bluuebunny/crossref_metadata_2025 huggingface dataset:
Microdialysis delivery of morphine sulfate to rat pontine reticular formation (PRF) decreases PRF adenosine levels
Authors: Nelson Ariana Marie , Ignasiak Diane , Baghdoyan Helen A. , Lydic Ralph
Published: June 2021

Opioids disrupt sleep even in the absence of pain and sleep disruption intensifies the perception of pain. The essential role of opioids in pain management encourages development of adjunctive therapies that preserve analgesia while avoiding the unwanted side effect of sleep disruption. Delivery of adenosine agonists to the PRF promotes sleep and analgesia (Anesthesiology 103: 1268, 2005) but no studies have determined whether opioids alter endogenous adenosine levels in the PRF. This study is testing the hypothesis that microdialysis delivery of morphine to rat PRF causes a concentration dependent decrease in PRF adenosine levels. During isoflurane anesthesia, the PRF of adult male Sprague‐Dawley rats (n=5) was dialyzed with Ringer.s solution containing 100 μM morphine. Compared to Ringer.s alone, morphine caused a significant (p<0.05) decrease in adenosine levels. Studies of additional rats (n=5) determined that co‐administration of naloxone (10 μM) prevented the decrease in adenosine caused by morphine. These results and ongoing concentration response studies support the interpretation that morphine decreases adenosine levels in a receptor‐dependent manner in the PRF, a brain region known to regulate sleep and nociception.

Supported by the National Institute of Health grants HL57120, HL40881, MH45361, and the U. of Michigan Department of Anesthesiology and Medical School Office of Student Programs.
Quantification of muscle lipid kinetics in obese and lean rabbits
Authors: Zhang Xiao‐jun , Chinkes David L , Aarsland Asle , Herndon David N , Wolfe Robert R
Published: June 2021

We compared lipid kinetics in obese and lean muscle in rabbits. A novel mathematical model was used to quantify muscle lipid kinetics by infusing U‐13C16‐palmitate and using palmitoyl‐carnitine as the precursor for lipid synthesis and oxidation, as we previously found that the palmitoyl‐carnitine enrichment is in equilibrium with that of palmitoyl‐CoA. Young rabbits were fed either a high‐fat diet (n = 6) or restricted food (n = 4). After 10‐weeks feeding the obese rabbits had greater body weight than the lean counterparts (5.3 ± 0.1 vs. 3.9 ± 0.2 kg, p<0.001). The tracer was infused for 8 h under general anesthesia; blood and leg muscle were sampled. Triglyceride (TG) content was greater in obese than in lean muscle (36.2 ± 2.8 vs. 8.8 ± 4.0 μmol /g, p<0.01), whereas muscle phospholipid (PL) contents were similar (2.1 ± 0.9 vs. 1.7 ± 0.7 μmol/g, p = 0.21). TG synthesis and oxidation rates in obese muscle were 0.0529 ± 0.0416 and 0.0582 ± 0.0345 μmol/g/h, which were greater (p<0.01) than the corresponding values of 0.0114 ± 0.0083 and 0.0098 ± 0.0039 μmol/g/h in lean muscle. In contrast, the synthetic rates of muscle PL were close (0.0141 ± 0.0058 vs. 0.0109 ± 0.0036 μmol/g/h, p = 0.22). In summary, the obese muscle contained more TG and comparable PL in comparison with lean muscle. The obese muscle had faster rates of TG synthesis and oxidation, suggesting that it consumed more lipid. This new model could be useful in the study of obesity.
Dietary kudzu root extract supplementation improves glucose tolerance and plasma lipid profiles in Spontaneously Hypertensive Rats (SHR) fed a basal, but not high, NaCl diet
Authors: Peng Ning , Prasain Jeevan K , Dai Yanying , Carlson Scott H , Wyss J. Micheal
Published: June 2021

The extract of Pueraria lobota (kudzu) root improves cardiovascular function and reduces mortality and morbidity in estrogen‐depleted female SHR. In the present study we tested the hypothesis that chronic dietary kudzu root extract also improves glucose handling and lipid profiles in this model. Female SHR were ovariectomized and maintained on a polyphenol‐free 1% NaCl diet with or without the addition of 0.2% kudzu root extract for 3 months. Thereafter, glucose tolerance was measured and plasma collected for lipid profiling. Dietary kudzu root extract significantly improved plasma glucose responses to the oral glucose challenge. The kudzu extract also lowered levels of APO‐E bound HDL and reduced the bound/unbound HDL ratio. In rats, HDL is the prominent cholesterol, and binding with APO‐E targets HDL for degradation. In contrast, in SHR fed a high (4%) NaCl diet the kudzu extract had no effect on glucose handling, although baseline glucose levels were significantly lower in the kudzu fed group. The high salt diet similarly increased all lipids to about the same level in treated and control rats, although plasma VLDL levels were lower in the kudzu group. These data suggest that chronic dietary kudzu supplementation exerts beneficial effects on glucose handling and lipid profiles in estrogen‐depleted female SHR on a basal salt diet. However, the high salt diet appears to eliminate the positive effects of dietary kudzu.
Impairment of Baroreflex Control of Heart Rate in Conscious OVE26 Diabetic Mice (Type 1)
Authors: Lin Min , Li Lihua , Epstein Paul N. , Cheng Zixi (Jack)
Published: June 2021

