bluuebunny/crossref_metadata_2025 huggingface dataset:
Dominant Negative Neuronal Nitric Oxide Synthase in Paraventricular Nucleus Augments Blood Pressure in Two Kidney‐One Clip Hypertensive Rats
Authors: Rossi Noreen F , Black Stephen M. , Chen Haiping , Maliszewska‐Scislo Maria , Augustyniak Robert A.
Published: June 2021

Angiotensin II (Ang II) increases blood pressure by both peripheral and central actions via AT1 receptor activation of brain circumventricular organs that lie outside the blood brain barrier. The subfornical organ projects to paraventricular nucleus (PVN) whose efferent projections influence sympathetic output. In other conditions of high sympathetic activity, nitric oxide (NO) within the PVN acutely attenuates sympathetic tone. We tested the hypothesis that 2K‐1C rats, a model of Ang II dependent hypertension, chronic blockade of NO generation within the PVN will augment blood pressure. Male Sprague Dawley rats were equipped with radiotelemetry transmitters at the time of renal artery clipping. After 6 wk, PVN were injected bilaterally with 250 nL with plasmids bearing RSV β‐galactosidase (125 ng/μL; β‐gal), the dominant negative construct for nNOS (250ng/μL; DNnNOS), or the wild type construct for nNOS (250 ng/μL; WTnNOS). Hemodynamic parameters were observed for 3 wk. After renal artery clipping, MAP progressively increased and did not differ among the groups. In the last week of observation, average MAP in the DNnNOS group (186.4±6.3 mmHg) was significantly higher than either the β‐gal (160.8±3.4 mmHg) or WTnNOS (156.9±1.5 mmHg) injected 2K‐1C rats (P< 0.001). Thus, chronic blockade of NO production by DNnNOS in PVN augments systemic arterial pressure in a model of Ang II dependent hypertension.
Varied functions of small, non‐coding RNAs in bacteria
Authors: Storz Gisela , Opdyke Jason A. , Fozo Elizabeth M. , Waters Lauren S. , Kawano Mitsuoki , Zhang Aixia
Published: June 2021

In recent years, a combination of computational and molecular approaches has led to the identification of >80 small, noncoding RNAs in E. coli. More and more small RNAs are also being discovered in other bacteria. Most of the characterized small RNAs have been shown to act by basepairing at the 5′ or 3′ ends of mRNAs. In some cases the region of complementarity is extensive while in other cases only short regions of complementarity are required for regulation. Basepairing can lead to increased or decreased message stability or can block or facilitate translation. The mechanisms by which basepairing between small RNAs and their mRNA targets leads to these varied regulatory outcomes will be discussed. Supported by the Intramural Program of the NICHD.
An HDL‐mimetic peptide improves cardiac performance and lipoprotein profiles in septic rodents
Authors: Zhang Zhenghao , Miller Andrew P , Zhang Yun , Palgunachari Mayakonda N , Anantharamaiah G.M. , White C. Roger
Published: June 2021

Background: Cardiac failure is a common cause of death in septic patients, and elevating plasma HDL reduces sepsis complications.

Objective: We tested the hypothesis that the apoA‐I mimetic peptide L‐4F improves cardiac function in rats undergoing cecal ligation and puncture (CLP).

Methods: Male Sprague‐Dawley rats were randomized to undergo CLP or sham surgery. CLP rats were further subdivided to receive saline (n=11) or L‐4F (10mg/kg, n=12) by ip. injection 6 hours after sepsis induction. Sham‐operated rats received saline (n=8).

Results: There was a significant decrease in right atrial pressure, left ventricular end‐diastolic volume, stroke volume and cardiac output 24hrs after CLP. L‐4F blunted changes in these parameters in CLP rats and improved ejection fraction. Lipoprotein profiles were altered by CLP surgery after 24 hrs. Total cholesterol (TC) was reduced from 96± 4 to 51± 4 mg/dl in CLP rats and was principally characterized by a reduction in HDL (80± 3 to 23± 3). L4F administration to CLP rats attenuated this shift in lipoproteins (TC: 67± 5/HDL: 43± 3).

Conclusion: L‐4F treatment improved cardiac performance in CLP rats compared to saline‐treated controls. This was associated with an increase in HDL cholesterol and a decrease in plasma and cardiac cytokines. It is hypothesized that HDL reduces sepsis complications by neutralizing pathogens, such as endotoxin, in CLP rats. Supported by NIH DK070040.
Reversal of Adrenergic Desensitization in Prenatally Stressed Rats by P38 Map Kinase Inhibitor, SB203580
Authors: Chen Fangping , Kan Hong , Xie Sherry , Beto Robert J , Warden Bradford E , Jain Abnash C , Finkel Mitchell S
Published: June 2021

We previously reported a defect in beta adrenergic responsiveness in cardiac myoctes isolated from pre‐natally stressed rats (PS) following restraint stress (R) (PS+R). We now report a reversal of the blunted beta adrenergic responsiveness in PS + R with the P38 MAP kinase inhibitor, SB203580, in vivo. Hemodynamics were measured in awake rats using polyurethane tubing inserted percutaneously into the left ventricle and the right femoral artery. Drugs were administered through a jugular venous catheter. PS + R had a significantly attenuated hemodynamic response to increasing doses of isoproterenol, including a reduction in both positive dp/dt (mmHg/s) max (19291¡À654 vs 14742¡À571, Control vs PS+R, respectively. p<0.05), and negative dp/dt (mmHg/s) max (−14834¡À649 vs −10786¡À482, Control vs PS+R, respectively. p<0.05) (n=8–12). SB 203580 treatment of PS+R for 24 hrs reversed the blunted isoproterenol response (19291¡À654 vs 20715 ¡À 515, and −14834¡À649 vs −15649 ¡À371; Control vs PS+R+SB, respectively. p<0.05) (n=8–12). Thus, continuous activation of P38 MAP kinase appears to be required to maintain myocardial dysfunction in this stress‐induced cardiomyopathy model.
Inducible expression of active Inhibitor‐1 enhances cardiac function and improves contractility after an ischemic insult
Authors: Nicolaou Persoulla A. , Zhou Xiaoyang , Rodriguez Patricia , Mitton Bryan , Pathak Anand , Robbins Jeffrey , Kranias Evangelia G.
Published: June 2021

