bluuebunny/crossref_metadata_2025 huggingface dataset:
Neuropeptide Y and age‐related development of hypertension in the rat
Authors: Mattar Louis M , Jackson Dwayne N , Shoemaker J Kevin
Published: June 2021

Essential hypertension is associated with increased sympathetic nerve activity in both animals and humans. We tested the hypothesis that neuropeptide Y (NPY) Y1 receptor activation was involved in the development of hypertension. In vivo infusions of NPY (15μg/kg) and Phenylephrine (PE, 10nmol/kg) to the intact hindlimb of spontaneously hypertensive rats (SHR) and their genetic control Wistar‐Kyoto (WKY) rats were performed in animals of 3, 5 and 7 weeks of age (n = 2–3 animals per strain per age). Femoral blood flow, blood pressure and heart rate were measured, while hindlimb vascular conductance (CHL), were calculated as an indicator of vascular responsiveness. Following phenylephrine (PE) infusions, CHL in the 7 week old SHR and WKY animals fell by 58.5% and 49.2% respectively (p<0.05). The response remained depressed in both strains for at least 100s (p<0.05). Compared with baseline, NPY infusion produced a 69% fall in CHL in the SHR that remained depressed for 100s (p<0.05). No effect of NPY was observed in the 7 week WKY and there was minimal response to NPY in either the SHR and WKY animals at 5 or 3 weeks. Neither PE nor NPY affected CHL in the 3‐week old animals of either strain. Thus, while PE elicited similar responses in the SHR and the WKY animals, the sensitivity to exogenous NPY became pronounced with age in the SHR. These data provide the first evidence that the development of hypertension in the SHR involves an upregulation of the NPY system.
Kinetics difference of regional sodium content change between medulla and cortex of kidney after furosemide injection
Authors: Liu Haiying , Kohler Martin G , Shen Xiaolan , Garcia Maria L , Alonso‐Galicia Maggie , Priest Birgit T , Williams Donwald S , Hargreaves Richard , Kaczorowski Gregory J
Published: June 2021

3D sodium images of living rat kidneys were obtained at 9.4 Tesla to track its regional kinetics in response to diuretics. In each experiment, diuretics infusion was started when the stable physiological condition was achieved under anesthesia (isofluorane), during which sodium images were continuously collected. Initial results showed that there existed a large corticomedullary sodium gradient under normal physiological condition and after administration of furosemide (6ug/min, BW=300g) this gradient was significantly attenuated within 10min. Also, we found that the sodium content collapsed much more quickly in medulla than in cortex, with characteristic time constants: 8.4+/−4.3 and 95+/−25mins (p<0.0027). Corresponding normalized changes of sodium signals in medullar and cortical regions are found to be: −0.71+/−0.02 and 1.41+/−0.07. In conclusion, in vivo experiments using furosemide given at clinically relevant doses through relatively slow infusion have revealed that sodium contents in medullary and cortical regions of rat kidney exhibited different temporal kinetics. These results are consistent with the mechanism of action of furosemide, i.e. inhibition of Na/K/2Cl co‐transporter in the thick ascending limb of Henle, which is responsible for establishing and maintaining a large sodium gradient across the kidney in the corticomedullary direction.
Renin‐Angiotensin System and NAD(P)H Oxidase Activation in the Rostral Ventrolateral Medulla are involved in the Further Blood Pressure Elevation on High Sodium Diet in Spontaneously Hypertensive rats
Authors: Koga Yasuaki , Hirooka Yoshitaka , Araki Shuichirou , Konno Satomi , Kishi Takuya , Sunagawa Kenji
Published: June 2021

Background: High salt diet (HS) causes sympathetic hyperactivity and increases blood pressure (BP) in spontaneously hypertensive rats (SHR). We previously demonstrated the reactive oxygen species (ROS) in the rostral ventrolateral medulla (RVLM) was increased more in SHR on a HS (8%) than a regular salt diet (0.5%: RS). This study aimed to elucidate whether the increased ROS in the RVLM of HS‐SHR is associated with central NAD(P)H oxidase activation and the pressor response on an HS diet is involved in renin‐angiotensin system (RAS) activation.

Methods and Results: Male SHR were fed an HS or RS diet for 6 weeks. Systolic BP was significantly higher in HS‐SHR than in RS‐SHR (244±15 mmHg vs 188±20 mmHg at 12 weeks old, n=8, P<0.05). The angiotensin II type1 receptor (AT1R) protein expression in the RVLM was significantly increased more in HS‐SHR than in RS‐SHR (5.1±0.5 vs 3.4±0.3, n=6, P<0.05). The AT1R antagonist (valsartan) microinjected bilaterally into the RVLM induced a significantly greater BP depression in HS‐SHR than in RS‐SHR (Δ MAP/basal MAP: −13.2±2.1 % vs −20.9±2.2% n=5, P<0.05). NAD(P)H oxidase activity was significantly higher in HS‐SHR than in RS‐SHR (1.53±0.10 vs 1.25±0.10, n=9, P<0.05).

Conclusions: These results suggest that the pressor response of SHR on an HS diet is mediated by RAS activation and the ROS increase is involved in the enhanced NAD(P)H oxidase activation in the RVLM.
NADPH oxidase‐derived reactive oxygen species regulate Ang II‐induced contractions via p38/MK2 pathway in the mouse mesenteric artery
Authors: Martinka Peter , Schmidt Sebastian , FÃ¤hling Michael , Steege Andreas , Gaestel Matthias , Persson Pontus BÃ¶rje , Patzak Andreas
Published: June 2021

