bluuebunny/crossref_metadata_2025 huggingface dataset:
Acute necrotizing pancreatitis shock and the sGC/cGMP system: methylene blue use as pharmacological tool
Authors: Capellini Verena Kise , Baldo Caroline Floreoto , Celotto Andrea Carla , Sônego Fabiane , Batalhão Marcelo Eduardo , Tirapelli Luís Fernando , Cárnio Evelin Capellari , dos Santos José Sebastião , Cunha Fernando de Queiróz , Evora Paulo Roberto Barbosa
Published: June 2021

Soluble guanylyl cyclase (sGC) is associated to endothelial barrier dysfunction and inflammatory reaction during acute pancreatitis. Thus, sGC/cGMP inhibition using methylene blue (MB) was investigated. Acute necrotizing pancreatitis was induced by retrograde infusion of sodium taurocholate 5% (1mL/kg) and enterokinase (8U/kg) in the pig pancreatic duct. Pigs were allocated in 4 groups (n=5): 1: control; 2: pancreatitis; 3: MB infusion post pancreatitis; and 4: MB infusion pre pancreatitis. Data underwent Two and One Way Analysis of Variance (ANOVA) followed by Tukey test with p<0.05. Hemodynamic changes begin 3 h after pancreatitis onset and exhibited mild alterations for either MB administrations. Plasma and ascitic amylase and lipase levels were greatly increased in groups 2, 3 and 4. Plasma nitric oxide (NO) was unaltered. Intra‐abdominal fluid collection had a 15‐fold volume increase on group 2. Groups 3 and 4 had less fluid accumulation. Preemptive MB was associated to 50% more free radical injury. For post‐treatment, neutrophil in pancreatic tissue had a 2‐fold increase. In summary, sGC inhibition by MB clinically did not affect hemodynamic pattern due to a non‐pivotal role of NO on this investigation. Moreover, early and late sGC inhibition may worsen free radical damage and magnitude of inflammatory process, respectively. Support: CAPES; FAEPA‐HC/FMRP.
Pyruvate‐enriched Ringer's resuscitation bolsters cytoprotective defenses in reperfused hindlimb in the setting of hemorrhagic shock
Authors: Flaherty Devin C , Hoxha Besim , Sun Jie , Simecka Jerry W , Olivencia‐Yurvati Albert H , Mallet Robert T
Published: June 2021

Hypothesis

Resuscitation with Ringer's solution containing pyruvate, an antioxidant and anti‐inflammatory metabolite, may mitigate hindlimb ischemia‐reperfusion injury in the setting of hemorrhagic shock.

Methods

Isoflurane‐anesthetized male goats were hemorrhaged to lower mean arterial pressure (MAP) to c. 50 mmHg, then the right femoral artery and vein were occluded for 90 min. Lactate‐ (LR) or pyruvate‐fortified Ringer's solution (PR) was infused iv (10 ml/min) from 30 min occlusion until 30 min after release of occlusion. At 4 h post‐occlusion, gastrocnemius was biopsied for analyses of antioxidative enzymes (spectrophotometry) and pro‐ and anti‐apoptotic proteins (actin‐normalized immunoblot). Results [*P < 0.05 vs. sham; †P < 0.05 vs. LR]: At 4 h post‐occlusion, MAP (mm Hg) was better maintained following PR (64 ± 6 †) than LR (44 ± 4). PR increased activities (mU/g) of antioxidant enzymes glutathione reductase (Sham 4.1 ± 0.2, LR 6.9 ± 0.7*, PR 9.8 ± 1.0* †) and glutathione peroxidase (Sham 11.3 ± 2.7, LR 14.0 ± 0.9, PR 17.4 ± 1.2* †). PR also stabilized muscle contents of the antiapoptotic protein Bcl‐XL (Sham 1.95 ± 0.02, LR 1.25 ± 0.12*, PR 1.77 ± 0.15 †) and the ratio of uncleaved (i.e., inactive)/cleaved (i.e., pro‐apoptotic) poly‐ADP ribose polymerase (PARP) (Sham 3.53 ± 0.57, LR 2.27 ± 0.23*, PR 3.90 ± 0.44 †).

Conclusions

Relative to conventional LR, resuscitation with PR more effectively stabilized MAP, bolstered the ischemic hindlimb's antioxidant and anti‐apoptotic phenotype. Funding: U.S. Dept. of Defense
Endocardial desmin in heart failure
Authors: Monreal Gretel , Nicholson Lisa M , Phillips Alistair B , Bauer John Anthony , Gerhardt Mark A
Published: June 2021

The cytoskeletal protein desmin is upregulated in ischemic heart failure (HF). To test the hypothesis that wall stress mechanisms influence desmin upregulation we examined its transmural distribution in HF. HF was induced in sheep (coronary embolization). LV EF and end‐systolic and ‐diastolic areas (ESA, EDA) were measured at baseline and 4‐ or 12mos later. Desmin content was measured in myocardium via histochemistry and image analysis. Multivariate regression with beta coefficient (β) assessed which parameter exerted the greatest influence on the outcome. *p=0.05. EF decreased from 55% to 24%*. ESA and EDA increased 235%* and 123%* at 12mos. Desmin content was greatest in the LV endocardium (+30% compared to epicardium), increased with HF progression (145%* and 259%* at 4‐ and 12‐mos), inversely correlated to EF (r=‐0.61*), and better predicted EF (β=‐1.18*) and ESA (β=0.89*) than meso‐ or epicardial. Transmural desmin better predicted cardiac dysfunction/remodeling than parallel measurements of fibrosis (r=0.32*) or myocyte hypertrophy (r=0.36*). Desmin upregulation shows a transmural endo‐>epicardial gradient in HF. Endocardial desmin content better correlates to worsening HF than meso‐ or epicardial desmin, suggesting that intra‐myocyte remodeling, likely related to mechanical stretch, are useful predictors of LV function and disease progression. Funding provided by AHA, FAER, NHF, and SCA.
Maternal High‐Fat Diet Causes Cardiac Dysfunction and Increased Endotoxin Sensitivity in Offspring in Mice
Authors: Huang Hong , Zhang Hongyan , Schanbacher Brandon , Giannone Peter , Bauer John
Published: June 2021

