bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of moisture tent on the ion content of nasal fluid in patients with cystic fibrosis
Authors: Roomans Godfried Maarten , Kozlova Inna , Hjelte Lena
Published: June 2021

Sleeping in a moisture tent was used as treatment for cystic fibrosis (CF) patients, in order to hydrate the viscous airway mucus. The efficiency of this method has been questioned. The effect of the procedure on the ion content of nasal airway surface liquid (ASL) in CF‐patients and healthy controls was studied. CF‐patients and controls spent a night (8h) in a moisture tent; samples of nasal ASL were taken before and after the stay in the tent, and then at each hour during 4h. Samples of nasal ASL were collected in Sephadex beads that were analyzed by X‐ray microanalysis. The ion concentrations in nasal ASL of CF patients (before entering the tent) were significantly higher than the levels in controls. During the stay in the tent, the ion content decreased markedly, both in CF‐patients and controls. After leaving the tent the ion levels in the nasal ASL increased to values higher than before entering the tent. The salt content of ASL is relevant in CF, since ASL contains anti‐bacterial proteins sensitive to the salt concentration. The decrease in ion content, caused by spending time in a moisture tent may be too short‐lived to have a significant effect. Supported by the Swedish Heart‐Lung Foundation.
Effect of hyperosmolar solutions on the permeability of airway epithelium
Authors: Roomans Godfried Maarten , Nilsson Harriet , Dragomir Anca , Ahlander Anders , Johannesson Marie
Published: June 2021

Though inhalation of hyperosmotic solutions has been used in treatment of cystic fibrosis (CF) or asthma patients, the mechanism behind the effect of this treatment is unclear. Possibly, the solutions open tight junctions (TJ), leading to increased airway surface liquid volume and easier removal of mucus. Normal or CF airway epithelial cell lines were exposed to hyperosmolar salt or sugar solutions (295–700 mOsm). Transepithelial resistance (TEER) was measured as indicator of the tightness of the epithelium. TEER was significantly higher in CF cell cultures compared to normal cells. Electrolyte solutions caused a significant but (partly) reversible decrease in TEER from 450 mOsm on; sugar solutions caused a decrease from 700 mOsm on. CF cells recovered better than control cells. Immunocytochemistry showed weaker staining for TJ proteins in samples exposed to hyperosmotic solutions. The ultrastructure revealed increased numbers of open TJ. Hyperosmotic solutions cause opening of TJ in airway epithelial cell cultures with electrolytes having stronger effects than non‐electrolytes; TJs are tighter in CF cells than in normal cells. Supported by the Swedish Research Council and the Heart‐Lung Foundation.
Hypoxic preconditioning ameliorates intestinal epithelial barrier defect and bacterial translocation in ischemia/reperfusion injury
Authors: Lu Yen Zhen , Wu Chi Chin , Chen Chau Fong , Yu Linda C.H
Published: June 2021

Intestinal epithelium and mucosal immune cells are defensive barriers against enteric microbial influx. Intestinal ischemia / reperfusion (I/R) induced mucosal injuries associated with bacterial translocation. Hypoxic preconditioning (HPC) protects heart and brain against ischemic insults. The aim of this study was to assess the effect of HPC in preventing intestinal I/R‐induced barrier defects. Male Wistar rats were raised either in normoxic air or in a hypobaric chamber (380 mmHg) for 21 days (17 hrs/day) for HPC. I/R rats received 20 min occlusion of superior mesenteric artery and 1 hour of reperfusion. Sham‐operated rats served as controls. In normoxic rats, I/R induced destruction of ileal mucosa, e.g. villi shortening and epithelial detachment, as well as increase of the bacterial colony forming units (CFU) in the liver and spleen. Rats exposed to HPC prior to I/R showed a reduction of mucosal damages and lowered CFU in internal organs. Moreover, elevated levels of myeloperoxidase activity and cytokine‐induced neutrophil chemoattractant‐1 were found in ileum of HPC I/R rats compared to those of normoxic I/R rats. In conclusion, HPC ameliorated I/R‐induced intestinal barrier defect and bacterial translocation. This protective mechanism may be partly attributed to enhanced neutrophil activation in the intestinal mucosa.
Imbalance angiogenesis regulators: A novel explanation for poor ulcer healing
Authors: Deng Xiaoming , Szabo Sandor , Khomenko Tetyana , Xiong Ximing , Sandor Zsuzsanna , Osapay Klara , Jadus Martin R. , Tostanova Ganna , Chen Longchuan
Published: June 2021

We tested the hypothesis that an imbalance in angiogenic and anti‐angiogenic factors may play an important role in development and delayed healing of duodenal ulcers induced by duodenal ulcerogens cysteamine or propionitrile in rats. Scrapings of proximal duodenal mucosa were collected to study gene expression of collagen XVIII by microarray and Real‐time PCR, proteolytic activity of matrix metalloproteinases (MMP) by zymography and ELISA, and levels of angiostatin, endostatin and VEGF by Western blot and ELISA. Additional groups of rats were daily given endostatin for 3 and 7 days after cysteamine to test the effect of endostatin on ulcer healing. The results showed increased endostatin and angiostatin accompanied by elevated MMP2 activity in the early or late stages of ulceration, when VEGF levels were also markedly increased. Endostatin treatment significantly delayed duodenal ulcer healing. Conclusions: 1) Both anti‐angiogenic factors angiostatin/endostatin and angiogenic factor VEGF were upregulated in experimental duodenal ulceration. 2) Excess of anti‐angiogenic factors, e.g., endostatin and angiostatin may be one of important mechanisms in impaired ulcer healing. 3) Thus, an imbalance of angiogenesis regulators seems to be a new element in ulcer pathogenesis and healing.

