bluuebunny/crossref_metadata_2025 huggingface dataset:
TLR‐pathway is involved in the biological activity of triptolide, the active compound from a botanical clinically shown to alleviate RA symptoms
Authors: Dey Moul , Premkumar Vg , Raskin Ilya
Published: June 2021

Triptolide is a diterpene triepoxide present in roots of Tripterygium wilfordii Hook F., with known anti‐inflammatory, immunosuppressive and anti‐cancer properties. Although it is known that triptolide inhibits NFκB activation downstream of TLRs, involvement of TLR cascade in triptolide activity was not reported.

Objective of the study was to investigate if triptolide suppresses expression of proinflammatory downstream effectors induced specifically by different TLR agonists.

The results suggest that the suppression of agonist‐induced NFκB activation and chemokine expression by triptolide is mediated by targeting the early signaling of TLRs, particularly that of TLR4 in RAW 264.7 cells. Triptolide down regulated the expression of TLR4 proteins and that of TRIF adapter proteins in MyD88‐independent pathway of TLR4. In addition gene expression profiles in response to triptolide treatment suggest that triptolide may have multiple cellular targets contributing to its strong anti‐inflammatory and immune suppressive properties.

This study reveals involvement of TLR signaling in triptolide activity and further increases understanding of how triptolide activity may down regulate NFκB activation during inflammatory conditions. Funding for this research came from multiple grants by National Institutes of Health, Bethesda, MD [to MD and IR] and in parts by Phytomedics Inc., Jamesburg, NJ [to IR].
The PON1 is a susceptibility gene for hypertension
Authors: Bakhit Dana Mohammed , Al‐Najai Mohammed , Vigilla Mary Grace B , Al‐Boudari Olyan , Muiya Paul , Meyer Brian F , Al‐Mohanna Futwan , Alshahid Maie , Dzimiri Nduna
Published: June 2021

The role of PON1 in cardiovascular disease is not fully understood yet. In this study, we tested the possibility that mutations in this gene may predispose individuals to acquiring hypertension. We first sequenced the coding regions of the gene to identify variants of interest in a total of 250 Saudi individuals, and then selected five SNPs showing a frequency of >10% in the general population [rs854560 (7761T>A), rs662 (16399A>G), rs854552 (25921C>T), rs3947577(26137A>G), rs3735590 (26350C>T)], for further studies involving 2138 hypertensive patients versus 656 controls all of Saudi origin. Two of the five studied variants, rs662 [Odds ratio(95% Confidence Interval = 1.21(1.07–1.37); p=0.003] and rs3917577[1.19(1.01–1.40); p=0.039], individually conferred risk for hypertension. Interestingly, the same variants were also significantly associated with hyperlipidaemia (rs662, p=0.031; rs3917577, p=0.013), but not with coronary heart disease or type 2 diabetes mellitus. No confounding effect of gender, age, or obesity was observed on these relationships. The other three SNPs displayed no particular trends with any of the vascular diseases examined. These results point to PON1 as a susceptibility gene for HTN, but not CAD in the Saudi population.

This work was supported through the Royal Cardiovascular Research Grant of the King Faisal Specialist Hospital and Research Centre
CYP3A4 Ser478‐phosphorylation by protein kinase (PK) A enhances its ubiquitination by UBC7/gp78 and thus its proteasomal degradation
Authors: Wang YongQiang , Guan ShenHeng , Acharya Poulomi , Burlingame Alma , Correia M. Almira
Published: June 2021

CYP3A4 degradation via the ubiquitin‐dependent 26S proteasomal pathway involves protein phosphorylation. We previously identified three CYP3A4 phosphorylation sites: S478 phosphorylated by undefined liver cytosolic kinases, and T264 and S420 by PKC. Heterologous expression of CYP3A4 wild type (wt) and/or its Ala‐mutants at each of these three residues in yeast revealed significant stabilization of CYP3A4S478A mutant that was synergized by coupling T264A and S420A mutations. The exact mechanism of such CYP3A4 stabilization and the kinases responsible for S478 phosphorylation remained unknown. Using in vitro CYP3A4 phosphorylation systems followed by LC‐MS/MS analyses, we now document PKA‐dependent CYP3A4 phosphorylation at S478 and three other previously unidentified sites: S116, S119 and S259. Furthermore, the isolation of a corresponding CYP3A2 phosphorylated peptide (LpSRQAILEPEK) from rat hepatocytes provides evidence for in vivo phosphorylation of this residue. Relative ubiquitination of CYP3A4wt and its S478A, T264A/S420A and S478D (phosphomimetic) mutants in an in vitro reconstituted UBC7/gp78‐system revealed that S478D was the most dominantly ubiquitinated CYP3A4 mutant. Indeed such dominant ubiquitination of CYP3A5, a natural “S478D mutant” of CYP3A4, was also observed after transfection in HEK293T cells. Additionally, LC‐MS/MS analyses of the UBC7/gp78‐ubiquitinated CYP3A4S478D mutant identified at the least two target ubiquitination sites: K282 and K115. Together, these findings reveal that PKA‐dependent S478‐phosphorylation enhances CYP3A4 proteasomal degradation by enhancing its ubiquitination. Supported by NIH grants GM44037 (MAC) and NCRR RR 01614 (ALB).
Regulation of IK by Captopril and ANG II via PI3K pathway in Hypertrophied Cardiac Myocytes
Authors: Alvin Zikiar , Zhao Aiqiu , Laurence Graham G. , Coleman Bernell R. , Haddad Georges E.
Published: June 2021