"The fetal origins hypothesis" has been recognized for decades. The relationship between nutritional uptake and fetal cardiovascular development has not been defined. We hypothesized that maternal high‐fat diet (HFD) affects innate immunity, and thereby predisposes the offspring to cardiovascular disease in adulthood. Female C57BL/6 mice were fed with either control diet (CD) or HFD while breeding and 3 weeks after delivery. Cardiac profiles were assessed in offspring at 5‐week with echocardiogram. Offspring were then given a single dose of bacterial endotoxin (LPS, 1mg/kg, ip). Plasma serum amyloid A (SAA) levels were measured with ELISA at 0, 2h, and 24h after treatment. There were no differences on total‐, LDL‐cholesterol, and triglycerides between control and prenatal HFD offspring. While basal heart rate, cardiac output, and LV fractional shortening (FS) showed no differences, offspring exposed to prenatal HFD had significant less isoproteronol (2.5 mg/kg, ip) induced increases of FS (p<0.05), indicating a reduced cardiac reserve capacity. An elevation of plasma SAA level was detected at 24h after LPS treatment (p<0.001), which was more significant in mice with prenatal HFD. We concluded that maternal HFD has permanent effect on cardiac function in offspring especially under stress. It also causes an increase in LPS sensitivity, which could be explained by altered innate immunity.
The effects of prenatal lipopolysaccharide on cardiac function
Authors: Han Bing , Huang Hong , Schanbacher Brandon L , Bauer John A , Giannone Peter J
Published: June 2021

Low birth weight has been strongly associated with cardiovascular disease in adulthood. The first report of poor maternal nutrition being associated with cardiovascular disorders in offspring was from Dr. David Barker who showed that people had an increased risk of death from cardiovascular disease if they were small at birth. There is also substantial evidence that maternal infection/inflammation are likely contributors to low birth weight. We tested the hypothesis that prenatal exposure of lipopolysaccharide (LPS) decreases cardiac function in adulthood.

Pregnant C57BL/6J mice were treated with a single dose of LPS (2 ug/mouse) i.p. or vehicle at gestation E16. Basal cardiac performance and responses to stress (isoproterenol, 2.5 mg/kg, ip) were determined in male offspring at 8 weeks with echocardiogram. Offspring were sacrificed and cardiac tissues were collected.

Compared with the control group, offspring of LPS‐treated dam had slower growth rate (body weight at 8w: 22.1±1.3 vs. 24.6±2.1g, p<0.05) with decreased cardiac output (CO, 21.8±3.9 vs. 25.7±2.1 ml/min, p<0.05), signifying an impaired cardiac contractility. In addition, offspring with prenatal LPS exposure seemed to have less increase of CO after stress test, suggesting a decreased cardiac reserve.

Our data suggest that prenatal inflammation has long term effects on cardiac function. Further mechanistic studies are undergoing.
Role of pendrin in iodide balance: going with the flow
Authors: Kim Young Hee , Pham Truyen Derek , Zheng Wencui , Hong Seongun , Baylis Christine , Pech Vladimir , Beierwaltes William H. , Farley Donna B. , Braverman Lewis E. , Verlander Jill W. , Wall Susan M.
Published: June 2021

Pendrin is expressed in the apical regions of B and non‐A, non‐B intercalated cells, where it mediates apical Cl−/HCO3− exchange. Pendrin is a robust I− transporter. So, we asked if I− restriction modulates pendrin abundance and if I− balance is altered in pendrin null mice. Pendrin abundance was evaluated with immunoblots, immunohistochemistry and immunogold cytochemistry with morphometric analysis. While pendrin abundance was unchanged when dietary I− intake was varied, I− balance differed in pendrin null and in wild type mice. Serum I− was lower, while I− excretion was higher in pendrin null relative to wild type mice, consistent with a role of pendrin in renal I− absorption. Increased H2O intake enhanced differences between wild type and pendrin null mice in I− balance, suggesting that H2O intake modulates pendrin abundance. Increasing water intake raised B cell apical plasma membrane pendrin abundance. Water intake did not modulate pendrin abundance by changing circulating renin or aldosterone concentration or by changing serum osmolality or circulating vasopressin. Moreover, in models of vasopressin escape serum Cl− was much lower in pendrin null than in wild type mice. We conclude that water intake modulates pendrin abundance. As water intake rises, pendrin becomes increasingly critical in the maintenance of Cl− and I− balance.
Filamin‐A modulates CFTR surface expression via Filamin‐A Ig repeat 21 and interacts with the scaffold protein RACK1
Authors: Liedtke Carole M , Xu Z , Smith Laura , Kohli Ekta , Misra Saurav
Published: June 2021

Filamin‐A (FlnA) binds to CFTR through a protein‐protein interaction that is disrupted by the missense mutation S13F in CFTR, causing unstable surface expression of CFTR. We hypothesized that the N‐terminal sequence of CFTR binds to immunoglobulin repeat 21 of FlnA (Ig21), based on previously studied binding preferences of Ig21. We confirmed binding of a CFTR N‐terminal peptide to Ig21 and solved the structure of the peptide bound to Ig21 in a novel staggered 2:1 complex, in which the single CFTR N‐terminal peptide completes an extended beta sheet spanning two parallel Ig21 domains. The structure reveals how the disease‐causing CF mutation S13F disrupts the FlnA:CFTR interaction. To test functional effects of Ig21, we delivered Ig21 into Calu‐3 human airway epithelial cells and examined CFTR apical membrane expression by biotinylation. Ig21 caused a dose‐dependent decrease in biotinylated CFTR. CoIP experiments demonstrated an interaction between CFTR and FlnA. We also detected RACK1 in IPs of FlnA and vice versa and colocalization of RACK1 and FlnA by IF. To study RACK1 binding to FlnA, FlnA or CFTR were IPed from Calu‐3 cells, washed under high stringent conditions, and incubated with recombinant RACK1. RACK1 was shown to bind to FlnA in a dose dependent manner, but not to CFTR. Our results suggest that the important FlnA:CFTR interaction is indeed mediated by FlnA‐Ig21 and that FlnA:RACK1 interaction may be important for RACK1‐mediated recruitment of PKCε to CFTR. Supported by the Ralph Wilson Medical Research Foundation (SM), NIH R01‐58598 and ‐67190 (CML) and an RDP grant from the Cystic Fibrosis Foundation (CML).
ADAM15 regulates endothelial permeability independently of sheddase activity
Authors: Sun Chongxiu , Wu Mack H. , Yuan Sarah Y.
Published: June 2021