(The present study was supported by a Department of Veterans Affairs, Veterans Health Administration Merit Review grant and by contributions from CPRC, Inc.)
CSR1 Inducing Cell Death Through Inactivation Of CPSF3
Authors: Luo Jianhua , Zhu Zehua , Yu Yan
Published: June 2021

CSR1, a newly characterized tumor suppressor gene located in the 8p21 region, undergoes hypermethylation of its promoter and exon 1 regions in over 30% of prostate cancers. Re‐expression of CSR1 inhibits cell growth and induces cell death, but the mechanisms of CSR1‐mediated tumor suppressor activity are not clear. In this study, a Yeast‐two hybrid screening identified proteins interacting with the C‐terminus of CSR1. The cleavage and poly‐adenylation specific factor 3 (CPSF3), an essential component for converting hnRNA to mRNA, bound with CSR1 C‐terminus with high affinity. Analysis of a series of GST‐CSR1 mutants found the binding motifs for CPSF3 located at sequence between amino acids 440–513. The interaction between CSR1 and CPSF3 induced CPSF3 translocation from nucleus to cytoplasm, resulting in nuclear inhibition of the polyadenylation activity both in vitro and in vivo. Down‐regulation of CPSF3 using siRNA induced cell death in a manner similar to those induced by CSR1 expression. CSR1 mutant unable to bind to CPSF3 did not alter CPSF3 subcellular distribution, and did not inhibit its polyadenylation activity nor induce cell death. In summary, CSR1 appears to induce cell death through a novel mechanism by hijacking a critical RNA processing enzyme.
The Pckcr Rat Model of Autosomal Recessive Polycystic Kidney Disease
Authors: Gattone Vincent H. , Sinders Rachel , Roberts Robin , Owens Dwight R.
Published: June 2021

The Pck gene in PCK rats is orthologous for the human PKHD1 gene responsible for Autosomal Recessive Polycystic Kidney Disease. This model was rederived from a colony at Charles River Japan and is now commercially available from Charles River Labortories, Wilmington MA. Basic aspects of its disease were previously described from private colonies. The present study provides a comprehensive characterization of the pathology. Breeder rats were fed Purina 5879 diet, While at IU, fed Teklad 7002 with water provided ad libitum. Male and female rats were evaluated from 4 – 35 weeks of age for renal and hepatic pathology and function. After 18 weeks of age, renal pathology (kidney weight, KW as % of body weight) and development of renal dysfunction (proteinuria, BUN and serum creatinine) was more severe in males, and liver pathology (LW as % of body weight and fibrocystic volume) was more severe in females. Both males and females exhibited some evidence of hyperlipidemia and increased total bilirubin, especially late. Some older females also exhibited elevated AST and ALT and had a tendency for a late stage increase in splenic size (suggesting the development of portal hypertension).
Proteomic profiles of liver microsomes from mice treated with the hepatotumorigenic conazole, triadimefon
Authors: Ortiz Pedro A. , Chen Pei‐Jen , Moore Tanya , Nesnow Stephen , Winnik Witold M.
Published: June 2021

Triadimefon is a triazole‐based conazole fungicide used in agriculture. This conazole is hepatotoxic and hepatotumorigenic in mice. As part of a large study aimed at determining the mechanism(s) of action of tumorigenic conazoles we have performed a comparative proteome analysis of liver microsomes from mice exposed to triadimefon. Liver microsomes from male CD‐1 mice exposed to tumorigenic doses of triadimefon for 4 days by intraperitoneal injection were prepared. Protein level changes were examined by a 2D difference gel electrophoresis (DIGE) and were identified by nanospray‐liquid chromatography‐tandem mass spectrometry (LC‐MSMS). Seventy‐eight proteins that have shown changes in expression have been identified; the pathways affected by these proteins are the metabolism of xenobiotics by cytochrome P450, proteasome, and the metabolism of glutathione, nitrogen, and glutamate. In addition, we have developed a reverse‐phase protein method to improve mass spectral detection and identification of microsomal cytochrome P450s and UDP‐glucuronosyltransferases proteins, which is being used to monitor changes of hydrophobic proteins that are poorly represented in 2‐D gels. Correlation of the conazole effects on protein expression with toxicology and genomic data will contribute to the elucidation of its mode of action.

This is abstract does not necessarily reflect EPA policy.
Freeze‐Frame Click‐Chemistry Synthesis on a Soluble Alpha‐7 Nicotinic Acetylcholine Receptor (nAChR) Ligand Binding Domain
Authors: Nemecz Akos , Talley Todd , Fotsing Joseph , Weide Timo , Grimster Neil , Ho Kwok‐Yiu , Fokin Valery V. , Sharpless K. Barry , Taylor Palmer
Published: June 2021

The cys‐loop family of ligand‐gated ion channels plays an important role in the chemical‐to‐electrical transduction of neuronal signaling. The well‐studied α7‐nAChR may be linked to nicotine addiction, and shows possible genetic linkages to such diseases as Alzheimer's and schizophrenia. The discovery of a homologous soluble snail acetylcholine binding protein (AChBP) provided a structural surrogate of the extracellular ligand‐binding domain; however, the binding protein, despite the requisite ligand properties, shows only 30% residue identity with the nicotinic receptor family. Thus, creating a soluble human extracellular domain with the residues of various receptor subtypes would greatly aid in finding selective drug targets. Accordingly, we systematically mutated soluble Aplysia californica towards the α7‐nAChR, simultaneously analyzing binding affinities. Key expressing constructs were subsequently characterized using classical α7‐nAChR selective ligands such as α‐bungarotoxin, (+)‐epibatidine, and methyllycaconitine. After the characterization and determination of a soluble template freeze‐frame “click” chemistry was applied using weak binding chemical building blocks to create a selective moderate affinity ligand.

