bluuebunny/crossref_metadata_2025 huggingface dataset:
Configurator Based Integrated Dynamic Simulation of Reverse Cholesterol Transport (RCT)
Authors: Adhikari Sam
Published: June 2021

Major constituents of RCT include acceptors such as high density lipo‐protien (HDL) and apoliproprotein A‐1 (ApoA‐1), and enzymes such as lechtin:cholesterol acyltransferase (LCAT), Phospholipid transfer protein (PLTP), hepatic lipase (HL), and cholesterol ester transfer protein (CETP).

Realistic quantitative modeling of RCT is extremely difficult through conventional methods. It requires a software configurator aided, database driven systems biology platform.

Our RCT quantitative model based dynamic, integrated simulation uses many complex models including kinetics to evaluate cholesterol efflux from the macrophages to ApoA‐1 via ABCA1 and ABCG1. The model is enhanced by the quantitative RCT effects of Caveolin, Sterol 27‐hydroxylase (CYP27A1), scavenger receptor SR‐B1 transport processes, and ABCG5/G8 hepatobiliary and intestinal sterol extraction gene.

The net RCT pathway is quantified with multiple parameters that can change depending on clinical in‐vivo or in‐vitro conditions. A standard relational database model holds all the objects of the quantitative simulation model. An optimization algorithm matches the aggregate model with the clinical RCT datasets. It automatically adjusts all the parameters till it can find the best solution.
Comparison of Human Adipose Derived Stem Cells with Human Bone Marrow Stem Cells in Myocardial Regenerative Therapy
Authors: Srivastava Sapna , Duong Minh , Chiu Ray , Shum‐Tim Dominique
Published: June 2021

Background

Cell therapy using Human Bone Marrow Stem Cells (HBMSCs) has been shown to improve heart function after a myocardial infarction. The harvesting technique involved with bone marrow stem cells is invasive and yields a low cell number. There is now an increasing interest in Human Adipose Derived Stem Cells (HADSCs) as they are abundant and readily accessible from liposuction material. The present study was undertaken to investigate if HADSCs are superior than HBMSCs in myocardial regenerative therapy.

Results

Both HADSCs and HBMSCs proliferated in a time dependent manner, however, the proliferative ability of HADSC was greater than the HBMSCs. In addition, both cells differentiated to the osteoblast lineage confirming their multipotency, when treated with induction medium. Furthermore, treatment of both cells with 5‐AC resulted in positive immunostaining of cardiac markers, troponinI and connexin 43, however the expression of these markers was enhanced in HADSCs. This was further confirmed by western blot analysis, however 5‐AC treatment did not exhibit cell contraction or multinucleation.

Conclusion

The data suggest that HADSCs may prove to be a more ideal type of cell for regenerative therapy in the future.
Tissue‐Specific Roles of MED1/TRAP220 Nuclear Receptor‐Interacting LxxLL Motifs in vivo
Authors: Zhang Xiaoting
Published: June 2021

Mediator recently has emerged as a central player in the direct transduction of signals from transcription factors to the general transcriptional machinery. In the case of nuclear receptors, in vitro studies have shown that the transcriptional coactivator function of the Mediator involves direct ligand‐dependent interactions of the MED1 (a.k.a. TRAP220) subunit, through its two classical LxxLL motifs, with the receptor AF2 domain. However, despite the strong in vitro evidence, there currently is no information regarding in vivo functions of the LxxLL motifs either in MED1 or in other coactivators. Towards this end, we have generated MED1 LxxLL motif‐mutant knockin mice. Interestingly, these mice are both viable and fertile, and do not exhibit any apparent gross abnormalities. However, they do exhibit severe defects in pubertal mammary gland development. This was manifested as a significant impairment in duct elongation and branching morphogenesis and a decrease in mammary epithelial cell proliferation in the mutant mice. Consistent with this phenotype, as well as a loss of the strong ligand‐dependent estrogen receptor (ER)α‐Mediator interaction, expression of a number of known ERα‐regulated genes was down‐regulated in MED1‐mutant mammary epithelial cells and could no longer respond to estrogen stimulation. Related, estrogen‐stimulated mammary duct growth in MED1‐mutant mice was also greatly diminished. Finally, additional studies show that MED1 is differentially expressed in different types of mammary epithelial cells and that its LxxLL motifs play a role in mammary luminal epithelial cell differentiation and progenitor/stem cell determination. Our results establish a key nuclear receptor‐ and cell‐ specific in vivo role for MED1 LxxLL motifs, through Mediator‐ERα interactions, in mammary gland development.
It's not my nerves! Augmented pressor response to stress in multiparous rats is not associated with increased sympathetic outflow.
Authors: Kaufman Susan , Levasseur Jody
Published: June 2021

There is epidemiological evidence that multiparity (multiple pregnancies) increases the long‐term risk for cardiovascular disease. We have shown that multiparous rats have an augmented pressor response to stress. This is due, at least in part, to an oxidative stress induced increase in vascular reactivity. We proposed that the augmented pressor response in the multiparous rats was also the result of a greater increase in sympathetic outflow. Age‐matched multiparous and virgin rats were implanted with telemetric devices to monitor renal sympathetic nerve activity (Telemetry Research) and blood pressure (DSI). One week later, they were exposed to 10 sec air jet. The stress‐induced increase in integrated mean amplitude of nerve activity was significantly higher in the virgin animals (Virgin: 3.2±1.6 uV to 36.5±12.2 uV; Multiparous: 0.8±0.6 uV to 8.9±6.2 uV; P<0.05). There was no difference between the groups with respect to the increase in spike frequency (Virgin: 28.2±7.8 to 36.6±10.0 spikes/sec; Multiparous: 16.6±2.8 to 28.6±1.4 spikes/sec; P>0.05). We conclude that, contrary to our hypothesis, the augmented pressor response of multiparous rats is due solely to increased vascular reactivity, not to a greater increase in sympathetic outflow. (HSFC)
Determining the dose‐dependent effects of long‐chain soluble polymers in rat spinotrapezius muscle
Authors: Connery Michael , Kameneva Marina V , Pittman Roland N
Published: June 2021

