bluuebunny/crossref_metadata_2025_split huggingface dataset:
N‐cadherin adhesion modulates RhoGTPase signaling to promote endothelial barrier integrity
Authors: Kruse Kevin Jonathan , Huang Fei , Ying Sun , Vogel Stephen , Malik Asrar , Komarova Yulia 
Published: November 2023

The vascular endothelium is comprised of a monolayer of endothelial cells (ECs) lining the blood vessels, which functions as a semi‐permeable barrier between blood and the surrounding tissue. Vascular Endothelial (VE)‐cadherin, which forms homotypic adhesion between ECs at cell‐cell contacts called adherens junctions (AJs) is the main adhesive molecule that restricts passage of protein rich fluids through the endothelial monolayer. Along with VE‐cadherin, ECs express Neural (N)‐cadherin, which is excluded from EC‐EC junctions and instead forms heterotypic adhesions between ECs and mural cells (such as pericytes or smooth muscle cells) at the abluminal surface of the endothelium. While the role of VE‐cadherin adhesion in regulating endothelial barrier function has been well established, the role of N‐cadherin in ECs remains mainly unknown. Recent evidence suggests that pericytes are critical to maintaining proper endothelial barrier function, however it is still unclear if this is solely due to pericyte recruitment to the endothelium or if N‐cadherin adhesion mediated signaling in ECs plays a role in strengthening the endothelial barrier. Using an EC specific, inducible knockout mouse model developed in our lab, we have shown that deletion of Cdh2 gene (N‐cadherin) results in increased permeability of pulmonary endothelial microvessels, suggesting that N‐cadherin adhesion restricts permeability in the endothelial barrier. To study N‐cadherin adhesion‐mediated signaling in endothelial monolayers, we have utilized surface chemistry techniques to mimic N‐cadherin homophilic interaction in vitro. Covalent attachment of N‐cadherin extracellular domain to an Ni‐NTA surface in an oriented manner induced formation of N‐cadherin adhesions proximal to tricellular junctions and promoted recruitment of VE‐cadherin to AJs. Depletion of N‐cadherin in ECs or denaturation of N‐cadherin extracellular domain abolished this effect, suggesting that N‐cadherin “outside‐in” signaling promotes assembly of VE‐cadherin adhesion. Using microscale biomimetic platforms combined with mass spectroscopy, we isolated and analyzed N‐cadherin complexes. We found that a dual Rac1 and RhoA activator, Trio, is recruited to N‐cadherin adhesion complexes to activate both Rac1 and RhoA signaling at AJs. This was consistent with increased acto‐myosin tension across VE‐cadherin analyzed with a FRET‐based biosensor as well as formation of lamellipodia protrusions above AJs. Furthermore, inhibition of ROCK signaling or depletion of Neuronal navigator 1 (Nav1) abolished recruitment of VE‐cadherin to AJs specifically in cells plated onto N‐cadherin but not gelatin‐coated surfaces. These data cumulatively demonstrate that N‐cadherin promotes assembly of VE‐cadherin adhesion by providing spatial control of RhoA and Rac1 activities through activation of Trio.Support or Funding InformationNIH R01 HL103922
Na‐driven Cl/HCO3 exchanger (NDCBE) activity is essential for establishing new steady‐state in intracellular pH during metabolic acidosis in murine hippocampal neurons and astrocytes
Authors: Salameh Ahlam , Boron Walter F 
Published: November 2023

