bluuebunny/crossref_metadata_2025 huggingface dataset:
Reactivity in resistant arteries of SHR treated with anti‐TLR4 antibody
Authors: Bomfim Gisele Facholi , Carvalho Maria Helena , Fortes Zuleica , Santos Rosangela Dos , Tostes Rita , Webb Clinton
Published: June 2021

Recent reports indicate that TLR4 participates in mediating the inflammatory responses in vascular diseases. Because hypertension is considered a chronic inflammatory disease, our hypothesis is that TLR4 expression is increased in arterioles of spontaneously hypertensive rats (SHR) and participates of vascular dysfunction present in hypertension. For this, SHR were treated with anti‐TLR4 antibody and we analyzed reactivity in mesenteric arterioles. TLR4 expression is increased in mesenteric arterioles from SHR with 15 weeks‐old (hypertensive phase) versus SHR 5 weeks‐old (normotensive phase). The treatment of SHR 15 weeks‐old for 15 days with losartan decreased TLR4 expression in mesenteric arterioles. Anti‐TLR4 treatment decreased blood pressure of SHR. In vascular reactivity, the maximum response of mesenteric arterioles to noradrenaline was decreased in SHR anti‐TLR4‐treated compared to untreated rats. The mechanisms involved are COX‐1 and COX‐2 dependent. There was no difference in relaxation response to vasodilatation agonists after the treatment. It was also found a decreased in IL‐6 secretion in SHR serum after anti‐TLR4 treatment. We suggest that TLR4 has a role in the low‐grade inflammatory process present in hypertension and in vascular dysfunction present in SHR.
Methyl palmitate is the perivascular adipose tissue‐derived relaxing factor
Authors: Lee Yuan‐Chieh , Chang Hsi‐Hsien , Chiang Chih‐Lung , Liu Chin‐Hung , Chen Mei‐Fang , Chen Po‐Yi , Lee Tony J.F.
Published: June 2021

The perivascular adipose tissue (PVAT)‐derived relaxing factor (PVATRF) is known to significantly regulate the vascular smooth muscle tone. Its chemical nature, however, remains unknown. We determined whether methyl palmitate or palmitic acid methyl ester (PAME) was the PVATRF. Release of PVATRF and PAME from aortic PVAT preparations of Wistar Kyoto rats was estimated using superfusion bioassay cascade technique and gas chromatography/mass spectrometry (GC/MS). The PVATRF and PAME were spontaneously and calcium‐dependently released from PVAT preparations. Both releases were inhibited by 4‐aminopyridine (2 mM) and tetraethylammonium (TEA) at 10 mM, but were not affected by TEA at 3 mM, glibenclamide (3 μM) or iberiotoxin (100 nM). Aortic vasorelaxations induced by PVATRF‐ and PAME‐containing Krebs' solutions were not affected after heating at 70°C, and were equally attenuated following hexane extractions. Culture mediums of differentiated adipocytes, but not those of fibroblasts, contained significant PAME and caused aortic vasorelaxation. These results indicate that PAME and PVATRF share similar characteristics in biochemistry and pharmacology. It is concluded that PAME is the PVATRF, causing aortic vasorelaxation by opening voltage‐dependent K+ channels on the smooth muscle cells (Supported by NSC, Tzu Chi Foundation & TCU).
Allopurinol Attenuates Left Ventricular Dysfunction In The Early Stage of Streptozotocin‐Induced Diabetes In Rats
Authors: Xu Yuan , Gao Xia , Xu Bo , Liu Hui‐min , Liu Yanan , Irwin Michael G , Xia Zhengyuan
Published: June 2021

Xanthine oxidase‐derived superoxide production and oxidative stress plays an important role in the development of diabetic complications (Diabetes 2002;51(4):1118–24) including diabetic cardiomyopathy. We hypothesized that treatment with the xanthine oxidase inhibitor allopurinol (ALP) may attenuate or prevent the development of left ventricle (LV) diastolic dysfunction, an early sign of diabetic cardiomyopathy (Cardiology 2002;98:33–9), in rats with streptozotocin (STZ)‐induced diabetes. Control Sprague‐Dawley (C) or STZ‐induced diabetic (D) rats (220±20g) were either untreated or treated with ALP (100 mg/kg/day) for four weeks starting at one week after STZ injection. In D rats, both plasma and cardiac tissue levels of 15‐F2t‐isoprostane, a specific index of oxidative damage, were increased (P<0.05 vs. C) at the 5th week, accompanied with significant increase in left ventricle (LV) weight/body weight ratio and decreases in LV end‐diastolic volume and stroke volume (all P<0.05 D vs. C). All these changes were either significantly attenuated or prevented by treatment with ALP. In addition, LV ejection fraction in D rats treated with ALP was higher than that in untreated D rats (P<0.05). It is concluded that ALP can attenuate hyperglycemia‐induced oxidative stress and subsequently present left ventricular dysfunction in early diabetes.
Triptolide‐Induced Expression of MicroRNA‐142‐3p Mediates Apoptosis of Pancreatic Cancer Cells by Inhibition of HSP70
Authors: MacKenzie Tiffany Noelle , Mujumdar Nameeta , Banerjee Sulagna , Sarver Aaron , Subramanian Subree , Sangwan Veena , Vickers Selwyn , Saluja Ashok
Published: June 2021

Triptolide inhibits cancer cell growth in vitro and blocks growth and metastatic spread in vivo. Our lab has shown that triptolide triggers apoptosis via inhibition of heat shock protein 70 (HSP70). As regulators gene expression, microRNAs (miRNAs) are increasingly recognized as powerful gatekeepers of apoptosis.

Objective

We would like to understand how triptolide affects miRNA expression and to identify miRNAs that mediate apoptosis. We hypothesize that triptolide inhibits HSP70 expression via a novel mechanism of inducing the expression of miR‐142‐3p.

Methods

We performed miRNA microarray analysis of human pancreatic cancer cells before and after triptolide treatment. We transfected miR‐142‐3p mimics and measured cell viability, caspase‐3 activation and HSP70 levels.