Support: R37‐GM18360; U01‐NS043063
NO‐dependent Molecular Mechanism in Relaxing Effect of Cilnidipine (CIL) in Human Internal Mammary Arteries (IMA)
Authors: Fan Li , Xiao Xiao‐qiu , Grove Kevin , Huang Yu , Yang Qin , Furnary Anthony , He Guo‐Wei
Published: June 2021

CIL, the novel long‐lasting L/N‐type dihydropyridine calcium channel blocker, induces both endothelium‐dependent and independent relaxation in human IMA. We investigated the underlying molecular mechanism on nitric oxide (NO)‐cyclic guanosine monophosphate (cGMP) pathway. The expression level of eNOS mRNA in IMA incubated with/without (n=5/6) 3 μ CIL for 30 min followed by 10 μM norepinephrine (NE) stimulation for 4 hrs was assayed by Real‐Time quantitative PCR. The phosphorylation of eNOS at Ser1177 (p‐eNOS) was determined by Western blotting in IMA (n=4) simulated by 10 μM NE for 15 min followed by incubation for 5 min with 3 μM CIL or 50 nM bradykinin. The CIL‐induced relaxation was examined in endothelium‐denuded (n=7) and ‐intact IMA (n=7) pretreated with/without LNNA (300 μM, n=8) and ODQ (3 μM, n=8). CIL 3 μM increased eNOS mRNA expression by 42.4% and p‐eNOS by 55.0% in the NE‐stimulated IMA. Endotheliumdenudation (47.7 ± 7.0%, p < 0.05), L‐NNA (48.6 ± 6.1%, p < 0.05), and ODQ (41.6 ± 3.8%, p< 0.01) remarkably reduced the CIL‐induced relaxation to 40 mM K+‐induced contraction, comparing to endothelium‐intact control (73.9 ± 6.4 %). Thus, CIL‐induced endothelium‐dependent relaxation in human IMA is mediated by NOcGMP signal pathway with strong molecular evidence.

Supported by St. Vincent Foundation, Hong Kong RGC (CUHK4383/03M) & CUHK (4450171, 4450231, 2041305).
Endogenous endothelin modulates arterial responses to vasoconstrictors, nitric oxide and EDHF
Authors: De Mey Jo G. R. , Lemkens Pieter , Hilgers Rob
Published: June 2021

Blockade of NO synthase is widely used to study endothelium‐dependent vasodilatation and to diagnose endothelial dysfunction, but can also promote production of endothelin‐1 (ET‐1). We tested the hypothesis that endogenous ET‐1 interferes with arterial contractile reactivity. Isolated rat mesenteric resistance arteries with (+E) or without endothelium (−E) were studied in the presence of indomethacin (1 μmol/L). Contractile responses to potassium (K+, 6 – 42 mmol/L) and phenylephrine (PHE, 0.16 – 20 μmol/L) did not differ between +E and −E. Acetylcholine (Ach, 1 μmol/L) caused small and large E‐dependent relaxations in the presence of K+ and PHE, respectively. L‐NAME (i) did not cause contraction, (ii) markedly increased contractile responses to K+ and PHE in +E, but not −E and (iii) abolished Ach‐induced relaxation during K+ but did not significantly affect it during PHE. The ETa‐ and ETb‐receptor antagonist bosentan (10 μmol/L) (i) did not cause contraction, (ii) reduced contractile responses in +E but not −E, (iii) abolished the E‐dependent procontractile effect of L‐NAME and (iv) significantly increased Ach‐induced relaxation during K+ and PHE in the absence and presence of L‐NAME. We conclude that endogenous ET‐1 increases vasoconstriction and inhibits EDHF‐like reactivity, especially during blockade of NO synthesis.

Supported by TIPharma contract# T2_108.
Participation of cycooxygenase‐2‐derived products in the development of hypertension in the SHR
Authors: Ibarra Maximiliano , De la Peña‐García Cesar , Gallardo‐Ortiz Itzell , Villalobos‐Molina Rafael , Torres‐Acosta María Fernanda
Published: June 2021

Several studies have suggested cyclooxygenase‐2 (COX‐2) plays a significant role in the development of renovascular hypertension. Therefore, the present study was designed to examine the effect of celecoxib a selective COX‐2 inhibitor on the development of hypertension in SHR. Four‐weeks‐old male SHR were divided into four groups: a) Control, b) Celecoxib 0.5 mg/kg/day, c) Celecoxib 1 mg/kg/day, and d) Captopril 30 mg/kg/day. SHR were treated by 10 weeks with these inhibitors in the drinking water. Body weight, heart rate (FC) and systolic blood pressure were evaluated. Kidneys were mounted into an isolated organ chamber in order to measure the contractile response to phenylephrine (alpha‐1 adrenoceptor agonist). At the end of the captopril and celecoxib treatment, systolic blood pressure was reduced in SHR treated (control: 169.7 ± 5.12 mm Hg, celecoxib 0.5 mg/kg: 147.9 ± 5.21 mm Hg, celecoxib 1 mg/kg: 138.7 ± 4.91 mm Hg and captopril 117. 22 ± 3.73). The body weight and heart rate were not modified by treatments. The vasoconstrictor renal response to phenylephrine was lower in SHR than WKY rats. Captopril and celecoxib treatments normalized the contractile response to phenylephrine. These results suggest cyclooxigenase‐2‐derived metabolites could be involved in the development of hypertension in the SHR.

Supported by PAPIIT IN218406
A comparative study of the protective effect of simvastatin and moxonidine against renovascular toxicity induced by smoking in rats
Authors: Nasr Magda A , El‐gowilly Sahar M , El‐Mas Mahmoud M
Published: June 2021

