bluuebunny/crossref_metadata_2025 huggingface dataset:
Erythrocytes Constitute the Main Blood Compartment for Aluminum
Authors: Adragna Norma C. , Collins Jodee , Arevalo Bernardo , Lauf Peter
Published: June 2021

Aluminum (Al), a trace metal with no biological function and toxic to humans and animals, is associated with hematological/metabolic alterations and neurodegenerative processes such as Alzheimer's disease (AD). Further evidence supports Al's role in the etiology of AD and validates use of readily accessible tissues such as peripheral blood. Basal Al concentration has been reported in plasma and serum, while studies in erythrocytes are scant. Here, we report optimal conditions for simultaneous determination of Al concentration in blood compartments from humans and sheep. Al was determined by electrothermal atomic absorption spectrophotometry in whole blood, plasma, serum and red blood cells (RBCs). Solvent type and composition, matrix modifier concentration and sample age were tested on the reproducibility of Al determination. Significant interindividual variability was observed in human blood samples in the different components possibly due to the inclusion of healthy donors and patients with AD in a single group. RBCs had larger variability in Al concentration and values were affected by the solvent used. The Al concentration in RBCs, plasma and serum was 70%, 30% and 25% of the total blood, indicating that RBCs constitute the major compartment for Al. Simultaneous determination of Al in all blood components under optimal conditions may help to clarify this biological toxin's role in degenerative diseases.
Detoxification of Biogenic Aldehydes in Parkinson¡¦s Disease
Authors: Wey Margaret Chia‐Ying , Fernandez Elizabeth , Hinojosa‐Laborde Carmen , Strong Randy
Published: June 2021

It has been recognized for almost 20 years that idiopathic Parkinson's disease (PD) is linked to diminished NADH dehydrogenase (mitochondrial complex I) activity. How chronic complex I inhibition leads to the characteristic pathological features of PD is largely unknown. PD is accompanied by elevation of neurotoxic biogenic aldehydes, including 4‐hydroxynonenal, a product of lipid peroxidation, and 3, 4‐dyhydroxyphenylacetaldehyde, a product of dopamine metabolism. We hypothesize that impaired complex 1 activity reduces the availability of the NAD+ co‐factor required by ALDHs to detoxify aldehydes. Aldh1a1 and Aldh2, are the only ALDH genes known to be expressed in midbrain brain dopamine neurons. Moreover, expression of ALDH1, the human homologue of Aldh1a1, is reduced in PD. Therefore, we tested the hypothesis that mice lacking both Aldh1a1 and Aldh2 would exhibit impaired motor function consequent to dopaminergic neurodegeneration. Compared to wild‐type controls, knockout mice exhibited significant age‐related decreases in the latency to fall during accelerated rotarod testing (no significant decrease at 7 months, 32% at 10 months, and 69% at 22 months). No age‐related deficits were observed in wild type controls. We will discuss neurochemical analyses of the nigrostriatal pathway in the double‐knockout mice.

Supported by the VA Office of Research & Development (EF and RS) and PHS: AG022307 (RS).
P. aeruginosa ExoY Disrupts Microtubules and Induces Endothelial Cell Gap Formation
Authors: Prasain Nutan , Stevens Troy
Published: June 2021

Microtubule architecture controls endothelial cell barrier function, as acute microtubule disruption leads to inter‐endothelial gap formation. The P. aeruginosa toxin, ExoY, is injected through a type III secretion system (TTSS) into the cytosol of endothelial cells, where it associates with microtubules, and produces a cytosolic cAMP pool that disrupts the endothelial barrier. However, it is unclear whether cytosolic cAMP reorganizes microtubules as a necessary prerequisite. To test this idea, confluent pulmonary microvascular endothelial cell monolayers were infected with two strains of P. aeruginosa that possess a: (1) functional TTSS, and only injects ExoY (ExoY+), and (2) functional TTSS, but injects a catalytically inactive ExoY (ExoYK81M) due to a single point mutation at the ATP binding site. After 4 hours of incubation, cells were stained for microtubule organization. Although microtubules were disrupted and inter‐endothelial gaps were formed in ExoY+‐treated cells, neither microtubules nor inter‐endothelial gaps were formed in ExoYK81M or vehicle control‐treated cells. However, stabilizing microtubules prior to infection prevented ExoY+‐induced microtubule disruption and inter‐endothelial gap formation. Collectively, these data suggest that ExoY generates a cytosolic cAMP pool that reorganizes microtubules and causes endothelial barrier disruption. [HL‐60024 & HL‐66299]
Does The Reactivity Of The Sympathetic Nervous System Contribute To The Morning Surge In Blood Pressure?
Authors: Head Geoffrey A , Chatzivlastou Kanella , Schlaich Markus , Lambert Gavin , Lambert Elisabeth
Published: June 2021

We determined whether the reactivity of the sympathetic nervous system (SNS) is related to the morning surge in blood pressure. Ambulatory blood pressure recordings were obtained from 33 subjects (14 males / 19 females ) and the amplitude, the rate of rise (RoR) and the rate x amplitude product (BPPower) of morning mean arterial pressure (MAP) were determined mathematically. Subjects were average age 41 + 4 years (range 18 ‐ 83), BMI 26+1 and 24% of whom were taking antihypertensive therapy. The reactivity of the SNS to an aversive stimulus was assessed on a separate day by microneurographic recording of multiunit, postganglionic muscle sympathetic nerve activity (MSNA), measured from the peroneal nerve. MAP and MSNA were measured concurrently at rest and during a cold pressor test (hand in ice water for 2 min). The cold pressor test increased MSNA and MAP by 24.0 + 2.4 mmHg. MSNA was adjusted for age and BMI. BPPower and RoR were positively correlated to the cold pressor induced increase in total MSNA (r=0.4, P=0.02) and MSN burst amplitude (r=0.5, P=0.01) but were not related to the increase in MSNA frequency. These results suggest that the CNS mechanisms influencing the increase in MSN burst amplitude during arousal may also be fundamental in determining the rate of blood pressure rise during the morning period.