Results

MicroRNA‐142‐3p, predicted to target HSP70, was found in our miRNA microarray analysis to increase immediately after treatment. This finding has been validated by qRT‐PCR. Overexpression of miR‐142‐3p decreases cell viability, triggers caspase‐3 activation and decreases HSP70 expression.

Conclusions

Triptolide triggers the expression of miR‐142‐3p. Whether altered miR‐142‐3p expression may manipulate chemosensitivity to triptolide would be interesting to investigate.

This research is funded in part by the PharmacoNeuroImmunology Training Grant: T32 DA07097
Development of novel claudin‐4 binder and its application in mucosal vaccine
Authors: Yagi Kiyohito , Yamane Seiji , Suzuki Hidehiko , Watari Akihiro , Kondoh Masuo , Uchida Hiroshi
Published: June 2021

Mucosal vaccines activate both mucosal and systemic immune responses, but mucosal administration is needle‐free and less painful. Thus, mucosal vaccination appears to be an ideal vaccination strategy. The effective delivery of antigens to mucosa‐associated lymphoid tissues (MALT) is critical to the development of potent mucosal vaccines. We previously found that claudin‐4‐targeting is a promising strategy for mucosal vaccination using a claudin‐4 ligand, the C‐terminal fragment of Clostridium perfringens enterotoxin (C‐CPE). In the present study, we identified the functional domain of C‐CPE, and we attempted to prepare a novel claudin binder with higher affinity for claudin using the functional mapping information of C‐CPE. Substitution of some amino acids with alanine increased affinity of C‐CPE to claudin‐4, and Kd values decreased from 455 pM to 46 pM. To investigate mucosal vaccination using the mutant C‐CPE, ovalbumin (OVA) was used as a model antigen. When mice were immunized with the C‐CPE mutant‐fused antigen, antigen‐specific serum IgG and nasal IgA increased 10‐fold when compared with mice immunized using C‐CPE‐fused antigen. Immunization with the C‐CPE mutant‐fused antigen OVA activated Th1‐ and Th2‐type responses more strongly than in mice immunized with C‐CPE‐fused antigen. These results indicate that the alanine‐substituted C‐CPE mutant is a promising lead for claudin‐targeted mucosal vaccines.
Selection of Food and Vitamins during Pregnancy to Prevent Hypervitaminosis A and Congenital Defects
Authors: Raja Dewan Saiful , Sultana Bahar
Published: June 2021

Introduction

A pregnant woman consumes a variety of food. Pregnancy is a critical period of life. Pregnant women need more than a person does on the average diet. Vitamin A is essential for growth of the developing embryo and fetus. Supplementing with certain vitamins can prevent congenital defects. On the other hand, excessive consumption of certain vitamins may cause congenital defects. Vitamin A is a potential teratogen.

Method

Multiple literatures from Web sites and textbooks are explored, and the information is summarized.

Discussion

Vitamin‐rich and nutritious food are expected by pregnant women. There are multiple sources of vitamin A. Vitamin A derivatives are sometimes used to treat acne. Acne is common among young women. Many women do not know the source of vitamin A in their food. Vitamin and food supplement is a fashion among rich and educated pregnant women. Certain groups of pregnant women used to take previtamin A‐rich–food like liver without knowing the consequences of vitamin‐A–related congenital defects.

Conclusions

It is advised to avoid vitamin A–rich previtamin food like liver and cod‐liver oil to prevent possible congenital defects. Vitamin A–derivative like Accutane is contraindicated during pregnancy.
The persistent sciatic artery: A case report
Authors: Patel Jitendra , Shah RK , Nirvan AB , Patel PR , Trivedi BD
Published: June 2021

A case of persistent sciatic artery in an adult female cadaver is presented. The persistent sciatic artery (PSA) is a rare continuation of the internal iliac artery and the axial artery of the embryo which, provide the major supply to the lower limb. During routine dissection in Anatomy Department of Smt. NHL Municipal Medical College, a large caliber artery was found in the gluteal region of an adult female cadaver. The artery was then further dissected in order to trace its course, relations, communications and termination. The artery was then identified as persistent sciatic artery (PSA). In present case, the PSA entered the gluteal region through the greater sciatic foramen superficial to the sciatic nerve. In the mid thigh, the artery passed laterally to the sciatic nerve and then continued as popliteal artery in the popliteal fossa. Neither the femoral nor the profunda femoris artery had any communication with the popliteal artery. On the right side, no such artery was found. Here, we have reported a very rare case of a large complete persistent sciatic artery with the co‐existence of hypoplastic femoral and profunda femoris arteries on left lower limb and normal anatomical arterial pattern in right lower limb of an adult female cadaver.
The ATPase motor of the Chd1 chromatin remodeler is negatively regulated by a pair of N‐terminal chromodomains
Authors: Bowman Gregory D. , Hauk Glenn , McKnight Jeffrey , Nodelman Ilana
Published: June 2021

ATP‐dependent chromatin remodeling complexes play a central role in the assembly, disassembly, and repositioning of nucleosomes. While the remodeling reaction is driven by a conserved ATPase motor, it is not known how this motor is regulated by auxiliary domains. We have solved the crystal structure of the Chd1 chromodomains coupled to the ATPase motor, which suggests that the chromodomains negatively regulate the ATPase motor. In the crystal, a conserved acidic helical linker between the chromodomains contacts a DNA‐binding surface on the ATPase motor, blocking DNA access to the motor. Naked DNA is a poor substrate for the Chd1 ATPase, and we find that disruption of this chromodomain‐ATPase interface permits naked DNA to stimulate ATPase activity to the same extent as nucleosome substrates. Small‐angle X‐ray scattering (SAXS) experiments suggest that in solution the chromodomains are not rigidly fixed against the ATPase motor but instead may occupy conformations distinct from that observed in the crystal structure. We propose that the chromodomains allow Chd1 to distinguish between nucleosomes and naked DNA by physically gating access to the ATPase motor.

