bluuebunny/crossref_metadata_2025 huggingface dataset:
The hereditary hemochromatosis protein, HFE, is present in substoichiometric concentrations with respect to transferrin receptors 1 and 2 in the liver
Authors: Chloupkova Maria , Zhang An‐Sheng , Enns Caroline A
Published: June 2021

Hereditary hemochromatosis is characterized by iron overload in parenchymal tissues, such as the liver. Mutations in the hereditary hemochromatosis protein, HFE, result in the most common type of this disease. Two binding partners of HFE, which also participate in regulation of iron homeostasis, have been identified: transferrin receptor 1 (TfR1) and 2 (TfR2). Recent efforts focused on understanding how HFE, TfR1, and TfR2 communicate to modulate hepcidin levels in the body, but basic questions such as the relative levels of these players have been overlooked. Thus, the aim of this study was to determine in vivo concentrations of HFE, TfR1, and TfR2 in human liver tissues. RT‐PCR analysis showed that the level of TfR2 mRNA was 100‐ and 200‐ fold higher than that of TfR1 and HFE, respectively. The molar concentration of TfR2 protein (1.95 nmoles/g protein) was about 4.5 times higher than that of TfR1 (0.43 nmoles/g protein) as judged by quantitative immunoblotting. The level of HFE protein was below 0.20 nmoles/g protein, indicating that HFE is present in substoichiometric concentrations with respect to both TfR1 and TfR2 in human liver tissue. This observation suggests that HFE might be the limiting factor during reorganization of HFE/TfR1 and HFE/TfR2 complexes in vivo in response to holo‐transferrin concentrations. This work was supported by NIH Grant RO1‐DK072166 (C.A.E.) and in part by MRF of Oregon ACEBD0082 (M.C.).
Recovery from DSS‐induced intestinal colitis is mediated by MIP2‐dependent neutrophil recruitment
Authors: Nanthakumar Nanda N , Meng Di
Published: June 2021

Conventionally raised mice successfully recover from the injury caused by DSS‐induced colitis. In contrast, bacteria‐depleted mice (BD mice), generated by treatment with broad‐spectrum oral antibiotics for two weeks, failed to recover from subsequent DSS‐induced injury, suggesting interactions between components of the normal gut microbiota of the host are required for recovery. Monocolonization by Bacteroides fragilis or commensal E. coli strain was able to prevent DSS‐induced mortality in BD mice. Since LPS is one component that is common to both these bacteria, we then determined if LPS alone would be sufficient to rescue BD mice from DSS‐induced mortality. Oral treatment with LPS with continuous antibiotic treatment was sufficient to reduce DSS‐induced mucosal injury and mortality in the wild type BD mice, but not in bacteria‐depleted TLR4−/− mice or MyD88−/− mice. The data suggest activation of TLR4‐dependent signals that enhance the recovery from DSS‐induced colitis in BD mice. We investigated the mechanistic basis for these observations and found that DSS treatment resulted in lower intestinal expression of MIP2 and correspondingly lower levels of neutrophil recruitment to the damaged mucosa. Administration of exogenous MIP2 eliminated DSS‐induced mortality in BD mice with a corresponding increase of neutrophils in the inflamed mucosa strongly suggesting a specific role for MIP2‐dependent neutrophil recruitment. These results support the idea that Gram‐negative commensal gut flora is necessary for the recovery from DSS‐induced colitis. The LPS‐mediated activation of TLR4‐MyD88 dependent signaling leads to up‐regulation of chemoattractants that recruit neutrophils, thereby enhancing the recovery from intestinal injury.
Source and level of protein fed to mice may affect their intestinal mucin production
Authors: Habiyambere Bupe Martha , Onyango Edward M
Published: June 2021

Mucins are large heavily glycosylated proteins which form the major component of mucus that protects epithelial surfaces of ducts in the body. The effect of the source and level of dietary protein on mucin production and intestinal mucosal morphology was investigated in mice. Thirty two male weanling ICR Swiss mice were randomized into 4 groups of 8 mice each. The four groups of mice were randomly assigned to four diets whose sources of protein were either from plant or animal origin and the level of each protein source was either 10% or 20% crude protein. Mice were provided the respective diets for 14 days and euthanized on the fifteenth day. Jejunal sections of their intestines were removed, fixed in methacarn, embedded in paraffin, sectioned and stained with alcian blue‐periodic acid Schiff (AB‐PAS) stain to visualize mucins and quantify the goblet cells. AB‐PAS stained jejunal sections showed an increase in mucus in mice fed diets containing proteins of animal origin. The number of goblet cells was higher in diets containing protein of animal origin (p<0.05). The increase in goblet cell number was greater for the higher protein level. The length of the villi did not seem to vary among the four different diets. It is concluded that the source and level of protein fed to mice may be influencing their intestinal mucin production.

Research was supported by East Tennessee State University.
The anti‐oxidative damage effect of semen cuscutae on the liver mitochondria of aging mice
Authors: Wei Xiao‐Dong , Wang Zhi‐Jun , Wang Shu‐Qiu , Ou Qin , Zhang Tao , Zhang Peng‐Xia , Wang Xian‐Gang , Kang Yu‐Ming
Published: June 2021

To evaluate the anti‐oxidation effect and the effect of ethanol extract of semen cuscutae (EESC) on the expression of mRNA of subunit CO I of complex IV of respiratory chain in mitochondria of D‐gal induced‐mice. Seventy mice were divided into 5 groups randomly: young, aging, and three different administration groups of 15, 30 and 45 days. The activity of manganese superoxide dismutase (Mn‐SOD), complex I and complex IV, and the content of MDA, the damage of mitochondrial DNA (mtDNA) and the expression of mRNA of subunit CO‐I were all examined for each group. Compared with young group, the activity of complex‐I, complex IV and SOD in mitochondrion of liver decreased, while the content of MDA and the expression level of mRNA of subunit CO‐I increased in aging group, and there were loss of mtDNA fragments in aging group. We found that EESC increases the activity of Mn‐SOD, complex I and complex IV, but decreases the loss of mtDNA and the expression of mRNA of subunit CO I.