Inhibitor‐1 (I‐1) is a critical regulator of cardiac function. Upon phosphorylation at T35, I‐1 inhibits Protein Phosphatase‐1 (PP1) and augments contractility. In fact, the depressed function in failing hearts is partially attributed to impaired PP1 regulation via I‐1. To define any beneficial effects of acute I‐1 expression, we generated a double transgenic (DTG) mouse model with inducible (Tet‐off) expression of active I‐1 (T35D). 8 weeks of I‐1 expression elicited maximal stimulation on cardiac function, assessed by in vivo catheterization. Specifically, the contraction and relaxation rates were increased by 21% and 32% respectively, as compared to wild‐type. This was specifically attributed to increased phospholamban phosphorylation, while there were no changes in Troponin I and the Ryanodine Receptor. To examine the potential therapeutic role of I‐1 in ischemia/reperfusion (I/R), we subjected hearts to global I/R ex vivo. The DTG mice continued to show higher contractility (by 27%), which was not accompanied by altered necrotic injury, assessed by infarct size and LDH efflux. However, apoptotic injury was significantly attenuated in the DTG hearts. In conclusion, expression of I‐1 enhances basal contractility and improves functional recovery after an ischemic insult, suggesting that I‐1 may represent a potential therapeutic target in myocardial infarction. (NIH HL64018 to EGK, AHA 0715500B to PN)
Periodic squat‐stand: A new approach for assessment of cardiac baroreflex function in humans
Authors: Zhang Rong , Claassen Jurgen , Okazaki Kazunobu , Diaz‐Arrastia Ramon , Levine Benjamin
Published: June 2021

A new approach for assessment of baroreflex function in humanss was developed in this study. Arterial pressure and R‐R interval were recorded in 10 healthy young (30 ± 5 yrs) and 8 elderly subjects (66 ± 6 yrs). Periodic squat‐stand maneuvers were performed at the frequencies of 0.05 and 0.1Hz respectively to produce large oscillations in arterial pressure to stimulate the baroreflex. Transfer function between changes in arterial pressure and R‐R interval was estimated to assess the cardiac baroreflex sensitivity (gain) and the temporal relations (phase) between these variables. During squat‐stand maneuvers, spectral power of oscillations in systolic blood pressure at the frequency of 0.05 Hz was 374 ± 191 mmHg2 in young and 561± 321 mmHg2 in the elderly, and at the 0.1Hz was 253 ± 119 mmHg2 in young and 203 ± 87 mmHg2 in the elderly. In response to these changes in arterial pressure, large oscillations in R‐R interval also were created at the frequencies of 0.05 and 0.1Hz respectively. When compared with the young, transfer function gain was reduced, while the phase became more negative in the elderly. In conclusion, periodic squat‐stand maneuvers produced large and coherent oscillations in arterial pressure and R‐R interval. Transfer function analysis between these variables provides a new and widely available method for assessment of dynamic baroreflex function in humans. Application of this method showed that not only was cardiac baroreflex sensitivity was reduced, but also the heart rate response to changes in arterial pressure was delayed in the elderly.
Marinobufagenin enhances cardiac contractility in mice with ouabain‐sensitive α1 Na,K‐ATPase (NKA)
Authors: Wansapura Arshani N , Fedorova Olga V , Lasko Valerie , Lingrel Jerry B , Bagrov Alexei Y , Lorenz John N
Published: June 2021

Endogenous Na+ pump inhibitors are thought to play important (patho)physiological roles, and occur in two different chemical forms in the mammalian circulation: cardenolides, such as ouabain, and bufadienolides, such as marinobufagenin (MBG). While all αNKA isoforms are sensitive to ouabain in most species, in rats and mice the ubiquitously expressed α1NKA is resistant to ouabain. We have previously shown that selective modification of the putative ouabain‐binding site of either the α1 or α2 NKA subunit in mice substantially alters the cardiotonic influence of exogenously applied cardenolides. To determine whether the ouabain‐binding site also interacts with MBG and if this interaction plays a functional role, we evaluated cardiovascular function in α1‐resistant/α2‐resistant (α1R/Rα2R/R), α1‐sensitive/α2‐resistant (α1S/Sα2R/R), and α1‐resistant/α2‐sensitive mice (α1R/Rα2S/S, wild type). Cardiovascular indices were evaluated in vivo by cardiac catheterization at baseline and during graded infusions of MBG. There were no differences in baseline measurements of targeted mice, indicating normal hemodynamics and cardiac function. As shown in the graph, MBG increased cardiac performance to a greater extent in α1S/Sα2R/R compared to α1R/Rα2R/R and wild type mice. These data indicate that MBG interacts with the ouabain‐binding site of the α1NKA subunit and can thereby influence cardiac inotropy.
Role of cardiac fibroblasts in diabetic cardiomyopathy
Authors: Stewart James A , Hutchinson Kirk R , Lord Kevin C , Varner Kurt J , Lucchesi Pamela A
Published: June 2021