This work was supported by the NIGMS.
TNF‐α‐induced suppressor of cytokine signaling‐3 expression via the JNK1/2, ERK1/2, NADPH oxidase and AP‐1 in human tracheal smooth muscle cells
Authors: Hsu Chih‐Kai , Yang Chuen‐Mao
Published: June 2021

Suppressor of cytokine signaling‐3 (SOCS‐3) is an intracellular protein that involves in a wide range of biological processes. Several studies have indicated that SOCS‐3 not only down‐regulates the cytokine‐stimulated signalings, but also exerts anti‐inflammatory responses. However, whether proinflammatory cytokines such as TNF‐α can induce the expression of SOCS‐3 in human tracheal smooth muscle cells (HTSMCs) is still unclear. Here, we report that TNF‐α‐induced SOCS‐3 protein, mRNA expression and promoter activity was attenuated by pretreatment with the inhibitors of JNK1/2 (SP600125), p38 (SB202190), MEK1/2 (U0126), NADPH oxidase [diphenylene iodonium chloride (DPI) and apocynin (APO)], and reactive oxygen species (ROS) scavenger [N‐acetyl‐L‐cysteine (NAC)] or transfection with siRNA. TNF‐α‐stimulated NADPH oxidase/ROS production was attenuated by pretreatment with the inhibitor of JNK1/2, MEK1/2, or NADPH oxidase. The involvement of NF‐κB and AP‐1 in TNF‐α‐induced SOCS‐3 expression was confirmed by the inhibitors of NF‐κB (Bay11‐7082) and AP‐1 (Tanshinone) or transfection with respective siRNAs. Taken together, our results suggested that TNF‐α‐induced NADPH oxidase/ROS production was mediated through the JNK1/2‐ and ERK1/2‐dependent NADPH oxidase pathway, in turn activated AP‐1, and ultimately induced SOCS‐3 expression in HTSMCs.
Evaluation of Voltage‐Activated Calcium Channel α‐1G Subunit Expression in Genetic Absence Epilepsy Rats
Authors: AKIN DEMET , KETENCI SEMA , SIRVANCI SERAP , ONAT FILIZ
Published: June 2021

T‐type voltage‐activated calcium channels play an essential role in the generation of absence seizures. It is well known that thalamocortical circuit is critically involved in the propagation of spontaneous spike and wave discharges (SWDs) in Genetic absence epilepsy rats from Strasbourg (GAERS).

Calcium channel α‐1G subunit expression was studied by immunohistochemical methods in the hippocampus (CA1, CA3, dentate gyrus), lateral and medial thalamic regions, globus pallidus and striatum of control Wistar rats (n=5) and GAERS (n=4). The brains were removed after perfusion and brain slices (40 mm) were incubated with antibodies against calcium channel α‐1G subunit. Peroxidase activity was visualized by 0.03 % 3‐3‐diaminobenzidine. We used semi‐quantitative scoring and analyzed the data with unpaired t test.

We found decreased α‐1G subunit immunolabeling in the lateral thalamic region of GAERS animals compared with control Wistar rats. No difference was observed in any of the other areas between Wistar rats and GAERS.

These results show that decreased calcium channel α‐1G subunit expression in lateral thalamic regions may play a role in the mechanisms underlying absence seizures. Further experiments will be performed to investigate the expression of other calcium channel subunits.

Supports: Istanbul Bilim University and Marmara University S.R.C.SAG‐A 090409‐0072
Chronic oxycodone exposure alters translational and signaling pathways in the rat brain stem
Authors: Korneeva Nadejda L , Schrott Lisa M
Published: June 2021

Oxycodone is a prescription opioid that is widely used because of its substantial analgesic efficacy. However, it is accompanied by numerous side effects such as nausea, constipation, mioisis and respiratory depression, which can decrease patient compliance with treatment, affect quality of life and potentially lead to coma or death. These adverse effects are mediated by neurons in brain stem nuclei. Understanding changes in gene expression and signaling following chronic exposure will be helpful in developing therapeutic strategies to mitigate these effects. We have analyzed the effect of chronic oxycodone exposure on MAPK and mTOR as well as translational pathways in rat brain stem. We observed increase in phosphorylation of p38 and mTOR but not ERK1/2 kinases by oxycodone. Since p38 kinase is activated in response to cytokines or stress, our results suggest that oxycodone exposure triggers the cellular response to chronic stress. Analysis of translational regulation in rat brain stem revealed both stimulatory and inhibitory effects of chronic oxycodone exposure on various steps of translation. The possible mechanism of translational regulation of specific mRNAs by chronic oxycodone exposure in rat brain stem will be discussed. Supported by Department of Emergency Medicine and Department of Pharmacology, Toxicology and Neuroscience, Louisiana State University Health Sciences Center; Shreveport.
SIRT2 inhibitors can induce significant increases in the gene expression of pro‐inflammatory factors in primary astrocyte cultures
Authors: Han Jin , Chen Heyu , Shao Jiaxiang , Nie Hui , Xia Weiliang , Ying Weihai
Published: June 2021

Multiple biological functions of SIRT2 have been reported, including affecting cell mitosis, cell motility, and oligodendrocyte differentiation. SIRT2 inhibitors have also been shown to reduce alpha‐synuclein‐induced neurotoxicity in cellular and Drosophila models of Parkinson's disease. However, there has been no study regarding the role of SIRT2 in neuroinflammation. In this study we determined the effects of SIRT2 inhibition on the gene expression of proinflammatory factors in primary murine astrocyte cultures. We used real‐time PCR assay to show that treatment of astrocytes with 10 micromolar AGK2 ‐‐‐ a selective SIRT2 inhibitor ‐‐‐ for 6 hours induced significant increases in the gene expression of pro‐inflammatory factors: The mRNA levels of TNF‐alpha, iNOS and IL‐6 were increased by 2–11 folds in the AGK2‐treated cells compared with controls. These observations suggest that SIRT2 could be an inhibitory factor of neuroinflammation, and a decrease in SIRT2 levels may lead to neuoinflammation in the brain. Due to the significant roles of neuroinflammation in multiple neurological diseases, our study has suggested that SIRT2 may become a therapeutic target for neurological diseases due to its effects on neuroinflammation (supported by a National Key Basic Research ‘973 Program Grant’ #2010CB834306, a Pujiang Scholar Program Award 09PJ1405900, and a Key Research Grant of Shanghai Municipal Educational Committee #09ZZ21).
A novel POK protein, ZNF499, induces BAX gene expression with apoptosis
Authors: Kim Min‐Young , Lee Kyung‐Mi , Hur Man‐Wook
Published: June 2021

