bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mitochondria‐Targeted Ogg1 and Aco‐2 Prevent Oxidant‐Induced Mitochondrial DNA Damage
Authors: Kim Seok‐Jo , Cheresh Paul , Tularisam Sandhya , Schumacker Paul , Weitzman Sigmund A , Bohr Vilhelm , Kamp David W. 
Published: June 2021

ObjectiveMitochondria‐targeted hOgg1 (mt‐hOgg1; a DNA base excision repair enzyme) and aconitase‐2 (Aco‐2; Kreb's cycle enzyme) each reduce oxidant‐induced alveolar epithelial cell (AEC) apoptosis but it is unclear whether protection occurs by preventing mtDNA damage.MethodsQuantitative PCR‐based measurement of mitochondrial DNA damage was assessed following transient transfection with wild‐type mt‐hOgg1, mt‐long α/β Ogg1 mutant (mt‐Lα/β Ogg1), Aco‐2 and siRNA for Aconitase‐2 (siAco‐2) in A549 cells with asbestos (5–25 μg/cm2) or H2O2 (100–250 μM) for 24 h. Apoptosis was assessed by cleaved caspase‐9/3 and DNA fragmentation.ResultsAsbestos and H2O2 induced mtDNA damage and apoptosis that was mitigated by overexpression of mt‐hOgg1, mt‐Lα/β Ogg1 or Aco2. Compared to scrambled siRNA, siAco‐2 enhanced mtDNA damage and apoptosis induced by asbestos or H2O2.ConclusionsOur data demonstrate a novel role for mt‐hOgg1 and Aco‐2 in preventing oxidant‐induced AEC mtDNA damage and apoptosis. Given the importance of AEC apoptosis in pulmonary fibrosis, we reason that the mt‐hOgg1/Aco‐2 effects on AEC mtDNA is an innovative target for preventing pulmonary fibrosis. Funding: NIH‐RO1 ES020357‐01A1 (DK) and VA Merit (DK)
Effect of surfactants on polystyrene nanoparticle (PNP) interactions with primary rat alveolar epithelial cell monolayers (RAECM)
Authors: Kim Yong Ho , Sipos Arnold , Fazlollahi Farnoosh , Borok Zea , Kim Kwang‐Jin , Crandall Edward D 
Published: June 2021

We investigated effects of apical 0.1% (w/v) Pluronic (P) or 1 mg/mL Infasurf (I) on flux of 20 nm carboxyl‐modified (negatively charged) PNP (20‐PNP) or 20 nm amidine‐modified (positively charged) PNP (20+ PNP) across RAECM, across which the bioelectric properties (transepithelial resistance (Rt) and equivalent short circuit current (Ieq)) were unchanged in the presence of these two surfactants. Results of flux measurements, along with hydrodynamic diameter (HD) measured by dynamic light scattering and zeta potential (ZP) measured in the presence and absence of P or I, are summarized below.

20+ PNP
20‐ PNP

Flux (pg/cm2/sec)
PNP alone
38±7
1.6±0.1

PNP with P
35±7
2.5±0.2

PNP with I
42±6
0.9±0.2

HD (nm)
PNP alone
543±28
156±7

PNP with P
374±13
80±3

PNP with I
316±9
278±9

ZP (mV)
PNP alone
37±10
−61±6

PNP with P
33±2
−29±2

PNP with I
−44±1
−82±4

These data indicate that there is remarkably little effect of P or I on PNP flux despite significant changes in measured HD and ZP. We conclude that PNP (and RAECM) exposure to either surfactant P or I surprisingly does not cause meaningful alterations in PNP translocation across RAECM, which may be explainable by the behavior of PNP in the extracellular microdomain adjacent to the apical alveolar epithelial cell membrane.(Funded by NIH and Hastings & Whittier Fdns.)
Nanodiamond (ND) interactions with primary rat alveolar epithelial cell monolayers (RAECM)
Authors: Kim Yong Ho , Sipos Arnold , Fazlollahi Farnoosh , Borok Zea , Kim Kwang‐Jin , Crandall Edward D 
Published: June 2021