NADPH‐oxidase (Nox) is activated by Ang II and plays a significat role in the generation of reactive oxygen species (ROS) in the vessels. It is however not known whether, vice versa, an increased ROS influences Ang II signalling, specifically in regard to its contractile properties. It is known that Ang II and ROS activate p38/MK2 pathway. Therefore, we hypothesize that increased oxidative stress in the VSMC activates p38/MK2 pathway and influences Ang II contractions. We increased ROS by increasing NADPH, the substrate for Nox, and analyzed whether the effects are age dependent. Small mesenteric arteries (SMA) from young (65 d) and older (130 d) mice were used for the isometric contraction studies. Ang II contractions were similar in both groups. NADPH treatment increased dose‐response (DR) contractions to Ang II only in the older group. L‐NAME + NADPH co‐treatment shifted the DR‐curves in the older group to the left. Inhibition of p38 MAPK and using MK2 −/− mice resulted in a significant reduction of the NADPH‐induced increase of the Ang II contractions. In conclusion, Nox‐derived ROS influences Ang II responses of the SMA in older mice. p38/MK2 pathway plays a critical role in this concept.
20‐HETE contributes to hypertension in postmenopausal rats.
Authors: Yanes Licy L. , Iliescu Radu , Sartori‐Valinotti Julio C. , Romero Damian G. , Stec David E. , Reckelhoff Jane F.
Published: June 2021

20‐HETE, a major metabolite of cytochrome P450 (CYP)‐4A, is a potent vasoconstrictor of renal arterioles, and has been shown to mediate hypertension in several animal models. The present study tested the hypothesis that 20‐HETE is involved in mediating hypertension in an animal model of postmenopausal hypertension, the old female spontaneously hypertensive rat (OF‐SHR). A CYP4A inhibitor of 20‐HETE production, 1‐aminobenzotriazole (ABT; 50 mg/kg per day i.p.), or vehicle, was administered to OF‐SHR, aged 16 mos, and young female SHR (YF‐SHR), aged 3 mos, (n=3–4 per group). Mean arterial pressure (MAP) was measured by radiotelemetry. ABT decreased MAP in OF‐SHR (164±5 vs. 151±6 mm Hg, p<0.05), whereas it had no effect in YF‐SHR (138±3 vs.136±1 mm Hg). CYP4A (isoform 2) mRNA expression was measured by real‐time RT‐PCR in kidney cortex and medulla of OF‐SHR and YF‐SHR. CYP4A2 mRNA expression was significantly upregulated in kidney cortex of OF‐SHR compared to YF‐SHR. In other studies administration of a specific 20‐HETE inhibitor, HET0016, also significantly decreased MAP in OF‐SHR. In summary, administration of the CYP4A inhibitor and a specific 20‐HETE inhibitor markedly attenuated hypertension in postmenopausal rats. Increases in renal vascular 20‐HETE constitute a new pathway that may be involved in mediating postmenopausal hypertension. (Supported by HL 51971).
D1 Dopamine Receptor Down‐regulates NADPH Oxidase Activity via a Protein Kinase C and Protein kinase A Cross‐talk in Human Kidney Cells
Authors: Han Weixing , Arnaldo Francis B , Jose Pedro A , Yu Peiying
Published: June 2021

D1‐like dopamine receptors inhibit NADPH oxidase activity via stimulation of protein kinase A (PKA) and inhibition of phospholipase D. We tested the hypothesis that a cross‐talk between PKA and protein kinase C (PKC) is involved in the regulation of NADPH oxidase activity in HEK‐293 cells heterologously expressing human D1 dopamine receptor (hD1R) (n=6/group). The D1‐like receptor agonist, fenoldopam (Fen., 1 μM), decreased NADPH oxidase activity (63±5.0% vs. vehicle=101.6±6.2%) that was completely blocked by the D1‐like receptor antagonist, Sch23390 (5μM) (84±6.2%), which, by itself, had no effect. The PKA inhibitor, H89 (10 μM), by itself, had no effect on NADPH oxidase activity but prevented (85.6±5.0%) the inhibitory effect of fenoldopam (P<0.01, vs. others, ANOVA Newman‐Keuls test). The D1R‐mediated decrease in oxidase activity (65±3.0%) was also completely reversed by PKC inhibitors, e.g., bisinolylmaleimide I (1 μM)(94±6.0%) and staurosporine (10 nM) (93.4±7.8%), which, by themselves, had no effect (P<0.001). Moreover, the PKC activator, phobol‐12‐myristate‐13‐acetate (PMA,1 μM), inhibited oxidase activity (72.7±8.1%), that was accompanied by increased PKCθS647 phosphorylation, effects that were also abolished by H89 (103±7.4%), which, by itself, had no effect (P<0.05). We conclude that D1R inhibition of NADPH oxidase activity is dependent on both PKA and PKC (PKCθS647) signaling. Our studies demonstrate for the first time that a cross‐talk between PKA and PKC signaling pathways plays an important role in the D1R negative regulation of NADPH oxidase activity in human kidney cells.
Oxidative Stress mediates soluble Flt‐1 induced vascular dysfunction in pregnant rats
Authors: Colson Drew , Gilbert Jeffrey S , Bridges Jason , Dukes Matthew P , Babcock Sara A , Ryan Michael J , Granger Joey P
Published: June 2021

Recent evidence indicates that both increased oxidative stress and an altered balance between pro‐ and anti‐angiogenic factors such as vascular endothelial growth factor (VEGF) and soluble VEGF receptor Flt‐1 (sFlt‐1) contribute to hypertension in preeclampsia. Thus, we hypothesized that infusion of sFlt‐1 to levels that mimic preeclampsia would increase blood pressure, and vascular and placental oxidative stress, and cause vascular endothelial dysfunction in the pregnant rat. sFlt‐1 infusion elevated plasma sFlt‐1 concentration (299±33 vs. 100±16 pg/ml; P<0.01) three fold and increased blood pressure (117±6 vs. 98±4 mm Hg; P<0.01) when compared to saline infused rats. sFlt‐1 infusion also increased superoxide in the placenta (222 ± 71 vs. 40 ± 9 RLU/min/mg; P < 0.05) and the vasculature (34 ± 8 vs. 12 ± 5; P < 0.05) compared to the vehicle infused controls. Wire myography revealed that percent relaxation of vascular rings to acetylcholine and sodium nitroprusside were both decreased (P<0.05) in the sFlt‐1 infusion group compared to the vehicle infusion group and this effect was attenuated by the superoxide dismutase mimetic Tiron (P<0.05). These data indicate that hypertension associated with elevated maternal concentrations of sFlt‐1 during pregnancy may be mediated, in part, by impaired vascular function as a result of increased oxidative stress.
Increased sensitivity to vagal stimulation in acutely overfed and chronically obese rabbits
Authors: Wilson Ana Kaye , Caffrey James L. , Carroll Joan F.
Published: June 2021