Early hypertrophy may be beneficial but sustained hypertrophic activation leads to myocardial dysfunction. Regulation of the repolarizing currents (IK and IK1) can be modulated by the activation of humoral factors, such angiotensin II (ANG II), which modulates protein kinases whose integrated responses leads to the early beneficial cardiac hypertrophy and later to heart failure. We have previously found that compensated cardiac hypertrophy is associated with alterations in K+ and Ca2+channels are mainly mediated through MAP kinase and PI 3‐kinase activation. The aim of this work is to assess the regulation of IK by ANG II/PI3 K pathway in volume overload rat ventricular myocytes, using patch‐clamp in whole‐cell configuration. Cells were stimulated from holding potential of −80mV, by 500msec, 10‐mV step voltages between −120mV +40mV. Cardiomyocytes from 3‐weeks‐shunted animals showed a significant reduction in IK current‐density. Treatment with captopril (0.5g/L/day in drinking water for 3 weeks) alleviated this difference seen between sham and shunt cardiomyocytes. Acute administration of ANG II (10−6M) to adult cardiomyocytes treated with captopril similarly reduced the IK current density in shams and shunts. Thus we conclude that captopril enhances IK current density and response to ANG II in hypertrophied myocytes of volume overload shunted rats.
Serotonin infusion via the iPrecio® micro infusion pump results in repeated reductions in blood pressure in the normotensive Sprague Dawley rat.
Authors: Davis Patrick , Fink Gregory D , Seitz Bridget , Watts Stephanie W
Published: June 2021

Chronic serotonin (5‐hydroxytryptamine; 5‐HT) infusion causes a prolonged fall in blood pressure (BP) in the Sprague Dawley (S.D.) rat. We have previously studied the hypotensive effects of 5‐HT using an infusion rate of 25μg/kg/min, administered s.c. continuously via osmotic pump for 7 days. We hypothesized that increasing delivery rates of 5‐HT to the S.D. rat would result in progressively greater reductions in BP. 5‐HT or Vehicle (Veh) were administered s.c. via the iPrecio® micro infusion pump for 48 hrs at an initial rate of 5μg/kg/min. The pumps were stopped for 48 hrs and then increasing rates of 5‐HT or Veh were delivered in increments of 10μg/kg/min to achieve a final rate of 45μg/kg/min. Delivery intervals were followed by an interval in which the pumps were stopped for 48 hrs. Mean arterial BP was monitored (DSI radiotelemetry). BP prior to 5‐HT infusion was 101 mm Hg, while BP prior to Veh infusion was 99mmHg. BP values during infusion of 5‐HT at rates of 5μg/kg/min, 15μg/kg/min, 25μg/kg/min, 35μg/kg/min, and 45μg/kg/min were 88 ± 1.6 mmHg, 76 ± 7.0 mmHg, 84 ± 5.8 mmHg, 86 ± 7.5 mmHg, and 88 ± 6.6 mmHg, respectively. BP did not change in Veh infused rats. This study demonstrates that a smaller rate of 5‐HT than we have previously employed is required to elicit a hypotensive effect in the S.D. rats. This is the first study to utilize the iPrecio® micro infusion pump to deliver 5‐HT chronically at increasing rates to the S.D. rat.
Impaired endothelial relaxation and enhanced endothelial contraction and microvascular ADMA in angiotensin II infused rats: effects of tempol
Authors: Wang Dan , Luo Zaiming , Welch William J , Aslam Shakil , Teerlink Tom , Wilcox Christopher S
Published: June 2021

Since angiotensin II (Ang II) causes endothelial dysfunction, we investigated if it increased microvascular reactive oxygen species (ROS) or asymmetric dimethylarginine ADMA) and switches endothelial function from vasodilator to vasoconstrictor pathways. Acetylcholine‐induced endothelium dependent responses of mesenteric resistance arterioles (MRAs) were assessed in a myograph and vascular nitric oxide (NO) and ROS by fluorescent probes in groups (n=6) of male rats infused for 14 days with Ang II (200 ng/kg/min) or vehicle alone or with oral tempol (2mmol/L, a radical scavenger). Ang II infusion increased blood pressure and plasma malondialdehyde (0.3±0.1 vs 0.6 ±0.02, p<0.01) and decreased endothelium derived relaxation (18±5 vs 54±6%, p<0.01) and hyperpolarizing (19±3 vs 29±3%, p<0.05) responses and NO activity (0.9±0.1 vs 1.6±0.2%, p<0.01) yet enhanced endothelium dependent contraction response (23±5 vs 5±5%, p<0.05) and ROS (0.82±0.05 vs 0.15±0.03 units; p<0.01). Ang II decreased the expression of dimethylarginine dimethylaminohydrolase‐2 and increased ADMA in MRAs (450±50 vs 260±35 pmol/mg protein, p<0.01) but not in plasma. Tempol prevented all these responses. In conclusion, Ang II infusion redirected endothelial responses from relaxation to contraction with a reduction in vascular NO and an increase in ROS and ADMA. These are dependent on ROS and could contribute to hypertension.
Glutamate‐stimulated, astrocyte‐derived carbon monoxide production is intracellular calcium concentration‐dependent in piglets
Authors: Xi Qi , Tcheranova Dilyara , Basuroy Shyamali , Parfenova Helena , Jaggar Jonathan H. , Leffler Charles W.
Published: June 2021

Glutamate‐stimulated, astrocyte‐derived carbon monoxide (CO) induces vasodilation by activating big conductance Ca2+‐activated potassium (BKCa) channels in arterial myocytes. Here, we examined the hypotheses that glutamate stimulates astrocyte‐derived CO production via heme oxygenase (HO)‐2 activation by elevating [Ca2+]i and Ca2+‐calmodulin complexes in piglets. The major findings are: (1) Exogenous glutamate stimulates Ca2+ oscillations and increases [Ca2+]i in primary‐cultured astrocytes. (2) A [Ca2+]i elevation increases CO production in astrocytes with an EC50 of 310 nM. (3) If [Ca2+]i is held constant, glutamate does not affect CO production. (4) Depleting intracellular Ca2+ stores in astrocytes with a sarco/endoplasmic reticulum Ca2+‐ATPases blocker, thapsigargin, decreases CO production, and blocks the glutamate‐induced increase in CO in astrocytes caused by glutamate and the exogenous HO substrate, hemin. (5) The calmodulin inhibitor, calmidazolium, blocks glutamate‐induced CO production in freshly isolated piglet astrocytes. Taken together, our data are consistent with the hypothesis that glutamate elevates [Ca2+]i in astrocytes, which triggers Ca2+‐calmodulin complex formation, leading to HO‐2 activation and CO production. (NIH 5R37HL042851 and 5R01HL034059).
Resveratrol Via Nitric Oxide Attenuates Angiotensin II Induced Acute Increase In Blood Pressure In Sprague Dawley Rats
Authors: Bhatt Siddhartha R. , Lokhandwala Mustafa F. , Banday Anees Ahmad
Published: June 2021