We investigated interactions of positively and negatively charged 5 nm ND (zeta potentials of 50 and −60 mV, respectively; provided by Carbodeon, Vantaa, Finland) with RAECM. Transepithelial resistance (Rt) and equivalent short circuit current (Ieq) of RAECM in the presence and absence of ND in apical fluid for up to 24 h did not change. We estimated cellular uptake and transepithelial trafficking of ND across RAECM over 24h spectrofluorometrically with excitation/emission wavelengths at 275/555 nm. Cell uptake of positively charged ND was ~3 times greater than that (~9 pg/μg cell protein) of negatively charged ND, while transepithelial trafficking of positively and negatively charged ND was about the same at ~20 pg/cm2/s. Trafficking of ND is ~10‐fold faster than that of negatively or positively charged quantum dots (core diameter 5 nm) and negatively charged polystyrene nanoparticles (PNP, 20 nm), but ~2‐fold slower than that of positively charged PNP (20 nm). These data indicate that positively or negatively charged ND do not affect barrier function of alveolar epithelium and suggest that ND surface charge affects accumulation in but not translocation across alveolar epithelium. Mechanisms underlying ND entry into/egress from alveolar epithelial cells remain to be determined.(Funded by NIH and Hastings & Whittier Fdns.)
Overexpression of Circadian CLOCK genes alters proinflammatory cytokine production in human alveolar epithelial cells
Authors: Parthasarathy Prasanna Tamarapu , Luong Tran , Lagishetty Venu , Phillips Oluwakemi , Cox Ruan , Castillo Annie , Abuelenen Toaa , Lockey Richard , Kolliputi Narasaiah 
Published: June 2021

Acute lung injury (ALI) is a devastating clinical syndrome manifested by inflammation and decreased lung compliance. Cytokines such as IL‐1b, IL‐6 and IL‐8 have been implicated in the process of inflammation and in chemokine stimulation associated with ALI. Recent studies suggest that circadian clock genes are involved in inflammation disorders; however, the role of these genes in ALI is yet unknown. Therefore, we investigated the role of clock genes on proinflammatory cytokine production in human alveolar epithelial cells (A549). A549 cells were transfected with core clock genes (Bmal1, CLOCK, Cry1/2, Per1, Per2) and subsequently treated with either IL‐1b or hydrogen peroxide. Supernatants were analyzed for the production of IL‐6 and IL‐8. There was a significant decrease in IL‐8 production in cells transfected with core CLOCK genes. However, the level of IL‐6 was unchanged in these cells. Further, A549 cells treated with IL‐1b or hydrogen peroxide after Cry1 and Per2 transfection showed a significant decrease in IL‐6 and IL‐8. Our data suggest that core clock genes may be protective by ameliorating cytokine production thereby offering a potential therapeutic to minimize the clinical severity and mortality of ALI.This work was supported by the AHA 09SDG2260957 and NIH R01 HL105932 grants to N.K.
Modeling the alveolar Ar after been squeezed by inspiring room air
Authors: Melendrez Jose Maria , Caucha Luis Jhony , Cruz Julio Cesar 
Published: June 2021