Conclusion

The EESC can increase the activity of antioxidase and the ability of eliminating free radical of mitochondrion in liver, and can decrease the content of free radical metabolite and the loss of mtDNA. It has a certain degree of anti‐aging effect. The more days of administration, the better the effect.
Lithium action on glucose transport in insulin‐resistant rat skeletal muscle: roles of insulin signaling and p38 MAPK
Authors: Macko Antoni Ryszard , Beneze Alan , Teachey Mary K. , Henriksen Erik J.
Published: June 2021

In insulin‐sensitive rat skeletal muscle, lithium activates glucose transport via a mechanism involving p38 mitogen‐activated protein kinase (p38 MAPK). The mechanism underlying lithium action on glucose transport in insulin‐resistant rat skeletal muscle is unknown. We therefore assessed the effects of lithium on basal and insulin‐stimulated glucose transport, insulin signaling (insulin receptor (IR), Akt, and glycogen synthase kinase‐3 (GSK‐3)), and p38 MAPK in soleus muscle from obese Zucker rats. Lithium (10 mM LiCl) increased basal glucose transport by 49% (p<0.05). Lithium did not induce basal IR tyrosine phosphorylation, but did enhance Akt ser473 phosphorylation (40%) and GSK‐3β ser9 phosphorylation (88%). Lithium potentiated insulin action on glucose transport (74%), Akt ser473 phosphorylation (39%), and GSK‐3β ser9 phosphorylation (36%), and elicited robust increases in p38 MAPK phosphorylation both in the absence (100%) or presence (88%) of insulin. Selective inhibition of p38 MAPK (using 10 µM A304000) completely blocked basal activation of glucose transport by lithium, and significantly reduced (42%) the lithium‐induced enhancement of insulin‐stimulated glucose transport. These results indicate that lithium enhances basal and insulin‐stimulated glucose transport and distal insulin signaling in insulin‐resistant skeletal muscle of obese Zucker rats via engagement of p38 MAPK.
Vascular Remodeling of Mesenteric Resistance Arteries in Diabetic Mice
Authors: Souza Flavia Moreira , Katz Paige S. , Stewart James A. , Vassallo Dalton V. , Lucchesi Pamela A.
Published: June 2021

Aims

Little is known about Type 2 diabetes‐induced structural remodeling of mesenteric resistance arteries. This study compared structural differences in small mesenteric resistance arteries in diabetic (db/db) and control (Db/db) mice.

Methods

Vessels were isolated from 16wk old mice and structural properties were assessed by pressure myography. Mean arterial pressure (MAP) was measured in vivo by telemetry. Western blot analysis and gelatin zymography were used to assess the expression and activity of matrix regulatory proteins.

Results

MAP was similar between control and db/db mice. Fasting blood glucose levels were higher in db/db mice (505±28 mg/dl) vs. control (115±10, p<0.001). The lumen diameter (121±5.5 μm vs. 77.8±5.5, p<0.001 at 75 mmHg) and media cross‐sectional area were significantly increased in db/db compared to control, respectively, suggesting hypertrophic outward remodeling. Cross‐sectional compliance was significantly larger in the diabetic arteries at 50 and 75 mmHg. The stress‐strain curve was shifted to the right in mesenteric arteries from diabetic mice compared with controls. The expression of the matrix regulatory proteins MMP‐9, MMP‐12, TIMP‐1, TIMP‐2, TGF‐β and PAI‐1 were increased in db/db arteries, while MMP‐2 activity was decreased.

Conclusions

These data suggest that diabetic mesenteric resistance arteries undergo hypertrophic outward remodeling, increased vessel compliance and reduced stiffness that was associated with ECM turnover secondary to an imbalance between pro and anti‐fibrotic factors. Supported by COBRE P20RR18766 and HL56046 (PAL) and CAPES (DV).
Distinct Mitochondrial Subpopulation Response in a Type 2 Diabetic Model
Authors: Dabkowski Erinne R , Williamson Courtney L , Frisbee Jefferson C , Brock Robert W , Hollander John M
Published: June 2021

Mitochondrial dysfunction plays a critical role in the pathogenesis of type 2 diabetes mellitus. Examination of mitochondria is complicated by the fact that two spatially distinct mitochondria populations exist in the cardiac myocyte, interfibrillar mitochondria (IFM) which situate between the myofibrils, and subsarcolemmal mitochondria (SSM) which exist beneath the sarcolemma. The goal of this study was to determine whether spatially distinct cardiac mitochondrial subpopulations are differentially affected by type 2 diabetic insult. Db/db mice were sacrificed at 20 weeks of age and mitochondrial subpopulations isolated. State 3 respiration was significantly lower in db/db SSM contributing to decreased RCR ratios (P<0.05). Electron transport chain complex activities (I, III, IV) were significantly decreased only in diabetic SSM as compared to control (P<0.05 for all three). Uncoupling protein 3 was significantly increased only in db/db SSM (P<0.05). Manganese superoxide dismutase (MnSOD) protein was significantly elevated in db/db IFM suggesting a potential spatially‐specific protective mechanism. Mitochondrial heat shock protein 70 (mthsp70), a mitochondrial import protein, was significantly decreased only in db/db SSM. These findings suggest that type 2 diabetic insult impacts spatially distinct cardiac mitochondria differently with SSM displaying greater dysfunctional profiles.
Increased GlcNacation and decreased caveolin‐3 in cardiac myocyte caveolae during diabetes mellitus
Authors: Kidd Michael W , Makino Aya , Villarreal Francisco J , Insel Paul A , Dillmann Wolfgang H , Roth David M , Patel Hemal H
Published: June 2021