Baroreflex control of heart rate (HR) is impaired in human diabetic mellitus. In this study, we used OVE26 mice as an experimental model to examine the effect of diabetes on the baroreflex control of HR. The beta cells in OVE26 mice were specifically damaged due to a calmodulin transgene regulated by the insulin promoter. The damage of beta cells resulted in a sustained high blood glucose level (over 500 mg/dl) and reduced insulin secretion since 30 days of age. OVE26 diabetic and FVB control mice (5–6 months) were anesthetized and the femoral artery and vein were catheterized. On the second day, reflex‐mediated HR responses to phenylephrine (PE)‐ and sodium nitroprusside (SNP)‐ induced arterial pressure (AP) changes were measured in conscious mice. Compared with FVB, we found in OVE26 mice: 1) mean AP (MAP) and HR were decreased (P <0.01); 2) PE‐induced MAP increases were comparable to those in FVB mice (p>0.05); 3) baroreflex‐mediated bradycardia was attenuated (P < 0.01); 4) SNP‐induced MAP decreases were reduced (p< 0.05); 5) baroreflex‐mediated tachycardia was attenuated (P < 0.01). We conclude that baroreflex control of HR is impaired in OVE26 diabetic mice. Our data provided a platform for the examination of functional changes and structural reorganization of the neural components within the baroreflex arc in OVE26 mice (Gu et al., 2008; Huang et al., 2008; Lin et al., 2008a, b; EB abstracts). NIH R01 HL‐79636.
Maternal Diabetes Alters the Activity of Ca2+ and Ca2+‐Activated K+ Channels Cardiac Motoneurons in the Nucleus Ambiguus (NA) of Neonatal Mice
Authors: Lin Min , Li Lihua , Epstein Paul N. , Wurster Robert D. , Liu Yeqi , Cheng Zixi (Jack)
Published: June 2021

Maternal diabetes induces baroreflex dysfunction in the offspring. The neural mechanism for such a change is not clear. Neonates from FVB control and OVE26 diabetic mothers (mated with FVB fathers) received tracer XRITC injection into the pericardial sac on postnatal days 7–9. Two days later, membrane voltages of XRITC‐labeled NA cardiac motoneurons were recorded using the whole‐cell patch technique in brainstem slices. Compared with control, maternal diabetes induced following changes in the cardiac motoneurons of neonates: 1) reduction of instantaneous spike frequency in response to current injections (n = 45, p<0.01), 2) broadening of the repolarization phase and increase of the after hyperpolarization (AHP) of action potentials (AP) (n = 45, p<0.05); 3) increase of the AP half‐width after calcium channel blocker cadmium (Cd2+), large‐ conductance Ca2+‐activated K+ (BK) channel blocker charybdotoxin (ChTX) or small‐conductance Ca2+‐activated K+ (SK) channel blocker apamin application (n = 11, p<0.05); 4) increase of the Cd2+, ChTX, and apamin blockade of the amplitude of AHP (n = 10, p<0.05). Our data suggests that maternal diabetes alters the activity of Ca2+ and Ca2+.Cactivated K+ channels, leading to a reduction of the excitability of NA motoneurons. We suggest that alteration of Ca2+ and Ca+2.Cactivated K+ channel activity may contribute to baroreflex dysfunction in the offspring of diabetic mothers.
Chronic AT1 receptor blockade normalizes NMDA‐mediated changes in renal sympathetic nerve activity from the paraventricular nucleus in heart failure
Authors: Kleiber Allison , Zheng Hong , Patel Kaushik P.
Published: June 2021

Increased renal sympathetic nerve activity (RSNA) after NMDA microinjection into the paraventricular nucleus (PVN) is potentiated in rats with chronic heart failure (HF), and normalized by exercise training, as is plasma angiotensin (Ang) II. We hypothesized that chronic Ang II receptor (AT1) blockade would also normalize PVN NMDA‐mediated RSNA changes in rats with HF. We used four groups of rats: Sham+Vehicle (Veh); HF+Veh; Sham+Losartan (Los; 50 mg/kg/day in the drinking water, 3 wk); and HF+Los. HF was induced by coronary artery ligation. Cardiac function in both HF groups was similar after Los treatment. The highest dose of NMDA (200 pmol) injected into the PVN increased RSNA from baseline in the HF+Veh group 93±13%, significantly higher than in the Sham+Veh group (45±2%). The response in the HF+Los group (47±2%) was significantly lower than the HF+Veh group and not different from either Sham group. Furthermore, Los treatment in rats with HF normalized expression of the NR1 subunit of the NMDA receptor in the PVN. These data support our hypothesis that chronic AT1 receptor blockade normalizes NMDA‐mediated changes in RSNA and NR1 expression within the PVN in rats with HF. (Support:NIH)
Prolonged amelioration of LV dysfunction after withdrawal of chronic CNS blockade of AT1 receptors in rats with CHF post MI
Authors: Huang Bing S. , Ahmad Monir , White Roselyn A. , Leenen Frans H. H.
Published: June 2021

In rats post‐MI, LV dysfunction is attenuated by CNS AT1 receptor blockade with icv infusion of losartan at doses which are ineffective when administered peripherally. The present study tested whether after withdrawal of the blockade these effects of icv losartan continue. As of 2 days after coronary artery ligation, Wistar rats were infused icv via osmotic minipumps with losartan (1 mg/kg/d) or vehicle, or injected sc daily with losartan (100 mg/kg/d), for 4 wks. Rats with sham ligation served as control. Echocardiography was performed at 4 wks. Two wks after withdrawal of losartan treatment, echo was repeated and LV function assessed with a Millar catheter.

These results indicate that in Wistar rats, in contrast to general blockade specific CNS blockade of AT1 receptors early post MI effectively ameliorates LV systolic dysfunction, and these effects remain for at least 2 weeks after withdrawal of icv losartan.

Supported by a grant from Heart Stoke Foundation of Ontario.
Characterization of the Thermoneutral Zone of the Laboratory Rat
Authors: Le Nhut‐Minh P , Brown Justin W
Published: June 2021

The thermoneutral zone (TNZ) is the range of ambient temperatures where metabolic rate is minimal and corresponds to minimal thermal stress. Housing an animal outside its TNZ affects its physiology and development. Much of the data used to determine the TNZ of rats is conflicting due to methodology that likely stressed the animal. The purpose of this study was to help characterize the TNZ of rats, including circadian changes, using a thermocline (15 °C to 40 °C) to determine selected ambient temperature (STa) while monitoring core temperature (Tc) using biotelemetry techniques. The data from 7 male SD rats illustrated that STa, Tc and motor activity (MA) exhibited a circadian pattern. The STa decreased by 4.1 °C (p<0.0001) from day to night, while Tc and MA increased by 0.5 °C and 13.9 m/hr, respectively (Tc p <0.001; MA p=0.011). Control animals (N=3 maintained at 22 °C) illustrated similar circadian changes despite not being able to behaviorally thermoregulate. The MA of thermocline rats was slightly more than control rats (NS) while Tc was constant. These preliminary data suggest changes in STa may counteract nocturnal heat generation from motor activity to maintain a constant Tc. Lastly, current housing guidelines recommend rats be maintained at ~22°C while 24 hr STa was 26.0±0.7 °C suggesting rats may be chronically cold stressed under current guidelines, which may need to be revised.
NO synthase inhibition and the uterine artery vasoconstrictor response to exercise in rabbit pregnancy
Authors: O'Hagan Kathleen P , Mangi Dana E , Murphy Robert J
Published: June 2021