The experiment of inhaling 1.9L of 79% argon in O2 was modeled previously (Cruz. Respir. Physiol. 86:1, 1991). To account for the argon inhaled, 1.501L, a large dead space, 0.425L, was necessary. Moreover, 1.9L of air was inhaled to reach TLC. Thus, the alveolar Ar that was inhaled previously was squeezed and pushed in to the end of Weibel's generations. We recently showed that the expired CO2, previously diluted in the alveoli, became more evident with the forced expiration and revealed the effect of alveolar CO2 diffusion from RV to ERV (Caucha's et al. FASEB J. 24:1063.5, 2012). Now, we modified Caucha's model to simulate the Ar expirogram, changing the number of generations assigned for convection, convection‐diffusion and diffusion in the seven parallel regions from apex to base of the lung. The Figure shows two kinds of experiments, the first one simulates the experiment of Cruz in 1991. This is depicted on the left scale (dashed curve) with a volume expired of 4.5L. The “sandwich” experiment is shown on the right scale (for clarity) with a volume expired of 6.4L. The total argon inhaled is now in the alveoli and 1.24L of Ar was exhaled. Thus, more Ar was diffused to RV to reach 0.234L in the model presented (error of 1.7%). Note that the curve shown is the one that would be obtained during expiration after the alveolar argon was diluted and squeezed by inhaling air. The alveolar plateau reached the ideal alveolar gas mixing, 0.20 [Amount of Ar/(Total Lung Capacity‐Dead space)] at 3.8L‐4.23L (horizontal dotted line). However, the slope of phase IV is negative.Supported in part by CEIS (Centro de Enseñanza, Investigación y Servicios).
Endostatin limits pulmonary endothelial repair via RhoA inactivation
Authors: Jian Ming‐Yuan , Creighton Judy 
Published: June 2021

Endothelial repair following vascular injury involves cell proliferation and migration. Endostatin regulates cell proliferation, and is essential for maintenance of a functional vascular barrier. However, elevated endostatin levels correlate with severity of inflammatory lung damage. The source of endostatin or its role in progression of vascular disease is not known. We reasoned endostatin release by lung endothelium during an injury event contributes to pulmonary vascular damage by limiting endothelial proliferation and migration; thus slowing barrier restoration. LPS increased endostatin expression in lung tissue in vivo and in pulmonary microvascular endothelial cells (PMVECs) in vitro. LPS caused translocation of endostatin from peri‐nuclear regions to cell membranes in PMVECs consistent with release of endogenously synthesized endostatin. LPS inhibits PMVEC proliferation and wound healing, an effect exacerbated by exogenous application of endostatin. Endostatin decreased RhoA activity in PMVECs implicating the RhoA pathway in endostatin signaling.Conclusioninflammation induces endostatin release from endothelium sufficient to limit lung vascular repair. Supported by HL102296 and HL 110803.
Cisplatin‐induced cell death in MCF‐7 breast cancer cells: Roles of ΔNErbB2 and pH regulatory ion transporters NHE1 and NBCn1
Authors: Sigurđsson Haraldur H. , Olesen Christina W. , Dybboe Rie , Lauritzen Gitte , Pedersen Stine Falsig 
Published: June 2021

We have shown that expression of a truncated constitutively active ErbB2 receptor (ΔNErbB2), which is associated with poor prognosis in breast cancer, greatly increased net acid extrusion in MCF‐7 breast cancer cells in a manner mediated by the Na+/H+ exchanger NHE1 and the Na+,HCO3− cotransporter NBCn1. Here, we explore the roles of NHE1 and NBCn1 in cisplatin‐induced cancer cell death. In both vector‐ and ΔNErbB2 cells, cisplatin treatment (25 μM, 18 h) induced cleavage of caspase‐9,‐7, PARP, and fodrin (a calpain substrate), and cysteine cathepsin release, all in a manner slightly augmented by NHE1 inhibition. Expression of the anti‐apoptotic Bcl‐2 protein was markedly reduced in ΔNErbB2‐ compared to vector cells. Cisplatin treatment increased p53‐ and PUMA levels in both cell types, and reduced the number of autophagic vesicles and increased p62 levels, most markedly in ΔNErbB2 cells. This reduction of autophagic flux was not significantly affected by inhibition of NHE1 or NBCn1. Finally, cisplatin treatment elicited marked perinuclear accumulation of NBCn1, but not of NHE1, and reduced the protein levels of both full length‐ and ΔN‐ErbB2. In conclusion, cisplatin treatment of MCF‐7 cells activates several death mechanisms, some of which are further exacerbated by NHE1 inhibition in agreement with previous findings that NHE1 inhibition sensitizes MCF‐7‐ΔNErbB2 cells to cisplatin‐induced death.Funding: Danish Cancer Society, Danish Research Council
Decreased calcium mobilisation and the lack of secretion in equine sweat gland cells
Authors: Robertson Jennifer , Ko Wing‐Hung , Bovell Douglas 
Published: June 2021