Cardiomyopathy, independent of the vascular effects of hypertension or coronary artery disease, occurs in patients with diabetes mellitus (DM), thus suggesting that factors within cardiac myocytes contribute to DM‐associated cardiac remodeling. Caveolin‐3 (Cav‐3), a myocyte‐specific protein, localizes numerous molecules within caveolae (flask‐like invaginations of the membrane) and can modulate cardiac remodeling; Knockout of Cav‐3 in mice results in a cardiomyopathic phenotype. To test the hypothesis that post‐translational modification of caveolar proteins occur in DM, we assessed hearts from control or streptozotocin‐induced DM mice using fractionation/immunoblot analysis and electron microscopy (EM). We found that diabetic hearts have reduced Cav‐3 expression in caveolar fractions and increased O‐GlcNacation (a post‐translational modification) of caveolar‐resident proteins. EM revealed that diabetic hearts had disruption of cytoarchitecture with disorganized, rounded mitochondria and fewer caveolar invaginations. DM is thus associated with enhanced Glc‐Nacation of caveolar proteins and decreased expression of Cav‐3 in caveolae. Thus, therapies designed to enhance Cav‐3/caveolae expression are potentially novel approaches to limit DM‐associated cardiac remodeling.
Local IGF‐I Prevents Sepsis‐Induced Muscle Atrophy
Authors: Lang Charles H. , Nystrom Gerald J. , Frost Robert A.
Published: June 2021

This study tests the hypothesis that local IGF‐I bioavailability regulates muscle protein balance and that muscle‐directed IGF‐I can selectively maintain muscle mass during sepsis. Increasing or decreasing bioavailable IGF‐I within muscle of mice by local injection of Leu24‐Ala31‐IGF‐I or IGFBP‐1, respectively, produced proportional changes in 4EBP1 and S6K1 phosphorylation ‐ surrogate markers of protein synthesis. In a second study, a time‐release pellet (5‐days) containing IGF‐I was implanted in the gastrocnemius and a placebo implanted in the contralateral muscle after sepsis. The gastrocnemius from septic mice with the placebo was atrophied and IGF‐I protein reduced. In contrast, local release of IGF‐I increased IGF‐I within the adjacent muscle to basal control levels, and was associated with proportional increases in muscle weight/protein. Locally‐directed IGF‐I increased the mass/protein of the exposed gastrocnemius in septic mice, and this was associated with increased phosphorylation of 4EBP1 and S6K1. Local IGF‐I selectively prevented the sepsis‐induced increase in muscle atrogin‐1 and IL‐6 mRNA. In conclusion, muscle‐directed IGF‐I prevents the characteristic sepsis‐induced atrophic response resulting from an increase in muscle protein synthesis and potentially from decreased proteolysis, without effecting metabolic processes in distant organs. (GM 38032)
Protein intake and nitrogen balance in male Japanese athletes
Authors: Yokota Yukari , Tatsuta Wakako , Kamei Akiko , Ebi Kumiko , Komatsu Yutaka , Kawahara Takashi , Okamura Koji
Published: June 2021

Recommendations for the requirements for protein intake amount usually to 1.2‐2.0 g/kg BW/day in athletes. The aim of this study was to compare the effects of a high protein diet (HP: 2.5 g/kg BW/day) and a normal protein diet (NP: 1.5 g/kg BW/day) on the nitrogen balance in male Japanese athletes. Subjects were 16 healthy, male Japanese athletes (20.9±2.2 years, height: 176.2±6.3 cm, body weight: 87±14.2 kg, %fat: 17.3±6.0 %) involved in 4 different sports. These 2 regimens were further divided to HP diet or NP diet. They were participated in 2 regimens in the crossover‐randomized design during 20 days (a 7‐day adaptation period and a 3‐day balance period). Dietary intake and life activity were assessed from food and life activity records during the experimental period. To determine the effects of a HP diet, urine and blood tests were conducted and the body composition was measured. Urine samples were collected on the final three consecutive days of each diet and analyzed for nitrogen. The nitrogen balance was calculated from the mean daily protein intake, mean urinary nitrogen losses. There were no significant changes in the body composition between the HP group and the NP group. Effects of the high protein diet could not be found in the urine (24hccr, NTx, Ca, ph, oxalate) and the blood (insulin, glucose, NTx) samples. Urinary nitrogen losses increased during the HP diet vs NP diet (35.7±30.0 vs 24.0±13.7 g/kg BW/day, p<0.05). These results suggest that male Japanese athletes might not be able to efficiently incorporate a protein intake of 2.5 g/kg BW/day into their body protein. However, kind of sport; level of training; timing and amount of protein consumed at one time have not been taken into consideration and must be further analyzed.
Chronic intermittent hypoxia and acetaminophen induce synergistic liver injury
Authors: Polotsky Vsevolod Y , Savransky Vladimir , Reinke Christian , Jun Jonathan , Bevans‐Fonti Shannon , Li Jianguo , Myers Allen C , Torbenson Michael
Published: June 2021

Obstructive sleep apnea (OSA) leads to chronic intermittent hypoxia (CIH) during sleep. OSA has been associated with liver injury. Our goal was to examine whether CIH increases liver injury induced by acetaminophen (APAP) treatment. C57BL/6J mice were exposed to CIH or intermittent air (IA) for 4 weeks while being treated with APAP i.p. (200 mg/kg) or normal saline daily. A combination of CIH and APAP caused liver injury with increases in serum alanine aminotransferase, aspartate aminotransferase (AST), gamma glutamyl transferase and total bilirubin levels, whereas CIH alone induced only elevation in serum AST levels. APAP alone did not affect serum levels of liver enzymes. Histopathology revealed hepatic necrosis and increased apoptosis in mice exposed to CIH and APAP, whereas the liver remained intact in all other groups. Mice exposed to CIH and APAP exhibited decreased hepatic glutathione and a five‐fold increase in nitrotyrosine levels. APAP or CIH alone had no effect on either glutathione or nitrotyrosine. A combination of CIH and APAP caused marked increases in monocyte chemoattractant protein‐1 and macrophage inflammatory protein‐2, which were not observed in mice exposed to CIH or APAP alone. We conclude that CIH and chronic APAP treatment lead to synergistic liver injury, which may have clinical implications for patients with OSA.