In rabbits, the uterine vasoconstrictor response to exercise is attenuated in late gestation. We asked whether this alteration in vascular control is related to enhanced production of nitric oxide (NO). Uterine artery blood flow (UtBF; Transonic) was measured in, and uterine artery conductance (UtC) was calculated for 10 rabbits during treadmill exercise (2 min at 10 m/min, 7% grade). Nonpregnant (NP) and pregnant (P) rabbits were studied on days 0 (NP) 10, 20 and 28 (term) of a 31 d gestation. Values are mean(SD). Rest UtBF [NP 2(1) to D28 17(7) ml/min] and UtC [NP 3(1) to D28 24(9) ml/min/mmHgx10−2] increased progressively through gestation. Exercise reduced UtBF and UtC, although the vasoconstrictor response was attenuated during gestation (NP Δ‐50(20)% vs D28 Δ‐19(18)% P<0.01). NO synthase inhibition with nitroG‐L‐arginine (5 mg/kg iv) reduced exercise and rest UtBF [rest NP 1(0.60); D28 15(7) ml/min) and UtC [rest NP 1.5(0.8); D28 20(11) ml/min/mmHgx10−2] in NP and P rabbits. However, the % change in UtC w/exercise was unaffected by NO block regardless of gestational status: NP: Δ‐38(20)% D28; Δ‐20(23)%; P=0.2 Intact vs NO block. While NO block lowers UtBF and UtC at rest and exercise, the vasoconstrictor response is not exaggerated w/NO block either at NP or pregnancy. NO is not responsible for the gestational attenuation of uterine vasoconstriction during exercise. Supported by R15 HL77128 and MWU.
mVps34 is Activated by an Acute Bout of Resistance Exercise
Authors: MacKenzie Matthew Graeme , Hamilton David Lee , Murray James T , Baar Keith
Published: June 2021

Resistance exercise (RE) training results in an increase in muscle mass and force production. Amino acids (AA) taken immediately before or after RE potentiate the effects of training on muscle mass. Therefore a protein that controls AA availability would be of interest in studying muscle growth. One candidate is the class 3 phosphatidylinositol 3OH‐kinase (PI3K) Vps34. Mammalian Vps34 (mVps34) is involved in both autophagy and AA signalling to mTOR (mammalian target of rapamycin) and its downstream target p70 S6 kinase (S6K1). To determine the role of mVps34 in mTOR activation following RE we measured mVps34 and S6K1 activity in the distal hindlimb muscles of rats 30 mins, 3, 6 and 18 hr after acute stimulation. In the tibialis anterior (TA) we found a 366.3±112.1 (p=0.04), 124.7±16 (p=0.01) and 129.2±0.1 (p=0.01) percent increase in S6K1 activity at the 30 min, 3 and 6 hr time points respectively. mVps34 activity increased only at the 3 and 6 hr time point in the TA muscle showing a 100.9±18.2 (p=0.01) and 36±8.8 (p=0.02) percent increase respectively. We have also shown an increase in S6K1 Thr389 phosphorylation and a 43±5.2 (p=0.02) percent increase in mVps34 activity in response to low Dexamethasone in C2C12 cells suggesting regulation through internal AA. We propose that a decrease in [AA] following RE activates mVps34 in order to replenish the AA pool maintaining mTOR activity.
Mechanisms of NFAT activation during CH
Authors: de Frutos Garcia Sergio , Alo Dominique , Bosc Laura Gonzalez
Published: June 2021

Physiological responses to CH include polycythemia, pulmonary arterial (PA) remodeling and vasoconstriction leading to pulmonary hypertension. We have recently demonstrated that NFATc3 is activated in PA from CH mice and it mediates CH‐induced PA remodeling with increased α‐actin expression. Our aim was to evaluate the mechanisms of CH‐induced NFAT activation in PA. 9xNFAT‐luciferase reporter mice were exposed to normoxia (N, 630 torr) or CH (380 torr) for 2 days. The animals were treated subcutaneously or by osmotic pumps with the calcineurin/NFAT inhibitor cyclosporin A (CsA, 25 mg/Kg/day); the non‐selective endothelin (ET) receptors antagonist PD145065 (0.1 mg/kg/day); the L‐type Ca2+ channel blocker diltiazem (100 mg/kg/day); the Rho kinase (ROK) inhibitor HA‐1077 (30 mg/kg/day) or vehicle (V). CH‐induced NFAT activation in PA was significantly inhibited by CsA and diltiazem, demonstrating the already established role of intracellular Ca2+ as an activator of calcineurin/NFAT pathway. ROK activity increases in CH and this pathway has been shown to activate NFAT in immune cells. Consistently, we found that HA‐1077 significantly attenuated CH‐induced NFAT activation. ET‐1 is an important factor in the development of pulmonary hypertension, also it is a potent activator of NFATc3 ex vivo but in the current study PD145065 failed to prevent NFAT activation. In conclusion, Ca2+ /calcineurin and ROK seem to mediate CH‐induced NFAT activation in PA. Supported by American Lung Association.
Effect of 4 days of intermittent hypoxia on oxidative stress in healthy men
Authors: Pialoux Vincent , Foster Glen E , Brugniaux Julien V , Duggan Cailean T , Hanly Patrick J , Poulin Marc J
Published: June 2021