The BTB‐POZ domain containing POK proteins regulate a variety of cellular events such as oncogenesis, cell cycle, differentiation, and development. The functions of most POK proteins are mostly uncharacterized. In this study, we isolated a new POK protein, ZNF499 (also called ZBTB45) by screening POK proteins with a potential of regulating the p53 pathway. We hypothesized that ZNF499 may activate genes of the p53 pathway. FACS data showed that ZNF499 induced G2‐M cell cycle arrest and inhibited cell proliferation. Quantitative PCR showed that ZNF499 activates p53 pathway genes such as p53, p21, BAX. Particularly, ZNF499 has an effect on transcription of p53 and BAX. Activation of BAX gene increased by ZNF 499 depends on the p53 which is also activated by ZNF499. Loss of function study of ZNF499 showed that ZNF 499 increases expression of apoptotic genes, p53 and BAX, not PUMA. Overall, our data suggested that ZNF499 is a potent transcription activator of BAX.
Early Heparin Administration Increases Plasma Tissue Factor Pathway Inhibitor and Attenuates Tissue Factor Mediated Thrombin Generation During Simulated Cardiopulmonary Bypass
Authors: Morizumi Sei , Hiramatsu Yuji , Matsuzaki Kanji , Sato Shoko , Abe Masakazu , Sakakibara Yuzuru , Matsushita Shonosuke
Published: June 2021

Background

We tested the hypothesis that early treatment with heparin prior to the emergence of tissue factor (TF) would boost plasma tissue factor pathway inhibitor (TFPI) levels and attenuate TF mediated thrombin generation during simulated cardiopulmonary bypass (CPB).

Methods

Four study groups were examined: Control group (heparin 3.75 U/ml blood, n=7), rTF group (heparin + recombinant human TF 1000 pg/ml, n=7), TFPI boost group (heparin, in pre‐heparinized donor blood, n=8), and TFPI boost + rTF group (heparin + rTF, in pre‐heparinized donor blood, n=7). In the two TFPI boost groups, 50U/kg of heparin was given to the donors intravenously five minutes before donation to elevate plasma TFPI levels.

Results

Pre‐heparinization increased total plasma TFPI levels by a factor of 8.0 when compared to non‐heparinized donor blood. Administration of rTF significantly enhanced the generation of prothrombin fragment (F1 {2) (p<0.0001). The boost of TFPI reduced F1+2 to Control group levels in TFPI boost + rTF group (p=0.0178), and in TFPI boost group (p<0.0001 ).

Conclusions

Heparin induced TFPI elevation results in attenuation of tissue factor mediated thrombin generation during simulated CPB. Early heparin administration prior to emergence of plasma TF may represent a novel strategy for the control of thrombin generation initiated by extrinsic coagulation pathway during CPB.
Novel Mechanisms Contributing to Fibrosis and Matrix Remodeling in Myxomatous Mitral Valve Disease
Authors: Hadley Thomas M , Hagler Michael A , Schaff Hartzell V , Suri Rakesh M , Miller Jordan D
Published: June 2021

Myxomatous mitral valve disease is characterized by fibrosis and matrix degradation and increases in transforming growth factor beta‐1 (TGFbeta‐1). Little is known, however, about changes in molecules that regulate TGFbeta‐1 signaling in myxomatous valves. Thus, we tested the hypothesis that TGFbeta‐1 promotes valvular fibrosis and matrix remodeling in myxomatous valves through canonical Smad2 signaling and permissive reductions in Smad inhibitors. Expression of TGFbeta‐1, TGFbeta‐1 target genes (connective tissue growth factor, matrix metalloproteinases 2/9), E3 ubiquitin‐protein ligases (Smurf 1/2, which degrade Smad2), and NAD(P)H oxidase catalytic subunits (Nox 2, 4, and 5) was measured using quantitative real‐time RT‐PCR. Phospho‐Smad2 levels were measured using western blotting. TGFbeta‐1 expression was higher in myxomatous valves (138.3 ± 12.5% of control, n = 24 per group; p < 0.05), while CTGF and MMP2 were increased by 5‐ and 2‐fold, respectively. Surprisingly, phospho‐Smad2 levels were unchanged between myxomatous and control tissue due to selective increases in Smurf2 expression in myxomatous valve tissue (165.2 ± 17.7% of control, p < 0.05), as Smurf1 expression was unchanged. Expression of Nox2 and Nox4—which can drive pro‐fibrotic and matrix remodeling gene expression in other tissues—were significantly elevated in myxomatous valves (211 ± 44% and 224 ± 35 % of control, respectively; p < 0.05). In conclusion, our data suggest that canonical TGFbeta‐1/Smad2 signaling is not required for progression of myxomatous valve disease, and that reducing Nox2‐and Nox4‐derived reactive oxygen species may be a novel intervention to slow progression of fibrosis and matrix remodeling in myxomatous valves.
Role of sex in telomere attrition with advancing age in human arteries
Authors: Morgan Garrett , Ives Stephen J , Lesniewski Lisa A , Andtbacka Robert H.I. , Noyes R. Dirk , Richardson Russell S. , Donato Anthony J.
Published: June 2021