ADAM15 (a disintegrin and metalloproteinase 15) is a transmembrane glycoprotein with proteolytic activity and integrin binding capability; both functions are important in vascular inflammation. The objective of this study was to explore the role and mechanisms of ADAM15 in regulating endothelial permeability during inflammatory stimulation by thrombin. We examined the effect of this molecule on endothelial barrier properties by measuring the transendothelial electric resistance (TER) in human umbilical vein endothelial cell (HUVEC) monolayers. The results showed that thrombin decreased TER and upregulated the expression of ADAM15 in a time‐dependent manner. Depletion of ADAM15 expression attenuated thrombin‐induced decrease in TER, whereas ADAM15 over‐expression augmented thrombin‐induced hyperpermeability. The permeability effect of ADAM15 did not seem to require its proteolytic activity, as cells expressing a catalytically dead mutant of ADAM15 (E350A) demonstrated the same response as the catalytically potent wild‐type control. Furthermore, over‐expression of ADAM15 did not cause any significant increase in the level of VE‐cadherin C‐terminal fragments, indicating that VE‐cadherin proteolysis or shedding was not involved in the ADAM15 response. Taken together, the data suggest that ADAM15 plays a signaling role in the pathophysiological regulation of endothelial permeability via a mechanism independent of its sheddase function. Supported by NIH HL61507, HL84542, HL70752, and HL73324.
Aldosterone regulates the ENaC Recycling compartment in CCD cells
Authors: Edinger Robert S , Wang Huamin , Butterworth Michael B , Balasubramani Manimalha
Published: June 2021

We have previously demonstrated that cultured CCD cells deprived of aldosterone develop a loss of Na+ reabsorptive activity and a loss of the cellular recycling compartment for ENaC. The recycling compartment and Na+ reabsorptive phenotype are restored over time by aldosterone. Restoration of the ENaC recycling pathway is associated with up‐regulation of multiple proteins in the endosomal compartment. Analysis of early endosomes isolated from CCD cells under these conditions using Two‐dimensional difference gel electrophoresis (DIGE) or Isobaric tag for relative and absolute quantitation (iTRAQ) indicates that aldosterone significantly up‐regulates 15‐20 distinct proteins. One major protein induced is Annexin 2 (Ann2). Ann2 has been implicated in both exocytic and endocytic pathways and has been described to form a functional complex with CFTR. Ann2 colocalizes with Rab11 endosomes and is more abundant in these endosomes with the addition of aldosterone. Knockdown of Ann2 in CCD cells results in a decrease of basal and forskolin induced ENaC activity. These results suggest that Ann2 localizes to recycling endosomes upon addition of aldosterone and facilitates the recycling of ENaC.
Regulation of the rat intestinal phosphate transporters by dietary phosphate
Authors: Giral‐Arnal Hector , Wilson Paul , Sutherland Eileen , Breusegem Sophia , Caldas Yupanqui , Barry Nicholas , Jiang Tao , Wang Xiaoxin , Levi Moshe
Published: June 2021

Elevated serum phosphate (Pi) remains a clinical challenge in patients with chronic kidney disease. However, Pi absorption mechanism in the small intestine is not fully understood. NaPi‐2b was identified as the main transporter responsible for intestinal Pi absorption. In mice NaPi‐2b expression concentrates mainly in the ileum. In contrast, rat NaPi‐2b mRNA and protein is expressed in jejunum and duodenum with negligible amounts in ileum, a pattern similar to humans. We report expression of another NaPi transporter showing a similar expression profile: Pit‐1. We characterized intestinal NaPi transport regulation by dietary Pi during chronic and acute alterations in dietary Pi. Chronic low Pi diet induces an increase in jejunal NaPi transport activity and increases in BBM NaPi‐2b protein and mRNA abundance. In contrast, we found an unexpected regulatory response in rats switched from a chronic low Pi diet to a high Pi diet acutely: Duodenum BBM NaPi uptake and NaPi‐2b protein is highly up‐regulated. Moreover, under this acute condition serum Pi is markedly increased as early as 2 hours after feeding. We propose that increased transcellular Pi transport mediated by NaPi‐2b is inducing the unfavorable increase of serum Pi under this condition. Moreover, Pit‐1expression in this tissue could also be playing a role in the intestinal Pi absorption in rats.
Constitutive endocytosis of NKCC2 in thick ascending limbs is dynamin dependent
Authors: Ares Gustavo Rosier , Ortiz Pablo A
Published: June 2021

The Na/K/2Cl cotransporter (NKCC2) mediates NaCl absorption by the epithelial cells of the thick ascending limb (TAL). Trafficking into (exocytosis) and out (endocytosis) of the apical membrane regulates surface NKCC2 levels. Dynamin‐2 is a ubiquitous protein that mediates endocytosis in many epithelial cells. It is not known whether NKCC2 undergoes constitutive endocytosis and whether dynamin‐2 is involved. We hypothesized that constitutive endocytosis of NKCC2 is mediated by dynamin‐2. We measured surface NKCC2 and the rate of NKCC2 endocytosis by biotinylation and Western blot. First, we studied whether dynamin‐2 is expressed in TAL. We found that dynamin‐2 is expressed in rat medullary TAL suspensions as a single band at the predicted molecular weight of 100 kDa. In control TALs we observed constitutive NKCC2 endocytosis over 30 min that averaged 10.1 ± 0.8 a.u/min. We tested whether the dynamin inhibitor (Dynasore) inhibits constitutive NKCC2 endocytosis. In the presence of Dynasore (100 μM), the rate of constitutive NKCC2 endocytosis averaged 2.8 ± 0.6 a.u/min ( n = 6, p < 0.01 vs control), a 72% inhibition. Acute (20 min) blockade of endocytosis with Dynasore increased surface NKCC2 by 66 ± 26% (p<0.05). The total pool of NKCC2 was not changed by Dynasore. We concluded that constitutive NKCC2 endocytosis is mediated by dynamin‐2. These are the first data showing NKCC2 endocytosis and a role for dynamin in TALs.
Ammonium transport in mouse colon: Role of NKCC1 and Rh glycoproteins
Authors: Worrell Roger T. , Engevik Melinda A.
Published: June 2021