Periodic occlusion of the upper airway in patients with obstructive sleep apnea (OSA) leads to intermittent hypoxia (IH). IH is characterized by repeated episodes of hypoxia/reoxygenation, which is known to increase the production of reactive oxygen species (ROS). Causal relationships between OSA, IH and oxidative stress have not been established because of confounding effects of medical co‐morbidities and obesity which are frequently associated with OSA. The aim of this study was to determine if IH changes oxidative stress in healthy volunteers. Nine men (29.3 ± 1.7 years; BMI, 25.0 ± 0.4 kg.m−2; mean±SD) were studied. Following baseline measurements breathing room air (day zero (D0)), all subjects were exposed to 4 days (D1 to D4) of IH while awake (2 min at PetO2 = 45 Torr and 2 min at PetO2 = 88 Torr) 6 hours.d−1. Lipid peroxidation (malondialdehides, MDA), DNA oxidation (8‐Hydroxy‐2′‐deoxyguanosine, 8‐OHdG) and oxidation protein products (AOPP) were assessed in plasma. MDA increased significantly between baseline (D0) and D1 (+39%: 3.48 ± 1.17 vs 4.85 ± 0.90 μmol.l−1) and then tended to return to baseline levels. 8‐OHdG increased significantly between D0 and D1 (+51%: 53.84 ± 54.63 vs 83.30 ± 45.49 μmol.l−1) and remained elevated until D4 (+67%: 53.84 ± 54.63 vs 89.71 ± 36.06 μmol.l−1). These findings demonstrate that IH increases oxidative stress and suggest that IH may contribute to ROS overproduction in OSA.

Supported by the Alberta Heritage Foundation for Medical Research and the Heart & Stroke Foundation of Canada.
Time‐lapse imaging reveals an extra‐cardiac contribution to the endocardium and cardiac jelly in avian embryos
Authors: Aleksandrova Anastasiia , Lansford Rusty D. , Little Charles D. , Rongish Brenda J.
Published: June 2021

Dynamic imaging of transgenic quail expressing histone 2B‐YFP under the control of the Tie1 promoter allowed endocardial progenitor cells to be followed through time and space during avian tubular heart morphogenesis. Simultaneous imaging of the nearby cardiac jelly/extracellular matrix (ECM) was performed as an indicator of the relative tissue motion during tubular heart formation. Monoclonal antibodies to fibronectin, fibrillin‐2, and laminin were fluorochrome‐conjugated and microinjected into the splanchnopleura of transgenic avian embryos at HH 8, prior to tubular heart formation. Time‐lapse recording was conducted until HH12, when cardiac looping is underway. Endocardial precursor cells were observed streaming into the anterior and posterior cardiac poles, as well as traversing the floor of the foregut to enter the heart via the dorsal mesocardium. Simultaneously, ECM fibrils were recruited from extracardiac sources to form portions of the cardiac jelly. In some cases, endocardial cells were observed to travel on aligned ECM fibrils to enter the heart. Ongoing cellular and ECM tracking will allow computation of endocardial cell movements relative to the surrounding ECM to determine the extent of cell autonomous motility versus tissue‐level convection during tubular heart morphogenesis. Supported by NIH HL085694 and the Mathers Foundation.

Grant Funding Source

Supported by NIH HL085694 and the Mathers Foundation.
Glycemic and Blood Pressure Responses to Acute Doses of Rebaudioside A, a Steviol Glycoside, in Subjects with Normal Glucose Tolerance or Type 2 Diabetes Mellitus
Authors: Maki Kevin C. , Curry Leslie L. , McKenney James M , Farmer Mildred V. , Reeves Matthew S. , Dicklin Mary R. , Gerich John E. , Zinman Bernard
Published: June 2021

Steviol glycosides, predominantly stevioside and rebaudioside A, extracted from the plant Stevia rebaudiana Bertoni, are used in several countries as food sweetening agents. This randomized, double‐blind, placebo‐controlled crossover trial examined the glycemic and blood pressure responses during meal tolerance tests to rebaudioside A consumed at three doses (500 mg, 750 mg, and 1000 mg) by men and women with normal glucose tolerance (n=45) or type 2 diabetes mellitus (n=48). Incremental areas under the concentration curves (pre‐meal to 240 min) for glucose, insulin, C‐peptide, and glucagon did not differ significantly for any dose of rebaudioside A tested vs. placebo or for all rebaudioside A doses combined vs. placebo. There were no significant differences between placebo and rebaudioside A in the effects on postprandial systolic or diastolic blood pressures. Rebaudioside A was well tolerated at all doses. These results suggest that acute consumption of up to 1000 mg of rebaudioside A has no clinically important acute effects on glucose homeostasis or blood pressure among individuals with normal glucose tolerance or type 2 diabetes mellitus. Funded by Cargill Corporation (Wayzata, MN).
Impact of enhanced nutrition program on child growth and diet in India
Authors: Avula Rasmi , Frongillo Edward A. , Arabi Mandana
Published: June 2021

The Indian Integrated Child Development Services (ICDS) provides supplemental food to children 6‐36 months. Evaluations have shown mixed effects on child nutrition. This study assessed the impact of program enhancements of local production of supplemental food, micronutrient supplementation, and improved program monitoring. Data came from 30 villages; half received the enhanced program. Multilevel linear regression was used to examine differences in changes in growth and nutrient intake between the enhanced and usual program groups. Children in enhanced program initially aged 12‐18 mo and 32‐36 mo gained, respectively 0.72 SD (p=0.02) and 0.63 unit SD (p=0.03) greater height‐for‐age. Similar results were observed for weight‐for‐age. Weight‐for‐height changes differed by almost 1 SD (p=0.01) among children who were initially 19‐25 mo. Significant differences were observed for energy intake among boys who were initially 12‐18 mo (137.45 kcal, p=0.03) with the enhanced program. A similar pattern was observed for protein and iron intake, but was explained by differences in energy intake. Older children benefited most from the enhanced program. Results from a parallel qualitative study support the plausibility of observed outcomes. The ICDS program would be more effective in improving child nutrition if it included these enhancements. Funded by UNICEF.
Stimulation of Toll‐Like Receptor 2 (TLR2) Results in Production of TGFß and Macrophage Chemotaxis Dependent on Hyaluronan (HA) and Its Receptor RHAMM
Authors: Savani Rashmin C. , Al‐Saadi Rajiha , Jiang Hongmei , Lam David T.
Published: June 2021