Arterial aging is an important risk factor for cardiovascular disease. Telomeres are non‐coding DNA repeats that protect coding DNA and shorten with each cell cycle in all mitotic tissues. Telomere length progressively declines over time in human leukocytes, but little is known regarding human arteries. Sixty‐two human arteries (~425 μM) were obtained during outpatient surgeries (35 males; 24–93yrs, 27 females; 21–79yrs), dissected and total genomic DNA extracted. Mean telomere length (mTL) was negatively correlated with age (r= −0.38, p< 0.01), as assessed by multiplex qPCR telomere length assay. The mTL for subjects in their 20's was 11% greater than those in their 30's, 31% greater than those in their 40's, 39% greater than those in their 50's (p= 0.03), 34% greater than those in their 60's (p= 0.07), and 56% greater than those in their 70's (p= 0.01). Age was a better predictor of mTL in women than in men (r= −0.58, p<0.01 and r=−0.27, p= 0.05, respectively). The predicted rate of decline in mTL was 9% per decade in all subjects, with a predicted decline of 12% in women and 7% in men. Furthermore, the predicted decline was 41% lower than that predicted from studies in human leukocytes using the same mTL assay. These results demonstrate that telomere length declines progressively with advancing age in human arteries and suggest that telomere length declines faster in women than in men.

Support: NIA‐AG029337, NHLBI‐HL09183
Regulation of Endothelial Nitric Oxide Production By Protein Methyltransferase's
Authors: Han Zhaosheng , Druhan Lawrence J , Cardounel Arturo J , Chicoine Louis G
Published: June 2021

Altered nitric oxide (NO) biosynthesis has been implicated in the pathogenesis of cardiovascular disease and evidence from clinical studies suggest that accumulation of endogenous nitric oxide synthase (NOS) inhibitors, asymmetric dimethylarginine (ADMA) and NG‐monomethy arginine (NMMA) contribute to reduced NO generation by competing with the substrate, L‐arginine for NOS. Free ADMA and L‐NMMA are derived from the proteolysis of methylated arginine residues on various proteins. The methylation is carried out by a group of enzymes referred to as protein‐arginine methyl transferase's (PRMT's). Recent studies from our laboratories demonstrate that in addition to generating free methylarginines, PRMT's can directly regulate NO production via direct methylation of eNOS protein. Incubation of purified eNOS (20 μg + cofactors) with PRMT1 (1 μg) and the methyl donor S‐adenosylmethionine (10 μM) demonstrated direct methylation of NOS and a 72% reduction in its catalytic activity. Consistent with these findings, pharmacological and molecular approaches to reduce endothelial PRMT1 activity resulted in a dose and time‐dependent increase in endothelial NO production with a greater than 50% increase observed at 48 hours post‐treatment. Results from these studies implicate PRMT‐dependent methylation of eNOS as a novel post‐translational modification involved in NO regulation and endothelial function.
Chronic effects of angiotensin II on NHE3 promoter involve several intracellular signaling pathways and Sp1/Egr‐1 binding site
Authors: Queiroz‐Leite Gabriella Duarte , Rebouças Nancy Amaral
Published: June 2021

Previous studies have shown that 24h exposure to 10−11 M angiotensin II (AII) stimulated NHE3 activity and increased NHE3 protein content and mRNA levels in OKP cells. The purpose of this study was to verify the effects of AII on the NHE3 gene promoter activity and its regulatory mechanisms. OKP cells were used in transient transfection assays. Four fragments of NHE3 promoter cloned into a vector containing the reporter gene Firefly luciferase were studied (−2095/+55, −152/+55, −85/+31 and −65/+31). The promoter activity of the fragment −65/+31 was significantly increased after 24h‐treatment with 10−11M AII. This increase was abolished by pretreatment with losartan (AT1R inhibitor). Two mutations were performed in this fragment. The mutation at the Sp1/Egr‐1 binding site completely abolishes the AII stimulatory effect, whereas the mutation at the AP2 binding site partially revert this effect. In order to verify the mechanisms involved in this stimulatory response, we used intracellular signaling pathways inhibitors. Our results showed that cytochrome P450, PI3K, PKA and MAPK pathways are involved in this process, while PLC and JAK/STAT pathways do not participate. These data reveal that AII chronic stimulatory effects on NHE3 proximal promoter occur through AT1R and are dependent on the Sp1/Egr‐1 binding site integrity. This response involves activation of different intracellular signaling pathways.

Financial Support: FAPESP and CNPq
Gene trapping uncovers gender‐specific mechanisms for upstream stimulatory factors 1 and 2 in angiotensinogen expression
Authors: Park Sungmi , Liu Xuebo , Davis Deborah R. , Sigmund Curt D.
Published: June 2021

A single‐nucleotide polymorphism (C/A) located within an E‐box sequence at the −20 position of the human angiotensinogen (AGT) promoter may regulate AGT transcriptional activation through differential recruitment of the transcription factors Upstream Stimulatory Factor (USF) 1 and 2. To study the contribution of USF1 vs USF2 binding to AGT gene expression, mice carrying a (−20C) human AGT transgene (hAGT) were bred with mice harboring a USF1 gene trap designed to knockdown USF1 expression [USF1Gt (XG830) Byg]. USF1 mRNA was significantly reduced in liver (9±1%), perigenital adipose (16±3%), kidney (17±1%), and brain (34±2%) in double‐transgenic mice of both genders. This decrease was confirmed by electrophoretic mobility shift assay. Quantitative chromatin immunoprecipitation (ChIP) analyses revealed a decrease in USF1 binding (2.2±0.6 vs. 0.9±0.1 fold of IgG) with retention of USF2 binding (7.2±0.4 vs. 5.9±0.5 fold of IgG) at the hAGT promoter in the liver of male liver. Surprisingly, hAGT expression was reduced in liver of female (45±6%) but not male mice (113%±3%). Treatment of USF1 knockdown males with intravenous adenoviral shRNA targeting USF2 resulted in reduced expression of USF1, USF2 and hAGT protein. In conclusion, both USF1 and USF2 are essential for AGT transcriptional regulation, and distinct gender‐specific mechanisms are involved in the activities of these transcription factors in vivo.
Bimodal mechanism of mRNA association with the endoplasmic reticulum (ER)
Authors: Jagannathan Sujatha , Chen Qiang , Reid David , Nicchitta Christopher
Published: June 2021