We have previously shown that NH4+ secretion occurs in the colonic crypt cell line, T84 (AJP 294:G429, 2008). In T84 cells, the secretory vector exceeds the absorptive vector thereby resulting in net NH4+ secretion. This study sought to extend the colonic cell line work to mouse colon. Absorptive NH4+ flux in isolated mouse distal colon (Jms) was 80±22 while secretory flux (Jsm) was 404±47, resulting in a net secretory NH4+ flux of 325±35 μg/cm2/hr. As was found in T84 cells, both NKCC1 and RhBG were implicated in contributing to this net secretion. Portal vein [NH4+] was ~1.5 fold higher in NKCC1−/− and ~1.24 fold higher in NKCC1+/− mice compared to WT mice indicative of decreased intestinal NH4+ secretion. Interestingly systemic blood [NH4+] was elevated ~2 fold in the NKCC1−/− mice suggesting a role for NKCC1 in hepatocyte NH4+ uptake. By real‐time RT‐PCR, both T84 cells and mouse colon express the ammonium transporters, RhBG and RhCG. Whereas both are expressed equally in T84 cells, the ratio of RhBG to RhCG varied along the length of the colon, with RhBG/RhCG being 9 in proximal colon and 2 in distal colon. RhBG levels were ~1.7 fold higher in NKCC1−/− verses WT colon yet RhCG levels were unchanged thus supporting a role for RhBG in colonic NH4+ secretion. These data support the ability of colonic epithelia to actively extrude NH4+ thereby limiting the interstitial accumulation of this toxic substance. Supported by NIH DK079979.
CFTR‐mediated Secretion is Supported by Different Basolateral Transporters in Juvenile Murine Proximal Colon
Authors: Gawenis Lara , Gawenis James
Published: June 2021

Multiple basolateral uptake mechanisms for Cl and HCO3 are stimulated to sustain transepithelial anion secretion during activation of CFTR by cyclic nucleotides. We hypothesized AE2 has a greater contribution in juvenile murine colon and that activity of NBCs are increased in adult colon. Short‐circuit current (Isc) responses were measured in muscle‐stripped proximal colon isolated from juvenile and adult wild‐type mice. In Cl and HCO3 containing Ringers, juvenile and adult proximal colon exhibited no differences in cAMP‐stimulated currents. However, juvenile proximal colon exhibited decreased sensitivity to the distilbene SITS, suggesting reduced activity of either NBC or AE2 in juvenile proximal colon. Blocking of coupled AE2/NHE1 (EIPA) reduced the Isc response to cAMP and SITS in juvenile but not adult proximal colon. Cl‐free Ringers significantly reduced the Isc response to cAMP in both adult and juvenile proximal colon. Under this condition, the SITS‐sensitive Isc was still reduced in juvenile, as compared to adult, proximal colon, further supporting the hypothesis that SITS‐sensitive NBC activity is lower in juvenile colon. Current studies are focused on whether these changes result from alterations in transporter expression or activity. We conclude that coupled NHE1/AE2 activity is increased and SITS‐sensitive NBC activity is decreased in juvenile proximal colon. Supported by AGA‐FDHN.
Role of Rh (rhesus) complex in K‐Cl cotransport (KCC) regulation in red blood cells (RBCs) of Rh and Rh‐associated glycoprotein (RhAg) knockout (KO) mice
Authors: Lauf Peter K. , Adragna Norma C. , Goossens Dominique , Cartron Jean‐Pierre
Published: June 2021

The Rh blood group is a macromolecular complex of protein subunits (D and CE), RhAG, a NH3/CO2 gas transporter (PNAS 101:17222,2004; FASEB J 22;1,2008), and accessory proteins (ICAM‐4/LW, CD47 and GPB). In the mild hemolytic human Rhnull disorder, the Rh complex is absent/severely deficient. Altered K fluxes have been found in Rhnull RBC (Blood 48: 457, 1976). A double blind study on male (m) or female (f) wild type (WT), and Rh−/− and Rhag−/− KO mice (Transf Clin Biol 13:164,2006) studied: Total, ouabain (O)‐sensitive Na/K pump, O‐insensitive+bumetanide (B)‐sensitive Na‐K‐2Cl cotransport (NKCC), and O‐ and B‐insensitive KCC ± the thiol alkylant N‐ethylmaleimide (NEM), staurosporine (STP, a kinase inhibitor) and Mg removal by A23187.

Results

1) Total, Na/K pump, NKCC and basal KCC influx were not different between groups. 2) NEM activated KCC by >100 % (p<0.001) in both m/f Rh−/− and RhAg−/−, compared to WT. KCC activation by both STP, and Mg depletion (except for m Rh−/−), was similar in m Rh−/−, m/f Rhag−/− RBCs and WT.