Supported by NHMRC project grant 317826
Rapid estrogen signaling via GPR30 in coronary artery smooth muscle
Authors: Han Guichun , Barman Scott A. , White Richard E.
Published: June 2021

There is no clear understanding of how coronary artery function is regulated by estrogen (E2), which can either relax or contract arteries via rapid, non‐genomic mechanisms involving classic estrogen receptors (ER). Our objective was to investigate non‐genomic E2 signaling mechanisms in coronary artery smooth muscle. In addition to ERα and ERβ, E2 may also stimulate a G protein‐coupled estrogen receptor, GPR30, in non‐vascular tissue; however, a potential role for GPR30 in coronary arteries is unknown. We found that G1, a selective GPR30 agonist, produces a concentration‐dependent relaxation of endothelium‐denuded porcine coronary arteries in vitro (EC50, 26.5 nM). Further, this response was inhibited by 1mM tetraethylammonium, an inhibitor of large‐conductance calcium‐activated potassium (BKCa) channels. Interestingly, ICI182,780, a non‐selective ER antagonist which can activate GPR30, also induced a concentration‐dependent relaxation of endothelium‐denuded coronary arteries (EC50, 137 nM) contracted with PGF2α, but was far less effective in relaxing arteries contracted with 30mM KCl. Immunoblot studies detected expression of GPR30 protein in smooth muscle from either porcine or human coronary arteries. These studies suggest a novel estrogen signaling mechanism in coronary arteries: stimulation of GPR30, leading to potassium efflux and membrane hyperpolarization. (Supported by HL73890)
INCREASED CARBONYLATION OF VENTRICULAR MYOSIN HEAVY CHAINS DURING DIABETES
Authors: Bidasee Keshore R , Rozanski George J , Nagai Ryoji , Horiuchi Seikoh , Stockdale Frank , Patel Kaushik , Shao ChunHong
Published: June 2021

The present study was undertaken to assess whether ventricular myosin heavy chains (MHC) become carbonylated during diabetes and whether formation of these adducts impairs MHC function and the force of cardiac contractions. Type 1 diabetes was induced in male Sprague‐Dawley rats using streptozotocin (STZ). Two weeks after STZ injection, diabetic rats were randomly divided into two groups. One group of diabetic rats was treated with aminoguanidine via drinking water while the other received no treatment. After eight weeks of diabetes basal cardiac ejection fraction, fractional shortening, rate of left ventricular pressure increase and myocyte evoked shortening decreased by 9%, 16%, 34% and 18%. MHC‐β was the dominant isozyme, accounting for 86% of total MHC proteins. Ca2+‐ and Mg2+‐actomyosin ATPase activities and peak actomyosin syneresis (strong myosin‐actin interactions) were reduced by 35%, 28%, and 72%. Trypsin digestion and mass spectrometric analysis revealed elevated levels of carbonyl adducts on MHC‐β and MHC‐α. Crosslinking pentosidine adducts were also found on MHC using adduct‐specific antibodies. Six weeks of aminoguanidine treatment blunted reductions in cardiac function and MHC activity. Aminoguanidine treatment also significantly reduced formation of carbonyl adducts on MHC without altering MHC ratio. These new data suggest that in addition to isozyme switching, carbonylation of MHC contributes to the reduction in the force of cardiac contractions during diabetes (Supported by ADA and NIH).
Adipose Expression and Regulation of Selenoprotein P in Obesity and Insulin Resistance
Authors: Zhang Yuanyuan , Chen Xiaoli
Published: June 2021

Oxidative stress and inflammation have been implicated in obesity and insulin resistance. As a selenium transporter, ubiquitously expressed selenoprotein P (SePP1) has been known to play a role in the regulation of antioxidant enzyme activity. However, its adipose expression and regulation in obesity and insulin resistance has not been explored. Herein, we investigate the adipose expression of SePP1 in obesity and insulin resistance and its regulation in response to oxidative and inflammatory molecules in 3T3L1 adipocytes. We showed that the expression of Sepp1 gene was significantly reduced in both adipose tissue and liver of ob/ob mice and high fat diet‐induced obese mice. Moreover, the expression of Sepp1 gene was decreased in isolated adipose cells from Zucker obese rats and insulin‐resistant patients. Our studies in 3T3‐L1 adipocytes demonstrated that both undifferentiated 3T3‐L1 fibroblasts and differentiated adipocytes were capable of synthesizing Sepp1 protein in the glycosylated and non‐glycosylated forms. The treatment of either TNFα or hydrogen peroxide for 24 hours caused a significant reduction in SePP1 gene expression in a dose‐dependent manner in 3T3L1 adipocytes. Thus, we demonstrate that the adipose expression of SePP1 is decreased in obesity and down‐regulated by oxidative and inflammatory molecules, suggesting a possible role of Sepp1 in oxidative stress/inflammation‐associated insulin resistance in adipocytes.
Diabetes‐induced hyperglycemia is associated with inhibition of insulin pathway without NF‐κB‐p65 activation in skeletal muscle.
Authors: Kelleher Andrew Ryan , Fairchild Timothy , Keslacy Stefan
Published: June 2021