TLR2 recognizes gram‐positive organisms and yeast, and promotes leukocyte recruitment. TGF β stimulates macrophage chemotaxis. Hyaluronan (HA), a glycosaminoglycan, also stimulates cell migration using Receptor for HA‐Mediated Motility (RHAMM). We hypothesized that TLR2 ligation leads to increased production of TGF β, which in turn stimulates macrophage chemotaxis in a RHAMM‐HA dependent manner. RAW 264.7 murine macrophages were stimulated with Pam3Cys, a specific TLR2 agonist. TGF β was measured using an ELISA. Chemotaxis was determined using a Boyden chamber, and cytoskeletal changes by phalliodin staining. Immunoblots with phospho‐specific antibodies examined signaling pathways. Antibodies to TGF β, RHAMM (R36), and HA‐binding peptide were used as blockers. Pam3Cys stimulation increased macrophage chemotaxis (optimal 5µM), a response that was blocked by anti‐TGFß and anti‐RHAMM antibodies, as well as HA‐binding peptide, but not by normal IgG or scrambled peptide. Pam3Cys also stimulated TGFß production and lamellaepodia. TGFß‐stimulated chemotaxis was also blocked by anti‐RHAMM Ab and HA‐binding peptide, but not by anti‐TLR2 Ab. TLR2 activation resulted in rapid phosphorylation of p38. We conclude that TLR2 signaling results in TGFß production and macrophage chemotaxis that is dependent on RHAMM:HA interactions. We speculate that RHAMM and HA play important roles in innate immune responses to bacterial challenge. Funded by NIH HL073986
Teaching procedural skills to medical students using unembalmed cadavers
Authors: DePhilip Robert , Khandelwal Sorabh
Published: June 2021

The Clinical Skills Immersion Experience (CSIE) is a required part of the third‐year medical school curriculum at The Ohio State University and teaches procedural skills through a series of authentic, hands‐on exercises. Of the 20 procedures taught in the program, five are performed on unembalmed cadavers in the anatomy morgue. Here we focus on how to prepare cadavers to train students in three procedures involving aspiration of fluid from body cavities. For thoracentesis, a cadaver is placed in a sitting position and fluid (1% phenoxyethanol tinted with food coloring) is injected into both pleural cavities. For pericardiocentesis, the pericardial cavity is accessed by reflecting skin, superficial fascia, pectoralis major and minor muscles, and removing a small section of the anterior chest wall. The pericardial sac is opened and a rubber balloon filled with 300 ml of phenoxyethanol fluid is inserted between the diaphragm and the heart. The balloon has been sealed by dipping in wax to allow repeated needle punctures. The abdominal cavity of the cadaver prepared for paracentesis is first expanded by insufflation as if for laparoscopic surgery. The expanded abdomen is then filled with 4‐5 liters of phenoxyethanol fluid. Students rate the training exercises using cadaver models among the highest of all procedures and comment frequently on the realism of the experience.
ATP hydrolysis in human Eg5 kinesin monitored by millisecond time‐resolved FTIR: real‐time dynamics of conformation and chemistry in vitro
Authors: Jun Bokkyoo , Kim Sunyoung
Published: June 2021

Human Eg5 (HsEg5), a kinesin motor protein, is capable of ATP hydrolysis and utilizes the energy to move along the microtubule and participate in the spindle pole segregation during mitosis in eukaryotic cells. Herein, we present time‐lapsed monitoring of an in vitro ATP hydrolysis reaction by HsEg5 with ms time resolution. To survey dynamic structural and chemical changes during ATP hydrolysis concomitantly, difference Fourier‐transform infrared spectroscopy was used to monitor HsEg5 catalysis, by triggering initiation of the reaction by UV‐photolysis of caged ATP. A HPO3− vibrational mode was observed in our time‐lapse infrared data at 10 s after ATP release. This mode is assigned to formation of Pi by HsEg5, consistent with steady‐state measurement of enzyme kinetics (Vmax = 0.11 s−1). In the carbonyl stretching, or amide I, vibration of the peptide backbone in the same time‐lapse spectra, four modes were observed, reflecting net changes that occur in the secondary structure of Eg5 in a temporal sequence. These amide I modes indicate four unique conformational changes, or 'switches,' were detected in HsEg5 catalysis that may correspond to different intermediates, such as ATP binding, isomerization, and bond cleavage. Moreover, there is a subset of conformational changes that precede Pi release and there is an independent set of conformational changes as occur in tandem with Pi release.
Adiponectin Induces Vascular Smooth Muscle Cell Differentiation via AMPK
Authors: Ding Min , Wagner Robert J. , Fetalvero Kristina M. , Kasza Zsolt , Powell Richard J. , Martin Kathleen A.
Published: June 2021

Adipocytes secrete adiponectin, an abundant serum hormone which is down‐regulated in obesity. Low adiponectin levels correlate with type 2 diabetes and cardiovascular disease. Studies in endothelial cells and cardiomyocytes suggest a cardioprotective role for adiponectin, but its role in vascular smooth muscle cell (VSMC) phenotype is unknown. VSMC de‐differentiation contributes to atherosclerosis and restenosis. We report that adiponectin induces differentiation in human coronary artery VSMC, and that the adiponectin receptors Adipo‐R1 and ‐R2 are expressed in these cells. Delivery of adiponectin to VSMC with an adenovirus (Ad‐Adpn) or plasmid promoted AMPK activation, mTOR inhibition and contractile protein expression in a dose‐dependent manner. These effects were not observed with control virus or plasmid. Conditioned media from Ad‐Adpn‐infected VSMC also induced differentiation in uninfected VSMC, which was inhibited by the AMPK inhibitor Compound C. Furthermore, the pharmacologic AMPK activator, AICAR, activated AMPK, inhibited mTOR and induced VSMC differentiation. We've previously shown that mTOR inhibition with the stent drug rapamycin promotes VSMC differentiation. Adiponectin may serve as an endogenous hormonal inhibitor of mTOR. Our data suggest that maintenance of VSMC contractile phenotype through AMPK activation may be a novel cardioprotective function of adiponectin.
Alteration of cochlear pericytes in response to noise trauma and the involvement of HIF‐1α and VEGF
Authors: Shi Xiaorui
Published: June 2021