Funded by NHLBI R01 HL088095
Do glycoprotein stains improve detection of epithelial discontinuities in fixed tissue?
Authors: Parikh Krupa V , Matthis Andrea , Montrose Marshall M
Published: June 2021

Small intestinal epithelial cells originate in crypts, migrate up the villus and are shed during normal cell renewal. Research on living tissues found that neighboring cells do not immediately migrate to close the gap left by a shed cell; rather, the gap is transiently filled with an impermeable substance that maintains the epithelial barrier. It is difficult to identify gaps in fixed specimens; a requirement prior to analyzing gap contents after microdissection. Our goals were to use new methods to distinguish gaps from goblet cells in histological sections, and to see if gaps contain glycoprotein. Frozen 5 µm mouse small intestine villi sections were fixed (ethanol) and stained using combinations of Hoechst 33258 (nucleus), DiI (membrane) and Hp Yellow (glycoprotein). Optimal staining required imaging sections first stained with DiI and iv Hoechst, then imaging after secondary staining with Hp Yellow. Gaps identified as epithelial cell positions lacking nuclei (unstained with iv. Hoechst 33258) were shown to have nuclei by secondary staining either with Hp Yellow or dipping slides in Hoechst 33258. Most "false gaps" had abundant glycoprotein, suggesting nuclei of goblet cells were selectively resistant to the Hoechst stain when applied to living cells. Results suggest that a fraction of gaps tallied in prior work were goblet cells, and goblet cells could not be unequivocally identified vs. gaps using the new stains.
Trafficking of Silica Nanoparticles Across the Alveolar Epithelial Barrier
Authors: Fazlollahi Farnoosh , Beier C. , Yacobi N. R. , Marchelletta R. , Kim Y. H. , Brutchey R. , Hamm‐Alvarez S. , Borok Z. , Kim K. J. , Crandall E. D.
Published: June 2021

Polystyrene nanoparticles (PNP) translocate across primary cultured rat alveolar epithelial cell monolayers (RAECM) in a surface charge‐ and size‐dependent manner. In this study, we investigated interactions of fluorescently labeled silica nanoparticles (SNP, 42 and 105 nm) with RAECM. Effects of apical 176 µg/mL SNP on transmonolayer resistance (Rt) and equivalent short‐circuit current (Ieq) were measured as functions of time. SNP fluxes (apical‐to‐basolateral) were determined in the presence and absence of the caveolin‐ or clathrin‐mediated endocytosis inhibitors methyl‐β ‐cyclodextrin (MβC) or chlorpromazine (CHL), respectively. Results showed that: i) SNP did not elicit changes in Rt and Ieq for up to 24 hr, ii) 42 nm SNP flux >105 nm SNP flux, iii) fluxes of negatively charged 20 and 100 nm PNP were 60‐80 fold less than fluxes of 42 and 105 nm SNP, iv) neither MβC nor CHL affected SNP fluxes, and v) confocal microscopy after exposure to SNP for 24 hr revealed intracellular localization of SNP. These data indicate that: (1) SNP do not injure RAECM, (2) translocation of SNP is transcellular and size‐dependent, (3) SNP translocation is much faster than comparably‐sized PNP translocation, and (4) SNP trafficking does not appear to take place via endocytosis pathways requiring clathrin or caveolin. We conclude that nanoparticle interactions with alveolar epithelium are at least in part composition‐dependent.

Funded by: NIH & Hastings Foundation
MicroRNA‐375 Regulates Alveolar Epithelial Cell Trans‐Differentiation through the Wnt/β‐catenin Pathway
Authors: Wang Yang , Bhaskaran Manoj , Zhang Honghao , Gou Deming , Guo Yujie , Chintagari Narendranath Reddy , Liu Lin
Published: June 2021

Alveolar epithelial type II cells (AEC II) are multifunctional cells in the lung. In addition to the secretory and synthetic functions, they have progenitor capacities. When alveolar epithelial type I cells (AEC I) are damaged under various disease conditions, cuboidal AEC II proliferate and trans‐differentiation into squamous ACE I, which helps to restore the normal air‐blood barrier. However, the molecular mechanisms of this trans‐differentiation process remain largely unknown. MicroRNAs are a recently discovered class of small noncoding RNA, which negatively regulate the expression of target genes at the post‐transcriptional level. Through microRNA microarray and real‐time PCR analyses, we found that microRNA‐375 (miR‐375) was significantly decreased during the trans‐differentiation of AEC II to AEC I. miR‐375 was expressed higher in adult lungs that fetal lungs and appeared to be enriched in AEC II. The Wnt/β‐catenin pathway was activated during alveolar epithelial cell differentiation. Overexpression of miR‐375 prevented the trans‐differentiation of AEC II to AEC I and inhibited the Wnt/β‐catenin signaling. In conclusion, our study shows that miR‐375 regulates the trans‐differentiation of AEC II to AEC I through the Wnt/β‐catenin signaling pathway.
Hydrogen Peroxide Stimulates ENaC: Role of Tyrosine Kinases and Anionic Phospholipids
Authors: Ma He‐Ping
Published: June 2021