Type 2 diabetes is associated with cardiomyopathy. Chronic hyperglycemia and activation of the renin‐angiotensin system lead to oxidative stress, excessive collagen accumulation and myocardial dysfunction. The cardiac collagen network is produced primarily by cardiac fibroblasts (CF). This study examined alterations in basal activation of signaling cascades and expression of pro‐fibrotic markers within the myocardium of diabetic (db/db) mice compared to non‐diabetic (Db/db) littermates. In vivo functional analysis with conductance catheters indicated decreased compliance (end diastolic pressure‐volume relationship) that correlated with increased collagen deposition in db/db hearts. Primary cultures of CF were isolated from both groups. Protein analysis demonstrated increased expression of type I collagen, TIMP‐2, TGF‐beta and PAI‐1 in db/db cells vs. Db/db. Moreover, iNOS and RAGE protein levels were elevated in db/db CF compared to Db/db. These changes occurred independently of glucose concentration in the media. This pattern of protein expression was associated with basal activation of ERK1/2 MAP kinases and SMAD 2/3. These studies demonstrate a distinct role for myocardial fibroblasts in establishing and maintaining a fibrotic environment in diabetic cardiomyopathy. Supported by HL056046, R01HL063318 and P20RR18766 (PAL).
Acute exercise‐induced heat shock proteins do not protect heart from hypoxia‐reoxygenation injury in adult rat
Authors: Tsujikawa Hiroto , Ogura Yuji , Kakigi Ryo , Kurosaka Mitsutoshi , Naito Hisashi , Watanabe Makino , Katamoto Shizuo , Okada Takao
Published: June 2021

It has been reported that heat shock proteins (HSPs) in heart induced by endurance exercise (EEx) training may contribute to cardio‐protective effects against ischemia‐reperfusion injury. On the other hand, some studies report that the HSPs are not essential to protect heart from oxidative stress and that cardiac function decreases after acute EEx in human. Therefore, we studied by using Langendorff‐perfused heart model if HSPs induction by acute EEx would contribute to cardio‐protective effects against hypoxia‐reoxygenation (H‐R) injury. Male Wistar rats (280–320g) were assigned to either a control (CON) or an acute EEx (3D) group (n=8/group). 3D animals performed 60 minutes treadmill running (∼20 m/min) for 3 days consecutively. Although cardiac HSP72 increased significantly in 3D group, pressure‐rate product of hearts at 30 minutes reoxygenation did not differ from CON group. In addition, the incidence rate of ventricular fibrillation or tachycardia in 3D group significantly increased compared with CON group (50% vs 13%, P<0.05). These results indicate that the acute EEx may cause cardiac dysfunction regardless of EEx‐induced HSP72 in rat.
Heart – Kidney Connection: Myocardial Infarction Induces Widespread Changes in Renal Cortical and Medullary Gene Expression together with Cortical and Medullary Fibrosis
Authors: Martin Fernando L , Huntley Brenda K , Harders Gerald E , Sandberg Sharon M , Heublein Denise M , Burnett John C
Published: June 2021

Studies in human myocardial infarction (MI) suggest that even in the absence of heart failure (HF) alterations in renal function may occur and contribute to poor outcomes. We hypothesized that even without preexisting renal disease, renal functional, structural and molecular changes would be present following MI.

Wistar rats were used, Sham(S;n=10) and MI(n=9). 3 weeks after MI, GFR was determined by inulin clearance. Blood was obtained for PRA and aldosterone. Cardiac function was assessed by echo. Genomewide microarray analysis was performed on kidney cortex (KC) and medulla (KM) (Affymetrix). EF decreased after MI (S:63±2, MI:43±6%, p<0.01) and LVEDd increased. PRA, aldosterone and blood pressure (BP) did not change. There was no HF as Na and water excretion was maintained. GFR tended to decrease. Pic Red staining in the KC and KM after MI showed greater fibrosis (KC S:1±0.2, MI:3±0.6%, p<0.001 and KM S:1±0.2, MI:19±6%, p<0.005). Microarray analysis showed that 303 genes changed in KM and 407 genes in KC after MI (1.5 fold, P<0.05) related to structure morphogenesis and metabolic processes(Z value>2). We conclude that experimental MI results in renal structural remodeling characterized by cortical and medullary fibrosis with a mild reduction in GFR and extensive modulation of molecular pathways related to renal growth and morphogenesis underscoring a heart‐kidney connection after MI.
Transmural Changes in Myocardial Tissue Volume During the Cardiac Cycle: Implications for Microvascular Flow Patterns with Ventricular Pacing
Authors: Ashikaga Hiroshi , Coppola Benjamin , Yamazaki Katrina , Villarreal Francisco , Omens Jeffrey , Covell James
Published: June 2021

The transmural gradient in average myocardial blood flow is well known. However, few techniques have the temporal and transmural resolution to examine tissue volume and by implication vascular volume changes during the cardiac cycle during normal activation and with epicardial ventricular pacing. We studied the time course of transmural tissue volume change (TVC) in the anterior left ventricular wall of normal canine heart (n=14) using implanted markers and 125 Hz cineradiography. TVC was calculated from marker locations (as a relative fraction, 1.00=no change), and capillary volume was an assumed fraction of the compressible tissue. Capillary diameter (CD) was estimated from capillary volume and length changes (from myofiber strain). During normal activation there was a significant transmural gradient in TVC 72ms (Early) after QRS (.99±.04 (SD) epi, .95±.05 endo), and at end systole (ES) (.97±.06 epi, .89±.08 endo). This corresponded to an Early gradient in CD: 5.6±.6 (μm, epi) to 4.9±1.0 (endo), and a gradient at ES: 5.1±.8 (epi) to 4.2±1.4 (endo). During ventricular pacing the Early TVC gradients reversed: .97±.02 (epi) and .99±.05 (endo), with reduced gradients at ES. Early CDs were 5.3±.4 (epi) and 5.3±.6 (endo). Thus, there is a transmural gradient of reduction in tissue volume and CD during systole, which changes with ventricular pacing. Changes in transmural blood flow may trigger local tissue remodeling associated with areas of early ventricular activation. Supported by HL43617 and HL32583.
Echocardiography under isoflurane anesthesia affects heart rate and function in commonly utilized mouse strains
Authors: Rojas Mauricio , Meredith Dane , Kylander Jackie , Barrick Delia , Corn Drew , Lockyer Pamela , Patterson Cam , Willis Monte S
Published: June 2021