Sweat secretion helps maintain skin surface ecosystems and allows humans and the Equidae to thermoregulate. A key component in sweat secretion is an increase in intracellular Ca2+ ([Ca2+])i from internal and external sources. Regulation of Ca2+ entry across the plasma membrane is initiated by emptying of the intracellular Ca2+stores and through a process known as store–operated Ca2+ entry (SOCE) via STIM and Orai proteins.We studied the role of SOCE in horse sweat gland cells from control animals and those that had developed anhidrosis, an inability to produce sweat.Western blot analysis and PCR confirmed the presence of STIM and Orai proteins in cell lines derived from control and anhidrotic animals. However, the results indicated a reduction in STIM1 mRNA and protein expression, as well as a decrease in the localisation of STIM1 in the ER of anhidrotic cells compared to control cells. The functionality of the SOCE pathway in both cell lines, in response to agonist stimulation was investigated using (FURA‐2AM) calcium imaging. Increases in [Ca2+]i could be abolished in the presence of pharmacological blockers of SOCE and that anhidrotic cells showed a significant decrease in the [Ca2+]i FURA‐2 ratio in response to agonists when compared to control cells.The results suggest that a reduction of STIM1 expression in the SOCE pathway may play a role in anhidrosis in horses.Support provided by the Caledonian University.
Purinergic signaling integrates local excitation and global inhibition signals that regulate neutrophil chemotaxis
Authors: Bao Yi , Chen Yu , Ledderose Carola , Li Linglin , Junger Wolfgang 
Published: June 2021

Neutrophil chemotaxis requires local excitatory signals at the front and inhibitory signals at the back of cells. The mechanisms that link these signals are unknown. We previously showed that chemoattractants induce ATP release and excitatory purinergic feedback mechanisms at the front. Here we show that pannexin‐1 (panx1) is responsible for ATP release and that chemotaxis requires translocation of panx1 to the front and A2a adenosine receptors (A2aR) to the back. Panx1 provides both, ATP and adenosine, to elicit the excitatory and inhibitory signals at the front and back of cells. Inhibition of panx1 with carbenoxolone (CBX) completely abolished chemotaxis, while inhibition of A2aR with the antagonist CSC reduced migration speed (Fig. 1). Our findings demonstrate that panx1 is the molecular link that integrates the excitatory and inhibitory purinergic feedback mechanisms that regulate chemotaxis at the front and back of cells.This work was funded by grants from the National Institutes of Health and the Congressionally Directed Medical Research Program.
Direct interaction with the Na+/H+ exchanger NHE1 regulates ERK1/2 activity
Authors: Johansen Jane , Hendus‐Altenburger Ruth , la Cour Jonas , Kragelund Birthe B. , Pedersen Stine F. 
Published: June 2021