Oxidative stress plays a major role in smoking‐induced renal injury. We conducted biochemical and functional studies to assess the protective effect of the lipid lowering drug simvastatin and the antihypertensive agent moxonidine against renovascular toxicity caused by smoking. Six groups of rats (n=8 each) were used: (i) control, (ii) cigarette smoke (CS, twice a day, 30 min each, 6 weeks), (iii) simvastatin (2×5 mg/kg/day, s.c), (iv) moxonidine (2×1 mg/kg/day, i.p.), (v) CS+simvastatin, and (vi) CS+moxonidine. CS exposure increased plasma urea and creatinine and renal malondialdyde and decreased renal glutathione peroxidase, superoxide dismutase and catalase activities. In the isolated perfused phenylephrine‐preconstricted kidney, CS attenuated vasodilator responses to carbachol or isoprenaline in contrast to no effect on papaverine vasodilation. Simvastatin or moxonidine treatment virtually abolished the deleterious effects of CS on lipid peroxidation, antioxidant activity, and urea and creatinine levels. Further, both protective drugs ameliorated the attenuating effect of smoking on carbachol renal vasodilations; isoprenaline vasodilation was improved only by moxonidine. Conclusions: while moxonidine and simvastatin are equipotent in correcting renal oxidative damage caused by smoking, moxonidine appears to be more effective in rectifying vascular reactivity abnormalities.
Resistance to endotoxic shock in mice lacking natriuretic peptide receptor‐A
Authors: Panayiotou Catherine Maria , Stidwill Raymond , Taylor Valerie , Singer Mervyn , Hobbs Adrian J.
Published: June 2021

Natriuretic peptides (NP) play key roles in cardiovascular homeostasis via guanylyl cyclase‐linked NP receptors (NPR). Plasma NP levels are increased in patients with septic (endotoxic) shock and correlate with the cardiac dysfunction observed. Yet, a definitive role for NP in the pathogenesis of septic shock has not been established. NO production by inducible NO synthase (iNOS) occurs in response to infection and aberrant regulation of iNOS underlies several cardiovascular disorders. Herein, we have used NPR‐A knockout (KO) mice to investigate a potential role for NP in regulating iNOS expression/activity and (cardio)vascular dysfunction during endotoxic shock.

In vivo lipopolysaccharide (LPS) administration caused a greater suppression of responses to U46619, ANP, ACh and SPER‐NO in aortae from WT versus NPR‐A KO mice. This differential vascular function was paralleled by reduced iNOS expression (lung: −38.5±6.3%*, aorta: −80.3±10.2%*; *P<0.05 v WT) and activity (plasma [NOx]: WT 597.7±35.0 μM, NPR‐A KO 387.2±22.4* μM). In accord, LPS‐induced hypotension was enhanced in WT versus NPR‐A KO mice (δmax MABP: WT − 29.7±3.8 mmHg, KO −10.5±6.3 mmHg*).

Thus, NPR‐A activation is an important determinant of iNOS expression and (cardio)vascular dysfunction during endotoxic shock and the NP system may represent a novel target in the treatment of this life‐threatening condition.

Supported by the Wellcome Trust
Flecainide, but not Ranolazine, Causes Use‐ and State‐dependent Slowing of Ventricular Conduction in Female Rabbit Hearts
Authors: Wu Lin , Li Hong , Rajamani Sridharan , Shryock John C. , Belardinelli Luiz
Published: June 2021

Background: Flecainide (Flec), an open state Na+ channel (NaCh) blocker, increases the QRS interval and mortality in post‐myocardial infarction patients. Ranolazine (Ran) interacts with the inactivated state of NaCh. This study compares the effects of Flec and Ran on the QRS interval of female rabbit isolated hearts paced at various rates and perfused with 4 mM and8 mM potassium solutions to mimic conditions whereby myocardium is depolarized.

Methods: Hearts were instrumented for recording of 12‐lead ECG and paced at rates of 1, 3 and 4 Hz after blockade of AV nodal conduction by thermoablation.

Results: Flec (0.1–30 μM, Fig A) and Ran (10–600 μM, Fig B) rate‐ and concentration‐dependently increased the QRS interval. Increases of K+ from 4 to 8 mM and rate of pacing augumented the effect of 2 μM Flec to increase the QRS interval, whereas 10 μM Ran did not increase the QRS interval under any condition tested (Table).

Conclusion: Compared to Flec, Ran, at therapeutic concentrations, did not prolong the QRS interval and thereby it should not slow ventricular conduction.
Red wine polyphenol resveratrol regulates Heme Oxygenase‐1 conversely for the disruption of Caveolin‐1/eNOS platform and improve left ventricular function in Ischemic Hypercholesterolemic Myocardium
Authors: Penumathsa SureshVarma , Koneru Srikanth , Samuel Samson Mathews , Zhan Lijun , Menon Venugopal P , Maulik Nilanjana
Published: June 2021

Impaired angiogenesis and vascular integrity due to reduced NO is a hallmark of the hypercholesterolemic (HC) myocardium. In this study, we investigated the mechanism behind impaired endothelial cell function in invivo myocardial infarction (MI) model of HC rats & further validated our results in Heme Oxygenase‐1 (HO‐1) transgenic (Tg) mice and examined the therapeutic efficacy of resveratrol (RSV) in HC. The rats were divided into 3 groups: control (C); rats maintained on a high cholesterol diet (5%) for 8 weeks (HC); & HC + oral administration of RSV (20mg/kg) for 2 weeks (RSV). Echocardiography showed improved ejection fraction & fractional shortening in the RSV compared to HC. Protein analysis showed increased expression of Cav‐1 & decreased phosphorylation of eNOS (p‐eNOS), HO‐1, VEGF along with reduced capillary & arteriolar density in HC compared to C & the converse results were observed on RSV treatment. These results showed that the increased Cav‐1 might result in increased Cav‐1/eNOS association, thus decreasing eNOS availability which might result in decreased VEGF expression in HC. As expected, HO‐1 Tg mice showed decreased expression of Cav‐1, increased p‐eNOS and VEGF compared to wild type. Thus our result showed that targeted disruption of Cav‐1/eNOS association by increased expression of HO‐1 play a vital role in NO production & VEGF expression thus reducing ventricular remodeling in HC myocardium.
The hypothalamic nonapeptides oxytocin and arginine vasopressin promote cardiac myocyte survival in rats with heart failure
Authors: Salikova Svetlana P , Stadnikov Alexander A
Published: June 2021