While many studies have described the advantages of performing echocardiography in mice using isoflurane anesthesia over other anesthetics, the direct comparison of isoflurane and conscious echocardiography in common mouse strains has not previously been determined. In the present study, we compared cardiac function in two commonly used mouse strains (C57BL/6 and 129S) at 8 weeks of age using high resolution two‐dimensional echocardiography (30MHz). C57BL/6J (N=12) and 129S (N=12) were echoed both under anesthesia (1.5% isoflurane in 100% oxygen) and consciously by four different operators within 1 week. We identified significant decreases in heart rate in anesthetized mice (both strains) compared to conscious mice (620+/−66 vs. 466+/−54). Since heart rate affects cardiac function, it was not surprising that a corresponding decrease in fractional shortening % was also identified in both strains (54%+/−11 vs. 35%+/−10) under the same conditions. Both anterior and posterior wall measurements as well as interventricular distances did not significantly differ between echo methods or individual operators. These findings demonstrate the utility of echocardiographic assessment of cardiac function in both conscious and isoflurane anesthetized mice in multiple commonly used mouse strains, highlighting the specific parameters affected by isoflurane.
The arrhythmogenic role of stretch‐activated channels in rat atrial myocytes
Authors: Youm Jae Boum , Leem Chae Hun , Han Jin , Earm Yung E
Published: June 2021

Since their first discovery in chick skeletal muscles, stretch‐activated channels (SACs) have been proposed as a probable mechano‐transducer of the mechanical stimulus at the cellular level. Channel properties such as ionic selectivity and stretch‐dependence have been studied both in the single‐channel and at the whole‐cell level. There is growing evidence that major stretch‐induced changes in electrical activity are mediated by activation of these channels. The involvement of SACs in stretch‐induced arrhythmias and diastolic depolarization was demonstrated using a specific blocker. Noma's group has created a cardiac cell model (Kyoto‐Model) to describe various cellular activities such as the action potential, Ca2+ transients and contractile force. We focus on the possible contribution of SACs to the generation of atrial rhythm disturbances by combining the Kyoto‐Model and experimental findings on SACs in rat atrial myocytes. The simulation shows that the SACs trigger the firing of action potentials by depolarizing the cell membrane to reach the threshold for the activation of voltage‐gated sodium channel and that the firing ceases when a significant rise in intracellular sodium concentration develops. The simulation also shows that the delayed increase in the intracellular calcium concentration triggers the calcium‐induced calcium release and that the subsequent activation of Na+/Ca2+ exchanger drives spontaneous firing of action potentials. These simulation results successfully demonstrate how the SACs can drive the generation of stretch‐induced arrhythmias such as atrial tachycardia and atrial fibrillation.
Ranolazine attenuates hydrogen peroxide‐induced increases in late sodium‐channel open probability and open time
Authors: Ma Jihua , Song Yejia , Wang Weiping , Zhang Peihua , Shryock John C , Belardinelli Luiz
Published: June 2021

Hydrogen peroxide (H2O2) increases late sodium current (INa) and is implicated in ischemia/reperfusion injury to myocardium. Inhibition of late INa is a mechanism underlying the cardioprotective effect of ranolazine (Ran). The objective of this study was to examine the hypothesis that Ran attenuates H2O2‐stimulated late INa by reducing the open probability and open time of Na+‐channel late openings. Single‐channel INa of guinea pig ventricular myocytes was measured using the cell‐attached patch‐clamp technique. H2O2 (300 μmol/L) significantly increased the mean open probability and open time of late Na+‐channel openings from 0.013±0.001 to 0.107±0.004 and from 0.689±0.015 to 1.486±0.016 ms, respectively. When tetrodotoxin (2 μmol/L) was included in the pipette solution, H2O2‐induced late channel openings were completely inhibited. The stimulatory effect of H2O2 on both open probability and open time was attenuated by Ran in a concentration‐dependent manner. Ran at 3 and 6 μmol/L significantly reduced H2O2‐induced increase of mean open probability by 60±3% and 95±1%, respectively, and H2O2‐induced increase of mean open time by 31±8% and 82±2%, respectively. The attenuations of H2O2‐induced late Na+‐channel openings by Ran at 3 and 6 μmol/L were comparable to those of lidocaine at 30 and 60 μmol/L. In conclusion, at concentrations within its therapeutic range, Ran attenuates H2O2‐induced late INa by reducing the open probability and open time of late Na+‐channel openings.
Duration of ventricular fibrillation affects first post‐shock activation time and defibrillation threshold
Authors: Dosdall Derek James , Osorio Jose , Robichaux Robert , Huang Jian , Ideker Raymond E
Published: June 2021

Background: Defibrillation waveforms and strategies have been developed using models of short duration ventricular fibrillation (SDVF). Significant metabolic changes occur after long duration ventricular fibrillation (LDVF) that may play an important role in the mechanisms of defibrillation. The objective of this study was to determine if defibrillation threshold (DFT) or the time to 1st post‐shock activation (PSA) differed after SDVF and LDVF.

Methods: A ramp up protocol was used to determine the defibrillation threshold (DFT) of a biphasic waveform in 9 pigs (30–35 kg) and 6 beagles (9–11 kg) after one episode each of SDVF (10 s) and LDVF (150 s). PSA time was recorded by intraventricular catheters in the both ventricles after successful defibrillation shocks.

Results: The DFT was higher for pigs after SDVF than LDVF (102±43 J and 79±34, respectively, p<0.05) but was not significantly different for beagles (8.3±1.5 and 13.3±9.3 J, respectively). The majority of the PSA after SDVF were recorded within 170 ms of the shock, (9/9 pigs, 4/6 beagles), while all PSAs after LDVF had a pause of greater than 275 ms.