mRNA localization is vital for the spatiotemporal control of cellular protein synthesis. Partitioning mRNAs between cytosol and ER is the most prominent mRNA localization process in cells, operating on the entire mRNA transcriptome. mRNAs encoding secretory pathway proteins are recruited to the ER membrane via the signal recognition particle (SRP) pathway, where mRNA binding to ER requires a translating ribosome. Previously, we have shown that mRNAs encoding ER‐resident proteins display ribosome‐independent interactions with ER, suggesting multiple pathways to regulate mRNA localization to ER. Here, we subjected microsomes derived from plasmacytoma cells to high salt/EDTA, which releases membrane‐bound ribosomes. We then analyzed the fraction of mRNA released to assess the interactions of candidate mRNAs encoding either ER resident proteins (mRNARes) or secretory pathway cargo proteins (mRNACargo) to the ER. While mRNARes are resistant to extraction by high salt/EDTA, mRNACargo are readily released. These results indicate that at least two modes of mRNA attachment to the ER are possible, and can be distinguished by the requirement for intact ribosome. As both cohorts of mRNAs encode proteins with N‐terminal signal peptide, these findings suggest that the SRP pathway may serve a primary function in directing mRNAcar to ER, while additional mechanisms could direct ER localization of mRNARes. NIH GM077382.
LOW INTENSITY RESISTANCE TRAINING IMPROVES METABOLIC PROFILE OF DIABETIC RATS
Authors: Macedo Fabricio N , Barreto André Sales , Mota Marcelo Mendonça , dos Santos Soares Ana Paula , Fontes Milene Tavares , da Silva Tharciano Luiz Teixeira Braga , de Melquiades Neto José Rezende , dos Santos Márcio Roberto Viana
Published: June 2021

The aim of this study was to evaluate the effects of RT on glucose, lipids, urea, creatinine, PCRhs, fructosamine and blood TBARS, and mass indexes heart, liver and muscle in diabetic rats induced by alloxan (40 mg/kg, iv). For all experiments we used male Wistar rats weighing 200–300g. The animals were divided into 3 groups (n=8 for each group), sedentary control (SC), sedentary diabetic (SD) and trained diabetic (TD) rats. The RT was performed in a squat‐training apparatus using 3x10 repetitions, 3 times a week for 8 weeks After measurement of the maximum weight lifted (1RM) the training load was set at 40% of 1RM and adjusted each 15 days. At the end of the RT protocol or time control, the animals were anesthetized with sodium thiopental (45 mg/kg i.p.) and sample blood and tissues were obtained. The RT was able to reduce blood glucose (from 381 ± 42 to 270 ± 17 mg/dL), plasma TBARS (1.4 ± 0.02 to 0.2 ± 0.05 nmol/ml) and liver mass index (0.0467 ± 0.0036 to 0.0391 ± 0.0034), and to increase HDL (from 16.25 ± 2.25 to 36.6 ± 6.7) and plantar muscle mass index (from 0.00295 ± 0.00026 to 0.00377 ± 0.00005) in relation to the SD. These results demonstrate that the low intensity RT is a useful non‐pharmacological intervention to treatment of metabolic complications from diabetes. Research Support: CAPES, Fapitec‐SE.
The effect of hyperglycemia on Achilles tendon structural properties in the rat
Authors: Moore M Scott , Gump Brian , Tedeschi Jamie , Peterson Amity , Kim Paul J , Broderick Thomas , Carroll Chad C
Published: June 2021

Diabetes is often associated with chronic foot ulcers leading to limb amputations. Ulcerations appear to develop, in part, because of increased pressures on the plantar aspect of the foot due to an equinus deformity (limitation of ankle joint dorsiflexion). Equinus deformity may be due to changes in the elastic properties of the Achilles tendon. Elevated blood glucose levels result in glycation induced collagen cross‐linking and studies of rat tail tendon suggest that hyperglycemia alters the content of tendon collagen and normal cross‐links. To our knowledge collagen content has not been assessed in the Achilles tendon of diabetic rats. Twenty male Sprague‐Dawley rats (8 weeks) were randomly assigned to control (n=7) or streptozotocin‐induced diabetic (n=11, 55 mg•kg body weight) groups. After 10 weeks tendons were evaluated for collagen content via HPLC. Weight gained was higher in the control rats (control: 162.7±14.9 g, diabetic: 62.3±10.1 g). Hyperglycemia did not alter the content of collagen in the Achilles tendon (control: 305.3±14.9, diabetic: 301.4±18.9 μg collagen/mg wet weight and control: 682.3±31.7 and 692.2±58.0 μg collagen/mg dry weight). It does not appear that changes in total collagen can explain the functional changes noted in the Achilles tendon of diabetics. Although total collagen was unaffected additional studies of cross‐link content and collagen I to III ratios are warranted.

Funding: Midwestern University
Measuring drug effects on exercise endurance under extreme environmental conditions in rats using motorized wheels
Authors: Radiloff Daniel R. , Wu Chan , Zhao Yulin , Hanna Gabi , Irwin David , Hamilton Karyn , Schroeder Thies
Published: June 2021

Objectives

Exercise capacity in laboratory animals is typically measured using treadmills which require large spatial allocation and have poor habituation efficacy, making it unsuitable for high throughput application. We present a novel rat exercise system involving motorized rodent wheels that has high habituation efficiency, and due to small spatial allocation allows for high throughput experimentation.

Methods

Female Sprague Dawley rats were habituated to maintain position in motorized wheels at low speed for 10 min daily. Wheel speed was increased incrementally over 10 days. On testing days, animals received i.p. injections of nifedipine (40 mpk), or iron sucrose (5 x 1 mpk daily) or vehicle. Animals were then exposed to escalating exercise burden under hypoxia. Exhaust signs included sliding, hanging, tail position, and time until self motivated movement after removal from wheel.