Parasympathetic modulation of heart rate (HR) declined immediately after initiation of an ad lib high fat diet (HFD), and remained depressed during 6–12 wks of developing obesity. Contributions from altered vagal transmission and end organ sensitivity were evaluated following 1 wk or 12 wks of HFD. HR responses were recorded during graded a) vagal nerve stimulations and b) left ventricular methacholine (MCh) infusions. After 1 wk of HFD, vagally mediated bradycardia at 8 Hz was 37.3% greater in overfed rabbits compared to lean controls (−110.9 ± 11.4 vs −80.8 ± 14.1 bpm, respectively, p=NS). After 12 wks of HFD, the bradycardia was 65.8% greater in the obese animals (−111.4 ± 13.9 vs −67.2 ± 8.70 bpm, p ≤ 0.05). MCh infusions were also consistently more effective in the obese rabbits. MCh reduced heart rate more in obese rabbits than in lean controls, with differences of 16.2 and 80.5 bpm, respectively, at low and high doses (0.5 and 3.0 μg/kg). The findings support the hypothesis that obesity lowers parasympathetic transmission and induces a compensatory increase in available neurotransmitter and/or cholinergic sensitivity within the sinoatrial node.
RhoA/ROCK signaling in substrate stiffness control of neonatal rat cardiomyocyte maturation
Authors: Jacot Jeffrey G , McCulloch Andrew D , Omens Jeffrey H
Published: June 2021

Cardiac myocytes mature and adapt to a changing mechanical environment in the first postnatal week. To test the hypothesis that the mechanical stiffness of the extracellular matrix affects this maturation, we plated neonatal rat ventricular myocytes (NRVMs) on polyacrylamide gels with elastic moduli varying from 1 to 50 kPa and cultured them for 7 days. We found that NRVMs generated greater mechanical force, had larger calcium transients, higher sarcoplasmic reticular (SR) calcium loads and greater expression of SERCA2a in cells grown on gels with an elastic modulus of 10 kPa (which is closest to the native resting myocardium) than on stiffer or softer substrates. Inhibition of the RhoA/ROCK pathway with hydroxyfasudil resulted in significantly greater force generation and calcium transients in NRVMs on the stiffest gels, but did not significantly affect NRVMs on soft gels. We conclude that extracellular elastic modulus affects the functional maturation of NRVMs, as measured by contractile force and calcium handling, and that activation of the RhoA/ROCK pathway is necessary for stiffness dependence on substrates stiffer than the native myocardium.
A novel approach to mobilize bone marrow stem cells with CXCR4 expression for enhanced tissue repair in rat cardiac allograft
Authors: Wang Yigang , Zhang Dongsheng , Zhao Tiemin , Xu Meifeng , Ashraf Muhammad
Published: June 2021

We tested whether bone marrow stem cells (BMC) overexpressing CXCR4 have higher migration potential. Female rats were lethally irradiated and substituted with male BMC with null/GFP (G1), or BMC overexpressing CXCR4/GFP (G2), or BMC treated with miRNA targeting CXCR4 (G3). After 30 days, donor hearts were transplanted into abdomen of irradiated rats and subjected to LAD occlusion. Following cardiac function was determined; hearts were harvested to analyze CXCR4, Nkx2.5, GATA‐4, GFP and Y‐chromosome positive cells. CXCR4+ cells were mobilized in the circulation in G2. CXCR4 was strongly expressed around the perivascular area in the transplanted hearts in G2. Myocytes and newly formed vessels expressing Y‐chromosome in donor heart were significantly increased in G2. These cells also expressed early cardiac markers in G2. Heart function was significantly improved in G2. Survival of the allografts was markedly increased in G2 (36.8 days) as compared with G1 (19.2 days), G3 (13.5 days). Donor heart function in G3 and upregulation of early cardiac markers were significantly reduced by miRNA. Overexpression of CXCR4 in BMC was extremely effective in mobilizing BMC to the damaged myocardium for myoangiogenesis and improved cardiac function.
Altered MAP kinases and Akt activation during compensated eccentric cardiac hypretrophy
Authors: Haddad Georges E. , Alvin Zikiar , Laurence Graham G. , Teos Leyla Y
Published: June 2021

The role of MAPK and PI 3K in the transduction of ANG II signaling during the development of cardiac hypertrophy was investigated. Eccentric cardiac hypertrophy was induced in adult rats by 3 weeks A‐V shunt. Hearts were retrogradely perfused for 20 min with either Tyrode alone (control), with ANG II (10−7M), or with respective kinase inhibitor (10−6M) in the presence or absence of ANG II (10−7M). Shunted hearts had a 33% increase in relative heart weight. Western blot analysis showed that shunted hearts had higher activation levels for ERK1 (56%), ERK2 (227%), p38 (75%) and Akt (67%). In normal hearts, ANG II increased ERK1, ERK2 and p38 MAPK phosphorylation by 57%, 196%, 94%, respectively. However in the hypertrophied hearts, ANG II did not. Treatment with either inhibitor for ERK1/2 (PD98059), p38 (SB203580) or Akt (LY294002) did not affect the activation level of the respective kinase in the normal hearts, but reduced it to sham ones in the hypertrophied hearts. In both groups, p38 MAPK inhibition induced a cross‐activation of ERK1/2, but reduced Akt activation in the shunt. However, Akt inhibition reduced ERK1/2 activation levels in the shunted hearts. Thus eccentric hypertrophy exhibits: (1) elevated MAPKs and Akt activity; (2) blunted activation of MAPKs to ANG II; and (3) altered cross‐activation between MAPK and Akt. This work was supported in part by grants GM08016‐36 from NIGMS/NIH and 2G12 RR003048 RCMI, NCRR/NIH.
Cardiovascular dynamics in healthy subjects with differing heart rate responses to tilt
Authors: Ramirez‐Marrero Farah A , Charkoudian Nisha , Zhong Liu , Eisenach John H , Joyner Michael J
Published: June 2021