The Na‐driven Cl/HCO3 exchanger (NDCBE) is highly expressed in CA1 hippocampal (HC) neurons but has not been reported to be present in astrocytes. NDCBE extrudes acid from the cytoplasm by mediating the exchange of 1 Na+ and 2 HCO3− (or 1 CO3=) from the extracellular compartment for 1 Cl− from the intracellular compartment. Previously, we showed that knocking‐out anion exchanger 3 (AE3), which is an acid loader expressed only in neurons but not astrocytes, reduces the ability of HC neurons and astrocytes to acid load the cytoplasm. We also showed that, after 70 sec in metabolic acidosis (MAc), intracellular pH (pHi) in neurons and astrocytes from AE3−/− mice decreases continuously over 7 min (i.e., fails to stabilize). We hypothesized that this failure to reach new steady‐state is due to a decreased ability of neurons and astrocytes to increase their acid‐extrusion rate (JE) sufficiently to balance the acid‐loading rate (JL). In the present study we investigate the role of NDCBE activity in facilitating sufficient JE to balance JL during MAc. We induced MAc by decreasing the extracellular [HCO3−] from 22 mM to 14 mM at fixed 5% CO2. This maneuver decreases extracellular pH from 7.4 to 7.2. We monitored the changes in pHi during MAc exposure using fluorescence imaging of the pH sensitive dye BCECF. We obtained co‐culture of neurons and astrocytes from the hippocampal CA1 regions from wild‐type (WT) and NDCBE‐knockout (NDCBE−/−) mice. Our results show that, for neurons, the initial steady‐state pHi is significantly higher in WT (7.25 ± 0.03) compared to NDCBE−/− (7.01 ± 0.01). However, the MAc‐induced acidification rate during the first 70 sec of the MAc (dpHi/dt)early is not significantly different between WT (−1.6 × 10−3 ± 0.8× 10−3 pH/s) and NDCBE−/− (−1.4 × 10−3 ± 0.9 × 10−3 pH/s). After 70 sec in MAc, the pHi of NDCBE−/− decreases continuously over 7 min, with a slope, (dpHi/dt)late, of −3.0 × 10−4 ± 0.6 × 10−4 pH/s, which is significantly faster than WT neurons (−0.9 × 10−4 ± 0.6 × 10−4 pH/s). For astrocytes, the knockout of NDCBE surprisingly reduces initial steady‐state from 6.99 ± 0.01 for the WT to 6.90 ± 0.01 for NDCBE−/− and significantly reduces (dpHi/dt)early from −3.3 × 10−3 ± 0.2 × 10−3 pH/s in the WT to −2.2 × 10−3 ± 0.1 × 10−3 in NDCBE−/−. The results also reveal that the absence of NDCBE reduces the astrocytes ability to stabilize pHi after 70 sec in MAc, with a (dpHi/dt)late of −2.0 × 10−4 ± 0.3 × 10−4 pH/s compared to the WT (dpHi/dt)late of −0.6× 10−4 ± 0.6 × 10−4 pH/s. Our results are consistent with the hypothesis that NDCBE activity is essential for braking the fall of pHi in both HC neurons and astrocytes during MAc exposure.Support or Funding InformationR01‐DK081567, R01‐NS018400 and T32 HL007913
Dysregulation of Pendrin and NBC‐e1 and Impaired HCO3− Secretion in the Kidneys of CFTR Knockout Mice: Role in the pathogenesis of metabolic alkalosis in cystic fibrosis
Authors: Kia Mujan Varasteh , Barone Sharon Lynn , Xu Jie , Zahedi Kamyar , Soleimani Manoocher 
Published: November 2023

IntroductionPatients with cystic fibrosis (CF) are prone to the development of metabolic alkalosis, which is manifested by an elevation of arterial blood pH and bicarbonate concentration and produces a physiologic diminution of respiratory effort that is detrimental in CF patients. The pathogenesis of metabolic alkalosis in CF remains unknown.HypothesisInactivation of CFTR will impair the ability of the kidney to excrete excess bicarbonate via dysregulation of HCO3− transporters, therefore resulting in the elevation of arterial pH and bicarbonate concentration and subsequently, generation of metabolic alkalosis in mutant mice subjected to an oral bicarbonate load.MethodsCFTR knockout (CF) mice with the intestinal rescue (over‐expressing wild type CFTR transgene in their intestine‐Science 1994) were examined for the changes in arterial pH and bicarbonate concentration as well as the expression of pendrin and NBC‐e1. Animals were subjected to an oral bicarbonate load (140 mM/lit bicarbonate in the drinking water) and examined after 3 days.ResultsBaseline parameters, including acid‐base status and urine pH were comparable in CF and WT mice. Compared to WT animals, CF mice demonstrated