Recent studies indicate that oxidative stress may contribute to the development of salt‐sensitive hypertension. To test a hypothesis that the renal epithelial sodium channel (ENaC) is a target of oxidative stress, we used patch‐clamp techniques to determine if ENaC in A6 distal nephron cells is regulated by hydrogen peroxide (H2O2). We found that H2O2 significantly elevated ENaC open probability and single‐channel current amplitude, but not the unit conductance. Both effects were abolished by herbimycin, a Src tyrosine kinase inhibitor, but not PP2, another Src tyrosine kinase inhibitor with a structure different from herbimycin, indicating that the Src family, possibly a specific isoform, mediates the stimulation of ENaC by H2O2. Confocal microscopy imaging data showed that herbimycin, but not PP2, blocked H2O2‐induced elevation of phosphatidylinositol‐3,4,5‐trisphosphate (PI(3,4,5)P3) in the apical membrane. Lipid‐protein overlay analysis demonstrated that H2O2 strongly elevated the bindings of both full‐length and the COOH (but not NH2) terminal segment of α‐ENaC to anionic phospholipids including phosphatidylinositol‐4,5‐bisphosphate (PI(4,5)P2) and PI(3,4,5)P3. Since we and others have previously shown that ENaC is stimulated by PI(4,5)P2 and PI(3,4,5)P3, increases in apical PI(3,4,5)P3 and α‐ENaC binding to anionic phospholipids may mediate H2O2‐induced elevation of ENaC open probability. Since the effect of H2O2 on ENaC single‐channel current amplitude was abolished by ouabain, a potent blocker of Na+‐K+‐ATPase, the elevated ENaC current amplitude may be due to an increased electrochemical driving force resulting from H2O2‐enhanced Na+‐K+‐ATPase activity. These data suggest that oxidative stress stimulates ENaC through Src‐mediated modification of the interactions between ENaC and anionic phospholipids.
USP2‐45 regulates ENaC endocytosis
Authors: Zhou Ruifeng , Snyder Peter M
Published: June 2021

The epithelial sodium channel (ENaC) is important for the maintenance of Na+ and volume homeostasis. Defects in its regulation cause hypertension and contribute to the pathogenesis of cystic fibrosis. ENaC surface expression is regulated by ubiquitination of each ENaC subunit. Previous work indicates that Nedd4‐2 catalyzes ENaC ubiquitination, which has two distinct effects on ENaC trafficking; ubiquitination increases ENaC endocytosis and increases its degradation in lysosomes, both which contribute to a reduction in ENaC surface expression. Recent work suggests that ENaC can be deubiquitinated by a ubiquitin‐specific protease, USP2‐45. Here we tested the hypothesis that USP2‐45 regulates ENaC trafficking. When coexpressed in HEK293T cells, we found that USP2‐45 produced a dose‐dependent increase in surface expression ofα, β, and γENaC, and reversed the Nedd4‐2 mediated decrease in ENaC surface expression. USP2‐45 coprecipitated with the cell surface pool of ENaC and decreased the rate of ENaC endocytosis. Moreover, USP2‐45 blunted the effect of Nedd4‐2 on ENaC endocytosis. In contrast, USP2‐45 had no effect on degradation of the surface pool of ENaC. Thus, USP2‐45 increases ENaC surface expression through a selective effect on ENaC endocytosis. This suggests a model in which USP2‐45 deubiquitinates ENaC at the cell surface but not in endosomes. Funded by the NIH.
Lip5 in the regulation of the Aquaporin‐2 water channel
Authors: Deen Peter MT , van Balkom Bas , Boone Michelle , Hendriks Giel , van der Sluis Peter
Published: June 2021

AVP stimulates AQP2 phosphorylation at S256 and its translocation to the apical membrane. Upon AVP removal, AQP2 is ubiquitinated at K270, internalized, and water reabsorption is reduced. To identify AQP2 interacting proteins, the AQP2 C‐terminus was used as a bait in a yeast‐2‐hybrid assay. The Lyst‐interacting protein 5 (Lip5) to identified to bind. Immunohistochemistry showed that Lip5 co‐localizes with AQP2 in collecting ducts and interacts with AQP2‐S256D and AQP2‐K270R, but not with AQP3 or AQP4. Shortening the C‐tail revealed that the proximal C‐tail (L230‐D243) in AQP2 is essential for Lip5 interaction. Also, Lip5 interacts with AQP2 via its N‐terminal microtubule interacting and trafficking (MIT) domain. Lip5 is implicated in sorting of other proteins to multivesicular bodies (MVB) and lysosomes, from where proteins can recycle to the plasma membrane or are targeted for lysosomal degradation. Indeed, knockdown of endogenous LIP5 by shRNAs reduced AQP2 degradation in cultured cells. In conclusion, (1) AQP2 is the first cargo protein shows to bind Lip5, (2) Lip5 is expressed in renal principal cells and specifically binds AQP2, independent of its PKA phosphorylation and ubiquitination state and (3) Lip5 plays a role in the targeting of AQP2 to MVBs and, possibly, in the decision process whether AQP2 is targeted for lysosomal degradation or recycling.
β1Pix suppresses the activity of the epithelial Na+ channel through 14‐3‐3 binding
Authors: Pavlov Tengis S , Chahdi Ahmed , Sorokin Andrey , Staruschenko Alexander
Published: June 2021

β1Pix has been identified as a p21‐activated kinase (Pak)‐interacting exchange factor and serves as a guanine nucleotide exchange factor (GEF) for Rac1 and Cdc42. Furthermore, it was shown that βPix can bind 14‐3‐3 proteins and act as a scaffolding protein. We demonstrate here that βPix is highly expressed in immortalized mouse collecting duct mpkCCDc14 cells. Additionally we have shown that β1Pix expression markedly decrease ENaC activity. Coexpression of β1Pix with ENaC decreased amiloride‐sensitive current density in CHO cells from 365 ± 35 to 158 ± 29 pA/pF. This effect did not occur through GEF activity of β1Pix because coexpression of Rac1 with ENaC markedly increased channel activity and coexpression of wild type and constitutively active (G12V) Cdc42 had no effect on ENaC activity. Moreover Δ602‐611 β1Pix construct which did not bind 14‐3‐3 but retained GEF activity had no effect on ENaC activity. In contrast, β1Pix (L238R,L239R) mutant which has no GEF activity decreased current density to the similar extent as wild type β1Pix. Thus, we can conclude that ß1Pix decreases ENaC activity via 14‐3‐3 recruitment but not through GEF activity.