The current study examines the hypothesis that the hypothalamic nonapeptides oxytocin (OT) and arginine vasopressin (AVP) are involved in morphogenic modulation of LV remodeling during development of heart failure in rats. Experimental heart failure (HF) has been induced in male Wistar rats (180–230g) by i.m. injections of 0.1 ml 1% mesatonum (phenylephrine) solution and subsequent swimming until exhaustion for 10 days. The small pieces of LV myocardium from the hearts of control and HF rats were explanted for 3 days in medium 199 supplemented with 20 % FBS. The explants were treated with OT (0.5·10−5 IU) or AVP (0.5·10−5 IU) for 72 hrs. The LV myocardium and the hypothalamic‐hypophyseal neurosecretory system (HHNS) were analyzed using light and electron microscopy. The presence of apoptotic cells were detected using TUNEL technique and p53 immunohistochemistry. We found that OT and AVP similarly reduced the number of necrotic and apoptotic cardiac myocytes compared to untreated explants (~4% and ~2% vs. ~15% and ~10%, respectively, P<0.05). However, although neurosecretory cells of the paraventricular and supraoptic nuclei of the hypothalamus were activated in rats with HF, there were signs of inhibition of release of OT and AVP from neurohypophysial terminals. Thus, we suggest that OT and AVP demonstrate a cardioprotective potential by preventing the cardiac myocyte death and that the inhibition of their release from the HHNS during development of heart failure might play a modulatory role in progression of LV remodeling.
Comparison of nasal proteins adducted by reactive metabolites of naphthalene (NA) in rat and rhesus macaque using 2 dimensional gel electrophoresis and MALDI TOF/TOF
Authors: Shields Christina E DeStefano , Morin Dexter , Buckpitt Alan
Published: June 2021

NA is a volatile pollutant classified by NTP as a ‘reasonably anticipated human carcinogen’ based on concentration‐dependent, rare olfactory epithelial neuroblastomas and respiratory (nasal) epithelial adenomas in rats (NTP 2000). However, epidemiologic studies have not provided a convincing link between exposure and cancer. Reactive metabolites of NA bind covalently to proteins in target cells; good correlations between adducted protein levels and injury have been observed (Drug Metab Rev 34:791, 2002). Accordingly, these studies compared adducted proteins in nasal epithelium of rats and rhesus macaques to develop biomarkers mechanistically associated with toxicity which could be applied to human populations. Rat and monkey nasal tissues were incubated with 14C ‐NA, proteins were resolved by 2D gel electrophoresis, and labeled proteins were imaged and subsequently identified. The adducted protein patterns between rat and rhesus macaque are remarkably similar. 45 adducted proteins identified in rat olfactory incubations include those involved in cell structure, transcription, protein synthesis/folding, ATP synthesis, and cell signaling; similar work is ongoing in monkeys. These studies suggest that several of these adducts may be good biomarker targets for assessing the potential for NA toxicity in humans. Supported by EPRI, NIEHS 04311, 04699 and RR00169.
The Human Equilibrative Nucleoside Transporter‐3 Is a Mitochondrial Transporter That Transports anti‐HIV Dideoxynucleoside Drugs
Authors: Govindarajan Rajgopal , Blonski Mary , Tse Chung‐Ming Ming , Wang Joanne , Unadkat Jashvant D.
Published: June 2021

The human equilibrative nucleoside transporter‐3 (hENT3) has been reported as a putative pH‐dependent lysosomal nucleoside transporter. However, our studies on immunolocalization and functional characterization of endogenous hENT3 in several different human cell lines suggest that it is a mitochondrial nucleoside transporter except in placental cells (JAR, JEG3) where endogenous hENT3 is also expressed on cell surface. Tagging the N‐terminus of hENT3 with YFP misdirected the chimeric protein to the lysosomes, while tagging HA at its C‐terminus directed the protein to cell surface and mitochondria with increased uptake of nucleosides via plasma membrane. When expressed in xenopus oocytes, hENT3‐mediated adenosine transport was stimulated by acidic pH (pH 6.5). Taken together with hENT3‐gene silencing data (in JAR cells), our results show that hENT3 transports nucleobases, nucleosides, nucleotides and anti‐HIV dideoxynucleoside drugs into cells as well as mitochondria. Based on these data, we propose that hENT3‐mediated transport of dideoxynucleoside drugs (e.g. didanosine) into cells and subsequently into mitochondria contributes to the mitochondrial toxicity of these drugs. Support by NIH GM054447.
Human MRP2 polymorphisms and their impact on substrate transport
Authors: Megaraj Vandana , Gerk Philip , Kim Richard , Vore Mary
Published: June 2021

Multidrug resistance protein 2 (MRP2/ABCC2) mediates the biliary excretion of endo‐ and xenobiotic organic anion conjugates. Single nucleotide polymorphisms (SNPs) in the MRP2 gene contribute to variability in drug response and adverse effects between individuals and different ethnic populations. We characterized the transport of wild type MRP2 (WT) and two MRP2 SNP variants, M4 (A1450T) and M5 (V417I) for the substrates LTC4 (leukotrieneC4), E23G (estradiol‐3‐glucuronide), TUDC (tauroursodeoxycholic acid) and E217G (estradiol‐17β‐lucuronide)

Methods

: WT, M4 and M5 were expressed in Sf9 insect cells and kinetic parameters (Km, μM; Vmax, pmol/mg−1.min−1; Hill coefficient [HC]) of ATP dependant transport of 3H‐LTC4, 3H‐E23G, 3H‐TUDC and 3H‐E217G determined in Sf9 plasma membrane vesicles. Transport activity of M4 and M5 was normalized to WT protein expression.

