bluuebunny/crossref_metadata_2025_split huggingface dataset:
GPCR proteomics: Mass spectrometric and functional analysis of histamine H1 receptor after baculovirus‐driven and in vitro cell free expression
Authors: Leurs Rob , Sansuk Kamonchanok , de Grip Wim , Booth Raymond , Hensbergen Paul 
Published: June 2021

During the past decades, MS analysis is standing out as the method of choices to determine protein‐protein interaction and ligand‐protein interactions. However, due to the low amount of purified protein obtained and the hydrophobic nature of GPCRs, limited information is available on ligand‐GPCR interaction. Here we reported the first successful use of MS analysis to directly reveal the primary structure of hH1R protein, obtained from cell free and/or baculovirus expression system. More than 80% of protein sequence, including five of TM domains, was covered by our optimized digestion procedure, prior to the MS analysis. This approach also allows us to further study the protein‐ligand interaction, by covalently labeling of the hH1R protein with irreversible histaminergic compounds. In combination with data obtained through homology modeling and site directed mutagenesis, these approaches will enhance our knowledge regarding the tertiary structure of this prototype GPCR and its interaction with small molecules.Partly supported by NIH grant R01 MH068655
Andrographolide induces the COX‐2 expression in MC3T3 – E1 cells via the ERK1/2 pathway
Authors: Perez Tania , Hidalgo Maria A , Carretta Maria D , Hancke Juan L , Burgos Rafael A 
Published: June 2021

Andrographolide is a labdane diterpene that possess antiinflamatory properties. This drug reduces iNOS, IL‐8 and COX‐2 expression in macrophages and neutrophils interfering with NF‐κB binding. COX‐2 are involved in the prostaglandins synthesis during inflammatory process, and selective COX‐2 inhibitors have been useful for rheumatoid arthritis and osteoarthritis treatment; however it is widely recognized that COX‐2 inhibition can also interfere with the bone repair, affecting the fracture healing. In this work we assess if androgrpholide can interfere with the COX‐2 expression induced by TNF‐α (proinflammatory cytokine) or TGF‐β (bone growth factor) in MC3T3 – E1 murine preosteoblastic cells. We analyzed the ERK1/2 and SMAD 2–3 phosphorylation and COX‐2 expression by western blot. By Q‐RT‐PCR we assess the effect of andrographolide on COX‐2, cbfa1 and NF‐κB mRNA level in cells treated with TNF‐α or TGF‐β. We demonstrated that andrographolide potentiates the COX‐2 expression and ERK1/2 phosphorylation induced by TNF‐α or TGF‐β in MC3T3 – E1 cells. Moreover we observed that andrographolide by itself was able to stimulate the ERK1/2 phosphorylation and COX‐2 expression in a dose dependant manner. We conclude that andrographolide can modulate directly the osteoblast activity inducing COX‐2 expression and potentiating other stimuli via ERK1/2 pathway.This work was supported by FONDEF D04I1240
Angiotensin receptor blocker (ARB) treatment causes paradoxical microvascular endothelial cell proliferation
Authors: Munzenmaier Diane H. , Greene Andrew S. 
Published: June 2021