Long‐chain soluble polymers (LSPs) have been shown to have profound hemodynamic effects at very low concentrations. At only 5 ppm in blood, LSPs increase blood flow and reduce vascular resistance. Despite studies showing therapeutic value in cases of hemorrhage, myocardial infarction, and atherosclerosis, the mechanism of action is unknown. The purpose of this study was to determine the dose‐dependency of LSPs on systemic and microcirculatory variables. Sprague‐Dawley rats were anesthetized and their spinotrapezius muscles isolated for transillumination. The experimental group (n=2) was given serial injections of the LSP polyethylene oxide (mol wt 4500 kDa) dissolved in saline to reach blood concentrations of 0.12, 0.4, 1.2 and 4 ppm. The control group (n=8) was given serial injections of the same volumes of saline. At 4 ppm, mean arterial pressure was lower in the LSP group (90 ± 2 mmHg vs 108 ± 17 mmHg). Interstitial PO2, as measured by phosphorescence quenching, was higher in the LSP group at 0.12 ppm (67 ± 3 mmHg vs 60 ± 4 mmHg) and 0.4 ppm (66 ± 2 mmHg vs 60 ± 4 mmHg). These findings are consistent with previous reports of this polymer.

Support: NHLBI HL18292 and HL079087
A new function for methyl CpG: Creating a C/EBPα binding site & mediating tissue‐specific gene expression
Authors: Chatterjee Raghunath , Rishi Vikas , Rozenberg Julian , Bhattacharya Paramita , Glass Kimberly , Zhao Jianfei , Vinson Charles
Published: June 2021

DNA methylation of the cytosine in the CpG dinucleotide is typically associated with gene inactivation. However, we observe that differentiation of primary mouse new‐born dermal fibroblasts into adipocytes activates genes with methylated promoters that are bound by the transcription factor CCAAT/enhancer‐binding‐protein‐α(C/EBPα). Experimental demethylation by either 5‐azacytidine treatment or by using siRNA to DNA methyltransferase1 (DNMT1) inhibits adipocyte differentiation. Differentiation inhibition caused by demethylation is mainly mediated by inhibition of both C/EBPα binding and activation of methylated promoters. Also, expressing of a dominant‐negative protein (A‐C/EBP) in the dermal fibroblasts that inhibits the DNA binding of C/EBP family members completely abolishes this differentiation. Specific C/EBP‐like and CRE‐like sequences are identified in tissue specific proximal promoters that are methylated in vivo, methylation enhances C/EBPα binding as observed by EMSA using pure protein, and need to be methylated for C/EBPα mediated activation as assayed by luciferase reporter. Similar results are observed when primary keratinocytes from new‐born mice are differentiated. These data identify a new function for methyl CpG, producing a DNA binding site for C/EBPα critical for activation of tissue specific gene expression.
Role of caveolin‐1 in angiotensin II‐induced hypertrophy and remodeling of cerebral arterioles
Authors: Houwen Frederick Keith , Chan Siu Lung , Baumbach Gary L
Published: June 2021

We examined effects of caveolin‐1 deficiency on structural alternations induced in cerebral arterioles by angiotensin II (Ang II). Caveolin‐1 deficient (Cav1−/−) and wild‐type (WT) mice were infused with either Ang II (1000 ng/kg/day) or saline via osmotic minipumps. After 4‐weeks of infusion, pressure and diameter were measured in cerebral arterioles before and during maximal dilatation (EDTA) through an open cranial window under anesthetized conditions. Cross‐sectional area of the vessel wall was measured histologically. Chronic infusion of Ang II increased cerebral arteriolar mean pressure in both WT (50±1 vs 39±1 mmHg in saline infused, P<0.05) and Cav1−/− mice (52±2 vs 34±1 mmHg, P<0.05). Ang II reduced external diameter (ED) and increased cross‐sectional area of the vessel wall (CSA) in WT mice (ED = 57±2 vs 63±2 μm; CSA = 411±38 vs 300±20 μm2; P<0.05), but not in Cav1−/− mice (ED = 64±2 vs 64±2 μm; CSA = 331±24 vs 305±16 μm2). These findings suggest that caveolin‐1 may play an important role in Ang II‐induced inward remodeling and hypertrophy of cerebral arterioles.

This work was supported by an award from the American Heart Association.
Regulation of adrenaline synthesis in the heart of the spontaneously hypertensive rat
Authors: Peltsch Heather Anne , Bizier Chad , Nguyen Phong , Tai T.C.
Published: June 2021

Phenylethanolamine N‐methyltransferase (PNMT) is the enzyme responsible for adrenaline synthesis. Adrenaline is involved in sympathetic control of blood pressure, and it augments cardiac function. Genetic mapping studies have linked PNMT to hypertension, and elevated PNMT levels are associated with higher blood pressure in hypertensive rats. The current study examined the expression of cardiac PNMT in the genetic rodent model of hypertension, and associated changes in the transcriptional regulators. RT‐PCR results show upregulation of PNMT in all chambers of the heart of spontaneously hypertensive rats (SHR) compared to Wistar‐Kyoto (WKY). RT‐PCR analyses of the previously identified key regulators of PNMT, Egr‐1, Sp1, AP2 and GR, show significant increases in all chambers of the SHR heart. Protein levels of Sp1, Egr‐1 and GR are elevated in the right atrium (RA) of SHR. In addition, AP2 shows increased binding to its cognate promoter sequence in SHR hearts. Plasma levels of corticosterone and MCP‐1 are significantly elevated in SHR, while IL‐1‐beta, IL‐12p70, IL‐18 and IFN‐gamma are significantly lower in SHR compared to WKY. These results show elevated PNMT gene expression in SHR that is likely mediated by altered transcriptional regulation of cardiac PNMT. Furthermore, SHR display systemic differences from WKY in hypothalamic‐pituitary‐adrenal axis activity and cytokine profiles in circulation.
Optimization of a fully implantable small animal infusion model involving multi‐pressure data collection
Authors: Gizzi Jay , Baird Theodore , O'Donohue Kyle , Yoder Josh , Grenwis Jessica , Bogie Heather
Published: June 2021