Results

Conclusion

The SNP in the nucleotide‐binding domain of M4 decreased the Vmax by 50% for LTC4, E23G and TUDC and decreased the HC for TUDC and E217G. The SNP in the transmembrane domain 2 of M5 minimally influenced the Vmax, but increased the Km 2–3 fold, increased the HC for TUDC and decreased the HC for E217G. (HD 58299)
Concomitant exposure to HIV and anti‐HIV drugs augment drug‐efflux functions in Human brain Microvascular endothelial cells (BBB‐ECs).
Authors: Roy Upal , Mondal Debasis
Published: June 2021

The central nervous system (CNS) is a sanctuary site for HIV‐1 and inability to transport highly active antiretroviral therapy (HAART) drugs through blood‐brain‐barrier (BBB) into the CNS is a serious clinical problem. Both the HIV‐1 protease inhibitors (HPIs) and nucleoside reverse transcriptase inhibitors (NRTIs) used in HAART, are effluxed from BBB‐endothelial cells (ECs) via ATP binding cassette (ABC) transporters such as P‐glycoprotein (P‐gp) and multidrug resistance proteins (MRPs). However, it is not known weather exposure to the virus and/or the anti‐HIV drugs can increase drug‐efflux functions. Our in vitro studies using BBB‐ECs showed that exposure to HIV‐1 significantly (p<0.01) upregulates both P‐gp gene expression (18 fold) and drug‐efflux function (2 fold) within 48 hrs. This P‐gp mediated drug‐efflux function was further increased (3 fold p<0.05) by concomitant exposure of the virus exposed BBB‐ECs to the anti‐HIV drugs Saquinavir and Zidovudine. Furthermore, specific involvement of P‐gp was confirmed using 3H‐Sequinavir retention in HIV‐1 exposed BBB‐ECs in the presence of P‐gp specific inhibitor (virapamil). The P‐gp induction in HIV‐1 infected microenvironments clearly indicated its role in drug inefficacy to the CNS. A thorough understanding of the regulation of ABC‐transporters in BBB‐ECs may implicate therapeutic strategies to inhibit HAART drug‐efflux from the brain.
GPCR proteomics: Mass spectrometric and functional analysis of histamine H1 receptor after baculovirus‐driven and in vitro cell free expression
Authors: Leurs Rob , Sansuk Kamonchanok , de Grip Wim , Booth Raymond , Hensbergen Paul
Published: June 2021

During the past decades, MS analysis is standing out as the method of choices to determine protein‐protein interaction and ligand‐protein interactions. However, due to the low amount of purified protein obtained and the hydrophobic nature of GPCRs, limited information is available on ligand‐GPCR interaction. Here we reported the first successful use of MS analysis to directly reveal the primary structure of hH1R protein, obtained from cell free and/or baculovirus expression system. More than 80% of protein sequence, including five of TM domains, was covered by our optimized digestion procedure, prior to the MS analysis. This approach also allows us to further study the protein‐ligand interaction, by covalently labeling of the hH1R protein with irreversible histaminergic compounds. In combination with data obtained through homology modeling and site directed mutagenesis, these approaches will enhance our knowledge regarding the tertiary structure of this prototype GPCR and its interaction with small molecules.

Partly supported by NIH grant R01 MH068655
Prediction of individual variation in thiopurine response phenotypes from genome‐wide association studies
Authors: Li Fang , Kalari Krishna R. , Fridley Brooke L. , Batzler Anthony , Jenkins Gregory D. , Schaid Daniel J. , Wang Liewei
Published: June 2021

Thiopurine drugs such as 6‐mercaptopurine (6‐MP) and 6‐thioguanine (6‐TG) are effective immunosuppressants and antineoplastic agents. We used a genome‐wide approach to identify genes that might contribute to individual variation in thiopurine response. Basal gene expression profiles, genome‐wide SNP data, and 6‐MP and 6‐TG Cell growth inhibition (GI50) values were generated for 200 Coriell Institute “Human Variation Panel” lymphoblastoid cell lines. Gene expression and genome‐wide SNPs were correlated with GI50 values to identify candidate genes that might contribute to thiopurine response. We identified 104 expression probes and 161 SNPs, as well as 315 gene probes and 579 SNPs that correlated significantly with GI50 values for 6‐MP and 6‐TG, respectively (P<10−5 and 10−6, respectively). Three genes, CD83, TXNDC13 and SNAP23, contained SNPs that contributed significantly to both 6‐MP and 6‐TG cytotoxicity and were also significantly associated with expression levels (P<10−4), suggesting a “three‐way model” involving genotype, gene expression and cytotoxicity. GBP5, a gene with an expression level that correlated negatively with 6‐MP and 6‐TG GI50 values, sensitized leukemia cells when overexpressed. Functional studies of the remainder of the candidate genes are being performed. These results may enhance our ability to predict thiopurine response phenotypes. NIH GM28157.
Integrated cellular bone homeostasis model for denosumab pharmacodynamics in multiple myeloma patients
Authors: Mager Donald E. , Verma Anshu
Published: June 2021

The purpose of this study is to integrate a cellular bone homeostasis model with the pharmacokinetics (PK) of denosumab, an inhibitor of receptor activator of nuclear factor‐κB ligand, to characterize the time course of serum N‐telopeptide (NTX), a bone resorption biomarker, following single escalating doses in multiple myeloma (MM) patients. Mean PK and serum NTX temporal profiles were extracted from the literature. Nonlinear denosumab PK profiles were well described by a model that includes rapid binding of the drug to its pharmacological target. These PK profiles were integrated into a previously reported theoretical cellular model of osteoblast‐osteoclast interactions. Serum NTX concentrations were linked to a resorbing active osteoclast (AOC) pool by a nonlinear transfer function. Reasonable fits were obtained for the NTX profiles from maximum likelihood estimation using the final model. Transfer function parameters, including the basal NTX level independent of AOC and the AOC concentration producing 50% of maximal NTX production, were estimated with good precision as 5.7nM and 1.2x10−5pM. In summary, an integrated cellular model was developed to quantitatively describe clinical biomarker data and may also provide a means for testing hypotheses on the role of cellular system variables in controlling drug response. This study was funded by Grant GM57980 and a postdoctoral fellowship from Amgen, Inc.
Single nucleotide polymorphism (SNP) discovery of human trace amine associated receptors TAAR1 and TAAR9 and their possible role in vasomotor tone
Authors: Tang Chih‐Min , Salem Rany , Siecke Neil , Fung Maple , O’Connor Daniel , Insel Paul
Published: June 2021