Conclusion

Thiol modification reveals a Rh‐complex role in KCC regulation, i.e. via protein phosphorylation or protein/protein interactions. However, unlike in human Rhnull RBCs, Na/K pump and passive K‐"leak" fluxes were normal, an apparent difference in the Rh complex‐K transport association between mice and man. Support in part by a WSU School of Medicine Grant.
Cardiac Heme Oxygenase 1 (HO‐1) mRNA expression in inbred rat strains after severe hemorrhage
Authors: OH Taesung , Calderon Mariam , Klemcke Harold
Published: June 2021

We have previously identified 8‐fold differences in survival time among inbred rat strains after severe hemorrhage (Klemcke et al., Shock 29: 748, 2008). It is probable that specific cellular mediators are associated with these differences. HO‐1 is an inducible, cytoprotective, anti‐inflammatory, and anti‐apoptotic protein and hence is one such candidate. To examine this possibility, we measured HO‐1 mRNA levels using real‐time PCR in cardiac tissue in three inbred rat strains (DA, Fawn Hooded Hypertensive (FHH), and Brown Norway (BN)) divergent in survival time after hemorrhage (BN > FHH > DA). Rats for each rat strain were randomized to three treatment groups (n=5/group): controls without surgery (C); surgically catheterized rats (Cath); and catheterized and hemorrhaged rats (Bled). Surgeries were conducted ~ 24 hr prior to hemorrhage and tissue collections. Tissues were collected 30 min after initiation of a 26 min hemorrhage, or 30 min post handling in C and Cath rats. There were no strain‐associated differences in HO‐1 mRNA levels for each treatment group. HO‐1 mRNA expression of DA and FHH was not different among treatment groups. However, in BN rats HO‐1 mRNA expression increased 2‐fold in bled rats (4371 ± 508 copies /20ng total RNA; mean ± SEM), compared to C and Cath groups respectively (1745 ± 508 and 2082 ± 508). These data indicate inbred rat strain‐dependent differences in HO‐1 mRNA response to hemorrhage.
The Phenotypes and Models Portal at RGD: a new interactive tool for physiologists linking genotype to phenotype and disease
Authors: Munzenmaier Diane H. , Dwinell Melinda , Nigam Rajni , Shimoyama Mary , Jacob Howard J.
Published: June 2021

Aim

The Rat Genome Database (RGD) has been developing tools and resources to aid investigators in the integration of genomic and phenomic data derived from rat research for the past 10 years. Recently, a new Phenotypes and Models Portal has been developed. Designed to be an entryway into RGD for physiologists, it allows access to phenotype data and disease model information for biomedical investigators doing rat research.

Methods

RGD has always contained valuable information for physiologists, though the focus has been primarily on genomic data, making it somewhat challenging for physiologists to navigate. This portal provides new types of phenotype data, as well as different ways to access existing data in RGD, allowing extraction of data relevant to an investigator's research interests by specific organ system function or identification of suitable rat models for specific diseases. The Phenotypes branch provides access to phenotype data for cardiovascular, pulmonary, blood, biochemistry, renal, vascular measurements and general morphological results. The Strains and Models branch houses strain information, includes strain "medical records" and links to genotype and phenotype data.

Conclusion

The Phenotypes and Models Portal promises to be an effective means to access and retrieve physiological data and disease model information for researchers using the rat as a model to study physiology and disease.
Inhibition of superoxide dismutase (SOD) partially attenuates anti‐diuretic and anti‐natriuretic responses to angiotensin II (AngII) in anesthetized rats
Authors: Khan Md Abdul H , Castillo Alexander , Islam Mohammed Toriqul , Majid Dewan SA
Published: June 2021

To evaluate the interaction between SOD and NADPH oxidase activity in AngII actions in the kidney, we assessed renal responses to intra‐arterial Ang II infusion (0.5 ng/kg/min for 45 min) before and during infusion of SOD inhibitor, diethyldithiocarbamate (DETC, 0.5 mg/kg/min) in rats (n=8) pretreated with enalaprilat (33 µg/kg/min). Total (RBF) as well as cortical (CBF) and medullary blood flows (MBF) were measured by Transonic and laser‐Doppler flowmetry. Prior to DETC, AngII caused reductions of 19±3% in RBF (baseline value, b, 6.0±0.3 mL/min/g), 16±4% in CBF (b, 358±26 PU/min/g), 11±1% in MBF (b, 161±27 PU/min/g), 32±4% in GFR (b, 0.90±0.05 mL/min/g), 33±3% in urine flow (V, b, 10±0.7 µL/min/g) and 51±3% in sodium excretion (UNaV, b, 1.3±0.1 µmol/min/g). DETC itself caused reductions of 14±3% in RBF, 9.5±2 % in CBF, 7.8±0.8% in MBF, 24±5% in GFR, 12±2% in V and 29±5% in UNaV. During DETC infusion, AngII caused similar decreases in RBF (18±4%), CBF (17±3%), MBF (12±2%) and GFR (24±5%) but significantly less decreases in V (12±2%) and UNaV (25±3%) as compared to AngII alone. Application of DETC (10−2 M) also reduces NADPH oxidase activity in renal cortical (573±43 to 67±4 RLU/mg protein, n=6) and medullary (1607±28 to 148±5 RLU/mg protein, n=6) tissue slices. These data suggest that a downregulation of NADPH oxidase in the condition of SOD inhibition is important to limit Ang II induced reduction in renal excretory function.
Interaction between α1B adrenoceptors and AT1 receptors in the control of renal haemodynamics in Sprague Dawley (SD) rats with pressure over load induced left ventricular hypertrophy
Authors: Rathore Hassaan Anwer , AbdulSattar Munavvar Zubaid , Abdullah NoorAzizan , Johns Edward J
Published: June 2021

This study examined the interaction between α1B adrenoceptors and AT1 receptors in the control of renal hemodynamics in rats with pressure overload ventricular hypertrophy. Groups (n=6 each) of male SD rats were subjected to sham surgery (controls) or supra renal aortic banding (AB). On day 45, rats were anaesthetized and prepared for measurement of mean arterial pressure (MAP) and renal cortical blood perfusion (CBP). The changes in CBP caused by intra renal administration of noradrenaline, phenylephrine, methoxamine and ANGII were determined before and after blocking renal α1B adrenoceptors using chlorethylclonidine (CEC). Data, means ± SEM were subjected to ANOVA with significance at P<0.05. Baseline MAP was higher in the AB group, 142±3 vs 105±2 mmHg, but CBP was lower, 624±28 vs 838±21 BPU (both P<0.05), than the control group. The low dose of CEC enhanced the vasoconstrictor responses to methoxamine in the AB group by 10% (P<0.05) compared to the vehicle phase. The high dose of CEC decreased the vasoconstrictor responses to all adrenergic agonists by 10‐20% in the control group versus 30‐60% in the AB group (all P<0.05) and to ANGII by 10% in the control and by 5% in AB group (both P<0.05). These data showed an increased functional importance of renal α1B adrenoceptors in pressure overload hypertrophy through a possible cross talk mechanism with renal AT1 receptors.
Interleukin‐10 Protects Against Vascular Dysfunction with Aging
Authors: Didion Sean P , Kinzenbaw Dale A , Faraci Frank M
Published: June 2021