Obesity‐induced inflammation is associated with the onset of diabetes. Whether obesity‐related increase in adipose tissue or hyperglycemia is the initiator of this inflammation is still unknown. Nuclear‐Factor kB (NF‐κB) is a critical transcription factor in the inflammatory process but its role in diabetes onset is unclear. We hypothesized that hyperglycemia is an early modulator of key proteins in the NF‐κB cascade. 10 Streptozotocin‐(STZ)‐treated rats were compared to10 control rats. Using total, cytosolic and nuclear protein extracts from hindlimb muscles: soleus (SOL), extensor digitorum longus (EDL), gastrocnemius (GM), and liver tissue, we assessed key proteins of NF‐κB and insulin pathway. STZ treatment decreased insulin to 3.9 ± 1.2 μU/ml and induced a concomitant hyperglycemia (296.5 ± 15.6mg/dL). SOL, EDL and GM mass decreased and liver mass increased in STZ‐treated animals. p65 expression in GM, and liver increased in diabetic rats, however, there was no IκB degradation or IKK phosphorylation. Muscle p65 remained bound to IκB and did not translocate or bind to DNA. Although insulin receptor (IR) and substrate‐1 (IRS‐1) expression were similar, STZ treatment abrogated their phosphorylation. Despite upstream inactivation of insulin pathway, STZ treatment did not acutely activate the NF‐κB cascade. Therefore, it is unlikely that hyperglycemia initiates obesity‐induced inflammation.
Chromium dinicocysteinate supplementation decreases blood glucose, glycosylated hemoglobin, CRP, MCP‐1 and lipid peroxidation, and increases adiponectin and vitamin C levels in Zucker Type 2 diabetic fatty rats
Authors: Bagchi Manashi , Jain Sushil K , Bagchi Debasis , Croad Jennifer L , Rains Justin L
Published: June 2021

Trivalent chromium (Cr3+) supplementation is widely used by the general public to improve normal protein, fat and carbohydrate metabolism. The present study examined the effect of oral supplementation with chromium nicotinate (CDN), chromium dinicocysteinate (CDNC), or L‐cysteine (LC) on fasting levels of blood glucose, glycosylated hemoglobin (HbA1), C‐reactive protein (CRP), monocyte chemoattractant protein‐1 (MCP‐1), adiponectin, vitamin C, and lipid peroxidation (LP) in Zucker diabetic fatty (ZDF) rats. Control buffer, CDN or CDNC (400 μg Cr/kg BW), and LC (0.93 mg/kg BW) was administered by gavage daily for 8 wks. Blood was collected by heart puncture using light anesthesia. With increasing age, ZDF rats had elevated blood levels of glucose, HbA1, CRP, MCP‐1 and decreased levels of adiponectin and vitamin C. Compared to control, CDNC supplementation showed a significant decrease in glucose, HbA1, CRP, MCP‐1 and LP. Compared to controls, only CDNC supplementation caused a significant increase in levels of adiponectin and vitamin C. There was no change in blood markers of liver and kidney function tests in CDNC supplemented group. This study demonstrates that chromium dinicocysteinate supplementation can lower the risk of glycemia and vascular inflammation in ZDF rats. Research was supported by InterHealth, Benicia, CA.
Expression of Epithelial sodium channel ENaC is regulated by posttranscriptional mechanisms
Authors: Perlewitz Andrea , Nafz Benno , Skalweit Angela , Fähling Michael , Persson Pontus Börje , Thiele Bernd Joachim
Published: June 2021

Epithelial sodium channel ENaC is essential in the regulation of sodium homeostasis for instance in the distal nephron and is involved in physiological processes like blood pressure control. Functional ENaC channels consist of three autonomously expressed subunits a, b and g. Their expression has been studied so far especially on the transcriptional and posttranslational level.

The aim of this study was to elucidate posttranscriptional mechanisms, in particular the role of untranslated regions (UTR) and their interaction with RNA‐binding proteins (RNA‐BP) for stability and translational efficiency of ENaC mRNAs. As a model we used kidney cell lines mpkCCD and HEK293 cultivated under the action of known regulators of ENaC expression, e.g. aldosterone and vasopressin. Regulative potential of UTRs was investigated by luciferase‐reportergene‐assays, RNA/protein interaction by UV‐crosslinking‐assays and identification and functional characterization of candidate RNA‐BPs by RNA‐affinity‐chromatography, MALDI‐TOF‐MS, polysome gradient analysis and co‐transfection experiments.

Our results show that posttranscriptional mechanisms are involved in the regulation of all three subunits under hormonal stimulation, but in particular in gENaC.
Structural and functional association between mitochondria and voltage‐gated K+ currents in the rat pulmonary (PAMs), but not mesenteric (MAMs) arterial myocytes
Authors: Smirnov Sergey V , Gordienko Dmitri V , Firth Amy L , Yuill Kathryn H
Published: June 2021

Mitochondria are one of the major oxygen sensors triggering hypoxic pulmonary vasoconstriction (HPV), a unique response of the pulmonary circulation to low oxygen. Various mitochondrial factors including reactive oxygen species, cytochrome c, ATP and magnesium, are potent modulator of voltage‐gated K+ currents (IKv) in PAMs. Here, cellular localization of mitochondria (labelled with MitoTracker Green) studied using confocal imaging was compared with the effect of two specific mitochondrial inhibitors, antimycin (a Complex III inhibitor) and oligomycin (an ATP synthase inhibitor) (1 µM) on IKv measured with the patch‐clamp technique. We found that mitochondria were located significantly closer to the cell membrane (labelled with Di‐8‐ANEPPS) in PAMs compared to MAMs. Conversely, sarcoplasmic reticulum (labelled with Brefeldin A BODIPY) was closer to the cell membrane in MAMs than in PAMs. Electrophysiological results showed that relative changes in activation, inactivation and IKv block induced by the inhibitors were significantly greater in PAMs than in MAMs. Pretreatment of PAMs with the cytoskeletal disrupter cytochalasin B (10 µM) altered mitochondrial distribution and significantly inhibited the effects of antimycin on IKv. Thus, structural and functional coupling between mitochondria and IKv involving cytoskeleton specifically exists in PAMs, which could play an important role in HPV. 2
The effects of obesity exercise program on Atherogenic Index and HOMA Index in obese middle aged women
Authors: Lee Hayan , Kim Donghee , Jang Seonwoong , Baek Kyoungyeop , Yang Meehyun , Kim Hyeok , Kwon Soyoung , Kim Daeyeol , Jung Hyemin , Cho Yoonshin , Choi Cheolseung
Published: June 2021