Responses to trace amines (tyramine, β‐phenylethylamine, tryptamine, and octopamine found in tissues in low nM concentrations) are mediated by trace amine‐associated receptors (TAARs), which are composed of nine G protein‐coupled receptors. Because little is known about genetic variations in TAARs, we assessed for SNPs in the coding region of TAAR1 and TAAR9 from 52 subjects [28 normotensive (NT)/ 24 hypertensive (HT), 42 male/10 female, 29 Caucasian/23 African American, mean age 47±2]. We identified six TAAR1 SNPs (three found only in HTs) and six TAAR9 SNPs including AAA‐>TAA in Lys61 that causes a premature stop codon. Analysis of the association of TAAR1/TAAR9 SNPs and venoconstriction in 50 healthy individuals [30 male/20 female, mean age 25±7] in response to tyramine infusion into dorsal hand veins revealed that subjects who have one of two TAAR9 SNPs (Asn3Asn or Ala278Thr) have more vasoconstriction than do wild‐type individuals. In spite of its high allelic frequency (22%), the TAAR9 codon 61 SNP was not associated with venoconstrictor responses. These results indicate that both TAARs are polymorphic but most SNPs (other than TAAR9 codon 61) are rare. The data suggest that normotensive and hypertensive subjects have differential expression of TAAR1 and TAAR9 SNPs and that particular TAAR9 SNPs (e.g., Asn3Asn and Ala278Thr) may contribute to enhanced venoconstrictive responses. (Supported by NIH grants)
Deciphering the association between up‐regulation of glucose regulated protein of Mr‐78 kDa (GRP78) and hypersensitivity to cisplatin employing comparative genomics
Authors: Gaddameedhi Shobhan , Chatterjee Satadal
Published: June 2021

We previously demonstrated an excellent association between the induction of GRP78 and hypersensitivity to alkylating/platinating agents. However, the mechanisms of this association are still not clear. GRP78 is a strong Ca++ binding chaperone protein residing in the endoplasmic reticulum (ER). Thus, GRP78 induction can potentially alter Ca++ homeostasis in the ER resulting in alteration in the Ca++‐dependent signaling pathways. We verified this hypothesis by examining the transcript levels using human cDNA microarrays and pathway specific macroarrays. A549 human lung cancer cells were exposed to 10 mM 2‐deoxyglucose or 25 nM thapsigargin or 250 nM tunicamycin for 48 h to induce GRP78, followed by 60 μM cisplatin for 2 h. We show that GRP78 induction alone causes a decrease in the transcript levels of DNA repair genes, DNA damage check point genes and an increase in that of apoptotic genes. Further, cisplatin treatment after GRP78 induction is augmenting the mitochondria‐mediated apoptotic cascades through the activation of caspase‐2 and down‐regulation of many genes involved in DNA repair. These results were further confirmed by examining relevant protein levels and enzyme activity. Our study will hence reveal insight into the molecular mechanisms of association between GRP78 induction and hypersensitivity to cisplatin that will ultimately allow strategies for transferring bench side results to the bed.
Correlation between global DNA methylation and retinoic acid and cisplatin sensitivity in testicular cancer stem cells
Authors: Beyrouthy Maroun J. , Kaur Mandeep , Spinella Michael J.
Published: June 2021

Human embryonal carcinoma (EC) are the cancer stem cells (CSC) of testicular germ cell tumors (TGCTs). TGCTs are also highly curable, even when widely metastatic. This study focuses on the use of the active derivative of vitamin A, retinoic acid (RA), and the EC cancer stem cell model to study RA‐mediated alterations in DNA methylation. Comparing global gene expression and DNA methylation in RA and cisplatin sensitive and resistant EC cells suggests that excessive gene silencing via DNA methylation is associated with RA and cisplatin resistance in EC. Further, RA induces global and local changes in DNA methylation in RA sensitive EC cells. This is associated with an abrupt decrease in DNA methyl transferase 3b (DNMT3b), but not other DNMTs. Ongoing studies, including overexpression and siRNA, will determine if DNMT3b and DNA methylation is important in self‐renewal and chemosensitivity of EC cells. We hypothesize that epigenetic reprogramming is an underlying mechanism for RA‐mediated tumor stem cell differentiation and loss of self‐renewal in EC.
Prenatal arsenic exposure altered liver gene expression associated with accelerated atherosclerosis
Authors: States J.Christopher , Singh Amar V , Miller Heather L , Piao Yulan , Ko Minoru S.H. , Knudsen Thomas B
Published: June 2021

Offspring of ApoE‐knockout mice exposed to arsenic in drinking water while pregnant exhibit accelerated and exacerbated atherosclerosis. Microarray analyses of liver mRNAs and microRNAs were performed to investigate the potential role of altered liver development in arsenic induced atherosclerosis. Liver RNAs from newborn and 10 week old mice were analyzed. Principal component analysis indicated that developmental trajectory was altered by arsenic exposure. A 51 gene signature of mRNAs altered by arsenic exposure at both ages was obtained. Nodes of interaction identified by Pathway Architect within this gene set include HSPA8, IgM and HNF4A. DAVID analysis of the overlap of arsenic exposure‐altered mRNAs and putative targets of altered microRNAs suggested that gluconeogeneis and glycolysis pathways were suppressed in newborn mice and pathways for protein export, ribosome, antigen processing and presentation, and complement and coagulation cascades were induced in 10 week old mice. These results support the hypthesis that in utero arsenic exposure induces changes in liver development that may alter liver inflammatory responses contributing to athero accelerated atherosclerosis. (Supported by PHS grants R01ES011314 & P30ES014443, UofL Center for Genetics and Molecular Medicine pilot grant, UofL Collaborative Planning and Development Grant and Intramural Research Program of NIA/NIH)
ANTECEDENT HYDROGEN SULFIDE ELICITS AN ANTI‐INFLAMMATORY PHENOTYPE IN POSTISCHEMIC MURINE SMALL INTESTINE: ROLE OF HEME OXYGENASE‐1
Authors: Yusof Mozow , Peyton Kelly , Kalogeris Theodore , Gaskin F. Spencer , Fay William , Durante William , Korthuis Ronald J.
Published: June 2021