Increased activity of extracellular signal regulated kinase (ERK)1/2 plays a central role in cancer pathology. More recently, the Na+/H+ exchanger NHE1 has also been assigned important roles in cancer development and chemotherapy resistance. Using a combination of fluorescence spectroscopy, NMR analysis, and chemical crosslinking experiments we characterized the interaction of human (h) NHE1 C‐terminal cytoplasmic distal tail (NHE1Cdt) and ERK2 in vitro, and found that NHE1Cdt and ERK2 physically interact with low affinity and with exploitation of D‐domains as identified from distinct changes in their chemical shifts upon titration with ERK2. The hNHE1Cdt contains two predicted D‐domains ‐ a typical feature of ERK‐interacting proteins ‐ located around amino acids 715 (D1) and 740 (D2), respectively. The functional effects of NHE1‐ERK interaction were evaluated by mutating the D‐domains in full‐length hNHE1 (D1D2 mutant) and expressing wt and mutant NHE1 in epithelial cells lacking endogenous NHE1. Wt‐ and D1D2 mutant hNHE1 appeared to target normally to the plasma membrane. D1D2 mutation abolished EGF‐induced ERK activation, and both D domains appeared to be required for full ERK activity. In contrast, net acid extrusion capacity after an acid load was similar in cells expressing D1D2 mutant‐ and wt hNHE1. In conclusion, ERK physically interacts with hNHE1 and this appears to regulate ERK activity.Funding: Danish Research Council
An N‐terminal Signal Peptide Increases Total Expression and Enhances Surface Trafficking of Olfactory Receptors (ORs)
Authors: Shepard Blythe D , Natarajan Niranjana , Protzko Ryan J , Acres Omar W , Pluznick Jennifer L 
Published: June 2021

ORs are G protein‐coupled receptors that govern the sense of smell. The majority of ORs are orphan receptors with no known ligand, largely because deorphanization typically requires OR expression on the surface of heterologous cells. Unfortunately, ORs are retained in the ER and degraded when heterologously expressed. We hypothesized that a 17‐amino acid leucine‐rich signal peptide (Lucy tag) would allow for OR surface trafficking. To test this, we added the tag to the N‐terminus of 15 ORs and assayed for surface expression by immunofluorescence (IF) of unfixed cells with an antibody against an extracellular epitope. Addition of the tag promoted surface expression of 6/15 ORs (vs. 3/15 without the tag) and increased total protein expression of all ORs as measured by immunoblotting and ELISA. When the Lucy tag was used together with a Rho tag and known OR chaperones (previously shown to aid in surface expression of a minority of ORs), all ORs reached the surface (15/15). By IF, we showed that the Lucy tag is cleaved from the immature protein and does not alter the mature protein. Consistent with this, the tag does not alter OR‐ligand binding or downstream signaling as assayed by a luciferase reporter assay for OR activation. We conclude that the Lucy tag, along with the Rho‐tag and OR chaperones, allows for the surface expression of all ORs tested, potentially allowing for future widescale deorphanization studies.Funding: NIDDK
Stress‐induced changes in gene expression associated with prostate cancer
Authors: Davalos Olinamyr , Flores Ivan , Gosselink Kristin L 
Published: June 2021

Prostate cancer is common among men in the U.S., and stress is prevalent in our society, but the relationship between stress and prostate cancer is not well defined. Stress modulation of the gonadotropin‐releasing hormone (GnRH) system, and the use of GnRH analogs in prostate cancer treatment, suggests this endocrine pathway as a possible link between these two conditions. In this study, our hypothesis was that stress would suppress GnRH and increase the expression of proliferation‐associated genes in prostate tissue. Adult male rats were acutely (30min × 1d) or repeatedly (30min × 21d) restrained and prostate tissues analyzed by PCR array to determine changes in cancer‐related gene expression. Acute and repeated stress caused significant (p<0.05) downregulation of different genes associated with cellular proliferation. Angiopoeitin‐2 and cadherin‐2 were reduced by acute stress, while caspase‐2 and ‐7, IGF binding protein‐3 and ‐7, and MAPKK1 were decreased following repeated stress. Telomerase‐associated protein‐1 was significantly decreased by both stress treatments. These data demonstrate that expression of genes involved in proliferation is downregulated in prostate tissue from stressed animals. Our findings may help identify biomarkers for prostate cancer, and mechanisms through which stress may contribute to prostate cancer progression.Support: 5U54RR022762–03S1, 8G12MD007592
Histone deacetylase‐dependent control of lung alveolar fluid clearance
Authors: Wujak Lukasz Andrzej , Becker Simone , Arnoldt Verena , Wygrecka Malgorzata , Seeger Werner , Morty Rory Edward 
Published: June 2021