Aim: Previously our laboratory has shown that cerebral angiogenesis in rats is stimulated by chronic treatment with the angiotensin II (ANG II) type 1 receptor (AT1R) antagonist, Losartan (LOS). Because the pro‐angiogenic actions of ANG II via AT1R have been well documented in the peripheral microvasculature, the finding that an AT1R antagonist stimulates cerebral angiogenesis is paradoxical. The aim of this study is to determine whether the in vivo findings are due to receptor blockade per se or whether LOS might exert nonspecific actions in the cerebral vasculature that could explain the in vivo paradox.Methods: Primary human cerebral and dermal microvascular endothelial cells (ECs), passage 3–8, were seeded into 96‐well plates (5000 cells/well) and allowed to adhere for 24 hrs. Cells were exposed to serum‐free media (SFM) alone and in the presence of LOS (10 μM), ANG II (1 μM) or both for 48 hours. Cell density was measured by a fluorescent cell proliferation assay (CyQUANT, Molecular Probes) on a plate reader at 480/520 nm.Results: Cell density increased with ANG II treatment of both cerebral (27.5%) and dermal ECs (8.9%) compared to SFM. LOS treatment elicited a similar response (cerebral: 27.1%, dermal: 19.0%). Treatment with both LOS and ANG II, which would simulate AT1R blockade, also stimulated EC proliferation (cerebral 33.7%, dermal: 15.2%).Conclusion: Since LOS alone, either in the presence or absence of agonist, stimulated cell proliferation in cells derived from both brain and skin, it would suggest that 1) LOS stimulates EC proliferation directly, not by receptor blockade, and 2) tissue differences are not detectable in cultured ECs derived from neural tissue compared to peripheral tissue. Future studies are needed to determine the mechanism for this effect and the cause of observed tissue differences in vivo.Supported by NIH HL29587 and HV28182
Arteriolar dilation in response to skeletal muscle contraction is reduced in Cx40‐deficient mice
Authors: Milkau Malte , de Wit Cor 
Published: June 2021

Active hyperemia requires substantial dilation in down‐ and upstream vessels. The ascending dilation may involve gap junctions (GJ) which allow the spread of a dilation into upstream vessels. GJ are composed of connexins (Cx) and of these Cx40 is crucial to support a conducted dilation initiated by endothelial agonists. We studied whether Cx40‐dependent communication contributes to arteriolar dilations during active hyperemia in the cremaster microcirculation in vivo using wildtype (wt) and Cx40‐deficient mice (Cx40ko).The nerve supplying the cremaster muscle was stimulated for 1 to 60 s at 10 or 100 Hz intermittently (burst 0.5, break 2.5 s). Arterioles were monitored by intravital microscopy and diameters measured off‐line. Responses were studied before and after L‐nitro‐arginine and indomethacin (LN+indo).The amplitude and the duration of the arteriolar dilation was graded with stimulation time and frequency. At 100 Hz the dilation amounted to 24% (1s), 56% (15s), and 83% of the maximal dilation (60s) in wt. 10 Hz stimulation produced about half of these responses. After LN+indo the dilation was only reduced after maximal stimulation (60s 100 Hz: 63%, p<0.05). In Cx40ko mice, the dilation was significantly reduced (15s: 32%; 60s: 58%) compared to wt. In mice carrying a replacement of Cx40 by Cx45 dilations were also reduced but to a lesser degree.We conclude that Cx40‐dependent communication contributes to arteriolar dilations in response to exercise. This function of Cx40 can only be partially replaced by Cx45. This suggests that the endothelium provides a Cx40‐dependent conduction pathway whose function during exercise cannot be compensated by other dilator mechanisms.
Pannexin protein expression in the rat middle cerebral artery
Authors: Chen Jie , Burns Alan R. , Phillips Sharon C. , Sokoya Elke M. 
Published: June 2021

Pannexins are a recently discovered family of proteins that could potentially be involved in cell‐cell communication. The expression pattern of pannexin 2 (Panx2) is thought to be predominantly confined to neuronal cell populations. In the present study, we report for the first time that pannexins are expressed in the cerebrovasculature. By using a combination of RT‐PCR, immunoblotting, immunohistochemistry and immunogold electron microscopy techniques, we characterized the expression pattern of pannexins in the rat middle cerebral artery (MCA). We report that pannexin 1 (Panx1) is weakly expressed in smooth muscle but not in endothelium, and that Panx2 is expressed in both endothelium and smooth muscle. The identification of pannexins in the rat MCA, indicates that pannexin expression is not limited to neuronal cell populations within the central nervous system. Ultrastructural studies using immunogold electron microscopy revealed that Panx2 is associated with both junctional (gap junctions) and nonjunctional areas of the plasma membrane. These data, suggest that Panx2 may play a role in supporting both cell‐cell coupling and hemichannel activity within the cerebrovasculature.Supported by NIH HL72954, EY017120, and HL070537.
Femoral artery‐vein shunt steals collateral flow and promotes collateral arteriogenesis
Authors: Yang Hsiao Tung , Bowles Douglas K , Terjung Ronald L , Laughlin Harold M 
Published: June 2021