Resveratrol is an antioxidant polyphenol which has been hypothesized to be the primary mediator of red wine's beneficial effects in cardiovascular diseases. However, its role in hypertension is not well established. We have previously shown that chronic resveratrol treatment attenuates development of hypertension in Spontaneously Hypertensive rats by decreasing oxidative stress and improving nitric oxide function. In this study we tested the acute effects of resveratrol on angiotensinII (AngII) induced increase in blood pressure in Sprague Dawley (SD) rats. Adult SD rats were first given a bolus dose of vehicle followed by a pressor dose of AngII (300ng/Kg). The rise in blood pressure was recorded. After 60–90 min the same animal was given a bolus dose of resveratrol 12mg/Kg followed by a pressor dose of AngII (300ng/Kg). To test the involvement of nitric oxide the rats were infused with 11micromol/Kg/min of NOS inhibitor L‐NAME for 25 min and the above treatment protocol repeated. Our results show that resveratrol significantly attenuated the AngII induced pressor response in comparison to vehicle pretreatment. This effect of resveratrol was completely prevented by pretreatment with L‐NAME. In conclusion, our results demonstrate that acute resveratrol treatment can attenuate AngII induced increase in blood pressure. Furthermore, nitric oxide appears to be a prominent mediator of this effect.
Enhanced cardiac Fas and mitochondrial dependent apoptosis in high fructose‐induced Metabolic Syndrome
Authors: Lee Yi‐Shin , Wu Liang‐Yi , Lee Shin‐Da
Published: June 2021

No information has been published regarding cardiac apoptosis in high fructose‐induced Metabolic Syndrome. The purpose of this study was to evaluate whether cardiac Fas‐dependent and mitochondria‐dependent apoptotic pathways are activated in high fructose‐induced Metabolic Syndrome. Ten high fructose induced Metabolic Syndrome (FIMS) and 10 normal diet control were studied at 3–4 months of age. The cardiac characteristics, myocardial architecture, and two major apoptotic pathways in the excised left ventricle from rats were measured by histopathological analysis, western blotting and TUNEL assays. Abnormal myocardial architecture, enlarged interstitial spaces, and more cardiac TUNEL‐positive apoptotic cells were observed in FIMS. The key components of Fas‐dependent apoptosis (Fas ligand, Fas death receptors, FADD, activated caspase 8, and activated caspase 3) and key components of mitochondria‐dependent apoptosis (Bax, Bax‐to‐Bcl2 ratio, cytosolic cytochrome c, activated caspase 9, and activated caspase 3) were significantly increased in FIMS hearts. Conclusions. Cardiac Fas‐dependent and mitochondria‐dependent apoptotic pathways are activated in high fructose‐induced Metabolic Syndrome, which might provide one of possible mechanism for developing heart diseases in patients with diet‐induced Metabolic Syndrome.
Salt modulates vascular response in eNOS‐null mice through adenosine A2A receptor: role of CYP450 epoxygenase and soluble epoxide hydrolase
Authors: Nayeem Mohammed A , Zeldin Darryl C. , Boegehold Mathew A , Falck John R
Published: June 2021

We analyzed the mechanism involved in relaxation in salt fed wild‐type and eNOS−/− mice. Using aortas from mice fed 4% (HS) or 0.45% NaCl (NS) diet for 4–5 wks, ACh and CRC for NECA and CGS 21680 were obtained with MS‐PPOH (epoxygenase blocker), AUDA (sEH blocker) and DDMS (CYP4A blocker). Relaxation to 10−7M ACh was greater in PE‐precontracted HS (+59.3±6.3%) compared to NS (+33.3±8.0%) eNOS+/+ aorta, whereas there were no response in HS (0.8±0.1%) and NS (1.5±0.3%) eNOS−/− mice. Relaxation to 10−6 M NECA was greater in HS eNOS−/− (+37.4 ± 3.2%) than in NS eNOS−/− (+7.4.0 ± 3.8%), Also, the relaxation were different in HS eNOS+/+ (30.4±2.4) and NS eNOS+/+ (+3.1 ± 1.3%) mice. Relaxation to 10−6 M CGS 21680 was greater in HS eNOS−/− (+45.4 ± 5.2%) than in NS eNOS−/− (+5.1 ± 5.0%). In HS eNOS−/−, the CGS 21680 response was reduced by MS‐PPOH (to +7.3 ± 3.2%; p<0.05), while no change was observed in NS eNOS−/−. In NS eNOS−/−, the CGS 21680 response was increased by AUDA (to +38.2± 3.3%) and DDMS (to +30.1 ± 4.1%; p<0.05). Compared to NS, HS showed increased CYP2J2 in both eNOS+/+ (35%; p<0.05) and eNOS−/− (61%; p<0.05) vessels, but decreased sEH in both eNOS+/+ (74%) and eNOS−/− (40%) vessels. Similarly, decreased CYP4A in HS both eNOS+/+ (35%; p<0.05) and eNOS−/− (34%). These data suggest that NS reduces vasodilation in eNOS+/+ and eNOS−/− mice via increased in sEH and CYP4A. However, in HS, activation of A2A AR triggers relaxation through CYP2J2.
Aging‐Related Decrease in Bax is Associated with Reduced Susceptibility to ACE Inhibitor‐Induced Remodeling in the Aorta of Spontaneously Hypertensive Rats
Authors: Biwer Lauren A , deBlois Denis , Hale Taben Mary
Published: June 2021