TGF‐β mediates acute lung injury (ALI) by impacting alveolar ion transport and thereby promoting pulmonary edema. The Na,K‐ATPase regulates ion transport and fluid balance and Na,K‐ATPase function is perturbed in ALI. It was hypothesized that TGF‐β impacts Na,K‐ATPase activity by altering subunit stoichiometry. Na,K‐ATPase activity and surface abundance were reduced in primary alveolar type II and A549 cells treated with TGF‐β, assessed by 86Rb+ uptake and cell surface biotinylation. The ATP1B1 subunit regulates surface stability, and hence alveolar fluid clearance (AFC) activity of the Na,K‐ATPase. PCR analysis revealed repression of ATP1B1 gene expression in lungs from ALI patients, in mice with bleomycin‐induced ALI model and in TGF‐β‐treated A549 cells. A Dual‐luciferase reporter assay revealed decreased ATP1B1 promoter activity in response to TGF‐ β. Enzyme inhibition and siRNA approaches demonstrated that ATP1B1 gene repression was mediated by epigenetic mechanisms and relied on HDAC2. TGF‐β induced phosphorylation of HDAC2 at S394 while chromatin immunoprecipitation confirmed binding of the HDAC2 to the ATP1B1 promoter. HDAC inhibition abrogated repression of the Atp1b1 gene in bleomycin‐induced ALI and drove edema resolution. In sum, HDAC2 mediated repression of the ATP1B1 gene, impairing Na,K‐ATPase function and AFC in ALI.German Research Foundation.
Targeted Proteomics: Endothelial Cell Membrane Response to TNF‐α
Authors: Prisco Anthony , Hoffmann Brian R , Kaczorowski Catherine C , Greene Andrew S 
Published: June 2021

Endothelial cells play a key role in regulating vascular function. One key role of the endothelium is to control the inflammatory response, limiting it to acute periods of defense. Produced by macrophages, one of the first cytokines produced in the inflammatory response is tumor necrosis factor alpha (TNFα). TNFα signaling to endothelial cells is essential for inducing the expression of proteins that recruit additional cells of the inflammatory response. TNFα signaling is complex, having two receptors that each signal through multiple pathways. In this study, we determine the effect of low dose TNFα on differential membrane protein expression in endothelial cells, identifying cell surface proteins that are upregulated, downregulated or uniquely expressed.We developed a novel proteomics technique, cell membrane associated enrichment (CMAE) for analyzing proteins contained on the outer cell surface and those closely associated with the cell surface. CMAE, targeting plasma membrane proteins with a surface amine group, combined with a more well‐established surface N‐glycoproteome enrichment (CSC) allowed increased coverage of the cell surface proteome. After CSC and CMAE results were combined allowing us to specifically target differentially regulated plasma membrane proteins in that were then validated by flow cytometry analysis of endothelial cells.
Team‐based learning format in the medical physiology teaching laboratory
Authors: Schmidt John William 
Published: June 2021

Case‐based problem solving that is structured according to the Team‐Based Learning (TBL) format (www.teambasedlearning.org) is being used in a first year medical school curriculum that combines anatomy, physiology and biochemistry in a single Human Biology course.Objectivehow effective is the TBL format when applied to multi‐disciplinary lab exercises? One lab exercise allows students to explore the role of alkaline phosphatase in the control of intestinal lumen pH. This exercise combines enzymology techniques with learning of physiology concepts related to homeostasis, receptor function, signal transduction and regulation of epithelial ion transport. This lab exercise has previously been taught to four student cohorts in a conventional group work format. In the TBL format, student understanding of the assigned reading is assessed at the start of the lab session by a multiple‐choice Individual Readiness Assessment Test (I‐RAT) using an electronic student response system. Teams then discuss their quiz answers while completing a Group RAT before the collection of data using a colorimetric enzyme assay. Most of the lab session group work is data collection followed by answering questions about the data and additional reading in two published journal articles. Effectiveness of the TBL format will be assessed by comparison of lab report scores and performance on multiple‐choice exam questions.