Conclusion: Different times of PSA and a change in DFT in pigs may indicate that there are significant differences in defibrillation after SDVF and LDVF. Further study of defibrillation after LDVF is warranted to determine if strategies and waveforms may be improved for clinically relevant durations of LDVF.
Parvalbumin immuno‐reactive somata in mouse barrels display a wide range of thalamocortical input
Authors: White Edward L. , Weinfeld Elizabeth
Published: June 2021

Electrophysiological studies indicate that parvalbumin (PV) containing neurons are fast spiking and strongly excited by thalamocortical (TC) afferents (Kawaguchi and Kabota, J. Neurophysiol 70:337, 1993; Beierlein et al. J. Neurophysiol 90:2987, 2003; Swadlow, 25:32, 2003; Bruno and Simons, J. Neurosci. 22:10966, 1975). This is a serial thin section analysis of synapses between degenerating TC afferents and PV‐labeled, non‐spiny neurons in layer IV of mouse PMBSF cortex in which lesion induced degeneration has proven reliable for quantification of TC synapses (White, 1989). 12 PV‐labeled somata (4 mice), of various shapes and sizes, received from 0 to 0.0544 TC synapses/μm2 of somatic membrane examined. We suggest that the PV‐labeled somata clustered at the high end of the range are equivalent to the FS cells described by Sun et al. (J. Neuroscience 26:1219, 2006) that, strongly excited by TC afferents, go on to provide powerful feedforward inhibition of spiny stellate cells. The remaining PV‐labeled somata formed few TC synapses, consistent with the contention (Porter et al., J. Neurosci 21:2699, 2001) that no clear relationship exists between the amount of TC input and the type of calcium binding protein within the postsynaptic neuron. Information on the numbers of TC synapses formed with the dendrites of PV‐labeled cells is needed for a more complete picture. Supported by the generosity of Suzanne B. Zlowtowski
Adrenergic receptors are differentially expressed in the rat cochlea
Authors: Khan Khalid Mohammad , Drescher Marian , Drescher Dennis , Hatfield James
Published: June 2021

Norepinephrine and nerve fibers containing dopamine β‐hydroxylase are present in the organ of Corti (OC) (ARO Abstr. 26: 12, 2003; 28: 226, 2005). However, the identity and localization of adrenergic receptors (ARs) in the cochlea are uncertain. Here we present the immunolocalization of α1, β1, and β2 AR subtypes in the cochlea of rat. For the α1 AR subtype, immunoreactivity (IR) was detected overlapping apical and basal sites of the inner hair cells (IHCs) in the basal turn, but no IR was detected in relation to the IHCs in the middle and apical turns. IR was observed in close association with the outer hair cells (OHCs) in middle and apical turns, occasionally overlapping Deiters’ cells. For the β1 AR subtype, IR was detected in close association with the IHCs in all turns. IR was also present in presumed nerve fibers at the base of the OHCs, but the OHCs themselves appeared devoid of IR. For the β2 AR subtype IR was associated with IHCs. IR was detected around the basal poles of the OHCs in the middle and apical turns, possibly associated with nerve fibers in all three turns. Within the spiral ganglion, type I afferent cell bodies showed IR for α1 and β1 ARs, whereas a subpopulation of cell bodies exhibited IR for the β2 AR. To our knowledge, this is the first report localizing α1 and β2 AR IR in the OC. ARs in the cochlea may modulate afferent neural activity, control OHC contractility, monitor intracellular Ca2+, and K+ transport.
Antifibrotic effect of globular adiponectin in human hepatocyte
Authors: Huh Joo Young , Ha Hunjoo
Published: June 2021

Adiponectin is a secreted 30‐kDa protein, which is mainly produced by adipose tissue. Adiponectin exhibits insulin‐sensitizing metabolic effects and anti‐atherogenic effects through its receptors (adiponectin receptor 1 and 2). Hypoadiponectinemia has recently been reported in liver cirrhosis. Further, adiponectin receptor 2, but not receptor 1, expression was decreased during the development of liver fibrosis. There is increasing evidence that hepatocytes in addition to stellate cells may play an important role in the development and progression of liver fibrosis. Thus, the objective of the present study was to determine the direct effect of adiponectin on extracellular matrix (ECM) protein expression of HepG2 cells as a first step to investigate the role of adiponectin in liver fibrosis. HepG2 cells were incubated with 10 ng/mL TGF‐β1 in the presence of globular adiponectin (0‐5 μg/mL) for up to 48 hours. TGF‐β1 significantly increased fibronectin, collagen I, and collagen IV mRNA and protein expression. Globular adiponectin inhibited TGF‐β1‐induced ECM upregulation in a dose‐dependent manner. These results suggest that adiponectin may provide a novel therapeutic strategy for the attenuation of liver fibrosis. (Supported by R01‐2006‐000‐10829‐0 and R15‐2006‐020 from KOSEF and BK 21)
Tamm‐Horsfall protein binds TGF‐ß2
Authors: Rhodes Diana C.J.
Published: June 2021