To study high flow‐stimulated collateral artery enlargement, a side to side femoral artery‐vein anastomosis was established in Yucatan pigs (~40 kg, n=7, limited to cage activity). The femoral artery was occluded proximally to the a‐v shunt by an Ameroid occluder placed between the a‐v shunt and a proximal transonic flow probe. Femoral artery blood flows (BF) were monitored daily to evaluate the magnitude and time‐course of BF change. Before femoral artery occlusion, BF through the proximal flow probe increased from ~400 to 700 ml/min, while BF through the distal flow probe (distal to the shunt) were ~100 to 200 ml/min. Following femoral artery occlusion (~ 2 wks post surgery), proximal BF reduced to only ~50 ml/min, while distal BF (i.e., arising from collateral dependent BF delivered distal to the shunt) gradually increased from ~200 to 400 ml/min by 4 wks post‐surgery. Thus, a significant­ly greater collateral‐depen­dent BF was shunted back to the central circulation over time. This placed increasing flow demands on the collateral circuit. Angiograms showed extensive collateral vessel development in the upper thigh area in the leg with the a‐v shunt, primarily arising from the profunda artery. We attribute this collateral vessel enlarge­ment to the apparent high shear stress induced in these vessels that would occur to support flow through the low‐resistance shunt created by the by‐pass. Thus, we conclude that an exceptional enlargement of the collateral circuit can be induced over a rather brief period of time, if the stimulus for collateral development is sufficient.Supported by NIH grant P01HL 52490‐12
Coordination of chromosome replication and cell cycle progression in Caulobacter
Authors: Shapiro Lucy 
Published: June 2021

The Caulobacter cell cycle is driven by sequential regulators, starting with the expression of DnaA in G1 and ending with the expression of the CcrM DNA methyltransferase at the end of replication. DnaA both allows replisome formation and is a transcription factor for over 40 replication genes. The timing of dnaA transcription is dependent on the methylation state of its promoter, which in turn is dependent on restricting the synthesis of CcrM to the end of the cell cycle. Replication initiation starts on a fully methylated chromosome. The dnaA gene, positioned near the replication origin, is only read when its promoter is in the fully methylated state. When the replication fork passes the dnaA gene, the dnaA promoter becomes hemi‐methylated and thus inactivated. This methylation‐based regulation of dnaA transcription modulates the temporal levels of DnaA during the cell cycle, providing a ratchet mechanism to govern sequential regulator expression.The bacterial cell also uses spatial information to coordinate cell cycle events. In Caulobacter, a novel system couples the placement of the FtsZ cytokinetic ring to the initiation of chromosome replication and the bipolar positioning of the duplicated origin regions. A ParA‐like protein, MipZ, forms a complex with the ParB partitioning protein near the origin of replication and moves with the duplicated origin regions to the cell poles. MipZ directly interferes with FtsZ polymerization, thereby restricting FtsZ ring formation to mid‐cell, the region of lowest MipZ concentration. The cellular positioning of MipZ thus serves the duel function of positioning the FtsZ ring in‐between the two nascent sister nucleoids and delaying formation of the cell division apparatus until chromosome segregation has initiated.
Model prediction of subendocardial perfusion in the presence of an epicardial coronary artery stenosis
Authors: van den Wijngaard Jeroen , Kolyva Christina , Siebes Maria , Dankelman Jenny , van Gemert Martin JC , Piek Jan J , Spaan Jos AE 
Published: June 2021