Orthostatic stress such as head‐up tilt (HUT) elicits a wide range of HR and BP responses among healthy individuals. In this study, we evaluated cardiovascular dynamics of subjects with different HR responses to HUT but without autonomic dysfunction. We measured BP (brachial artery) and HR (ECG) during 5 min of 60° HUT in 76 healthy young individuals, while norepinephrine (NE) was assayed during the final 30 s. We then chose 30 individuals based on the extremes of HR responses to HUT: 15 with the highest (ΔHR≥ 20 bpm) and 15 with the lowest response (ΔHR ≤10 bpm). Age and BMI were not different between groups; however, gender distribution was different (high: 5F, 10 M; low: 10F, 5M). Peak HR during HUT was 87.2±10.4 bpm in the high and 69.1±14.1 bpm in the low group (P< 0.05). Baseline HR was not different between groups. High HR responders had lower SBP at baseline (120.9±9.4 vs. 129.4±11.0 mmHg) and during HUT (119.9±9.7 vs. 131.4±13.1 mmHg). High HR responders also had higher plasma NE response to HUT (ΔNE: 156.9±82.1 vs. 89.0±48.0 pg/ml; P< 0.05). Higher HR with lower BP during HUT is consistent with normal baroreflex mechanisms of integration. Although inter‐individual variability appears to be a fundamental part of cardiovascular regulation, the mechanisms of these differences and the gender discrepancy requires further investigation.

Supported by K23 RR17520, HL46493, NS32352, KL2 RR024151‐01.
Neural Precursor cells (NPCs) derived from cervical and upper thoracic spinal cord
Authors: Kim Yong‐Jung , Yoo Young‐Mi , Yoo Hyun‐TAE , Yoo Chan‐Jong , Lee Uhn
Published: June 2021

Spinal cord injuries result in cellular loss that accompanies the destruction of descending motor and ascending tracts and leads to permanent damage with limited prospects for spontaneous repair. Neural stem cells are promising alternative to fetal tissues for cell therapies, primarily due to their self‐renewal and pluripotentially. The use of human derived neural stem cells has the additional advantage of yielding translational information that is relevant to clinical therapies.

NPCs were taken from passages 5 through 13 and seeded into monolayer. The single cells began to cell division, growing into centipede shaped morphology at 5 days. When cells grew at approximately 80% confluence, the parts of cells formed a small cluster and became a large cluster through excellent cell division. Large clusters were center and slowly pulled adjacent cell and rolled up like carpet, and formed more lager cluster.
Effective Treatment of Smell Loss with Theophylline
Authors: Henkin Robert Irwin , Velicu Irina , Schmidt Loren
Published: June 2021

While patients with smell loss in the US number in the millions there has been no effective method to either evaluate or treat the majority of them. These patients exhibit several causes for their loss including post viral infection, head injury and allergic rhinitis. Their semll loss has been measured objectively by psychophysical techniques and subjectively by quantitative evaluation of loss. We described the major biochemical defect causing these sensory losses‐decreased cAMP and cGMP in nasal mucus; this defect inhibits growth and development of olfactory receptor cells. To improve patients’ sensory loss we attempted to correct their observed biochemical defect by increasing cAMP and cGMP though use of the phosphodiesterase inhibitor theophylline which we gave orally to 309 patients at doses of 200–600 mg for 2–8 months. Psychophysical measurements of smell function improved in over 70%. Subjective responses improved in only 144 (47%); among these responders were 27% who improved 51% and 20% who improved 84%. 165 (53%) did not improve‐nonresponders‐ who improved 0.5%. Responders had significantly less severe smell loss than did nonresponders but there were no differences in age or blood levels of theophylline. These results indicate that patients with smell loss of several etiologies with this biochemical defect can be treated effectively with theophylline to improve smell fucntion.
Neuronal Birth Dating in the Radial Nerve Cords of Holothuria glaberrima: A Search For Neurogenesis
Authors: Maldonado‐Soto Angel Ricardo , Miguel‐Ruiz Jose Enrique San , Garcia‐Arraras Jose Enrique
Published: June 2021

Echinoderms are able to regenerate various tissues when these have been injured or lost. Studies by our group have shown that these organisms can readily regenerate injured nerves. This study sets out to determine to what extent neurogenesis occurs when their nervous system is injured. Radial nerve cords (RNC) of sea cucumbers, Holothuria glaberrima, were transected. Animals were divided into groups, each of which was injected with BrdU during 1 of 8 weeks of regeneration. On day 62 post‐injury (dpi) all the animals were sacrificed, the injured nerves dissected, sectioned in a cryostat, and subjected to indirect immuno‐fluorescence. Proliferating cells were detected with a primary antibody against BrdU. Neuronal elements were detected using antibodies against the neuronal transcription factor Nurr1 or the neuropeptide GFSKLYFamide. Our results indicate that neurons are present in the injured nerve at 62 dpi. Cell division within the injured nerve was restricted to the first 3 weeks of regeneration. A number of the dividing cells gave rise to neurons. However, most BrdU‐labeled cells did not label as neurons. These results indicate that neurons arose in the regenerating RNC, with cell division accounting for a small percentage of these. Mechanisms other than division are thus involved. Future studies will focus on the elucidation of these mechanisms, as well as in the characterization of the cells involved.
A mathematical model of collective cell motility and pattern formation
Authors: Szabo Andras , Czirok Andras
Published: June 2021

Cell motility and its guidance through cell‐cell contacts is instrumental in vasculogenesis, when multicellular sprouts invade into avascular areas and eventually create a polygonal pattern. Sprout elongation depends on a continuous supply of endothelial cells, streaming along the sprout towards its tip.