Supported by NIH, AHA and ASN.
Decreases in labile intracellular zinc (Zni) contribute to LPS‐induced apoptosis in cultured sheep pulmonary artery endothelial cells (SPAEC)
Authors: Thambiayya Kalidasan , Wasserloos Karla , St Croix Claudette M , Pitt Bruce R
Published: June 2021

After iron, zinc is the most abundant intracellular metal and Zni quota is maintained in a narrow range (100‐500 uM) by the activity of zinc importers (SLC39A1‐14) and zinc exporters (SLC30A1‐10). Although virtually all zinc is bound to protein, a chelatable pool of Zni is capable of participating in intracellular signaling and functions. We used chemical and genetic techniques to reveal a role for decreases in Zni to affect LPS mediated apoptosis in SPAEC. Early (0.5 to 4.0 hrs) after a proapototic dose (100 ng/ml) of LPS, we noted a decrease in Zni as revealed by the zinc sensitive fluorophore, FluoZin‐3, via FACS. Confirmation of this relative decrease after LPS was obtained via: a) EPI fluorescence microscopy in live cells that revealed a 60% decrease in Zni ; and b) 40% decrease of activity of zinc sensitive metal transcription element (MRE) fused to luciferase reporter that was previously transfected into SPAEC. The functional consequences of decreases in Zni were ascertained by: a) rescuing SPAEC from LPS induced increases in caspase 3/7 by addition of exogenous zinc to normalize potential LPS induced decreases in Zni; and b) mimicking effect of LPS on caspase 3/7 activity with zinc chelator, TPEN. We conclude that LPS results in a change in net activity of zinc transporters resulting in decrease in Zni and this decrease is an important intracellular signaling pathway transducing the apoptotic effects of LPS on SPAEC.
Modeling of gender differences in respiratory function during exercise using Quantitative Human Physiology
Authors: Carter Cory Blake , Hodnett Benjamin Lucas , Hester Robert
Published: June 2021

Quantitative Human Physiology (QHP) is an open‐source XML model of human physiology based on Quantitative Circulatory Physiology (QCP), a previous model created to incorporate over 4000 simultaneous equations. The QHP model has been expanded to include a female (F) subject, allowing investigation of gender differences on human physiology. For this model, respiratory function was examined in a 78‐kg male (M) subject and a 60‐kg F subject. The protocol involved having the subjects perform 30‐min treadmill exercise protocol (2% incline at 2 miles/hour) and comparing respiratory parameters to pre‐exercise conditions. Respiratory parameters were analyzed as percent increases, due to the different baseline parameters between the M and F subjects. Pre‐exercise alveolar ventilation (Va) for M and F was 4.4 L/ min and 3.5 L/min, respectively. Following 30 min of exercise, M Va increased 305% whereas F Va increased 394% (Figure). Interestingly, post‐exercise Va was similar between the M and F (17.8 L/min vs. 17.3 L/min). This model will investigate the physiological mechanisms underlying these differences in respiratory function between M and F, including the role of differences in pulmonary blood flow and blood gas exchange. Supported by NSF EPSCoR and NIH # HL‐51971.
Nitrosothiols regulate antioxidant capacity in red blood cells
Authors: Rogers Stephen Colin , Corcuera Daniella , McLaughlin Dylan , Patel Krupali , Ross Jerlinda , Doctor Allan
Published: June 2021

Hypoxic RBCs are less resilient to oxidative stress. This results from constrained glycolytic flux through the hexose monophosphate pathway (HMP), the RBCs sole source of nicotinamide adenine dinucleotide phosphate (NADPH). NADPH plays an essential role in maintaining thiol‐based anti‐oxidant defense by regenerating glutathione (GSH) from the glutathione disulphide. We hypothesized that pre‐exposure of RBCs to exogenous nitrosothiol would recover thiol‐based oxidant defence under hypoxic conditions by inhibiting the EMP enzyme glyceraldehyde 3‐phosphate dehydrogenase and enhacing HMP glycolytic flux. Human erythrocytes pre‐incubated ± L‐cysteine NO (L‐CysNO; SNO:Hb 1:250) under oxygenated conditions, were subjected to controlled oxidant (superoxide) loading following hypoxic exposure. The sufficiency of antioxidant defense was determined by measurement of reduced RBC membrane thiol and serial quantification of GSH and NADPH redox couples. The loss of reduction potential and reduced membrane thiol observed in control hypoxic RBCs was restored in the L‐CysNO pre‐treated hypoxic RBCs. These data indicate that nitrosothiols may abrogate hypoxia‐induced vulnerability to oxidative stress.
MiR‐21 plays a Role in Smooth Muscle Cell Proliferation induced by Hypoxia
Authors: Sarkar Joy , Turaka Prasanna , Gou Deming , Ramchandran Ramaswamy , Zhou Guofei , Raghavan Aarti , Raj Usha
Published: June 2021

Chronic hypoxia is an important contributing factor to the development of pulmonary hypertension. Hypoxia triggers pulmonary vascular smooth muscle cell (PVSMC) remodeling from a differentiated state to a proliferative, dedifferentiated state. cGMP‐dependent protein kinase type 1 (Prkg1) is involved in the regulation of vasomotor tone and remodelling. MicroRNAs play important roles in various biologic processes in cells, where they regulate apoptosis, proliferation, differentiation, development & metabolism. However, the role of microRNAs in hypoxia‐mediated Prkg1 down‐regulation in smooth muscle cells and SMC proliferation is unknown. The objective of the study was to characterize miRNA regulation in PVSMC response to hypoxia, affecting cell proliferation. This is important in understanding the molecular mechanisms triggered by cell exposure to low oxygen tension in Persistent Pulmonary Hypertension (PPH) in neonates. We used human PVSMCs as an in vitro model system and studied the effects of hypoxic (3% oxygen) exposure. Cells were processed for mRNA, protein and microRNA studies. We found reduced mRNA and protein expression of SMC markers such as α‐Smooth muscle actin and calponin as well as Prkg1. Quantitative PCR analyses showed that 24h hypoxia treatment induced miR‐21 upregulation. Our studies suggest that miR‐21 plays a role in hypoxia‐induced smooth muscle cell proliferation. This work was supported by R01 HL075187‐05A1
Increased COX‐2 expression and PGE2 production in pressurized renal medullary interstitial cells
Authors: Norregaard Rikke , Donohue Kaitlin E. , Jensen Anja M. , Roberts Angela L. , Chen Jie , Poppas Dix P. , Felsen Diane , Frøkiær Jørgen
Published: June 2021

Pressure plays an important role during many physiological processes, and abnormal pressures may induce changes which favour onset and progression of disease in many organs. In vivo, renal medullary interstitial cells (RMIC) are subjected to pressure as a result of ureteral obstruction, which may influence the expression of COX‐2.