Objective: With coronary disease the subendocardium is most vulnerable to ischemia which will occur when Diastolic Time Fraction (DTF) is to short and/or coronary pressure (Pc) to low. Since recent clinical techniques permit measurement of subendocardial perfusion our objective is to predict the effects of hemodynamic conditions on the induction of ischemia.Methods: Using data from microsphere experiments a layered model of the myocardial wall was developed in which each layer is represented by a hyperemic resistance and autoregulatory resistance in series. Myocardial perfusion distribution during hyperemia was predicted for different degrees of coronary stenosis, at different levels of DTF.Results: A reference DTF was chosen (corresponding to HR=90) at which perfusion was rather evenly distributed at normal Pc = 90 mmHg. At this reference condition the effect of the stenosis on perfusion distribution was homogenous. However, at shorter or longer DTF, the subendocardium was the first or last to suffer from shortage of perfusion, the maginitude of which depended on stenosis degree.Conclusions: For larger values of DTF as normal for patients with medication and with the applied pharmacological stress tests the possible occurrence of subendocardial ischemia at physical exercise is underestimated.
The effect of glutathione on the gastric H,K‐ATPase in vitro
Authors: Nagatani Gerald , Okamoto Curtis 
Published: June 2021

OBJECTIVEThe gastric H,K‐ATPase is a heterodimeric P‐type ATPase transporter responsible for acid secretion in the stomach. Glutathione (GSH) is an antioxidant that has been shown to regulate the activity of another P‐type ATPase transporter, the sarcoplasmic/endoplasmic reticulum Ca‐ATPase. Here we examine a potential role that glutathione might play in the regulation of the gastric H,K‐ATPase in vitro.METHODSGastric microsomes, purified by standard subcellular fractionation techniques from hog stomach parietal cells, were used in all assays. Para‐nitrophenylphosphate (pNPP) hydrolysis due to H,K‐ATPase activity was measured in the presence of varying concentrations of GSH. GSH‐treated microsomal proteins were separated by SDS‐PAGE and Coomassie‐stained and immunoblotted for the α‐subunit (HKα) or the β‐subunit (HKβ) with their respective monoclonal antibodies. HKα was immunoprecipitated and immunoblotted with a monoclonal GSH antibody. The interaction of glutathione‐S‐transferase (GST) with HKα was tested using a blot overlay assay on microsomal proteins transferred to a nitrocellulose membrane as well as a cosedimentation assay with microsomes and GST.RESULTSH,K‐ATPase acitivty as measured by pNPP hydrolysis was inhibited in a dose dependent manner by GSH. Treatment of gastric microsomes with 10 mM GSH resulted in a loss of HKα, but not HKβ, immunoreactivity as shown by Western blots and Coomassie staining. HKα is glutathiolated as shown by immunoblotting immunoprecipitated HKα with the GSH antibody. HKα is bound by GST in overlay assays. GST cosediments with gastric microsomes.CONCLUSIONGlutathione not only affects H,K‐ATPase activity and HKα protein levels, but also appears to be a post‐translational modification on HKα. GST may bind to HKα via the glutathione modification.
Caloxin 1c2‐ A Novel Engineered Selective Inhibitor of Plasma Membrane Ca2+ ‐ Pump Isoform 4, Effects on Coronary Artery
Authors: Szewczyk Magdalena M. , Pande Jyoti , Kuszczak Iwona , Grover Ashok K. 
Published: June 2021