The purpose of this study was to determine if Tamm‐Horsfall protein (THP), an abundant protein in urine, interacts with transforming growth factor‐beta (TGF‐ß). Binding between THP and TGF‐ß2 (Genzyme, Cambridge, MA) was evaluated by two techniques, an ELISA and a resonant mirror biosensor (IAsys, NeoSensors Ltd., Durham, UK). The ELISA experiments revealed binding between THP and TGF‐ß2 (KD≈3 × 10−6 M) that was inhibited by soluble THP in a dose dependent manner. In the IAsys experiments, TGF‐ß2 bound to immobilized THP with a KD≈1 × 10−7 M. This binding was specifically inhibited by soluble THP. The above experiments were performed with THP from one female volunteer. A single male THP sample bound TGF‐ß2 1000‐fold less strongly, even though both THP samples bound C1q (another THP ligand) similarly. In light of the significant role of TGF‐ß in the progression of renal fibrosis, the interaction between this profibrogenic factor and an abundant urinary protein, THP, warrants further investigation, including evaluation of biochemical differences between THP samples influencing TGF‐ß2 binding activities. (TGF‐β2 was a generous gift from Genzyme Corp., Cambridge, MA.)
The Effects of Aging and Caloric Restriction upon Rat CA3 Hippocampus: A Three‐Dimensional Ultrastructural Study
Authors: Donohue Howard Stephen , Linville Constance , Newton Isabel , Adams Michelle , Brunso‐Bechtold Judy
Published: June 2021

We have previously shown an age‐related decline in glutamate receptor subunit levels in hippocampal CA3 that is altered by caloric restriction (CR). Specifically, CR beginning at 4 months of age results in a decline in NMDA and AMPA receptor subunits by 10 months of age to levels that remain stable across life‐span. By 28 months of age, subunits in ad libitum (AL) fed rats have declined to levels significantly lower than observed in CR animals. Moreover, cognitive performance in 28 month‐old CR rats is significantly better than in age‐matched AL animals. Because NMDA and AMPA receptor subunit levels as well as neural function have been correlated with area and complexity of hippocampal excitatory synapses, we used serial 3‐D reconstruction to assess size and morphology of postsynaptic densities (PSDs) in CA3 of young‐AL, aged‐AL and aged‐CR male F344×BN rats. Preliminary analysis reveals no difference in mean PSD surface area among groups. We also are quantifying the effects of age and CR on the overall synapse population in CA3 as well as on those with functionally‐relevant morphologies such as perforated synapses and multiple spine boutons.
Glomerular cell density and number in OVE26 diabetic mice
Authors: Carlson Edward C , Teiken Jennifer , Kees Eric , Audette Janice , Laturnus Donna , Zheng Shirong , Epstein Paul N
Published: June 2021

We recently showed that in OVE26 transgenic diabetic mice, glomerular volume was increased while podocyte density (pNV) and number (pN) were reduced, particularly in older animals. In the current study, similar LM and TEM morphometric techniques were applied to OVE26 and age‐matched FVB control mice (60, 150, or 450 days) in an effort to determine whether endothelial and mesangial cell density (eNV, mNV) and number (eN, mN) also may be altered and how the data may relate to total glomerular cell density and number. At 60 days, mNV, eNV, mN, and eN were not statistically distinguishable in OVE26 and controls. Likewise, at 150 days, eNV and mNV were not significantly altered in the diabetics. However, at 150 days, both mN and eN were significantly increased (~143% and ~62% respectively), and in 450 day‐old diabetic animals, mNV, mN and eN were all significantly increased (~78%, ~179%, and ~71% respectively) relative to age‐matched FVB mice. These data indicate that in older OVE26 mice, mesangial and endothelial cells numbers increase leading to significant increases in total glomerular cell number, and that glomerular hypertrophy is accompanied by both mesangial and endothelial cell proliferation.
Developmental dynamics of pelvis formation in the chick
Authors: Pomikal Christine , Günther Johannes , Wicke Waltraut , Streicher Johannes
Published: June 2021

While the knowledge about body axis and limb development especially in terms of pattern formation and genetic background has enormously grown, the establishment of the axio‐appendicular junction ‐ the development of the girdles ‐ is vaguely comprehended. The present study examines pelvis morphogenesis in the chick, with respect to axio‐appendicular linkage, origin and number of elements, and the attachment process. Specimens from developmental stage HH1 27 to 34 were serially sectioned, 3D‐reconstructed and analyzed2. In contrast to amphibians3 and mammals4 the chick pelvis ab initio locates close to the body axis, although the primary skeletal element in contact ‐ the femur ‐ suggests an appendicular origin. The pelvis anlage does not reorient from the developmental to the final position as observed in other vertebrates3,4. Ossification commences later and follows a heterochronic pattern. The formation of the synsacrum, the synostotic junction with the ilium, and the omission of ventral closure significantly distinguishes pelvis formation in the chick. These result demonstrate substantial differences in the ontophyletic organization of axio‐appendicular linkage in vertebrates.
Cross‐linking histones and DNA to prevent transient site exposure
Authors: Fukuto Jennifer , Haushalter Karl A
Published: June 2021

Although the mechanism of base excision by DNA glycosylases has been well‐studied in free DNA, it is unknown how these DNA repair enzymes gain access to DNA damage densely packed in chromatin. One possibility is that DNA glycosylases gain access to nucleosomal DNA when it spontaneously unwraps from the histone octamer surface. To investigate this model, we are creating nucleosome core particles with DNA covalently bonded to the histone octamer via disulfide crosslinks to prevent transient site exposure. Recombinant histones have been prepared with appropriate cysteine substitutions and 3′‐thiol linker containing DNA has been synthesized. When the DNA and histones are cross‐linked together, the resulting complex can be used to determine how preventing dynamic unwrapping of the DNA affects the rate of base excision repair from nucleosome core particles.
RecF Recognizes DNA Damage in Concert with RecR
Authors: Makharashvili Nodar , Koroleva Olga , Korolev Sergey
Published: June 2021