The collective motility and cell patterning was studied in vitro with 2D cultures. When grown on a suitable ECM substrate at high cell density, non‐contact inhibited cells such as BAEC, MDCK or human gliomas all exhibit streaming – adjacent cells moving in similar directions and thereby creating multicellular streams. At lower cell densities many cell lines (C6 gliomas, C2C12 myoblasts, 3T3 fibroblasts) form multicellular linear arrangements, similar to the multicellular vasculogenic sprouts.

To explain these phenomena, we augmented the cellular Potts model with two features: (i) a cell polarization with an intrinsic persistence time, and (ii) an asymmetric cell‐cell attraction whereby surfaces of elongated cells become preferred adhesion substrates. We demonstrate that these two effects are sufficient to explain the observed abundance of streaming and sprouting behavior in cell cultures and in vivo.
Mechanistic Insights into the NEDD8 Conjugation Cascade
Authors: Schulman Brenda A
Published: June 2021

Post‐translational modification by ubiquitin‐like proteins (ublps) such as ubiquitin, NEDD8, ISG15, and SUMO has emerged as a predominant cellular regulatory mechanism, with important roles in the immune response, development, vesicular trafficking, cell division and cancer. How is a ublp coordinated with its particular targets? These ublps are attached to their targets by parallel but distinct enzymatic cascades that sequentially involve an E1 activating enzyme, an E2 conjugating enzyme, and an E3 ligase. The E1 enzyme sits at the top of the cascade and selects its ublp and activates the ublp C‐terminus by adenylation. The E1 then forms a thioester intermediate between the E1's catalytic cysteine and the ublp's C‐terminus and transfers the ublp to an E2's catalytic cysteine. Finally, an E3 facilitates transfer of the ublp from the E2 to the target. The ubiquitin pathway involves one E1, tens of E2s, hundreds of E3s, and thousands of protein targets with in diverse biological functions. By contrast, the NEDD8 involves one E1, one E2, and few E3s, ultimately directing the ublp NEDD8 to relatively few targets. We have exploited the minimalist nature of the NEDD8 pathway to simplify the identification of protein‐protein interactions specific for a particular ublp's conjugation cascade. Recent progress toward understanding selective conjugation of NEDD8 and other ublps will be discussed.
ERAD: mechanism and physiolgcial function
Authors: Hampton Randolph Y.
Published: June 2021

The HRD pathway degrades a large variety of misfolded lumenal and transmembrane ER proteins. We study how the normal protein Hmg2, an isozyme of HMG‐CoA reductase undergoes regulated HRD pathway degradation, to better understand both cellular measurement of sterol pathway activity and the mechanisms the underlie ERAD. Entry of Hmg2 into the HRD quality control pathway is regulated by cellular levels of the sterol pathway molecule FPP, and the Hmg2p transmembrane domain is required for FPP‐regulated degradation. The TMD has an embedded motif known as the SSD, or sterol sensing domain, found in mammalian HMGR and other sterol‐pertinent proteins. Although this motif is most often implicated in the recognition of of sterols, it is clear for Hmg2p that FPP mediated HRD pathway entry depends on the SSD, suggesting that the motif is required for recognition of this non‐sterol signal. To understand regulated Hmg2p ubiquitination, and subsequent extraction from the ER membrane we have devised in vivo and in vitro approaches to study the biochemical requirements and the underlying mechanisms. Both HRD dependent ubiquitination and Cdc48‐dependent retrotranslocation are tractable for in vitro analysis, and allow evaluation of current models and discovery of new features of regulated Hmg2p degradation. The status of these studies will be discussed and integrated into our current models of regulated ER quality control.
MAIN AND ACCESSORY RENAL ARTERIES ‐ A MORPHOLOGICAL STUDY
Authors: Lall Kumud , Dhar Puspha
Published: June 2021

The present work was undertaken to document the incidence of accessory renal arteries in the kidney specimens obtained from subjects of Indian origin.

Comprehensive dissection carried out in the dissection hall of Anatomy Department of All India Institute of Medical Sciences (India) on forty cadavers over a period of five years revealed a single main renal artery on either side in 80% of the specimens. The mean length of the main renal artery was 31.05 ± 12 and 25.0 ± 9.5 mm on the right and left side respectively. Multiple (accessory) renal arteries were observed in 20% of the specimens with unilateral anomaly (15%) being more commonly encountered than bilateral anomaly (5%). The mean length of the accessory renal artery was 36.4 ± 10 and 36.6 ± 11 mm on the right and left side respectively. The accessory renal artery when present, invariably crossed the anterior aspect of the ureter.

Familiarity about the possible variations in the renal arterial pattern are especially important for the personnel dealing wit kidney retrieval and transplantation, various endourologic procedures and innumerable interventional techniques. In the majority of such situations, it is the comprehensive knowledge of the renal arterial pattern which remains the key issue in determining the technical feasibility of surgical interventions as well as the post operative management.
Apoptotic/Recombinogenic Endonuclease G is Regulated by Promoter Methylation and Histone Acetylation.
Authors: Wang Xiaoying , Tryndyak Volodymyr , Apostolov Eugene O , Yin Xiaoyan , Shah Sudhir V , Pogribny Igor P , Basnakian Alexei G
Published: June 2021