Aging produces vascular inflammation and dysfunction in blood vessels. We tested the hypothesis that interleukin‐10 (IL‐10), a potent anti‐inflammatory cytokine, protects against aging‐induced vascular dysfunction. Responses of carotid arteries from young (7‐8 mo) and old (19‐20 mo) wild‐type and IL‐10 deficient mice (IL‐10‐/‐) were examined in vitro. In arteries from wild‐type mice, acetylcholine (an endothelium‐dependent agonist) produced relaxation that was not altered at this age. In contrast, relaxation to acetylcholine in arteries from IL‐10 deficient mice was markedly impaired with age. For example, relaxation of the carotid artery to 10 μmol/L acetylcholine was 87±3 and 48±2% in old wild‐type versus old IL‐10 deficient mice, respectively (P<0.01). Tempol (1 mM), a scavenger of superoxide, did not affect responses in young or old wild‐type or young IL‐10 deficient mice, but restored vasodilation to acetylcholine to normal in old IL‐10 deficient mice. Relaxation of the carotid artery to nitroprusside was not altered in any group. These findings provide the first evidence that age‐related and superoxide‐mediated endothelial dysfunction occurred much earlier and to a greater extent with IL‐10 deficiency. Our findings suggest a novel role for IL‐10 in oxidative stress and age‐related vascular dysfunction.
Comparison of sex hormones in ischemic‐reperfusion injury in isolated rat hearts
Authors: Balonan Lino , Das Rapti , Ho Susan , Wong Tak Ming
Published: June 2021

To compare potential protective effects of testosterone and estrogen on myocardium, we exposed isolated rat hearts to 30 minutes of ischemia followed by 120 minutes of reperfusion. One group of rats served as normal controls (sham). The experimental group underwent either bilateral orchidectomy (Gx) or ovariectomy (Ovx). After one week post‐surgery, each group was further divided, and received either a 60‐day release implant containing testosterone (Gx+T; Ovx+T) or 17 β‐estradiol (Ovx+E; Gx+E). There was no statistically significant difference in the infarct size among these groups of rats.

However, when noradrenaline (10−7M) was administered during the ischemic period, the infarct size was significantly greater in both the Gx (50.66 ± 1.80) and Ovx (52.03 ± 2.36) groups than in sham cohorts (38.63 ± 1.88 and 44.09 ± 1.68, respectively). Hormone replacement in these groups significantly reduced the infarct size to levels comparable to the corresponding sham groups (Gx: 42.06 ± 1.81 versus 38.63 ± 1.88 following testosterone treatment and 42.11 ± 1.51 versus 38.63 ± 1.88 following estrogen treatment; Ovx: 41.3 ± 2.185 versus 44.09 ± 1.68 with estrogen and 43.73 ± 1.66 with testosterone).

Our results suggest testosterone imparts cardioprotection and plays an important role in determining the extent of cardiac injury in response to adrenergic stimulation during myocardial ischemia.
Investigating the Forces that Drive Membrane Protein Folding
Authors: Bowie James Ulrich
Published: June 2021

Understanding the forces that drive protein folding is essential for many of the central quests of structural biology including structure prediction, design and deciphering evolution. Nevertheless, we still have a rudimentary understanding of membrane protein folding energetics. I will describe tools we have developed for studying the factors that stabilize bacteriorhodopsin. Application of these methods suggests that packing forces, rather than hydrogen bonding, dominate membrane protein stabilization. Given the focus on dispersion forces, it is possible that membrane proteins might utilize packing more effectively than soluble proteins. We find that membrane proteins are not necessarily more efficiently packed, but manage to bury more surface area than soluble proteins.
Atomic force and fluorescence microscopy imaging of cardiomyocyte plasma membrane, insulin receptor and caveolin‐3
Authors: McAndrew Elizabeth G , Xue Jianjing , Wold Loren E , Vesenka James , Davidoff Amy J
Published: June 2021

Impaired insulin signaling is a component of diabetic cardiomyopathy. Our hypothesis is that myocardial insulin resistance involves a dissociation of insulin receptors (IR) from caveolae and its intracellular targets. Our aim was to develop techniques to image the spatial relationship between IR and caveolae using atomic force microscopy (AFM) and immunohistochemistry (IHC). We used an embryonic rat cardiac cell line (H9c2) to evaluate various fixation protocols and determined that 4% formaldehyde (rather than glutaldehyde, methanol or combinations), maintained the integrity of the plasma membrane. We tested a variety of AFM tips (standard, tungsten, carbon coated) in both contact and tapping modes on hydrated and dehydrated cells. We now can scan cells in solution, with a resolution of up to 10 nm. To validate our methods and reagents we conducted IHC and immunoblotting. IHC images included fixed and living cells. Future studies will use AFM techniques with gold conjugated antibodies for the IR and caveolin 3, in cells cultured in normal and high extracellular glucose. Ultimately, we intend to evaluate cell surface receptor distributions on adult rat ventricular myocytes. Resolving the localization of IR and caveolae in intact cells and comparing normal and diabetic‐like groups could further our understanding of the pathogenesis of insulin resistance in diabetic cardiomyopathy. APS Undergraduate Summer Research Fellowship (EGM)
Chronic Wy‐14,643 administration attenuates myocardial respiratory capacity and contractile function
Authors: Zungu Makhosazane , Stanley William C , Essop M.Faadiel
Published: June 2021