Coronary artery smooth muscle expresses two isoforms of plasma membrane Ca2+ pumps: PMCA4 and PMCA1. The previously reported peptide inhibitor caloxin 1b1 was obtained by using extracellular domain 1 (amino acids 115–131) of PMCA4 as the target. Here, we report its mutagenesis by creating a phage display library of caloxin 1b1‐like peptides. The library was screened by affinity chromatography with the PMCA purified from the human erythrocyte ghosts that contain mainly PMCA4 isoform. This yielded caloxin 1c2 with the following key properties: a) Ki = 2.3 ± 0.3 μM which corresponds to a 20x higher affinity for PMCA than caloxin 1b1, and b) it is isoform selective since it has 10x higher affinity for PMCA4 than for PMCA1, 2 or 3. It had the following functional effects on the coronary artery smooth muscle: a) it increased the basal tone of the de‐endothelialized arteries: the increase being similar at 10, 20 or 50 μM, and b) it enhanced the increase in the force of contraction at 0.05 but not at 1.6 mM extracellular Ca2+ when Ca2+‐extrusion via the Na+‐Ca2+‐exchanger and the sarco/endoplasmic reticulum Ca2+ pump were inhibited. We conclude that PMCA4 is pivotal to Ca2+ homeostasis in coronary artery smooth muscle. We anticipate caloxin 1c2 to aid in understanding the role of PMCA4 in signal transduction and Ca2+ homeostasis due to its isoform selectivity and ability to act when added extracellularly.Supported by Heart & Stroke Foundation of Ontario.
Mathematical model of interactions between cell volume regulation and transport in cortical TAL cells
Authors: Nieves‐Gonzalez Aniel , Clausen Chris , Layton Harold E , Moore Leon C 
Published: June 2021

We developed a mathematical model of a cortical thick ascending limb (cTAL) cell to study interactions between ion transport and cell volume (CV) regulation. Because the NKCC2 transporter mediates apical Na+ uptake, and its activity is regulated by CV‐dependent kinases, we hypothesize that transport‐related CV changes should result in compensatory regulation of NKCC2 activity and Na+ reabsorption. The cTAL model is based on mass conservation and includes models of Na+‐K+‐ATPase, the NKCC2 (A‐isoform) and KCC1 cotransporters, and passive ion fluxes. CV‐sensitive regulation of the NKCC2 and KCC1 activities is represented with empirical functions. Model parameters were chosen to yield potentials, cytosolic [ion], and ionic fluxes consistent with published data. To test our hypothesis, we simulated published experiments which measured the relationship between short‐circuit current (Isc) and luminal [NaCl]. Simulations were done with and without NKCC2 and KCC1 regulation. The agreement between the predicted and measured responses was significantly improved, particularly at lumen [NaCl] > 75 mM, by inclusion of NKCC2 and KCC1 regulation. Moreover, the gain that provided the best fit to the Isc curve yielded regulatory volume decreases consistent with published data. These findings support the concept that CV regulatory responses modulate Na+ transport in cTAL cells.Supported by NIH grant DK‐42091.
Thyroid hormone effect on activity levels and tissue mitochondrial biogenesis and function in young and old rats
Authors: Klaus Katherine A , Walrand Stephane , Morse Dawn , Schimke Jill M , Short Kevin R , Novak Colleen , Levine James A , Nair K. Sreekumaran 
Published: June 2021