A 2‐week treatment with angiotensin converting enzyme inhibitor (ACEI) in young adult SHR reverses hyperplasia via apoptosis of aortic smooth muscle cells. The present study examined whether the same effect is observed in older rats. Male SHR (young: 11wks, mature: 26wks) were treated with either tap water or enalapril. Mature rats showed a significant increase in aortic cross‐sectional area, media thickness, lumen diameter, and media to lumen ratio but no change in collagen deposition. Enalapril significantly reduced blood pressure equivalently in young and mature SHR. Aortic medial cell number per unit length was significantly reduced in young, but not mature rats. Expression of pro‐apoptotic protein Bax was significantly reduced in aorta of mature rats. The present study demonstrates that vascular susceptibility to ACEI induced structural remodeling is altered significantly over a relatively short period (15wks) early in adulthood. This may provide a rationale for treating hypertension at an early stage; inasmuch that enhanced vascular remodeling may reduce arterial hypertrophy and stiffening, ultimately leading to improved long‐term protection against hypertensive target organ damage.
Role of Rho‐Kinase Activity and Expression in the Ductus Arteriosus in the Chicken Embryo
Authors: Cadar Adrian G , Sirsat Tushar S , Dzialowski Edward M
Published: June 2021

Incubation O2 levels have significant effects on the O2‐induced contraction of the chicken ductus arteriosus (DA) and its eventual closure. Incubation in hyperoxia speeds up closure while hypoxic incubation delays closure of the chicken ductus arteriosus. We hypothesized that one mechanism for this may be timing differences in the maturation of the Rho‐kinase pathway involved in calcium sensitization and myosin light chain phosphatase inhibition. To examine this, embryos were incubated in hypoxia (15%O2), normoxia (21%O2) and hyperoxia (30%O2) and used on embryonic day 18 and as internally and externally pipped embryos. The role of Rho‐kinase in O2‐induced contraction was measured by pre‐contracting the vessel with O2 and then exposing vessels to step‐wise increase in the Rho‐kinase inhibitor Y‐27632. On day 18 of incubation, Y‐27632 relaxed the O2 −induced DA significantly more in hyperoxia than normoxia or hypoxia suggesting that Rho‐kinase activity increases earlier in the DA from hyperoxic incubated embryos. To examine changes in DA gene expression within the pathway, RhoA mRNA levels from hypoxic, normoxic and hyperoxic embryos was measured using rtPCR. Preliminary data suggest that incubation in chronic hyperoxia increases RhoA mRNA levels. Taken together with the in vitro contractile response to Y‐27632 this suggests that development in high O2 up regulates RhoA gene expression. Development in a hyperoxic environment speeds up the maturation of the Rho kinase pathway in the chicken DA. This work was funded by the American Heart Association South Central Affiliate.
Bioengineering heart valves using autologous endothelial progenitor cell‐seeded bioscaffolds
Authors: Pollard Ricquita DeAnn , Raghavan Devanathan , Jordan James , Yoo James , Williams Koudy
Published: June 2021

Current valve replacement procedures often require repeat operations. Presently, the options available for heart valve replacements have limited durability and slow integration and often require lifelong anticoagulation. We hypothesize that tissue engineered heart valves (TEHVs) using autologous endothelial progenitor cell (EPC)‐derived endothelial cells (ECs) to cover a bioscaffold will provide an implant that does not require anticoagulation, produces minimal host immune response, and has the potential to remodel as the patient grows. Autologous EPCs were isolated from the peripheral circulation by random blood sampling and affinity column separation using an arterial‐venous shunt containing a column of antibodies recognizing the CD‐133 surface marker. Decellularized pig pulmonary heart valves were seeded with EPC‐derived ECs under static conditions or under pulsatile flow using a bioreactor, which then enhances cell coverage while maintaining biomechanical properties. Re‐endothelialization of decellularized pulmonary heart valve scaffolds may enhance the functionality and longevity of TEHVs. It is postulated that these bioengineered valves will integrate into host tissue faster, remodel as the patient grows and respond to in vivo physiological signaling, producing a longer lasting valve implant.
The expression of leukotriene B4 type‐1 receptor, BLT1, is facilitated by AML1 in leukocytes
Authors: Hashidate Tomomi , Murakami Naoka , Shimizu Takao , Nakamura Motonao
Published: June 2021

Leukotriene B4 (LTB4) is a lipid mediator that activates leukocytes through a specific G protein‐coupled receptor, BLT1. BLT1 plays important roles in host defense and pathogenesis of inflammatory and immune diseases. BLT1 is exclusively expressed in peripheral leukocytes, suggesting that its expression is stringently regulated. However, the precise mechanism of BLT1 expression is not fully understood. Here, we show that acute myeloid leukemia 1 (AML1/Runx1) is involved in the enhancement of BLT1 expression in leukocytes. In retinoic acid (RA)‐stimulated HL‐60 cells, the transcription of the BLT1 gene is significantly activated. RA did not directly modulate the BLT1 promoter, suggesting an enhancer(s) in other loci. DNase I‐hypersensitivity analyses revealed an activated region, termed AE‐BLex, at the intron‐I:exon‐II boundary. AE‐BLex acts as an enhancer for the BLT1 promoter and possesses two AML1 recognition sites. We demonstrated that the enhancement of BLT1 expression during the RA‐induced differentiation of HL‐60 cells is due to a loosening of the chromatin structure around AE‐BLex, which leads to the incremental binding of AML1. The AML1/AE‐BLex complex was confirmed in other BLT1‐expressing leukemia cell lines and human peripheral leukocytes. Thus, AML1 enhances BLT1 expression by binding to AE‐BLex, which is accessible in leukocytes.
Substituted cysteine accessibility mutagenesis of P2X2 receptors suggests the position of the gate with structural implications for closed‐open transitions
Authors: Khakh Baljit S. , Kracun Sebastian
Published: June 2021

P2X receptors are trimeric ligand‐gated cation channels that transition from closed to open states upon binding ATP. The crystal structure of the closed zebrafish P2X4.1 receptor reveals that the ion‐conducting pathway is formed by three transmembrane domain 2 (TM2) alpha‐helices. However, the transitions in TM2 that accompany channel opening are incompletely understood and remain unresolved. In the present study we used site‐directed mutagenesis (SDM) and electrophysiology to quantify gated access to Cd2+ at substituted cysteines in TM2 of P2X2. Our closed state data are consistent with the zfP2X4.1 structure, with I332 and T336 positioned one helical turn apart lining the channel wall on approach to the gate. Our open state data reveal gated access to deeper parts of the pore (T339, V343, D349 and L353), suggesting the closed channel gate is between T336 and T339. We also found unexpected interactions between native C348 and D349C that result in tight Cd2+ binding deep within the intracellular vestibule in the open state. Our data suggest that the channel gate opens near T336/T339 and is accompanied by movement of the pore‐lining regions, which narrow towards the cytosolic end of TM2. Such transitions would relieve the barrier to ion flow and render the intracellular vestibule less splayed during channel opening in the presence of ATP.