We hypothesized that chronic in vivo Wy‐14,643 treatment exerts direct effects on myocardial mitochondrial respiratory function and contractile function. Wy‐14,643 was administered to Wistar rats for 14 consecutive days and compared with matched controls. State 3 mitochondrial respiration was lower in the Wy‐14,643 group while state 4 respiration was significantly increased. In agreement, oligomycin‐insensitive proton leak was increased and the respiratory control index significantly decreased. The rate of ADP phosphorylation decreased by 44.9 ± 1.9% (p≤0.05 vs. control). Left ventricular systolic and developed pressures were lower while rate pressure product declined by 36 ± 2.6% (p≤0.01 vs. control) after Wy‐14,643 treatment. We show that chronic in vivo Wy‐14,643 administration to rats impairs myocardial mitochondrial oxidative phosphorylation and increases proton leak, contributing to a decreased rate of ATP synthesis and contractile function. These factors should be considered when employing Wy‐14,643 for both in vitro and in vivo experiments.
Influence of left ventricular diastolic function on pulmonary function in healthy individuals
Authors: Lalande Sophie , Sierra‐Johnson Justo , Miller Wayne L , Johnson Bruce D
Published: June 2021

The aim of this study was to determine if left ventricular (LV) diastolic function influences pulmonary function (PF) in healthy subjects and to determine if they are independently related after accounting for age, gender, BMI and blood pressure (BP). Echocardiograms and PF tests were performed in 103 healthy subjects (22‐76 yr). The ratio of peak early (E) and late (A) mitral inflow velocities and the ratio of peak early (E') and late (A') mitral annular velocities were obtained by pulsed wave Doppler and tissue Doppler imaging. The first stage of diastolic dysfunction, often observed in healthy aging, is characterized by reduced E and E', and greater A and A' resulting in a reversal of E/A and E'/A'. PF measures included forced vital capacity (FVC), carbon monoxide diffusing capacity (DLCO) and forced mid‐expiratory flow (FEF25‐75). Age, gender and BMI accounted for >60% of the interindividual variation between diastolic function and PF. After accounting for age, gender, BMI and BP, E' was positively associated with FVC (additional R2=0.03, P<0.01). A was negatively and A' positively associated with DLCO (additional R2=0.03, R2=0.01, P<0.05) and FVC (additional R2=0.03, R2=0.03, P<0.01). E/A was positively associated with DLCO (additional R2=0.02, P<0.01). In conclusion, LV diastolic function may influence PF in healthy subjects, with the greatest interactions being observed in older males. NIH HL71478
Flow‐Induced Changes in Lymphatic Endothelial Barrier Function
Authors: Kurtz Kristine M , Breslin Jerome W
Published: June 2021

The purpose of this study was to determine how shear stress affects barrier function of cultured lymphatic endothelial cells. We determined the transendothelial electrical resistance (TER) of cultured human dermal microlymphatic endothelial cells (HMLEC‐d) to evaluate quantitative, real‐time changes in barrier function, during no‐flow and various flow conditions. Cells were exposed to continuous or pulsing (2 s on, 2 s off) flow at various shear stresses (2.5, 5, 9, 19, or 38 dynes/cm2) for 30 min with periods of no‐ or low‐ (0.5 dynes/cm2) flow in between. We observed a shear‐dependent increase in TER that was reversed upon turning flow off or to low at 30 min. The increase in TER with 9 dynes/cm2 was abolished by blockade of actin dynamics with 10 μM phalloidin, and significantly inhibited by blockade of Rac with 50 μM NSC23766 (9.1 ± 1.1 % increase for control vs. 2.5 ± 2.0 % with NSC23766, P<0.05). Blockade of PKA (10 μM H‐89), or ROCK (5 μM Y‐27632) did not inhibit the response. A mathematical model based on our impedance data shows that the flow‐induced changes in TER were primarily due to altered current flow between cells and not beneath cells. These results suggest that lymphatic endothelial cells dynamically alter their morphology and barrier function in response to changes in shear stress by a mechanism dependent upon Rac‐mediated actin dynamics.
The Role of Dynamin Family Members in Membrane Fission
Authors: Mears Jason A , Ray Pampa , Fang Shunming , Lackner Laura , Nunnari Jodi , Hinshaw Jenny E
Published: June 2021

The dynamin family of proteins are required for numerous membrane fission and remodeling events throughout the eukaryotic cell. Dynamin is involved in the final stages of fission during clathrin‐mediated endocytosis, caveolae internalization, and vesiculation from the Golgi and recycling endosome, while Drp1 (dynamin‐related protein 1) is necessary for mitochondrial division. During membrane fission, dynamin or Drp1 is believed to wrap around the constricted site to facilitate vesiculation or organelle division. In support of this model purified dynamin has been shown to self‐assemble into spirals (50 nm diameter) and readily form dynamin‐lipid tubes, which constrict and fragment upon addition of GTP. The three‐dimensional structures of dynamin in the constricted and non‐constricted states were solved by cryo‐electron microscopy methods. Placement of the GTPase and Pleckstrin Homology crystal structures into the cryo‐EM densities revealed a twisting motion that suggests a corkscrew model for dynamin constriction. We are currently examining the structure and function of the Drp1 homologue in yeast, Dnm1, to determine if a common mechanism of action exists among dynamin family members. In collaboration with Dr. Jodi Nunnari (UC Davis), we have shown that Dnm1 assembles into large spirals with a diameter of ~110 nm, which is a similar diameter to the observed mitochondrial constriction sites seen in vivo. Dnm1 also assembles onto lipid and forms Dnm1 decorated tubes that constrict significantly upon GTP addition. A preliminary 3D map of Dnm1‐lipid tubes reveals a slightly different architecture to dynamin. For example, the Dnm1 map lacks a strong inner radial density near the lipid bilayer, which correlates well with the absence of a pleckstrin homology domain in Dnm1. Overall, these results suggest that although dynamin family members share common mechanochemical properties, the structure of each member vary to fit their unique function.
Osteological analysis of nonhuman remains: understanding the growth and developmental patterns of post‐crania
Authors: Lee Rosanna , Macsiaz Rachael , Hong Simon , Caceres Noel , Márquez Samuel
Published: June 2021