a significant increase in blood HCO3− concentration (21.87 +/− 0.8 in wt vs. 26.83 +/− 0.9 mEq/l in CF mice, p<0.004) and pH (7.33 +/− 0.01 in wt vs. 7.42 +/− 0.02 in CF mice, p<0.003) and impaired kidney HCO3− excretion (urine pH 8.10 +/− 0.09 in wt vs. 7.35 +/− 0.09 in CF‐KO mice, p<0.002) following a 3‐day oral bicarbonate load. Immunofluorescence labeling demonstrated profound reduction in the apical expression and increased cytoplasmic and basolateral localization of pendrin in the intercalated cells in CF mice (Fig. 1). Moreover, an unexpected aberrant induction of the Na+:HCO3− co‐transporter NBCe1 (kidney NBC1) was detected in principal cells of the cortical collecting duct in CF mice (Fig. 2).ConclusionWe propose that patients with cystic fibrosis are prone to the development of metabolic alkalosis secondary to the dysregulation of pendrin and NBC‐e1, specifically during volume depletion, which is a common occurrence in CF patients.Support or Funding InformationMerit Review Award from Veterans Administration
Structure ‐ function relationship of the human mitochondrial thiamin pyrophosphate transporter (MTPPT; SLC25A19): Critical roles for Ser34, Lys 291 and His137
Authors: Sabui Subrata , Subramanian Veedamali S , Ghosal Abhisek , Kapadia Rubina , Said Hamid M 
Published: November 2023

The water‐soluble vitamin B1 (thiamin) plays critical role in cellular energy metabolism. Mammalian cells lack the ability to synthesize thiamin, and thus, must obtain the vitamin from their surrounding via transport across cell membrane. Once in the cytoplasm, free thiamin is enzymatically converted to thiamin pyrophosphate (TPP); the bulk of this TPP is then taken up by the mitochondria by a carrier‐mediated process that involves the MTPPT (product of SLC25A19 gene). Previous studies from our laboratory and others have characterized different physiological and biological aspects of the MTPPT, but little is currently known about its structure‐function relationship. Using protein‐docking modeling (www.sbg.bio.ic.ac.uk/phyre2 and http://www.dockingserver.com/web) and site‐directed mutagenesis, we examined in the current investigation the role of amino acid residues that are predicted (by the docking model) to be involved in the function of the human MTPPT (hMTPPT; i. e., Thr29, Arg30, Ile33, Ser34, Asp37 and Phe298); we also examined the role of conserved (among species) positively charged amino acids (i. e., His82, His137, Lys231 and Lys291) in hMTPPT function of uptake of the negatively charged TPP. Our results showed that of the six predicted residues, Ile33, Ser34, Asp37 appeared to be critical for hMTPPT function as their individual mutation led to a significant reduction in carrier‐mediated MTPP uptake (with no change in protein stability/translational efficiency of the transporter). The cause of impairment in the case of Ile33 and Asp37 appeared to be due to inefficient delivery of the mutant protein to the mitochondria as indicated by confocal imaging and by Western blot analysis. With regards to the role of the conserved positively charged residues, only His137 and Lys291 were found to be important for hMTPPT function as indicated by the inhibition in MTPP uptake upon their mutation. This inhibition in MTPP uptake was again found to be due to inefficient delivery of the protein to the mitochondria (again with no change in protein stability/translational efficiency of the transporter). Results of this investigation show an important role for Ser34, H137 and Lys291 in the function of the hMTPPT.Support or Funding InformationSupported by grants from the NIH (AA018071 and DK56061) and the DVA
Effects of Varied Ionic Concentrations on The Feeding Ability of Cordylophora caspia, An Invasive Hydroid
Authors: Cruise Jessica D. , Hofer Sarah J. , Busch Jennifer L. , Folino‐Rorem Nadine C. 