Collecting multiple, intra‐subject pressures provides a broader physiological picture for many research applications, while potentially reducing animal use. Research decisions often hinge on cardiovascular (CV) data that previously could only be obtained from a large animal model due to the lack of appropriate, fully implantable solutions enabling simultaneous measurement of multiple pressures in small animals. DSI has optimized possibilities for multi‐channel pressure evaluation by developing the dual pressure HD‐S21 transmitter. Emulating a study pioneered by MPI Research with two independent pressure transmitters, DSI conducted a follow‐up investigation to demonstrate the advantages obtained by using the HD‐S21. Two male CD® IGS rats were implanted with the HD‐S21 to monitor LVP, MAP, ECG and core temperature. iPRECIO® infusion pumps, also used in the MPI validation, delivered the same IV infusion program. While the initial (MPI) validation assessed verapamil and dobutamine infusion effects on temporally contiguous measures of LVP and MAP via independent devices, the HD‐S21 enabled simultaneous and continuous monitoring of all parameters from a single device. Together, the HD‐S21 and iPRECIO® provide pharmacology research the first fully implantable and easily validated small animal infusion model optimized to wirelessly record a comprehensive parameter battery with high sensitivity.
Mitochondrial subpopulation‐specific proteomic alterations in the type 2 diabetic heart
Authors: Croston Tara Lyne , Dabkowski Erinne R , Williamson Courtney L , Baseler Walter A , Hollander John M
Published: June 2021

Diabetic cardiomyopathy is the leading cause of mortality in patients who suffer from diabetes mellitus (DM). Mitochondria dysfunction contributes to the cardiac complications associated with type 2 DM. Cardiomyocytes contain two spatially distinct mitochondrial subpopulations, interfibrillar mitochondria (IFM) and subsarcolemmal mitochondria (SSM). The goal of this study was to determine whether the proteomic profiles of the two cardiac mitochondrial subpopulations were influenced differently by type 2 diabetic insult. Db/db mice and WT controls were sacrificed at 18 weeks of age and cardiac mitochondrial subpopulations were isolated. Proteomic analysis revealed a decrease in proteins involved in the electron transport chain (ETC) complexes in the SSM. Specifically, ubiquinol cytochrome c reductase core protein 2 (complex III) and cytochrome c oxidase subunit VIIa1 (complex IV) were downregulated in db/db SSM. Further, peroxiredoxin V (PRDX5), an antioxidant, and adenine nucleotide transporter 1 (ANT1), an inner mitochondrial membrane transport protein, were also significantly decreased only in db/db SSM. These results suggest that the SSM are at greater risk for protein loss as a result of type 2 DM, which may contribute to the pathogenesis of diabetic cardiomyopathy.

(Supported by NIH DP2 DK083095, AHA0815406D, AHA0855484D)
Increased contractile responses in corpora cavernosa of heart failure rats
Authors: Carneiro Fernando S , Biancardi Vinicia C , Carneiro Zidonia N , Stern Javier E , Webb R Clinton , Tostes Rita C
Published: June 2021

Introduction

Heart failure (HF) is a risk factor for erectile dysfunction (ED). HF patients experience a decreased libido, frequency of coitus and sexual performance. The mechanisms underlying the association of HF and ED are unknown. We hypothesize that HF increases cavernosal reactivity.

Methods and Results

HF was induced, in Wistar male rats (8 week‐old), by left coronary artery ligation for 6 weeks. Ejection fraction (Sham: 85.5±3.3 vs HF: 33.9±3.0, %) and fractional shortening (Sham: 63.9±3.8 vs HF: 17.3±1.7, %) were measured to confirm the presence of HF. HF rats displayed increased cavernosal contractile responses to KCl (Sham: 0.55±0.07 vs HF: 0.82±0.06, mN/mg) and phenylephrine in the absence (Sham: 0.71±0.05 vs HF: 0.97±0.05, mN/mg) or in the presence of L‐NAME (Sham: 0.70±0.05 vs HF: 1.14±0.06, mN/mg). Sympathetic‐mediated contractile responses were increased in cavernosal strips of HF rats in the absence (Sham: 0.55±0.09 vs HF: 1.03±0.08, mN/mg) and in the presence of L‐NAME (Sham: 0.67±0.1 vs HF: 1.33±0.1, mN/mg). HF rats displayed increased cavernosal relaxation to sodium nitroprusside (Sham: 57.8±3.4 vs HF: 76.5±5.7, % of relaxation), but no changes were observed in nonadrenergic‐noncholinergic‐ (NANC)‐induced relaxation.

Conclusion

Our results suggest that increased contractility of the cavernosal tissue in HF rats may represent the mechanism of ED associated with HF.
Effect of Prenatal Glucocorticoid Treatment on Adrenal Phenylethanolamine N‐methyltransferase Gene Expression in the Adult Wistar‐Kyoto Rat
Authors: Nguyen Phong , Peltsch Heather , Crispo James , de Wit Jose , Ubriaco Gino , Ansell Dominique , Wall Vanessa , Eibl Joseph , Tai T.C.
Published: June 2021