Results

>90% of rats ran proficiently within 10 days of habituation. Nifedipine significantly decreased time run to exhaust. Iron sucrose on the other hand significantly enhanced exercise performance under hypoxia vs. control.

Conclusion

We established a novel, ergonomic, and efficient system to measure exercise capacity in rats under hypoxia for high throughput experimentation. This system has application in various disciplines, including exercise physiology, environmental medicine, and drug development.
Inflammation and muscle damage markers of football athletes during heavy physical training with DHA supplementation
Authors: Dobson Justin P , Oliver Jonathan M , Riechman Steven E , Crouse Stephen F
Published: June 2021

Docosahexaenoic acid (DHA) is a C22:6 omega‐3 fatty acid that has been shown to reduce inflammation. Our study examined serum inflammatory (GM‐CSF, IL‐6, IL‐8, TNF‐α, interferon‐γ, CRP) and muscle damage (creatine kinase, LDH, ALT, AST, alkaline phosphatase) markers following 2 g/d of supplemented, purified algal DHA (E;n=24) or placebo (P;n=28) in American collegiate football athletes during preseason training. Fasted blood was drawn three different times: 1) Summer (S) before starting supplements (T1); 2) August, pre‐camp (PRE) after 35 d supplementation (T2); and 3) August, post‐camp (POST) after 52 d supplementation (T3). A 2x3 ANCOVA with repeated measures (covaried for race; CRP for weight change). A significant group x time interaction was observed in IL‐8 (p<0.05). Other markers of inflammation showed no differences between groups (p>0.05). POST CRP was significantly higher than PRE (p<0.001). POST levels of creatine kinase, LDH, ALT, AST, alkaline phosphatase were significantly (p<0.001) increased compared to S and PRE in both groups. Preseason training can elevate muscle damage markers. DHA supplementation did affect IL‐8 levels during preseason training of collegiate football athletes.

Funding provided by Martek Biosciences Corp., Columbia, MD and The Sydney & J.L. Huffines Institute for Sports Medicine & Human Performance
Effect of exercise training on vascular reactivity in inbred strains of mice
Authors: Kim Seung Kyum , Avila Joshua , Massett Michael Peter
Published: June 2021

The aim of this study was to characterize the strain‐dependent differences in vascular reactivity in aorta from sedentary and exercise‐trained mice. Male mice (n ≈ 8/strain) from 5 inbred strains (129S1, 129X1, C57BL/6J, FVB, PWD) were assigned to either sedentary or exercise training (EX) groups. EX mice completed a 4‐wk treadmill running protocol (5 days/wk, 60 min/day, 60% of max speed). After training, aortic rings were suspended for isometric tension recording. Exercise training had no effect on vascular reactivity in any strain. Significant strain effects were found for maximal responses to phenylephrine (PE) and potassium chloride (KCl). For each agent, aorta from PWD had the lowest (PE = 5.4 ± 2.0 mN, KCl = 12.0 ± 2.6 mN) and aortas from 129‐substrains had the highest (PE = 16.4 ± 3.5 mN, KCl = 17.9 ± 3.3 mN) responses. Relaxation responses to acetylcholine were also different across strains, with aortas from the 129‐substrains having the lowest (73.0 ± 16.8%) and aorta from the FVB having the highest (90.6 ± 12.6%) responses. Relaxation responses to sodium nitroprusside were not different across strains. Strain‐dependent differences in sensitivity were observed for all agents tested. The large difference in maximal responses and sensitivity to vascular agents across mouse strains suggests that genetic background significantly affects vascular responsiveness.

Supported by NIH grant HL085918 to MPM
The mechanism of L‐Glutamine‐mediated protection requires the activation of cell surface receptors
Authors: Niederlechner Stefanie , Baird Christine , Wischmeyer Paul
Published: June 2021

Introduction

L‐Glutamine (GLN), integrin/Src and epidermal growth factor receptor (EGFR) pathway can induce cellular protection.

Hypothesis

GLN activates the O‐glycosylation pathway, HSF‐1 and HSP expression via cell swelling by the activation of integrin/Src and EGFR signaling to prevent apoptosis.

Methods

IEC‐6 cells treated with GLN (up to 20 mM), with or without the integrin inhibitor GRGDSP (50 μM), inactive control peptide GRGESP (50 μM), Src‐kinase inhibitor PP‐2 (75 μM), or EGFR inhibitor AG1478 (10 μM). Cell survival via MTS assay 24h post‐HS (44ºCx50min). O‐glycosylated proteins, HSF‐1, HSPs, Bax, cleaved caspase‐3 and PARP levels determined via Western blot, cell area size via fluorescence microscopy after non‐lethal HS (43ºCx45min).

Results

GRGDSP significantly attenuated GLN‐mediated increases in cell area size, HSF‐1 and O‐GlcNAc levels after HS (p< 0.05; n>3). GLN‐mediated increases in HSPs were decreased by GRGDSP and PP2 by 70%–100% after HS (p< 0.05; n=4). GRGDSP, PP2, and AG1478 attenuated GLN's protection by 70%–100% (p< 0.05; n=4). GRGDSP increased GLN‐mediated decreases in cleaved caspase‐3, cleaved PARP and Bax levels after HS, indicating that GLN lost its protection (p<0.05; n=3). GRGESP control peptide had no effect on GLN‐mediated protection pathways.