The RecFOR recombination mediator complex detects single‐stranded gapped DNA and recruits RecA recombinase at the damage site through an unknown mechanism. Our structural and biochemical studies suggest that an interaction between a symmetric RecF dimer and an asymmetric broken DNA structure initiates recruitment of RecR and ‐O proteins to the site. We demonstrate that an ATP‐dependent dimerization of RecF is essential for its other activities, DNA binding and interaction with RecR. In the presence of ATP, RecF alone binds DNA relatively weakly. No preferential binding towards single stranded (ss)/double stranded (ds) DNA junctions was observed, while there was a slight preference for ssDNA. RecR drastically changes RecF's interaction with DNA. It substantially increases RecF's affinity towards dsDNA while affinity to ssDNA remains the same. RecR also alters RecF‐mediated ATP hydrolysis; the rate increases in the presence of ssDNA, while it decreases in the presence of dsDNA. Our findings suggest a dynamic multi‐step mechanism of damage site recognition by the RecFR complex as the first step in the recombination mediation reaction.
Studies of African Swine Fever Virus Polymerase X/DNA complexes in the presence of mismatched base pairs
Authors: Benitez Benedetta Alessandra Sampoli , Arora Karunesh , Balistreri Lisa , Schlick Tamar
Published: June 2021

The African Swine Fever Virus (ASFV) is an encapsulated deoxyvirus known to induce a lethal infection in domestic pigs. The double‐stranded DNA viral genome encodes a total of 151 proteins, including a minimal DNA repair system, with a repair polymerase, pol X, which catalyzes a single‐nucleotide filling in gapped DNA. Pol X, is the smallest naturally occurring DNA‐directed DNA polymerase described so far. It belongs to the X family of polymerases and shares sequence and structure similarity with human DNA polymerase β (pol β). However, where pol β is a moderate fidelity enzyme, pol X is much more error tolerant. The key feature of pol X is that the insertion rate of the G:G mispair is comparable to that of the four Watson‐Crick base pairs. Using molecular dynamics simulations (MD) we previously suggested an induced fit‐mechanism for pol X. The goal of this study is to understand at an atomic level, how pol X can accommodate mismatched base pairs more frequently compared to pol β. MD simulations of pol X/gapped DNA complexes in the presence of mismatched pairs were performed for 10.5 ns each using CHARMM package. Our results show that a conformational change, consistent with a transition from open to closed state, is achieved in the G:G mispair, only when the incoming nucleotide is in the syn configuration. Simulations results for other mismatched base pairs results will be presented and compared to the available kinetic data.
Poly(A)‐Binding Protein Affects the Kinetics of Tobacco Etch Virus Pseudoknot RNA Binding to Wheat germ Translation Initiation Factor eIF4F
Authors: Khan Mateen A , Gallie Daniel R , Goss Dixie J
Published: June 2021

Previous studies indicated that the pseudoknot (PK) of tobacco etch virus (TEV) RNA is necessary to promote cap‐independent translation (Zeenko and Gallie, 2005, J.B.C; 280, 26813‐26824). We investigated the effect of poly(A)‐binding protein (PABP) on the PK1 RNA binding to eIF4F in the absence and presence of eIF4B. Equilibrium studies of PK1 RNA binding to eIF4F showed ~ 2‐fold stronger affinity in the presence of PABP. Addition of PABP and eIF4B to the eIF4F enhances binding affinity ~3.5‐fold as compared to eIF4F binding to PK1 RNA. Binding of eIF4F·PABP and eIF4F·4B·PABP to PK1 RNA is both enthalpy and entropy‐favorable. Further, kinetic analysis of eIF4F, eIF4F·PABP and eIF4F·4B·PABP with PK1 RNA and the temperature dependence of this reaction were measured and compared. The stopped‐flow fluorescence anisotropy data showed that the observed rate constant for the binding of PK1 RNA increased linearly with an increase in eIF4F, eIF4F·PABP or eIF4F·4B·PABP initiation factor concentration. The association rate constant (kon) for PK1 binding was ~ 2.8‐fold faster for eIF4F·4B·PABP than eIF4F. The dissociation rate constant (koff) was ~1.5‐fold slower for eIF4F‐PK1 in the presence of eIF4B and PABP. The activation energy of the PK1 RNA binding reaction was markedly reduced in the presence of PABP and eIF4B. The enhancement of kon, lowering of koff, and reduction in energy barrier for the formation of eIF4F·4B·PABP‐PK1 complex suggests a more stable platform for efficient translation of protein synthesis. These results demonstrate the first direct kinetic measurements of plant translation initiation factors binding to an internal ribosome entry site (IRES).
Ferritin L and H subunits are differentially regulated on a post‐transcriptional level
Authors: Sammarco Mimi C. , Ditch Scott , Banerjee Ayan , Grabczyk Ed
Published: June 2021

Ferritin plays a key role in the storage and release of iron. The protein is a multimer composed of 24 ferritin L (FTL) and ferritin H (FTH) subunits, in ratios that vary in different cell types. Because the subunits are not functionally interchangeable, both L and H units are critical for maintaining iron homeostasis and protecting against iron overload. FTL and FTH are regulated primarily at a post‐transcriptional level in response to cellular iron. Individual regulation of FTL and FTH is of much interest, and although transcriptional differences between FTL and FTH have been shown, differences in their post‐transcriptional regulation have not been evaluated. We report here that FTL and FTH are differentially regulated in 1% oxygen on a post‐transcriptional level. We have designed a novel cell‐based assay system, sensitive enough to detect differences between FTL and FTH iron regulatory elements (IREs) that a standard electrophoretic mobility shift assay does not. The FTL IRE is the primary responder in the presence of an iron donor in hypoxic conditions, and this response is reflected in endogenous FTL protein levels. These results provide evidence that FTL and FTH subunits respond independently to cellular iron concentrations and underscore the importance of evaluating FTL and FTH IREs separately.
Characterization of Pokeweed Antiviral Protein's interaction with eukaryotic initiation factors and an S/R loop oligo
Authors: Friedland Diana Elaine , Goss Dixie J. , DeGrazia Jeannine , Baldwin Amy E.
Published: June 2021

Plant viruses have numerous invasion strategies thus plants have devised ways to continuously defend themselves against pathogenic attack. Ribosome‐inactivating proteins (RIPs) are naturally occurring cytotoxic N‐glycosidase agents and are found in numerous species. They inhibit protein synthesis by depurinating A4324 on the conserved sarcin/ricin loop (SRL) of the 28S rRNA. Pokeweed Antiviral Protein (PAP) is a type I RIP that has long been recognized as an antiviral agent. It is also able to inhibit infection without loss of host cell translation.