Prenatal exposure to glucocorticoids (GCs) programs for hypertension later in life. The current study examined the impact of prenatal GC exposure on the post‐natal regulation of the gene encoding for phenylethanolamine N‐methyltransferase (PNMT), the enzyme involved in the biosynthesis of the catecholamine, epinephrine. PNMT has been linked to hypertension and is elevated in animal models of hypertension. Results from this study show that systolic, diastolic and mean arterial blood pressure are elevated in male offspring (5 to 17 weeks of age) from dams treated with dexamethasone (DEX, +15%) or corticosterone (CORT, +12%) compared to male offspring from saline‐treated (SAL) dams. RT‐PCR analysis shows that adrenal PNMT mRNA is 1.5‐fold higher in DEX and CORT males compared to SAL males. RT‐PCR analysis of transcriptional regulators of the PNMT gene show that prenatal GC exposure increased mRNA levels of Egr‐1 (2.5‐fold), AP‐2 (1.9‐fold) and GR (2.0‐fold). In contrast, Western blot analyses show increased expression of PNMT protein (1.44‐fold, DEX), along with increased Egr‐1 (1.5 fold, DEX and CORT), AP‐2 (1.4 fold, CORT), and GR (1.7 fold, DEX and CORT) compared to SAL males. These results suggest that increased PNMT gene expression via altered transcriptional activity is a mechanism by which prenatal GC insult may program for hypertension later in life.
Effect Of PPADS On Mesenteric Arterial And Portal Venous Flow Responses To Splanchnic Nerve Stimulation
Authors: Martin Doug , Kjellsen Barton
Published: June 2021

Our previous work suggested that splanchnic nerve stimulation may exert differential effects on mesenteric arterial and portal venous hemodynamics. This study tested the hypothesis that ATP contributes to the differences in mesenteric arterial and portal venous responses to splanchnic nerve stimulation.

Spontaneously hypertensive rats (SHR) were anesthetized with urethane/chloralose and fitted with catheters to record arterial pressure. The rats were also instrumented for splanchnic nerve stimulation and for mesenteric arterial and portal venous blood flow recording. Frequency response curves (1–15 Hz) were constructed before and after treatment with pyridoxal‐phosphate‐6‐azophenyl‐2′,4′‐disulfonate (PPADS; 20 mg/kg), an ATP P2X receptor antagonist.

Splanchnic nerve stimulation caused frequency dependent decreases in mesenteric arterial and portal venous blood flow. Treatment with PPADS caused a rightward displacement of the frequency response curves for both mesenteric arterial flow and portal venous flow. The magnitude of this displacement was less than that observed following omega conotoxin treatment.

Collectively, these data suggest that ATP contributes to both mesenteric arterial and portal venous responses to splanchnic nerve stimulation. Supported by NIH #2RO1HL63053‐ARRA (DM) and USD SSOM bridge funds.
Heterogeneous response of cytokine levels of rat white adipose tissue in cachexia
Authors: Batista Miguel L , Neves Rodrigo X , Donatto Felipe F , Seelaender Marília CL
Published: June 2021

The white adipose tissue (WAT) is severely affected by cachexia, a paraneoplastic inflammatory syndrome, in which cytokines, such as TNF‐α and IL‐10 are known to play an important role. Recently, the IL‐10/ TNF‐α ratio has been recently postulated as a marker for the assessment of the degree of inflammation, which correlates with disease‐associated morbidity and mortality. We measured (ELISA) the local concentration of these cytokines in three WAT depots: retroperitoneal (RPAT), epididymal (EAT) and mesenteric (MEAT) of Walker 256 tumour‐bearing rats in a time‐interval of 0 (T0), 7 (T7) and 14 (T14) days after tumour‐cell inoculation (5x10−5 cells/mL). Higher concentration of TNF‐α was found on T14 of RPAT (2.4‐fold) and of EAT (6.9‐fold) of cachectic rats, both compared with T0. EAT and RPAT IL‐10 levels were reduced on T14 (87.6 and 67.2%, respectively) compared to T0. Regarding the IL‐10/ TNF‐α ratio, there was a decrease in EAT (97.2%) and RPAT (91.4%) after 14 days (T14), compared with T0. The results suggest a heterogeneous local response of both TNF‐α and IL‐10 expression, with a more accentuated reduction in IL‐10 levels of EAT than others depots. We conclude that the degree of inflammation of WAT is dependent of anatomical localization of the pad.
CTP: Phosphocholine cytidyltransferase alpha (CCTalpha) siRNA induce cell death in p53 negative and positive lung cancer cells
Authors: Marijani Rukia , Abonyo Barack
Published: June 2021

CCT is a rate limiting enzyme required for the synthesis of phosphatidylcholine through the CDP‐choline pathway, which is critical for cell proliferation. Anti‐cancer drugs have therefore been designed based on their ability to inhibit this enzyme especially CCTα isoform. The purpose of this study was to explore non toxic molecular techniques of inhibiting CCTα expression or activity which results in the killing of lung cancer cells. Briefly, p53 positive, A549 and negative, H1299 lung epithelial cells were transfected with 0–800 nM of CCTα siRNA sequences (A, B, and C) or control siRNA for 24 hrs and cell viability and toxicity analyzed by Cell Titer Blue (CTB) as well as lactosedehydrogenase (LDH) assay. Western Blot analysis showed that all the CCTα siRNA sequences significantly inhibited CCTα protein expression while Light Microscopy depicted that CCTα siRNA transfected cells were completely shrunken and had nuclei filled with dark granular structures similar to apoptotic bodies. At high concentrations of siRNA the cells were completely degraded showing signs of cell lyses. CTB and LDH assays revealed that CCTα siRNA transfected cells were significantly less viable. These results demonstrate that CCTα siRNAs knocked down CCTα protein resulting in cell death, implying that the sequences could be useful as anticancer agents.
Physiological Significance of Mitochondrial Uncoupling Protein 1 in the Prevention of Reactive Oxygen Species and Control of Substrate Oxidation
Authors: Jastroch Martin , Keipert Susanne , Meyer Carola W , Kutschke Maria , Heldmaier Gerhard , Klaus Susanne , Oelkrug Rebecca
Published: June 2021

The established function of mitochondrial uncoupling protein 1 in mammalian brown adipose tissue is the dissipation of proton motive force as heat. Uncoupling of respiration may also prevent mitochondrial superoxide production and ROS formation, but the involvement of UCP1 in this process is highly disputed. We here studied in isolated mitochondria a) whether the presence of endogenous uncoupling protein 1 in brown adipose tissue affects superoxide production, in particular during cold acclimation and beta oxidation, b) whether ectopically expressed UCP1 in skeletal muscle is functional and manipulates superoxide, and c) how substrate oxidation adapts to the presence of UCP1.