This research was supported by the NIH.
Identification of keratin 10 in a novel threadlike‐structure, Bonghan ducts and corpuscles
Authors: Kim Sorim , Lee Seul Ki , Jang Soo Hwa , Choi Jae Hong , Lee Byung Cheon , Lee So Yeong , Ryu Pan Dong
Published: June 2021

Keratin 10 (K10) is one of the major keratins involved in keratinoncytes differentiation and keratinization. In the present study, we examined the expression of K10 in the novel thread‐like structures (NTS) composed of Bonghan ducts and corpuscles located on the surface of the rat abdominal organs. The K10 mRNA and protein was identified by RT‐PCR and Western blot analysis. The K10 immunoreactivities were detected in a follicle‐like structure that covers several cells inside the NTS. Taken together, NTS expresses K10, in particular, in its internal structure. This is the first report demonstrating the expression of K10 in the NTS and may help to understand the structure and function of the NTS.
Characterization of a novel Ca2+‐activated Cl channel activator.
Authors: Lee Hyo Eun , Lee Jun‐Ho , Shin Minkyu , Hong Moochang , Kim Yangseok , Bae Hyunsu
Published: June 2021

Nelumbinis Semen is a traditional medicine that has been used for hundreds of years in East‐Asia. Nelumbinis Semen is used to treat insomnia, anxiety and women's post‐menstrual‐pause depression. In this study, the signal transduction mechanism of Nelumbinis Semen was investigated in Xenopus oocytes using two‐electrode voltage‐clamp technique. Nelumbinis Semen produced a large outward current at membrane potentials more positive than −20 mV when it was applied to the exterior of oocytes, but not when injected intracellularly. The effect of Nelumbinis Semen was concentration‐dependent and reversible. The effect of Nelumbinis Semen was completely blocked by bath application of the Ca2+‐activated Cl− channel blocker niflumic acid and by intracellular injection of the calcium chelator BAPTA or the IP3 receptor antagonist heparin. Moreover, the effect was partially blocked by bath‐applied U‐73122, a phospholipase C (PLC) inhibitor and by intracellularly injected GTPγS, a non‐hydrolyzable GTP analogue. Whereas, it was not altered by pertussin toxin (PTX) pretreatment. These results indicate that interaction of Nelumbinis Semen with membrane component at the extracellular side leads to Ca2+‐activated Cl channel opening and this process involves PLC activation, the release of Ca2+ from the IP3‐sensitive intracellular store and PTX‐insensitive G protein activation.

This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government(MEST) (No. 2009‐0063466).
Angiotensin‐II induced protein kinase D activation via src kinase and protein kinase C mediates aldosterone secretion in bovine adrenal glomerulosa cells
Authors: Olala Lawrence Ogweno , Bollag Wendy B.
Published: June 2021

Objective

To study the mechanism of angiotensin II (AngII) induced protein kinase D (PKD) activation in modulating aldosterone secretion in bovine adrenal glomerulosa (BAG) cells.

Methods

Adenoviral vectors were used to overexpress S738/742A PKD mutant which is not phosphorylatable by protein kinase C (PKC), Y463F PKD mutant, and Src wildtype as well as a dominant negative K296R/Y528F Src mutant. Supernatants were stored for aldosterone assay, and the cells processed for immunoblot analysis. Samples were immunoprecipitated with either PKD, or anti‐phosphotyrosine antibodies and reciprocally blotted. Blots were quantified and statistically significant differences were determined when p<0.05.

Results

Overexpression of non‐phosphorylatable S738/742A and the Y463F PKD mutants downregulated PKD activation and decreased acute (30‐minute) aldosterone secretion. The dominant negative K296R/Y528F Src mutant, which blocks Src activation, attenuated PKD phosphorylation and aldosterone secretion. AngII induced an increase in tyrosine phosphorylation of PKD presumably at the tyrosine 463 residue.

Conclusion

Angiotensin II (AngII) activates protein kinase D in BAG cells via a mechanism involving Src kinase and PKC, to underlie increased aldosterone production.

Funding Support

National Institutes of Health/National Heart, Lung and Blood Institute

American Heart Association
SorLA: a membrane raft protein in glial cells
Authors: Salgado Iris , Serrano Melissa , García José , Martínez Namyr , Maldonado Hector , Pagán Carlos Báez , Lasalde‐Dominicci José A.
Published: June 2021

SorLA is a sorting protein in neurons with relevance to Alzheimer's disease (AD). It shares roles with the caveolins, markers of the lipid membrane rafts microdomains. To further our knowledge on sorLA's expression and traffic, we studied sorLA expression in various cultured glia and its relation to caveolin‐1 (cav1), a caveolar microdomain marker. RT‐PCR and immunoblots demonstrated sorLA expression in rat cells: C6 glioma, primary cultures astrocytes (PCRA) and 1321N1 human astrocytoma cells. Differentiation of C6 cells to an astrocyte phenotype (C6 diff) led to a significant decrease in sorLA's mRNA and protein expression. Sucrose density gradient revealed cofractionation of sorLA with cav1 in light density membrane raft fractions of undifferentiated C6, C6 diff and 1321N1 cells. Immunofluorescences analysis of C6 and PCRA revealed a cellular distribution akin to sorLA's staining pattern in neurons. In C6 and PCRA cells a significant percent of sorLA colocalized with cav1, mostly in perinuclear compartments, cytoplasmic vesicles and plasmalemma. These findings establish that sorLA is expressed in glia and is a membrane raft protein involved with cav1 in glial intracellular membrane microdomains trafficking. This study supports a role for glia and its caveolar membrane raft microdomains to the sorting functions of sorLA in the neurodegenerative cascade of AD, unveiling new venues for disease treatment.
Promyelocytic Leukemia Tumor Suppressor Factor (PML) as a Biochemical Signal for Integration of Synaptic Plasticity
Authors: Tejada‐Simon Maria Victoria , Butler Karlis , Oni Emmanuel
Published: June 2021