Apoptotic endonuclease G (EndoG) is a DNase/RNase that produces double‐stranded DNA breaks and promotes recombination. EndoG can be induced by cytotoxic stimuli, however, the mechanisms of transcriptional regulation of this protein is unknown. The present study was initiated by the analysis of promoter sequences of all known human apoptotic endonucleases. This analysis revealed that EndoG is the only endonuclease that contains a CpG island, a potential site for DNA methylation, in the promoter region. We then compared three human prostate cancer cell lines and showed that EndoG was highly expressed in 22Rv1 and LNCaP, and not expressed in PC3 cells. The expression of EndoG in these cell lines inversely correlated with EndoG promoter methylation and directly correlated sensitivity of the cells to cisplatin or etoposide. The silencing of EndoG by siRNA decreased DNA fragmentation measured by TUNEL assay. The global hypomethylation by 5‐aza‐2′‐deoxycytidine induced EndoG mRNA and protein expression in PC3 cells and increased the cells' sensitivity to cisplatin and etoposide. In addition, TSA, a histone deacetylase inhibitor, induced EndoG expression in 22Rv1 cells, while it had no such effect in PC3 cells. These data indicate that EndoG is regulated by methylation of its gene promoter, and partially by histone acetylation, and that it is essential for DNA damage‐mediated cell death.
Partition of mismatch recognition between MutSα and MutSβ
Authors: TIAN LEI , Gu Liya , Wang Issac , Li Guo‐Min
Published: June 2021

DNA mismatch repair plays an important role in genome maintenance. MutS α (MSH2–MSH6) and MutS β (MSH2–MSH3) are two major mismatch repair proteins in mammalian cells that preferentially recognize base‐base and small (1‐2 nucleotides) insertion/deletion (ID) mispairs, respectively. Normally, cells maintain a MutSα:MutSβ ratio at ~ 10:1. Overexpressing MSH3 alters the MutSα:MutSβ ratio, leading to a mutator phenotype. To determine the molecular basis by which mispairs are differentially recognized by MutSα and MutS β, purified proteins were tested for interactions, independently or competitively, with a G‐T or an ID heteroduplex using gel shift and stopped‐flow kineticanalyses. Although MutS α bound both the G‐T and ID substrates, the affinity of the protein to the G‐T duplex is ~ 1.5‐fold higher than that to the ID duplex. Surprisingly, MutS β could also interact with the G‐T substrate, but the binding affinity was only ~ 1/3 of the activity that the protein had for the ID substrate. These relative affinities measured in these equilibrium binding and competition experiments were confirmed by direct measurement of association and dissociation kinetic constants by stopped‐flow spectroscopy. The order of binding activities among these proteins and DNA substrates is MutS β ‐ID > MutSα‐G‐T > MutSα‐ID > MutS β ‐G‐T. Our findings therefore explain why cells maintain a high MutSα:MutSβ ratio.
NF90 binds novel human non‐coding RNAs
Authors: Mathews Michael B , Parrott Andrew M
Published: June 2021

Proteins in the nuclear factor 90 (NF90) family of dsRNA binding proteins are abundantly expressed in a broad spectrum of vertebrate tissues and participate in many aspects of RNA metabolism. NF90, also known as DRBP76 and NFAR1, forms heterodimeric complexes with NF45 protein. We detected several small non‐protein‐coding RNAs that associate with NF90 in vivo. They include snaRs (small NF90‐associated RNAs), a novel family of RNAs that are unique to the African Great Apes and display a restricted tissue distribution in humans.

The genes for 3 of these RNA species are located within the proximal promoters of protein‐coding genes and are transcribed by RNA polymerase III (Pol III) in the opposite direction to their ‘host’ Pol II genes. snaRs are highly structured RNAs, ∼117 nucleotides in length. The snaR‐G1 and snaR‐G2 genes are in the proximal promoters of chorionic gonadotropin genes CG □ 1 and 2, respectively, in locations shared by chimpanzee orthologs. The gene encoding a novel tRNAimet precursor is 242 bp upstream of the 5′‐end of NF45 mRNA, in a highly conserved position in vertebrates. The binding of NF90 to a pre‐tRNAimet transcript whose gene resides in the NF45 promoter is suggestive of an intricate feedback control mechanism. We speculate that NF90 regulates the transcription of specific protein‐coding genes through interaction with small non‐coding RNAs transcribed by Pol III.

Supported by NIH grant AI034552.
Automated planar patch clamp: Multiple applications for miniaturized devices
Authors: Haarmann Claudia , Haythornthwaite Alison , Brueggemann Andrea , Stoelzle Sonja , Farre Cecilia , George Michael , Fertig Niels
Published: June 2021

The Port‐a‐Patch is a miniaturized patch clamp rig which uses a planar borosilicate glass chip for performing electrophysiological recordings. Suction applied from the underside of the chip is used to attract a single cell to the recording site without the need for optical visualization. The Port‐a‐Patch has been successfully used for whole cell, perforated patch, and cell attached recordings of a variety of ion channels with high success rates (typically 60‐80%). Fast application of ligand is possible using the external perfusion system. Recordings from recombinant GABAA receptors (α1β2γ2) expressed in HEK293 cells will be shown. Examples of voltage‐gated ion channels recorded using the Port‐a‐Patch will also be shown, including NaV1.5 stably and Shaker K+ transiently expressed in HEK293 cells. The effect of temperature on the amplitude and kinetics of hERG channels will also be presented.

One of the newest and most exciting aspects of planar patch clamping using the Port‐a‐Patch is the ability to perfuse the internal membrane of the cell, whilst maintaining a continuous recording during the solution exchange. The internal perfusion system for the Port‐a‐Patch allows the exchange of up to 8 different solutions. Exemplar traces will be shown for regulation of TRP V1 by internal as well as external regulators. Such ease with which the internal solution may be exchanged is a unique feature to Nanion products.
Association of ATP‐sensitive K+ channels with adenosine A1 receptors
Authors: Garg Vivek , Hu Keli
Published: June 2021