Published: November 2023

The objective of our study was to observe the effects of KCl concentrations on nematocyst functionality as it related to feeding ability in the invasive hydroid Cordylophora caspia (phylum Cnidaria, class Hydrozoa). Nematocysts are stinging organelles encapsulated in cnidocytes on the tentacles. When tentacles are tactilely stimulated by prey, cnidocytes experience a rapid uptake of water and ions, resulting in a buildup of osmotic pressure that ultimately forces the nematocyst from the cell, rendering the prey immobile. This discharge mechanism makes nematocyst function potentially susceptible to variation in environmental NaCl and KCl concentrations.Cordylophora caspia can thrive in a variety of osmotic environments, from freshwater to brackish habitats. We assessed the effects of different ionic environments on nematocyst discharge by adding 5 mM KCl to fresh and brackish water. Two genotypes of Cordylophora caspia (one native to fresh water and one native to brackish water) were collected and cultured in the laboratory. The freshwater genotype was native to a salinity of 10 mM NaCl (0.6 ppt), and the brackish genotype was native to a salinity of 270 mM NaCl (16 ppt). Colonies from each genotype were cultured at their native salinity (controls) and at the native salinity of the other genotype in the absence and presence of additional 5 mM KCl.Our results showed that treatment‐induced effects varied as a function of salinity, rather than genotype. When compared to control groups lacking additional KCl, colonies of both genotypes cultured in fresh water + 5 mM KCl displayed a significantly reduced ability to stun and capture prey, indicative of impeded nematocyst discharge. Conversely, no colonies cultured in brackish water + 5 mM KCl differed significantly from controls in ability to stun and capture prey. Colonies with a reduced ability to stun and capture prey regained their ability to do so when returned to water with no added KCl.These data suggest that an impermanent reduction in nematocyst functionality occurs when 5 mM KCl is added in a freshwater environment. Understanding the physiology of nematocyst discharge has applications for taxonomy and species differentiation in Cordylophora spp., and these results may be used as a model for osmotic studies in other aquatic organisms.
Further Characterization of Anuran‐Like Aquaporins in the Urinary Bladder of Urodeles, Notopthalmus viridescens and Cynops pyrrhogaster
Authors: Hillyard Stanley , Shibata Yuki , Ishii Ryo , Nagai Takatoshi , Suzuki Masakazu 
Published: November 2023

Recent Bayesian analysis of tetrapod aquaporin gene clusters by Finn et al (PLOS1, November 26, 2014) suggests anuran‐specific aquaporins that mediate water transport across the ventral skin (AQPa2S) and urinary bladder (AQPa2U) are more closely related to AQP6 found in mammals and reptiles. Furthermore, AQP6/a2 aquaporins are syntenic with AQP5. We used RT‐PCR to show both AQP6/a2U and AQP5 homologs are expressed in the urinary bladder of the newts, Notophthalmus viridescens (nv) and Cynops pyrrhogaster (cp). Full length amino acid sequences showed 72% and 71% identity between AQP6/a2U of nv and anuran species Hyla japonica and Xenopus tropicalis (xt). Amino acid identity of AQP5 between nv and cp was 95% while identity between both urodeles and xt was 69%–70%.Consensus phosphorylation sites for PKA were identified for AQP6/a2U and AQP5 for both nv and cp. AQP5 could be immunolocalized in the apical membrane of bladder epithelial cells in rehydrating newts. These results indicate an absorptive role for AQP5 in the urodele urinary bladder and suggest that this role may originate in the ancestor of urodeles and anurans.Support or Funding InformationSupported by a Grant‐in‐Aid for Scientific Research (26440165) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
Evolution of the Cytochrome c Oxidase Subunit 4 Paralogs
