumin (CUR), the active principle of turmeric root (Curcuma longa), can form Michael adducts. To test the hypothesis that the electrophilic CUR activates TRPA1 and desensitizes in presence of physiological levels of calcium, we have performed whole‐cell voltage‐clamp analysis on both human TRPA1 heterologously expressed in HEK293 cells (hTRPA1) and native mouse vagal neurons. In nominally calcium‐free solutions to minimize calcium‐dependent indirect activation of TRPA1, CUR increased TRPA1 currents in hTRPA1 cells in a concentration‐dependent manner (1 – 30 μM) but did not activate recombinant TRPM8, TRPV1, or TRPV4 channels. In 7/11 vagal neurons from wild type mice, CUR (30 μM) produced inward currents (11.6 ± 5.4 pA/pF) that were largely reversed by the non‐specific ion channel blocker, ruthenium red (10 μM) or the selective blockers, AP‐18 (20 μM) and HC‐030031 (20 μM). By contrast, 6 capsaicin‐sensitive neurons from TRPA1‐deficient mice did not respond to CUR. CUR‐dependent currents in hTRPA1‐HEK cells desensitized completely and exhibited marked tachyphylaxis. The data demonstrate that CUR causes activation and subsequent desensitization of native and recombinant TRPA1 ion channels of multiple mammalian species.
Effect of the estrous cycle on the muscle reflex in female rats
Authors: Koba Satoshi , Watanabe Tatsuo
Published: June 2021

The level of estrogen during proestrus and estrus phases (P‐E) in the estrous cycle of most mammalian females is higher than that during metestrus and diestrous phases (M‐D). Muscle contraction stimulates thin fiber muscle afferents and reflexly evokes sympathoexcitation. Previous literature has shown that estrogen attenuates the muscle reflex in female cats. We examined the effect of the estrous cycle on the muscle reflex in female rats. The estrous cycle was judged by vaginal smear. Electrically induced 30 s hindlimb muscle contraction in decerebrate rats during P‐E (n=6) evoked less (P<0.05) increases in mean arterial pressure as compared to rats during M‐D (n=6) (+8 ± 4 vs. +25 ± 4 mmHg; PE vs. M‐D). The renal sympathetic nerve activity (RSNA) response to 1 min intermittent (1–4 s stimulation to relaxation) bouts of static contraction in rats during P‐E (n=6) was also significantly less than that in rats during M‐D (n=6) [+35 ± 10 vs. +96 ± 15 arbitrary unit (a.u.)]. The RSNA elevation seen during 30 s tendon stretch, a stimulus of mechanically sensitive muscle afferents, was significantly less in rats during P‐E (n=12) than that in rats during M‐D (n=10) (+161 ± 26 vs. +266 ± 36 a.u.). Collectively, we suggest that estrous cycle influences the muscle reflex through an effect on mechanically sensitive muscle afferents. Estrogen may play a role in attenuating the muscle reflex seen during P‐E. Supported by KAKEN 21590255.
Sensitivity to low pH in mouse glomus type I cells correlates with ASIC3 expression
Authors: Lu Yongjun , Whiteis Carol A , Chapleau Mark W , Sluka Kathleen , Abboud Francois M
Published: June 2021

We have recently reported that acid‐sensing ion channels (ASICs) are expressed in glomus cells of rat carotid bodies (CB) and that their activation at low extracellular pH results in rapid early depolarization which is inhibited by amiloride (Circ Res 101:109‐1019, 2007). Moreover, the ASIC mediated inward current and depolarization at low pH are enhanced in glomus cells of spontaneously hypertensive rat (SHR) which overexpress ASIC3 in their carotid bodies (Circ Res 105:536‐545, 2010). In this study we tested the hypothesis that ASIC3 contributes to responses to low pH in mouse carotid bodies (CBs). We contrasted calcium transients Δ[Ca2+]i during extracellular acidosis (pH 6.0) and cyanide (CN 1mM) in three groups of mice: ASIC3 null (KO), wild type (WT), and ASIC3 transgenic (Tg) mice. Clusters of glomus cells were dissociated from the carotid bodies, loaded with Fluo4/AM (a single wavelength Ca2+ probe), imaged with a CCD camera (Photometrics Cool SNAP‐cf) and analyzed with software (MetaMorph 6.1). Baseline values [Ca2+]i were 57±2, 56±3, and 55±5 nM in KO, WT and Tg respectively. The results shown below indicate that in KO there was a significant reduction in the percentage of cells responding to low pH compared to CN. The magnitude of the Ca2+ transients during low pH was significantly lower than during CN in KO mice but significantly higher in Tg mice (p < 0.01).

Low pH 6.0
CN (1mM)

KO
WT
Tg
KO
WT
Tg

Total cells
207
205
32
118
129
32

% responsive
41%†
75%
69%
71%
81%
59%

Δ[Ca2+]i nM
9±1*
10±1*
15±1†*
12±1
14±1
7±1

p < 0.05 (KO or Tg vs. WT);

p < 0.01 (pH6.0 vs. CN)

Contrasting responses to low pH to those with CN allowed us to confirm the specificity of the correlation between pH responsiveness and ASIC3 expression. The results indicate that ASIC3 is an important determinant of acid sensitivity in mouse carotid body chemoreceptors. (HL14388).
Bradycardic response induced by stimulation of α4β2 nicotinic receptors
Authors: Li Yi‐Fan , LaCroix Carly , Freeling Jessica
Published: June 2021

Our recent studies revealed that, in a dual‐phase response induced by non‐selective nicotinic stimulation, the bradycardic phase of the response was blocked by a selective agonist to α4β2 nicotinic cholinergic receptors (nAchRs). This study was to further investigate the functional role of this subtype of nAchRs in regulation of heart rate. In urethane‐anesthetized male Swiss Webster mice, the intravenous injection of an α4β2 nAchRs agonist, varenicline, induced a quick and pronounced bradycardic response without sympathetic responses. This bradycardic response was prolonged after the administration of a cholinesterase inhibitor, neostigmine and eliminated by a muscarinic receptor antagonist, atropine, suggesting that this response is associated with acetylcholine (Ach) action and is mediated via muscarinic receptors. These data suggest that α4β2 nAchRs may be located in the intracardial ganglia where the receptors play a role in regulation of Ach release.