We have recently shown that ATP‐sensitive potassium (KATP) channels are largely localized to caveolin‐enriched microdomains. It is known that KATP channels in the heart are activated by stimulation of adenosine A1 receptors and adenosine A1 receptors are localized in the caveolae of cardiac myocytes. The present study was designed to determine whether KATP channels associate with adenosine A1 receptors and whether this interaction depends on intact caveolae. Co‐immunoprecipitation experiments were conducted in caveolin‐rich fractions from both adult and neonatal rat cardiomyocytes. Immunoblot analysis of the samples showed the pore‐forming subunit of KATP channels Kir6.2 and adenosine A1 receptors were detected in the anti‐caveolin‐3 or anti‐Kir6.2 immunoprecipitates from the caveolin‐enrich fractions, but not detected in the samples without primary antibody. When adult rat cardiomyocytes were pretreated with the cholesterol depleting agent methy‐β‐cyclodextrin (M βCD, 10 mM) for 30 min prior to homogenization, MβCD diminished caveolin‐3 that co‐precipitated with Kir6.2 and adenosine A1 receptors, while the levels of Kir6.2 and adenosine A1 receptors were also reduced. We concluded that cardiac KATP channels and adenosine A1 receptors associate with each other in caveolin‐rich membrane microdomains and this association depends on the level of cholesterol.
Zinc‐mediated inhibition of ClC‐3 current is pH‐dependent
Authors: Matsuda James J , Iyer Krishna S , Collins Malia M , Lamb Fred S
Published: June 2021

ClC‐3 is a Cl/H antiporter required for endosomal reactive oxygen production. Cl and H transport become uncoupled at low (<6.0) extracellular pH and ClC‐3 behaves as an acid‐activated anion conductance. Zinc (Zn) has been shown to block ClC‐1 anion channels in a pH‐dependent manner. We used whole cell patch‐clamp recording to characterize the effect of Zn on ClC‐3 current in HEK293 cells overexpressing the channel using an adenoviral construct. Current was activated at pH 4.0 (pH 7.35; 24.7 ± 2.3, pH 4.0; 112 ± 22 pA/pF, HP = ‐40 mV, TP = +100 mV). Zn inhibited current at pH 7.35 (10 μM Zn; 15.4 ± 3.3 (38%), 100 μM Zn; 4.8 ± 1.5 pA/pF = 81% inhibition). At pH 4.0 currents were less sensitive to Zn (100 μM; 53.4 ± 9.4 (47%), 1mM; 28.9 ± 5.8 pA/pF = 74% inhibition). Mutation of glutamate 224, the "fast gate" to alanine (E224A) resulted in currents that were more active at pH 7.35, not further activated by pH 4 and were completely resistant to Zn (control; 48.3 ± 2.7, pH 4.0; 45.3 ± 4.0, 100 μM Zn at pH 7.35; 47.3 ± 1.1). Deletion of extracellular sequence between transmembrane helices B and C, which contains 4 cysteine residues (C103_P130del), also completely prevented inhibition by Zn. Thus Zn inhibits ClC‐3 via pH‐dependent interaction with the extracellular surface of ClC‐3. Supported by NIH HL62483.
Lipid and protein interactions at the C‐terminal part of TRPM4
Authors: Owsianik Grzegorz , Schildermans Karin , Waelkens Etienne , Voets Thomas , Nilius Bernd
Published: June 2021

TRPM4 is a Ca2+‐impermeable Ca2+‐activated cation channel that undergoes a fast desensitization by intracellular Ca2+ ([Ca2+]i). The desensitization of TRPM4 to [Ca2+]i is attenuated by phosphatidylinositol (4,5)‐biphosphate (PIP2) and requires a pleckstrin homology‐like domain localized in the C terminus of the channel. Our data suggest that some of enzymes involved in the PIP2 metabolism, such as a phospholipase C, a PIP2‐regenerating kinase, and a PIP2‐depleting phosphatase might be functionally associated with TRPM4. To examine direct interaction of TRPM4 with phosphoinositides as well as formation of a signaling complex with other proteins, we fused the C‐terminal part of TRPM4 to GST and expressed in E. coli. The purified fusion protein was able to interact with membrane lipids in lipid‐protein overlay experiments. In order to identify interacting protein partners, we performed GST‐pulldown experiments with protein lysates from cells endogenously expressing TRPM4 channels. The mass spectrophotometric analysis revealed several putative interactions at the C terminus of TRPM4 that will be reconfirmed and further tested at a functional level. Supported by Interuniversity Poles of Attraction Program IUAP Nr.3P4/23, Excellentiefinanciering EF/95/010, Research Foundation‐Flanders (G.0565.07).
ZAC1 is up‐regulated by hypertonicity and decreases sorbitol dehydrogenase expression allowing accumulation of sorbitol in kidney cells
Authors: Lanaspa Miguel Angel , Andres‐Hernando Ana , Dai Yue , Li Nanxing , Berl Tomas , Rivard Christopher John
Published: June 2021

The zinc‐finger protein ZAC1 was determined to be markedly upregulated under hypertonic stress in inner medullary collecting duct (IMCD3) cells by Affymetrix genechip analysis (219‐fold, p<0.001). This change in message was verified by Quantitative PCR and by Western blot for protein. In mouse and human kidney tissues, ZAC1 expression was substantial in the papilla and absent in the cortex. ZAC1 protein expression was localized to the nucleus in cells under hypertonic stress by immunocytochemical analysis. Since a previous study in the hippocampal cell line HW3‐5 reported an inverse correlation between the expression of ZAC1 and sorbitol dehydrogenase (SDH), ‐a key enzyme involved in sorbitol degradation that is down regulated with hypertonicity. We examined if the decrease in SDH expression under hypertonic stress is mediated by the up‐regulation of ZAC1. Stable IMCD3 clones silenced for ZAC1 to varying levels demonstrated a direct effect on SDH expression. Silencing ZAC1 expression under hypertonic conditions caused maintenance of high SDH protein expression which prevented sorbitol accumulation in IMCD3 cells. Taken together, these data indicates that ZAC1 is up‐regulated under hypertonic stress and negatively regulates expression of SDH allowing for accumulation of sorbitol as a compatible organic osmolyte.
Functional expression of background K+ channels in rat carotid body cells.
Authors: Kim Donghee , Cavanaugh Eric J , Kim Insook , Carroll John L
Published: June 2021