Authors: Moyes Christopher D , Porplycia Danielle , McDonald Jared 
Published: November 2023

The largest subunit of cytochrome c oxidase exists as two paralogs in most vertebrates: a constitutive COX4‐1 and hypoxia‐inducible COX4‐2. In primates and rodents, COX4‐2 is a hypoxia‐responsive gene encoding a protein with its ATP‐binding site disrupted by a disulfide bridge, precluding COX from the allosteric regulation that is exerted through the this site in COX4‐1. In an effort to understand the evolution of the gene, we examined role of the paralogs in fish. COX4‐2 of fish differs from mammals in three respects. First, the fish COX4‐2 gene does not appear to be hypoxia responsive at either the mRNA or protein level. This was shown in mammalian cells transfected with fish promoter constructs, and hypoxia treatments of cultured fish cells and whole animals. We used fish species from multiple lineages and differing in hypoxia tolerance, exposed to different degrees and durations of low oxygen. Second, the fish COX4‐2 protein lacks the structural features (paired CYS residues) that distinguish COX4‐2 from COX4‐1 in mammals. This pair of CYS residues in COX4‐2 protein appears only in the lineage of vertebrates that encompasses rodents and primates. Third, COX4‐2 expression patterns are quite different in fish. They show higher constitutive expression of COX4‐2 in all tissues, and demonstrate changes in COX4‐2 expression with development. Immunohistochemistry of tissues expressing both COX4 paralogs revealed that some tissues displayed both proteins in most cells, whereas other tissues showed cell‐specific patterns. In heart, individual myocytes expressed both COX4 paralogs, although select non‐myocytes expressed only COX4‐1. The paralog patterns also changed with size/age. Heart was dominated by COX4‐1 mRNA and protein in small fish but transitioned to predominately mRNA and COX4‐2 in large fish. The distinctions in COX4‐2 between mammalian models (rodents, humans) and other vertebrates (other mammalian lineages, reptiles, fish) bring into question the genetic origins of the paralog pair and its functional evolution during vertebrate diversification.Support or Funding InformationSupported by NSERC Canada
Pulmonary Endothelial Heterogeneity in Response to Infection‐induced Cytotoxic Supernatant
Authors: Morrow Kyle Adam , Balczon Ron , Frank Dara W , Stevens Troy Adam 
Published: November 2023

Here, we tested the hypothesis that Pseudomonas aeruginosa type 3 secretion system effectors ExoY and ExoU induce release of endothelial‐derived cytotoxic factors that have differential cytotoxic effects when applied to pulmonary endothelium. Pulmonary microvascular endothelial cells (PMVECs) were grown 12–24 hours post‐confluence and inoculated at a multiplicity of infection of 20:1 with P. aeruginosa possessing either ExoY (ExoY+) or ExoU (PA103). Bacterial delivery of ExoY and ExoU induced time‐dependent PMVEC gap formation that was paralleled by release of endothelial‐derived proteins, including oligomeric tau, into the extracellular space. Supernatant was collected, centrifuged for 10 minutes at 4500 × g, and filter‐sterilized. Transfer of filtered supernatant onto confluent naïve PMVECs induced apparent inter‐endothelial gap formation and destabilized pre‐formed endothelial networks in Matrigel. When filtered supernatant was transferred onto naïve pulmonary artery endothelial cells, there was a marked decrease in the cytotoxic effects when compared with PMVECs. Thus, we provide the first evidence for pulmonary endothelial heterogeneity when faced with potential disease propagation due to infection‐induced cytotoxic supernatant.Support or Funding InformationThis work was supported by American Heart Association postdoctoral fellowship grant 14POST18080004 (K.A.M.) and National Institutes of Health grants HL‐60024 and HL‐66299 (T.S. and R.B.)