Osteological analyses of human and nonhuman bones are essential in the forensic assessment of unknown commingled remains. T. Dale Stewart was one of the first to recognize the resemblance between human and nonhuman remains when he reported the similarity between human and North American black bear tarsal bones, reinforcing the importance and difficulty of discerning human from nonhuman bones. We report findings on commingled skeletal remains uncovered from a construction site in New York City. Preliminary assessment of the bones narrowed them to a cow distal femur, a deer fibula and a pig humerus. Further analysis of the cow femur showed a flattened plateau on the anterior surface, characteristic of a sub‐adult cow. In addition, it lacked the sloped trochlea apparent in adult femurs. At the joint surface, four condylar‐like bony projections were present, consistent with the removed epiphysis of a sub‐adult. A comparison of the characteristic anatomy of found bones with bones from the terrestrial mammalian collection representing 7 genera and 9 species from the Division of Mammalogy at the American Museum of Natural History confirmed our analysis. Our study demonstrates that knowledge of the origin of bones, as well as the developmental stages of the specimens, is critical towards the assessment of unknown remains for a just resolution and sound testimony in medical and legal investigations.

Grant Funding Source

Do not have funding
An Anatomically Informed Approach to Transcutaneous Injection Laryngoplasty
Authors: Pearson Bill , Noordzij Pieter
Published: June 2021

Injection larygoplasty medializes deinnervated vocal folds (VFs) by introducing bulking material into the thyroarytenoid muscle. Transcutaneous larygoplasty is preferable to a transoral approach in certain compromised patients, but accessing the VFs with this approach is a challenge for the inexperienced clinician. A geometric approach to consistently and accurately guide the needle to various locations along the VF was developed using cross sectional anatomy software. Pigment was injected into the VF guided by this approach without visualization in 4 female and 3 male cadavers. The vocal folds were then dissected and results recorded. Injections were effective in 11 of 12 tries when VF lengths were within the standard deviation of the mean and in abduction.

Grant Funding Source

n/a
Local Regulation of Free PI(4,5)P2 Levels by MARCKS Protein
Authors: Sheetz Michael P. , Sable Julia
Published: June 2021

The lipid phosphatidylinositol 4,5‐bisphosphate (PI(4,5)P2) has an important role in regulating membrane‐cytoskeleton‐adhesion and many other cellular processes. Although synthesis and degradation may locally regulate levels of PI(4,5)P2 in many cases, an alternative mechanism could be buffering by cationic proteins, like MARCKS . We found that increasing MARCKS protein levels by about 50% caused a decrease in membrane‐cytoskeleton adhesion by two‐fold, consistent with a drop in free PI(4,5) P2 levels. The drop in PI(4,5) P2 levels was reversed by over‐expression of phosphatidylinositol phosphate 5‐kinase type Iα (PIP5KIα) or by phosphorylation of MARCKS by PKC activation. Biochemical analyses of MARCKS‐/‐ fibroblasts showed elevated rates of turnover of both PI(4)P and PI(4,5)P2 as well as a decrease in PA turnover. Although total cellular level of PI(4,5)P2 did not change in MARCKS‐/‐ cells, the plasma membrane level of PI(4,5)P2 decreased by two fold, consistent with the loss of a buffer. All of these data suggest that there is compartmental regulation of PI(4,5)P2 in the cell that is not only controlled by the balance between synthesis and degradation, but also via sequestration by MARCKS protein.
Inhibition of Ras‐expressing Keratinocytes by De‐epidermized Dermis
Authors: Andrews Capri Michele , Vaughan Melville B.
Published: June 2021

Cancer cells secrete matrix‐digesting enzymes allowing invasion and migration to other locations. Ras cells secrete similar enzymes, but their targets may be different than basement membranes. Our previous research showed that ras keratinocytes, when plated on a dermal layer, invade prior to basement membrane formation, and then appear to stop. Therefore, it can be determined if the basement membrane is a factor crucial in inhibiting invasion. To test this hypothesis, ras‐overexpressing keratinocytes were plated on de‐epidermized dermis (DED), consisting of a dermal compartment and a basement membrane. Plating on the dermis‐side was used as a basement membrane‐negative control. The tissues were fixed at 1, 7, 14, and 21 days for both frozen and paraffin sections. Invasion of keratinocytes was expected in the dermis‐side samples due to the absence of a basement membrane. Surprisingly, no invasion occurred in either condition until the 21‐day mark, where a correlation between invasion and the degradation of the basement membrane became evident. Epidermal differentiation, previously shown to be randomized in the earlier model, will be assessed to determine whether the environment can instruct cells.

Grant Funding Source

Research, Creative, and Scholarly Activities Grant
Twist and E47 regulate Snail promoter activity in palatogenesis
Authors: YU Wenli , Serrano Maria , Svoboda Kathy
Published: June 2021

Purpose

Snail is a key transcription factor in EMT (epithelial‐mesenchymal transition). Twist and E‐47 are bHLH (basic helix‐loop‐helix) transcription factors, which binds to E‐boxes and induce EMT. Several E‐boxes were present in Snail promoter region. Previously we showed downregulation Twist or Snail blocked EMT in palatogenesis. The objective of this study is to explore the interactions between these transcription factors and examine if Twist and E47 regulate Snail expression.

Methods

1.7K, 1.5K, 1.0K, 0.5k Snail promter‐luciferase constructs were co‐transfected with Twist, E47 overexpressing plasmids in MDCK cells. SV40‐Renilla luciferase plasmid was transfected to control the efficiency of transfection. Expression of Firefly and Renilla luciferases were analyzed 24h post‐transfection. Dual‐luciferase reporter assay was used to measure ‐ promoter activity according to the manufacturer's instructions.

Results

In MDCK cells, Twist and E47 were required for different promoter fragment activation. Twist activated the 1.7K promoter to 12.5 fold and E47 activated the 1.0K promoter to 9 fold over controls. When cells overexpressed both Twist and E47, the 1.0K promoter activity increased to 10 fold.

Conclusion

Twist and E47 may be the upstream factors that regulate Snail expression by binding to the Snail promoter to induce EMT.

Grant Funding Source

March of Dime