The number of obese people has been steadily rising over one and a half times since recent ten years in Korea. Obesity accompanied by increased body fat could lead to hyperlipidemia, hyperinsulunemia, and hypertension, contributing to metabolic syndrome. The study of metabolic syndrome‐related blood lipids predisposing to atherosclerosis have taken attention more. To know the effects of obesity program on Atherogenic Index(AI) and HOMA Index in the obese middle aged women. Obese middle aged women(age: 46.4±6, BMI: 33.1±2.9, %Fat: 33.5±3.5) had done power walking and jogging on the treadmill for aerobic exercise(50‐60 %VO2max for first 1‐6 weeks, 60‐70 %VO2max for later 7‐12 weeks), weight training for resistant exercise(50‐60 %1RM for first 1‐6 weeks, 60‐80 %1RM for later 7‐12 weeks) for a hour. AI and HOMA Index extrapolated from blood samples were analysed at the beginning of exercise, 6 weeks after beginning exercise, and 12 weeks after beginning exercise. The variables were analyzed using one‐way ANOVA. AI decreased at 12 weeks after beginning exercise(p<.05) and HOMA Index decreased at 12 weeks after beginning exercise(p<.05). Insulin decreased at 12 weeks after beginning exercise but were not statistically significant. Therefore, the maintenance of normal weight and doing exercise is beneficial to preventing the metabolic syndrome and arteriosclerosis.
TRPV1‐MEDIATED MODULATION OF SYNAPTIC INPUTS IN IDENTIFIED KIDNEY‐RELATED SYMPATHETIC PREMOTOR NEURONS IN THE ROSTRAL VENTROLATERAL MEDULLA
Authors: Derbenev Andrei , Stocker Sean D. , Smith Bret N.
Published: June 2021

The central nervous system regulates arterial blood pressure (ABP), largely by controlling actions of the sympathetic nervous activity which includes control of kidney. Synaptic balance in the neurons of the rostral ventrolateral medulla (RVLM) is a critical component in the central pathways mediating sympathetic cardiovascular regulation. Transient receptor potential vanilloid type 1 (TRPV1) are expressed in the RVLM, but cellular effects of TRPV1 activation in the RVLM are not known. Whole‐cell patch‐clamp recordings in brainstem slices were used to characterize synaptic activity of kidney‐related RVLM neurons identified using the transynaptic retrograde viral vector PRV‐152 and to test the hypothesis that activation of TRPV1 enhances synaptic inputs to these neurons. Immunohistochemical studies showed that TRPV1 receptors are distributed through RVLM and colocalized with kidney‐related RVLM neurons. Kidney‐related RVLM neurons fired action‐potentials at rest and received abundant synaptic input that could be modulated by TRPV1 agonists. Application of the TRPV1 receptor agonist, capsaicin (1 μM), increased the frequency of spontaneous IPSCs from 1.6±0.6 Hz to 4.6±1 Hz and EPCSs from 4±0.4 to 8.7±1 Hz, respectively. Capsaicin also increased action‐potential frequency in kidney‐related sympathetic premotor RVLM neurons from 3.1±0.5 Hz to 6±0.6 Hz (p<0.05). Modulation of RVLM neurons by TRPV1 agonists represents a novel means of modulating ABP and, by inference, hypertension.

Supported by AHA 0865395D and NIH HL091293‐01A1
A short‐term free‐fall landing enhances local bone formation without showing of woven bone
Authors: Lin Hsin‐Shih , Huang Tsang Hai , Yang Ai Lun , Mao Shih Wei , Tai Yuh Shiou , Yang Rong Sen
Published: June 2021

Objective

The purpose of this study was to investigate the effects of a mechanical loading course (free‐fall landing) on local bone metabolism.

Methods

Thirty‐two female Wistar rats (7‐week‐old) were randomly assigned into three groups, which were the L30 (n=11), L10 (n=11) and CON (n=10) groups. Animals of the L30 and L10 group underwent 5‐day free fall landing from a height of 40cm for 30 and 10 times per day, respectively. Landing ground reaction force (GRF) was recorded on day 1 and day 5. Methods of dynamic histology, tissue geometric and biomaterial measurements were performed on animals' ulnae. One‐way ANOVA was used for statistical analysis. When statistical significance was shown (p<0.05), pair‐wise comparisons were made using the Fisher's LSD method.

Results

In dynamic histological analysis, no ulna revealed formation of woven bone in all animals. Periosteal as well as endosteal mineral apposition rate (MAR, μm/day) was significantly higher in L30 group as compared to the CON group. And, periosteal bone formation rate per unit of bone surface (BFR/BS, μm2/μm3/year) was significantly higher in the L10 and L30 groups.

Conclusions

In conclusion, a 5‐day free‐fall landing training produce a slight but significant higher bone formation on ulnae of female young rats. As compared with other mechanical loading models, this free‐fall landing revealed no signs of damages (no woven bone formation) to landing site and would be a more realistic model to mimic mechanical stimulus induced by various physical activities.

Grant supported from the National Science Council (NSC‐94‐2314‐B‐006‐033, Taiwan).
Role of iron‐sulfur cluster in E. coli DinG helicase
Authors: Ren Binbin , Ding Huangen
Published: June 2021

The E. coli DinG protein encoded by the DNA damage‐inducible gene (dinG) is a member of the helicase superfamily 2. It has the DNA‐dependent ATPase activity and ATP‐dependent helicase activity. In this study, we report that the purified DinG protein contains a [4Fe‐4S] cluster with a midpoint redox potential ‐390±23 mV (pH 8.0). More importantly, the DinG helicase activity is inactivated when its [4Fe‐4S] is reduced and reactivated when the cluster is re‐oxidized, suggesting that the redox state of the [4Fe‐4S] cluster in DinG can modulate helicase activity. Unlike the [4F‐4S] cluster of E. coli dihydroxyacid dehydratase, the [4Fe‐4S] cluster in DinG is resistant to the oxidative stress caused by hydrogen peroxide and remains active. On the other hand, the DinG helicase activity is completely abolished by nitric oxide (NO) through modification of its 4Fe‐4S cluster and almost fully restored when the NO‐modified [4Fe‐4S] cluster is reassembled. The results suggest that the [4Fe‐4S] cluster in DinG may act as a sensor of the cellular redox potential and that the NO modification of the iron‐sulfur cluster in DinG and other DNA repair enzymes may contribute to the NO‐mediated genomic instability.
A novel p21Cip1 phosphorylation/degradation pathway playing a role in the ERK‐mediated cell proliferation signal
Authors: Hwang Chae Young , Lee Cheolju , Kwon Ki‐Sun
Published: June 2021