Investigation of the Cardiotonic Steroids, Marinobufagenin and Resibufogenin, in the Acute Respiratory Distress Syndrome
Authors: Abbas Mir Mohammad Kamran , Chen Qingzheng , Das Aayudh , Oliver Joel , Jiang Weiwu , Moorthy Bhagavatula , Patel Bela , Morin Kristi , Puschett Jules 
Published: November 2023

The bufodienolides are a group of “cardiac glycosides” also termed the “cardiotonic steroids”, which are produced largely in the adrenal gland, but also in the placenta, as well as, perhaps, in the brain. They circulate in the blood and are excreted into the urine. They were originally discovered in the skin and the venom of the common toad (Bufo marinus), hence their appellation. Their major action is that of the inhibition of the ubiquitous enzyme, Na+/K+ ATPase. Acute respiratory distress syndrome (ARDS) is a life‐threatening disorder with a prevalence rate of 64 cases per 100,000 person‐years and a mortality rate of 40–52%. It is most often encountered in severely ill patients with a background of sepsis, trauma, complicated severe pneumonia and burns. Aside from improvements in ventilator therapy, there have occurred no new therapeutic strategies that improve survival. In an animal model of ARDS involving the production of the disease by exposing the animals (rats) to > 95% oxygen for 48–60 hours, serum levels of marinobufagenin (MBG) rose to 99.7 +/− 17.5 (SE) pg/ml from those determined in sham rats (mean: 25.7 +/− 2.3 SE pg/ml) (p<0.02). One hour after the hyperoxia period was completed, the intraperitoneal administration of 160 ug of resibufagenin (RBG), an antagonist of MBG, in a third group of animals, resulted in a decline of mean serum MBG to 17.2 +/− 2.7 pg/ml (p< 0.001). The mean values for MBG of the sham and hyperoxia + RBG groups differed (p= 0.003). In 18 patients with ARDS, recruited from the ICU of the Memorial‐Hermann Hospital in Houston, TX, the mean urinary excretion level of MBG/mg creatinine was 760 +/− 233 pg/mg creatinine compared to that obtained in 19 control patients admitted to the ICU for other indications was 169 +/− 74 pg/mg creatinine (p= 0.01).We conclude that: 1) Elevated levels of MBG serve to diagnose ARDS and 2) RBG may prove to provide “rescue” therapy for this disorder.Support or Funding InformationJules B. Puschett, M.D. Research Endowment Fund
S‐Nitrosocysteine Alters Outward Potassium Current in Isolated Glomus Cells of Rat Carotid Body under Normoxic and Hypoxic Conditions
Authors: Getsy Paulina M. , Coffee Gregory A. , Sawczak Victoria E. , Pye Richard L. , Zaman Khalequz , Lewis Stephen J. 
Published: November 2023

Glomus (type I) cells in the carotid body (CB) respond to low blood oxygen (O2) by depolarizing, opening voltage‐gated calcium channels, and releasing excitatory transmitters. These neurotransmitters activate chemosensory afferent terminals of the carotid sinus nerve (CSN), which stimulate the brainstem to increase ventilation. Our lab shows that circulating blood levels of S‐Nitrosocysteine (SNO‐L‐CYS) increase in response to hypoxia in the rat. Our objective was to determine whether SNO‐L‐CYS acts directly on the rat glomus cell to modulate its response to hypoxia. To test this we isolated glomus cells from postnatal (P) P11–P14 male and female rat pups and plated them on poly‐D‐Lysine coated coverslips. After a 2–3 hour incubation period at 37°C, 5% CO2, whole‐cell perforated patch clamp recordings were performed to characterize glomus cell current‐voltage relationship in response to 1μM SNO‐L‐CYS under normoxic (21% O2), and hypoxic (5–8% O2) conditions. Our results show that at 21% O2, 1 μM SNO‐L‐CYS increases the maximum outward steady‐state potassium current (n = 3). Under hypoxic conditions, we discovered two populations of glomus cells; (1) those whose steady‐state maximum potassium current was inhibited by hypoxia and further inhibited after administration of 1 μM SNO‐L‐CYS while in hypoxia (n=8), and (2) those whose steady‐state maximum potassium current was inhibited by hypoxia and then increased after administration of 1 μM SNO‐L‐CYS while in hypoxia (n=9). The SNO‐L‐CYS effect is washed out in recovery at 21% O2. This data suggests that under hypoxic conditions SNO‐L‐CYS has a dual‐ modulatory effect. It can act on glomus cells to further increase cell depolarization and transmitter release, leading to an increase in CSN firing. In addition, it can act on glomus cells to suppress depolarization and neurotransmitter release, thus returning the glomus cell to its resting state. Having two populations of glomus cells that respond differently to SNO‐L‐CYS may allow highly regulated carotid body firing under hypoxic conditions.Support or Funding InformationNIH PO1‐HL1018710