The promyelocytic leukemia protein (PML) is a tumor suppressor factor associated with acute promyelocytic leukemia. It is involved in very important cellular processes. Recent studies suggest that PML also exerts some functions outside of the haemopoietic cells. In the brain, PML expression was found to be restricted to the neural progenitor cells of the developing neocortex, and protein ablation caused an impairment of cell differentiation and a decrease in cortical thickness, indicating that PML might be important for neurogenesis. It is well established that neural progenitor cells are also present in the hippocampus, and that hippocampal neurogenesis is required for hippocampus‐dependent learning. Thus, we hypothesize that lack of PML in the developing brain will likely lead to neurodevelopmental defects that impair neuronal plasticity and function in the adult animal affecting cognition. To test this hypothesis we examined the effect of PML ablation on 1) protein expression levels, 2) behavior, and 3) neuronal cytoarchitecture. Studying the expression of different proteins involved in synaptic plasticity and neurogenesis (Rac, Cdc42, ERK, PI3K, AKT, and mTOR) will provide a better understanding on the role of PML in the brain and how it regulates neurogenesis within the cortex and hippocampus, affecting neuronal structure and function. Additionally, performance on certain learning tasks will suggest whether PML might be relevant for cognition and mental health. Taken together, our findings point to PML as having an important role in brain function.

Funding by NIH‐NS048037, UH‐I098045 (M.V.T.S.).
Effects of Adenovirus AMP activated protein kinase (AMPK) on Scopolamine‐induced Memory Impairment in Rats
Authors: Kim SooJeong , Lee Jun‐Ho , Liu Jian , Kim Yong , Lee Hyo Eun , Shin Min‐Kyu , Hong Moo‐Chang , Kim Yangseok , Bae Hyunsu
Published: June 2021

AMP‐activated protein kinase (AMPK) is an important regulator of energy metabolism. AMPK is activated in cellular stresses such as ischemia, glucose depletion, and exercise, when intracellular levels of AMP increase. In this study, we investigated the neuroprotective effect of AMPK on scopolamine‐induced memory impairment in rats. We constructed a c‐Myc‐tagged AMPK wild type alpha subunit‐encoding adenovirus (Ad‐AMPK‐WT) and a dominant negative adenovirus (N157A, Ad‐AMPK‐DN), which were injected into the hippocampus of Sprague‐Dawley rats with sterotaxic apparatus. The cognition improve effect of AMPK was studied using Morris water maze test in scopolamine‐induced amnesia animal models. The Ad‐AMPK‐WT injected rats significantly improved escape latency of the Morris water maze. Moreover, the activity of acetylcholinesterase was significantly higher in the Ad‐AMPK‐WT injected rats than in the Ad‐AMPK‐DN injected rats. These data demonstrated that AMPK might be useful in the treatment of cognitive dysfunction, and its beneficial effects are mediated via protective effect of hippocampal neuron. This work was supported by the Korea Science and Engeneering Foundation (KOSEF) grant funded by the Korea government (MEST) (no. 2009‐0063466)
Putative role for insulin in glucose regulation by avian kidneys
Authors: Sparr Richard , Braun Eldon J. , Sweazea Karen L.
Published: June 2021

Birds have naturally high blood glucose concentrations. Prior studies of mourning doves (MODO) have shown that supraphysiological doses of insulin mildly, but significantly, decrease blood glucose levels. However, glucose uptake was not increased into tissues typically associated with insulin‐mediated glucose uptake (i.e. skeletal muscle, adipose tissue or cardiac muscle), nor was uptake increased in kidney, liver or brain tissues. These findings demonstrate that MODO are resistant to insulin‐mediated glucose uptake. Therefore, the goal of the present study was to test the hypothesis that insulin exerts a hypoglycemic role by augmenting glucose excretion by the avian kidney. Using a polyclonal rabbit antibody against the alpha‐subunit of the insulin receptor, western blots were performed on isolated kidneys from adult male MODO. The results supported the hypothesis that the avian kidney expresses the insulin receptor. In contrast to birds, others have shown that expression of the insulin receptor in mammals is downregulated with insulin resistance. As mentioned, glucose uptake into MODO kidneys is not enhanced in response to insulin. The presence of the insulin receptor in MODO kidneys, therefore, suggests a potential role for insulin in the regulation of glucose homeostasis through excretion. Further studies are aimed at elucidating this role of insulin.
Evidence of baroreflex in tegu lizards
Authors: Bicego Kenia Cardoso , Dantonio Valter , Toledo Ana Gabriela , Gargaglioni Luciane Helena
Published: June 2021

Tegu lizards have hearts with a ventricule anatomically and functionally undivided. Thus, blood pressures are equal in the systemic and pulmonary circulations leading to a blood flow distribution dependent on their relative vascular resistances. There is evidence for the baroreflex control in some species of reptiles, but no conclusive data exist for tegus. The present study aimed to verify the presence of a functional baroreflex in the tegu lizard, Tupinambis merianae, during winter (hibernation; June‐August) and spring (activity; October‐December). Arterial blood pressure (ABP) and heart rate (HR) were determined after systemic injections of sodium nitroprusside (SNP; 2.5, 5, 10, 25 μg/kg) and phenylephrine (PE; 5, 10, 25, 50 μg/kg). Experiments were conducted at 24–27oC in both seasons to eliminate temperature effect on ABP and HR. Basal ABP and HR were, respectively: 35.5 ± 1.3 mmHg and 10 ± 1.5 bpm (winter) and 34.7 ± 1.4 mmHg and 13.3 ± 0.7 bpm (spring). All doses of SNP caused significant increases in ABP with respective decreases in HR in both seasons. PE induced a dose dependent increase in ABP, a response that was more prominent during spring, but did not affect HR regardless season. The results indicate the presence of a hypotensive baroreflex in T. merianae during winter and spring. The lack of reflex response of HR to the PE injections remains to be clarified. Supported by FAPESP, CNPq. INCT Fisiologia Comparada.
IGFBP‐2 Expression in MCF‐7 Cells is Regulated by the PI3K/AKT/mTOR Pathway through Sp1 Induced Increase in Transcription
Authors: Mireuta Matei , Darnel Andrew , Pollak Michael
Published: June 2021

Insulin‐like growth factor binding protein 2 (IGFBP‐2) has been implicated in the pathophysiology of neoplasia. The PI3K/AKT/mTOR pathway has recently been implicated as a predominant regulator of IGFBP‐2 at the protein level in MCF‐7 breast cancer cells.