We show that UCP1 mitigated mitochondrial superoxide rates in both tissues. In brown adipose tissue, increases of superoxide during cold acclimation and beta oxidation were blunted as compared to UCP1 knockout mice. Uncoupling alters oxidation capacities differently depending on cell‐type: While the presence of UCP1 in brown adipose tissue allows cold‐induced increase in oxidative capacity, skeletal muscle UCP1 depresses substrate oxidation. Our results demonstrate the physiological involvement of UCP1 in the regulation of mitochondrial superoxide and provide models to further investigate how uncoupling controls oxidative capacity by altering superoxide and/or cellular ATP/ADP ratios.
Stimulation of P2X7 receptors causes Ca2+‐ and PKC‐mediated reactive oxygen species production in murine macrophages
Authors: Martel‐Gallegos Guadalupe , Casas‐Pruneda Griselda , Ortega Filiberta , Sanchez‐Armass Sergio , Perez‐Cornejo Patricia , Arreola Jorge
Published: June 2021

Stimulation of macrophages with extracellular ATP or Bz‐ATP induces Reactive Oxygen Species (ROS) production. We tested the idea that in J774 macrophages activation of P2X7 receptors (P2X7R) by ATP leads to NADPH oxidase assembly and ROS production via Ca2+ entry and PKC activation. We used whole‐cell voltage clamp to measure P2X7R‐mediated currents, Fura‐2 to determine [Ca2+]i, 2′,7‐dichlorodihidrofluorescein to assay ROS production, PCR and western blot for P2X7R expression and phospho‐specific antibodies to determine activation of PKC isoforms. Stimulating J774 cells with Bz‐ATP (100 μM) induced an inward current at −80 mV that was sensitive to A438079 (10 μM), a blocker specific for P2X7R. ROS production induced by Bz‐ATP was also inhibited by A438079, PKC inhibitors and by removing extracellular Ca2+. Similarly, the increase in [Ca2+]i seen with Bz‐ATP was inhibited by zero external Ca2+ and A438079. Bz‐ATP induced PKC α activation. Importantly, Bz‐ATP induced a transient activation of PKC δ, while PKC ζ displayed an oscillatory pattern and was inhibited by A438079, confirming the participation of P2X7R. In summary, we propose that purinergic stimulation in macrophages induced ROS production due to Ca2+ entry via P2X7R and subsequent PKC activation which may, in turn, induce NADPH oxidase assembly and activation.

Supported by grants 79897 and 105457 to JA and P. P‐C
The double life of Set1‐MLL in the initiation of transcription
Authors: Lin Justin , Carey Michael
Published: June 2021

The Set1/MLL1‐4 complexes represent the mammalian trithorax family of histone‐lysine methyltransferases. The Set1a/b‐containing complexes are responsible for H3K4 trimethylation, a modification which correlates with both actively transcribed and poised promoters. We show here, using an immobilized template assay, that Set1‐MLL is recruited to a pre‐initiation complex (PIC) on a chromatinized template by the model activator GAL4‐VP16. We observe that this recruitment can occur via a direct interaction with GAL4‐VP16, but is stimulated by the dual presence of the PIC components PAF and Mediator. To further understand the downstream consequences of Set1 function, we assembled synthetically modified H3K4me3 chromatin. The modification stimulated activator‐dependent in vitro transcription. Using a discovery‐based assay involving MuDPIT to identify H3K4‐specific factors captured on the immobilized template, we found that the chromatin remodeler CHD1 is enriched on H3K4me3 templates, but is recruited in an activator‐dependent manner, downstream of Mediator and MLL. In summary, preliminary findings suggest that the initial recruitment of Set1‐MLL is can occur both independent of or cooperatively with a functional PIC, the consequence of which is CHD1 recruitment and the stimulation of transcription. The work described above is supported by a grant from the NIH.
Neuroprotective effects of exogenous melatonin on spontaneously hypertensive rats with focal cerebral ischemia
Authors: Kim Joo‐heon , Lee Sang‐kil , Lee Seunghoon , Chang Kyu‐tae , Hong Yonggeun
Published: June 2021

Hypertension is involved in the exacerbation of stroke; thus, in the present study, we investigated the neuroprotective effects of melatonin against focal cerebral ischemia in spontaneously hypertensive (SHR). We examined whether hypertension influences focal cerebral ischemia. Following middle cerebral artery occlusion‐reperfusion (MCAo), melatonin was injected subcutaneously each day at 7:00 and 19:00 for four weeks. The brains of the rats were then isolated and sectioned at 2‐mm intervals from the frontal to the occipital pole to yield six coronal slices. The slices were then immersed for 30 min in 2% TTC at 37°C. Gene expression was then analyzed, and the infarct volume was quantified using Image‐J analysis software. The infarct volume in the SHR group was significantly increased compared to the normal vehicle group (p<.01). Exposure to melatonin decreased the infarct volume compared to vehicle exposure. NOS expression was decreased in the melatonin‐treated group after MCAo; however, BDNF gene expression in the melatonin‐treated group was increased, as compared to that in the vehicle group. These results suggest that melatonin can prevent neurological injury in cases of hypertension‐assisted focal cerebral ischemia.
Characteristics of early‐stage experimental middle cerebral occlusion‐reperfusion
Authors: Park Sookyoung , Lee Sang‐kil , Chang Kyu‐tae , Hong Yonggeun
Published: June 2021

Middle cerebral artery occlusion (MCAo)‐reperfusion microsurgery induces focal cerebral ischemia (FCI) and neurological deficiency. Recently, it was reported that integral membrane proteins known as caveolins (cav) may be involved in the pathogenesis and mechanism of cerebral ischemia. Therefore, in this study we established the behavioral features and investigated the expression of cav and signaling molecules in rat brains subjected to MCAo. All rats were evaluated for neurological and motor dysfunction using the modified neurological severity score (mNSS), gait analysis, and beam‐walking test at 24, 48, and 72 h after MCAo. Next, the brains of the rats were cut into 2‐mm thick coronal sections, which were subjected to TTC staining for confirmation of the infarct volume. On assessing the behavior of the MCAo rats, neurological deficiency, motor dysfunction, and impaired balance were detected (p<.05). Moreover, the gene expression of cav, nNOS, BDNF, and Trk family was decreased, whereas iNOS gene expression was increased in the MCAo group, as compared to the control group, at 24 h after microsurgery (p<.05). Also, the cerebral infarct volumes were increased significantly in the MCAo group, as compared to the control group (p<.05). These results imply that cav and signaling molecules, especially iNOS, are a strong causative factor in the early stages of brain damage after FCI.