p21Cip1 is an inhibitor of cell cycle progression that promotes G1 phase arrest by direct binding to cyclin‐dependent kinase and proliferating cell nuclear antigen. Here we demonstrate that mitogenic stimuli, such as epidermal growth factor treatment and oncogenic Ras transformation, induce p21Cip1 downregulation at the post‐translational level. We show that ERK2 interacts with and phosphorylates p21Cip1, promoting p21Cip1 nucleocytoplasmic translocation and ubiquitin‐dependent degradation. Phosphopeptide analysis of in vitro ERK2‐phosphorylated p21Cip1 revealed two phosphorylation sites, Thr57 and Ser130. Both the phosphorylation‐deficient T57A/S130A p21Cip1 mutant and the NES mutant, that are resistant to ERK2‐mediated downregulation, retain the ability to potently inhibit the G1/S transition. These results indicate that ERK2 activation transduces mitogenic signals, at least in part, by phosphorylation, translocation, and degradation of the cell cycle inhibitory protein, p21Cip1, suggesting an alternative mechanism of Ras‐ERK signaling‐mediated cell proliferation. This work was supported by grants M10503010002, M10601000164, and M10642040003.
The HAND and SANT Domains Regulate the In vitro and In vivo Activity of the ISW2 Chromatin Remodeling Complex
Authors: Hota Swetansu Kumar , Hemeryck Christine S , Pugh B Franklin , Bartholomew Blaine
Published: June 2021

The catalytic subunit of ISW2 chromatin remodeling complex consists of the conserved DEXD/HELIC domain and HAND, SANT and SLIDE domain. The role of HAND and SANT domain were analyzed by specific deletion of the entire domain or mutation of key amino acid residues. HAND and SANT domain are found to be dispensable for ISW2 complex integrity and binding to nucleosomal substrate however are critical for efficient nucleosome remodeling. SANT domain was found to be required for efficient ATP hydrolysis and remodeling of equally spaced dinucleosomes. Deletion of HAND domain affected a subset Isw2 regulated genes; however, SANT domain affected a larger set of gene characteristic of a dominant negative phenotype. Further the role of these domains were investigated by mutating conserved ISWI specific, surface accessible basic amino acids, forming a basic path and predicted to interact with extranucleosomal DNA, to alanine. Multiple mutations in HAND or SANT domain did not affect complex assembly or binding but did affect the rate of nucleosome remodeling. In vivo de‐repression of transcription was observed at several Isw2 specific loci with either deletion or mutations in HAND or SANT domains. Consistently nucleosome position changes were observed upon domain deletion at STE6 promoter region indicating that conserved ISWI specific basic amino acid residues in these domains regulate ISW2 activity and gene expression through changes in nucleosome position.

This work was supported by Public Health Service Grant GM70864
Histone acetylation modulates the binding and remodeling activity of two different SWI/SNF complexes
Authors: Chatterjee Nilanjana , Shogren‐Knaak Michael , Bartholomew Blaine
Published: June 2021

The coordination of chromatin remodeling with chromatin modification is key to the regulation of chromatin structure and function. Recent studies implicated a functional link between histone acetylation and the SWI/SNF class of chromatin remodeling enzymes. This functional interplay is mediated via highly conserved acetyl‐lysine binding modules, the bromodomains. The presence of bromodomain (s) in SWI/SNF and RSC therefore strongly suggests that recognition of histone acetylation might play a major role in chromatin targeting and / or in their chromatin remodeling function. Here the nucleosome binding and remodeling activities of the two yeast SWI/SNF complexes were dissected out separately and the impact of histone tail acetylation on each of these processes was studied systematically. We show that histone H3 tail acetylation and not H4 augments nucleosome binding by actually enhancing the binding affinity of RSC and SWI/SNF complexes for nucleosomes. Further, H3 tail acetylation mediated enhanced binding of SWI/SNF mimics SWI/SNF recruitment by transcription activator Gal4‐VP16. Finally, kinetic analyses under saturating enzyme conditions revealed that rates of remodeling and ATP hydrolysis are both stimulated independent of nucleosome binding upon acetylation of H3 tails.

Thus acetylation additionally modulates the catalytic activity of SWI/SNF complexes besides providing a unique recognition surface.

This work was supported by Public Health Service grant GM 48413
Nuclear protein extraction from frozen porcine myocardium
Authors: Kuster Diederik , Merkus Daphne , Dekkers Dick , Duncker Dirk Jan , Verhoeven Adrie
Published: June 2021

Extraction of nuclear proteins from cells or tissues is an important tool for studying gene regulation and the responsible transcription factors. Here we present a homogenization and fractionation protocol for the preparation of nuclear extracts from frozen porcine left ventricular (LV) tissue. This nuclear extraction procedure gave a highly reproducible protein yield (7.3 ± 0.3% of total protein; mean ± SE, n= 11), a six‐fold enrichment of the nuclear marker B23, and more than 40% recovery of B23. The nuclear extracts were essentially devoid of contamination with cytosolic, myofilament and histone proteins as shown by Western blotting. Using this nuclear extraction method we show that upon treatment of animals with the β‐adrenergic agonist dobutamine, the amount of tyrosine phosphorylated STAT3 increased 6.9 ± 2.1 fold (n = 4, P = 0.03) in nuclear extracts from LV tissue, but only a minor fraction of the total PY‐STAT3 appeared in the nucleus. This nuclear extraction method appears to be well‐suited to nuclear proteome analysis of frozen heart tissue collected in biobanks.