This study explored the effect of acoustic trauma on cochlear pericytes. Transmission electron microscopy revealed that pericytes on the capillaries of the stria vascularis were closely associated with the endothelium in control guinea pigs and mice. Foot processes of the pericytes were tightly positioned adjacent to endothelial cells. Exposure to wide‐band noise at the level of 120 dB for 3 hours per day for two consecutive days produced a significant hearing threshold shift and structurally damaged blood vessels in the stria vascularis. The serum protein, IgG, leaked from capillaries of the stria vascularis. Pericytes lost their tight association with endothelial cells. A pericyte structural protein, desmin, substantially increased after noise exposure in guinea pigs and mice, with a corresponding increase in pericyte coverage of vessels. The increased expression of desmin was associated with the induction of hypoxia‐induced factor (HIF‐1α) and the upregulation of vascular endothelial growth factor (VEGF). Inhibition of HIF‐1αactivity decreased VEGF expression in the vessels of the stria vascularis. Blocking VEGF activity significantly attenuated the expression of desmin in the pericytes. Taken together, these data demonstrate that cochlear pericytes were markedly affected by acoustic trauma and displayed an abnormal morphology. HIF‐1α activation and VEGF upregulation are important factors for the alteration of pericyte structural protein desmin.

Supported by NIDCD DC 008888 and NIDCD DC 005983
Role of the proximal tubule AT1A receptor in sodium transport regulation
Authors: Riquier Anne DM , McDonough Alicia A , Kou Dow‐Ning , Coffman Thomas M , Gurley Susan B
Published: June 2021

Chronic AngII infusion raises blood pressure (BP) in mice which depends on renal AngII receptors (AT1R). AngII is anti‐natriuretic while high BP is natriuretic.

Aim 1

To determine how AngII infusion affects Na+ transporter abundance and phosphorylation (by immunoblot of homogenates). AngII infusion (1 μg/kg/min x 14d) increases systolic BP ~30 mmHg. Versus sham infused (=1.0), AngII decreased total NHE3 to 0.7±0.1, NHE3‐P (at Ser552) to 0.8±0.1 without altering NHE3 ‐P/total ratio. NaPi2 and myosin VI were unchanged. Distal convoluted tubule (DCT) total NCC increased to 2.2±0.3, NCC‐P (at Thr60) to 5.4 ± 1.6 and NCC‐P/total ratio to 2.2±0.5 fold.

Aim 2

To determine the role of proximal tubule (PT) AT1R in regulation of Na+ transporters in mice lacking PT AT1R (KO). BP decreased 10mmHg in KO vs. WT and baseline expression of NHE3, NaPi2, and NCC were unchanged in KO vs. WT. AngII infusion increased BP 20mmHg less than in WT. AngII infusion in KO decreased: total NHE3 to 0.55±0.1, NHE3‐P to 0.66±0.1 (no change in ‐P/total NHE3 ratio), myosin VI to 0.56±0.1, NaPi2 to 0.64±0.1. NCC total and NCC‐P were unchanged in the AngII infused KO.

Conclusions

1) increased NCC contributes to AngII induced high BP while decreased PT NHE3 compensates for high BP in WT, 2) AngII dependent increase in NCC depends on PT AT1R, and 3) the removal of PT AT1R facilitates pressure natriuresis by further decreasing PT transporters during AngII infusion.
Angiotensin II and Oxidative Stress Increases JNK and IRS‐1ser307 Activity and Inhibits Insulin Signal Transduction in Rats with Insulin Resistance Induced by Low Salt Intake
Authors: Coelho Michella Soares , Negri Heloisa B. , Oliveira Ivone B. , Ferreira Daniele N. , Shimizu Maria HM. , Casarini Dulce E. , Prada Patricia O. , Dolnikoff Miriam S. , Saad Mario JA. , Heimann Joel C.
Published: June 2021

Low salt diet (LS) increases angiotensin II (AII) and oxidative stress (TBARS), which may enhance JNK and IRS1ser307 phosphorylation (PP) decreasing insulin signaling (INS‐S).

Objective

To evaluate if losartan (LOS) improves INS resistance by decreasing JNK and IRS1ser307 PP.

Methods

Twelve‐week‐old rats were fed a LS (0.15% NaCl) or normal salt diet (NS: 1.3%) since weaning. Groups: LS+LOS (30mg/kg/day), LS+vehicle, NS+LOS and NS+vehicle. Blood pressure (BP), blood glucose (G), plasma INS and TBARS, HOMA, and tissue AII were evaluated. The INS‐S and inflammatory signaling (IS) were determined by Western blot and immunoprecipitation. AT1 receptor gene expression was determined by RT‐PCR.

Results (mean±SEM, P<0.05, n=8)

BP and AT1 did not differ among groups. INS, HOMA and TBARS were higher in LS (INS: 712±60) than in NS (257±40 pmol/l). G and TBARS were lower and INS was higher in LS+LOS (TBARS: 4±0.5) than in LS (6±1 nmol/ml). Liver AII was higher in LS than in NS. INS receptor and Akt PP were higher and JNK (120±3) and IRS1ser307 PP (100±6 AU) were lower in the liver of LS+LOS compared to LS. IkBα PP was lower in LS (20±2) than in NS (100±4 AU) suggesting an activation of IS in LS. Similar results were observed in the muscle.

Conclusions

INS resistance induced by LS is associated with alterations in regulation of INS‐S, activated IS, and higher tissue AII and TBARS. In LS, LOS improves INS‐S by decreasing JNK and IRS1ser307 PP with no change in BP.

FAPESP
Dual effect of Alternariol on Acetylcholinestrase and Monoamine oxidase Extracted from Different Parts of Rat Brain
Authors: Osman Mohamed Yousry Hassan , Osman Hassan Mohamed Yousry
Published: June 2021

Alternariol is a metabolite product of different strains of alternaria tenuis. It's structure has a similarity to cannabinol derivatives .It has a chemical formula 3,4,5 trihydroxy 6‐ methyl dibenzox ‐pyrone.