Funding: BioGreen21 Program (Code No. 20070401‐034‐006‐009‐02‐00), Rural Development Administration, Korea
Skeletal muscle lipid metabolism changes in mice selected for high maximal metabolic rate
Authors: Wone Bernard , Donovan Edward R. , Hayes Jack P.
Published: June 2021

Exercise and fasting both increase free fatty acids, and elevated unsaturated free fatty acids are up‐regulators of lipid metabolism in skeletal muscles. We examined fatty acids in skeletal muscles in lines of Mus musculus artificially selected for high maximal metabolic rate (high‐MMR). These mice were not exercised and standard laboratory rat chow was available ad libitum. After 7 generations of directional selection for increased mass‐independent MMR, high‐MMR mice showed a 10.2% increase in mass‐independent MMR and a concomitant 2.8% increase in mass‐independent basal metabolic rate compared to control mice. Free fatty acids in the gastrocnemius muscle differed significantly between high‐MMR and control mice. For mice selected for high‐MMR, there was a significant increase in linoleic and oleic acids—consistent with elevated fatty acid oxidation in the skeletal muscle. There was no difference in muscle phospholipid fatty acid composition between high‐MMR and control mice. These findings suggest that selection for high‐MMR resulted in elevated fatty acid oxidation in the skeletal muscles. Most importantly, the results suggest that there is a genetic component to elevated unsaturated free fatty acids in skeletal muscles. Supported by NSF IOS 0344994.
Gene expression basis for flexibility of intestinal maltase activity in young house sparrows
Authors: Karasov William , Gatica‐Sosa Claudia , Brzandęk Pawel , Caviedes‐Vidal Enrique
Published: June 2021

House sparrows (Passer domesticus) undergo a natural diet switch from insects to starch‐containing seed diet during development. Correlated with that is an increase in intestinal maltase activity that is partly genetically programmed (occurs even in absence of dietary carbohydrate) and partly diet induced (activity doubled by inclusion of 25% dietary starch) (Brzek et al. 2009 J. Exp. Biol. 212:1284) and completely reversible (Kohl et al 2009 Integ. and Comp. Biol. 49:e93). To test if the observed maltase flexibility has a gene expression basis we assessed using RT‐qPCR the maltase‐glucoamylase (MG) mRNA of mid‐intestine samples. Nestlings were raised from day 4 after hatch on either a starch free or a 25% starch diet. At age 12 days some were switched to the alternate diet and others remained on the original diet (hence 4 diet groups). Birds at ages 12 days and 30 days were tested for maltase activity and MG mRNA. Cq values normalized to β ‐actin and GDPH showed that the up‐ and down‐regulation of maltase activity by dietary carbohydrate correlated with a significant increase and decrease in MG mRNA (P<0.05), which is consistent with transcriptional regulation as found in rodents. These changes seem specific, because in the same tissues mRNA for SGLT1, the active glucose transporter, was not increased on 25% starch diet. Funded by NSF IOS 0615678 to WHK and UNSL‐CyT 22Q751 & FONCYT 25561 to EC‐V.
Innate immune function in mice selected for high metabolic rate
Authors: Downs Cynthia J , Wone Bernard , Donovan Edward R , Hunter Kenneth W , Hayes Jack P
Published: June 2021

Innate immune function is potentially energetically expensive. We tested the hypothesis, posited by life history theory, that trade‐offs exist among energetically expensive activities (e.g., innate immune function and metabolic rate). We assessed innate immune function by injecting mice with lipopolysaccharide (LPS), and we measured basal metabolic rates (BMR) and maximal metabolic rates (MMR). Mice were artificially selected either for high MMR (MAX lines) or for high MMR and low BMR (COR lines). All selection was on mass‐independent metabolism. After 7 generations of selection, mass‐adjusted BMR was highest in MAX mice, intermediate in COR mice, and lowest in non‐selected controls (CONT) (0.61, 0.57, and 0.59 ml O2/min, respectively). Immune challenged (i.e., LPS injected) mice had higher BMR than mice injected with saline (0.88 and 0.80 ml O2/min, respectively), and during the immune challenge BMR was most elevated in COR mice. COR mice also had the greatest aerobic scope (MMR‐BMR). Collectively, these results suggest a possible trade‐off between cost of mounting an innate immune response and ability to elevate MR during aerobic activity (i.e., aerobic scope). Support: NSF IOS 0344994.
Angiotensin IV Induces Phosphorylation Of Extracellular Signal‐Regulated Kinase 1/2 Via The AT4 Receptor In 3T3‐L1 Adipocytes
Authors: Jungwirth Julie Ann , Phillips Erin E. , Daley Brian J. , Enderson Blaine L. , Karlstad Michael D.
Published: June 2021