To further examine this regulation, rats were subjected to unilateral ureteral obstruction (UUO) for 6 and 12h; showed increased COX‐2 mRNA levels in IM, whereas COX‐2 protein expression was increased only after 12hUUO. COX‐1 mRNA and protein level were unchanged. To explore the direct effect of pressure on the expression and activity of COX‐2 cultured RMIC were subjected to pressures of 60 mmHg over time (2‐12 h) using a novel pressure apparatus. QPCR confirmed increased COX‐2 mRNA after 2h. COX‐2 protein expression was increased following 60 mmHg of pressure for 6h. COX‐1 mRNA and protein levels were unchanged. PGE2 excretion from the RMIC was increased, when cells were subjected to pressure of 60 mm Hg for 6h, which was prevented by a selective COX‐2 inhibitor. Importantly, TUNEL and MTT assay studies showed that applying pressure to the RMIC does not affect cell viability, apoptosis, and proliferation.

We demonstrated that in vitro application of pressure recapitulates the effects on RMIC found after in vivo UUO. This directly implicates pressure as an important regulator of renal COX‐2.
Sex dependent differences in collecting duct renin during high salt intake
Authors: Rands Vicky F. , Martin Victoria , Kavanagh Kimberley , Lowenburg Ben , Planchard Jeffrey , Botros Fady , Prieto‐Carresquerro Minolfa
Published: June 2021

Collecting duct (CD) renin is upregulated during Ang II‐dependent hypertension. In this study we examined if CD renin exhibits sex differences during changes in salt intake in 19 Sprague‐Dawley rats distributed in 4 groups [1) Controls male (n=5); 2) Controls female (n=5); HS male (n=5); and HS female (n=4) fed a normal and HS diet (8% NaCl). Baseline systolic blood pressure (SBP) was not different between males and female rats (males: 116±3 vs. female: 119±2 mmHg) nor after 2‐weeks fed a HS diet (male 126±2 vs. female: 128±2 mmHg). Although renin mRNA and protein levels examined by qRT‐PCR and Western blot in renal medullary tissues were similar in male and female rats under control conditions [Controls male: mRNA= 1.0±0.4 AU; protein=1.0±0.1 DU vs. Controls female: mRNA=0.3±0.1AU; protein=0.8±0.1 DU)]; HS diet intake significantly increased the CD renin transcript and protein levels in females but not in male rats [mRNA=(HS male: 1.5±0.1 vs. HS female: 3.4±0.6 AU); protein= (HS male: 1.0±0.3 DU vs. HS female: 1.8 ±0.7 DU). These data suggest that although there is not sex difference in CD renin gene expression under basal conditions, HS intake appears to stimulate renin in distal nephron segment only in female rats. Future studies are planned to determine whether HS intake during chronic Ang II infusion leads to sexual dimorphisms of blood pressure during Ang II‐dependent hypertension.
Lung ischemia‐reperfusion injury stimulates airway biosensors through oxidative stress
Authors: Li Huafeng , Du Lei , Mei Kai , Folz Rodney , Yu Jerry
Published: June 2021

Lung ischemia‐reperfusion injury (LIRI) stimulates airway vagal chemosensors. We hypothesize that the stimulation is mediated by increased production of oxygen free radicals (OFRs). Two questions are asked: 1) Does increased production of OFRs by injecting a mixture of xanthine/xanthine oxidase into the receptive field stimulate airway chemosensors? 2) Can reduction of OFRs by intravenous infusion of superoxide dismutase (SOD) prevent LIRI induced chemosensor stimulation? Rabbits were anesthetized, the lungs were mechanically ventilated, the chest was opened, and airway chemosensor activities were recorded from the vagus nerve. LIRI was produced by closing (30 min) and then releasing (15 min) a snare around the pulmonary artery. Chemosensors activity increased sharply after local injection of xanthine/xanthine oxidase and peaked within 10 sec. The increased activity recovered within a min or so. The results demonstrate that the chemosensors are sensitive to increased OFRs. Chemosensor discharge (21±10 imp/min; baseline) increased to 56±36 imp/min (n=9; P=0.022) during the first min of reperfusion. Such increased activities after LIRI did not exist when SOD was infused intravenously for 20 min before reperfusion. Thus, antioxidants blocked the sensory response to LIRI. Our results support the hypothesis that LIRI activates airway chemosensors, which is mediated by increased production of OFRs.
Microinjection of kynurenic acid induces airway smooth muscle relaxation in ferrets
Authors: Neziri Burim , Krasniqi Shaip , Jakupaj Muharrem , Ahmetaj Halil , Devaja Shqipe
Published: June 2021