We recently demonstrated that preconditioning with an exogenous hydrogen sulfide donor (H2S) donor (NaHS‐PC) 24 h prior to ischemia and reperfusion (I/R) causes postcapillary venules to shift to an anti‐inflammatory phenotype in C57Bl/6 WT mice such that these vessels fail to support increased postischemic leukocyte rolling (LR) and adhesion (LA). The objective of the present study was to determine whether heme oxygenase‐1 (HO‐1) is an effector of the anti‐inflammatory effects noted during I/R in mice preconditioned with NaHS. I/R induced marked increase in LR and LA, effects that were prevented by NaHS‐PC. Treatment with the HO inhibitor SnPPIX, but not the inactive protoporphyrin CuPPIX, just prior to I/R prevented the anti‐inflammatory effects of antecedent NaHS. We then evaluated the effect of NaHS as a preconditioning stimulus in mice that were genetically deficient in HO‐1 (HO‐1 −/− in the H129 background with appropriate WT controls). NaHS‐PC was ineffective in HO‐1−/− mice. Our data provides a novel effector mechanism by which H2S preconditioning is protective in the microcirculation. Supported by AA14945 and DK43785.
Targeting of calpains by NSAIDs results in reduced cellular migration
Authors: Silver Kristopher , Lillich James D , Freeman Lisa C
Published: June 2021

Chronic treatment with non‐steroidal anti‐inflammatory drugs (NSAIDs) inhibits intestinal epithelial cell migration (IEC‐6). Microarray analysis of IEC‐6 monolayers treated with NSAIDs revealed that mRNA expression of calpain 8, a member of a family of cysteine proteases, is substantially down‐regulated. qRT‐PCR experiments and analysis of protein expression using Western blotting showed that mRNA and protein levels of calpain 2, in addition to calpain 8, are reduced upon treatment with 10 μM indomethacin and NS‐398, NSAIDs that inhibit cell migration. SC‐560, which does not affect cell migration, had no effect on either mRNA or protein levels of any calpain. Functional experiments examining the effect of these NSAIDs on cell migration in the absence or presence of ALLM and ALLN, compounds that inhibit calpains, demonstrated that the effects of indomethacin and NS‐398, but not SC‐560, on migration of IEC‐6 monolayers are additive with ALLM and ALLN. Furthermore, suppression of calpain protein expression using siRNA techniques inhibited migration of IEC‐6 monolayers. These results demonstrate that inhibition of epithelial cell migration by NSAIDs is at least partially the result of interruption of calpain activity.
Immunomodulatory effects of beta‐glucan from Coriolus versicolor on the phagocytosis of macrophages: involvement of SR‐B1 expression
Authors: Son Eunwha , Lim Jung‐Dae , Park Sulkyoung , Kang Nam‐Sung , Do Hang , Pyo Suhkneung
Published: June 2021

Coriolus versicolor has been known to have effective immunomodulatory effects in Asia. Most of these studies have demonstrated that it appears to have significant antimicrobial, antiviral and antitumor properties when used as a supplement to chemotherapy and/or radiotherapy. To further understand the immunomodulatory effects of beta‐glucan, we examined tumoricidal activity, phagocytosis and nitric oxide (NO) production in beta‐glucan‐treated Raw264.7 cells. Our data showed that the treatment of beta‐glucan from C. versicolor induced a remarkable increase of phagocytosis and tumoricidal activity by macrophages. In addition, reverse transcription‐polymerase chain reaction (RT‐PCR) and Griess Reagent assay showed the increase of iNOS mRNA expression and slight induction of NO production at 1ug/ml concentration. We also examined whether SR‐A1 and SR‐B1 are involved in phagocytosis. The present data demonstrated that mRNA expression of SR‐A1 and SR‐B1 was increased respectively. In addition, the increase of SR‐B1 protein expression was observed. Moreover, beta‐glucan treatment resulted in increase of the expression of CK2 and Ikaros in Raw264.7 cells. Taken together, these results suggest that beta‐glucan from C. versicolor enhances phagocytosis via SR‐B1 expression and immunomoulatory effects of beta‐glucan might be related to CK2 and ikaros activation.
In vitro effects of immunomodulators on lymphocyte proliferation and macrophage function in young and old mice.
Authors: Kim Kyungho , Choi Hyesook , Joo Haemi , Lee Hyerim , Park Sulkyoung , Kim Jihye , Jang Seon‐A , Pyo Suhkneung , Son Eunwha
Published: June 2021

The immune system may play an important role in aging and the changes in immune status are associated with treatment of various immunomodulators. We examined the effects of various immunomodulators on in vitro lymphocyte proliferation and macrophage function in young (8 wks) and aged (82 wks) C57BL/6 male mice. The effects of Acrylamide (ACR) and ascorbic acid (AA) on the proliferation of splenocytes from young and old mice were assessed by XTT assay. ACR treatment resulted in the decrease of LPS‐induced proliferation of B lymphocytes and splenocytes in both groups. However, AA enhanced ConA‐induced proliferation of T lymphocytes and splenocytes in both groups. The immunomodulating activity of beta‐glucan was observed only in young mice. The significant decrease of thymus weight and the increase of tumoricidal activity and phagocytosis in macrophages were observed in old mice. In contrast, the tumoricidal activity of microglia was not altered. Our data suggest that the immunostimulating activity of old mice is higher than young mice, whereas the immunoresponsiveness is lower. Moreover, the immunoresponsiveness of LPS‐induced B lymphocytes treated with ACR and ConA‐induced T lymphocytes treated with AA were more significant in old mice. Overall, our results suggest that various immunomodulators have differential effects on young and aged mice and age need to be considered to select proper immunomodulator.