Objective

To elucidate the specific molecular mechanisms that underlie this regulation.

Results

We show that the PI3K/AKT/mTOR pathway regulates IGFBP‐2 protein levels by modulating IGFBP‐2 mRNA abundance in MCF‐7 cells. This change is achieved by regulating transcription through a critical region present in the first 200 bp upstream of the transcription initiation site where Sp1 transcription factor binds and drives transcription. Manipulations of the PI3K/AKT/mTOR pathway lead to changes in posttranslational modifications and nuclear abundance of Sp1 transcription factor.

Methods used

QRT‐PCR, ELISA, Western Blot, Luciferase reporter assays, Immunofluorescence, Chromatin immunoprecipitation.

Conclusion

This work provides a mechanistic explanation for the observed effects of the PI3K/AKT/mTOR pathway on IGFBP‐2 levels in MCF‐7 cells.

Matei Mireuta was supported by the graduate studentship from the Weekend to End Breast Cancer of the Jewish General Hospital Foundation.
Nesfatin‐1 Stimulates Stress Hormone Secretion
Authors: Samson Willis K , Yosten Gina LC , Ferguson Alastair V
Published: June 2021

Cellular actions of nesfatin‐1 on oxytocin (OT), vasopressin (AVP) and corticotropin releasing factor (CRF) neurons (J Neuroendocrinol 20:255,2008), the activation of early gene expression in those types of neurons following stress (Brain Res 1300:114,2009), and the ability of nesfatin‐1 to elicit fear and anxiety behaviors (Psychopharmacol 201:115,2008; AJP 297:R330,2009) suggested a role for nesfatin‐1 in the stress response. In conscious, unrestrained male rats i.c.v. administration of 180 pmole nesfatin‐1 resulted in significant increases in plasma levels of AVP (vehicle: 1.9±0.5, n=8; nesfatin‐1: 10.0±3.4 pg AVP/ml, n=8, p = 0.018) and ACTH (vehicle: 6.0±1.4; nesfatin‐1: 28.5±7.7 pg ACTH/ml; p<0.01) 10 minutes later. Plasma OT levels were unchanged. The actions of nesfatin‐1 on food intake and cardiovascular function are dependent upon activation of POMC neurons (AJP 297:R330,2009) and roles for OT in those actions have been reported. We suggest that nesfatin‐1 activates stress hormone secretion by releasing AVP and CRF but that the ability of nesfatin‐1 to activate OT neurons reflects an action to release OT centrally and not into the peripheral plasma.
Distribution of G alpha subunit mRNA in rat adrenal cortex and adrenal medulla
Authors: Parker Lindsay M , Goodchild Ann K
Published: June 2021

Mineralocorticoid release from adrenal cortex and catecholamine release from adrenal medulla contribute to the regulation of blood pressure. Release is controlled by actions of several G protein coupled receptors (GPCRs) including angiotensin and melanocortin receptors in the cortex and PACAP and opioid receptors in the medulla. Activation of a GPCR releases the Gá subunit (11 types) that activates downstream signaling components. Stimulatory Gás and inhibitory Gái/o/z subunits act via the cAMP signaling pathway. The aim of this study was to assess distribution of stimulatory and inhibitory Gá subunits in adrenal cortex and medulla to determine the primary signaling pathways utilized. The relative amount of Gá subunit mRNA was calculated in adrenal cortex and adrenal medulla of Wistar Kyoto rats (n=5) using quantitative RT‐PCR. All Gá subunits tested were detected in the cortex and medulla, with a greater abundance of each in the medulla. Abundance was normalized relative to Gz (least abundant subunit) as follows: Gs>Gi2>Gi3>Gi1>Go>Gz in cortex and Gs>>Gi2>Gi3>Go>Gi1>Gz in medulla. Gs and Gi2 were most abundant in both cortex and medulla, suggesting they are the primary stimulatory and inhibitory subunits involved in GPCR‐mediated signaling in adrenal glands. In situ hybrization is currently underway to examine distribution of these subunits in adrenal glands from normotensive and hypertensive animals.
Does cerebral blood flow & oxygenation limit maximal exercise performance in healthy humans?
Authors: Louvaris Zafeiris , VOGIATZIS IOANNIS , DIMITRIS ATHANASOPOULOS , VASILIS ANDRIANOPOULOS , PANAGIOTIS ALEXOPOULOS , HARRIETH WAGNER , HABAZETTL HELMUT , ROUSSOS CHARIS , WAGNER PETER , ZAKYNTHINOS SPYROS
Published: June 2021

Recent reports suggest that changes in cerebral oxygenation may play a pivotal role in limiting maximal exercise capacity in humans.

We investigated whether a critical change in cerebral oxygen blood flow and oxygenation may occur from modest to supra‐maximal exercise in hypoxia compared to normoxia.

Cerebral blood perfusion was measured by near infrared spectroscopy using indocyanine green dye in 11 cyclists during an incremental discontinuous protocol to the limit of tolerance.