Beside extensive study central mechanisms and neuronal networks that control airway constriction and relaxation remain unclear and poorly understood. In the present study realized in anesthetized, paralyzed and artificially ventilated ferrets we sought to determine whether nicotinic acetylcholine receptors (nAChRs) dominate respiratory drive under control conditions due to suppression of N‐methyl‐D‐aspartate (NMDA) receptors. We observed that blockade of cholinesterase activity by administration of eserine is followed by increased cholinergic outflow and airway smooth muscle tone (ASMT). Microinjection of kynurenic acid (KYNA) as NMDA receptor blockers, into the region of the ventrolateral medulla where airway related vagal preganglionic neurons (AVPNs) are located, significantly decreased the reflex changes in tracheal tone. KYNA, a product of tryptophan metabolism has neuroprotective and neuroinhibitory properties that have been attributed to its action as competitive antagonist at the glycine site on NMDA receptors. Furthermore, KYNA non‐competitively inhibits alpha7 nicotinic acetylcholine presynaptic receptors (nAChRs), inhibiting glutamate release, and regulates the expression of alpha4beta2 nAChR. In our physiological experiments performed under in vivo conditions we conclude that stimulation or inhibition of AVPNs by eserine or KYNA was manifested with increase or decrease, respectively, of pressure in tracheal segment (Ptseg), lung resistance (RL), or dynamic compliance (Cdyn). Our results indicate that nAChRs might be targets for KYNA and suggest a functionally significant cross reaction between the nicotinic cholinergic system and kynurenine pathway in AVPNs and respiratory drive.
Variability in measures of exhaled breath Na+ and K+, influence of cardiac output and saliva
Authors: Cassuto Nicholas A. , Wheatley Courtney M. , Foxx‐Lupo William T. , Snyder Eric M.
Published: June 2021

The assessment of inflammatory markers, ions, and cations in exhaled breath condensate (EBC) is being used more frequently in diseases such as asthma and cystic fibrosis. Previous work has demonstrated significant variability in the measures of exhaled Na+ and K+ in healthy subjects and in patients with cystic fibrosis. We sought to determine if the previously‐described variability in exhaled Na+ and K+ was due to differences in cardiac output (Q) or saliva Na+. To determine this, we collected EBC three times over 1.5hrs with the simultaneous sampling of saliva Na+ and assessment of Q (using acetylene rebreathing) in 7 healthy subjects (age=25±4yrs., ht.=177±7cm, wt.=70±25kg, BMI=22±7kg/m2). Exhaled Na+ averaged 2.1±0.8mmol/l and exhaled K+ averaged 0.14±0.04mmol/l. The coefficients of variation for exhaled ions across three measures averaged 30% for exhaled Na+ and 32% for exhaled K+. Within subjects, the relationship (Pearson correlation) between exhaled Na+ and K+ with Q averaged 0.61 and 0.45, respectively, and the relationship between exhaled Na+ with saliva values averaged 0.85 (p<0.05). Between subjects, the relationship between exhaled Na+ and K+ with Q averaged 0.52 and 0.38 (p<0.05 for both), respectively, and that between exhaled Na+ with saliva values averaged 0.72 (p<0.05). These results suggest that both cardiac output and saliva may contribute to the variability in exhaled ion measurement.
Identification and characterization of the IRD motif, a novel domain identified in the Ioc3 subunit of the ISW1a chromatin remodeling complex
Authors: Olufemi Lola , Bartholomew Blaine
Published: June 2021

ISW1 forms two distinct chromatin remodeling complexes in S. cerevisiae, ISW1a and ISW1b. ISW1a is composed of Isw1 and Ioc3 while, ISW1b is comprised of Isw1, Ioc2, and Ioc4. Isw1, functions as the catalytic subunit of the complex. Similar to drosophila ISWI, Isw1 contains HAND, SANT, and SLIDE domains. Ioc2 has an atypical PHD motif that is often found in chromatin associated proteins and Ioc4 has a PWWP motif, a putative DNA binding domain, which is often found in enzymes that function in transcription regulation. Ioc3 has no conserved domains that have been previously characterized in S. cerevisiae; neither does it bear any similarity to proteins which their function has been defined.

The aim of this work was to determine the domain architecture of Ioc3 of the ISW1a chromatin remodeling complex. This work explicitly identifies the regions of Ioc3 responsible for maintenance of complex integrity, modulation of directionality during remodeling, and nucleosome spacing. More significantly, the IRD domain (ISW1a‐Regulator of Directionality domain), a novel domain within Ioc3 that has not been previously characterized in S. cerevisiae was identified. The IRD domain was found to be required for the proper regulation of ISW1a remodeling vitro and is a distinguishing feature between the ISW1a and ISW1b complexes.

This work was supported by Public Health Service grant GM 70864 from the National Institute of Health.
Accessory subunit of ISW2, Itc1 senses extranucleosomal DNA for regulating nucleosome remodeling
Authors: Prasad Punit , Bartholomew Blaine
Published: June 2021

ATP‐dependent chromatin remodeling complexes utilize the energy from ATP hydrolysis to bring about changes in the chromatin structure and make DNA accessible to regulatory factors. One such ATP‐dependent chromatin remodeling enzyme from Saccharomyces cerevisiae is ISW2. ISW2, a four subunit complex, consists of Isw2, the catalytic subunit, Itc1, the largest accessory subunit and two histone fold subunits Dpb4 and Dls1. ISW2 complex is well characterized in terms of its function in vivo and in vitro, which makes it a model remodeling enzyme to study biological functions. This work aims at defining the role of Itc1 subunit in nucleosome remodeling by ISW2. Systematic C‐terminal truncations of Itc1 were created with FLAG epitope tag for affinity purification of native complexes. Nucleosome binding affinity was reduced with shortening of the C‐terminus indicating the role of Itc1 in stabilizing the interaction of ISW2 with nucleosomes. Remodeling activity of most of these mutants was unaffected except one which resulted in nucleosome positioning defect. This mutant ISW2 complex was named as DNP (Defect in Nucleosome Position) ISW2. The defect in nucleosome positioning is not due to the loss of complex integrity or change in stoichometry of subunits. Further characterization of DNP ISW2 by site‐directed mapping of nucleosome positions showed that the truncation of the C‐terminus of Itc1 resulted in loss of ability to sense extranucleosomal DNA to effectively remodel nucleosomes.

This work was supported by Public Health Service grant GM 70864 from the National Institute of Health