This work was supported by American Heart Association grant No. 0835256N and NIH grant No. 1R03AG033291. Varenicline compound was provided by Pfizer Inc.
Age‐related hypertension is associated with impairments in kidney dopamine D1 and angiotensin II AT1 receptor functions
Authors: Chugh Gaurav , Lokhandwala Mustafa , Asghar Mohammad
Published: June 2021

The kidney dopamine D1 (D1R) and angiotensin II AT1 (AT1R) receptors by regulating sodium transporters maintain sodium homeostasis and blood pressure (BP). We have reported that the aging process is associated with changes in kidney function and increase in BP. Therefore, we hypothesized that impairments in kidney D1R and AT1R functions contribute to altered sodium balance and high BP in aging. To test this hypothesis, we measured BP and D1R and AT1R functions in adult (3‐month) and old (21‐month) Fischer 344 X Brown Norway F1 rats (a rat aging model). The D1R and AT1R functions were determined by administering D1‐like receptor agonist (SKF‐38393) and AT1 receptor antagonist (candesartan) respectively. We found that systolic, diastolic and mean arterial blood pressures were higher in old rats. SKF‐38393 increased urinary sodium excretion (UNaV) and fractional sodium excretion (FENa) in adult but not in old rats. While candesartan increased UNaV in both adult and old rats, the magnitude of natriuretic response was greater in old rats. Candesartan‐mediated increase in FENa over basal was also higher in old rats. Although the basal UNaV was similar in both groups, basal FENa was lower in old rats. These studies demonstrate the presence of diminished D1R and exaggerated AT1R functions in the aging kidneys, which may contribute to sodium retention and increase in blood pressure in old rats (NIH/NIA AG25056, AG29904).
Capture and Analysis of Newly Synthesized RNA –A Novel Enabling Technology to Study High Resolution Gene Expression.
Authors: Kumar Kapil , Agnew Brian
Published: June 2021

Newly transcribed RNA contains valuable information of the transcriptome that cannot be obtained from total RNA pool. Development of enabling technologies using affinity nucleosides will help accelerate research that depends on high resolution gene expression. We describe an enabling tool based on click chemistry, which begins with metabolic incorporation of an ethynyl uridine (EU) into newly synthesized RNA. The purified total EU‐RNA is reacted with biotin‐azide using copper catalyzed click chemistry followed by binding to streptavidin beads. The captured nascent RNA can be analyzed by RT‐qPCR, micro arrays & sequencing. Toxic effects of EU on cell health and transcriptome were investigated. Our results show that nascent RNA can be captured and analyzed via RTqPCR and RNA‐seq. No toxic effects on cell health were seen. Microarray analyses show no alteration of the global transcriptome. For the first time, this method enables pulse‐chase of RNA resulting in capture of new RNA and its identification. The main limitation of all other approaches is that true mRNA turnover cannot be studied. Using our approach, we show RNA turnover rates for several mRNA transcripts and correlation to relative abundance.
cAMP‐mediated processing of FGF23 : involvement of O‐glycosylation and furin.
Authors: Bhattacharyya Nisan , Wiench Malgorzata , Andreopoulou Panagiota , Connolly Brian , Bugge Thomas , Gafni Rachel I. , Collins Michael T.
Published: June 2021

The phosphate and vitamin‐D regulating hormone, FGF23, circulates in both an active intact (iFGF23) and an inactive C‐terminal (cFGF23) form. Post‐translational processing of FGF23 involves furin and the glycosyltransferase, GalNac‐T3. Subjects with fibrous dysplasia of bone (FD), which is caused by activating mutations in the cAMP‐regulating protein Gsalpha, have elevated levels of c‐ vs. iFGF23. To test the hypothesis that Gsalpha/cAMP signaling regulates FGF23 processing, we evaluated FGF23 levels, FGF23 glycosylation, and GalNac‐T3 and furin activity. HEKF, a HEK293 clonal cell‐line that is over‐expressing FGF23 and MLO‐Y4 cells, a mouse osteocyte cell line were used. Cells were transfected with mutant Gsalpha; and/or treated with dibutyryl cAMP (db‐cAMP) and/or forskolin. Both db‐cAMP and forskolin treatment resulted in increased c‐ vs: iFGF23 levels. Results from deglycosylation experiments indicated that i‐ and cFGF23 are glycosylated, and this process is modulated by db‐cAMP and forskolin. cAMP treatment resulted in decreased GalNac‐T3/FGF23 association in co‐immunoprecipitation experiments, and increased furin levels. No FGF23 cleavage was observed when furin (−/−) LoVo cells were transfected with FGF23, indicating the involvements of furin activity in FGF23 processing. Our results demonstrate that FGF23 processing can be regulated by Gsalpha/cAMP and both furin and GalNac‐T3 activities are regulated and important for FGF23 processing.