The effect of the alternariol on both acetylcholinestrase (AChE) and monoamineoxidase ( MAO) isozymes which were extracted from whole brain male albino rats and it's different five parts namely : frontal cortex, basal ganglia, cerebellum, pons , medulla oblongata was studied. Kinetic studies were done to determine the type of inhibition of AChE and MAO isozyme, and the enzyme ‐inhibitor dissociation constant (Ki) by alternariol.

The results indicated that alternariol inhibited both AChE and MAO isozymes of the cortex and cerebellam more than the extracts of the other parts of the brain .These parts are responsible for perception , motor, sensony, psychic activities, coordination of movements and balance.

The inhibition of these isozymes increased with increasing the amount of alternariol added to the assay mixture, i.e., the inhibition was dose dependent and of the competitive type. The values of Ki for alternariol ‐ AChE isozymes

varied from 12.0 to 15.0 mmol/L and for alternariol ‐ MAO isozymes varied from 28 to 30 mmol/L.

The difference in the degree of inhibition of the extracts of these parts of the brain could be attributed to the slight difference in the structure ( arrangement of the amino acids) of these isozymes ( isozyme phenomenon) and to the distinct gene loci. The inhibition of AChE and MAO by alternariol may save ACh and biogenic amines which are of

great importance for the patients suffering from Alzheimer and dementia.
Role of Acetylation in the NFkB Dependent Transcription of Proinflammatory Cytokines in Stimulated Human Macrophages
Authors: Ramaswami Sitharam , Ghosh Chandra C , Juvekar Ashish P , Le Hong Dung , Soe Kevin , Vancurova Ivana
Published: June 2021

Proinflammatory cytokines such as tumor necrosis factor‐alpha (TNF‐α), interleukin‐8 (IL‐8) and IL‐6 play an important role in activating cells of the immune system and thus aid in the initiation of inflammatory response. One of the major sources of these proinflammatory mediators are the macrophages at the site of injury. In these cells, the regulation of transcription of these cytokines is mediated by the transcription factor NFκB. Our results indicate that the inhibitor of NFκB, IκBα, differentially regulates the in vivo p65 NFκB binding to promoter regions of these cytokines. The histone deacetylase inhibitor (HDACi), trichostatin A (TSA), enhances cytokine transcription at lower concentrations, while at higher concentrations TSA inhibits cytokine transcription by increasing the nuclear levels of IκBα. The inhibitory effect of nuclear IκBα is partially rescued by decreased deacetylation, which seems to be cytokine specific. Understanding mechanisms by which the nuclear IκBα regulates p65 NFκB recruitment to distinct promoters will contribute to the development of more specific therapies for inflammatory disorders characterized by excessive cytokine release.
ANTIMICROBIAL ACTIVITIES OF SOME PYRIMIDINO AND ADENOSINO‐ 1,2,4 – TRIAZOLE‐ 3 – THIONES
Authors: Rifati Arleta , Govori Sevdije , Bytyçi Arlinda , Ibrahimi Hamide , Kryeziu Fadil
Published: June 2021

5‐amino ‐7‐methyl‐ [1,2,4] triazolo [4,3‐c] pyrimidine ‐ 3(2H) ‐thione (1), 5‐iminobis [1,2,4] triazolo [4,3‐c:4',3′‐f] pyrimidine ‐ 3,7 (2H,5H) ‐ dithione (2), and 4 ‐ amino ‐ 3,4 ‐ dihydroxy ‐ 5 ‐ (hydroxymethyl tetrahydrofuran‐2‐y) ‐1,7 ‐ dihydro ‐ 8H [1,2,4] triazolo [3,4‐b] purine ‐8 (1H) thione (3), were synthesized and evaluated for in vitro antibacterial activity against Staphylococcus aureus , Escherichia coli , Klebsiella pneumonie and Candida albicans . The assessment of the antimicrobial action of the synthesized compounds was performed using the disk diffusion and microdilution methods. During the research work of microbiological activity of these compounds (1‐3), is proved that these compounds obviously inhibit the growth of Candida albicans and Klebsiella pneumonie .
The well‐characterized TRPV1 antagonist BCTC is an agonist of TRPA1: new pharmacology of an old compound.
Authors: Bhattacharya Anindya , Ao Hong , Banke Tue , Wickenden Alan D
Published: June 2021

TRPA1 is a nociceptor that is activated by allicin, cinnamaldehyde, allyl isothiocynate (AITC), gingerol, carvacrol, farnesyl thiosalicylic acid (FTS), disulfiram, chlordantoin, and 15d‐PGJ2. Formalin and cyclophosphamide, two irritants used to study pain behavior, also activate TRPA1. Recently, it was shown that menthol, the well‐known TRPM8 agonist, also activates human TRPA1 (Xiao et al., J Neurosci 28:9640, 2008). Here we demonstrate that BCTC, a compound that has been used extensively in the literature as a TRPV1 antagonist, is an agonist for TRPA1. BCTC concentration dependently activated TRPA1 in a calcium influx assay (FLIPR) with a pEC50 of 5.2 ± 0.03 and 4.3 ± 0.02 at the rat and human recombinant TRPA1, respectively. Differences in potency of BCTC between rat and human TRPA1 were not due to differential expression of TRPA1, as AITC & 15d‐PGJ2 were equipotent while FTS was more potent at the human TRPA1. BCTC‐induced calcium flux was attenuated by the TRPA1 antagonist, HC030031 (Tocris). 100 μM HC030031 caused a 2.6‐fold shift (rTRPA1) and a 5‐fold shift (hTRPA1) of the BCTC concentration response curve. The same concentration of HC030031 displayed a higher shift with AITC (32 fold; hTRPA1) and FTS (12 fold; hTRPA1) as agonists indicating non‐identical interaction sites of HC030031 with BCTC, AITC and FTS. In addition, BCTC was also able to activate the hTRPA1 current in a whole‐cell patch clamp assay.
Lead Exposure and Cardiovascular Dysregulation in Children
Authors: MacKenzie James A. , Gump Brooks B. , Roosa Kristen , Bendinskas Kestas , Dumas Amy , Morgan Robert , Parsons Patrick
Published: June 2021