This work was supported by a grant from the Netherlands Heart Foundation (NHS2005B234).
GL‐9 regulates the C. elegans hypoxia inducible factor (HIF‐1) by two mechanisms, with differing requirements for EGL‐9 prolyl hydroxylase activity
Authors: Shao Zhiyong , Zhang Yi , Powell‐Coffman Jo Anne
Published: June 2021

Animals have to sense and respond to environmental changes in order to survive. For example, when oxygen levels drop below the levels required to sustain normal physiological demands, the hypoxia inducible factor transcription factors (HIFs) implement most of the changes in gene expression that allow animals to survive. HIF heterodimeric complexes include a constitutive beta subunit and an oxygen sensitive alpha subunit. The EGL‐9 / PHD2 dioxygenases catalyze the prolyl hydroxylation of HIFalpha subunits and promote its degradation. This pathway is conserved from Caenorhabditis elegans to humans. We have investigated EGL‐9 function in C. elegans. We found that in addition to regulating HIF‐1 stability, EGL‐9 also suppresses HIF‐1 activity (Shen et al. 2006 Genetics 174:1205). To more fully understand the dual functions of EGL‐9, we have generated and characterized mutations in the endogenous egl‐9 gene and in transgenes. We found that deletion of the conserved MYND domain in EGL‐9 has little effect on either its hydroxylase activity or its ability to suppress HIF‐1 transcriptional activity. Further, we find that inhibition of EGL‐9 catalytic function do not abolish EGL‐9‐mediated repression of HIF‐1 transcriptional activity. We conclude that EGL‐9 regulates HIF‐1‐mediated gene expression by two distinct mechanisms that are separable. This work is funded by NIH GM078424.
The Protein Interaction of Saccharomyces cerevisiae Glutaredoxin5 with SPT10 and its in vivo Function
Authors: Oh Young‐Mee , Kim Il‐Han
Published: June 2021

Glutaredoxins are members of a superfamily of thiol disulfide oxidoreductases involved in maintaining the redox state of target proteins. Using a yeast two‐hybrid system, we isolated a Grx5‐binding protein. The protein was identified as SPT10 protein. SPT10 protein has been suggested to involve in chromatin maintenance and transcriptional regulation. We investigated the protein interaction between Grx5 and SPT10 and found that the Cys165 of SPT10 and the conserved cysteine (Cys60) of Grx5 are essential for their interaction, suggesting formation of dimer between them via disulfide bond. To address the physiological function of the protein interactions, we constructed single or double yeast mutants lacking Grx5, SPT10, and both proteins, and examined their physiology. The growth retardation of delta Grx5 became profound by deleting SPT10. Moreover, all of three typed mutants (deltaGRx5, deltaSPT10, and deltaGrx5deltaSPT10) did not grow in non‐fermentable media. Taken together, these observations suggest that the dimer between Grx5 and SPT10 is required for the respiratory growth.*This work was financially supported by KRF (C00434).
SM 22α regulates proliferation of vascular smooth muscle cells and inhibits neointimal formation after balloon injury in rats
Authors: Dong Liu‐hua , Wen Jin‐kun , Miao Sui‐bing , Zheng Bin , Han Mei
Published: June 2021

Smooth muscle 22 alpha (SM22α), a specific marker of contractile VSMCs, plays a role in inhibiting the phenotypic modulation of VSMCs. In this report, we found that overexpression of SM22α inhibited PDGF‐induced proliferation with concurrent blockade of the cell cycle progression in VSMCs. Despite of PDGF treatment, VSMCs infected with Ad‐SM22α were arrested in the G0/G1 phase, and showed increase in expression of the CDK inhibitors p21 and p27. Conversely, knockdown SM22α expression by specific small interfering RNA (siRNA) resulted in acceleration of cell cycle progression even if under serum deprivation. The evidences from an experiment in vivo showed that overexpression of SM22αinhibited neointimal formation induced by balloon injury. These findings suggest that SM22α abolishes pro‐proliferative signaling cascades and maintains the quiescent state of VSMCs. Furthermore, overexpression of SM22α resulted in a robust reduction in the activity of MEK/ERK1/2, but no change in the activity of JNK and p38 MAPK. Similarly, the activity of Raf was also reduced in SM22α‐overexpressed cells. The previous studies have demonstrated that Ras links PDGF receptor to Raf‐MEK/ERK1/2 pathway, mediating signal cascades in response to PDGF. We found that there was increase in binding of SM22α to Ras in SM22α‐overexpressed VSMCs, leading to negative regulation of Ras‐Raf‐MEK/ERK1/2 signaling pathway.
Who's Afraid of the Big, Bad, Misfolded Protein?
Authors: Murphy M , Pollnow C , Misplon M , Pollen J , Stack M , Stack D , Manogaran A L
Published: June 2021

Fatal prion diseases, such as Creutzfeldt‐Jakob disease in humans, are associated with the conversion of the normally folded mammalian PrP protein to a misfolded prion form that aggregates. Understanding how prions behave has been greatly facilitated by the study of prions in Saccharomyces cerevisiae, or baker's yeast. In yeast, the translation release factor, Sup35, misfolds to form the [PSI+] prion. Although the Sup35 protein has a significantly different primary sequence from PrP, its prion form behaves similarly to human prions. The N‐terminus of Sup35 is Q/N‐rich and responsible for prion formation. Determining the structure of this region has proven difficult since the N‐terminus forms aggregates instead of the ordered crystals required for structural studies. However, a small seven amino acid sequence (GNNQQNY) from Sup35's N‐terminus was crystallized by Nelson et al. (2005). We have constructed a 3D physical model of GNNQQNY as part of the MSOE SMART Teams program using 3D printing technology. GNNQQNY's structure suggests that multiple prion molecules assemble into strong fibrous aggregates through tightly structured interlocking parallel beta sheets called a cross‐β spine. The structure of GNNQQNY suggests a potential model of how human prions assemble, which could potentially help in developing therapies that prevent aggregation. Supported by a grant from NIH‐NCRR‐SEPA.
Proliferation of pulmonary smooth muscle cells is stimulated by lung microvascular endothelial cells exposure to hypoxia via JAKs/STATs pathway
Authors: Wang Guansong , Wang Changzheng , Qian Guisheng , Yang Yu , Li Shujun , Zhang Zhiyuan
Published: June 2021

Rationale

It is well known that hypoxia can regulate the proliferation and Apoptosis of many kinds of pulmonary cells, the effect of lung microvascular endothelial cells (LMVECs) on pulmonary arterial smooth muscle cells (PASMCs), and the related mechanism of signal transduction is unclear.