During both normoxic and hypoxic (FIO2: 0.12) conditions brain blood flow significantly increased reaching identical levels, at maximal (42±5 versus 42±2 ml/min/100g) and supra‐maximal (40±5 versus 40±4 ml/min/100g) exercise. Deoxy‐hemoglobin concentration (deoxy‐Hb: an index of oxygen extraction) was greater in hypoxia compared to normoxia at maximal (13.1±1.3 vs. 2.3±0.5 μmol/L) and supra‐maximal (11.9±1.5 vs. 2.5±0.5 μmol/L) exercise. Neither in hypoxia nor in normoxia deoxy‐Hb changed significantly from modest (60% max) to supra‐maximal exercise (13.8±1.6 vs. 11.9±1.5 and 1.0±1.5 versus 2.5±0.5 μmol/L, respectively). Importantly, supra‐maximal normoxic deoxy‐Hb (2.5±0.5 μmol/L) was lower than that recorded at 60% max exercise (13.8±1.6 μmol/L) in hypoxia.

Cerebral blood flow and oxygenation do not appear to be the signals that terminate exercise in humans.
Influence of contractile work and muscle fiber recruitment on skeletal muscle blood flow in humans
Authors: Richards Jennifer Clarke , Crecelius Anne R. , Kirby Brett S , Dinenno Frank A.
Published: June 2021

Contractile work has been considered the primary determinant of skeletal muscle oxygen consumption (VO2), which is tightly coupled with blood flow (BF). Recently it was observed that within matched work conditions, the BF response following a single contraction increased with a greater degree of muscle fiber recruitment (MFR). The purpose of this study was to determine whether the primary determinant of skeletal muscle BF was contractile work or MFR during exercise. Accordingly, we measured the forearm BF response (FBF; Doppler ultrasound) during 4 minutes of rhythmic handgrip exercise (RHE) in 9 young males. Each workload (low and high) consisted of two trials matched for total work by varying the load (% maximal voluntary contraction; %MVC) and the contraction: rest ratio (low: 20%1:7 vs 10%1:3, high: 20%1:3 vs 10%1:1). Greater MFR was attained by increasing %MVC. VO2 was calculated from FBF and deep venous blood samples. Greater MFR significantly increased the FBF response at both low and high workloads (164±16 vs 139±16 p=.03 and 270±24 vs 240±20 p=.01 (ml min−1)). Greater MFR did not significantly affect VO2 (207±19 vs 177±23 p=0.3, and 327±44 vs 305±37 p=0.4(ml min−1)) at any workload, however, the ratio of ΔFBF: ΔVO2 was unchanged. Our preliminary data suggest contractile work is not the primary determinant of FBF and greater MFR may have an independent influence on FBF during RHE in humans.

Supported by HL‐087952
Effect of Surface Charge of Silver and Gold Nanoparticles on the Cytotoxicity in Human Lung Carcinoma, A549 Cells
Authors: CHOI SEON YOUNG , PARK JIN HO , JANG SOO HWA , LEE HOSUB , LEE KANGTAEK , RYU PAN DONG , YANG SUNG IK , JOO SANG WOO , LEE SO YEONG
Published: June 2021

The cytotoxicity of oppositely‐charged gold (Au) and silver (Ag) nanoparticles (NPs) was compared using 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2H‐tetrazolium bromide (MTT), lactate dehydrogenase, and fluorescence activated cell sorter (FACS) measurements. NP uptake into the A549 cells was characterized by transmission electron microscopy. According to the MTT viability assay, positively charged NPs were found to be more toxic compared to negatively charged NPs. FACS analysis demonstrated that the cells treated with both Ag and Au NPs possessing positive or negative charges showed an increase in the sub‐G1 phase suggesting that the decrease in cell viability is due to the induction of apoptotic effects in the cells. These results indicate that the surface charge effects of Ag and Au NPs on cytotoxicity is crucial for the potential application of Ag and Au NPs in biological system.

This work is supported by National Research Foundation of Korea (2009‐0066580)
Bacterial co‐expression as a convenient method for the production and purification of core histones
Authors: Dutnall Robert , Anderson Megan , Huh Joon H , Ngo Thien , Lee Alice , Hernandez Genaro , Pang Joy , Perkins Jennifer
Published: June 2021

Co‐expression of proteins in bacteria offers an important strategy for producing multiprotein complexes for biochemical and biophysical studies. We have found that co‐expression of histones H2A and H2B (from yeast, chicken or Drosophila) leads to production of soluble heterodimeric H2AH2B complexes. Drosophila histones H3 and H4 can also be produced as a soluble (H3H4)2 heterotetrameric complex if they are co‐expressed with the histone chaperone Asf1. The soluble H2AH2B and (H3H4)2 can be purified by simple chromatographic techniques and have similar properties to endogenous histones. Our methods should facilitate histone production for studies of chromatin structure and regulatory proteins that interact with histones. We describe a simple strategy for constructing co‐expression plasmids, based on the T7 RNA polymerase system, which is applicable to other systems. It offers several advantages for quickly creating plasmids to express two or more proteins and for testing different combinations of proteins for optimal complex production, solubility or activity.
Effect of glucose tolerance on muscle atrophy in streptozotocin‐induced diabetic rats
Authors: Park Inhee , Lee Seunghoon , Lee Youngjeon , Hong Yonggeun
Published: June 2021

Diabetic mellitus (DM) is characterized by an increased plasma glucose level, and reduced glucose uptake can activate protein degradation machinery leading to autophagy in skeletal muscle fibers. Thus, in this study we investigated the effect of glucose tolerance on numerous markers related to muscle atrophy in skeletal muscles obtained from streptozotocin‐induced diabetic rats. Male Sprague‐Dawley rats (n=8, eight‐week‐old) were divided into two experimental groups: diabetic (group‐DM, induced i.p. by streptozotocin at 60 mg/kg) and group‐C (treated with vehicle for eight weeks). The blood glucose level in the rats was determined each week using blood samples obtained by venipuncture of a tail vein. After eight weeks, the rats were sacrificed by decapitation and skeletal muscle tissue was collected for RNA isolation. The differential expression of muscle atrophy‐related genes such as MuRF1 and MAFbx was then analyzed. MuRF1 expression was significantly increased in the skeletal muscles of the diabetic rats. Our results indicate the expression of specific muscle atrophy‐related genes increased in diabetic‐induced muscle atrophy.

Funding: BioGreen21 Program (Code No. 20070401‐034‐006‐009‐02‐00), Rural Development Administration, Korea