Triiodothyronine (T3) has a major impact on the regulation of energetics and mitochondrial (mt) function. We determined whether chronic T3 administration alters the age‐related decline in mt function. Ten young (Y, 8 mo) and ten old (O, 27 mo) F344/BN rats were randomized to receive T3 (200 mg/d) or placebo for 14d via subcutaneous osmotic pump. Because OT3 rats decreased food consumption by 39%, we added ten pair fed rats (OPF). T3 treatment caused a significant weight loss (Y, −11.97%; O, −17.49%), while energy expenditure increased in both Y (50.86%) and O (54.40%), but decreased in PF (17.55%). Fat and muscle loss were similar in Y and O in response to T3 (fat, −52.53%(Y) vs −51.69% (O); muscle, −21.50% (Y) vs −21.03% (O) in soleus and −16.70% (Y) vs −17.35% (O) in plantaris), whereas these changes were minor with OPF (fat, −10.4%; soleus −1.69% ; plantaris 7.69%). While mt ATP production and citrate synthase activity in heart and soleus muscle were lower in O, these measures increased equally in Y and O in response to T3. In the soleus, mt DNA abundance increased in Y (54.39%) and O (23.36%) and UCP3 expression increased 10‐fold in both age groups. Gene transcripts levels of PGC1α, COX3&4 and NADH4, UCP2& 3 increased in soleus of T3 rats. In conclusion, T3 similarly increased energy expenditure, weight loss and mt biogenesis in both Y and O rats, despite differences in food intake.NIH grant PO1 AG09531 and Mayo Foundation
Elevated serum advanced glycation end products and poor grip strength in older community‐dwelling women
Authors: Dalal Mansi Sunil , Ferrucci Luigi , Sun Kai , Fried Linda , Varadhan Ravi , Walston Jermey , Guralnik Jack , Semba Richard 
Published: June 2021

Background:Aging is characterized by loss of muscle strength, but its etiology is not well understood. Advanced glycation end products (AGEs) are bioactive compounds that are implicated in the pathogenesis of diabetes, cardiovascular disease, and renal failure. However, their relationship with skeletal muscle has not been well characterized.Objective:The objective of the study was to examine the relationship between serum AGEs, circulating receptor for AGEs (sRAGE), endogenous secretory RAGE (esRAGE), and grip strength in older women.Methods:Serum AGEs, sRAGE, esRAGE, and grip strength were measured in 559 women, age ≥65 years, in the Women's Health and Aging Study I.Results:The mean (SD) grip strength among women in the highest quartile of serum AGEs compared with the lower three quartiles was 18.2 (6.4) and 20.1 (6.2) kg, respectively (P = 0.004). Women in the highest quartile of serum AGEs had decreased grip strength (beta = −1.31, SE = 0.61, P = 0.03) in a multivariate linear regression model, adjusting for age, race, BMI, cognitive dysfunction, and depression. Serum sRAGE and esRAGE were not significantly associated with grip strength.Conclusion:Women with high serum AGEs have greater muscle weakness. Further studies are needed to determine whether AGEs, a potentially modifiable risk factor, are associated with physical performance and disability in older adults.
Prolonged Exercise Training does not Preserve Mitochondrial Enzyme Activity in Senescent Rats
Authors: Betik Andrew C , Thomas Melissa M , Wright Kathryn J , Hepple Russell T 
Published: June 2021

Skeletal muscle aerobic function (VO2max) and mitochondrial oxidative capacity decline with aging, particularly in senescence (SEN). Since exercise training improves muscle VO2max and oxidative capacity in young adult skeletal muscle, our objective was to determine if treadmill training from late middle age (LMA) into SEN would minimize the declines in skeletal muscle aerobic function and oxidative capacity seen in SEN. 62 male rats at late middle age (29 mo) were randomly separated into control and exercise groups. Exercise consisted of 60 m/day treadmill running, 5 d/week for 7 weeks and then decreased to 4 d/week for a total of 5 and 7 mo (until 34 and 36 mo of age respectively). Although running capacity was significantly higher in the trained groups (13% at 34 mo and 21% at 36 mo), muscle VO2max at matched muscle O2 delivery was not significantly greater in the trained groups compared to sedentary controls at either age. Similarly, citrate synthase and cytochrome oxidase activity in the plantaris, soleus and the heart muscle were not different in the trained vs the sedentary groups, suggesting no benefit of training for muscle oxidative capacity. The superior exercise capacity suggests a better overall level of function of the senescent rats; however, contrary to our hypothesis and prior observations in LMA rats, treadmill exercise training between LMA and SEN does not benefit muscle aerobic function or mitochondrial oxidative capacity.Supported by CIHR (AB, RT), CIHR Provincial Training Program in Bone and Joint Health (AB), AHFMR (RT) and NSERC (MT).