Objective

To investigate the effect of hypoxic LMVECs on PASMCs growth and the related signal pathway.

Methods

Rat LMVECs were randomly divided into 3 groups: normal group (N), 8 h hypoxia (H8), and 8 h hypoxia plus SU5416(H8+SU). Rat PASMCs were cultured with LMVECs medium from N, H8 and H8+SU. The gene expression of VEGF and TGFβ1 were detected with real‐time PCR. The protein levels of VEGF and TGFβ1 in their supernatant were detected with ELISA. The phophorylation of JAK2 and STAT3 was detected with Western blotting or EMSA.

Results

1. PASMCs proliferation was stimulated by hypoxic LMVECs medium. 2. VEGF and TGFβ1 mRNA and protein levels was increased in hypoxic medium. 3. The phosphorylation of JAK2 and STAT3 was activated in PASMCs cultured with hypoxic medium. 4. TGFβ1 neutralizing antibodies added to hypoxic medium could decrease the expression of p‐JAK2 and p‐STAT3, and suppress PASMCs proliferation. On the other hand, the growth of PASMCs was repressed during the phosphorylation of STAT3 was inhibited by AG490 and STAT3 inhibitor peptide.

Conclusion

Growth factors, especially TGFβ1, released from LMVECs exposure to hypoxia activate cocultured PASMCs proliferation through JAK2‐STAT3 pathway.

This abstract is funded by NSFC No.30770928, 973 Program No.2006CB504100, PLA grant No.06G083, and TMMU grant 2006HG04.
Proteomic Profiling of the Normal Human Mitral Valve
Authors: Suri Rakesh Mark , Srinivasan Manivannan , Abdul Rahiman Abdul Jaleel , Schaff Hartzell V. , Nair K. S.
Published: June 2021

Background

Structural and functional mitral valve abnormalities are commonly encountered in the general population. In order to determine which functional protein classes may be important to normal valve function we performed the first reported comprehensive proteomic profiling of structurally normal human mitral valves.

Methods

We harvested mitral valve leaflets from 5 patients who underwent orthotopic cardiac transplantation for cardiomyopathy. The morphologic appearance of mitral valves was normal with only mild functional mitral regurgitation on echocardiogram. High resolution 2D gel electrophoresis was performed to separate proteins. Gel spots were excised from 2 representative samples and protein identification was carried out using liquid chromatography tandem mass spectrometry.

Results

We identified 190 unique proteins with >99% probability and at least 2 unique peptides. The proteins were from various cellular locations, including cell surface, cytoskeleton, endoplasmic reticulum, extracellular matrix, mitochondrion, nucleus, cytoplasm, and plasma membrane. Functional clustering revealed that the identified proteins were involved in major molecular pathways including, energy along with fatty acid metabolism, calcium signaling acute phase response & oxidative stress, and anti‐apoptosis pathways.

Conclusion

Proteomic profiling of the normal human mitral valve demonstrates a pattern of expression suggesting multiple biological functions. Further quantification of protein levels and comparison with diseased mitral valves will provide new insights into the etiology and therapy of mitral valve disease in humans.
STRAW‐BASED SPECIMEN CRYOPRESERVATION & BANKING
Authors: Cohen Laurent
Published: June 2021

When archiving serum, DNA, or other types of samples at very low temperatures for clinical trials, epidemiology or other purposes, there is a choice of methodologies and technologies. This poster compares the most common methods from the standpoints of science and economics. The parameters for consideration are the storage temperature (e.g., liquid nitrogen vs. ‐80ºC; LN2 liquid vs. vapor phase), the type of container (e.g., upright vs. chest freezer), and packaging (e.g., vials vs. straws) used for the repository. Different configurations were assessed according to the following criteria: "Quality" preservation as measured by stability of markers and cell survival rates, "Safety" assessed from the standpoint of bio‐containment and cross‐contamination risk, "Efficiency" quantified by the number of aliquots per container unit, "Traceability" of the samples throughout the chain of custody, as well as the level of "workflow automation" and general "ease‐of‐use". A comprehensive cost model was developed to evaluate the overall cost per sample for any storage configuration. The model includes capital equipment, maintenance, "fuel" (electricity or LN2), technician labor, and additional air conditioning required. This poster presents the results and outlines the scientific and economic benefits of a straw‐based, integrated and automated solution.
Internucleosomal DNA fragmentation in apoptosis may be a cooperative activity between DNA fragmentation factor and the Ca2+‐Mg2+‐dependent endonuclease DNAS1L3
Authors: errami youssef , Zerfaoui Mourad , Zhang Jianhua , Xu Ming , Boulares Hamid
Published: June 2021