Increasing lead (Pb) exposure is associated with increased blood pressure in humans and animal studies suggest this is due to increased vascular resistance. We looked at cardiovascular reactions to acute stress (AS) in 9‐11 year old children (N = 134) with low blood Pb levels (≤3.76 μg/dL). Pb levels were significantly associated with increased total peripheral resistance (TPR) responses and diminished stroke volume and cardiac output responses to AS. This vascular pattern of response to AS tasks may predict future hypertension. Pb was also associated with sympathetic nervous system (SNS) withdrawal during AS and SNS activation at rest, and the Pb‐SNS relationship during AS was not significant when baseline SNS was included as a covariate. In addition, serum aldosterone was negatively associated with blood Pb and positively associated with SNS reactivity to AS. Inclusion of aldosterone as a covariate also eliminated the Pb‐SNS reactivity relationship. Our data suggests two independent mediational pathways underlying the Pb‐TPR association. One involves SNS activation at rest and the other involves decreased circulating aldosterone levels, and both ultimately decrease the capacity to elicit a SNS response to AS. The effects described here were significant for Pb levels considered well below the 10 μg/dL threshold set by the CDC for deleterious effects. Supported by grants ES10190 and ES09815 (NIH).
Yeast Elc1 plays an important role in global genomic repair but not in transcription coupled repair
Authors: LeJeune Danielle T , Chen Xuefeng , Ruggiero Christine , Berryhill Shannon , Ding Baojin , Li Shisheng
Published: June 2021

Transcription coupled repair (TCR) is a nucleotide excision repair (NER) pathway that is dedicated to repair in the transcribed strand of an active gene. The genome overall NER is called global genomic repair (GGR). Elc1, the yeast homolog of the mammalian elongation factor elongin C, has been shown to be a component of a ubiquitin ligase complex that contains Rad7 and Rad16, two factors that are specifically required for GGR. Elc1 has also been suggested to be present in another ubiquitin ligase complex that lacks Rad7 and Rad16 and is involved in UV‐induced ubiquitylation and subsequent degradation of RNA polymerase II. Here we show that elc1 deletion increases UV sensitivity of TCR‐deficient cells but does not affect the UV sensitivity of otherwise wild type and GGR‐deficient cells. Cells deleted for elc1 show normal NER in the transcribed strand of an active gene but have no detectable NER in the non‐transcribed strand. Elc1 does not affect UV‐inducedmutagenesis when TCR is operative, but plays an important role in preventing the mutagenesis if TCR is defective. Furthermore, the levels of Rad7 and Rad16 proteins are not significantly decreased in elc1 cells, and overexpression of Rad7 and Rad16 individually or simultaneously in elc1 cells does not restore repair in the non‐transcribed strand of an active gene. Our results suggest that Elc1 has no function in TCR but plays an important role in GGR. Furthermore, the role of Elc1 in GGR may not be subsidiary to that of Rad7 and Rad16.
Expression of Polygalacturonase from Lygus hesperus
Authors: Browning Jenny , Booth Rachell E
Published: June 2021

Tarnished plant bug, Lygus hesperus, causes a significant amount of damage to a wide array of cultivated plants during feeding, resulting in considerable economic losses. Polygalacturonase (PG), secreted by the Lygus bug during feeding, is the primary cause of plant damage. Polygalacturonase is a cell wall degrading enzyme, which catalyzes the hydrolysis of the glycosidic linkages of pectin (polygalacturonic acid). Polygalacturonase inhibitor proteins (PGIPs) are glycoproteins located in plant cell walls that selectively bind and inhibit pathogen PGs. In order to screen PGIPs for optimum inhibition, we have cloned and expressed functional polygalacturonase in a Drosophila expression system. Two polygalacturonase (PG) genes, LhPG4‐1 and LhPG4‐4 produced by the tarnished plant bug, Lygus hesperus, were ligated into the expression vector pMT/BiP/V5‐His A. Positive clones were transfected into Drosophila S2 cells and expression was induced at 24 hours with copper sulfate. PG protein was detected with western blotting and activity detected with a standard reducing sugar assay. Partially purified PG will be used to screen potential PG inhibitory proteins. Funding support provided by the Welch Foundation and Texas State University‐San Marcos.
Aerobic capacity of African American males: Association with serum levels of IGF‐1, IGF‐1 binding protein and prostate cancer cell growth
Authors: Sridhar Rajagopalan , Bond Vernon , Cousins Valerie Marie , Jackson Anwar , Millis Richard , Zhang Renshu
Published: June 2021

Prostate cancer incidence is disproportionately high in blacks. High levels of insulin‐like growth factor‐1 (IGF‐1) and low levels of insulin‐like growth factor binding protein‐3 (IGFBP‐3) are associated with increased risk for prostate cancer. This study examined levels of IGF‐1 and IGFBP‐3 in African American males with low (N=8) and high (N=9) levels of aerobic fitness. The growth rate of LNCaP human prostate cancer cells were examined in culture medium fortified with serum from different individuals. RPMI‐1640 medium was supplemented with 10% serum from each individual for comparing the relative abilities of sera to support the growth of androgen sensitive LNCaP cancer cells. Cell growth was estimated using the MTT assay for cell proliferation. The serum concentrations of testosterone, IGF‐1, and IGFBP‐3 were measured using ELISA kits. Serum levels of IGF‐1 were significantly higher in the low fitness group compared to the high fitness group (p<0.05). Serum levels of IGFBP‐3 in the high fitness group were significantly higher compared to those in the low fitness group (p<0.01). Growth rates of LNCaP cells were significantly higher in medium supplemented with sera from men in the low fitness group compared to those from high fitness group (p<0.05). There was no significant intergroup difference in serum testosterone levels. These results suggest that the rate of prostate cancer cell growth may be higher in sedentary men than in physically active men.

(Supported in part by USMRMC grant DAMD17‐03‐1‐0123 and grant 2G12 RR003048 from RCMI program of NCRR, NIH and grant R25 HL003679 from HLBI, NIH)