Angiotensin II acting via the AT1 receptor has an inhibitory effect on the insulin‐signaling pathway in adipocytes by activation of extracellular signal‐regulated kinase (ERK 1/2) and subsequent serine phosphorylation of IRS‐1. The objective of this study was to determine if Ang IV is involved in downstream insulin‐signaling pathways by phosphorylation of ERK 1/2 via the AT4 receptor in 3T3‐L1 adipocytes. We initially confirmed the expression of AT4 receptors by RT‐PCR in 3T3‐L1 adipocytes. Adipocytes were incubated with various combinations of Ang IV (10−10M) for 10 min, AT4 receptor inhibitor, Norleual (10−10M) for 1 hr, and insulin (10−7M) for 5 min. Cell lysates were resolved by SDS‐PAGE electrophoresis and immunoblotted with anti‐phospho‐ERK 1/2. Ang IV and insulin alone or in combination increased the phosphorylation of ERK 1/2 as compared with controls. AT4 induced phosphorylation of ERK 1/2 was reversed by pretreatment with the novel AT4 receptor antagonist, Norleual. Norleual attenuated Ang IV phosphorylation of ERK1/2, whereas it did not affect ERK 1/2 phosphorylation with insulin or the combination of insulin and Ang IV. This study shows that Ang IV is involved in the insulin‐signaling cascade by increasing phosphorylation of ERK 1/2, which is inhibited by AT4 receptor blockade and is independent of the insulin receptor in adipocytes.
Isoleucine administration downregulates the genes for gluconeogenic rate‐limiting enzymes
Authors: Oikawa Nodoka , Ishii Shunsuke , Saito Kenji , Zhao Zhi‐Lei , Kato Hisanori , Sugahara Kunio , Yoshizawa Fumiaki
Published: June 2021

Isoleucine, a branched chain amino acid, plays an important role in the improvement of glucose metabolism as evidenced by the reduction of gluconeogenesis in liver, along with an increase of glucose uptake in the muscle. This study evaluated the effect of isoleucine on the expression of hepatic gluconeogenic rate‐limiting enzymes in fasted rats. The expressions of phosphoenolpyruvate carboxykinase (PEPCK) mRNA and glucose‐6‐ phosphatase (G6Pase) mRNA in isoleucine‐administered rat liver were decreased compared with that in the fasted rat liver. These ﬁndings imply that isoleucine downregulates the expression of gluconeogenic rate‐limiting enzyme genes and inhibits glucose production in the liver. We also compared the gene expression profiles in the liver of isoleucine‐administered rats and fasted rats by DNA microarray. The gene expression data, although preliminary, indicated that there were changes in the expression of genes for enzymes associated with glycolysis or gluconeogenesis. In addition, pathway analysis using Ingenuity Pathways Analysis software identified biological interaction networks among PEPCK regulated genes.
The effects of exogenous hormones on maximal and submaximal responses to high intensity interval training
Authors: Zierke Jennifer Marie , Turner Rebekah Joy , Parker Daryl , Quintana Roberto
Published: June 2021

High intensity interval training (HIIT) leads to VO2max improvements in 2‐weeks (Talanian, 2007). Long term (≥ 4 months) exogenous hormone (EH) use can decrease VO2max by 11% (Casazza, 2002). It is unknown whether EH impairs exercise training induced adaptations.

PURPOSE

The purpose of this study was to investigate the effects of seven HIIT sessions over 2 weeks on maximal and submaximal (submax) physiological responses to exercise in women taking EH.

METHODS

Sixteen healthy women of average fitness, VO2max = 40.29±7.51, volunteered for this study (n=8 EH, n=8CON). Prior to participating in the study, the EH women were taking a monophasic or triphasic oral contraceptive pill for ≥ 6 months and CON were regularly menstruating. VO2max and submax testing (10% below VT1) were performed on a cycle ergometer pre and post HIIT. Statistical comparisons were made between EH and CON to determine the physiological adaptations that occurred with HIIT.

RESULTS

CON had a significant decrease in submax HR (148±6.1vs136±4.7bpm, p=0.02), and an increase in O2pulse (10.36±.81vs10.98±.81 ml/bpm, p=0.02). The EH had a significant increase in dBP (59.7±3.0vs65.2±3.12mmHg). There were no other significant differences between groups.

CONCLUSION

EH use alters submax cardiovascular training responses without affecting VO2max. EH use blunts HR, dBP, and O2pulse adaptations to HIIT.
ABOUT EVALUATION OF ADAPTATION POSSIBILITIES OF STUDENTS
Authors: Slivkina Natalia , Sattiklishov Berik , Dalenov Erbulat , Tell Leonid
Published: June 2021

The aim of our research was to study the adaptive capacity of students. Analysis rhythmograms found that among students of 1st ‐year predominate the parasympathetic tone. By the 2th year the % of adolescents with a predominance of parasympathetic influences is reduced, while increasing the number of students with a mixed tone. Then have been assessed the functional state of the organism. 32,9% freshmen were in the stage adaptation disruption and only 10,9% found satisfactory adaptation. Among sophomores cases of adaptation disruption decreased to 24%, but cases with a satisfactory adaptation increased to 16%. As students 3rd year, the frequency of disruption of adaptation was 28.9%. In our view, our findings are explained by the fact that first‐year students faced with the increasing psychological and emotional pressures, changing location and living conditions, the usual way of life and previously formed stereotypes that imposes high demands on adaptability of the organism and often leads to failure of adaptation. To the second year the body organism can adapt to change of customary conditions
Heat acclimation reduces gut permeability, endotoxin translocation, and inflammatory/anti‐inflammatory cytokine cascades in humans
Authors: Kuennen Matthew Ryan , Gillum Trevor , Christmas Kevin , Kulovitz Michelle , Alumbaugh Brent , Dokladny Karol , Schneider Suzanne
Published: June 2021

Purpose

To examine human gut barrier responses to a 7‐day heat acclimation (HA) protocol.

Methods

Five men exercised to a core temperature of 39.20 ± 0.04°C for 7 days to develop HA. Blood drawn pre (Pre) and post (Post) exercise on day 1 and day 7 of HA was assayed for endotoxin, TNF‐a, Il‐6, Il‐10, intracellular (iHSP70) and extracellular HSP70 (eHSP70). Additional measures of gut permeability (lactulose sugar test) were performed at baseline (prior to exercise/heat exposure) and on days 1 and 7 of HA.