Apoptosis or programmed cell death is a key process involved in animal development and tissue homeostasis. Internucleosomal DNA fragmentation (INDF) is a hallmark of apoptosis, in which genomic DNA is first degraded into large 50 kb fragments and then into repeats of 200 bp. Mechanism by which INDF occurs is still unclear. Our studies revealed that this process could involve two endonucleases working cooperatively. The DNA fragmentation factor (DFF), is induced on cleavage of DFF45 (the inhibitory and chaperone subunit of DFF40) by caspase‐3. Several cell lines undergo key apoptotic events including cleavage of DFF45 and of chromatin into large 50 kb fragments but failed to display clear INDF. Such failure did not correlate with the expression levels of DFF40 or 45. Apoptotic cells derived from DFF45−/− mice exhibited neither 50 kb nor INDF. Suggesting that caspase‐3 activation, cleavage of DFF45, and the consequent activation of DFF40 are necessary but not sufficient for INDF. We identified DNAS1L3, a Ca2+‐Mg2+‐dependent endonuclease, as an important apoptotic INDF‐mediating enzyme. Here, we show, that DNAS1L3 translocates from the endoplasmic reticulum (ER) to the nucleus upon etoposide exposure. And interestingly, DFF40 and DFF45 were required for DNAS1L3‐mediated INDF. Altogether, our results show that DNAS1L3 translocates to the nucleus upon apoptotic stimulus and cooperates with DFF40 to mediate INDF.
Antifibrillatory actions of HBI‐3000 in the conscious canine model of sudden cardiac death
Authors: Lee Jullia , Lucchesi Benedict R
Published: June 2021

The antifibrillatory actions of HBI‐3000 (sulcardine sulfate) were studied in a conscious canine model of sudden cardiac death (SCD). Female purpose‐bred beagles were anesthetized, and left ventricular anterior wall ischemic injury was induced by a 90 minute occlusion followed by reperfusion of the left anterior descending coronary artery. Three to seven days later, the dogs were randomized into two groups: Group 1 (n = 8) received 0.9% NaCl solution for injection while Group 2 (n = 7) received 15 mg/kg of HBI‐3000 intravenously. The dogs were subjected to anodal current‐induced injury of the circumflex coronary artery, resulting in transient interruptions in coronary artery blood flow and posterolateral myocardial ischemia. The electrocardiogram confirmed the presence of regional myocardial ischemia and ventricular fibrillation (VF). Control animals developed premature ventricular complexes (PVC) followed by ventricular tachycardia, which terminated in VF in four of the eight dogs and one expired overnight. PVC was also observed in HBI‐3000 treated animals, but only one of the seven animals developed VF in SCD (p < 0.05 vs. control). The data indicate that HBI‐3000 is effective in reversing polymorphic ventricular arrhythmias and has favorable antifibrillatory actions in the post‐infarcted canine heart subjected to a remote ischemic event.

Supported by HUYA Bioscience International.
Platelet Activity In Elevated Plasma Seretonin Level
Authors: Ziu Endrit , Kilic Fusun
Published: June 2021

The serotonin transporter (SERT) on the platelet surface is the major mechanism for the uptake of plasma serotonin (5‐hydroxytryptamine; 5HT), which subsequently is stored in platelet dense granules. The 5HT uptake capacity of the platelets depends on the number of SERT molecules on the plasma membrane.

Our earlier studies demonstrated the presence of a dynamic relationship between plasma 5HT elevation, loss of surface SERT, and depletion of platelet 5HT. However it is not clear whether decreased SERT density on the platelet plasma membrane is a cause or consequence of increased plasma 5HT levels.

In the current study, we addressed this issue by using two model systems: (a) 5HT‐pretreated platelets isolated from untreated C57BL/6J mice, and (b) platelets isolated from C57BL/6J mice in which plasma 5HT was elevated chronically by osmotic minipump. Our data in both model systems show a profound loss of surface SERT expression in platelets, depletion of platelet 5HT content and enhanced aggregation response to fibrillar collagen, demonstrating that 5HT‐depleted platelets appear to aggregate more readily. Furthermore, the aggregation rate of platelet is rescued by blocking the uptake ability of platelet SERT with serotonin reuptake inhibitor (SSRI), SERT blocker. These studies explicitly indicated that SERT down‐regulation by high extracellular 5HT in isolated platelets occurs in vivo and impacts platelet function against further aggregation in a protective feedback mechanism.

Overall, these studies addressed that the intracellular 5HT concentration has a significant role in promoting hypercoagulation than the plasma 5HT‐level. The importance of understanding the structure, function, and regulation of SERT is underscored by the observations that plasma 5HT is elevated in the plasma, either locally or globally, during atherosclerosis, hypertension, stroke, and other cardiovascular diseases.
Fast age‐related on‐transient contraction kinetics during alpha1‐ adrenergic phenylephrine stimulation in rat thoracic aortae artery
Authors: Padilla Javier , Castillo Maria del Carmen , Guevara Gustavo , López Jorge , Castillo Carlos
Published: June 2021

Does the threecomponent empirical exponential mathematical model (3C) fit properly the aorta phenylephrine (phen) induced contraction data (APCD), identifying three "expected" phases (3Ps: P1, P2, and P3) of developing Force (P1F, P2F, and P3F) of contraction and kinetically characterize (K) them in terms of time constant (tau in s: P1Ftau, P2Ftau, and P3Ftau) in rat thoracic aorta artery (TAA)? and, are there significant kinetic (tau) differences between young (Y) and old (O) Wistar rats?. Y (230‐280 g bw; 8 weeks old, n= 8 ) and O (200‐250 g bw; 16‐18 weeks old, n= 7) groups TAA tissue rings bath on‐transient isometric (OnTI) contraction data of response, were fitted by means of nonlinear regression techniques with 3C to assess their kinetics (tau, MeanResponseTime in s) from a baselineonset to offset stimulation with 1 · 106 M phen.The 3C properly fitted, OnTI APCD in Y and O data; i) identifying 3FPs: the rapid one response (P1F), the moderately fast response (P2F), and the slow response (P3F); ii) K them as follows: P1Ftau < P2Ftau< P3Ftau in either Y or O arteries, and; the values from the 3FPs (except from P1Ftau), and MRT resulted significantly different between Y and O for P1Ftau (Y_7.17±3.04 similar to O_9.10±3.60, s), P2Ftau (Y_ 98.94±13.34< O_69.96±14.12, s), P3Ftau (Y_291.68± 12.70< 202.51±14.52,s), and MRT (Y_137.37±26.66> O_116.77±16.46, s). These findings showed fast age‐related OnTI contraction kinetics phen induced in TAA.