Oxygen‐sensing glomus cells of the carotid body (CB) express a number of K+ channels that responds to hypoxia. TASK‐like K+ channels are expressed in CB cells and have been suggested to be particularly important for hypoxia‐induced depolarization. However, it is still unclear whether the TASK‐like K+ channels in CB cells are functional correlates of TASK‐1, TASK‐3, TASK‐1/TASK‐3 heteromer, or another K+ channel. To address this issue, we studied the properties of active K+ channels in resting CB cells isolated from 6‐7 day‐old rats, and compared them with those of cloned K+ channels. Single channels were recorded from cell‐attached and inside‐out patches of CB cells. In symmetrical 150 mM KCl, ~15‐pS (TASK‐1‐like) and ~32‐pS (TASK‐3‐like) channels were present together in 90% of patches, and were inhibited by acid (pH 6.0) and methanandamide (5 μM), similar to those of cloned TASK‐1 and TASK‐3. In addition to TASK‐like channels, another acid‐sensitive K+ channel with a unitary conductance of ~30‐pS was present in ~15% of patches. Mechano‐ and fatty acid‐sensitive channels with properties of TREK‐1 and TREK‐2 were also recorded in a few patches. These results show that TASK‐like channels in CB cells are functional correlates of both TASK‐1 and TASK‐3. TREK‐1, TREK‐2 and an unidentified acid‐sensing K+ channel also present in CB cells should contribute to the background K+ current. (Funded by NIH HL054621)
The Effect of Inhaled L‐arginine on Pulmonary Hypertension Induced by Hypoxia or Intravenous Arginase Infusion
Authors: Vrancken K , Power G G , Job L , Wilson S M , Blood A B
Published: June 2021

Background

Failure of the pulmonary vasculature to produce nitric oxide (NO) results in elevated pulmonary vascular resistance (PVR). L‐arginine (L‐arg) deficiency may be etiologic in some types of pulmonary hypertension (PH).

Objective

To test the hypotheses that inhalation of L‐arg will attenuate PH induced by hypoxia or an intravenous infusion of arginase, and that depletion of plasma L‐arg will result in elevated PVR.

Design/Methods

15 healthy newborn lambs 5‐10 days of age were anesthetized and maintained on a mechanical ventilator. Pulmonary arterial (PAP) and venous pressure and cardiac output were measured. 12 lambs were exposed to 6 sequential 10‐min periods of hypoxia. Inhaled L‐arg or saline was administered prior to the 4th hypoxic episode. In an additional 3 sheep, intravenous arginase was infused. Inhaled L‐arg was then administered for 15 min.

Results

Repeated episodes of hypoxia resulted in a rapid selective onset of PH. L‐arg inhalation caused no significant difference in PAP, PVR, or exhaled NO as compared to saline controls. Arginase resulted in a rapid significant increase in PAP (p <0.001), and in PVR (p < 0.05). PAP and PVR both decreased gradually during L‐arg inhalation, returning to control values by the end of the arginase infusion.

Conclusions

Plasma L‐arg is vital for the maintenance of low PVR. Preliminary results indicate that L‐arg inhalation may be effective in decreasing PH caused by L‐arg deficiency.
The apolipoprotein mimetic peptide 4F prevents sepsis‐induced changes in HDL composition in rodents
Authors: Zhang Zhenghao , Datta Geeta , White C. Roger
Published: June 2021

Clinical studies suggest that sepsis complications are related to both a reduction in plasma HDL and a decrease in HDL functional properties. Our previous studies show that administration of the apoA‐I mimetic peptide 4F to rats undergoing cecal ligation and puncture (CLP) surgery reduces inflammatory injury and improves survival. The goal of the current study was to test the hypothesis that protective effects of 4F are related to an improvement in HDL composition. Male Sprague‐Dawley rats were randomized to undergo CLP or sham surgery. CLP rats were further subdivided to receive saline (n=10) or 4F (10mg/kg, n=10) by ip injection 6 hours after sepsis induction. Sham‐operated rats received saline (n=10). Plasma total cholesterol (TC) and HDL were significantly reduced and lipid hydroperoxide (LOOH) content increased in CLP rats 24 hrs post‐surgery compared to shams. 4F treatment in CLP rats blunted these changes. Compositional analysis of isolated HDL fractions revealed a significant decrease in apoA‐I, apoA‐IV, apoA‐II and apoC and an increase in apoE. The concentration of a protein that was identified by mass spectrometry as serum amyloid A was also increased. 4F normalized these changes in HDL‐associated proteins. It is proposed that the attenuation of sepsis‐induced changes in HDL‐associated proteins by 4F enhances the functional properties of HDL in CLP rats.
Macrophage Uptake of Unopsonized Staphylococcus aureus Strains: High‐Throughput Analysis by Scanning Cytometry
Authors: DeLoid Glen , Sulahian Timothy , Imrich Amy , Kobzik Lester
Published: June 2021

Lung macrophages phagocytose unopsonized particles and pathogens via innate immune receptors such as scavenger receptors (SR). Signaling pathways for SR‐mediated phagocytosis of unopsonized bacteria likely govern the fate of ingested pathogens, but are poorly characterized. To allow high‐throughput screening (HTS) analysis, we developed a scanning cytometry assay to quantify phagocytosis of S. aureus by adherent human AM‐like macrophages. We compared the effects of receptor, signaling and cell process inhibitors (n = 7) on phagocytosis by human blood‐derived macrophages of a panel of live unopsonized S. aureus strains, (Wood, ATCC 29523, RN6390), as well as killed Wood strain, and latex beads. Differential fluorescent labeling of internalized vs. bound bacteria and image analysis of collapsed stacks of confocal sections in triplicate wells provided quantitation for ~500 cells per inhibitor per sample from three normal individuals. We found surprising heterogeneity of inhibition patterns among S. aureus strains, as well as between live and killed Wood strain, and between all S. aureus forms and unopsonized latex beads. The classic antagonist of SRs polyinosine inhibited uptake of latex beads and killed Wood strain, but had no effect on uptake of any live S. aureus strains. The data confirm the utility of the HTS assay, and suggest that receptor(s) other than SRs mediate uptake of live unopsonized S. aureus.

Supported by NIH ESO11008 and ESO11903