Results

HA Day 1
HA Day 7

Pre
Post
Pre
Post

Endotoxin (pg/ml)
7.8±1.8
10.7±2.3*
4.8±0.7^
7.7±2.7^

Lactulose (fold Δ from baseline)
‐
11.1±6.4*
‐
6.7±3.3

TNF‐a (pg/ml)
10.9±0.9
13.2±1.5*
10.4±1.0
10.5±1.1#

Il‐6 (pg/ml)
1.3±0.4
6.8±1.7*
1.2±0.5
2.9±0.6*#

Il‐10 (pg/ml)
7.5±2.7
20.3±5.3*
7.5±1.5
10.5±2.4#

eHSP70 (ng/ml)
0.7±0.4
1.5±0.8*
1.4±0.4
1.3±0.5

iHSP70/actin (pixels)
0.3±0.1
0.6±0.1*
0.2±0.07
0.9±.04*#

Data are mean ± SE.

difference within conditions, p<.05.

difference between conditions, p<.05.

difference between conditions, p <.07

Conclusions

Both direct (endotoxin) and indirect (lactulose sugar test) measures support a reduction in exercise‐induced gut permeability following the 7‐day HA protocol. Reductions in gut permeability were associated with increased exercise HSP70 accumulation and attenuated exercise cytokine responses. This may be a potential mechanism by which HA confers protection against exertional heat illness.
A claudin‐4 modulator enhances the mucosal absorption of peptide
Authors: Uchida Hiroshi , Kondoh Masuo , Hanada Takeshi , Takahashi Azusa , Hamakubo Takao , Yagi Kiyohito
Published: June 2021

Objective

Paracellular pathway is a principal route for absorption of biologics (peptides, proteins and nucleic acids); however, tight junctions (TJs) restrict passing of biologics across epithelium. Claudin is known as a key component in TJs‐barrier. We previously found that a claudin‐4 modulator, C‐terminal fragment of Clostridium perfringens enterotoxin (C‐CPE), enhanced jejunal absorption of dextran. In this study, we investigated whether a claudin‐4 modulator is a potent mucosal absorption‐enhancer of a peptide drug.

Methods

Mucosal absorption was evaluated by rat jejunal, pulmonary and nasal administration of human parathyroid hormone derivative, hPTH(1–34) and C‐CPEs. Plasma hPTH(1–34) levels were measured by RIA. Binding of C‐CPEs to claudin‐4 was assayed by ELISA and BIAcore analysis.

Results

Pre‐treatment with C‐CPE enhanced mucosal absorption of hPTH(1–34). However, the jejunal and pulmonary absorption‐enhancing effects were not observed by co‐treatment with C‐CPE. The deletion of 10 amino acids at the N‐terminal of C‐CPE increased its solubility by 30‐fold. Moreover, the truncated C‐CPE bound to claudin‐4, modulated the TJ‐barrier and enhanced jejunal absorption of dextran. Co‐treatment of the truncated C‐CPE also enhanced jejunal and pulmonary absorption of hPTH(1–34).

Conclusion

Claudin‐4 modulator may be a promising absorption‐enhancer of a peptide drug.
Anti‐proliferative Effect of Kv1.3 in A549 Human Lung Adenocarcinoma in vitro and in vivo
Authors: Jang Soo Hwa , Choi Sun Young , Ryu Doug‐Young , Ryu Pan Dong , Lee So Yeong
Published: June 2021

Voltage‐gated potassium channels (Kv) are widely expressed in the plasma membranes of numerous cells and contribute to a variety of cellular functions in both excitable neuronal cells and non‐excitable epithelial cells. Recently, it has been demonstrated that Kv channels are associated with the proliferation of several types of cancer cells. In the present study, we investigated the effects of suppressed expression of Kv1.3 on cell proliferation and cell cycle distribution in human lung adenocarcinoma, A549 cells. Treatment with margatoxin (MgTX), a selective blocker of Kv1.3, and shRNA against Kv1.3, significantly blocked A549 cell proliferation. In addition, selective inhibition of Kv1.3 significantly increased expression level of p21Waf1/Cip1 and significantly decreased the expression level of Cdk4 and cyclin D3. We also applied the MgTX into a xenograft model using nude mice and MgTX caused a reduction of tumor volume when it was injected into tumor tissues. These results suggest that Kv1.3 may serve as a novel therapeutic target for lung adenocarcinoma therapy. This work is supported by National Research Foundation of Korea (2009‐0066580)
Cortistatin Inhibits Intestinal Secretion in the Guinea‐pig Ileum In Vitro
Authors: Liu Sumei
Published: June 2021

Background and Aims

Cortistatin (CST) is a novel neuropeptide with high structural homology with somatostatin. We previously found that CST is expressed by enteric neurons and acts as an inhibitory neurotransmitter in the submucosal plexus. The aim of the present study was to evaluate the effect of CST on neurogenic intestinal secretion.

Methods

Submucosal/mucosal preparations from guinea pig ileum were mounted in Ussing flux chambers for measurement of short‐circuit current (Isc) as an indicator of mucosal secretion. Transmural electrical field stimulation (EFS) was used to evoke neural‐mediated intestinal secretion.

Results

Application of CST‐14 (rat), CST‐17 (human), CST‐29 (rat), or CST‐29 (human) to the submucosal side of the preparation decreased baseline Isc in a concentration‐dependent manner with an IC50 (in nM) of 90.5, 52.1, 102.9 or 71.6, respectively. The somatostatin‐2 receptor (SSTR‐2) antagonist CYN 154806 significantly suppressed CST‐14r, CST‐17h, CST‐29r, or CST‐29h‐evoked ΔIsc. The neuronal blocker tetrodotoxin significantly reduced CST‐14r, CST‐17h, CST‐29r, or CST‐29h‐evoked ΔIsc. CSTs produced a concentration‐dependent reduction of the EFS‐evoked secretory responses, which was antagonized by CYN 154806. Immunoreactivity (IR) of CST‐17 was found in submucosal neurons. CST‐17‐IR was localized in cholinergic secretomotor neurons but was absent from non‐cholinergic secretomotor neurons.

Conclusions

The results suggest that CSTs act at SST‐2 receptors in the submucosal plexus to suppress secretomotor neuronal excitability and neurogenic intestinal secretion.