Conclusion

Integrin/Src and EGFR signaling seem to be vital components of GLN's protective mechanism. R01 GM078312
p300 acetyltransferase activity differentially regulates the localization and activity of the FOXO homologues in skeletal muscle
Authors: Senf Sarah Marie , Sandesara Pooja B. , Judge Andrew Robert
Published: June 2021

The Forkhead Box O (FOXO) transcription factors are both necessary and sufficient for skeletal muscle atrophy. Therefore understanding the mechanisms of FOXO regulation in skeletal muscle is of significant importance. The objective of the current study was to determine if FOXO signaling is regulated in skeletal muscle via the acetyltransferase (HAT) activities of p300/CBP. Gene transfer of rat soleus muscle with a dominant‐negative (d.n.) p300, which lacks HAT activity and inhibits endogenous p300 HAT activity, increased FOXO reporter activity and induced transcription from the promoter of the atrophy‐related gene, atrogin‐1. Conversely, gene transfer of either WT p300 or WT CBP repressed FOXO activation and atrogin‐1 in response to muscle disuse, and in C2C12 cells in response to acute starvation. Co‐transfection of FOXO1, FOXO3a or FOXO4 with the p300 constructs further identified p300 HAT activity to differentially regulate the activity of the FOXO factors. Decreased HAT activity potently increased FOXO3a transcriptional activity, while increased HAT activity repressed FOXO3a activity and prevented its nuclear localization in response to nutrient deprivation. In contrast, p300 increased FOXO1 nuclear localization. In summary, this study provides the first evidence to support the acetyltransferase activities of p300/CBP in regulating FOXO signaling in skeletal muscle.
FoxO1‐mediated disruption of the Akt/mTOR signaling pathway in skeletal myotubes
Authors: DeLong Alissa D , Pratt Rachael , McLoughlin Thomas J
Published: June 2021

Our previous research has demonstrated that FoxO1 blunts skeletal muscle hypertrophy in vivo and this blunting is mediated, in part, by suppression of mTOR signaling. The aim of this research is to elucidate potential mechanisms involved in FoxO1‐mediated regulation of skeletal muscle hypertrophy. C2C12 mouse skeletal muscle cells that have been stably transfected with an empty control or a tamoxifen inducible vector to promote FoxO1 activation were grown into mature myotubes. Myotubes were exposed to media supplemented with 10 mM leucine (purported activator of mTOR) and tamoxifen for 30 min, 1, 2, and 4 hrs and analyzed for phosphorylated Akt (S473) and p70s6k (T389) by western blot. Our findings show that i) FoxO1 activation dramatically increases Akt phosphorylation compared to control cells, ii) leucine, a purported specific activator of mTOR, amplified Akt phosphorylation in both cell types across the time course and iii) despite the dramatic amplification in Akt activity observed with FoxO1 activation, this signaling was not transferred downstream as phosphorylation of p70s6k appeared muted in both untreated and leucine treated conditions. This preliminary data suggests that FoxO1 activation results in a disconnect between Akt signaling and activation of downstream targets in skeletal myotubes. Research is ongoing to elucidate the mechanism by which this disconnect in signaling is manifested by FoxO1. This research was supported by an ACSM Foundation Research Grant from the American College of Sports Medicine Foundation.
Ontogenetic expression of connexins by mouse pre‐Bötzinger complex neurons
Authors: Kelty Jonathan , Cornillie Sean
Published: June 2021

Current evidence is inconclusive concerning the potential importance of interneuronal coupling via gap junctions in the generation/patterning of rhythmic activity by an inspiration‐related network in the medulla's pre‐Bötzinger complex. Accordingly, this study used immunohistochemistry to examine the ontogenetic expression of connexins (Cx), one of the types of proteins from which gap junctions are formed, in the mouse pre‐Bötzinger complex. Specifically, we examine the distribution of Cx 26, 32, 36, 45 and 47. Our data demonstrate that neurons in the region containing the pre‐Bötzinger complex express some of these proteins at various points during pre‐natal and post‐natal development.
Angiotensin AT1 receptor subtypes AT1A and AT1B mRNAs are expressed in tyrosine hydroxylase immunoreactive (TH‐ir) neurons in the rat caudal nucleus of the solitary tract (NTS)
Authors: Bathina Chandra , Nedungadi Thekkethil , Cunningham J. T. , Mifflin Steve
Published: June 2021

Identifying the gene expression profiles in cells in the caudal NTS is a major challenge due to the heterogeneity of cellular populations in this region. We utilized quick immunohistochemical labeling coupled with laser capture microdissection to identify and capture TH‐ir neurons from the caudal NTS. Six groups of 10–13 TH‐ir caudal NTS neurons were captured from adult, male Sprague‐Dawley rats and total RNA was extracted from each group. mRNA levels of TH and the angiotensin II receptor subtypes AT1A and AT1B were assessed by quantitative real time reverse transcription polymerase chain reaction using GAPDH as the housekeeping gene. The 2−ΔCt for TH averaged .171 ± .028. mRNA of both AT1A and AT1B subtypes of the angiotensin AT1 receptor was expressed in the TH neurons. The 2−ΔCt for AT1A and AT1B averaged .023 ± .004 and .025 ± .003 respectively. Our data show the existence of AT1A and AT1B receptor subtype mRNAs in catecholaminergic neurons in the caudal NTS.
Activity in neurons from the nodose ganglia after treatment with CP 55,940 and cholecystokinin
Authors: Rose Juliane M , Freeman Kimberly G , Edwards Gaylen L
Published: June 2021

Previous work has shown that endocannabinoids increase feeding, while cholecystokinin (CCK) has an anorexigenic effect on food intake. Receptors for these hormones are located on cell bodies of vagal afferent nerves in the nodose ganglia. An interaction of CCK with endocannabinoid receptors has been suggested (J. Neurosci. 24:2708, 2004). The purpose of these studies is to explore the effect of pretreatment with a cannabinoid agonist, CP 55,940 (CP), on nodose neuron activation by CCK. To determine the effect of CP and CCK on neuron activation, rats were anesthetized and nodose ganglia were extirpated. The neurons were dissociated and plated. The cells were treated with media, CP, CCK, or CP followed by CCK. Immunohistochemistry was performed to stain the cells for cFos as a measure of cell activation. Neurons were identified using neurofilament immunoreactivity. The neurons on each slip were counted using fluorescence imaging, and the number of neurons that were c‐Fos and neurofilament positive were counted. Pretreatment with CP 55,940 decreased the percentage of neurons expressing cFos‐immunreactivity in response to CCK. This observation suggests that endocannabinoids inhibit CCK activation of nodose ganglion neurons. (Supported by Univ of Georgia Research Foundation and College of Veterinary Medicine)