Here we report in vitro fluorescence studies of PAP‐eIFiso4G binding revealing an interaction with a Kd of approximately 100 nM. This interaction enhances the cap binding affinity of PAP 3‐fold. Our experiments have shown that PAP binds to cap with a similar affinity to that of the wheat germ cap binding protein eIFiso4E. ATP and GTP compete with cap for PAP binding; both nucleotides and the cap analog show little salt dependence in binding, suggesting that the interactions are specific for these nucleotides. Binding to an oligonucleotide that contains the S/R sequence was slightly weaker than cap binding. We have also determined that PAP directly interacts with eIFiso4E. The biological significance of PAP‐cap binding remains unclear, however this may serve as a means to target RNA or, since ATP binds to the same site, this may be a protective mechanism for host cell capped RNA. The interaction with initiation factors suggests that additional proteins may aid PAP in accessing particular RNA and distinguishing structural elements of the RNA.

This work was supported in part by a Research Opportunity Award (NSF MCB 0413982 and a CUNY Collaborative Incentive Research Grant 80209‐07‐12).
The URE2 IRES of yeast: physical characterization and regulation by eIF2A
Authors: Merrick William C. , Reineke Lucas C. , Cao Yu , Caprara Mark G.
Published: June 2021

Previous studies established that the cellular URE2 mRNA contains an IRES element in the region encoding the N‐terminus of Ure2p. We have determined the minimal IRES element for this mRNA. It is a stem of about 10 base pairs and with a loop of about 24 nucleotides. The size of the loop is a determinant of efficiency with smaller loops leading to reduced levels of expression. To date, all mutations that increase the stability of the stem reduce the level of expression. At the same time, limited mutations that destabilize the stem also show reduced expression. Thus, it would appear that an optimal balance of stability has been selected naturally in yeast.

All of the IRES constructs tested are effectively repressed by eIF2A. We conclude that the regulation by eIF2A is not due to any cis‐acting element in the mRNA, but rather to the process of internal initiation. We are currently attempting to determine if there are trans‐acting factors that influence the expression from the URE2 IRES element or other portions of the URE2 mRNA that enhance or repress IRES‐mediated expression. The regulation of eIF2A is also being examined. Studies of the eIF2A mRNA indicate that most/all stresses lead to a dramatic reduction in eIF2A mRNA levels. However, we do not see uniform reductions in eIF2A protein and are currently evaluating whether post‐translational modifications may be associated with those circumstances. (NIH GM068079 and T32 GM08056)
Remodeling in the Developing Lung – Role for the EGF Receptor
Authors: LeCras Tim D.
Published: June 2021

The alveolar phase of lung development occurs predominantly after birth in humans and rodents. Studies in animal models have shown that insults that impact hormonal and growth factor signaling in the neonatal lung can disrupt morphogenesis and cause alterations in lung structure and function that persist into adulthood. Clinical data in humans that exemplify this include children with bronchopulmonary dysplasia (BPD) and asthma. Long‐term outcome studies have suggested that even infants with mild BPD, or who recover from BPD, may be at increased risk for asthma and chronic obstructive pulmonary disease later in life. Children with asthma have a more rapid decline in lung function as they age compared to normal individuals. While the pathogenesis is unclear, lung remodeling is thought to play an important role in declining lung function in these diseases. Clinical studies have associated increases in the EGF receptor (EGFR) and its ligands, including TGF‐α, with lung remodeling in pediatric and adult lung diseases, including BPD and asthma. Work from our group has shown that elevated EGFR signaling in lung specific TGF‐α transgenic mice can disrupt pre‐ and postnatal lung morphogenesis and cause fibrosis and remodeling. Even transient increases in EGFR signaling during the alveolar phase disrupted lung morphogenesis and caused chronic lung disease.
Post‐natal age enhances somal calcium responses of peripheral chemoreceptor neurons in response to extracellular ATP
Authors: Nunes Ana Rita , Gauda Estelle B. , Donnelly David F
Published: June 2021

ATP, acting through P2X receptors, mediates hypoxia‐induced excitation in the carotid body (CB). The magnitude of excitation increases over the first 3 weeks after birth. We hypothesized that this increase is due, in part, to enhanced responses of P2X receptors. The CB was harvested, intact, with the petrosal ganglion and chemoreceptor neurons of SD rats aged P12‐P17 (n=5, younger) and P20‐24 (n=2, older). Chemoreceptor neurons were labelled by applying calcium green dextran crystals (3 Kd, Molecular Probes) to the CB. The change in fluorescent (°F) from baseline in response to increasing concentrations of ATP was obtained for 57 and 36 neurons in the younger and older animals, respectively. At each concentration of ATP, the percent of positively responding cells was greater for the older than the younger animals: 45% vs 30% at 250 μM; 67% vs 21% at 500 μM; and 34% vs 6% at 1000 μM of ATP. Furthermore, the magnitude °F was greater in the older vs the younger animals with 11 vs 5% of the cells, having > 20% °F in response to 500 μM of ATP and 11 vs 3% in response to 1000 μM of ATP in the older vs younger age group, respectively. The °F in response to ATP was blocked by pre‐incubation with suramin (0.1 μM) nonspecific blocker, or TNP‐ATP (0.5 μM ) PX3 receptor blocker. Thus, a greater sensitivity of the P2X receptors on chemoafferents to ATP may in part account for maturation of hypoxic chemosensitivty.

NIH HL072748; SFRH / BD / 39473 / 2007